Handbook of Experimental Pharmacology 224Arnold von EckardsteinDimitris Kardassis Editors High Density LipoproteinsFrom Biological Understanding to Clinical ExploitationHandbook of Experimental PharmacologyVolume 224Editor-in-ChiefW. Rosenthal, JenaEditorial BoardJ.E. Barrett, PhiladelphiaV. Flockerzi, HomburgM.A. Frohman, Stony Brook, NYP. Geppetti, FlorenceF.B. Hofmann, MünchenM.C. Michel, IngelheimP. Moore, SingaporeC.P. Page, LondonA.M. Thorburn, Aurora, COK. Wang, BeijingMore information about this series athttp://www.springer.com/series/164Arnold von Eckardstein • Dimitris KardassisEditorsHigh Density LipoproteinsFrom Biological Understandingto Clinical ExploitationEditorsArnold von EckardsteinUniversity Hospital ZurichInstitute of Clinical ChemistryZurichSwitzerlandDimitris KardassisUniversity of Crete Medical SchoolIraklion, CreteGreeceISSN 0171-2004 ISSN 1865-0325 (electronic)ISBN 978-3-319-09664-3 ISBN 978-3-319-09665-0 (eBook)DOI 10.1007/978-3-319-09665-0Springer Cham Heidelberg New York Dordrecht LondonLibrary of Congress Control Number: 2014958300# The Editor(s) and the Author(s) 2015Open Access This book is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction in anymedium, provided the original author(s) and source are credited.All commercial rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation,broadcasting, reproduction on microfilms or in any other way, and storage in data banks. Duplicationof this publication or parts thereof is permitted only under the provisions of the Copyright Law of thePublisher’s location, in its current version, and permission for commercial use must always be obtainedfrom Springer. Permissions for commercial use may be obtained through RightsLink at the CopyrightClearance Center. Violations are liable to prosecution under the respective Copyright Law.The use of general descriptive names, registered names, trademarks, service marks, etc. in thispublication does not imply, even in the absence of a specific statement, that such names are exemptfrom the relevant protective laws and regulations and therefore free for general use.While the advice and information in this book are believed to be true and accurate at the date ofpublication, neither the authors nor the editors nor the publisher can accept any legal responsibility forany errors or omissions that may be made. The publisher makes no warranty, express or implied, withrespect to the material contained herein.Printed on acid-free paperSpringer is part of Springer Science+Business Media (www.springer.com)PrefaceIn both epidemiological and clinical studies as well as the meta-analyses thereof,low plasma levels of high-density lipoprotein (HDL) cholesterol (HDL-C)identified individuals at increased risk of major coronary events. Observationalstudies also found inverse associations between HDL-C and risks of ischemicstroke, diabetes mellitus type 2, and various cancers. In addition, HDLs exertmany effects in vitro and in vivo which protect the organism from chemical orbiological harm and thereby may interfere with the pathogenesis of atherosclerosis,diabetes, and cancer but also other inflammatory diseases. Moreover, in severalanimal models transgenic overexpression or exogenous application of apolipopro-tein Α-I (apoA-I), the most abundant protein of HDL, decreased or prevented thedevelopment of atherosclerosis, glucose intolerance, or tissue damage induced byischemia or mechanical injury.However, as yet drugs increasing HDL-C such as fibrates, niacin, or inhibitors ofcholesteryl ester transfer protein have failed to consistently and significantly reducethe risk of major cardiovascular events, especially when combined with statins.Moreover, mutations in several human genes as well as targeting of several murinegenes were found to modulate HDL-C levels without changing cardiovascular riskand atherosclerotic plaque load, respectively, into the opposite direction asexpected from the inverse correlation of HDL-C levels and cardiovascular risk inepidemiological studies. Because of these controversial data, the pathogenic role,and, hence, the suitability of HDL as a therapeutic target, has been increasinglyquestioned. Because of the frequent confounding of low HDL-C with hypertrigly-ceridemia, it has been argued that low HDL-C is an innocent bystander of (post-prandial) hypertriglyceridemia or another culprit related to insulin resistance orinflammation.These complex relationships are depicted in Fig. 1. It is important to note thatprevious intervention and genetic studies targeted HDL-C, i.e., the cholesterolmeasured by clinical laboratories in HDL. By contrast to the pro-atherogenic and,hence, disease causing cholesterol in LDL (measured or estimated by clinicallaboratories as LDL cholesterol, LDL-C) which after internalization turnsmacrophages of the arterial intima into pro-inflammatory foam cells, the cholesterolin HDL (i.e., HDL-C) neither exerts nor reflects any of the potentially anti-atherogenic activities of HDL. By contrast to LDL-C, HDL-C is only a nonfunc-tional surrogate marker for estimating HDL particle number and size withoutvdeciphering the heterogeneous composition and, hence, functionality of HDL. HDLparticles are heterogeneous and complex macromolecules carrying hundreds oflipid species and dozens of proteins as well as microRNAs. This physiologicalheterogeneity is further increased in pathological conditions due to additionalquantitative and qualitative molecular changes of HDL components which havebeen associated with both loss of physiological function and gain of pathologicaldysfunction. This structural and functional complexity of HDL has prevented clearassignments of molecules to the many functions of HDL. Detailed knowledge ofstructure–function relationships of HDL-associated molecules is a prerequisite totest them for their relative importance in the pathogenesis of HDL-associateddiseases. The identification of the most relevant biological activities of HDL andtheir mediating molecules within HDL, as well as their cellular interaction partners,is pivotal for the successful development of anti-atherogenic and anti-diabetogenicdrugs as well as of diagnostic biomarkers for the identification, treatment stratifica-tion, and monitoring of patients at increased risk for cardiovascular diseases ordiabetes mellitus but also other diseases which show associations with HDL.This Handbook of Experimental Pharmacology on HDL emerged from theEuropean Cooperation in Science and Technology (COST) Action BM0904 entitled“HDL—from biological understanding to clinical exploitation” (HDLnet: http://cost-bm0904.gr/). This COST Action was run from 2010 to 2014 and involvedmore than 200 senior and junior scientists from 16 European countries. HDLnet hasbeen a scientific network dedicated to the study of HDL in health and disease, to theidentification of targets for novel HDL-based therapies, and to the discovery ofbiomarkers which can be used for diagnostics, prevention, and therapy of cardio-vascular disease. HDLnet fostered the cooperation and interaction of EuropeanHDL-researchers, the exchange of information and materials, the training andmacro-vascular diseases micro—vascular diseasesneuro-degenerativediseasescancerreduced prognosis in infection or other acute serious illnessescause?(potentially treatable)reverse causality?(not treatable)Innocent bystander?(not treatable)lipid efflux and transportsignalling effectsdetoxificationanti-oxidationinsulin resistancenegative acute phase reactionCatabolismPoor healthimmunefunctionscellmig-rationvascularbiologydiabetesmellituscholesterolhomeostasiscell Sur-vivalcell proli-ferationoxi-dationcellfunc-tionscelldiffer-entiationhyperinsulinismInflammation, smokinghypertriglyceridemiasomething else?Fig. 1 Possible pathophysiological relationships of low HDL cholesterol with its associateddiseasesvi Prefacepromotion of early career scientists, the gain of technological know-how, and thedissemination of old and new knowledge on HDL to the scientific and medicalcommunity as well as the lay public. In this setting, the chapters of this handbookhave been written by cooperative and interactive efforts of many senior scientists ofthe HDLnet consortium and colleagues from the United States. It is published asopen access through COST funding so that the knowledge on HDL can be spreadwithout limitation.As the chairman and vice-chairman of HDLnet, the editors of this Handbook ofExperimental Pharmacology issue like to thank not only the authors of the22 chapters of this handbook but all members of the COST Action for their engagedparticipation and cooperation. We thank Ms. Zinovia Papatheodorou (seniorAdministrative Officer of the grant holder FORTH, Heraklion) for excellent grantadministrative work in HDLnet, the Science Officers Dr. Magdalena Radwanskaand Dr Inga Dadeshidze, the Administrative Officers Ms Anja van der Snickt andMs Jeannette Nchung (all from COST Office, Brussels, Belgium), as well as the DCRapporteur, Prof. Marieta Costache (Bucharest, Romania), for their excellentsupport and sustained interest in our Action. We gratefully acknowledge AndreaBardelli and Giulia Miotto from COST Publications Office for their help inpublishing this book as an open access Final Action Publication (FAP). Finallywe wish to thank Prof. Martin Michel for his interest and guidance as well asSusanne Dathe and Wilma McHugh from Springer who supported us with patienceand enthusiasm in the production of this book.Zurich Arnold von EckardsteinIraklion Dimitris KardassisPreface vii.AcknowledgementThis publication is supported by COSTCOST is supported by the EU Framework Programme Horizon 2020COST—European Cooperation in Science and Technology is an intergovernmen-tal framework aimed at facilitating the collaboration and networking of scientists andresearchers at European level. It was established in 1971 by 19 member countries andcurrently includes 35 member countries across Europe, and Israel as a cooperatingstate.COST funds pan-European, bottom-up networks of scientists and researchersacross all science and technology fields. These networks, called “COST Actions”,promote international coordination of nationally funded research.By fostering the networking of researchers at an international level, COST enablesbreak-through scientific developments leading to new concepts and products, therebycontributing to strengthening Europe’s research and innovation capacities.COST’s mission focuses in particular on:• Building capacity by connecting high-quality scientific communities throughoutEurope and worldwide• Providing networking opportunities for early career investigators• Increasing the impact of research on policy makers, regulatory bodies, andnational decision makers as well as the private sectorThrough its inclusiveness policy, COST supports the integration of researchcommunities in less research-intensive countries across Europe, leverages nationalresearch investments, and addresses societal issues.Over 45,000 European scientists benefit from their involvement in COST Actionson a yearly basis. This allows the pooling of national research funding and helpscountries’ research communities achieve common goals.ixAs a precursor of advanced multidisciplinary research, COST anticipates andcomplements the activities of EU Framework Programmes, constituting a “bridge”towards the scientific communities of emerging countries.Traditionally, COST draws its budget for networking activities from successiveEU RTD Framework Programmes.COSTMission: COST aims to enable breakthrough scientific developments leadingto new concepts and products. It thereby contributes to strengthening Europe’sresearch and innovation capacities.x AcknowledgementContentsPart I Physiology of HDLStructure of HDL: Particle Subclasses andMolecular Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Anatol Kontush, Mats Lindahl, Marie Lhomme, Laura Calabresi,M. John Chapman, and W. Sean DavidsonHDL Biogenesis, Remodeling, and Catabolism . . . . . . . . . . . . . . . . . . . 53Vassilis I. Zannis, Panagiotis Fotakis, Georgios Koukos, Dimitris Kardassis,Christian Ehnholm, Matti Jauhiainen, and Angeliki ChroniRegulation of HDL Genes: Transcriptional, Posttranscriptional,and Posttranslational . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113Dimitris Kardassis, Anca Gafencu, Vassilis I. Zannis,and Alberto DavalosCholesterol Efflux and Reverse Cholesterol Transport . . . . . . . . . . . . . 181Elda Favari, Angelika Chroni, Uwe J.F. Tietge, Ilaria Zanotti,Joan Carles Escolà-Gil, and Franco BerniniFunctionality of HDL: Antioxidation and Detoxifying Effects . . . . . . . . 207Helen Karlsson, Anatol Kontush, and Richard W. JamesSignal Transduction by HDL: Agonists, Receptors, andSignaling Cascades . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229Jerzy-Roch NoferPart II Pathology of HDLEpidemiology: Disease Associations and Modulators ofHDL-Related Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259Markku J. SavolainenBeyond the Genetics of HDL: Why Is HDL Cholesterol InverselyRelated to Cardiovascular Disease? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285J.A. Kuivenhoven and A.K. GroenxiMouse Models of Disturbed HDL Metabolism . . . . . . . . . . . . . . . . . . . . 301Menno Hoekstra and Miranda Van EckDysfunctional HDL: From Structure-Function-Relationshipsto Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337Meliana Riwanto, Lucia Rohrer, Arnold von Eckardstein, and Ulf LandmesserPart III Possible Indications and Target Mechanisms of HDL TherapyHDL and Atherothrombotic Vascular Disease . . . . . . . . . . . . . . . . . . . . 369Wijtske Annema, Arnold von Eckardstein, and Petri T. KovanenHDLs, Diabetes, and Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . . . 405Peter Vollenweider, Arnold von Eckardstein, and Christian WidmannHigh-Density Lipoprotein: Structural and Functional Changes UnderUremic Conditions and the Therapeutic Consequences . . . . . . . . . . . . . 423Mirjam Schuchardt, Markus Tölle, and Markus van der GietImpact of Systemic Inflammation and Autoimmune Diseaseson apoA-I and HDL Plasma Levels and Functions . . . . . . . . . . . . . . . . 455Fabrizio Montecucco, Elda Favari, Giuseppe Danilo Norata,Nicoletta Ronda, Jerzy-Roch Nofer, and Nicolas VuilleumierHDL in Infectious Diseases and Sepsis . . . . . . . . . . . . . . . . . . . . . . . . . . 483Angela Pirillo, Alberico Luigi Catapano, and Giuseppe Danilo NorataHigh-Density Lipoproteins in Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . 509Olivier MeilhacTherapeutic Potential of HDL in Cardioprotection and Tissue Repair . . . . 527Sophie Van Linthout, Miguel Frias, Neha Singh, and Bart De GeestPart IV Treatments for Dyslipidemias and Dysfunction of HDLHDL and Lifestyle Interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569Joan Carles Escolà-Gil, Josep Julve, Bruce A. Griffin, Dilys Freeman,and Francisco Blanco-VacaEffects of Established Hypolipidemic Drugs on HDL Concentration,Subclass Distribution, and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . 593Monica Gomaraschi, Maria Pia Adorni, Maciej Banach, Franco Bernini,Guido Franceschini, and Laura CalabresiEmerging Small Molecule Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617Sophie Colin, Giulia Chinetti-Gbaguidi, Jan A. Kuivenhoven,and Bart Staelsxii ContentsApoA-I Mimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631R.M. Stoekenbroek, E.S. Stroes, and G.K. HovinghAntisense Oligonucleotides, microRNAs, and Antibodies . . . . . . . . . . . 649Alberto Dávalos and Angeliki ChroniIndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691Contents xiiiPart IPhysiology of HDLStructure of HDL: Particle Subclassesand Molecular ComponentsAnatol Kontush, Mats Lindahl, Marie Lhomme, Laura Calabresi,M. John Chapman, and W. Sean DavidsonContents1 HDL Subclasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Molecular Components of HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.1 Proteome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.1.1 Major Protein Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.1.2 Protein Isoforms, Translational and Posttranslational Modifications . . . . . . . . 192.2 Lipidome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.2.1 Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.2.2 Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.2.3 Neutral Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27A. Kontush (*) • M. Lhomme • M.J. ChapmanNational Institute for Health and Medical Research (INSERM), UMR-ICAN 1166, Paris, FranceUniversity of Pierre and Marie Curie - Paris 6, Paris, FrancePitié – Salpétrière University Hospital, Paris, FranceICAN, Paris, Francee-mail: anatol.kontush@upmc.fr; m.lhomme@ican-institute.org; john.chapman@upmc.frM. LindahlDepartment of Clinical and Experimental Medicine, Linköping University, Linköping, Swedene-mail: mats.lindahl@liu.seL. CalabresiDepartment of Pharmacological and Biomolecular Sciences, Center E. Grossi Paoletti,University of Milan, Milan, Italye-mail: laura.calabresi@unimi.itW.S. DavidsonDepartment of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH45237, USAe-mail: davidswm@ucmail.uc.edu# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_133 The Structure of HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.1 Introduction/Brief History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.2 HDL in the Test Tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.2.1 Discoid HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.2.2 Spherical rHDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333.3 “Real” HDL Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41AbstractA molecular understanding of high-density lipoprotein (HDL) will allow a morecomplete grasp of its interactions with key plasma remodelling factors and withcell-surface proteins that mediate HDL assembly and clearance. However, theseparticles are notoriously heterogeneous in terms of almost every physical,chemical and biological property. Furthermore, HDL particles have not lentthemselves to high-resolution structural study through mainstream techniqueslike nuclear magnetic resonance and X-ray crystallography; investigators havetherefore had to use a series of lower resolution methods to derive a generalstructural understanding of these enigmatic particles. This chapter reviewscurrent knowledge of the composition, structure and heterogeneity of humanplasma HDL. The multifaceted composition of the HDL proteome, the multiplemajor protein isoforms involving translational and posttranslationalmodifications, the rapidly expanding knowledge of the HDL lipidome, thehighly complex world of HDL subclasses and putative models of HDL particlestructure are extensively discussed. A brief history of structural studies of bothplasma-derived and recombinant forms of HDL is presented with a focus ondetailed structural models that have been derived from a range of techniquesspanning mass spectrometry to molecular dynamics.KeywordsHDL • Composition • Structure • Heterogeneity • Proteomics • Lipidomics •Proteome • Lipidome • Post-translational • ModificationsHigh-density lipoprotein (HDL) is a small, dense, protein-rich lipoprotein ascompared to other lipoprotein classes, with a mean size of 8–10 nm and density of1.063–1.21 g/ml (Kontush and Chapman 2012). HDL particles are plurimolecular,quasi-spherical or discoid, pseudomicellar complexes composed predominantly ofpolar lipids solubilised by apolipoproteins. HDL also contains numerous otherproteins, including enzymes and acute-phase proteins, and may contain smallamounts of nonpolar lipids. Furthermore, HDL proteins often exist in multipleisoforms and readily undergo posttranslational modification. As a consequence ofsuch diverse compositional features, HDL particles are highly heterogeneous in theirstructural, chemical and biological properties. This chapter reviews current knowl-edge of the composition, structure and heterogeneity of human plasma HDL.4 A. Kontush et al.1 HDL SubclassesHuman plasma HDLs are a highly heterogeneous lipoprotein family consisting ofseveral subclasses differing in density, size, shape and lipid and protein composi-tion (Table 1).Differences in HDL subclass distribution were first described by Gofman andcolleagues in the early 1950s by using analytic ultracentrifugation (De Lalla andGofman 1954), the gold standard technique for HDL separation. Two HDLsubclasses were identified: the less dense (1.063–1.125 g/mL), relatively lipid-rich form was classified asHDL2 and the more dense (1.125–1.21 g/mL), relativelyprotein-rich form as HDL3. The two major HDL subclasses can be separated byother ultracentrifugation methods, such as rate-zonal ultracentrifugation(Franceschini et al. 1985) or single vertical spin ultracentrifugation (Kulkarniet al. 1997). Ultracentrifugation methods are accurate and precise but requireexpensive instruments, time and technical skills. A precipitation method has beenproposed for HDL2 and HDL3 separation and quantitation (Gidez et al. 1982),which is inexpensive and easier, but with a high degree of interlaboratoryvariability. HDL2 and HDL3 can be further fractionated in distinct subclasseswith different electrophoretic mobilities by non-denaturing polyacrylamide gradi-ent gel electrophoresis (GGE) (Nichols et al. 1986), which separates HDLsubclasses on the basis of particle size. Two HDL2 and three HDL3 subclasseshave been identified and their particle size characterised by this method: HDL3c,7.2–7.8 nm diameter; HDL3b, 7.8–8.2 nm; HDL3a, 8.2–8.8 nm; HDL2a, 8.8.–9.7 nm; and HDL2b, 9.7–12.0 nm. The equivalent subclasses of HDL with similarsize distribution may be preparatively isolated by isopycnic density gradient ultra-centrifugation (Chapman et al. 1981; Kontush et al. 2003).Agarose gel electrophoresis allows analytical separation of HDL according tosurface charge and shape into α-migrating particles, which represent the majorityof circulating HDL, and preβ-migrating particles, consisting of nascent discoidaland poorly lipidated HDL. Agarose gels can be stained with Coomassie blue or withanti-apolipoprotein A-I (apoA-I) antibodies, and the relative protein content of thetwo HDL subclasses can be determined (Favari et al. 2004). The plasma preβ-HDLconcentration can be also quantified using a sandwich enzyme immunoassay (Miidaet al. 2003). The assay utilises a monoclonal antibody which specifically recognisesapoA-I bound to preβ-HDL. The agarose gel and the GGE can be combined into a2-dimensional (2D) electrophoretic method, which separates HDL according tocharge in the first run and according to size in the second run. Gels can be stainedwith apolipoprotein-specific antibodies, typically with anti-apoA-I antibodies,allowing the detection of distinct HDL subclasses (Asztalos et al. 2007). This isby far the method with the highest resolving power: up to 12 distinct apoA-I-containing HDL subclasses can be identified, referred to as preβ (preβ1 and preβ2), α(α1, α2, α3 and α4) and preα (preα1, preα2, preα3) according to their mobility andsize (Asztalos and Schaefer 2003a, b).According to the protein component, HDL can be separated into particlescontaining apoA-I with (LpA-I:A-II) or without apoA-II (LpA-I) by anStructure of HDL: Particle Subclasses and Molecular Components 5electroimmunodiffusion technique in agarose gels; plasma concentration of LpA-Iand LpA-I:A-II can be determined from a calibration curve (Franceschiniet al. 2007).More recently, a nuclear magnetic resonance (NMR) method for HDL subclassanalysis has been proposed, based on the concept that each lipoprotein particle of agiven size has its own characteristic lipid methyl group NMR signal (Otvoset al. 1992). According to the NMR signals, three HDL subclasses can be identified:large HDL (8.8–13.0 nm diameter), medium HDL (8.2–8.8 nm) and small HDL(7.3–8.2 nm); results are expressed as plasma particle concentration. The relativeplasma content of small, medium and large HDL (according to the same cut-off)can also be determined by GGE, dividing the absorbance profile into the three sizeintervals (Franceschini et al. 2007).HDL subfractionation on an analytical scale has been generally accomplished bythe different techniques in academic laboratories; however, clinical interest in HDLheterogeneity has been growing in the last 10 years and a number of laboratory testsfor determining HDL subclass distribution are now available, including GGE, NMRand 2D gel electrophoresis (Mora 2009). Whether evaluation of HDL subfractionsis performed by academic or commercial laboratories, there are a number of factorsthat confound the interpretation of the results of such analyses. The number andnomenclature of HDL subclasses are not uniform among the different techniques;Table 1 Major HDLsubclasses according todifferent isolation/separation techniquesDensity (ultracentrifugation)HDL2 (1.063–1.125 g/mL)HDL3 (1.125–1.21 g/mL)Size (GGE)HDL2b (9.7–12.0 nm)HDL2a (8.8–9.7 nm)HDL3a (8.2–8.8 nm)HDL3b (7.8–8.2 nm)HDL3c (7.2–7.8 nm)Size (NMR)Large HDL (8.8–13.0 nm)Medium HDL (8.2–8.8 nm)Small HDL (7.3–8.2 nm)Shape and charge (agarose gel)α-HDL (spherical)Preβ-HDL (discoidal)Charge and size (2D electrophoresis)Preβ-HDL (preβ1 and preβ2)α-HDL (α1, α2, α3 and α4)Preα-HDL (preα1, preα2, preα3)Protein composition (electroimmunodiffusion)LpA-ILpA-I:A-II6 A. Kontush et al.moreover, each subclass contains distinct subpopulations, as identified by, e.g. 2Delectrophoresis. In addition, whereas some methodologies measure HDL subclassconcentrations, others describe the percent distribution of the HDL subclassesrelative to the total or characterise the HDL distribution by average particlediameter. As a consequence, there is little relation among HDL subfractionationdata produced by different analytical techniques. A panel of experts has recentlyproposed a classification of HDL by physical properties, which integrates terminol-ogy from several methods and defines five HDL subclasses, termed very large,large, medium, small and very small HDL (Rosenson et al. 2011). The proposednomenclature, possibly together with widely accepted standards and qualitycontrols, should help in defining the relationship between HDL subclasses andcardiovascular risk as well as in assessing the clinical effects of HDL modifyingdrugs.2 Molecular Components of HDL2.1 Proteome2.1.1 Major Protein ComponentsProteins form the major structural and functional component of HDL particles.HDL carries a large number of different proteins as compared to other lipoproteinclasses (Table 2). HDL proteins can be divided into several major subgroups whichinclude apolipoproteins, enzymes, lipid transfer proteins, acute-phase responseproteins, complement components, proteinase inhibitors and other proteincomponents. Whereas apolipoproteins and enzymes are widely recognised as keyfunctional HDL components, the role of minor proteins, primarily those involved incomplement regulation, protection from infections and the acute-phase response,has received increasing attention only in recent years, mainly as a result of advancesin proteomic technologies (Heinecke 2009; Hoofnagle and Heinecke 2009;Davidsson et al. 2010; Shah et al. 2013). These studies have allowed reproducibleidentification of more than 80 proteins in human HDL (Heinecke 2009; Hoofnagleand Heinecke 2009; Davidsson et al. 2010; Shah et al. 2013) (for more details seethe HDL Proteome Watch at http://homepages.uc.edu/~davidswm/HDLproteome.html). Numerous proteins involved in the acute-phase response, complement regu-lation, proteinase inhibition, immune response and haemostasis were unexpectedlyfound as components of normal human plasma HDL, raising the possibility thatHDL may play previously unsuspected roles in host defence mechanisms andinflammation (Hoofnagle and Heinecke 2009).Importantly, the composition of the HDL proteome may depend on the methodof HDL isolation. Indeed, ultracentrifugation in highly concentrated salt solutionsof high ionic strength can remove some proteins from HDL, whereas other methodsof HDL isolation (gel filtration, immunoaffinity chromatography, precipitation)provide HDL extensively contaminated with plasma proteins or subject HDL tounphysiological conditions capable of modifying its structure and/or compositionStructure of HDL: Particle Subclasses and Molecular Components 7Table 2 Major components of the HDL proteomeProtein Mr, kDa Major functionNumber of proteomicstudies in which theprotein was detectedaApolipoproteinsApoA-I 28 Major structural and functionalapolipoprotein, LCAT activator14ApoA-II 17 Structural and functionalapolipoprotein13ApoA-IV 46 Structural and functionalapolipoprotein14ApoC-I 6.6 Modulator of CETP activity,LCAT activator12ApoC-II 8.8 Activator of LPL 12ApoC-III 8.8 Inhibitor of LPL 14ApoC-IV 11 Regulates TG metabolism 6ApoD 19 Binding of small hydrophobicmolecules11ApoE 34 Structural and functionalapolipoprotein, ligand for LDL-Rand LRP13ApoF 29 Inhibitor of CETP 8ApoH 38 Binding of negatively chargedmolecules8ApoJ 70 Binding of hydrophobic molecules,interaction with cell receptors11ApoL-I 44/46 Trypanolytic factor of human serum 14ApoM 25 Binding of small hydrophobicmolecules12EnzymesLCAT 63 Esterification of cholesterol tocholesteryl esters4PON1 43 Calcium-dependent lactonase 12PAF-AH(LpPLA2)53 Hydrolysis of short-chain oxidisedphospholipidsGSPx-3 22 Reduction of hydroperoxides byglutathioneLipid transfer proteinsPLTP 78 Conversion of HDL into larger andsmaller particles, transport of LPS5CETP 74 Heteroexchange of CE and TG andhomoexchange of PL between HDLand apoB-containing lipoproteins3Acute-phase proteinsSAA1 12 Major acute-phase reactant 10SAA4 15 Minor acute-phase reactant 10Alpha-2-HS-glycoprotein39 Negative acute-phase reactant 9(continued)8 A. Kontush et al.(e.g. extreme pH and ionic strength involved in immunoaffinity separation). Thus,proteomics of apoA-I-containing fractions isolated from human plasma by anon-denaturing approach of fast protein liquid chromatography (FPLC) reveal thepresence of up to 115 individual proteins per fraction, only up to 32 of which wereidentified as HDL-associated proteins in ultracentrifugally isolated HDL (Collinset al. 2010). Indeed, co-elution with HDL of plasma proteins of matching size isinevitable in FPLC-based separation; the presence of a particular protein across arange of HDL-containing fractions of different size isolated by FPLC on the basis oftheir association with phospholipid would however suggest that such a protein isindeed associated with HDL (Gordon et al. 2010). Remarkably, several of the mostabundant plasma proteins, including albumin, haptoglobin and alpha-2-macroglob-ulin, are indeed present in all apoA-I-containing fractions isolated by FPLC (Col-lins et al. 2010), suggesting their partial association with HDL by a non-specific,low-affinity binding.Table 2 (continued)Protein Mr, kDa Major functionNumber of proteomicstudies in which theprotein was detectedaFibrinogenalpha chain95 Precursor of fibrin, cofactor inplatelet aggregation10Complement componentsC3 187 Complement activation 9Proteinase inhibitorsAlpha-1-antitrypsin52 Inhibitor of serine proteinases 11Hrp 39 Decoy substrate to preventproteolysis10Other proteinsTransthyretin55 Thyroid hormone binding andtransport12Serotransferrin75 Iron binding and transport 10VitaminD-bindingprotein58 Vitamin D binding and transport 10Alpha-1B-glycoprotein54 Unknown 9Hemopexin 52 Heme binding and transport 8aOut of total of 14 proteomic studies according to Shah et al. (2013). Only proteins detected inmore than 50 % of the studies are listed, together with seven others previously known to beassociated with HDL (apoC-IV, apoH, LCAT, PAF-AH, GSPx-3, PLTP, CETP)CE cholesteryl ester, CETP cholesteryl ester transfer protein, GSPx-3 glutathione selenoperoxidase3,Hrp haptoglobin-related protein, LDL-R LDL receptor, LCAT lecithin/cholesterol acyltransferase,LPL lipoprotein lipase, LpPLA2 lipoprotein-associated phospholipase A2, LRP LDL receptor-relatedprotein, PAF-AH platelet-activating factor acetyl hydrolase, PL phospholipid, PLTP phospholipidtransfer protein, PON1 paraoxonase 1, SAA serum amuloid A, TG triglycerideStructure of HDL: Particle Subclasses and Molecular Components 9ApolipoproteinsApolipoprotein A-I is the major structural and functional HDL protein whichaccounts for approximately 70 % of total HDL protein. Almost all HDL particlesare believed to contain apoA-I (Asztalos and Schaefer 2003a, b; Schaeferet al. 2010). Major functions of apoA-I involve interaction with cellular receptors,activation of lecithin/cholesterol acyltransferase (LCAT) and endowing HDL withmultiple anti-atherogenic activities. Circulating apoA-I represents a typical amphi-pathic protein that lacks glycosylation or disulfide linkages and contains eightalpha-helical amphipathic domains of 22 amino acids and two repeats of11 amino acids. As a consequence, apoA-I binds avidly to lipids and possessespotent detergent-like properties. ApoA-I readily moves between lipoproteinparticles and is also found in chylomicrons and very low-density lipoprotein(VLDL). As for many plasma apolipoproteins, the main sites for apoA-I synthesisand secretion are the liver and small intestine.ApoA-II is the second major HDL apolipoprotein which represents approxi-mately 15–20 % of total HDL protein. About a half of HDL particles may containapoA-II (Duriez and Fruchart 1999). ApoA-II is more hydrophobic than apoA-I andcirculates as a homodimer composed of two identical polypeptide chains (Shimano2009; Puppione et al. 2010) connected by a disulfide bridge at position 6 (Breweret al. 1972). ApoA-II equally forms heterodimers with other cysteine-containingapolipoproteins (Hennessy et al. 1997) and is predominantly synthesised in the liverbut also in the intestine (Gordon et al. 1983).ApoA-IV, an O-linked glycoprotein, is the most hydrophilic apolipoproteinwhich readily exchanges between lipoproteins and also circulates in a free form.ApoA-IV contains thirteen 22-amino acid tandem repeats, nine of which are highlyalpha-helical; many of these helices are amphipathic and may serve as lipid-bindingdomains. In man, apoA-IV is synthesised in the intestine and is secreted into thecirculation with chylomicrons.ApoCs form a family of small exchangeable apolipoproteins primarilysynthesised in the liver. ApoC-I is the smallest apolipoprotein which associateswith both HDL and VLDL and can readily exchange between them. ApoC-I carriesa strong positive charge and can thereby bind free fatty acids and modulate activitiesof several proteins involved in HDL metabolism. Thus, apoC-I is involved in theactivation of LCAT and inhibition of hepatic lipase and cholesteryl ester transferprotein (CETP). ApoC-II functions as an activator of several triacylglycerol lipasesand is associated with HDL and VLDL.ApoC-III is predominantly present in VLDLwith small amounts found in HDL. The protein inhibits lipoprotein lipase (LPL) andhepatic lipase and decreases the uptake of chylomicrons by hepatic cells. ApoC-IVinduces hypertriglyceridemia when overexpressed in mice (Allan and Taylor 1996;Kim et al. 2008). In normolipidemic plasma, greater than 80 % of the protein residesin VLDL, with most of the remainder in HDL. The HDL content of apoC-IV is muchlower as compared to the other apoC proteins.ApoD is a glycoprotein mainly associated with HDL (McConathy andAlaupovic 1973). The protein is expressed in many tissues, including liver andintestine. ApoD does not possess a typical apolipoprotein structure and belongs to10 A. Kontush et al.the lipocalin family which also includes retinol-binding protein, lactoglobulin anduteroglobulin. Lipocalins are small lipid transfer proteins with a limited amino acidsequence identity but with a common tertiary structure. Lipocalins share a structur-ally conserved beta-barrel fold, which in many lipocalins bind hydrophobic ligands.As a result, apoD transports small hydrophobic ligands, with a high affinity forarachidonic acid (Rassart et al. 2000). In plasma, apoD forms disulfide-linkedhomodimers and heterodimers with apoA-II.ApoE is a key structural and functional glycoprotein component of HDL despiteits much lower content in HDL particles as compared to apoA-I (Utermann 1975).The major fraction of circulating apoE is carried by triglyceride-containinglipoproteins where it serves as a ligand for apoB/apoE receptors and ensureslipoprotein binding to cell-surface glycosaminoglycans. Similar to apoA-I andapoA-II, apoE contains eight amphipathic alpha-helical repeats and displaysdetergent-like properties towards phospholipids (Lund-Katz and Phillips 2010).ApoE is synthesised in multiple tissues and cell types, including liver, endocrinetissues, central nervous system and macrophages.ApoF is a sialoglycoprotein present in human HDL and low-density lipoprotein(LDL) (Olofsson et al. 1978), also known as lipid transfer inhibitor protein (LTIP)as a consequence of its ability to inhibit CETP. ApoF is synthesised in the liver andheavily glycosylated with both O- and N-linked sugar groups. Such glycosylationrenders the protein highly acidic and results in a molecular mass some 40 % greaterthan predicted (Lagor et al. 2009).ApoH, also known as beta-2-glycoprotein 1, is a multifunctional N- andO-glycosylated protein. ApoH binds to various kinds of negatively chargedmolecules, primarily to cardiolipin, and may prevent activation of the intrinsicblood coagulation cascade by binding to phospholipids on the surface of damagedcells. Such binding properties are ensured by a positively charged domain. ApoHregulates platelet aggregation and is expressed by the liver.ApoJ (also called clusterin and complement-associated protein SP-40,40) is anantiparallel disulfide-linked heterodimeric glycoprotein. Human apoJ consists oftwo subunits designated alpha (34–36 kDa) and beta (36–39 kDa) which sharelimited homology (de Silva et al. 1990a, b) and are linked by five disulfide bonds.The distinct structure of apoJ allows binding of both a wide spectrum of hydropho-bic molecules on the one hand and of specific cell-surface receptors on the other.ApoL-I is a key component of the trypanolytic factor of human serum associatedwith HDL (Duchateau et al. 1997). ApoL-I possesses a glycosylation site and sharesstructural and functional similarities with intracellular apoptosis-regulatingproteins of the Bcl-2 family. ApoL-I displays high affinity for phosphatidic acidand cardiolipin (Zhaorigetu et al. 2008) and is expressed in pancreas, lung, prostate,liver, placenta and spleen.ApoM is an apolipoprotein found mainly in HDL (Axler et al. 2007) whichpossesses an eight stranded antiparallel beta-barrel lipocalin fold and a hydrophobicpocket that ensures binding of small hydrophobic molecules, primarily sphingo-sine-1-phosphate (S1P) (Ahnstrom et al. 2007; Christoffersen et al. 2011). ApoMreveals 19 % homology with apoD, another apolipoprotein member of the lipocalinStructure of HDL: Particle Subclasses and Molecular Components 11family (Sevvana et al. 2009), and is synthesised in the liver and kidney. The bindingof apoM to lipoproteins is assured by its hydrophobic N-terminal signal peptidewhich is retained on secreted apoM, a phenomenon atypical for plasmaapolipoproteins (Axler et al. 2008; Christoffersen et al. 2008; Dahlback and Nielsen2009).ApoO, a minor HDL component expressed in several human tissues (Lamantet al. 2006), is present in HDL, LDL and VLDL, belongs to the proteoglycan familyand contains chondroitin sulphate chains, a unique feature distinguishing it fromother apolipoproteins. The physiological function of apoO remains unknown(Nijstad et al. 2011).Minor apolipoprotein components isolated within the density range of HDL arealso exemplified by apoB and apo(a), which reflect the presence of lipoprotein(a) and result from overlap in the hydrated densities of large, light HDL2 andlipoprotein (a) (Davidson et al. 2009).EnzymesLCAT catalyses the esterification of cholesterol to cholesteryl esters in plasmalipoproteins, primarily in HDL but also in apoB-containing particles. Approxi-mately 75 % of plasma LCAT activity is associated with HDL. In plasma, LCATis closely associated with apoD, which frequently co-purify (Holmquist 2002). TheLCAT gene is primarily expressed in the liver and, to a lesser extent, in the brainand testes. The LCAT protein is heavily N-glycosylated. The tertiary structure ofLCAT is maintained by two disulfide bridges and involves an active site covered bya lid (Rousset et al. 2009). LCAT contains two free cysteine residues at positions31 and 184.Human paraoxonases (PON) are calcium-dependent lactonases PON1, PON2and PON3 (Goswami et al. 2009). In the circulation, PON1 is almost exclusivelyassociated with HDL; such association is mediated by HDL surface phospholipidsand requires the hydrophobic leader sequence retained in the secreted PON1.Human PON1 is largely synthesised in the liver but also in the kidney and colon(Mackness et al. 2010). Hydrolysis of homocysteine thiolactone has been proposedto represent a major physiologic function of PON1 (Jakubowski 2000). The name“PON” however reflects the ability of PON1 to hydrolyse the organophosphatesubstrate paraoxone together with other organophosphate substrates and aromaticcarboxylic acid esters. Catalytic activities of the enzyme involve reversible bindingto the substrate as the first step of hydrolytic cleavage. PON1 is structurallyorganised as a six-bladed beta-propeller, with each blade consisting of four beta-sheets (Harel et al. 2004). Two calcium atoms needed for the stabilisation of thestructure and the catalytic activity of the enzyme are located in the central tunnel ofthe enzyme. Three helices, located at the top of the propeller, are involved in theanchoring of PON1 to HDL. The enzyme is N-glycosylated and may contain adisulfide bond. PON2, another member of the PON family, is an intracellularenzyme not detectable in serum despite its expression in many tissues, includingthe brain, liver, kidney and testis. The enzyme hydrolyses organophosphatesubstrates and aromatic carboxylic acid esters. PON3 possesses properties which12 A. Kontush et al.are similar to those of PON1, such as requirement for calcium, N-glycosylation,secretion in the circulation with retained signal peptide and association with HDL.PON3 displays potent lactonase, limited arylesterase and no PON activities and ispredominantly expressed in the liver.Platelet-activating factor acetyl hydrolase (PAF-AH) equally termedlipoprotein-associated phospholipase A2 (LpPLA2) is a calcium-independent,N-glycosylated enzyme, which degrades PAF by hydrolysing the sn-2 ester bondto yield biologically inactive lyso-PAF (Mallat et al. 2010). The enzyme cleavesphospholipid substrates with a short residue at the sn-2 position and can thereforehydrolyse proinflammatory oxidised short-chain phospholipids; however, it is inac-tive against long-chain non-oxidised phospholipids. PAF-AH is synthesisedthroughout the brain, white adipose tissue and placenta. Macrophages representthe most important source of the circulating enzyme (McIntyre et al. 2009). PlasmaPAF-AH circulates in association with LDL and HDL particles, with the majority ofthe enzyme bound to small, dense LDL and to lipoprotein (a) (Tselepis et al. 1995).The crystal structure of PAF-AH reveals a typical lipase alpha/beta-hydrolase foldand a catalytic triad (Samanta and Bahnson 2008). The active site is close to thelipoprotein surface and at the same time accessible to the aqueous phase. Twoclusters of hydrophobic residues build a lipid-binding domain ensuring associationwith lipoproteins.Plasma glutathione selenoperoxidase 3 (GSPx-3), also called glutathione per-oxidase 3, is distinct from two other members of the GSPx family termed GSPx-1and GSPx-2 which represent erythrocyte and liver cytosolic enzymes. All GSPxenzymes protect biomolecules from oxidative damage by catalysing the reductionof hydrogen peroxide, lipid peroxides and organic hydroperoxide, in a reactioninvolving glutathione. Human GSPx-3 is a homotetrameric protein containingselenium as a selenocysteine residue at position 73. Human GSPx-3 is synthesisedin the liver, kidney, heart, lung, breast and placenta. In plasma, GSPx-3 is exclu-sively associated with HDL (Chen et al. 2000).Lipid Transfer ProteinsPhospholipid transfer protein (PLTP) belongs to the bactericidal permeability-increasing protein (BPI)/lipopolysaccharide (LPS)-binding protein (LBP)/Pluncsuperfamily of proteins. PLTP is synthesised in the placenta, pancreas, lung,kidney, heart, liver, skeletal muscle and brain. In the circulation, PLTP is primarilyassociated with HDL and converts it into larger and smaller particles. PLTP alsoplays a role in extracellular phospholipid transport and can bind LPS. The proteincontains multiple glycosylation sites and is stabilised by a disulfide bond. PLTP is apositive acute-phase reactant with a potential role in the innate immune system.CETP equally belongs to the BPI/LBP/Plunc superfamily and contains multipleN-glycosylation sites. It is primarily expressed by the liver and adipose tissue. Inthe circulation, CETP shuttles between HDL and apoB-containing lipoproteins andfacilitates the bidirectional transfer of cholesteryl esters and triglycerides betweenthem. The structure of CETP includes a hydrophobic tunnel filled with twocholesteryl ester molecules and plugged by an amphiphilic phosphatidylcholineStructure of HDL: Particle Subclasses and Molecular Components 13(PC) molecule at each end (Qiu et al. 2007). Such interactions additionally endowCETP with PC transfer activity. CETP may also undergo conformational changes toaccommodate lipoprotein particles of different sizes (Qiu et al. 2007).Acute-Phase Response ProteinsPositive acute-phase response proteins, whose plasma concentrations are markedlyelevated by acute inflammation, form a large family of HDL-associated proteins(Vaisar et al. 2007; Heinecke 2009). Under normal conditions, the content of suchproteins in HDL is however much lower as compared to apolipoproteins. On theother hand, plasma levels of several HDL apolipoproteins, such as apoA-I andapoA-IV, are reduced during the acute-phase response (Navab et al. 2004); suchproteins can therefore be considered as negative acute-phase response proteins.Serum amyloid A (SAA) proteins, major acute-phase reactants, are secretedduring the acute phase of the inflammatory response. In humans, three SAAisoforms, SAA1, SAA2 and SAA4, are produced predominantly by the liver.Hepatic expression of SAA1 and SAA2 in the liver is induced during the acute-phase reaction, resulting in increase in their circulating levels by as much as 1,000-fold from basal concentrations of about 1–5 mg/l (Khovidhunkit et al. 2004). Bycontrast, SAA4 is expressed constitutively in the liver and is therefore termedconstitutive SAA. SAA1, the major member of this family, is predominantly carriedby HDL in human, rabbit and murine plasma (Hoffman and Benditt 1982; Marhauget al. 1982; Cabana et al. 1996). In the circulation, SAA1 does not exist in a freeform and associates with non-HDL lipoproteins in the absence of HDL (Cabanaet al. 2004).LBP is an acute-phase glycoprotein capable of binding the lipid A moiety ofLPS of Gram-negative bacteria and facilitating LPS diffusion (Wurfel et al. 1994).LBP/LPS complexes appear to interact with the CD14 receptor to enhance cellularresponses to LPS. LBP also binds phospholipids, thereby acting as a lipid exchangeprotein (Yu et al. 1997), and belongs to the same BPI/LBP/Plunc protein superfam-ily as PLTP and CETP.Fibrinogen is a common acute-phase protein and a cofactor in platelet aggrega-tion synthesised by the liver, which is converted by thrombin into fibrin duringblood coagulation. Fibrinogen is a disulfide-linked heterohexamer which containstwo sets of three non-identical chains (alpha, beta and gamma).Alpha-1-acid glycoprotein 2, equally termed orosomucoid-2, belongs to thecalycin protein superfamily which also includes lipocalins and fatty acid-bindingproteins. The protein appears to modulate activity of the immune system during theacute-phase reaction. In plasma, the protein is N-glycosylated and stabilised bydisulfide bonds.Alpha-2-HS-glycoprotein (fetuin-A) promotes endocytosis, possesses opsonicproperties and influences the mineral phase of bone. The protein shows affinity forcalcium and barium ions and contains two chains, A and B, which are held togetherby a single disulfide bond. Alpha-2-HS-glycoprotein is synthesised in the liver andsecreted into plasma.14 A. Kontush et al.Complement ComponentsSeveral complement components associate with HDL. Complement component3 (C3) plays a central role in the activation of the complement system through bothclassical and alternative activation pathways. C3 exists in a form of two chains, betaand alpha, linked by a disulfide bond. C4 is a key component involved in theactivation of the classical pathway of the complement system, which circulates as adisulfide-linked trimer of alpha, beta and gamma chains. C4b-binding proteincontrols the classical pathway of complement activation and binds as a cofactor toC3b/C4b inactivator, which then hydrolyses complement fragment C4b. C9 is apore-forming subunit of the membrane attack complex that provides an essentialcontribution to the innate and adaptive immune response by forming pores in theplasma membrane of target cells.Vitronectin is another HDL-associated protein involved in complement regula-tion. The protein belongs to cell-to-substrate adhesion molecules present in serumand tissues, which interact with glycosaminoglycans and proteoglycans and can berecognised by members of the integrin family. In complement regulation,vitronectin serves as an inhibitor of the membrane-damaging effect of the terminalcytolytic pathway. Vitronectin is largely expressed in the liver but also in visceraltissue and adrenals. The presence of vitronectin in HDL raises the possibility thatcertain HDL components can be derived from non-cellular sources or cells distinctfrom those that synthesise apoA-I in the liver and intestine (Heinecke 2009).Proteinase InhibitorsA family of proteins in HDL contains serine proteinase inhibitor domains (Vaisaret al. 2007). Serine protease inhibitors (serpins) are important regulators ofbiological pathways involved in inflammation, coagulation, angiogenesis andmatrix degradation. HDL-associated serpins are exemplified by alpha-1-antitrypsin which in the circulation is exclusively present in HDL (Karlssonet al. 2005; Ortiz-Munoz et al. 2009). Alpha-2-antiplasmin, a serpin that inhibitsplasmin and trypsin and inactivates chymotrypsin, is another key proteinase inhibi-tor which circulates in part associated with HDL. HDL also carries inter-alpha-trypsin inhibitor heavy chain H4 and bikunin, two components of inter-alpha-trypsin inhibitors consisting of three of four heavy chains selected from the groups1 to 4 and one light chain selected from the alpha-1-microglobulin/bikunin precur-sor or Kunitz-type protease inhibitor 2 groups. The full complex inhibits trypsin,plasmin and lysosomal granulocytic elastase. Inter-alpha-trypsin inhibitor heavychain H4 is produced in the liver and is cleaved by kallikrein to yield 100 and35 kDa fragments in plasma, and the resulting 100 kDa fragment is furtherconverted to a 70 kDa fragment. Bikunin is a light chain of the inter-alpha-trypsininhibitor, which is produced via proteolytic cleavage of the alpha-1-microglobulin/bikunin precursor protein together with alpha-1-microglobulin and trypstatin.Other proteolysis-related proteins which are detected on HDL includehaptoglobin-related protein (Hrp), kininogen-1, prothrombin, angiotensinogenand procollagen C-proteinase enhancer-2 (PCPE2). Hrp contains a crippled cata-lytic triad residue that may allow it to act as a decoy substrate to prevent proteolysis.Structure of HDL: Particle Subclasses and Molecular Components 15Kininogen-1 (alpha-2-thiol proteinase inhibitor) plays an important role in bloodcoagulation and inhibits the thrombin- and plasmin-induced aggregation ofthrombocytes. Prothrombin is a precursor of thrombin, a key serine protease ofthe coagulation pathway. Angiotensinogen is a an alpha-2-globulin that is pro-duced constitutively mainly by the liver and represents a substrate for renin whoseaction forms angiotensin I. PCPE2 binds to the C-terminal propeptide of type I or IIprocollagens and enhances the cleavage of the propeptide by bone morphogeneticprotein 1 (BMP-1, also termed procollagen C-proteinase).Other Protein ComponentsHDL equally transports distinct proteins displaying highly specialised functions.The metabolic purpose of such association is unclear; it might prolong the residencetime of a protein or represent a mechanism for protein conservation in the circula-tion. For example, plasma retinol-binding protein, which delivers retinol from theliver stores to peripheral tissues, co-isolates with HDL3 (Vaisar et al. 2007). Inplasma, the complex of retinol-binding protein and retinol interacts withtransthyretin, thereby preventing loss of retinol-binding protein by filtrationthrough the kidney glomeruli. As a corollary, transthyretin, a homotetramericthyroid hormone-binding protein, is equally present on HDL (Hortin et al. 2006;Vaisar et al. 2007; Davidson et al. 2009). Serotransferrin, an iron-transportglycoprotein largely produced in the liver, is also in part associated with HDL.Hemopexin, an iron-binding protein that binds heme and transports it to the liverfor breakdown and iron recovery, equally co-isolates with HDL3 (Vaisaret al. 2007).HDL also carries proteins involved in the regulation of various biologicalfunctions, such as Wnt signalling molecules, which participate in cell-to-cellsignalling (Neumann et al. 2009), and progranulin, a precursor of granulinswhich play a role in inflammation, wound repair and tissue remodelling.In addition, HDL transports lysosomal proteins, such as prenylcysteine oxidase,which is involved in the degradation of prenylated proteins (Vaisar et al. 2007).Other minor abundance proteins reported to be associated with HDL are albumin,alpha-1B-glycoprotein, alpha-amylase, vitamin D-binding protein and plateletbasic protein (Vaisar et al. 2007; Davidson et al. 2009).In addition to proteins, HDL carries a large number of small peptides in the massrange from 1 to 5 kDa (Hortin et al. 2006). These peptides are present in HDL at lowconcentrations of about 1 % of total HDL protein, with some representingfragments of larger proteins, such as apoB, fibrinogen and transthyretin (Hortinet al. 2006). The association of small peptides with HDL as a vehicle may representa pathway for peptide delivery or scavenging, in order to slow renal clearance andproteolysis (Hortin et al. 2006).Heterogeneity in HDL ProteinsHDL proteins are non-uniformly distributed across HDL subpopulations. Indeed,proteomic analysis of five HDL subpopulations isolated from normolipidemicsubjects by isopycnic density gradient ultracentrifugation identifies five distinct16 A. Kontush et al.patterns of distribution of individual protein components across the HDL densitysubfractions (Davidson et al. 2009) (Fig. 1). The most interesting of thesedistributions identifies small, dense HDL3b and 3c as particle subpopulations inwhich seven proteins occur predominantly, notably apoJ, apoL-1, apoF, PON1/3,PLTP and PAF-AH. Activities of HDL-associated enzymes (LCAT, PON1,PAF-AH) are equally elevated in small, dense HDL3c (Kontush et al. 2003;Kontush and Chapman 2010). The HDL3c proteome also contains apoA-I; apoA-II; apoD; apoM; SAA 1, 2, and 4; apoC-I; apoC-II; and apoE (Davidson et al. 2009).Consistent with these data, apoL-I (Hajduk et al. 1989), apoF (He et al. 2008; Lagoret al. 2009), apoJ (de Silva et al. 1990a, b; Bergmeier et al. 2004), PON1 (Kontushet al. 2003; Bergmeier et al. 2004), apoA-IV (Bisgaier et al. 1985; Ohta et al. 1985),apoM (Wolfrum et al. 2005), apoD (Campos and McConathy 1986) and SAA1/2 (Benditt and Eriksen 1977; Coetzee et al. 1986) are known to preferentiallyco-isolate with dense HDL3. Furthermore, small, dense HDL may also representa preferential carrier for human CETP (Marcel et al. 1990). On the other hand,apoE, apoC-I, apoC-II and apoC-III preferentially localise to large, light HDL2(Schaefer et al. 1979; Cheung and Albers 1982; Schaefer and Asztalos 2007;Davidson et al. 2009) (Fig. 1). Importantly, these associations can in part beconfirmed using an alternative approach of gel filtration subfractionation of HDLparticles (Gordon et al. 2010).The low HDL content of the majority of these proteins of less than one copy perHDL particle suggests internal heterogeneity of the HDL3c subfraction. Thisconclusion is further consistent with the isolation of a unique particle containingthe trypanosome lytic factor apoL-I, plus apoA-I and Hrp, in the HDL3 densityrange (Shiflett et al. 2005).In a similar fashion, apoF co-isolates with dense HDL (mean density, 1.134 g/ml)which also contains apoA-I, apoC-II, apoE, apoJ, apoD and PON1 (He et al. 2008).Complexes formed by PON1 with human phosphate-binding protein and apoJrepresent another example of protein–protein interactions occurring within theHDL particle spectrum (Vaisar 2009).Specific protein–protein interactions should thus drive the formation of suchcomplexes in the circulation. In support of such a mechanism, PLTP in humanplasma resides on lipid-poor complexes dominated by apoJ and proteins implicatedin host defence and inflammation (Cheung et al. 2010).In addition and as mentioned above (Sect. 1), immunoaffinity technique allowsseparating HDL particles containing only apoA-I (LpA-I) from those containingboth apoA-I and apoA-II (LpA-I:A-II) (Duriez and Fruchart 1999). ApoA-I istypically distributed approximately equally between LpA-I and LpA-I:A-II,whereas virtually all apoA-II is in LpA-I:A-II (Duriez and Fruchart 1999). LpA-Iand LpA-I:A-II contain approximately 35 and 65 % of plasma apoA-I, respectively(James et al. 1988). On the other hand, approximately half of HDL particles containapoA-II (Wroblewska et al. 2009). In addition to apoA-II, the two subclasses maydiffer in their content of other proteins, as exemplified by PON1 which preferablyassociates with LpA-I (Moren et al. 2008).Structure of HDL: Particle Subclasses and Molecular Components 17012341020ApoL-I Pon1PLTP ApoJ Pon 3 ApoF PAF-AH0510152080120ApoBapo(a)Average Number of Peptides Identified036912apoDapoMSAA 1+20510152025apoEapoC-II0612456075ApoA-IapoA-II SAA4apoC-I2b 2a 3a 3b 3cClass AClass BClass CClass DClass EExclusively dense HDL3Preferentially dense HDL3No preferential distributionPreferentially light HDL2Exclusively light HDL2Fig. 1 Abundance pattern of proteins across healthy normolipidemic HDL subpopulations. ClassA: exclusively present in small, dense HDL3b and 3c. Class B: enriched in small, dense HDL3band 3c. Class C: equally abundant across HDL subpopulations. Class D: enriched in large, lightHDL2b and 2a. Class E: exclusively present in large, light HDL2b and 2a [modified from(Davidson et al. 2009)]18 A. Kontush et al.LpA-I particles can be further subfractionated according to size. The number ofapoA-I molecules in such subpopulations is increased from two to three to four withan increase in the particle size (Gauthamadasa et al. 2010). On the other hand, theentire population of LpA-I:A-II demonstrates the presence of only two apoA-Imolecules per particle, while the number of apoA-II molecules varies from onedimeric apoA-II to two and then to three. Upon compositional analyses of individ-ual subpopulations, LpA-I:A-II exhibits comparable proportions for major lipidclasses across subfractions, while LpA-I components show significant variability(Gauthamadasa et al. 2010).Another important subpopulation of HDL particles is formed by apoE-containing HDL. The presence of apoE facilitates expansion of the lipid core as aresult of the accumulation of cholesteryl ester, with formation of large, lipid-richHDL; these particles represent an excellent ligand for the LDL receptor (Hatterset al. 2006).The diversity of molecules which bind to HDL suggests that the lipoprotein canserve as a versatile adsorptive surface for proteins and peptides to form complexesplaying roles not only in lipid metabolism but equally in acute-phase response,innate immune response, complement activation, plaque stability and proteolysisinhibition (Heinecke 2009). As the abundance of the most of HDL-associatedproteins is below 1 mol/mol HDL (i.e. less than 1 copy per HDL particle), itremains however unclear as to how they are distributed among minor HDLsubpopulations of potentially distinct origin and function. Specific proteins maytherefore be confined to distinct HDL subpopulations of distinct origin and func-tion, which are differentially distributed across the HDL particle spectrum(Davidson et al. 2009). The HDL fraction as a whole therefore appears to represent“a collection of individualised species with distinct functionalities that happen tohave similar physicochemical properties” (Shah et al. 2013) and are primarilydefined by specific protein–protein interactions facilitated by the presence ofphospholipid, rather than “a transient ensemble of randomly exchanging proteins”(Gordon et al. 2013; Shah et al. 2013).2.1.2 Protein Isoforms, Translational and PosttranslationalModificationsIn line with the evolvement of proteomics, a large number of proteins have beenidentified in HDL as described in the previous section (Vaisar et al. 2007). Inaddition, most proteins are expressed as different isoforms due to co- and posttrans-lational modifications (Karlsson et al. 2005; Candiano et al. 2008). This makes theproteome of HDL both complex and dynamic, which most likely result in variousHDL particles with different protein composition in respect to the environment.Common posttranslational modifications (PTMs) such as glycosylations, truncationsand phosphorylations change the charge and/or the size of the protein, which can beutilised for separation of the isoforms, andmodern mass spectrometric techniques canbe used to detect, characterise and nowadays also measure even small massdifferences in proteins. However, although isoforms of major HDL proteins havebeen known for decades (Zannis et al. 1980; Hussain and Zannis 1990), surprisinglyStructure of HDL: Particle Subclasses and Molecular Components 19little is still known on how these variations of the HDL proteome affect the function-ality. The following is a comprehensive review of isoforms patterns described incommon human HDL apolipoproteins, apoA-I, apoA-II, apoC-III and SAA, and theirpossible functional relevance.ApoA-I (pI 5.3/28 kDa, accession no. P02647) is normally found as differentcharge isoforms; besides the major isoform (70–75 % of total apoA-I) and theslightly more basic pre-apoA-I (5–10 % of total apoA-I), also two more acidicisoforms are generally detected by isoelectric focusing (IEF) and 2D gel electro-phoresis (2-DE) (Contiero et al. 1997; Karlsson et al. 2005). The nature of theseacidic isoforms is still unclear. An early report suggested deamidation of Gln or Asnresidues, resulting in a +1 charge shift, which could be formed during the analyticalprocedure (Ghiselli et al. 1985). At the same time, a few reports indicated theimportance of acidic apoA-I in vivo; increased levels of acidic apoA-I, whiledecreased levels of the major form, were found in LDL from obese subjects,especially in women (Karlsson et al. 2009). Also, higher degree of deamidatedapoA-I has been shown in relation to diabetes (Jaleel et al. 2010) and acidic apoA-Imay be more vulnerable to methionine oxidation (Fernandez-Irigoyen et al. 2005).In 2-DE HDL protein patterns also 30–35 kDa variants of apoA-I are usuallydetected (Karlsson et al. 2005). Although mass spectrometry (MS) analysis wasin agreement with O-glycosylation at two potential sites (Thr78 or Thr92), this hasnot been confirmed by others, and it is generally regarded that apoA-I is notN-linked or O-linked glycosylated. In contrast, non-enzymatically glycation ofapoA-I has been found in association to diabetes and believed to affect apoA-Ifunctions, such as LCAT activation (Fievet et al. 1995; Nobecourt et al. 2007; Parket al. 2010).Another potentially important PTM of apoA-I is truncation. During atheroscle-rotic inflammation, apoA-I might be N-terminally and C-terminally truncated byreleased proteases. Specific cleavage sites at Tyr42, Phe57, Tyr216 and Phe253 forchymase have been identified that in reconstituted HDL reduces its ability topromote cholesterol efflux (Lee et al. 2003; Usami et al. 2013). Low amounts ofC-terminally truncated apoA-I can be measured in normal serum (Usamiet al. 2011) and fragmented apoA-I is a feature in plasma from children withnephrotic syndrome, a condition linked to higher risk of atherosclerosis (Santucciet al. 2011). Truncation has also been implicated in apoA-I dimerisation as studiedin apoA-I Milano (R197C) and apoA-I Paris (R175C) (Calabresi et al. 2001; Favariet al. 2007; Gursky et al. 2013). Notably, apoA-I with an apparent molecular massof 50 kDa, consistent with dimeric apoA-I, has been found in patients withmyocardial infarction (Majek et al. 2011) but also appears to be present in HDLfrom healthy individuals (Karlsson et al. 2005). Finally, oxidatively modifiedapoA-I have been extensively studied during the recent years by the help of MStechniques, as described in detail in several reviews elsewhere (e.g. Nicholls andHazen 2009; Shao 2012). It has been proposed that myeloperoxidase-mediatedinflammation results in oxidation of apoA-I. Specific sites have been identifiedfor methionine oxidation, for nitrated/chlorinated tyrosines and for lysines modifiedby reactive carbonyls. Importantly, the modifications have been coupled to20 A. Kontush et al.functional impairment in apoA-I activity such as ABCA1-mediated cholesterolefflux and are linked to cardiovascular disease.ApoA-II (pI 5.0/8.7 kDa, accession no. P02652) is mostly found as two isoformsin HDL that differ slightly according to pI and molecular mass, probably due toO-linked glycosylation/sialylation (Karlsson et al. 2005; Halim et al. 2013). Similarto apoA-I, the protein is produced as a more basic pro-form (Hussain and Zannis1990). In contrast, apoA-II appears to be quickly processed to the mature form, asthe pro-form is not found in the circulation. In addition to glycosylation, phosphor-ylation at Ser68, C-terminal truncated variants (des-Gln and des-Thr-Gln) and acysteinylated variant has been detected in the circulation (Jin and Manabe 2005;Nelsestuen et al. 2008; Zhou et al. 2009). ApoA-II also forms a homodimer atCys29 that is abundant in plasma (Gillard et al. 2005; Jin and Manabe 2005).Overall, more than ten different variants of apoA-II are present in humans, butthe physiological relevance of this heterogeneity is unclear. However, sialylatedapoA-II appear to be selectively associated to HDL3 (Remaley et al. 1993; Karlssonet al. 2005), and elevated levels of modified apoA-II isoforms have been linked topremature delivery in pregnant women (Flood-Nichols et al. 2013).ApoC-III (pI 4.7/8.8 kDa, accession no. P02656) is generally found as threecharge isoforms depending on O-linked glycosylation (GalGalNAc) at Thr94 withor without sialylation; disialylated apoC-III2, monosialylated apoCIII1 andnon-sialylated apoC-III0 (Karlsson et al. 2005; Bruneel et al. 2008). An early reportshowed that glycosylation is not necessary for apoC-III secretion and does notaffect its relative affinity to different lipoprotein particles (Roghani and Zannis1988), and, as judged by gel electrophoresis and MS analysis of HDL and plasma,the non-sialylated variant is least abundant, usually less than 5 % of total apoC-IIIin normal individuals (Wopereis et al. 2003; Bruneel et al. 2008; Mazur et al. 2010;Holleboom et al. 2011). In addition to glycosylation, apoC-III can also beC-terminal truncated (des-Ala and des-Ala-Ala), which further increases the num-ber of isoforms (Bondarenko et al. 1999; Jin and Manabe 2005; Nicolardiet al. 2013a). Interestingly, novel results strongly suggest that glycosylation ofapoC-III is an important event in the regulation of lipid metabolism (Holleboomet al. 2011; Baenziger 2012). Thus, apoC-III is exclusively glycosylated by GalNActransferase 2 (GALNT2) (Holleboom et al. 2011; Schjoldager et al. 2012), andheterozygotes with a loss-of-function mutation in GALNT2 present with an alteredapoC-III isoform pattern with more of the non-sialylated variant and less of themonosialylated variant, while the total apoC-III plasma concentration was about thesame as compared to wild-type controls (Holleboom et al. 2011). This is then linkedto reduced inhibition of lipoprotein lipase and improved triglyceride clearance. Inline, the production rate of apoC-III1 and -III2 is more strongly correlated withplasma triglyceride levels than apoC-III0 (Mauger et al. 2006), increased apoC-III1/apoC-III0 ratio has been found in diabetic subjects (Jian et al. 2013), and HDL3from subjects with low HDL-C is characterised by higher levels of monosialylatedapoC-III than subjects with high HDL-C (Mazur et al. 2010). The evaluation ofapoC-III isoforms is complicated by the fact that apoC-III0 can be separated into anon-glycosylated form and glycosylated but non-sialylated forms (BruneelStructure of HDL: Particle Subclasses and Molecular Components 21et al. 2008; Holleboom et al. 2011; Nicolardi et al. 2013a). Moreover, a recent MSstudy of 96 serum samples showed that 30 % of the individuals displayed an apoC-III pattern with additional glycosylated variants, characterised by fucosylation(Nicolardi et al. 2013b). The relevance of these glycosylated non-sialylated variantsof apoC-III, as of the C-terminal truncated forms, is yet unclear, but may explainsomewhat contradictory results showing higher relative levels of non- and less-sialylated apoC-III in obese subjects than in lean subjects (Harvey et al. 2009;Karlsson et al. 2009), although obesity is generally associated with high triglyceridelevels.SAA exists in a form of SAA1 (pI 5.9/11.7 kDa, accession no. P0DJI8) andSAA2 (pI 8.3/11.6 kDa, accession no. P0DJI9). The two proteins display about93 % sequence homology, and depending on natural variation in alleles, SAA1 isseparated into five isoforms, SAA1.1 to SAA1.5, and SAA2 is separated into twoisoforms SAA2.1 and SAA2.2 (often also denoted as alpha, beta, etc.), with one tothree amino acid difference between the isoforms. In addition, both N-terminal andC-terminal truncated variants of SAA1/SAA2 are detected in serum (Ducretet al. 1996; Kiernan et al. 2003; de Seny et al. 2008). By using a combined 2-DE/MS and SELDI-TOF approach, eight isoforms were identified in HDL after LPSinfusion in healthy individuals; besides native SAA1.1 and SAA2.1, N-terminaltruncations (des-R, -RS and -RSFF) of each variant were also found (Levelset al. 2011). Interestingly, a subgroup, based on HDL protein profile, characterisedby elevated antioxidative PON1 activity showed a delayed response of SAA to LPSin particular for the most truncated (des-RSFF) variants. Otherwise, very little isknown about differential physiological relevance of the SAA isoforms. However,SAA2.1 but not SAA1.1 has been shown to promote cholesterol efflux frommacrophages (Kisilevsky and Tam 2003). Today, contradictory results make itunclear whether the increased level of SAA in inflammation, believed to replaceapoA-I in HDL, actually is a mechanism in atherosclerosis or merely is a marker forinflammation (de Beer et al. 2010; Chiba et al. 2011; Kisilevsky and Manley 2012).Future differential quantitative MS analysis of the highly homologous SAAisoforms such as described by Sung et al. (2012) may resolve this controversy.Sensitive MS techniques have revealed a large number of proteins associated toHDL. Most of them are also expressed as different isoforms depending on transla-tional and posttranslational modifications. This leads to a need to developMS-based methods for specific and reliable quantification of protein isoforms.With appropriate standards, measurements can be performed with low coefficientof variation and with a specificity superior to, e.g. immunoassays. Consequently,such applications in the field of HDL are being presented by using, e.g. multiplereaction monitoring and top-down proteomics with high-resolution MS (Mazuret al. 2010; Sung et al. 2012). Another interesting and fairly simple approach is touse ratio determinations, e.g. modified/native protein expression (Nelsestuenet al. 2008). As these measures are concentration-independent, they bear a potentialto reduce individual variations. Furthermore, such MS approaches are not onlyuseful for PTMs but also of value to understand the impact of protein variationscaused by genetic polymorphism. For example, a recent study of heterozygotes22 A. Kontush et al.with an apoA-I mutation (K131Del) showed that, in contrast to what could beexpected, the mutant protein was more abundantly expressed in HDL than thenative protein (Ljunggren et al. 2013). Herein four HDL proteins that are allexpressed as different isoforms have been discussed: two (apoA-I and apoC-III)in which PTMs have been shown to be important for lipid metabolism and two(apoA-II and SAA) in which the role of PTMs is still unclear. In light of the vitalimportance of carboxylations and truncations in the processes of haemostasis,which also involves other PTMs such as phosphorylation, hydroxylation, glycosyl-ation and sulphation, it appears highly unlikely that the diversity in the “HDLome”would not be relevant for lipid metabolism and cardiovascular disease. Therefore,characterisation of PTMs is probably one of the most challenging but also one of themost important tasks in order to understand the complex function of HDL.2.2 LipidomeThe real power of lipidomic technologies involving mass spectrometry results fromtheir ability to provide quantitative data on individual molecular species of lipidsand on low-abundance lipid molecules. The pioneering study of Wiesner andcolleagues published in 2009 (Wiesner et al. 2009) provided reference values forthe lipidome of HDL isolated from healthy normolipidemic controls by FPLC. In anattempt to further characterise HDL composition and address its inherent heteroge-neity, we recently reported the phospho- and sphingolipidome of five major HDLsubpopulations isolated from healthy normolipidemic subjects (Camontet al. 2013).2.2.1 PhospholipidsPhosphatidylcholine is the principal plasma phospholipid that accounts for 32–35 mol % of total lipids in HDL (Wiesner et al. 2009) (Table 3). PC is a structurallipid, consistent with its even distribution across HDL subpopulations (Fig. 2).Major molecular species of PC are represented by the 16:0/18:2, 18:0/18;2 and16:0/20:4 species (Lhomme et al. 2012). As compared to other lipoproteins, HDL isenriched in PC containing polyunsaturated fatty acid moieties (Wiesner et al. 2009).LysoPC is an important phospholipid subclass in HDL (1.4–8.1 mol % of totallipids; Table 3). It is derived from the regulated degradation of PC byphospholipases, including LCAT, consistent with the preferential association ofthe latter with HDL particles (Kontush et al. 2007). More specifically, LCAT wasreported earlier to associate mainly with small, dense HDL particles, which are alsoenriched in lysoPC by approximately twofold as compared to large, light HDL(Camont et al. 2013) (Fig. 2). LysoPC is also produced by the hydrolytic action ofLp-PLA2 on oxidised PC or by secreted PLA2 under pro-atherogenic conditions,such as oxidative stress and inflammation, and constitutes therefore a potentialbiomarker of inflammation. Major molecular species of HDL lysoPC containsaturated fatty acid moieties of predominantly 16 and 18 carbon atoms, reflectingLCAT preference for 16 and 18 carbon atom long PCs (Lhomme et al. 2012). AsStructure of HDL: Particle Subclasses and Molecular Components 23considerable amounts of serum lysoPC are also associated with albumin (Wiesneret al. 2009), HDL contamination by the both compounds is typical for FPLCisolation. However, in HDL isolated by isopycnic density gradient ultracentrifuga-tion, lysoPC content in HDL is two- to tenfold lower (Camont et al. 2013; Stahlmanet al. 2013).Phosphatidylethanolamine (PE) is moderately abundant in HDL (0.7–0.9 mol % of total lipids; Table 3), and its content tends to increase with increasingHDL hydrated density (Wiesner et al. 2009; Camont et al. 2013) (Fig. 2). PEprincipal molecular species are represented by the 36:2 and 38:4 fatty acid residuesin HDL (Kontush et al. 2007).Table 3 Major components of the HDL lipidomeLipid class HDL content in mol % of total lipidsPhospholipids 37.4–49.3Phosphatidylcholine 32–35PC-plasmalogen 2.2–3.5LysoPC 1.4–8.1Phosphatidylethanolamine 0.70–0.87PE-plasmalogen 0.54–0.87Phosphatidylinositol 0.47–0.76Cardiolipin 0.077–0.201Phosphatidylserine 0.016–0.030Phosphatidylglycerol 0.004–0.006Phosphatidic acid 0.006–0.009Sphingolipids 5.7–6.9Sphingomyelin 5.6–6.6Ceramide 0.022–0.097Hexosyl Cer 0.075–0.123Lactosyl Cer 0.037–0.060S1P d18:1 0.015–0.046S1P d18:0 0.007SPC d18:1 0.001Neutral lipids 46.7–54.0Cholesteryl esters 35–37Free cholesterol 8.7–13.5Triacylglycerides 2.8–3.2Diacylglycerides 0.17–0.28Minor lipidsFree fatty acids 16:0, 18:0, 18:1aIsoprostane-containing PC ND (IPGE2/D2-PC (36:4))aData are shown for HDL obtained from normolipidemic healthy subjects according to Deguchiet al. (2000), Kontush et al. (2007), Wiesner et al. (2009), Camont et al. (2013), Stahlmanet al. (2013), Pruzanski et al. (2000), Sattler et al. (2010), Argraves et al. (2011)SPC sphingosylphosphorylcholine, S1P sphingosine-1-phosphate, IPGE2 isoprostaglandin E2a no quantitative data available, major molecular species identified24 A. Kontush et al.Plasmalogens contain a vinyl ether-linked fatty acid essential for their specificantioxidative properties (Maeba and Ueta 2003). PC-plasmalogens are the mostabundant species in HDL (2.2–3.5 mol %) but represent less than 10 % of total PC(Stahlman et al. 2013). On the contrary, PE-plasmalogens and PE are equallyabundant in HDL (0.6–0.9 mol %; Table 3). PC- and PE-plasmalogens contain0.0350.0450.0550.0650.0750.0850.095Ceramide0.00350.0055LysoPCPEPI0.40.81.21.6Mol % of total lipids2b 2a 3a 3b 3cClass BClass CClass DClass E0.0000.010PSPAS1P0.020.080.140.20Class APG4.55.56.57.5Free cholesterolSphingomyelin111621048CEPCTAG1222324252Highly enriched in dense HDL3Enriched in dense HDL3Equally abundantEnriched in light HDL2Highly enriched in light HDL2Fig. 2 Abundance pattern of lipids across healthy normolipidemic HDL subpopulations. Class A:highly enriched in small, dense HDL3b and 3c (>1.5-fold relative to HDL2b). Class B: enriched insmall, dense HDL3b and 3c (1.2–1.5-fold relative to HDL2b). Class C: equally abundant acrossHDL subpopulations (<1.2-fold variations between HDL2b and HDL3b+ 3c). Class D: enrichedin large, light HDL2b (1.2–1.5-fold relative to HDL3b + 3c). Class E: highly enriched in large,light HDL2b (>1.5-fold relative to HDL3b + 3c). S1P, sphingosine-1-phosphateStructure of HDL: Particle Subclasses and Molecular Components 25mainly polyunsaturated species: 38:5 and 36:2 in PC-plasmalogens (Stahlmanet al. 2013) and 18:0/20:4 and 16:0/20:4 in PE-plasmalogens (Wiesner et al. 2009).Phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol(PG), phosphatidic acid (PA) and cardiolipin are negatively chargedphospholipids present in HDL (Table 3) which may significantly impact on its netsurface charge (Rosenson et al. 2011; Lhomme et al. 2012). The content of theselipids can thereby modulate lipoprotein interactions with lipases, membrane proteins,extracellular matrix and other protein components; indeed, such interactions arelargely charge-dependent.PI, similarly to PE, is moderately abundant in HDL (0.5–0.8 mol %; Table 3) andtends to be enriched in small, dense HDL (Fig. 2). Major molecular species of PI inHDL include the 18:0/20:3 and 18:0/20:4 species (Lee et al. 2010).PS is a minor negatively charged phospholipid component of HDL (0.016–0.030 mol %; Table 3). This phospholipid was very recently reported to be highlyenriched (34-fold) in the small, dense HDL3c subpopulation relative to large, lightHDL2 (Fig. 2) (Camont et al. 2013) as well as in small discoid preβ HDL and smallnascent HDL formed by ABCA1 (up to 2.5 mol % of total lipids) (see Kontush andChapman 2012 for review). Interestingly, small, dense HDL also displayed potentbiological activities which correlated positively with PS content in HDL (Camontet al. 2013). This lipid could therefore, in part, account for enhanced functionalityof HDL3c.PA, a second messenger, is both a common metabolic precursor and an enzy-matic product of phospholipid metabolism. This negatively charged lipid is presentin very low abundance in HDL (0.006–0.009 mol %; Table 3) but, similarly to PS, isenriched in small, dense HDL (by more than threefold) (Camont et al. 2013)(Table 3). This observation might reflect preferential association of PA withapoL-I which is equally enriched in small, dense HDL (Kontush and Chapman2012).PG is a metabolic precursor of cardiolipin present in HDL in very low amounts(0.004–0.006 mol %; Table 3). PG tends to be enriched in small, dense particles(Camont et al. 2013) (Fig. 2).Cardiolipin is a minor anionic phospholipid present in trace amounts in HDL(0.08–0.2 mol %; Table 3). This lipid with potent anticoagulant properties maycontribute to the effects of lipoproteins on coagulation and platelet aggregation(Deguchi et al. 2000).Together, these data indicate that although negatively charged lipids representminor HDL constituents (0.8 mol % of total lipids), they are highly enriched insmall, dense HDL, consistent with the elevated surface electronegativity of thissubpopulation (Rosenson et al. 2011).Isoprostanes are well established as biomarkers of oxidative stress and arepredominantly associated with HDL (see Kontush and Chapman 2012 for review).Major molecular species of isoprostane-containing PCs include 5,6-epoxy-isoprostaglandine A2-PC (EIPGA2-PC) 36:3, 5,6 EIPGE2-PC 36:4, IPGE2/D2-PC 36:4, IPGF-PC 36:4, IPGE2/D2-PC 38:4 and IPGF-PC 38:4 (Pruzanski et al. 2000)(Table 3).26 A. Kontush et al.2.2.2 SphingolipidsSphingomyelin, a structural lipid which enhances surface lipid rigidity (Ryeet al. 1996; Saito et al. 2000), is the major sphingolipid in circulating HDL (5.6–6.6 mol % of total lipids) (Wiesner et al. 2009; Camont et al. 2013; Stahlmanet al. 2013) (Table 3), which largely originates from triacylglyceride-richlipoproteins and only to a minor extent from nascent HDL (Nilsson and Duan2006). Major molecular species of sphingomyelin are the 16:0 and 24:1 species(Lhomme et al. 2012). Unlike negatively charged PL, sphingomyelin is depleted byup to 30 % in small, dense relative to large, light HDL (Kontush et al. 2007; Camontet al. 2013) (Fig. 2). This result may, in part, reflect the low abundance ofsphingomyelin in nascent HDL, a metabolic precursor of HDL3c, and suggestdistinct metabolic pathways for HDL subpopulations.Among lysosphingolipids, S1P is particularly interesting as this bioactive lipidplays key roles in vascular biology (Lucke and Levkau 2010). More than 90 % ofcirculating sphingoid base phosphates are found in HDL and albumin-containingfractions (Table 3) (Kontush and Chapman 2012). Interestingly, S1P associatespreferentially with small, dense HDL particles (up to tenfold enrichment comparedto large, light HDL) (Kontush et al. 2007) (Fig. 2) consistent with the high contentin apoM, a specific carrier of S1P, in small, dense particles (Davidson et al. 2009).Other biologically active lysosphingolipids carried by HDL are represented bylysosphingomyelin and lysosulfatide (Lhomme et al. 2012).Ceramide is a sphingolipid intermediate implicated in cell signalling, apoptosis,inflammatory responses, mitochondrial function and insulin sensitivity (Lipina andHundal 2011). This lipid is poorly transported by HDL, which carries only25 mol % of total plasma ceramide (Wiesner et al. 2009), and constitutes onlybetween 0.022 and 0.097 mol % of total HDL lipids (Wiesner et al. 2009; Argraveset al. 2011; Camont et al. 2013; Stahlman et al. 2013) (Table 3). Similarly tosphingomyelin, this product of sphingomyelin hydrolysis is enriched in large, lightHDL (Fig. 2), suggesting common metabolic pathways for these lipids. Thishypothesis is however not supported by the pattern of major molecular species ofceramide observed in HDL, which are the 24:0 and 24:1 species (Wiesneret al. 2009; Stahlman et al. 2013).Lipidomic data on glycosphingolipids, gangliosides and sulfatides are scarce(Lhomme et al. 2012). Hexosyl and lactosyl species constitute the majorglycosphingolipids in plasma lipoproteins (Scherer et al. 2010) (Table 3).2.2.3 Neutral LipidsUnesterified (free) sterols are located in the surface lipid monolayer of HDLparticles and regulate its fluidity. HDL sterols are dominated by cholesterol,reflecting the key role of lipoproteins in cholesterol transport through the body.Other sterols are present in lipoproteins at much lower levels as exemplified byminor amounts of lathosterol, ergosterol, phytosterols (β-sitosterol, campesterol),oxysterols and estrogens (largely circulating as esters) (Kontush and Chapman2012). Free cholesterol, whose affinity for sphingomyelin is now well established,tends to preferentially associate with large, light HDL (Fig. 2).Structure of HDL: Particle Subclasses and Molecular Components 27Cholesteryl esters (CE) are largely (up to 80 %) formed in plasma HDL (Fig. 2),as a result of transesterification of PL and cholesterol catalysed by LCAT. Thesehighly hydrophobic lipids form the lipid core of HDL and contribute up to 36 mol %of total HDL lipid (Wiesner et al. 2009; Camont et al. 2013; Stahlman et al. 2013)(Table 3). Most of HDL CE is accounted for by cholesteryl linoleate (Lhommeet al. 2012). A pioneering work on CE molecular species distribution across HDLsubpopulations using gas chromatography showed very similar profiles betweenHDL2 and HDL3 particles (Vieu et al. 1996).HDL-associated triacylglycerides (TAG) are dominated by species containingoleic, palmitic and linoleic acid moieties (Lhomme et al. 2012) and representaround 3 mol % of total HDL lipids (Vieu et al. 1996; Kontush et al. 2007; Wiesneret al. 2009; Camont et al. 2013; Stahlman et al. 2013) (Table 3). Similarly to CE,TAG species profile is conserved between HDL2 and HDL3 (Vieu et al. 1996).Minor bioactive lipids present in HDL include diacylglycerides (DAG),monoacylglycerides (MAG) and free fatty acids (Lhomme et al. 2012). In thestudy of Vieu et al., sn-1,2 and sn-1,3 isomers of DAG species were identified inHDL (Vieu et al. 1996). More recently, detailed characterisation of DAG molecularspecies in HDL revealed 16:0/18:1 and 18:1/18:1 as major species, estimating theircontent at 0.2 mol % of total lipids (Stahlman et al. 2013) (Table 3).HDL composition in free fatty acids shows a predominance of palmitic, stearicand oleic acid-containing species (Lhomme et al. 2012).Together, available lipidomic studies have already provided detailedcharacterisation of HDL isolated using two complementary techniques, FPLC(Wiesner et al. 2009) and ultracentrifugation (Kontush et al. 2007; Camontet al. 2013). The latter studies demonstrated preferential association of negativelycharged lipids (PS, PA) and S1P with small, dense HDL together with preferentialassociation of sphingomyelin and ceramide with large, light particles, consistentwith distinct metabolic origins and potent biological activities of small, dense HDL(Camont et al. 2013). These data illustrate the power of lipidomics to providecrucial information on the metabolism and function of lipoproteins relevant forthe development of cardiovascular disease, which can in turn deliver novelbiomarkers of cardiovascular risk.3 The Structure of HDL3.1 Introduction/Brief HistoryWhen it was becoming clear that plasma levels of HDL were inversely correlatedwith cardiovascular disease, numerous laboratories were already doing pioneeringwork on the structure of human plasma HDL (Edelstein et al. 1972; Jonas 1972;Laggner et al. 1973; Atkinson et al. 1974; Schonfeld et al. 1976; Tardieuet al. 1976). Knowing little about the nature of the stabilising proteins, Scanu andcolleagues used detailed compositional studies to build models of human HDL328 A. Kontush et al.particles (Shen et al. 1977; Scanu 1978). Concepts derived from these studies withrespect to fundamental packing of the protein and various lipid components stillapply today. After the sequences of the major HDL proteins, apolipoprotein A-I andapoA-II, were reported in the 1970s (Brewer et al. 1972, 1978), Segrest (Segrestet al. 1974) and others (McLachlan 1977) quickly noted periodically repeating unitsthat, when mapped on a helical wheel plot, indicated the presence of amphipathicalpha helices. With hydrophobic faces mediating lipid interactions and polar facesinteracting with water, these structures turned out to be responsible for thedetergent-like ability of these proteins to solubilise lipids into stabile lipoproteinparticles. At around the same time, Jonas and colleagues were learning how tocombine purified apolipoproteins with lipids under control of detergents to producerecombinant forms of HDL (Matz and Jonas 1982). Furthermore, the work ofNichols et al was exploring new ways to characterise these particles by nativeelectrophoresis (Nichols et al. 1983). Because the electron microscopy work ofForte indicated that they likely have a discoid shape (Forte and Nordhausen 1986),these particles were referred to as reconstituted (r)HDL discs. Their compositionalsimplicity and homogeneity quickly made them a mainstay for HDL structuralstudies.Segrest et al. and others (Wlodawer et al. 1979) proposed in the late 1970s thatthe α-helices of apoA-I were arranged around the circumference of discoidal HDLwith the long axis of the helices perpendicular to the acyl chains (Fig. 3). Thisbecame known as the “belt” or “bicycle wheel” model. Alternatively, otherinvestigators theorised that the 22-amino acid helical repeats were an ideal lengthto traverse the bilayer edge with the helices parallel to the acyl chains (Jonaset al. 1989; Nolte and Atkinson 1992). The intervening proline residues werethought to induce hairpin turns that punctuated the repeats. This “picket fence”model was favoured during the early 1990s because of supporting infrared(IR) spectroscopy studies (Brasseur et al. 1990) and the fact that the modelaccounted nicely for the experimentally observed size classes of apoA-I containingHDL (Wald et al. 1990). Furthermore, the plausibility of the picket fence model wassupported by Phillips et al. using molecular modelling techniques (Phillipset al. 1997).However, the publication of the first successful X-ray crystal structure of alipid-free fragment of apoA-I by Borhani et al. (1997) brought fresh energy to thedebate. The crystal structure showed a tetramer of highly α-helical apoA-Imolecules arranged in a ring-shaped complex with no evidence of hairpin turnsthat would be prevalent in the picket fence. The belt model picked up moresupport with methodologically updated IR experiments performed by Koppakaet al. (1999) that contradicted the earlier IR studies supporting the picket fence inrHDL discs. Over the next few years, several laboratories reported results thatsupported the belt orientation using methodologies like fluorescence energy transfer(Li et al. 2000) and lipid-based fluorescence quenching (Panagotopulos et al. 2001).With the question of helical orientation largely addressed, much of the focus for thefirst decade of the 2000s centred on determining the spatial relationships betweenStructure of HDL: Particle Subclasses and Molecular Components 29apoA-I molecules on these discs. This effort was aided by the detailed “double belt”computer model put forth in the classic paper by Segrest (Segrest et al. 1999).The preceding paragraphs quickly summarise a tremendous amount of workbetween 1970 and 2000 aimed at understanding the structure of HDL and itsrecombinant forms, and by no means does it do justice to all contributions. Formore complete discussions of this period of HDL structural research, the reader isreferred to the classic review series by Brouillette (Brouillette and Anantharamaiah1995; Brouillette et al. 2001). In addition, due to space limitations, this section willnot focus on the structure of lipid-free apolipoproteins nor their proposedmechanisms for lipid binding and particle generation. The reader is directed tothe recent review by Phillips (Phillips 2013) for an excellent discussion of theseissues. For the purposes of this section, we will focus on work done over the last14 years that has built upon the concept of the double belt model in bothreconstituted and “real” circulating plasma HDL.Fig. 3 The double belt model for a rHDL discoidal particle and its various refinements. Theclassic LL 5/5 double belt model for apoA-I as proposed by Segrest (Segrest et al. 2000) is shownin panel (a). Each amphipathic helical domain is shown in a different colour with helix 5 in greenat the front. This model did not include the N-terminal 43 a.a. The “belt and buckle” model (b)does not form a continuous circle around the particle but has its N-terminal 43 a.a. folding backacross the belts (40). The “looped belt” (c) features a localised separation between residues133 and 146 (helix 5) (41). The “solar flares” model (d) encapsulates the lipid with an antiparalleldouble belt but has disorganised domains that resemble solar flares erupting between residues165 and 180. The double superhelix model (e) maintains similar intermolecular contact among themolecules of apoA-I as the double belt, but the lipid is stabilised as an elongated micelle and nointramolecular contact is made between the N- and C-termini of apoA-I30 A. Kontush et al.3.2 HDL in the Test Tube3.2.1 Discoid HDLDiscoid HDLs probably exist in human plasma but are extremely short-lived due tothe fact that they are excellent substrates for LCAT (Jonas 2002). HDL discs havebeen detected in sequestered compartments like peripheral lymph and interstitialfluid which likely have lower LCAT activity (Sloop et al. 1983). As stated above,discs can be reconstituted in vitro, and these particles exhibit many of the traits ofnative HDL such as LCAT activation (Jonas and McHugh 1984), lipid transfer(Davidson et al. 1995) and receptor binding (Liadaki et al. 2000).The most well-studied rHDL discs consist of two apoA-I molecules, are ~96 Å indiameter and ~47 Å in thickness and contain 150–200 phospholipid molecules(Jonas 1986; Jonas et al. 1989). This “benchmark” particle was used as a basis forthe original double belt molecular model (Segrest et al. 1999). In this model, tworing-shaped apoA-I molecules encapsulate a lipid membrane leaflet in an antiparal-lel orientation (Segrest 1977; Segrest et al. 1999; Brouillette et al. 2001; Klonet al. 2002a) in which helix 5 of both apoA-I molecules directly oppose each other(Fig. 3a). In this configuration, there are two possible interfaces between themolecules depending on how they are stacked: left to left (LL) and right to right(RR). Computer analysis indicated that an LL interface in which helix 5 (morespecifically, glycine 129) of both molecules are directly opposed produced thehighest weighted score of potential salt bridges between the apoA-I molecules(Segrest et al. 1999). This is called the LL5/5 (G129j) registry and, interestingly,is the same orientation found in the Borhani crystal structure (Borhani et al. 1997).This orientation was experimentally validated in three studies that utilised chemicalcross-linking to derive the molecular orientation of the two apoA-I molecules inthese particles. Two from our laboratory were highly consistent with the 5/5orientation across most of the molecule, although certain cross-links were alsoconsistent with a second 5/2 registry (Davidson and Hilliard 2003; Silvaet al. 2005). Cross-linking studies by Bhat et al. (2005) were also consistent withthe general 5/5 belt model for most of the molecule, but these investigatorsproposed that the N- and C-terminal 40–50 residues doubled back on the molecule(Fig. 3b). This refinement was called the “belt and buckle”. Electron paramagneticresonance studies performed by Martin and colleagues (Martin et al. 2006) on 96 ÅrHDL particles were also consistent with a 5/5 antiparallel double belt orientationover most of the particle circumference. However, they observed that the spincoupling signatures of residues 134–145 (within helix 5) were consistent with alooping region that causes a localised opening between the parallel belts. This“looped belt” was proposed to be a potential site by which LCAT could gain accessto the cholesterol and phospholipid acyl chains that are otherwise buried in the lipidbilayer (Fig. 3c). Using hydrogen/deuterium exchange (HDX) experiments, Wu andcolleagues proposed an alternative refinement of the double belt model whichincluded the full-length apoA-I (Wu et al. 2007). This “solar flares” model againfeatured a 5/5 double belt for most of the particle, but the N-termini were modelledas globular nodules (Fig. 3d). The HDX profile of the region between 165 andStructure of HDL: Particle Subclasses and Molecular Components 31180 suggested that this region loops off the particle edge and could be a site forLCAT interaction. This argument was bolstered by results showing that addedLCAT could alter the exchange patterns of these residues and that mutations inthis region affected LCAT activity. The conclusions of this study were called intosome question by a more recent HDX study performed by Chetty et al. on similarparticles that failed to support the existence of the looped solar flares (Chettyet al. 2013). They showed that almost all of apoA-I was helical in the discs withthe exception of the first 6 and last 7 residues. Interestingly, the region betweenresidues 125 and 158 exhibited bimodal exchange kinetics, suggesting that thisregion flits between a helical and less ordered structure. This is consistent with thelooped belt idea (Martin et al. 2006), but the putative solar flares region(Wu et al. 2007) was not found to be particularly dynamic in this study. The authorssuggested that this region is responsible for absorbing changes in particle size byunfolding under conditions when lipid surface area is limiting. This is consistentwith previous studies from our laboratory which used lipid-based fluorescencequenching agents to show that this same region changes its exposure to lipidwhen the particle diameter is reduced (Maiorano et al. 2004).The recent publication of a high-resolution (2.2 Å) crystal structure of a lipid-free truncation mutant of apoA-I (Δ185–243) also strongly supports the double beltmodel (Mei and Atkinson 2011). Although lipid-free, the dimeric apoA-I formed acurved structure derived from long, kinked alpha helices. Interestingly, much of thestructure resembled the 5/5 double belt, even down to a similar degree of curvature.The model also suggested increased temperature factors in helix 5 hinting that therecould be a separation of the helices at this site, consistent with the looped belt idea.This advance should prove to be a valuable guide for future modelling of both thelipid-free and lipid-bound forms of apoA-I.The models discussed above conform to the generally accepted idea that theserHDLs are discoid in shape. However, Hazen and colleagues proposed a model thatdeparted from this idea (Wu et al. 2009). Based on small-angle neutron scattering(SANS), they proposed that antiparallel apoA-I molecules form a double superhe-lix (DSH) that encapsulates a cigar-shaped micelle of phospholipids (Fig. 3e). Thus,the particles resemble an ellipsoid rather than a disc. Despite the different shape, theoverall molecular interactions along the interface of the two apoA-I molecules arethe same as the 5/5 double belt model. However, unlike in the planar disc, the N-and C-termini are not predicted interact in the DSH. The viability of this modelremains under debate as molecular dynamics studies suggest that this model rapidlycollapses to a disc-shaped structure (Jones et al. 2010). Even simulations done bythe group that proposed the model seem to indicate that the elongated structurecollapses to an oblate spheroid, though they argue it remains micellar (Gogoneaet al. 2010). Going forward, this model must be squared with observations of disc-like shapes by electron microscopy (Zhang et al. 2011), EPR data (Joneset al. 2010), and the quantised changes in particle size that can be readilydemonstrated by reconstituting different lipid to protein ratios (Wald et al. 1990).In addition to experimental work, significant progress has been made usingmolecular dynamics to simulate the structure of rHDL discs. The earliest32 A. Kontush et al.simulations by Klon et al. utilised apoA-I lacking the N-terminal 43 amino acids(Klon et al. 2002b). The short 1 ns simulations were performed using the 5/5 doublebelt model, encapsulating simulated phospholipids in a standard ~96 Å particle as astarting point. The simulations provided four major conclusions. First, the overallstructure remained intact over the course of the simulation, arguing the physicalplausibility of the structure. Second, specific salt bridges were found to be impor-tant with regard to the molecular registry of the two apoA-I molecules. Third, thepunctuating proline residues were found to induce peri-helical “kinks” that alsoplayed a role in molecular registry. Finally, the motions of the molecule suggestedthat apoA-I may have the potential to change its molecular registry. This conceptwas first proposed in the fluorescence resonance energy transfer experiments of Liet al. (2002), and the concept was later supported by certain chemical cross-links(Silva et al. 2005). This idea of molecular registry shifts on the HDL particle surfaceraises fascinating implications with respect to alternative interactions with the widearray of HDL docking proteins.Further simulations in which lipids were added to or subtracted from the systemshowed that apoA-I can twist into a saddle-shaped structure in response to lipidremoval (Gu et al. 2010). These results were supported by independent studies(Miyazaki et al. 2010) and in subsequent temperature jump studies using full-lengthapoA-I (Jones et al. 2011). Segrest and colleagues concluded that apoA-I maintainsminimal surface bilayers and responds to changes in surface lipid content bywinding or unwinding of this twisted saddle shape. It is important to emphasise,however, that these twisted models of dimeric apoA-I maintain similar intermolec-ular contacts as the planar double belt model and thus are consistent with thefluorescence, EPR and cross-linking data described above.3.2.2 Spherical rHDLAlthough the majority of HDL particles in human plasma are spherical, theseparticles are extremely understudied from a structural point of view. SphericalHDL particles contain a neutral lipid core composed of cholesteryl ester andtriglyceride and thus lack a particle “edge” to constrain the apolipoproteins as inthe discs. The helical domains have the potential to spread across the phospholipidsurface penetrating past the phosphate group to interact with the acyl chains.Segrest et al. (1994) argued early on that if apoA-I exists in a belt-like model indiscoidal particles, the fundamental interactions of apoA-I helices with the phos-pholipid acyl chains should not change significantly in spheres. Brasseur (Brasseuret al. 1990) and Borhani (Borhani et al. 1997) have also argued the similarity inapoA-I structure between the two shapes. These arguments were supported bycircular dichroism and fluorescence studies showing that apoA-I secondary struc-ture content and the exposure of Trp residues do not undergo gross changes when areconstituted discoidal particle converts to a sphere in the presence of LCAT (Jonaset al. 1990). In addition, careful NMR studies suggested that the changes inconformation of apoA-I between reconstituted discs and spheres are primarilylimited to the apoA-I N-terminus (Sparks et al. 1992).Structure of HDL: Particle Subclasses and Molecular Components 33Using the cross-linking approach, we studied the structure of reconstitutedapoA-I-containing spherical particles generated by incubating rHDL discs withLCAT under controlled conditions (Silva et al. 2008). These spheres contained alargely cholesteryl ester core and were of roughly similar diameter to the “bench-mark” discs described above. Cross-linking experiments showed that these spherescontained 3 molecules of apoA-I, vs. only two in the discs. Quite surprisingly, thecross-linking pattern derived in the spheres was almost identical to that of the discs.Our solution to the problem of how the exact same molecular interactions occurbetween two apoA-Is in a disc vs. three in a sphere was the “trefoil model” (Fig. 4).In this scheme, kinks were added in helix 5 and 10 of apoA-I. Two antiparallel beltsas per the double belt model are then bent 120 on these kinks and a third molecule,bent the same way, was inserted. This formed a three-dimensional cage-likestructure in which the helical domains in each of the three apoA-I moleculesmade exactly the same salt bridge interactions postulated for the original doublebelt. This cage can be envisioned to encapsulate a neutral lipid ester core andsupport the surface polar lipids in the intervening open spaces, or lines, between themolecules.Studying the exact same particles using SANS, Wu and colleagues came to adifferent conclusion (Wu et al. 2011). The low-resolution molecular envelope of theprotein component was consistent with three different architectures: (1) two apoA-Imolecules in a double belt with the third as a separate hairpin, called the helicaldimer with hairpin (HdHp), (2) three separate hairpins (3Hp), or (3) an integratedtrimer (iT) which is similar to the trefoil arrangement. Based on chemical cross-linking, the authors argued that the data was most consistent with the HdHp model.Despite the differences in the HdHp and the trefoil arrangements from a conceptualpoint of view, the models are quite similar from a molecular perspective in that theinteractions in both models are predicted to be similar. All contacts in the trefoil areintermolecular where the interactions within the hairpined apoA-I are intramolecu-lar, but the same sequences interact in all molecules. Thus, the epitopes on the HDLparticles should be similar in both models and therefore might be expected toFig. 4 The development of the trefoil model for a spherical particle from the double belt. Theclassic LL 5/5 double belt for apoA-I is shown in panel (a) coloured as in Fig. 3. (b) Kinks areintroduced within helix 5 and helix 10 and the molecules are bent 60 out of the plane of the disc. Athird molecule of apoA-I, bent the same way, can now be introduced. (c) The trefoil modeldemonstrates similar intermolecular contacts between all three apoA-I molecules34 A. Kontush et al.interact similarly with HDL docking proteins. It should also be noted that analternative spherical model that is related to the trefoil organisation was proposedby Gursky (Gursky 2013) that invokes different hinge domains as extrapolatedfrom the crystal structure of the lipid-free apoA-I fragment (Mei and Atkinson2011). Distinction between these models awaits the development of strategies thatallow for the higher resolution determination of cross-link span on particlescontaining more than two molecules of apoA-I.Catte et al. made initial explorations into the structure of reconstituted sphericalparticles using a combination of atomistic and course-grained molecular dynamicssimulations (Catte et al. 2008). The simulated particle consisted of a saddle-shapedrHDL particle with two molecules of apoA-I in which surface phospholipids werereplaced in silico with cholesteryl ester (CE) molecules. During the simulations, theCE is sequestered into a core that was well covered by the surface protein andphospholipid, forming a prolate ellipsoid shape. The overall helicity and salt bridgeorientation between the resident apoA-I molecules remained similar to the discmodels, though there was an increase in overall salt bridge number. Despite anuncertain relationship with experimentally derived spherical particles, thesesimulations are consistent with the idea that apoA-I structures in discs and spheresare generally similar. The same group later simulated particles that more closelyresembled those appearing in plasma (Segrest et al. 2013). Their data suggested thatspheres created under artificial conditions in the test tube may represent kineticallytrapped species with artificially high ratios of surface lipids with respect to thescaffold proteins. However, again, the resident apoA-I particles exhibited overallsimilar structures.3.3 “Real” HDL ParticlesReconstituted HDL discs and spheres have been a critical tool for understanding thefundamental arrangements of apoA-I on the HDL particle surface. The homogene-ity of these preparations allowed the use of traditional spectrophotometricapproaches such as circular dichroism, NMR, EPR, etc. However, the ultimategoal has always been to understand HDL in its natural plasma form. As outlined inother sections of this chapter, human plasma HDL as isolated by density gradientultracentrifugation is heterogeneous in almost every property: protein composition,lipid composition, particle size, particle shape and particle charge. This heteroge-neity precludes the use of most traditional structural techniques. For example,circular dichroism studies of a mixed system in which apoA-I is present either inseveral different conformations or with other apolipoproteins will produce averageddata that will likely not be applicable to any of the particles present in the sample.Even attempts to simplify these particles, such as by immunoisolating only thoseparticles that contain apoA-I but not apoA-II (LpA-I), still result in preparationsStructure of HDL: Particle Subclasses and Molecular Components 35with unacceptable compositional and size heterogeneity for informative structuralstudies. For these reasons, our understanding of HDL structure in authentic plasmaHDL particles has lagged far behind the reconstituted forms.The use of mass spectrometry as a structural tool has finally allowed this barrierto be breached. With the mass accuracy and resolution of modern massspectrometers, it is possible to identify cross-links that are specific for a givenprotein, even in the presence of many others. Taking advantage of this, we studied5 density subfractions of normal human plasma HDL as delineated by ultracentri-fugation. In order to simplify the particles to the extent possible, we isolated thoseparticles containing predominantly apoA-I (Huang et al. 2011). Surprisingly, wenoted cross-linking patterns that were remarkably similar to those found in thereconstituted spheres and discs, irrespective of the particle size or density. Thisstrongly suggested that the general features of apoA-I structure are related amonglipid-associated forms of all sizes, shapes and origin. Careful measurements of theprotein/lipid components indicated a range 3–5 apoA-I molecules per particle,depending on size. In fact, an important outcome of this study was the realisationthat the surface of HDL particle is dominated by protein. For example, we deter-mined that apoA-I accounts for 87 % of the surface of an LpA-I3c particle. This onlyreduces to 71 % in the larger LpA-I2b particles. This is in agreement with theassertions of Segrest et al. (2013) whose compositional calculations indicate thatapoA-I is much more tightly packed on native spheres than it is on typicalreconstituted particles. Figure 5 shows one proposal for how 4 and 5 apoA-Is canbe accommodated by changing the hinge bend angles of the trefoil whilemaintaining the same intermolecular interactions, and hence cross-linking pattern.Furthermore, we proposed that HDL particle size is modulated via a twistingmotion of the resident apoA-Is, consistent with the MD studies of the Segrest lab(Catte et al. 2008). Combining these data, we developed the first detailed molecularmodels for native LpA-I HDL particles, exemplified by that for LpA-I2b in Fig. 6. Inthis model, apoA-I forms an 11 nm cage stabilising ~170 molecules of phospholipidat a physiological packing density. The cutaway shows a cavity of correct volumefor the ~143 CEs and ~23 TGs measured in the core. These are the first detailed,molecular-scale models of native plasma HDL to be proposed.Taking another MS approach, Chetty et al. used HDX to study spherical LpA-Iparticles isolated from human plasma by ultracentrifugation and further isolated byhigh-resolution gel filtration chromatography (Chetty et al. 2013). These particleswere approximately 10 nm in diameter and contained on average 5 molecules ofapoA-I per particle as measured by cross-linking. Comparing these directly to abenchmark rHDL disc, they found that the total helical content was similar betweenthe native spheres and the reconstituted discs. Strikingly, the HDX profiles werealso quite similar between the two with both showing near-complete helicity withthe exception of the first and last 6 or so residues. Like the discs, the plasma spheresshowed bimodal HDX kinetics indicating a possible loop region in helix 5 ofapoA-I. Interestingly, the length of this partially disordered segment was similar36 A. Kontush et al.to that of rHDL discs of 8 nm in diameter, suggesting that apoA-I on these spheres ismore tightly packed than in the 96 Å discs. This is consistent with higher number ofapoA-I molecules on the surface. Overall, these studies reinforce the idea that theglobal apoA-I organisation does not change significantly between particles ofdifferent shape or origin; however, more subtle conformational adaptations mayoccur in localised regions of the protein in response to changes in either particlediameter or surface packing density.Fig. 5 Models of apoA-I in human plasma HDL particles of various size and number of apoA-Imolecules. (a) The trefoil model is shown as a schematic with each molecule of apoA-I in adifferent colour. (b) LpA-I2b contains 5 apoA-I molecules, but shows a similar cross-linkingpatterns to reconstituted particles that have only 2 and 3 molecules of apoA-I. This figure showsthat more apoA-I molecules can be added to the trefoil framework by increasing the hinge bendangle and adding more apoA-I molecules. (c) A possible model for LpA-I2a that has four apoA-Imolecules on average. (d) Further reductions in HDL particle size may be accomplished by atwisting action of the resident apoA-I moleculesStructure of HDL: Particle Subclasses and Molecular Components 37Fig. 6 Molecular model of a human plasma LpA-I2b particle. (a) Five molecules of apoA-I areshown in an organisation shown schematically in Fig. 5b. Phospholipid headgroups (tan) arepresent in the intervening spaces (lunes) between the apoA-I belts (purple). (b) A cross section of thesame particle showing the helical belts floating atop the acyl chains of the phospholipids (brown).Note the large internal cavity that is of sufficient volume to contain the number of triglycerides andcholesteryl ester molecules that have been measured in these particles (Huang et al. 2011). (Colourfigure online)38 A. Kontush et al.Conclusions and PerspectivesHDL, a lipoprotein class historically and classically defined and isolated on thebasis of its hydrated density alone, is a highly heterogeneous particle familyconsisting of multiple subclasses differing in hydrated density, size, shape andlipid and protein composition. HDL-associated proteins have been considereduntil now to predominate in determining the particle structure and biologicalfunction of all HDL particles. Indeed, HDL is distinguished from other lipopro-tein classes by its cargo of a large number (>80) of different proteins whoseabundance may vary by more than 1,000-fold and which fall into several majorsubgroups, including apolipoproteins, diverse enzymes, lipid transfer proteins,acute-phase response proteins, complement components and proteinaseinhibitors; additional functions, such as host defence and inflammation, cannotbe excluded. Many of the individual proteins are present as distinct isoforms dueto co- and posttranslational modification, thereby adding a further dimension tothe complex and dynamic HDL structure.It would be erroneous however to consider that the proteome(s) of HDLparticles solely determines their respective functions, particularly as cuttingedge lipidomic technologies have recently revealed significant—and indeeddeterminant—roles of specific HDL lipid components in particle function. Inthis regard, negatively charged phospholipids, and notably molecular species ofphosphatidylserines, are intimately associated with the cellular cholesterolefflux, antioxidative, antiapoptotic and anti-inflammatory actions of smalldense HDL3 particles and equally modulate platelet activity (Camontet al. 2013). In addition, earlier studies have suggested that both core neutrallipids in HDL (cholesteryl esters and triacylglycerols) and surface lipids canimpact surface fluidity and, in turn, antioxidative and other functions (Zerrad-Saadi et al. 2009; Camont et al. 2013).Increasingly then, a new concept is emerging, one in which specific proteinand lipid components interact closely to form individual HDL particle species,such as overall compositional and structural features translating into expressionof highly defined biological functions (Camont et al. 2011; Shah et al. 2013).Clearly we are at the beginning of a new era in our understanding of HDLstructure and function, with the promise of exciting mechanistic insights intofunction, which, in turn, will translate into new therapeutic strategies to harnessthe anti-atherosclerotic potential of HDL.The central thesis that specific protein–protein interactions may drive theformation of lipoprotein complexes in the HDL particle spectrum is equallyemergent. Indeed, the diversity of molecules which bind to HDL suggests thatthe lipoprotein can serve as a versatile adsorptive surface for proteins to formcomplexes playing roles not only in lipid metabolism but equally in the acute-phase response, innate immune response, complement activation, proteolysisinhibition and plaque stability.More comprehensive understanding of HDL structure is critical to futureprogress in HDL biology. A molecular understanding of HDL-bound apoA-Iwill allow further insight into its interactions with key plasma remodellingStructure of HDL: Particle Subclasses and Molecular Components 39factors, such as LCAT, PLTP or CETP, and in addition with cell-surface proteinsthat mediate HDL assembly and clearance, such as ABCA1 and scavengerreceptor B-I. A firm handle on these interactions would contribute to therapeuticmanipulation that could enhance the protective features of HDL with respect tocardiovascular disease, or perhaps other pathologies (Gordon et al. 2011).Because HDL and its recombinant analogues have been resistant to traditionalhigh-resolution structural approaches such as NMR or X-ray crystallography,investigators have been forced to apply a series of lower resolution techniques tothe problem. As described above, these approaches have led to a generalunderstanding of apoA-I structure in HDL that can be summarised by thefollowing statements: (1) apoA-I pairs with itself in a double belt-like arrange-ment in almost all of its lipidated states; (2) apoA-I interacts via intermolecularsalt bridges that commonly stabilise the registry in the 5/5 orientation, thoughthere may be a possibility of dynamic shifts to other orientations; and (3) despitea similar general organisation among different types of HDL particles, specificregions within the scaffold can undergo conformational changes depending onparticle size or perhaps composition—possible sites include the N- andC-termini and central region in helix 5 or between residues 165 and 180.These conformationally dynamic sites are attractive possibilities for the interac-tion of lipid-modifying docking proteins such as LCAT and CETP.Now that these conformationally dynamic sites within apoA-I have beenproposed, it will be important to specifically define their importance for keyHDL remodelling reactions. This will require well-designed site-directed muta-genesis experiments in reconstituted particles and antibody blockingexperiments in native plasma particles. Furthermore, cross-linking studies holdmuch promise for directly mapping the sites of interaction for plasmaremodelling factors on HDL-bound apoA-I.Finally, yet another key unanswered question relates to the unknown effectsof other HDL structural proteins on apoA-I structure. ApoA-II is commonlyfound with apoA-I on many HDL particles and would be expected to have asignificant impact not only on properties such as protein packing on the particlesurface but also on the conformation of apoA-I in a functionally significant way.For example, does apoA-II perform distinct structural and protein docking rolesvs. apoA-I? How does each of these proteins determine the components of thebound lipidome in individual particle species? Resolution of these and otherquestions will continue to require the use of both reconstituted and native,authentic HDL particles combined with the appropriate application of multiplestructural techniques (including continued attempts at X-ray crystallography).It is poignant to consider that in 2014 we still lack critically important insightsinto HDL structure as it relates to the intravascular metabolism andatheroprotective function of these pseudomicellar, quasi-spherical particles.Had sufficient effort been invested in solving these unknowns heretofore, thenperhaps our costly forays—and failures—into HDL-targeted therapeutics overthe past 15 years could at least partially have been avoided.40 A. Kontush et al.Acknowledgements This article results from individual contributions of all the co-authors. Thehighly complex world of HDL subclasses is presented by Laura Calabresi. The multifacetedcomposition of the HDL proteome is discussed by Anatol Kontush. Protein isoforms, includingtranslational and posttranslational modifications, are reviewed by Mats Lindahl. Rapidly growingknowledge of the HDL lipidome is discussed by Marie Lhomme. W. Sean Davidson presents abrief history of structural studies of HDL, critically reviews existing models of HDL structure, andtogether with M. John Chapman draws conclusions and discusses future perspectives.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAhnstrom J et al (2007) Hydrophobic ligand binding properties of the human lipocalin apolipo-protein M. J Lipid Res 48(8):1754–1762Allan CM, Taylor JM (1996) Expression of a novel human apolipoprotein (apoC-IV) causeshypertriglyceridemia in transgenic mice. J Lipid Res 37(7):1510–1518Argraves KM et al (2011) S1P, dihydro-S1P and C24:1-ceramide levels in the HDL-containingfraction of serum inversely correlate with occurrence of ischemic heart disease. Lipids HealthDis 10:70Asztalos BF, Schaefer EJ (2003a) High-density lipoprotein subpopulations in pathologicconditions. Am J Cardiol 91(7A):12E–17EAsztalos BF, Schaefer EJ (2003b) HDL in atherosclerosis: actor or bystander? Atheroscler Suppl 4(1):21–29Asztalos BF et al (2007) Role of LCAT in HDL remodeling: investigation of LCAT deficiencystates. J Lipid Res 48(3):592–599Atkinson D et al (1974) The structure of a high density lipoprotein (HDL3) from porcine plasma.Proc R Soc Lond B Biol Sci 186(083):165–180Axler O et al (2007) An ELISA for apolipoprotein M reveals a strong correlation to totalcholesterol in human plasma. J Lipid Res 48(8):1772–1780Axler O et al (2008) Apolipoprotein M associates to lipoproteins through its retained signalpeptide. FEBS Lett 582(5):826–828Baenziger JU (2012) Moving the O-glycoproteome from form to function. Proc Natl Acad SciUSA 109(25):9672–9673Benditt EP, Eriksen N (1977) Amyloid protein SAA is associated with high density lipoproteinfrom human serum. Proc Natl Acad Sci USA 74(9):4025–4028Bergmeier C et al (2004) Distribution spectrum of paraoxonase activity in HDL fractions. ClinChem 50(12):2309–2315Bhat S et al (2005) Intermolecular contact between globular N-terminal fold and C-terminaldomain of ApoA-I stabilizes its lipid-bound conformation: studies employing chemicalcross-linking and mass spectrometry. J Biol Chem 280(38):33015–33025Bisgaier CL et al (1985) Distribution of apolipoprotein A-IV in human plasma. J Lipid Res 26(1):11–25Bondarenko PV et al (1999) Mass spectral study of polymorphism of the apolipoproteins of verylow density lipoprotein. J Lipid Res 40(3):543–555Borhani DW et al (1997) Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation. Proc Natl Acad Sci USA 94(23):12291–12296Brasseur R et al (1990) Mode of assembly of amphipathic helical segments in model high-densitylipoproteins. Biochim Biophys Acta 1043(3):245–252Structure of HDL: Particle Subclasses and Molecular Components 41Brewer HB Jr et al (1972) Amino acid sequence of human apoLp-Gln-II (apoA-II), an apolipo-protein isolated from the high-density lipoprotein complex. Proc Natl Acad Sci USA 69(5):1304–1308Brewer HB Jr et al (1978) The amino acid sequence of human APOA-I, an apolipoprotein isolatedfrom high density lipoproteins. Biochem Biophys Res Commun 80(3):623–630Brouillette CG, Anantharamaiah GM (1995) Structural models of human apolipoprotein A-I.Biochim Biophys Acta 1256(2):103–129Brouillette CG et al (2001) Structural models of human apolipoprotein A-I: a critical analysis andreview. Biochim Biophys Acta 1531(1–2):4–46Bruneel A et al (2008) Two dimensional gel electrophoresis of apolipoprotein C-III and MALDI-TOF MS are complementary techniques for the study of combined defects in N- and mucintype O-glycan biosynthesis. Proteomics Clin Appl 2:1670–1674Cabana VG et al (1996) HDL content and composition in acute phase response in three species:triglyceride enrichment of HDL a factor in its decrease. J Lipid Res 37(12):2662–2674Cabana VG et al (2004) Influence of apoA-I and apoE on the formation of serum amyloidA-containing lipoproteins in vivo and in vitro. J Lipid Res 45(2):317–325Calabresi L et al (2001) Limited proteolysis of a disulfide-linked apoA-I dimer in reconstitutedHDL. J Lipid Res 42(6):935–942Camont L et al (2011) Biological activities of HDL subpopulations and their relevance tocardiovascular disease. Trends Mol Med 17(10):594–603Camont L et al (2013) Small, dense high-density lipoprotein-3 particles are enriched in negativelycharged phospholipids: relevance to cellular cholesterol efflux, antioxidative, antithrombotic,anti-inflammatory, and antiapoptotic functionalities. Arterioscler Thromb Vasc Biol 33(12):2715–2723Campos E, McConathy WJ (1986) Distribution of lipids and apolipoproteins in human plasma byvertical spin ultracentrifugation. Arch Biochem Biophys 249(2):455–463Candiano G et al (2008) High-resolution 2-DE for resolving proteins, protein adducts andcomplexes in plasma. Electrophoresis 29(3):682–694Catte A et al (2008) Structure of spheroidal HDL particles revealed by combined atomistic andcoarse-grained simulations. Biophys J 94(6):2306–2319Chapman MJ et al (1981) A density gradient ultracentrifugal procedure for the isolation of themajor lipoprotein classes from human serum. J Lipid Res 22(2):339–358Chen N et al (2000) Physiologic concentrations of homocysteine inhibit the human plasma GSHperoxidase that reduces organic hydroperoxides. J Lab Clin Med 136(1):58–65Chetty PS et al (2013) Comparison of apoA-I helical structure and stability in discoidal andspherical HDL particles by HX and mass spectrometry. J Lipid Res 54(6):1589–1597Cheung MC, Albers JJ (1982) Distribution of high density lipoprotein particles with differentapoprotein composition: particles with A-I and A-II and particles with A-I but no A-II. J LipidRes 23(5):747–753Cheung MC et al (2010) Phospholipid transfer protein in human plasma associates with proteinslinked to immunity and inflammation. Biochemistry 49(34):7314–7322Chiba T et al (2011) Serum amyloid A facilitates the binding of high-density lipoprotein from miceinjected with lipopolysaccharide to vascular proteoglycans. Arterioscler Thromb Vasc Biol 31(6):1326–1332Christoffersen C et al (2008) The signal peptide anchors apolipoprotein M in plasma lipoproteinsand prevents rapid clearance of apolipoprotein M from plasma. J Biol Chem 283(27):18765–18772Christoffersen C et al (2011) Endothelium-protective sphingosine-1-phosphate provided byHDL-associated apolipoprotein M. Proc Natl Acad Sci USA 108(23):9613–9618Coetzee GA et al (1986) Serum amyloid A-containing human high density lipoprotein 3. Density,size, and apolipoprotein composition. J Biol Chem 261(21):9644–965142 A. Kontush et al.Collins LA et al (2010) Integrated approach for the comprehensive characterization of lipoproteinsfrom human plasma using FPLC and nano-HPLC-tandem mass spectrometry. Physiol Geno-mics 40(3):208–215Contiero E et al (1997) Apolipoprotein AI isoforms in serum determined by isoelectric focusingand immunoblotting. Electrophoresis 18(1):122–126Dahlback B, Nielsen LB (2009) Apolipoprotein M affecting lipid metabolism or just catching aride with lipoproteins in the circulation? Cell Mol Life Sci 66(4):559–564Davidson WS, Hilliard GM (2003) The spatial organization of apolipoprotein A-I on the edge ofdiscoidal high density lipoprotein particles: a mass specrometry study. J Biol Chem 278(29):27199–27207DavidsonWS et al (1995) Effects of acceptor particle size on the efflux of cellular free cholesterol.J Biol Chem 270(29):17106–17113Davidson WS et al (2009) Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance to antioxidative function. Arterioscler Thromb Vasc Biol 29(6):870–876Davidsson P et al (2010) Proteomics of apolipoproteins and associated proteins from plasma high-density lipoproteins. Arterioscler Thromb Vasc Biol 30(2):156–163de Beer MC et al (2010) Impact of serum amyloid A on high density lipoprotein composition andlevels. J Lipid Res 51(11):3117–3125De Lalla OF, Gofman JW (1954) Ultracentrifugal analysis of serum lipoproteins. MethodsBiochem Anal 1:459–478de Seny D et al (2008) Monomeric calgranulins measured by SELDI-TOF mass spectrometry andcalprotectin measured by ELISA as biomarkers in arthritis. Clin Chem 54(6):1066–1075de Silva HV et al (1990a) Purification and characterization of apolipoprotein J. J Biol Chem 265(24):14292–14297de Silva HV et al (1990b) A 70-kDa apolipoprotein designated ApoJ is a marker for subclasses ofhuman plasma high density lipoproteins. J Biol Chem 265(22):13240–13247Deguchi H et al (2000) Cardiolipin is a normal component of human plasma lipoproteins. ProcNatl Acad Sci USA 97(4):1743–1748Duchateau PN et al (1997) Apolipoprotein L, a new human high density lipoprotein apolipoproteinexpressed by the pancreas. Identification, cloning, characterization, and plasma distribution ofapolipoprotein L. J Biol Chem 272(41):25576–25582Ducret A et al (1996) Characterization of human serum amyloid A protein isoforms separated bytwo-dimensional electrophoresis by liquid chromatography/electrospray ionization tandemmass spectrometry. Electrophoresis 17(5):866–876Duriez P, Fruchart JC (1999) High-density lipoprotein subclasses and apolipoprotein A-I. ClinChim Acta 286(1–2):97–114Edelstein C et al (1972) On the subunit structure of the protein of human serum high densitylipoprotein. I. A study of its major polypeptide component (Sephadex, fraction 3). J Biol Chem247(18):5842–5849Favari E et al (2004) Depletion of pre-beta-high density lipoprotein by human chymase impairsATP-binding cassette transporter A1- but not scavenger receptor class B type I-mediated lipidefflux to high density lipoprotein. J Biol Chem 279(11):9930–9936Favari E et al (2007) A unique protease-sensitive high density lipoprotein particle containing theapolipoprotein A-I Milano dimer effectively promotes ATP-binding Cassette A1-mediated cellcholesterol efflux. J Biol Chem 282(8):5125–5132Fernandez-Irigoyen J et al (2005) Oxidation of specific methionine and tryptophan residues ofapolipoprotein A-I in hepatocarcinogenesis. Proteomics 5(18):4964–4972Fievet C et al (1995) Non-enzymatic glycosylation of apolipoprotein A-I and its functionalconsequences. Diabete Metab 21(2):95–98Flood-Nichols SK et al (2013) Longitudinal analysis of maternal plasma apolipoproteins inpregnancy: a targeted proteomics approach. Mol Cell Proteomics 12(1):55–64Structure of HDL: Particle Subclasses and Molecular Components 43Forte TM, Nordhausen RW (1986) Electron microscopy of negatively stained lipoproteins.Methods Enzymol 128:442–457Franceschini G et al (1985) Effects of storage on the distribution of high density lipoproteinsubfractions in human sera. J Lipid Res 26(11):1368–1373Franceschini G et al (2007) Effects of fenofibrate and simvastatin on HDL-related biomarkers inlow-HDL patients. Atherosclerosis 195(2):385–391Gauthamadasa K et al (2010) Speciated human high-density lipoprotein protein proximity profiles.Biochemistry 49(50):10656–10665Ghiselli G et al (1985) Origin of apolipoprotein A-I polymorphism in plasma. J Biol Chem 260(29):15662–15668Gidez LI et al (1982) Separation and quantitation of subclasses of human plasma high densitylipoproteins by a simple precipitation procedure. J Lipid Res 23(8):1206–1223Gillard BK et al (2005) Plasma factors required for human apolipoprotein A-II dimerization.Biochemistry 44(2):471–479Gogonea V et al (2010) Congruency between biophysical data from multiple platforms andmolecular dynamics simulation of the double-super helix model of nascent high-densitylipoprotein. Biochemistry 49(34):7323–7343Gordon JI et al (1983) Biosynthesis of human preproapolipoprotein A-II. J Biol Chem 258(22):14054–14059Gordon SM et al (2010) Proteomic characterization of human plasma high density lipoproteinfractionated by gel filtration chromatography. J Proteome Res 9(10):5239–5249Gordon SM et al (2011) High density lipoprotein: it’s not just about lipid transport anymore.Trends Endocrinol Metab 22(1):9–15Gordon SM et al (2013) Multi-dimensional co-separation analysis reveals protein-proteininteractions defining plasma lipoprotein subspecies. Mol Cell Proteomics 12(11):3123–3134Goswami B et al (2009) Paraoxonase: a multifaceted biomolecule. Clin Chim Acta 410(1–2):1–12Gu F et al (2010) Structures of discoidal high density lipoproteins: a combined computational-experimental approach. J Biol Chem 285(7):4652–4665Gursky O (2013) Crystal structure of Delta(185–243)ApoA-I suggests a mechanistic frameworkfor the protein adaptation to the changing lipid load in good cholesterol: from flatland tosphereland via double belt, belt buckle, double hairpin and trefoil/tetrafoil. J Mol Biol 425(1):1–16Gursky O et al (2013) Structural basis for distinct functions of the naturally occurring Cys mutantsof human apolipoprotein A-I. J Lipid Res 54(12):3244–3257Hajduk SL et al (1989) Lysis of Trypanosoma brucei by a toxic subspecies of human high densitylipoprotein. J Biol Chem 264(9):5210–5217Halim A et al (2013) LC-MS/MS characterization of O-glycosylation sites and glycan structures ofhuman cerebrospinal fluid glycoproteins. J Proteome Res 12(2):573–584Harel M et al (2004) Structure and evolution of the serum paraoxonase family of detoxifying andanti-atherosclerotic enzymes. Nat Struct Mol Biol 11(5):412–419Harvey SB et al (2009) O-glycoside biomarker of apolipoprotein C3: responsiveness to obesity,bariatric surgery, and therapy with metformin, to chronic or severe liver disease and tomortality in severe sepsis and graft vs host disease. J Proteome Res 8(2):603–612Hatters DM et al (2006) Apolipoprotein E structure: insights into function. Trends Biochem Sci 31(8):445–454He Y et al (2008) Control of cholesteryl ester transfer protein activity by sequestration of lipidtransfer inhibitor protein in an inactive complex. J Lipid Res 49(7):1529–1537Heinecke JW (2009) The HDL proteome: a marker - and perhaps mediator - of coronary arterydisease. J Lipid Res Supp 50:S167–S171Hennessy LK et al (1997) Isolation of subpopulations of high density lipoproteins: three particlespecies containing apoE and two species devoid of apoE that have affinity for heparin. J LipidRes 38(9):1859–186844 A. Kontush et al.Hoffman JS, Benditt EP (1982) Changes in high density lipoprotein content following endotoxinadministration in the mouse. Formation of serum amyloid protein-rich subfractions. J BiolChem 257(17):10510–10517Holleboom AG et al (2011) Heterozygosity for a loss-of-function mutation in GALNT2 improvesplasma triglyceride clearance in man. Cell Metab 14(6):811–818Holmquist L (2002) Selective extraction of lecithin:cholesterol acyltransferase (EC 2.3.1.43) fromhuman plasma. J Biochem Biophys Methods 52(1):63–68Hoofnagle AN, Heinecke JW (2009) Lipoproteomics: using mass spectrometry-based proteomicsto explore the assembly, structure, and function of lipoproteins. J Lipid Res 50(10):1967–1975Hortin GL et al (2006) Diverse range of small peptides associated with high-density lipoprotein.Biochem Biophys Res Commun 340(3):909–915Huang R et al (2011) Apolipoprotein A-I structural organization in high-density lipoproteinsisolated from human plasma. Nat Struct Mol Biol 18(4):416–422Hussain MM, Zannis VI (1990) Intracellular modification of human apolipoprotein AII (apoAII)and sites of apoAII mRNA synthesis: comparison of apoAII with apoCII and apoCIIIisoproteins. Biochemistry 29(1):209–217Jakubowski H (2000) Calcium-dependent human serum homocysteine thiolactone hydrolase. Aprotective mechanism against protein N-homocysteinylation. J Biol Chem 275(6):3957–3962Jaleel A et al (2010) Identification of de novo synthesized and relatively older proteins: acceleratedoxidative damage to de novo synthesized apolipoprotein A-1 in type 1 diabetes. Diabetes 59(10):2366–2374James RW et al (1988) Protein heterogeneity of lipoprotein particles containing apolipoprotein A-Iwithout apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated fromhuman plasma. J Lipid Res 29(12):1557–1571Jian W et al (2013) Relative quantitation of glycoisoforms of intact apolipoprotein C3 in humanplasma by liquid chromatography-high-resolution mass spectrometry. Anal Chem 85(5):2867–2874Jin Y, Manabe T (2005) Direct targeting of human plasma for matrix-assisted laser desorption/ionization and analysis of plasma proteins by time of flight-mass spectrometry. Electrophoresis26(14):2823–2834Jonas A (1972) Studies on the structure of bovine serum high density lipoprotein using covalentlybound fluorescent probes. J Biol Chem 247(23):7773–7778Jonas A (1986) Reconstitution of high-density lipoproteins. Methods Enzymol 128:553–582Jonas A (2002) Lipoprotein structure. In: Vance DE, Vance JE (eds) Biochemistry of lipids,lipoproteins and membranes. Elsevier, Amsterdam, pp 483–504Jonas A, McHugh HT (1984) Reaction of lecithin: cholesterol acyltransferase with micellarsubstrates. Effect of particle sizes. Biochim Biophys Acta 794(3):361–372Jonas A et al (1989) Defined apolipoprotein A-I conformations in reconstituted high densitylipoprotein discs. J Biol Chem 264(9):4818–4824Jonas A et al (1990) Apolipoprotein A-I structure and lipid properties in homogeneous,reconstituted spherical and discoidal high density lipoproteins. J Biol Chem 265(36):22123–22129Jones MK et al (2010) Assessment of the validity of the double superhelix model for reconstitutedhigh density lipoproteins: a combined computational-experimental approach. J Biol Chem 285(52):41161–41171Jones MK et al (2011) “Sticky” and “promiscuous”, the Yin and Yang of apolipoprotein A-Itermini in discoidal high-density lipoproteins: a combined computational-experimentalapproach. Biochemistry 50(12):2249–2263Karlsson H et al (2005) Lipoproteomics II: mapping of proteins in high-density lipoprotein usingtwo-dimensional gel electrophoresis and mass spectrometry. Proteomics 5(5):1431–1445Karlsson H et al (2009) Protein profiling of low-density lipoprotein from obese subjects. Proteo-mics Clin Appl 3(6):663–671Structure of HDL: Particle Subclasses and Molecular Components 45Khovidhunkit W et al (2004) Effects of infection and inflammation on lipid and lipoproteinmetabolism: mechanisms and consequences to the host. J Lipid Res 45(7):1169–1196Kiernan UA et al (2003) Detection of novel truncated forms of human serum amyloid A protein inhuman plasma. FEBS Lett 537(1–3):166–170Kim E et al (2008) Expression of apolipoprotein C-IV is regulated by Ku antigen/peroxisomeproliferator-activated receptor gamma complex and correlates with liver steatosis. J Hepatol 49(5):787–798Kisilevsky R, Manley PN (2012) Acute-phase serum amyloid A: perspectives on its physiologicaland pathological roles. Amyloid 19(1):5–14Kisilevsky R, Tam SP (2003) Macrophage cholesterol efflux and the active domains of serumamyloid A 2.1. J Lipid Res 44(12):2257–2269Klon AE et al (2002a) Comparative models for human apolipoprotein A-I bound to lipid indiscoidal high-density lipoprotein particles. Biochemistry 41(36):10895–10905Klon AE et al (2002b) Molecular dynamics simulations on discoidal HDL particles suggest amechanism for rotation in the apo A-I belt model. J Mol Biol 324(4):703–721Kontush A, Chapman MJ (2010) Antiatherogenic function of HDL particle subpopulations: focuson antioxidative activities. Curr Opin Lipidol 21(4):312–318Kontush A, Chapman MJ (2012) High-density lipoproteins: structure, metabolism, function andtherapeutics. Wiley, New YorkKontush A et al (2003) Small, dense HDL particles exert potent protection of atherogenic LDLagainst oxidative stress. Arterioscler Thromb Vasc Biol 23(10):1881–1888Kontush A et al (2007) Preferential sphingosine-1-phosphate enrichment and sphingomyelindepletion are key features of small dense HDL3 particles: relevance to antiapoptotic andantioxidative activities. Arterioscler Thromb Vasc Biol 27(8):1843–1849Koppaka V et al (1999) The structure of human lipoprotein A-I. Evidence for the “belt” model. JBiol Chem 274(21):14541–14544Kulkarni KR et al (1997) Quantification of HDL2 and HDL3 cholesterol by the vertical autoprofile-II (VAP-II) methodology. J Lipid Res 38(11):2353–2364Laggner P et al (1973) Studies on the structure of lipoprotein A of human high density lipoproteinHDL3: the spherically averaged electron density distribution. FEBS Lett 33(1):77–80LagorWR et al (2009) Overexpression of apolipoprotein F reduces HDL cholesterol levels in vivo.Arterioscler Thromb Vasc Biol 29(1):40–46Lamant M et al (2006) ApoO, a novel apolipoprotein, is an original glycoprotein up-regulated bydiabetes in human heart. J Biol Chem 281(47):36289–36302Lee M et al (2003) Apolipoprotein composition and particle size affect HDL degradation bychymase: effect on cellular cholesterol efflux. J Lipid Res 44(3):539–546Lee JY et al (2010) Profiling of phospholipids in lipoproteins by multiplexed hollow fiber flowfield-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry. JChromatogr A 1217(10):1660–1666Levels JH et al (2011) High-density lipoprotein proteome dynamics in human endotoxemia.Proteome Sci 9(1):34Lhomme M et al (2012) Lipidomics in lipoprotein biology. In: Ekroos K (ed) Lipidomics:technologies and applications. Wiley, Weinheim, pp 197–231Li H et al (2000) Structural determination of lipid-bound ApoA-I using fluorescence resonanceenergy transfer. J Biol Chem 275(47):37048–37054Li HH et al (2002) ApoA-I structure on discs and spheres. Variable helix registry and conforma-tional states. J Biol Chem 277(42):39093–39101Liadaki KN et al (2000) Binding of high density lipoprotein (HDL) and discoidal reconstitutedHDL to the HDL receptor scavenger receptor class B type I. Effect of lipid association andAPOA-I mutations on receptor binding. J Biol Chem 275(28):21262–21271Lipina C, Hundal HS (2011) Sphingolipids: agents provocateurs in the pathogenesis of insulinresistance. Diabetologia 54(7):1596–160746 A. Kontush et al.Ljunggren S et al (2013) APOA-I mutations, L202P and K131DEL, IN HDL from heterozygoteswith low HDL-cholesterol. Proteomics Clin Appl 8(3–4):241–250Lucke S, Levkau B (2010) Endothelial functions of sphingosine-1-phosphate. Cell PhysiolBiochem 26(1):87–96Lund-Katz S, Phillips MC (2010) High density lipoprotein structure-function and role in reversecholesterol transport. Subcell Biochem 51:183–227Mackness B et al (2010) Human tissue distribution of paraoxonases 1 and 2 mRNA. IUBMB Life62(6):480–482Maeba R, Ueta N (2003) Ethanolamine plasmalogen and cholesterol reduce the total membraneoxidizability measured by the oxygen uptake method. Biochem Biophys Res Commun 302(2):265–270Maiorano JN et al (2004) Identification and structural ramifications of a hinge domain in apolipo-protein A-I discoidal high-density lipoproteins of different size. Biochemistry 43(37):11717–11726Majek P et al (2011) Plasma proteome changes in cardiovascular disease patients: novel isoformsof apolipoprotein A1. J Transl Med 9:84Mallat Z et al (2010) Lipoprotein-associated and secreted phospholipases A in cardiovasculardisease: roles as biological effectors and biomarkers. Circulation 122(21):2183–2200Marcel YL et al (1990) Distribution and concentration of cholesteryl ester transfer protein inplasma of normolipemic subjects. J Clin Invest 85(1):10–17Marhaug G et al (1982) Characterization of amyloid related protein SAA complexed with serumlipoproteins (apoSAA). Clin Exp Immunol 50(2):382–389Martin DD et al (2006) Apolipoprotein A-I assumes a “looped belt” conformation on reconstitutedhigh density lipoprotein. J Biol Chem 281(29):20418–20426Matz CE, Jonas A (1982) Micellar complexes of human apolipoprotein A-I with phosphatidyl-cholines and cholesterol prepared from cholate-lipid dispersions. J Biol Chem 257(8):4535–4540Mauger JF et al (2006) Apolipoprotein C-III isoforms: kinetics and relative implication in lipidmetabolism. J Lipid Res 47(6):1212–1218Mazur MT et al (2010) Quantitative analysis of intact apolipoproteins in human HDL by top-downdifferential mass spectrometry. Proc Natl Acad Sci USA 107(17):7728–7733McConathy WJ, Alaupovic P (1973) Isolation and partial characterization of apolipoprotein D: anew protein moiety of the human plasma lipoprotein system. FEBS Lett 37(2):178–182McIntyre TM et al (2009) The emerging roles of PAF acetylhydrolase. J Lipid Res Supp 50:S255–S259McLachlan AD (1977) Repeated helical pattern in apolipoprotein-A-I. Nature 267(5610):465–466Mei X, Atkinson D (2011) Crystal structure of C-terminal truncated apolipoprotein A-I reveals theassembly of high density lipoprotein (HDL) by dimerization. J Biol Chem 286(44):38570–38582Miida T et al (2003) Analytical performance of a sandwich enzyme immunoassay for pre beta1-HDL in stabilized plasma. J Lipid Res 44(3):645–650Miyazaki M et al (2010) Static and dynamic characterization of nanodiscs with apolipoprotein A-Iand its model peptide. J Phys Chem B 114(38):12376–12382Mora S (2009) Advanced lipoprotein testing and subfractionation are not (yet) ready for routineclinical use. Circulation 119(17):2396–2404Moren X et al (2008) HDL subfraction distribution of paraoxonase-1 and its relevance to enzymeactivity and resistance to oxidative stress. J Lipid Res 49(6):1246–1253Navab M et al (2004) The oxidation hypothesis of atherogenesis: the role of oxidizedphospholipids and HDL. J Lipid Res 45(6):993–1007Nelsestuen GL et al (2008) Top-down proteomic analysis by MALDI-TOF profiling:concentration-independent biomarkers. Proteomics Clin Appl 2(2):158–166Neumann S et al (2009) Mammalian Wnt3a is released on lipoprotein particles. Traffic 10(3):334–343Structure of HDL: Particle Subclasses and Molecular Components 47Nicholls SJ, Hazen SL (2009) Myeloperoxidase, modified lipoproteins, and atherogenesis. J LipidRes Supp 50:S346–S351Nichols AV et al (1983) Characterization of discoidal complexes of phosphatidylcholine, apoli-poprotein A-I and cholesterol by gradient gel electrophoresis. Biochim Biophys Acta 750(2):353–364Nichols AV et al (1986) Nondenaturing polyacrylamide gradient gel electrophoresis. MethodsEnzymol 128:417–431Nicolardi S et al (2013a) Mapping O-glycosylation of apolipoprotein C-III in MALDI-FT-ICRprotein profiles. Proteomics 13(6):992–1001Nicolardi S et al (2013b) Identification of new apolipoprotein-CIII glycoforms with ultrahighresolution MALDI-FTICR mass spectrometry of human sera. J Proteome Res 12(5):2260–2268Nijstad N et al (2011) Overexpression of apolipoprotein O does not impact on plasma HDL levelsor functionality in human apolipoprotein A-I transgenic mice. Biochim Biophys Acta 1811(4):294–299Nilsson A, Duan RD (2006) Absorption and lipoprotein transport of sphingomyelin. J Lipid Res 47(1):154–171Nobecourt E et al (2007) The impact of glycation on apolipoprotein A-I structure and its ability toactivate lecithin:cholesterol acyltransferase. Diabetologia 50(3):643–653Nolte RT, Atkinson D (1992) Conformational analysis of apolipoprotein A-I and E-3 based onprimary sequence and circular dichroism. Biophys J 63(5):1221–1239Ohta T et al (1985) Studies on the in vivo and in vitro distribution of apolipoprotein A-IV in humanplasma and lymph. J Clin Invest 76(3):1252–1260Olofsson SO et al (1978) Isolation and partial characterization of a new acidic apolipoprotein(apolipoprotein F) from high density lipoproteins of human plasma. Biochemistry 17(6):1032–1036Ortiz-Munoz G et al (2009) HDL antielastase activity prevents smooth muscle cell anoikis, apotential new antiatherogenic property. FASEB J 23(9):3129–3139Otvos JD et al (1992) Development of a proton nuclear magnetic resonance spectroscopic methodfor determining plasma lipoprotein concentrations and subspecies distributions from a single,rapid measurement. Clin Chem 38(9):1632–1638Panagotopulos SE et al (2001) Apolipoprotein A-I adopts a belt-like orientation in reconstitutedhigh density lipoproteins. J Biol Chem 276(46):42965–42970Park KH et al (2010) Fructated apolipoprotein A-I showed severe structural modification and lossof beneficial functions in lipid-free and lipid-bound state with acceleration of atherosclerosisand senescence. Biochem Biophys Res Commun 392(3):295–300Phillips MC (2013) New insights into the determination of HDL structure by apolipoproteins:thematic review series: high density lipoprotein structure, function, and metabolism. J LipidRes 54(8):2034–2048Phillips JC et al (1997) Predicting the structure of apolipoprotein A-I in reconstituted high-densitylipoprotein disks. Biophys J 73(5):2337–2346Pruzanski W et al (2000) Comparative analysis of lipid composition of normal and acute-phasehigh density lipoproteins. J Lipid Res 41(7):1035–1047Puppione DL et al (2010) Mass spectral analyses of the two major apolipoproteins of great apehigh density lipoproteins. Comp Biochem Physiol Part D Genomics Proteomics 4(4):305–309Qiu X et al (2007) Crystal structure of cholesteryl ester transfer protein reveals a long tunnel andfour bound lipid molecules. Nat Struct Mol Biol 14(2):106–113Rassart E et al (2000) Apolipoprotein D. Biochim Biophys Acta 1482(1–2):185–198Remaley AT et al (1993) O-linked glycosylation modifies the association of apolipoprotein A-II tohigh density lipoproteins. J Biol Chem 268(9):6785–6790Roghani A, Zannis VI (1988) Mutagenesis of the glycosylation site of human ApoCIII. O-linkedglycosylation is not required for ApoCIII secretion and lipid binding. J Biol Chem 263(34):17925–1793248 A. Kontush et al.Rosenson RS et al (2011) HDL measures, particle heterogeneity, proposed nomenclature, andrelation to atherosclerotic cardiovascular events. Clin Chem 57(3):392–410Rousset X et al (2009) Lecithin: cholesterol acyltransferase–from biochemistry to role in cardio-vascular disease. Curr Opin Endocrinol Diabetes Obes 16(2):163–171Rye KA et al (1996) The influence of sphingomyelin on the structure and function of reconstitutedhigh density lipoproteins. J Biol Chem 271(8):4243–4250Saito H et al (2000) Inhibition of lipoprotein lipase activity by sphingomyelin: role of membranesurface structure. Biochim Biophys Acta 1486(2–3):312–320Samanta U, Bahnson BJ (2008) Crystal structure of human plasma platelet-activating factoracetylhydrolase: structural implication to lipoprotein binding and catalysis. J Biol Chem 283(46):31617–31624Santucci L et al (2011) Protein-protein interaction heterogeneity of plasma apolipoprotein A1 innephrotic syndrome. Mol Biosyst 7(3):659–666Sattler KJ et al (2010) Sphingosine 1-phosphate levels in plasma and HDL are altered in coronaryartery disease. Basic Res Cardiol 105(6):821–832Scanu AM (1978) Ultrastructure of serum high density lipoproteins: facts and models. Lipids 13(12):920–925Schaefer EJ, Asztalos BF (2007) Increasing high-density lipoprotein cholesterol, inhibition ofcholesteryl ester transfer protein, and heart disease risk reduction. Am J Cardiol 100(11A):S25–S31Schaefer EJ et al (1979) The composition and metabolism of high density lipoprotein subfractions.Lipids 14(5):511–522Schaefer EJ et al (2010) Marked HDL deficiency and premature coronary heart disease. Curr OpinLipidol 21(4):289–297Scherer M et al (2010) Sphingolipid profiling of human plasma and FPLC-separated lipoproteinfractions by hydrophilic interaction chromatography tandem mass spectrometry. BiochimBiophys Acta 1811(2):68–75Schjoldager KT et al (2012) Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells. Proc Natl Acad SciUSA 109(25):9893–9898Schonfeld G et al (1976) Structure of high density lipoprotein. The immunologic reactivities of theCOOH- and NH2-terminal regions of apolipoprotein A-I. J Biol Chem 251(13):3921–3926Segrest JP (1977) Amphipathic helixes and plasma lipoproteins: thermodynamic and geometricconsiderations. Chem Phys Lipids 18(1):7–22Segrest JP et al (1974) A molecular theory of lipid-protein interactions in the plasma lipoproteins.FEBS Lett 38(3):247–258Segrest JP et al (1994) The amphipathic alpha helix: a multifunctional structural motif in plasmaapolipoproteins. Adv Protein Chem 45:303–369Segrest JP et al (1999) A detailed molecular belt model for apolipoprotein A-I in discoidal highdensity lipoprotein. J Biol Chem 274(45):31755–31758Segrest JP et al (2000) Detailed molecular model of apolipoprotein A-I on the surface of high-density lipoproteins and its functional implications. Trends Cardiovasc Med 10(6):246–252Segrest JP et al (2013) MD simulations suggest important surface differences betweenreconstituted and circulating spherical HDL. J Lipid Res 54(10):2718–2732Sevvana M et al (2009) Serendipitous fatty acid binding reveals the structural determinants forligand recognition in apolipoprotein M. J Mol Biol 393(4):920–936Shah AS et al (2013) Proteomic diversity of high density lipoproteins: our emerging understandingof its importance in lipid transport and beyond. J Lipid Res 54(10):2575–2585Shao B (2012) Site-specific oxidation of apolipoprotein A-I impairs cholesterol export by ABCA1,a key cardioprotective function of HDL. Biochim Biophys Acta 1821(3):490–501Shen BW et al (1977) Structure of human serum lipoproteins inferred from compositional analysis.Proc Natl Acad Sci USA 74(3):837–841Shiflett AM et al (2005) Human high density lipoproteins are platforms for the assembly of multi-component innate immune complexes. J Biol Chem 280(38):32578–32585Structure of HDL: Particle Subclasses and Molecular Components 49Shimano H (2009) ApoAII controversy still in rabbit? Arterioscler Thromb Vasc Biol 29(12):1984–1985Silva RA et al (2005) A mass spectrometric determination of the conformation of dimericapolipoprotein A-I in discoidal high density lipoproteins. Biochemistry 44(24):8600–8607Silva RA et al (2008) Structure of apolipoprotein A-I in spherical high density lipoproteins ofdifferent sizes. Proc Natl Acad Sci USA 105(34):12176–12181Sloop CH et al (1983) Characterization of dog peripheral lymph lipoproteins: the presence of adisc-shaped “nascent” high density lipoprotein. J Lipid Res 24(11):1429–1440Sparks DL et al (1992) The conformation of apolipoprotein A-I in discoidal and sphericalrecombinant high density lipoprotein particles. 13C NMR studies of lysine ionization behavior.J Biol Chem 267(36):25830–25838Stahlman M et al (2013) Dyslipidemia, but not hyperglycemia and insulin resistance, is associatedwith marked alterations in the HDL lipidome in type 2 diabetic subjects in the DIWA cohort:impact on small HDL particles. Biochim Biophys Acta 1831(11):1609–1617Sung HJ et al (2012) Large-scale isotype-specific quantification of Serum amyloid A 1/2 bymultiple reaction monitoring in crude sera. J Proteomics 75(7):2170–2180Tardieu A et al (1976) Structure of human serum lipoproteins in solution. II. Small-angle x-rayscattering study of HDL and LDL. J Mol Biol 101(2):129–153Tselepis AD et al (1995) PAF-degrading acetylhydrolase is preferentially associated with denseLDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme. Arterioscler Thromb Vasc Biol 15(10):1764–1773Usami Y et al (2011) Detection of chymase-digested C-terminally truncated apolipoprotein A-I innormal human serum. J Immunol Methods 369(1–2):51–58Usami Y et al (2013) Identification of sites in apolipoprotein A-I susceptible to chymase andcarboxypeptidase A digestion. Biosci Rep 33(1):49–56Utermann G (1975) Isolation and partial characterization of an arginine-rich apolipoprotein fromhuman plasma very-low-density lipoproteins: apolipoprotein E. Hoppe Seylers Z PhysiolChem 356(7):1113–1121Vaisar T (2009) Thematic review series: proteomics. Proteomic analysis of lipid-proteincomplexes. J Lipid Res 50(5):781–786Vaisar T et al (2007) Shotgun proteomics implicates protease inhibition and complement activa-tion in the antiinflammatory properties of HDL. J Clin Invest 117(3):746–756Vieu C et al (1996) Identification and quantification of diacylglycerols in HDL and accessibility tolipase. J Lipid Res 37(5):1153–1161Wald JH et al (1990) Structure of apolipoprotein A-I in three homogeneous, reconstituted highdensity lipoprotein particles. J Biol Chem 265(32):20037–20043Wiesner P et al (2009) Lipid profiling of FPLC-separated lipoprotein fractions by electrosprayionization tandem mass spectrometry. J Lipid Res 50(3):574–585Wlodawer A et al (1979) High-density lipoprotein recombinants: evidence for a bicycle tiremicelle structure obtained by neutron scattering and electron microscopy. FEBS Lett 104(2):231–235Wolfrum C et al (2005) Apolipoprotein M is required for prebeta-HDL formation and cholesterolefflux to HDL and protects against atherosclerosis. Nat Med 11(4):418–422Wopereis S et al (2003) Apolipoprotein C-III isofocusing in the diagnosis of genetic defects inO-glycan biosynthesis. Clin Chem 49(11):1839–1845Wroblewska M et al (2009) Phospholipids mediated conversion of HDLs generates specific apoA-II pre-beta mobility particles. J Lipid Res 50(4):667–675Wu Z et al (2007) The refined structure of nascent HDL reveals a key functional domain forparticle maturation and dysfunction. Nat Struct Mol Biol 14(9):861–868Wu Z et al (2009) Double superhelix model of high density lipoprotein. J Biol Chem 284(52):36605–36619Wu Z et al (2011) The low resolution structure of ApoA1 in spherical high density lipoproteinrevealed by small angle neutron scattering. J Biol Chem 286(14):12495–1250850 A. Kontush et al.Wurfel MM et al (1994) Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins andacts as a cofactor in the neutralization of LPS. J Exp Med 180(3):1025–1035Yu B et al (1997) Lipopolysaccharide binding protein and soluble CD14 catalyze exchange ofphospholipids. J Clin Invest 99(2):315–324Zannis VI et al (1980) Isoproteins of human apolipoprotein A-I demonstrated in plasma andintestinal organ culture. J Biol Chem 255(18):8612–8617Zerrad-Saadi A et al (2009) HDL3-mediated inactivation of LDL-associated phospholipidhydroperoxides is determined by the redox status of apolipoprotein A-I and HDL particlesurface lipid rigidity: relevance to inflammation and atherogenesis. Arterioscler Thromb VascBiol 29(12):2169–2175Zhang L et al (2011) Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy. J Lipid Res 52(1):175–184Zhaorigetu S et al (2008) ApoL1, a BH3-only lipid-binding protein, induces autophagic cell death.Autophagy 4(8):1079–1082Zhou W et al (2009) An initial characterization of the serum phosphoproteome. J Proteome Res8(12):5523–5531Structure of HDL: Particle Subclasses and Molecular Components 51HDL Biogenesis, Remodeling, andCatabolismVassilis I. Zannis, Panagiotis Fotakis, Georgios Koukos, DimitrisKardassis, Christian Ehnholm, Matti Jauhiainen, and Angeliki ChroniContents1 Biogenesis of HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561.1 ATP-Binding Cassette Transporter A1 (ABCA1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571.1.1 Structure of apoA-I and Its Interactions with ABCA1 In Vitro . . . . . . . . . . . 571.1.2 Interaction of apoA-I with ABCA1 In Vivo Initiates the Biogenesisof HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581.1.3 Unique Mutations in apoA-I May Affect apoA-I/ABCA1 Interactionsand Inhibit the First Step in the Pathway of HDL Biogenesis . . . . . . . . . . . . . 601.2 Lecithin/Cholesterol Acyltransferase (LCAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631.2.1 Interactions of Lipid-Bound ApoA-I with LCAT . . . . . . . . . . . . . . . . . . . . . . . . . . 631.2.2 ApoA-I Mutations that Affect apoA-I/LCAT Interactions . . . . . . . . . . . . . . . . . 64V.I. Zannis (*) • P. FotakisMolecular Genetics, Whitaker Cardiovascular Institute, Boston University School of Medicine,Boston, MA 02118, USADepartment of Biochemistry, University of Crete Medical School and Institute of MolecularBiology and Biotechnology, Foundation of Research and Technology of Hellas, Heraklion,Crete 71110, Greecee-mail: vzannis@bu.eduG. KoukosMolecular Genetics, Whitaker Cardiovascular Institute, Boston University School of Medicine,Boston, MA 02118, USAD. KardassisDepartment of Biochemistry, University of Crete Medical School and Institute of MolecularBiology and Biotechnology, Foundation of Research and Technology of Hellas, Heraklion,Crete 71110, GreeceC. Ehnholm • M. JauhiainenNational Institute for Health and Welfare, Public Health Genomics Unit, Biomedicum,Helsinki FI-00251, FinlandA. ChroniInstitute of Biosciences and Applications, National Center for Scientific Research “Demokritos”,Athens 15310, Greece# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_2531.3 ApoA-I Mutations May Induce Hypertriglyceridemiaand/or Hypercholesterolemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681.3.1 Potential Mechanism of Dyslipidemia Resulting from apoA-IMutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711.4 ApoE and apoA-IV Participate in the Biogenesis of HDL Particles Containingthe Corresponding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721.5 Clinical Relevance of the Aberrant HDL Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742 Remodeling and Catabolism of HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742.1 ATP-Binding Cassette Transporter G1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762.2 Phospholipid Transfer Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782.3 apoM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792.4 Hepatic Lipase and Endothelial Lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802.5 Cholesteryl Ester Transfer Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 822.6 Scavenger Receptor BI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832.6.1 Role of SR-BI in HDL Remodeling Based on Its In Vitro Interactionswith Its Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832.6.2 In Vivo Functions of SR-BI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842.7 Role of Ecto-F1-ATPase/P2Y13 Pathway in Hepatic HDL Clearance . . . . . . . . . . . . . 862.8 Transcytosis of apoA-I and HDL by Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 862.9 The Role of Cubilin in apoA-I and HDL Catabolism by the Kidney . . . . . . . . . . . . . . 873 HDL Subclasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 883.1 The Origin and Metabolism of Preβ-HDL Subpopulations . . . . . . . . . . . . . . . . . . . . . . . . 883.2 Complexity of HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894 Sources of Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91AbstractIn this chapter, we review how HDL is generated, remodeled, and catabolized inplasma. We describe key features of the proteins that participate in theseprocesses, emphasizing how mutations in apolipoprotein A-I (apoA-I) and theother proteins affect HDL metabolism.The biogenesis of HDL initially requires functional interaction of apoA-Iwith the ATP-binding cassette transporter A1 (ABCA1) and subsequentlyinteractions of the lipidated apoA-I forms with lecithin/cholesterolacyltransferase (LCAT). Mutations in these proteins either prevent or impairthe formation and possibly the functionality of HDL.Remodeling and catabolism of HDL is the result of interactions of HDL withcell receptors and other membrane and plasma proteins including hepatic lipase(HL), endothelial lipase (EL), phospholipid transfer protein (PLTP), cholesterylester transfer protein (CETP), apolipoprotein M (apoM), scavenger receptorclass B type I (SR-BI), ATP-binding cassette transporter G1 (ABCG1), the F1subunit of ATPase (Ecto F1-ATPase), and the cubulin/megalin receptor.Similarly to apoA-I, apolipoprotein E and apolipoprotein A-IV were shown toform discrete HDL particles containing these apolipoproteins which may haveimportant but still unexplored functions. Furthermore, several plasma proteinswere found associated with HDL and may modulate its biological functions. Theeffect of these proteins on the functionality of HDL is the topic of ongoingresearch.54 V.I. Zannis et al.KeywordsHDL biogenesis • HDL remodeling • HDL catabolism • HDL phenotypes •Apolipoprotein A-I mutations • Apolipoprotein E • Apolipoprotein A-IV • ATP-binding cassette transporter A1 (ABCA1) • Lecithin/cholesterol acyltransferase(LCAT) • HDL subclasses • Preβ- and α-HDL particles • Dyslipidemia •Hypertriglyceridemia • Lipoprotein lipase (LPL) • Hepatic lipase (HL) • Endo-thelial lipase (EL) • Phospholipid transfer protein (PLTP) • Apolipoprotein M •Cholesteryl ester transfer protein (CETP) • Scavenger receptor class B type I(SR-BI) • ATP-binding cassette transporter G1 (ABCG1) • Ecto-F1-ATPase •Cubilin • Transcytosis • Clinical phenotypesAbbreviationsABCA1 ATP-binding cassette transporter A1ABCG1 ATP-binding cassette transporter G1apoA-I Apolipoprotein A-IapoA-IV Apolipoprotein A-IVapoB Apolipoprotein BapoE Apolipoprotein EapoJ Apolipoprotein JapoM Apolipoprotein MBHK cells Baby hamster kidney cellsCaco-2 cells Human epithelial colorectal adenocarcinoma cellsCAD Coronary artery diseaseCD36 Cluster of differentiation 36CE Cholesterol esterCETP Cholesteryl ester transfer proteinCHO cells Chinese hamster ovary cellseNOS Endothelial nitric oxide synthaseEL Endothelial lipaseEM Electron microscopyFED Fish eye diseaseFLD Familial LCAT deficiencyFPLC Fast protein liquid chromatographyHA-PLTP High-activity PLTPHDL High-density lipoproteinHDL-A-IV apoA-IV-containing HDLHDL-C HDL cholesterolHDL-E apoE-containing HDLHEK293 cells Human embryonic kidney 293 cellsHDL Biogenesis, Remodeling, and Catabolism 55HepG2 cells Liver hepatocellular carcinoma cellsHL Hepatic lipaseIDL Intermediate density lipoproteinLA-PLTP Low-activity PLTPLCAT Lecithin/cholesterol acyltransferaseLDL Low-density lipoproteinLDLr Low-density lipoprotein receptorMPO MyeloperoxidasePLA1 Phospholipase A1PLTP Phospholipid transfer proteinRCT Reverse cholesterol transportrHDL Recombinant HDLSNP Single-nucleotide polymorphismSR-BI Scavenger receptor class B type ISREBPs Sterol regulatory element-binding proteinsTC Total cholesterolVEGF-A Vascular endothelial growth factor AVLDL Very low-density LipoproteinWT Wild type1 Biogenesis of HDLThe biogenesis of HDL is a complex process and involves several membrane boundand plasma proteins (Zannis et al. 2004a). The first step in HDL biogenesis involvessecretion of apoA-I mainly by the liver and the intestine (Zannis et al. 1985).Secreted apoA-I interacts functionally with ABCA1, and this interaction leads tothe transfer of cellular phospholipids and cholesterol to lipid-poor apoA-I. Thelipidated apoA-I is gradually converted to discoidal particles enriched inunesterified cholesterol. The esterification of free cholesterol by the enzyme leci-thin/cholesterol acyltransferase (LCAT) (Zannis et al. 2006a) converts the discoidalto spherical HDL particles (Fig. 1).The absence or inactivating mutations in apoA-I, ABCA1, and LCAT preventthe formation of apoA-I-containing HDL (Daniil et al. 2011). For this reason, weclassify the apoA-I, ABCA1, and LCAT interactions that will be discussed in thischapter as early steps in the biogenesis of HDL. Following a similar pathway, apoEand apoA-IV can also synthesize HDL particles that contain these proteins (Dukaet al. 2013; Kypreos and Zannis 2007). The first part of this review providesimportant information on the unique properties of apoA-I that permits it to acquirelipids via interactions with ABCA1 and LCAT. It also provides examples of howspecific mutations in apoA-I disrupt specific steps in the pathway of HDL biogene-sis and generate distinct aberrant HDL phenotypes. The HDL phenotypes describedhere can serve as molecular markers that could be used for the diagnosis, prognosis,56 V.I. Zannis et al.and potential treatment of HDL abnormalities or dyslipidemias associatedwith HDL.1.1 ATP-Binding Cassette Transporter A1 (ABCA1)1.1.1 Structure of apoA-I and Its Interactions with ABCA1 In VitroapoA-I contains 22 or 11 amino acid repeats which, according to the models ofNolte and Atkinson (1992), are organized in amphipathic a-helices (Segrestet al. 1974). Based on the crystal structure of apoA-I in solution (Borhaniet al. 1997, 1999), a belt model was proposed to explain the structure of apoA-Ion discoidal HDL particles (Segrest et al. 1999). Various models have beenproposed to explain the arrangement of apoA-I on spherical HDL particles basedon structural work and cross-linking(Wu et al. 2011; Silva et al. 2008; Huanget al. 2011). Details on the structure of apoA-I and HDL are provided in chapter“Structure of HDL: Particle Subclasses and Molecular Components.”ABCA1 is a ubiquitous protein that belongs to the ABC family of transportersand is expressed abundantly in the liver, macrophages, brain, and various othertissues (Langmann et al. 1999; Kielar et al. 2001). ABCA1 is localized only on thebasolateral surface of the hepatocytes (Neufeld et al. 2002); it is also found onendocytic vesicles and was shown to travel between late endocytic vesicles and thecell surface (Neufeld et al. 2001). ABCA1 promotes efflux of cellular phospholipidsFig. 1 Schematic representation of the pathway of the biogenesis of HDL containing apoA-I orapoE or apoA-IVHDL Biogenesis, Remodeling, and Catabolism 57and cholesterol to lipid-free or minimally lipidated apoA-I and otherapolipoproteins, but not to spherical HDL particles (Wang et al. 2000; Remaleyet al. 2001).Studies in HeLa cells that expressed an ABCA1 green fluorescence fusionprotein showed the intracellular trafficking of ABCA1 complexed to apoA-I(Neufeld et al. 2001, 2002). Other studies showed that in macrophages ABCA1associates with apoA-I in the coated pits, is internalized, interacts with intracellularlipid pools, and is re-secreted as a lipidated particle (Takahashi and Smith 1999;Smith et al. 2002; Lorenzi et al. 2008). A similar pathway that leads to transcytosishas been described in endothelial cells (Cavelier et al. 2006; Ohnsorg et al. 2011).A series of cell culture and in vitro experiments investigated the ability of apoA-I mutants to promote ABCA1-mediated efflux of cholesterol and phospholipids andto cross-link to ABCA1. These mutants had amino-terminal deletions, carboxy-terminal deletions that removed the 220-231 region, carboxy-terminal deletions thatmaintained the 220-231 region, and double deletions of the amino- and carboxy-terminal regions (Chroni et al. 2003).These studies presented in Chroni et al. (2003) showed that wild-type (WT) -ABCA1-mediated cholesterol and phospholipid efflux was not affected by amino-terminal apoA-I deletions, but it was diminished by carboxy-terminal deletions inwhich residues 220-231 were removed. Efflux was not affected by deletion of thecarboxy-terminal 232-243 region, and it was restored to 80 % of WT control bydouble deletions of both the amino- and carboxy-termini (Zannis et al. 2004a,2006a; Chroni et al. 2003; Reardon et al. 2001). Lipid efflux was either unaffectedor moderately reduced by a variety of point mutations or deletions of internalhelices 2–7. The findings indicated that different combinations of central helicescan promote lipid efflux (Chroni et al. 2004a, b). Chemical cross-linking/immunoprecipitation studies showed that the ability of apoA-I mutants to promoteABCA1-depended lipid efflux is correlated with the ability of these mutants to becross-linked efficiently to ABCA1 (Chroni et al. 2004b). Cross-linking betweenapoA-I and ABCA1 and cholesterol efflux was also affected by mutations inABCA1 that are found in patients with Tangier disease. The majority of theABCA1 mutants cross-link poorly to WT apoA-I and have diminished capacity topromote cholesterol efflux (Bodzioch et al. 1999; Fitzgerald et al. 2002). A notableexemption is the ABCA1[W590S] mutant which cross-linked stronger to apoA-Ithan to WT ABCA1 but had diminished capacity to promote cholesterol efflux andto promote formation of HDL (Bodzioch et al. 1999; Fitzgerald et al. 2001, 2002).We suggested that this ABCA1 mutation may have altered the environment of thebinding site of ABCA1 in such a way that the binding of apoA-I is strong but notproductive and thus prevented efficient lipid efflux (Chroni et al. 2004b).1.1.2 Interaction of apoA-I with ABCA1 In Vivo Initiatesthe Biogenesis of HDLInactivating mutations in ABCA1 found in patients with Tangier disease areassociated with very low levels of total plasma and HDL cholesterol, diminishedcapacity to promote cholesterol efflux, formation of preβ-migrating particles, and58 V.I. Zannis et al.abnormal lipid deposition in various tissues (Daniil et al. 2011; Brunhamet al. 2006b; Orso et al. 2000; Assmann et al. 2001; Timmins et al. 2005).Analysis of the serum of carriers with ABCA1 mutations by two-dimensionalgel electrophoresis and western blotting using a rabbit polyclonal anti-humanapoA-I antibody showed that subjects homozygous or compound mutant hetero-zygous for ABCA1 fail to form α-HDL particles, but instead they form preβ- andother small-size particles (Fig. 2).As explained later, such particles are found in the plasma of mice expressingC-terminal mutants and may be created by mechanisms that involve nonproductiveinteractions between ABCA1 and apoA-I (Chroni et al. 2007; Fotakis et al. 2013a).Several ABCA1 mutations in humans that alter the functions of ABCA1 areassociated with increased susceptibility to atherosclerosis (Singaraja et al. 2003).Specific amino-acid substitutions found in the Danish general population wereassociated with increased risk for ischemic heart disease and reduced life expec-tancy through unknown mechanisms (Frikke-Schmidt et al. 2008). Inactivation ofthe ABCA1 gene in mice leads to low total serum cholesterol levels, lipid deposi-tion in various tissues, impaired growth, and neuronal development and mimics thephenotype described for patients with Tangier disease (Orso et al. 2000). In additionABCA1-deficient mice exhibit moderate increase in cholesterol absorption inresponse to high cholesterol diet (McNeish et al. 2000; Aiello et al. 2002). Therole of ABCA1 on the lipid content of bile salts and cholesterol secretion is not clear(Vaisman et al. 2001; Groen et al. 2001).Bone marrow transplantation experiments indicated that ABCA1 plays animportant role in the control of macrophage recruitment to the tissues (Van Ecket al. 2002, 2006).The contribution of ABCA1 in the pathogenesis of atherosclerosis in mice ispresented in chapter “Mouse Models of Disturbed HDL Metabolism” (Hoesha andVan Eck). In men and mice, the majority of HDL is produced by the liver (Brunhamet al. 2006a). When the liver and intestinal ABCA1 genes were inactivated in mice,Fig. 2 Two-dimensional gel electrophoresis of plasma obtained from homozygotes or compoundheterozygotes ABCA1-deficient human subjects with Tangier disease as indicated [Adapted fromDaniil et al. (2011)]HDL Biogenesis, Remodeling, and Catabolism 59HDL was not found in plasma, indicating that the liver and the intestine are the onlysites that contribute to the production of HDL (Timmins et al. 2005). Followingintestinal-specific inactivation of the ABCA1 gene in mice, the HDL that wasgenerated by the liver accounted for 70 % of the HDL found in WT mice (Brunhamet al. 2006a). In mice that do not express hepatic ABCA1, the HDL concentration inthe lymph was greatly diminished despite the fact that the intestine contributes 30 %to the synthesis of HDL. This implies that the HDL that is produced in the intestineis secreted directly into the plasma (Brunham et al. 2006a). This is further supportedby the finding that in mice that do not express intestinal ABCA1, the apoA-I andcholesterol concentration of lymph was not affected (Brunham et al. 2006a).In liver-specific or whole-body ABCA1 knockout mice, the plasma HDL cata-bolism and the fractional catabolic rate of HDL by the liver and to a lesser extent bythe kidney and the adrenal is increased (Timmins et al. 2005; Singaraja et al. 2006).In ABCA1-deficient mice, lipidated apoA-I particles or preβ-HDL failed to matureand are rapidly catabolized by the kidney (Timmins et al. 2005).1.1.3 Unique Mutations in apoA-I May Affect apoA-I/ABCA1Interactions and Inhibit the First Step in the Pathway of HDLBiogenesisThe in vivo interactions of apoA-I with ABCA1 were studied systematically byadenovirus-mediated gene transfer of WT and mutant apoA-I forms. Similar studieswere performed to probe the interactions of lipid-bound apoA-I with LCAT. Four tofive days postinfection plasma was collected and analyzed for lipids and lipoproteinsand by two-dimensional gel electrophoresis to identify the HDL subpopulations. Theplasma was fractionated by density gradient ultracentrifugation and fast protein liquidchromatography (FPLC), and the HDL fraction was analyzed by electron microscopy(EM) to assess the size and shape of HDL (Chroni et al. 2003; Reardon et al. 2001).Also the hepatic mRNA levels of apoA-I were determined to ensure that there wascomparable expression of the WT and the mutant apoA-I forms (Zannis et al. 2004a;Chroni et al. 2003, 2007; Reardon et al. 2001).We have studied most recently the effect of two sets of point mutations in the218-222 and 225-230 region of apoA-I that affects apoA-I/ABCA1 interactions onthe biogenesis of HDL. Adenovirus-mediated gene transfer of these mutants inapoA-I/ x apoE/ mice (Fotakis et al. 2013a, b) showed that compared to theWT apoA-I, the expression of an apoA-I[L218A/L219A/V221A/L222A] mutantdecreased plasma cholesterol, apoA-I, and HDL cholesterol levels and generatedpreβ- and α4 HDL subpopulations (Fotakis et al. 2013a) (Table 1 and Fig. 3a–g).To eliminate the involvement of apoE in the generation of apoE-containing HDLparticles (Kypreos and Zannis 2007), the apoA-I[L218A/L219A/V221A/L222A]mutant was expressed in apoA-I/ x apoE/ mice via adenovirus-mediated genetransfer (Fotakis et al. 2013a). In this mouse background, the FPLC fractionation ofthe plasma showed the near absence of an HDL cholesterol peak. Density gradientultracentrifugation of the plasma showed small amount of the apoA-I in HDL3 andin d< 1.21 g/ml fractions (Fig. 3a). EM analysis showed the presence of discoidalparticles along with larger particles corresponding in size to IDL/LDL (Fig. 3b).60 V.I. Zannis et al.Table 1 Plasma lipids and hepatic mRNA levels of apoA-I/ or apoA-I/ x apoE/ miceexpressing WT and mutant forms of apoA-I in the presence and absence of LCAT as indicatedProtein expressedTotalcholesterol(mg/dL)Triglycerides(mg/dL)RelativeapoA-ImRNA(%)apoA-Iplasmalevels(mg/dL)WT apoA-I in apoA-I/ mice 278 74 78 24 100 26 260 40apoA-I[L218A/L219A/V221A/L222A] in apoA-I/ mice45 14 50 20 95 24 41 5WT apoA-I in apoA-I/ xapoE/ mice1,343 104 294 129 100 13 –apoA-I[L218A/L219A/V221A/L222A] in apoA-I/ x apoE/mice778 52 18 2 92 23 –apoA-I[L218A/L219A/V221A/L222A] plus LCAT in apoA-I/x apoE/ mice754 122 37 10 90 3 –Fig. 3 (a–g) ApoA-I mutations and their effect on the HDL phenotypes. Analysis of plasma ofapoA-I/ x apoE/ mice infected with adenoviruses expressing the WT apoA-I or the apoA-I[L218A/L219A/V221A/L222A] mutant, as indicated, by density gradient ultracentrifugation andSDS-PAGE (a, e). EM analysis of HDL fractions 6–7 obtained from apoA-I/ x apoE/ miceexpressing the WT apoA-I or the apoA-I[L218A/L219A/V221A/L222A] mutant, as indicated,following density gradient ultracentrifugation of plasma (b, f). SDS gel electrophoresis showingapoprotein composition of fractions 6 and 7 (c) used for the EM analysis in panel b.Two-dimensional gel electrophoresis of plasma of apoA-I/ x apoE/ mice infected withadenoviruses expressing WT apoA-I or the apoA-I[L218A/L219A/V221A/L222A] mutant, asindicated (d, g)HDL Biogenesis, Remodeling, and Catabolism 61These particles may originate from apoB-48 containing lipoproteins that are foundin the HDL fractions (Fig. 3c). Two-dimensional gel electrophoresis showed thepresence of only preβ-HDL particles (Fig. 3d). Control experiments showed thatWT apoA-I when expressed in apoA-I/ x apoE/ mice floated predominantly inthe HDL2/HDL3 region and generated spherical particles and normal preβ- andα-HDL subpopulation (Fig. 3e–g).Co-expression of the apoA-I[L218A/L219A/V221A/L222A] mutant and humanLCAT in apoA-I/ x apoE/ mice did not correct the plasma apoA-I levels anddid not correct the aberrant HDL phenotype.The findings shed light to previous studies which showed that carboxy-terminaldeletion mutants that lacked the 220-231 region of apoA-I prevented the biogenesisof normal α-HDL particles but allowed the formation of preβ-HDL particles(Chroni et al. 2003, 2007).Studies discussed in a later section showed that naturally occurring pointmutations in apoA-I when expressed in mouse models activate LCAT insufficientlyand in some instances lead to the accumulation of discoidal HDL particles inplasma (Koukos et al. 2007a, b; Chroni et al. 2005a). A characteristic feature ofthese mutations is that they could be corrected in vivo by the co-expression of themutant apoA-I and LCAT.The phenotype produced by the apoA-I[L218A/L219A/V221A/L222A]mutations is distinct from all previously described phenotypes and cannot becorrected by overexpression of LCAT. In addition, the mutant protein had reducedcapability to promote the ABCA1-mediated cholesterol efflux. Although otherinterpretations are possible, the in vivo and in vitro data suggest that the interactionof the apoA-I [L218A/L219A/V221A/L222A] mutant with ABCA1 results indefective lipidation of apoA-I that leads to the generation of preβ-HDL particlesthat are not a good substrate for LCAT. If this interpretation is correct, the normallipidation of apoA-I may require a precise initial orientation of the apoA-I ligandwithin the binding site of ABCA1 that is similar to that described for enzyme-substrate interactions. Such a configuration would allow correct lipidation of apoA-I that could subsequently undergo cholesterol esterification and formation of matureHDL, whereas incorrectly lipidated apoA-I becomes a poor substrate for LCAT.In addition to the unique role of the L218/L219/V221/L222 residues in thebiogenesis of HDL, the same residues are also required to confer trans-endothelialtransport capacity (Ohnsorg et al. 2011) and bactericidal activity (Biedzka-Sareket al. 2011) to apoA-I. These overall properties suggest that the L218/L219/V221/L222 residues represent an effector domain for several activities of apoA-I.The phenotype generated by the expression of the apoA-I[F225A/V227A/F229A/L230A] mutant was similar to that obtained with the apoA-I[L218A/L219A/V221A/L222A] mutant. However co-expression of the apoA-I[F225A/V227A/F229A/L230A] mutant and human LCAT corrected the abnormal HDLlevels, created normal preβ- and α-HDL subpopulations, and generated sphericalHDL particles (Fotakis et al. 2013b).62 V.I. Zannis et al.1.2 Lecithin/Cholesterol Acyltransferase (LCAT)1.2.1 Interactions of Lipid-Bound ApoA-I with LCATPlasma LCAT is a 416 amino acid long plasma protein that is synthesized andsecreted primarily by the liver and to a much lesser extent by the brain and the testis(Warden et al. 1989; Simon and Boyer 1970). LCAT interacts with discoidal andspherical HDL and catalyzes the transfer of the 2-acyl group of lecithin orphosphatidylethanolamine to the free hydroxyl residue of cholesterol to formcholesteryl ester, using apoA-I as an activator (Fielding et al. 1972; Zanniset al. 2004b). It also catalyzes the reverse reaction of esterification of lysolecithinto lecithin (Subbaiah et al. 1980). The esterification of free cholesterol of HDLin vivo converts the discoidal to mature spherical HDL particles (Jonas 2000;Chroni et al. 2005a). Regarding the mechanism of activation of LCAT by apoA-I,it has been proposed that residues R130 and K133 play an important role in theformation of an amphipathic presentation tunnel located between helices 5-5 in thedouble belt model. Such a tunnel could allow migration of the hydrophobic acylchains of phospholipids and the amphipathic unesterified cholesterol from thebilayer to the active site of LCAT that contains sites for phospholipase activityand esterification activity (Jones et al. 2009). The esterification of the cholesterolconverts the 3.67 residues per turn helices to an idealized 3.6 residues per turn helix(Borhani et al. 1997).Mutations in LCAT are associated with two phenotypes. The familial LCATdeficiency (FLD) is characterized by the inability of the mutant LCAT to esterifycholesterol on HDL and LDL and the accumulation of discoidal HDL in the plasma.The fish eye disease (FED) is characterized by the inability of mutant LCAT toesterify cholesterol on HDL only. Both diseases are characterized by low HDLlevels (Santamarina-Fojo et al. 2001) and formation of preβ- and α4subpopulations.Analysis of plasma of patients with complete LCAT deficiency (homozygous orcompound heterozygous carriers of functional LCAT mutations) bytwo-dimensional electrophoresis shows mostly the presence of small α-HDL sub-population (Daniil et al. 2011) (Fig. 4a, b). Analysis of plasma of LCAT-deficientmice by two-dimensional electrophoresis shows the presence of preβ- and small-size α4 HDL particles (Fig. 4c). Expression of human LCAT by adenovirus-mediated gene transfer generated large-size α-HDL subpopulations (Fig. 4d). Incontrast, expression of a LCAT mutant generated preβ-HDL along with small-sizeα4, α3, and α2 HDL subpopulations (Fig. 4e).Sera obtained from LCAT heterozygotes had increased capacity to promoteABCA1-mediated cholesterol efflux and decreased capacity to promote ABCG1-and SR-BI-mediated cholesterol efflux from macrophages as compared with seraobtained from normal subjects. These properties were attributed to the increasedpreβ- and decreased α-HDL subpopulations in the sera of the LCAT heterozygotes(Calabresi et al. 2009b). Heterozygosity for LCAT mutations in the Italian popu-lation was not associated with increased preclinical atherosclerosis (Calabresiet al. 2009a).HDL Biogenesis, Remodeling, and Catabolism 63The LCAT activation ability of apoA-I is inhibited following treatment withMPO (Shao et al. 2008). In addition myeloperoxidase modification alters severalfunctions of HDL and generates pro-inflammatory HDL particles (Undurtiet al. 2009).1.2.2 ApoA-I Mutations that Affect apoA-I/LCAT InteractionsSeveral naturally occurring apoA-I mutations that produce pathological phenotypeshave been described (Zannis et al. 2006b; Sorci-Thomas and Thomas 2002;Miettinen et al. 1997b). It has been estimated that structural mutations of apoA-Ioccur in 0.3 % of the Japanese population and may affect the plasma HDL levels(Yamakawa-Kobayashi et al. 1999). From a total of 46 natural mutations of apoA-I,25 are associated with low HDL levels, and 17 of these mutants reduce the capacityof apoA-I to activate LCAT (Sorci-Thomas and Thomas 2002; Zannis et al. 2006b).These mutations are clustered predominantly in or at the vicinity of helix 6 of apoA-Iand some of them predispose to atherosclerosis (Huang et al. 1998; Miller et al. 1998;Miettinen et al. 1997b; Miccoli et al. 1996). Here we describe how some represen-tative mutations of this category affect the biogenesis and maturation of HDL.Fig. 4 (a–e) Two-dimensional gel electrophoresis from plasma obtained from one homozygoteand one compound heterozygote for LCAT deficiency as indicated (a, b) (Daniil et al. 2011).Two-dimensional gel electrophoresis of plasma obtained from plasma of control LCAT/ mice(a) and LCAT/ mice infected with an adenovirus expressing either the WT human LCAT (d) orhuman LCAT[T147I] found in a patient with fish eye disease (e). LCAT cDNA probes of WT andLCAT mutants were provided by Dr. J.A. Kuivenhoven (Medical Center of Groningen)64 V.I. Zannis et al.Previous studies showed that hemizygotes (compound heterozygotes for anapoA-I null allele and an apoA-I(L141R)Pisa allele) had greatly decreased plasmaapoA-I levels and near absence of HDL cholesterol. Plasma from hemizygotescontained preβ1-HDL and low concentration of small particles with alpha electro-phoretic mobility (Miccoli et al. 1997). Heterozygotes for apoA-I(L141R)Pisa hadapproximately half-normal values for HDL cholesterol and plasma apoA-I (Miccoliet al. 1996; Pisciotta et al. 2003). Three male hemizygote patients and one hetero-zygote patient developed coronary stenosis (Miccoli et al. 1996).Other studies also showed that heterozygotes for apoA-I(L159R)FIN mutationhad greatly reduced plasma levels of HDL cholesterol and apoA-I (Miettinenet al. 1997a) that was mainly distributed in the HDL3 region and had abnormalelectrophoretic mobility (Miettinen et al. 1997b; McManus et al. 2001). They alsohad small-size (8–9 nm) HDL particles and decreased plasma and HDL cholesterylester levels (Miettinen et al. 1997b; McManus et al. 2001). Human HDL containingapoA-I(L159R)FIN had increased fractional catabolic rate compared to normalHDL, indicating increased catabolism of the mutant apoA-I protein (Miettinenet al. 1997a, b). Only one affected patient with this mutation had clinicallymanifested atherosclerosis (Miettinen et al. 1997b).To explain the etiology and potential therapy of genetically determined lowlevels of HDL resulting from natural apoA-I mutations, we have studied the in vitroand in vivo properties of the naturally occurring mutants, apoA-I[L141A]Pisa andapoA-I[L159R]FIN (Koukos et al. 2007a). In vitro studies showed that bothmutants were secreted efficiently from cells, had normal ability to promoteABCA1-mediated cholesterol efflux, but greatly diminished capacity to activateLCAT (0.4–2 % of WT apoA-I). Adenovirus-mediated gene transfer showed thatcompared to WT apoA-I, expression of either of the two mutants in apoA-I-deficient (apoA-I/) mice greatly decreased total plasma cholesterol and apoA-Ilevels as well as the CE/TC ratio compared to WT apoA-I (Table 2). Anotherchange that was associated with differences between the WT apoA-I and either ofTable 2 Plasma lipids and hepatic mRNA levels of apoA-I/ mice expressing WT and themutant forms of apoA-I in the presence and absence of LCAT as indicatedProtein expressedCholesterol(mg/dl) CE/TCTriglycerides(mg/dl)RelativeApoA-ImRNA(%)Apo A-IProtein(mg/dl)apo A-I WT 148 11 0.78 0.01 63 1 100 32 186 34apoA-I (L141R)Pisa 23 0.4 0.44 0.03 11 2.8 88 9 17 4apoA-I(L141R)Pisa + LCAT184 53 0.68 0.01 41 0.3 91 2 224 7apoA-I (L159R)FIN 16 5 0.13 0.04 25 4 216 32 25 9apoA-I(L159R)FIN + LCAT224 22 0.73 0.01 53 15 63 9 190 20apo A-I (R159L)Oslo 43 13 0.23 0.01 36 4 117 30 66 31apo A-I (R160L)Oslo 250 47 0.082 0.01 62 11 60 1 127 26HDL Biogenesis, Remodeling, and Catabolism 65the two mutants was the greatly decreased HDL cholesterol peak as determined byFPLC fractionation of the plasma. Density gradient ultracentrifugation of plasmashowed great reduction of the amount of apoA-I that floated in the HDL region ofthe apoA-I[L141R]Pisa mutant as compared to WT apoA-I (Fig. 5a, d). EM analysisFig. 5 (a–m) ApoA-I mutation that influence activity of LCAT. Analysis of plasma of apoA-I/mice infected with adenoviruses expressing the WT apoA-I or the apoA-I[L141]Pisa mutant aloneor in combination with human LCAT by density gradient ultracentrifugation and SDS-PAGE (a, d,g) as indicated. EM analysis of HDL fractions 6–7 obtained from apoA-I/ mice expressing theWT apoA-I or the apoA-I[L141R]Pisa or mutant alone or in combination with human LCAT,following density gradient ultracentrifugation of plasma, as indicated (b, e, h). Two-dimensionalgel electrophoresis of plasma of apoA-I/ mice infected with adenoviruses expressing WT apoA-I or the apoA-I[L141q]Pisa or mutant alone or in combination with human LCAT, as indicated (c, f,i). Western blot analysis of plasma from apoA-I/ mice infected with adenoviruses expressingeither the control protein, GFP, or the WT apoA-I or the apoA-I(L141R)Pisa alone or in combina-tion with human LCAT, as indicated at the top of the figure (j). Schematic representation showingthe pathway of biogenesis of HDL (k) and how the apoA-I(L141R)Pisa and apoA-I(L159R)FINmutants affect the esterification of cholesterol of the pre-HDL particles and prevent their conver-sion to discoidal and spherical HDL, thus promoting their catabolism (l). Schematic representationshowing the inability of the apoA-I(R160L)Oslo mutant to convert the discoidal to spherical HDLparticles (m)66 V.I. Zannis et al.of the HDL fractions obtained by density gradient ultracentrifugation showed thepresence of a large number of spherical HDL for WT apoA-I but only a fewspherical HDL particles for the apoA-I[L141R]Pisa mutant (Fig. 5b, e).Two-dimensional gel electrophoresis of the plasma showed the formation ofsmall amount of preβ-HDL and large amount of α1, α2, α3, α4 HDL subpopulationsfor the WT apoA-I and only preβ- and small-size α4 HDL subpopulations for theapoA-I[L141R]Pisa mutant (Fig. 5c, f). Similar results were observed for apoA-I[L159R]FIN (Koukos et al. 2007a).Coinfection of apoA-I/ mice with adenoviruses expressing either of the twomutants and human LCAT normalized the plasma apoA-I, the total plasma choles-terol levels, and the CE/TC ratio (Table 2) and increased the HDL cholesterol peakand the amount of apoA-I that floated in the HDL region (Fig. 5g). It also generatedlarge amount of spherical HDL (Fig. 5h) and restored the normal preβ- and α-HDLsubpopulations (Fig. 5i). Similar results were observed for apoA-I[L159R]FIN(Koukos et al. 2007a).Another interesting naturally occurring apoA-I mutation is the apoA-I[R160L]Oslo. Previous studies showed that heterozygotes of apoA-I[R160L]Oslohave approximately 60 and 70 % of normal HDL and apoA-I levels, respectively,from preβ1-and small-size α-HDL particles and have a 30 % reduction in theirplasma LCAT activity (Leren et al. 1997). Gene transfer of the apoA-I[R160L]Oslomutant in apoA-I/ mice resulted in low plasma cholesterol and apoA-I levels(Table 2) and generated discoidal particles with α4 electrophoretic mobility. Theaberrant phenotype could be corrected by co-expression of this mutant withhuman LCAT (Koukos et al. 2007b).Similar but not identical phenotypes were produced by expressing thebioengineered apoA-I[R160V/H162A] and apoA-I[R149A] mutants and the natu-rally occurring mutants apoA-I[R151C]Paris and apoA-I[L144R]Zaragosa (Haaseet al. 2011; Chroni et al. 2005a; Koukos et al. 2007b). This phenotype could becorrected by co-expression of the mutant with human LCAT.The last two mutations have not been associated with incidence of atherosclero-sis in humans.The apoA-I mutations discussed here offer a valuable tool to dissect the molec-ular events which lead to the biogenesis of HDL and possibly to understand thetypes of molecular interactions between apoA-I and LCAT which lead to theactivation of the enzyme.In our case, residues R149, R153, and R160 were reported to create a positiveelectrostatic potential around apoA-I. Mutations in these residues reduced drasti-cally the ability of rHDL particles containing these apoA-I mutants to activateLCAT in vitro (Roosbeek et al. 2001). Based on the “belt” model for discoidalrHDL, these residues are located on the hydrophilic face of the apoA-I helices anddo not form intramolecular salt bridges in the antiparallel apoA-I dimer that coversthe fatty acyl chain of the discoidal particle. This arrangement allows in principlethese apoA-I residues to form salt bridges or hydrogen bonds with appropriateresidues of LCAT and thus contribute to LCAT activation.HDL Biogenesis, Remodeling, and Catabolism 67To explain the low HDL levels and the abnormal HDL phenotype of the apoA-I/ mice expressing the apoA-I(L141R)Pisa, we analyzed the relative abundance ofthe endogenous mouse LCAT following gene transfer of the apoA-I(L141R)Pisamutant alone or in the presence of LCAT. This analysis showed a dramatic increaseof the mouse LCAT in mice expressing the apoA-I(L141R)Pisa mutant as comparedto mice expressing the WT apoA-I. Coinfection of apoA-I/ mice with the apoA-I(L141R)Pisa mutant and human LCAT restored the mouse LCAT to normal levels(those observed in the presence of WT apoA-I) (Fig. 5j). The depletion of theendogenous LCAT in mice expressing the mutant forms of apoA-I could be theresult of rapid degradation of endogenous mouse LCAT bound to minimallylipidated apoA-I mutants possibly by the kidney.The ability of the apoA-I[L141R]Pisa and apoA-I(L159R)FIN mutants to besecreted efficiently from cells and to promote ABCA1-mediated cholesterol effluxsuggests that the functional interactions between apoA-I and ABCA1 that lead tothe lipidation of apoA-I are normal and the low apoA-I and HDL levels caused bythese two mutants are the result of fast removal of the lipidated nascent HDLparticles from the plasma compartment. This interpretation is supported by theincreased catabolic rate of HDL containing apoA-I(L159R)FIN (Miettinenet al. 1997b) and the accumulation of proapoA-I in the plasma of hemizygotes forapoA-I(L141R)Pisa (Miccoli et al. 1996). Accumulation of proapoA-I has beenpreviously observed in patients with Tangier disease (Zannis et al. 1982) that arecharacterized by increased catabolic rate of HDL (Assmann et al. 2001). It has beenalso shown previously that cubilin, a 600 KDa membrane protein, binds both apoA-I and HDL and promotes their catabolism by the kidney (Kozyraki et al. 1999;Hammad et al. 1999).Previous studies showed that preβ-HDL is an efficient substrate of LCAT(Nakamura et al. 2004b). In the presence of excess LCAT, the esterification ofthe cholesterol of the newly formed preβ-particles appears to prevent their fastcatabolism and allows them to proceed in the formation of discoidal and sphericalHDL. In the case of the apoA-I[R160L]Oslo, the HDL pathway appears to beinhibited in the step of the conversion of the discoidal to spherical HDL particles.Figure 5k–m depicts the normal pathway of the biogenesis of HDL (Fig. 5k) and thedisruption of this pathway by the apoA-I(L141R)Pisa and apoA-I(L159R)FIN(Fig. 5l) and apoA-I[R160L]Oslo mutants (Fig. 5m).1.3 ApoA-I Mutations May Induce Hypertriglyceridemiaand/or HypercholesterolemiaA series of apoA-I mutations had resulted in severe hypertriglyceridemia (Chroniet al. 2004a; 2005b) (Table 3). The most recently studied case was apoA-I[D89A/E91A/E92A] mutant where the charged residues were substituted by alanines. Thecapacity of the apoA-I[D89A/E91A/E92A] mutant to promote ABCA1-mediatedcholesterol efflux and activate LCAT in vitro was approximately 2/3 of that of WTapoA-I (Kateifides et al. 2011).68 V.I. Zannis et al.In vivo studies using adenovirus-mediated gene transfer in apoA-I-deficientmice showed that compared to WT apoA-I, the apoA-I[D89A/E91A/E92A] mutantincreased plasma and HDL cholesterol, reduced the CE/TC ratio, and caused severehypertriglyceridemia (Table 3) (Kateifides et al. 2011). Following density gradientultracentrifugation of plasma, approximately 40 % of the apoA-I mutant wasdistributed in the VLDL/IDL region. In contrast, the WT apoA-I was distributedin the HDL2/HDL3 region (Fig. 6a). Whereas the WT apoA-I formed sphericalHDL (Fig. 5b), the apoA-I[D89A/E91A/E92A] mutant formed mostly sphericaland few discoidal HDL particles as determined by EM (Fig. 6b). Two-dimensionalgel electrophoresis showed that WT apoA-I formed normal preβ- and α-HDLsubpopulations, whereas the apoA-I[D89A/E91A/E92A] mutants formed preβ-and α4 HDL subpopulations (Fig. 6c) (Kateifides et al. 2011).Co-expression of apoA-I[D89A/E91A/E92A] mutants and human lipoproteinlipase in apoA-I-deficient mice abolished hypertriglyceridemia (Table 3),redistributed apoA-I in the HDL2/HDL3 regions (Fig. 6d), restored in part theα1,2,3,4 HDL subpopulations (Fig. 6f), but did not change significantly the choles-terol ester to total cholesterol ratio (Table 3) or the formation of discoidal HDLparticles (Fig. 6e) (Kateifides et al. 2011).The findings indicate that residues D89, E91, and E92 of apoA-I are important forplasma cholesterol and triglyceride homeostasis as well as for the maturation of HDL.The lipid, lipoprotein, and HDL profiles generated by another mutant, apoA-I[K94A/K96A], where the charged residues were changed to Alanines, were similarto those of WT apoA-I, indicating that the observed changes on the HDL phenotypewere unique for the charged residues D89, E91, and E92 (Kateifides et al. 2011).Expression of a deletion mutant, apoA-I[Δ89-99], in apoA-I/ mice, increasedplasma cholesterol levels, increased the plasma preβ-HDL subpopulation,generated discoidal HDL particles, but did not induce hypertriglyceridemia (Chroniet al. 2005b).Table 3 Plasma lipids and hepatic mRNA levels of apoA-I/ mice expressing WT and themutant forms of apoA-I as indicatedProteinexpressedCholesterol(mg/dL) CE/TCTriglycerides(mg/dL)Relativeapo A-ImRNA (%)Plasmaapo A-I(mg/dL)apo A-I/ 33 6 – 42 7 – –WT apoA-I 268 55 0.72 0.06 70 11 100 32 283 84apo A-I [D89A/E91A/E92A]497 139 0.36 0.31 2,106 1,629 101 24 235 106apo A-I [D89A/E91A/E92A]+ hLPL122 56 0.44 0.14 49 16 41 6 99 18apo A-I [Δ(62–78)]220 16 – 986 289 130 5 265 36apo A-I [E110A/E111A]520 45 – 1,510 590 69 23 204 27HDL Biogenesis, Remodeling, and Catabolism 69Fig. 6 (a–h) Effects of apoA-I mutations on the induction of dyslipidemia. Location of the apoA-Imutations that cause hypertriglyceridemia as indicated. Analysis of plasma of apoA-I/ miceinfected with adenoviruses expressing the apoA-I[D89A/E91A/E92A] mutant alone or in combi-nation with human LPL, as indicated, by density gradient ultracentrifugation and SDS-PAGE (a,d). EM analysis of HDL fractions 6–7 obtained from apoA-I/ mice expressing the apoA-I[D89A/E91A/E92A] mutant alone or in combination with LPL, as indicated, following densitygradient ultracentrifugation of plasma (b, e). Two-dimensional gel electrophoresis of plasma ofapoA-I/ mice infected with adenoviruses expressing the apoA-I[D89A/E91A/E92A] mutantalone or in combination with LPL, as indicated (c, f). Schematic representation of the solventinaccessible interhelical charged interactions of apoA-I dimers arranged in an antiparallel70 V.I. Zannis et al.1.3.1 Potential Mechanism of Dyslipidemia Resulting from apoA-IMutationsThe apoA-I[D89A/E91A/E92A] mutant has two similar characteristics with two othermutants in different regions of apoA-I, the apoA-I[Δ(61-78)] and the apoA-I [E110A/E111A] (Chroni et al. 2004a; 2005b), that cause hypertriglyceridemia (Table 3). Thefirst characteristic is that all three mutants caused accumulation of apoA-I in the VLDL/IDL region. As shown previously, the accumulation of apoA-I in the lower densitiesaffects the in vitro lipolysis of the VLDL/IDL fraction by exogenous lipoprotein lipase(Chroni et al. 2004a; 2005b). The second characteristic is that the three apoA-I mutantshave lost negative-charged residues that are present in the WT sequence. The E78,D89, and E111 residues have the ability to form solvent inaccessible salt bridges withpositively charged residues present in the antiparallel apoA-I molecule of a discoidalHDL particle (Segrest et al. 1999) (Fig. 6g).In these arrangements of the apoA-I molecules on the HDL particle, residuesE78 in helix 2, D89 in helix 3, and E111 in helix 4 can form solvent inaccessible saltbridges with residues R188 in helix 8, R177 in helix 7, and H155 in helix 6, respec-tively, of the antiparallel strand. The affinity of all three mutants for triglyceride-rich lipoprotein particles is further supported by binding studies to triglyceride-richemulsion particles (Gorshkova and Atkinson 2011).It is interesting that in the 11/3 α-helical wheel residues E78, D89, and E111 areall located in wheel position 2. With the exception of R188, all other five residuesinvolved in salt bridges are conserved in mammals.The lipid and lipoprotein abnormalities observed in this mutant suggest that theincreased abundance of apoA-I in the VLDL/IDL region may create lipoproteinlipase insufficiency that is responsible for the induction of hypertriglyceridemia.The persistence of discoidal particle following the lipoprotein lipase treatmentindicates a direct effect of the [D89A/E91A/E92A] mutation in the activation ofLCAT in vivo. Previous studies showed that discoidal and small-size HDL particlesand LCAT associated with them may be catabolized fast by the kidney and thuslead to LCAT insufficiency and reduced plasma HDL levels (Koukos et al. 2007a;Timmins et al. 2005; Miettinen et al. 1997a).It is conceivable that loosening of the structure of apoA-I around the D89 or E92area due to the substitution of the original residues by alanines may provide newsurfaces for interaction of HDL with other proteins or lipoprotein particles such asVLDL in ways that inhibit triglyceride hydrolysis. Furthermore, the accumulationof discoidal HDL as well as the formation of preβ- and small α4 HDL particles asshown by the in vivo experiments indicate that replacement of D89, E91, and E92by A has a direct impact on the activation of LCAT.The preceding analyses described in Figs. 3, 4, 5, and 6 demonstrate that expres-sion of mutant apoA-I forms in different mouse models disrupted specific steps alongthe pathway of the biogenesis of HDL and generated discrete lipid and HDLFig. 6 (continued) orientation in the belt model of rHDL (g). The pathway of HDL biogenesis.Superimposed on the pathway are defects that inhibit different steps of this pathway (h)HDL Biogenesis, Remodeling, and Catabolism 71phenotypes (Zannis et al. 2006b). The phenotypes generated included inhibition ofthe formation of HDL (Chroni et al. 2003, 2007; Fotakis et al. 2013a, b), generation ofunstable intermediates (Koukos et al. 2007a), inhibition of the activation of LCAT(Chroni et al. 2005a; Koukos et al. 2007b), and increase in plasma cholesterol orincrease in both plasma cholesterol and triglycerides (Chroni et al. 2004a, 2005b;Kateifides et al. 2011) (Fig. 6h).1.4 ApoE and apoA-IV Participate in the Biogenesis of HDLParticles Containing the Corresponding ProteinsUsing adenovirus‐mediated gene transfer in apoA‐I‐ or ABCA1‐deficient mice, weobtained unequivocal evidence that apoE of any phenotype participates in thebiogenesis of apoE‐containing HDL particles (HDL-E) using a similar pathwaythat is used for the biogenesis of apoA-I-containing HDL particles (Kypreos andZannis 2007). In the initial experiments, gene transfer of an apoE4‐expressingadenovirus increased both HDL and the triglyceride‐rich VLDL/IDL/LDL fractionand generated discoidal HDL particles (Fig. 7a). Control experiments showed theabsence of discoidal or spherical HDL-size particles in the plasma of apoA-I-deficient mice. The involvement of ABCA1 which was established by gene transferof apoE in ABCA1/ mice prior to and after treatment with apoE4, and indicatedthat apoE4 could not promote formation of HDL particles in ABCA1/ miceFig. 7 EM analysis of apoA-I/ and ABCA1/ mice following infection with adenovirusexpressing human apoE4 as indicated (a, b). EM analysis of apoA-I/ mice following infectionwith adenovirus expressing human apoE4 and LCAT (c). FPLC profile of apoA-I/ miceexpressing apoE4 and LCAT (d). Analyses of the plasma of apoA-I/ mice following infectionwith adenovirus expressing human apoA-IV by density gradient ultracentrifugation andSDS-PAGE (e), EM (f), and two-dimensional gel electrophoresis (g)72 V.I. Zannis et al.(Fig. 7b). Other experiments in apoA-I/ x apoE/ mice established that residues1-202 of apoE are sufficient to promote biogenesis of apoE-containing HDL(Vezeridis et al. 2011). Coinfection of apoA‐I/ mice with a mixture ofadenoviruses expressing both apoE4 and human LCAT converted the discoidal tospherical HDL (Fig. 7c), suggesting that LCAT is essential for the maturation of thediscoidal apoE‐containing HDL to spherical particles (Kypreos and Zannis 2007).The LCAT treatment also cleared the triglyceride‐rich lipoproteins and increasedthe HDL cholesterol peak as determined by FPLC (Fig. 7d).These findings suggest that in contrast to apoA-I where the C-terminal domain isrequired for the biogenesis of HDL (Chroni et al. 2007), the carboxy-terminaldomain of apoE is not required for HDL formation. Overall, the findings indicatethat apoE has a dual functionality. In addition to its documented role in theclearance of triglyceride‐rich lipoproteins, it participates in the biogenesis ofHDL-E in a process that is similar to that of apoA‐I.HDL-E thus formed may have antioxidant and anti‐inflammatory functionssimilar to those described for apoA‐I‐containing HDL, which may contribute tothe atheroprotective properties of apoE (Mineo et al. 2003; Plump et al. 1992;Schaefer et al. 1986; Navab et al. 2000). ApoE‐containing HDL may also haveimportant biological functions in the brain (Li et al. 2003).A similar set of gene transfer experiments in apoA-I/ and apoA-I/ x apoE/mice also established that similar to apoE, apoA-IV also participates in the biogene-sis of apoA-IV containing HDL (HDL-A-IV) and requires for this purpose theactivity of ABCA1 and LCAT (Duka et al. 2013).Gene transfer of apoA-IV in apoA-I/ mice did not change plasma lipid levels.Density gradient ultracentrifugation showed that apoA-IV floated in the HDL2/HDL3 region (Fig. 7e), promoted the formation of spherical HDL particles asdetermined by electron microscopy (Fig. 7f), and generated mostly α- and a fewpreβ-HDL subpopulations as determined by two-dimensional gel electrophoresis(Fig. 7g). When expressed in apoA-I/ apoE/ mice, apoA-IV increasedplasma cholesterol and triglyceride levels and shifted the distribution of theapoA-IV protein in the lower density fractions. This treatment likewise generatedspherical particles and α- and preβ-like HDL subpopulations. Co-expression ofapoA-IV and LCAT in apoA-I/ mice restored the formation of HDL-A-IV.Spherical and α-migrating HDL particles were not detectable following genetransfer of apoA-IV in ABCA1/ or LCAT/ mice (Duka et al. 2013). Theability of apoA-IV to promote biogenesis of HDL may explain previously reportedanti-inflammatory and atheroprotective properties of apoA-IV.In vitro studies showed that lipid-free apoA-IV and reconstituted HDL-A-IVpromoted ABCA1- and scavenger receptor BI (SR-BI)-mediated cholesterol efflux,with the same efficiency as apoA-I and apoE (Chroni et al. 2005c; Duka et al. 2013).HDL Biogenesis, Remodeling, and Catabolism 731.5 Clinical Relevance of the Aberrant HDL PhenotypesGenome-wide association studies indicated that specific gene loci were associatedwith low or high HDL cholesterol and triglyceride levels and could in principleaffect the risk for coronary artery disease (CAD) (Teslovich et al. 2010). Prospec-tive population studies have also shown that HDL has a protective role against CAD(Gordon et al. 1989). The beneficial functions of HDL are also supported by theatheroprotective effect of apoA-I overexpression in transgenic mice (Rubinet al. 1991; Paszty et al. 1994) or rabbits (Emmanuel et al. 1996) or followingadenovirus-mediated gene transfer in mice (Belalcazar et al. 2003; Benoitet al. 1999; Tangirala et al. 1999). The studies described above provide molecularmarkers that could be used for the diagnosis, prognosis, and potential treatment ofHDL abnormalities or dyslipidemias associated with the biogenesis and remodelingof HDL. Diagnostic phenotypes such as those depicted in Figs. 2a–c, 3a–d, 4a, b,5d–f, and 6a–c can be used to assess defects in apoA-I, ABCA1, and LCATrespectively.The HDL phenotypes observed in human patients carrying the apoA-I[L141R]Pisa and apoA-I[L159R]Fin mutations resemble closely the phenotypesobserved in apoA-I-deficient mice expressing these mutants and indicate the valid-ity of the gene transfer studies in mice to establish defects in HDL biogenesis. It ispossible that phenotypes generated by mutagenesis of apoA-I may exist in thehuman population and can be detected by one or more of the analyses describedpreviously. The correction of the aberrant HDL phenotypes by treatment withLCAT suggests a potential therapeutic intervention for HDL abnormalities thatresult from specific mutations in apoA-I or conditions that result in low HDL levels.Additional supporting evidence has been obtained by adenovirus-mediated genetransfer of human LCAT in squirrel monkeys. This treatment increased two-fold theHDL levels without affecting apoA-I levels, increased the size of HDL, anddecreased apoB levels (Amar et al. 2009).The potential contribution of apoA-I mutations to hypertriglyceridemia inhumans is interesting. Hypertriglyceridemia resulting from apoA-I mutations maybe further aggravated by other genetic and environment factors such as diabetes andthyroid status. The contribution of apoA-I mutations to hypertriglyceridemia couldbe addressed in future studies in selected populations of patients with hypertrigly-ceridemia of unknown etiology.2 Remodeling and Catabolism of HDLFollowing synthesis by the liver and the intestine, HDL is remodeled by variousplasma proteins and is subsequently catabolized in the plasma by cell receptors andother plasma proteins (Fig. 8a–d).Turnover studies showed that the mean plasma residence time of radiolabeled125I-HDL2 and HDL3 was 6 days for normal subjects (Schaefer et al. 1979) and0.22 days for patients with Tangier disease (Schaefer et al. 1981). Using stable74 V.I. Zannis et al.isotope turnover kinetic measurements, the resident time was estimated to be 4–5days. The fractional catabolic rates (FCR) were expressed as pools per day, and thesecretion rates that were determined by this method for men and women weresimilar. The FCR were not affected significantly by diet, diabetes, or LDL receptordefects, but it was increased in subjects with increased glucose tolerance (Marshet al. 2000). HDL remodeling affects the structure and metabolic turnover of HDLand generates a dynamic mixture of discrete HDL subfractions that vary in size,shape, apolipoprotein, and lipid composition and functions (Siggins et al. 2007;Fielding and Fielding 2007; Xu and Nilsson-Ehle 2007; Harder and McPherson2007).Remodeling of HDL by the action of hepatic lipase (HL) and endothelial lipase(EL) involves hydrolysis of residual triglycerides and some phospholipids of HDL(Maugeais et al. 2003a; Santamarina-Fojo et al. 2004), leads to the conversion ofHDL2 to HDL3 and preβ-HDL (Ishida et al. 2003; Krauss et al. 1974; Breckenridgeet al. 1982; Brunzell and Deeb 2001), and accelerates the catabolism of HDL.Preβ-HDL formation also requires the functions of apolipoprotein M (apoM)(Wolfrum et al. 2005).Portion of the cholesteryl esters formed by the actions of LCAT can be trans-ferred to VLDL/IDL/LDL by the cholesteryl ester transfer protein (CETP)Fig. 8 (a–d) Schematic representation of the pathway of HDL remodeling by the action of hepaticand endothelial lipase (a), CETP (b), PLTP (c), and SR-BI, ABCG1, and HDL holoparticle uptakeby F1-ATPase (d)HDL Biogenesis, Remodeling, and Catabolism 75(Hopkins and Barter 1980; Barter et al. 2003) The phospholipid transfer protein(PLTP) can transfer the phospholipids from VLDL/IDL to the HDL particle duringlipolysis to generate HDL2 and can also convert HDL3 particles to HDL2 andpreβ-HDL (Tall et al. 1983; Lusa et al. 1996). HDL-binding proteins/receptors ortransporters have been documented at all steps of HDL metabolism and involve theSR-BI, which is mostly expressed in hepatocytes, macrophages, and steroidogenictissues and mediates selective CE uptake by the cells and tissues and cholesterolefflux (Gu et al. 1998; Krieger 1999; Rohrl and Stangl 2013; Pagler et al. 2006a);the ABCG1, which mediates cholesterol efflux (Nakamura et al. 2004a); the ecto-F1-ATPase subunit, which mediates HDL holoparticle uptake by the liver (Martinezet al. 2003, 2007); and the cubilin/megalin receptors for removal of apoA-I andpreβ-HDL by the kidney (Martinez et al. 2007).2.1 ATP-Binding Cassette Transporter G1HDL can be remodeled following interactions with ABCG1, a 67 kDa protein,which is a member of the ABC family of half transporters. ABCG1 is expressed inthe spleen, thymus, lung, and brain (Savary et al. 1996; Croop et al. 1997;Nakamura et al. 2004a) and was reported to be localized on plasma membrane,the Golgi, and recycling endosomes (Wang et al. 2006; Vaughan and Oram 2005;Sturek et al. 2010a; Xie et al. 2006a). The expression of ABCG1 is induced by LXRagonists in macrophages and the liver or by cholesterol loading in macrophages(Klucken et al. 2000; Venkateswaran et al. 2000; Wang et al. 2006).Overexpression of ABCG1 promotes cholesterol efflux from different cells toHDL but not to lipid-free apoA-I (Wang et al. 2004, 2006; Nakamuraet al. 2004a; Vaughan and Oram 2005) (Fig. 8d). HDL obtained from CETP-deficient subjects or patients treated with the CETP inhibitors torcetrapib oranacetrapib was shown to have enhanced ability to promote ABCG1-dependentcholesterol efflux from macrophages (Matsuura et al. 2006; Yvan-Charvetet al. 2007, 2010). ABCG1-mediated cholesterol efflux to HDL is abolished bymutations in the ATP-binding Walker A motif indicating that the ATP-bindingdomain in ABCG1 is essential for both lipid transport activity and protein traffick-ing (Vaughan and Oram 2005). In addition, ABCG1 was shown to promote effluxof 7-ketocholesterol and related oxysterols from macrophages and endothelial cellsto HDL, thus protecting cells from apoptosis (Terasaka et al. 2007; Li et al. 2010).In macrophages, ABCG1, and not SR-BI or ABCA1, has been shown recently to beprimarily responsible for free cholesterol mobilization to rHDL (Cuchelet al. 2010a).Studies in ABCG1-deficient mice also suggested that ABCG1 plays a criticalrole in the efflux of cellular cholesterol to HDL (Kennedy et al. 2005). Studies usingintraperitoneal injection of mice with [3H]cholesterol-labeled J774 macrophageswith either increased or reduced ABCG1 expression, as well as primarymacrophages lacking ABCG1 expression, and measurement of the macrophage-derived [3H]cholesterol levels in plasma and feces, showed that ABCG1 plays a76 V.I. Zannis et al.critical role in promoting macrophage reverse cholesterol transport in vivo (Wanget al. 2007). Nevertheless, the studies in ABCG1-deficient and ABCG1 transgenicmice showed that plasma lipids, HDL, and other lipoprotein levels were notaffected (Kennedy et al. 2005; Out et al. 2007; Burgess et al. 2008). Other studiesshowed that loss of ABCG1 gene in mice results in massive lipid accumulation inhepatocytes and in macrophages within multiple tissues, with the more markedaccumulation in pulmonary macrophages (Kennedy et al. 2005; Out et al. 2006,2007; Burgess et al. 2008; Baldan et al. 2006; Ranalletta et al. 2006; Wojciket al. 2008). In addition, ABCG1 has been suggested to mediate cholesterol effluxto HDL particles from other cell types than macrophages, including adipocytes(Zhang et al. 2010) and human placental endothelial cells, where it may facilitatethe transfer of maternal cholesterol to the fetus (Stefulj et al. 2009).Previous studies indicated that there is little or no specificity of ABCG1 for theacceptor of cholesterol since LDL, HDL2, HDL3, phospholipid/apoA-I particles ofvarious sizes and small unilamellar particles can function as acceptors for choles-terol from cells in an ABCG1-mediated manner (Sankaranarayanan et al. 2009;Favari et al. 2009). However, a recent study by us showed that the ABCG1-mediated efflux of cholesterol to rHDL containing different apoA-I mutants isdiminished by deletion of the carboxyl-terminal domain 185-243 from full-lengthapoA-I (Daniil et al. 2013). Analysis of rHDL used in these studies suggested thatthe impairment of ABCG1-mediated cholesterol efflux is not due to majordifferences in particle composition or size between rHDL particles containingWT apoA-I or apoA-I[Δ(185-243)].The mechanism by which ABCG1 promotes sterol efflux to extracellularacceptors has not been resolved. The earlier studies failed to detect specific HDLassociation in BHK or HEK293 cells overexpressing the human ABCG1 (Wanget al. 2004; Sankaranarayanan et al. 2009). Also the initial studies had suggestedthat ABCG1 is localized to both the plasma membrane and internal membranestructures (Vaughan and Oram 2005; Wang et al. 2006; Xie et al. 2006b), whilemore recent studies suggested that ABCG1 is localized to endosomes and recyclingendosomes (Sturek et al. 2010b; Tarling and Edwards 2011). It has been proposedthat ABCG1 could transport sterols across the bilayer of endosomes before theirfusion with the plasma membrane and thus redistribute these sterols to the outerleaflet of the plasma membrane and facilitate their subsequent efflux of sterols toHDL or other acceptors (Tarling and Edwards 2011; Vaughan and Oram 2005).However, the similar pattern of lipid-free and rHDL-bound apoA-I mutants topromote ABCA1- and ABCG1-mediated cholesterol efflux is compatible with atransient localization of ABCG1 in the plasma membrane that will allow itsinteraction with lipoprotein acceptors (Chroni et al. 2003, 2004b; Daniil et al.2013). The similar cholesterol efflux capacity of lipid-free and lipidated apoA-Imutants could favor a model where lipid-free apoA-I is lipidated in an ABCA1-mediated process, changes its conformation, and subsequently accepts more choles-terol from membrane pools generated by ABCA1 or ABCG1.The different capacity of rHDL-associated apoA-I[Δ(185-243)] mutant to pro-mote ABCG1-mediated cholesterol and 7-ketocholesterol efflux (Daniil et al. 2013)HDL Biogenesis, Remodeling, and Catabolism 77may be related to the looser association of 7-ketocholesterol with the plasmamembrane compared to cholesterol (Kan et al. 1992).The finding that lipid-free and lipid-bound apoA-I[Δ(185-243)] has reducedcapacity to promote ABCA1- and ABCG1-mediated cholesterol efflux, respec-tively, may have physiological significance since proteolysis of HDL-associatedapoA-I in vivo may affect its ability to promote cholesterol efflux frommacrophages. In this regard, proteolysis of apoA-I by metalloproteinases presentin the arterial wall or alveolar macrophages (Russell et al. 2002; Galis et al. 1995)produces various fragments that correspond to apoA-I cleaved after residues 191 or188 and are compatible in size with apoA-I[Δ(185-243)] (Lindstedt et al. 1999;Eberini et al. 2002). This may explain the accumulation of foam cells containinghigh cholesterol levels in alveolar macrophages of ABCG1-deficient mice(Kennedy et al. 2005; Out et al. 2006).A recent study using high-density genotyping arrays containing single-nucleotide polymorphisms suggested an association between HDL cholesterollevels in humans and ABCG1 (Edmondson et al. 2011). Recent genetic associationstudies in humans identified functional variants in ABCG1 associated withincreased risk of coronary artery disease (Xu et al. 2011; Schou et al. 2012),suggesting an important role of ABCG1 in the protection from atherosclerosisand cardiovascular disease.2.2 Phospholipid Transfer ProteinPhospholipid transfer protein (PLTP) transfers phospholipids, diacylglycerol (Raoet al. 1997), free cholesterol (Nishida and Nishida 1997), R-tocopherol (vitamin E)(Kostner et al. 1995), and lipopolysaccharide among lipoproteins and betweenlipoproteins and cells (Hailman et al. 1996; Levels et al. 2005) (Fig. 8c). In vitrostudies have identified a number of functions for PLTP in HDL metabolism (Albersand Cheung 2004; Siggins et al. 2007). PLTP displays two major functions incirculation: (1) it transforms HDL particles in a conversion or fusion processwhereby small HDL3 particles are fused leading to the generation of large fusedHDL particles and preβ-HDL that can participate in cholesterol removal from cells(Vikstedt et al. 2007a), and (2) it transfers post-lipolytic VLDL surfacephospholipids to HDL (Albers and Cheung 2004; Siggins et al. 2007). Functionsof PLTP which may influence the formation of atherosclerotic lesions include thegeneration of acceptors for lipid efflux from cells, regulation of plasma HDL levels,protection of lipoproteins from oxidation, and regulation of production of athero-genic lipoproteins (Jiang et al. 2001).In human plasma, two distinct forms of PLTP are present, one with high activity(HA-PLTP) and the other with low activity (LA-PLTP) (Oka et al. 2000;Karkkainen et al. 2002). It was reported that phospholipid transfer activity is aprerequisite for efficient PLTP-mediated HDL enlargement (Huuskonenet al. 2000) and that enrichment of triglyceride in the HDL core could promotesuch fusion (Rye et al. 1998). Of these two forms, only the high specific activity78 V.I. Zannis et al.PLTP promotes macrophage cholesterol efflux via fusion of HDL particles thatleads to the release of lipid-poor preβ-mobile apoA-I particles which act as efficientcholesterol acceptors (Vikstedt et al. 2007a). The mechanisms by which LA-PLTPis generated and its physiological functions are currently unknown. However, sinceapoE is able to interact with PLTP, and apoE-containing proteoliposomes canactivate inactive or low active PLTP, the presence of apoE in PLTP complexes isexpected to enhance PLTP activity. This is consistent with the suggestion that apoEmay play a role in regulating the PLTP activity level in plasma (Janis et al. 2005).HA- and LA-PLTP forms are surface-active proteins, and the low active form wasdemonstrated to dock more strongly onto a phospholipid monolayer surface ascompared to HA-PLTP form (Setala et al. 2007). It is therefore possible thatLA-PLTP form could play other important lipid transfer-independent functionssuch as signaling on cell surface as suggested (Albers et al. 2012).Although the role of PLTP in lipoprotein metabolism and atherogenesis has beenintensively studied in gene-targeted mouse models and using in vitro experiments,the physiological role of PLTP in human metabolism is far from being resolved.Genetic approach has provided some evidence that genetic variation at the PLTPlocus affects its phospholipid transfer activity and HDL particle size and mighthighlight its relevance in cholesterol efflux process (Vergeer et al. 2010a). PLTP-deficient mice have a marked decrease in HDL and apoA-I (Jiang et al. 1999) butreduced atherosclerosis in the background of apoE/ or apoB-transgenic mice(Jiang et al. 2001). It has also been reported that macrophage-derived PLTPcontributes significantly to total plasma PLTP activity and deficiency of PLTP inmacrophages leads to reduced atherosclerosis in LDLr/ mice (Vikstedtet al. 2007b). There is also an interesting interaction between PLTP and CETPsince it was demonstrated that purified PLTP enhances cholesteryl ester transferfrom HDL3 to VLDL (Tollefson et al. 1988), although PLTP has no cholesterolester transfer activity of its own. Moreover, CETP transgenic/PLTP KO mice havesignificantly lower plasma CETP activity as compared to that of CETP transgenicmice (Kawano et al. 2000). Currently, the physiological relevance of this PLTP-CETP interaction in HDL metabolism is poorly understood.2.3 apoMApolipoprotein M (apoM), which is also involved in HDL remodeling (Fig. 8a), is a26 kDa glycoprotein that belongs to the lipocalin protein superfamily and has beenshown to bind lipophilic ligands in its hydrophobic binding pocket (Xu andDahlback 1999; Nielsen et al. 2009; Dahlback and Nielsen 2009; Hu et al. 2010).It is secreted by the liver and to a lesser extent by the kidney and associates withHDL through its retained N-terminal signal peptide (Christoffersen et al. 2008a;Axler et al. 2008) and to a lesser extent with other lipoproteins. ApoM is involved inthe recycling of small lipophilic ligands via the multi-ligand receptor megalin(Nielsen et al. 2009) and has been shown to participate in the remodeling andmaturation of HDL in plasma.HDL Biogenesis, Remodeling, and Catabolism 79Studies in humans and in mice overexpressing or lacking apoM have shown apositive association between plasma apoM levels and total as well as HDL and LDLcholesterol concentrations (Axler et al. 2007; Plomgaard et al. 2009; Christoffersenet al. 2008b). Lack of hepatocyte nuclear factor-1α (HNF-1α) (Shih et al. 2001) orinhibition of apoM expression in C57BL/6 mice injected with small interferingRNA for apoM (Wolfrum et al. 2005) is characterized by diminished concentrationof preβ-HDL particles and presence of large-size HDL particles. In vitroexperiments using plasma obtained from WT mice, apoM knockout, and apoMtransgenic mice showed that apoM increases the formation of preβ-HDL particlesfollowing incubation of the plasma at 37 C (Christoffersen et al. 2008b).These studies also indicate that apoM-containing HDL particles isolated fromhuman plasma and the plasma of apoM transgenic mice have increased capacity tostimulate cholesterol efflux from macrophage foam cells and are more efficient inprotecting against LDL oxidation (Wolfrum et al. 2005; Christoffersenet al. 2008b). Cell culture studies indicated that expression of apoM in cellstransfected with ABCA1 increases the size of preβ-HDL (Mulya et al. 2010).Reduced plasma apoM levels have been reported in animal models of diabetesand some patients with diabetes and metabolic syndrome (Plomgaard et al. 2009;Dullaart et al. 2009; Xu et al. 2006), indicating potential involvement of apoM inthe development of diabetes. Genetic linkage studies in Chinese populations havealso associated two single-nucleotide polymorphisms (SNPs) located in the apoMproximal promoter region (SNP T-778C and SNP T-855C) with the development ofcoronary artery disease (Xu et al. 2008; Jiao et al. 2007) and one of them (theT-778C) associated with susceptibility to coronary artery disease.Adenovirus-mediated gene transfer of apoM in LDL-receptor-deficient mice orhepatic overexpression of apoM in apoM transgenic mice partially protected themice from atherosclerosis development (Christoffersen et al. 2008b; Wolfrumet al. 2005). Data accumulating until now strongly suggest a protective role forapoM, and the protection might be mediated via HDL.2.4 Hepatic Lipase and Endothelial LipaseHDL is first remodeled in the circulation and subsequently catabolized by cells andtissues. Hepatic lipase (HL) and endothelial lipase (EL) are two plasma lipasesplaying an important role in HDL remodeling (Fig. 8a). HL and EL have specificityprimarily for phospholipids and to a lesser extend for triglycerides of apoB-containing lipoprotein remnants and large HDL (Maugeais et al. 2003a;Santamarina-Fojo et al. 2004). HL-deficient mice exhibit elevated levels of largeHDL particles enriched in phospholipids and apoE (Homanics et al. 1995) andreduced atherosclerosis in the background of apoE/ mice (Karackattu et al. 2006;Mezdour et al. 1997). In contrast, overexpression of HL in mice reduced plasmaHDL levels (Braschi et al. 1998). A rat liver perfusion of human native HDL2 ortriglyceride-enriched HDL promoted the formation of the preβ1-HDL subspeciesand a reduction of the α-HDL2 (Barrans et al. 1994). These changes were attributed80 V.I. Zannis et al.to the triglyceride lipase activity of HL (Barrans et al. 1994). Characterization ofpreβ1-HDL showed that these particles contain one to two molecules of apoA-I,associated with phospholipids, and some free and esterified cholesterol (Guendouziet al. 1999). When compared to triglyceride-rich HDL2, remnant-HDL2 had lost onthe average one molecule of apoA-I, 60 % of triglycerides, and 15 % ofphospholipids. The estimated composition supported the hypothesis that HL hadsplitted the initial particle into one preβ1-HDL and one remnant-HDL2. Remnant-HDL2 had different composition and properties from HDL3, suggesting that HLdid not promote the direct conversion of HDL2 to HDL3 (Guendouzi et al. 1999).Analysis of HL transgenic rabbits suggested that HL reduces the size of α-migratingHDL and increases the rate of catabolism of apoA-I (Kee et al. 2002). Cell studiesshowed that HL promotes selective HDL3 cholesterol ester uptake independentfrom SR-BI and that proteoglycans are needed for the HL action on selective CEuptake (Brundert et al. 2003). Earlier studies in mice deficient in both HL and ELsuggested an additive effect of HL and EL on plasma HDL levels but not onmacrophage-mediated reverse cholesterol transport in mice (Brown et al. 2010).However, a recent study demonstrated that targeted inactivation of both HL and ELin mice promoted macrophage-to-feces RCT and enhanced HDL antioxidantproperties (Escola-Gil et al. 2013).HL-deficient patients have elevated plasma concentrations of cholesterol in theHDL and β-VLDL and increased concentration of triglycerides and phospholipidsin the LDL and HDL (Breckenridge et al. 1982). Analyses carried out in completeand partial HL-deficient subjects as well as in normotriglyceridemic and hyper-triglyceridemic controls suggested that HL activity is important for physiologicallybalanced HDL metabolism (Ruel et al. 2004). However, the presence of HL maynot be necessary for normal HDL-mediated reverse cholesterol transport processand is not associated with pro-atherogenic changes in HDL composition andmetabolism (Ruel et al. 2004). In addition, another Mendelian randomizationstudy showed that subjects with loss-of-function genetic variants of HL haveelevated levels of HDL cholesterol, but are not associated with risk of ischemiccardiovascular disease and therefore may not be protected against ischemic cardio-vascular disease (Johannsen et al. 2009).Endothelial lipase (EL) has phospholipase activity (mostly PLA1 activity) andlow levels of triglyceride lipase activity (Jaye et al. 1999). Overexpression of EL inmice markedly decreased plasma HDL cholesterol and apoA-I levels, had a modesteffect on apoB-containing lipoproteins, and increased 2.5–3-fold the uptake of theHDL by the kidney and the liver (Ishida et al. 2003; Maugeais et al. 2003a). Incontrast, the EL deficiency in mice increased HDL cholesterol levels (Ishidaet al. 2003; Ma et al. 2003) and reduced atherosclerosis in the background ofapoE/ mice (Ishida et al. 2004). Analysis of atherosclerosis prone LDLR/ xApoB(100/100) mice suggested that EL and the HDL cholesterol levels wereregulated by SREBPs and VEGF-A (Kivela et al. 2012). Overexpression of EL inmice markedly decreased plasma HDL cholesterol and apoA-I levels and had amodest effect on apoB-containing lipoproteins (Maugeais et al. 2003b; Ishidaet al. 2003). Furthermore, the HDL phospholipid and cholesteryl ester contentHDL Biogenesis, Remodeling, and Catabolism 81decreased, while HDL triglyceride content increased (Nijstad et al. 2009) and thefree cholesterol content remained unaltered. Fast protein liquid chromatographyanalysis and agarose gel electrophoresis showed that the expression of EL resultedin the generation of small preβ-HDL particles (Nijstad et al. 2009). In addition,overexpression of EL increased the selective uptake of hepatic HDL cholesterylester by SR-BI as well as hepatic holoparticle uptake. This resulted in a dramaticincrease in the uptake of the HDL protein, but not the cholesteryl ester moieties,into the kidneys (Nijstad et al. 2009). These data support a model in whichEL-mediated phospholipid hydrolysis of HDL destabilizes the particle, resultingin the shedding of poorly lipidated apoA-I from the particle surface, which arepreferentially cleared by the kidneys and via increased selective uptake by SR-BI.Several genetic EL variants have been reported to be associated with plasmaHDL-C levels (deLemos et al. 2002; Edmondson et al. 2009), and genome-wideassociation studies have shown that single-nucleotide polymorphisms (SNPs) nearLIPG (EL) are associated with plasma HDL-C levels (Kathiresan et al. 2008a,2009; Teslovich et al. 2010). However, the relationship of genetic variation in theEL locus with the risk for coronary artery disease remains uncertain (Vergeeret al. 2010b). A newer study showed that carriers of an EL mutant characterizedby complete loss of function had significantly higher plasma HDL cholesterollevels compared to carriers having partial loss-of-function mutations (Singarajaet al. 2013). Apolipoprotein B-depleted serum from carriers of HL with completeloss of function had significantly enhanced capacity to promote cholesterol efflux ascompared to apoB-depleted serum obtained from HL carriers with partial loss offunction (Singaraja et al. 2013). In the same study, it was reported that carriers ofcertain EL mutations exhibited trends toward reduced coronary artery disease infour independent cohorts (Singaraja et al. 2013).2.5 Cholesteryl Ester Transfer ProteinCholesteryl ester transfer protein (CETP) promotes the transfer of cholesteryl estersfrom HDL to VLDL, IDL, and LDL in exchange for triglycerides (Fig. 8b). It wasestimated that 66 % of the cholesteryl esters of HDL return to the liver by the actionof CETP, indicating an important role of CETP in reverse cholesterol transport(Barter et al. 2003), and 33 % by the action of SR-BI (Fielding and Fielding 2007).Deficiency in CETP in humans is associated with increased plasma levels of HDL(hyperalphalipoproteinemia) (Inazu et al. 1990; Maruyama et al. 2003) anddecreased levels of small preβ1-HDL particles (Arai et al. 2000; Asztalos 2004).An early study showed that the hyperalphalipoproteinemia and high plasma HDLcholesterol levels in a Japanese family without incidence of atherosclerosis was theresult of deficiency in CETP (Koizumi et al. 1985). Inhibition of CETP activity byCETP inhibitors increased HDL cholesterol levels and the size of HDL particle anddecreased LDL cholesterol levels in human subjects, but did not increaseatheroprotection (Landmesser et al. 2012; Brousseau et al. 2004; de Groothet al. 2002). However, subsequent studies indicated that heterozygous mutations82 V.I. Zannis et al.in CETP increase the risk for CAD (Hirano et al. 1995, 1997; Zhong et al. 1996).The effect of CETP on the HDL pathway was also studied in mice expressing thehuman CETP gene. CETP transgenic mice have a significant decrease in apoA-Iand HDL levels (Melchior et al. 1994) and increased preβ-HDL levels (Franconeet al. 1996) and are susceptible to atherosclerosis in the background of apoE/ orLDLr/ mice (Plump et al. 1999).2.6 Scavenger Receptor BI2.6.1 Role of SR-BI in HDL Remodeling Based on Its In VitroInteractions with Its LigandsSR-BI is an 82 kDa membrane glycoprotein consisting of a large extracellulardomain, two transmembrane domains, and two cytoplasmic amino and carboxy-terminal domains (Krieger 1999). SR-BI is primarily expressed in the liver, steroido-genic tissues, and endothelial cells but is also found in other tissues (Actonet al. 1996), and it binds a variety of ligands including HDL, LDL, VLDL, andmodified lipoproteins. SR-BI has also been shown to affect chylomicron metabolismin vivo and bind non-HDL lipoproteins in vitro (Out et al. 2004b, 2005; Krieger1999, 2001; Acton et al. 1994, 1996; Murao et al. 1997). The most importantproperty of SR-BI is considered its ability to act as the HDL receptor (Fig. 8d).It has been shown that SR-BI binds to native HDL and discoidal reconstitutedHDL containing apoA-I or apoE, through their apolipoprotein moieties (Krieger2001; Chroni et al. 2005c; Liadaki et al. 2000; Xu et al. 1997). When it is bound toHDL, SR-BI mediates selective uptake of cholesteryl ester, triglycerides,phospholipids, and vitamin E from HDL to cells (Acton et al. 1996; Greeneet al. 2001; Thuahnai et al. 2001; Stangl et al. 1999; Gu et al. 1998, 2000b;Urban et al. 2000). It also promotes bidirectional movement of unesterified choles-terol (Ji et al. 1997; Gu et al. 2000a). Interactions of HDL with SR-BI areresponsible for mobilization of free cholesterol from the whole body(Ji et al. 1997; Gu et al. 2000a; Cuchel et al. 2010b). SR-BI-mediated HDLholoparticle endocytosis may also be involved in SR-BI-mediated selective CEuptake under certain conditions in some types of cells (Pagler et al. 2006b; Ahraset al. 2008). To understand the molecular interaction of SR-BI with HDL, SR-BImutants which display altered biological functions were generated by in vitromutagenesis. A SR-BI[M158R] mutant does not bind HDL (Gu et al. 2000a). ASR-BI[Q402R/Q418R] mutant also does not bind HDL, but in contrast with the firstmutant, it binds LDL (Gu et al. 2000a; b). A SR-BI[G420H] mutant has normalselective cholesteryl ester uptake but reduced cholesterol efflux to HDL andreduced hydrolysis of internalized cholesteryl esters (Parathath et al. 2004). Cellculture cholesterol efflux studies using rHDL containing mutated apoA-I and theseSR-BI mutants showed that the greater reduction of cholesterol efflux in cellsexpressing WT SR-BI was with the mutants apoA-I[D102A/D103A] and apoA-I[R160V/H162A] (21 % and 49 %, respectively) (Liu et al. 2002).HDL Biogenesis, Remodeling, and Catabolism 83Follow-up in vivo studies showed apoA-I-deficient mice infected with an ade-novirus expressing the apoA-I[D102A/D103A] had an HDL phenotype that resem-bled that of WT apoA-I (Chroni et al. 2005a). In contrast, mice expressing theapoA-I[R160V/H162A] had a phenotype similar to that described for apoA-I[R160L]Oslo that could be corrected by co-expression of the apoA-I[R160L]Oslomutant and human LCAT (Chroni et al. 2005a). Following density gradient ultra-centrifugation, the apoA-I[R160V/H162A] mutant that floated in the HDL regionwas decreased relatively to WT apoA-I, and it was shifted toward the HDL3 region(Chroni et al. 2005a).When the mutant SR-BI[M158R] was examined, several apoA-I mutants testedhad reduced efflux and bound less tightly compared to WT apoA-I with theexception of rHDL that contained the mutant apoA-I[A160V/H162A]. The bindingof this mutant was almost as tight to the cells that expressed SR-BI[M158R] mutantas it was for the cells that expressed WT SR-BI (Liu et al. 2002). Based on thesefindings, it was suggested that efficient SR-BI-mediated cholesterol efflux requiresnot only direct binding (Gu et al. 2000a) but also the formation of a productivecomplex between SR-BI and the rHDL particle (Liu et al. 2002).2.6.2 In Vivo Functions of SR-BIExpression of SR-BI in the liver was shown to be critical for the control of plasmalevels of HDL cholesterol (HDL-C) (Leiva et al. 2011; Zhang et al. 2007), and itsexpression in the steroidogenic tissues is important for synthesis of steroidhormones (Landschulz et al. 1996; Krieger 1999). Transgenic mice expressingSR-BI in the liver had decreased apoA-I and HDL cholesterol levels and increasedclearance of VLDL and LDL (Wang et al. 1998; Ueda et al. 1999). SR-BI-deficientmice had decreased HDL cholesterol clearance (Out et al. 2004a), twofoldincreased plasma cholesterol, and presence of large-size abnormal apoE-enrichedparticles that were distributed in the HDL/IDL/LDL region (Rigotti et al. 1997).The in vivo phenotypes generated by overexpression or deficiency of SR-BI areconsistent with its in vitro functions to promote selectively lipid transport fromHDL to cells and efflux of free cholesterol from cells. SR-BI has also been shown toaffect chylomicron metabolism in vivo (Out et al. 2004b; 2005).Deficiency of SR-BI in mice reduced greatly the cholesteryl ester stores ofsteroidogenic tissues and decreased the secretion of biliary cholesterol by approxi-mately 50 %. However, the SR-BI deficiency did not affect the secretion of the poolsize of bile acids or the fecal secretion of bile acids and the intestinal cholesterolabsorption (Rigotti et al. 1997; Mardones et al. 2001). These findings establishedthat two important functions of SR-BI are the transfer of the CE of HDL to the liverand subsequent incorporation into the bile for excretion (Rigotti et al. 1997;Mardones et al. 2001) and the delivery of cholesteryl esters to the steroidogenictissues where it is utilized for synthesis of steroid hormones (Ji et al. 1999). Further-more SR-BI controls the concentrations and composition of plasma HDL (Krieger2001; Wang et al. 1998; Ueda et al. 1999; Rigotti et al. 1997; Webb et al. 2002) andprotects different mouse models from atherosclerosis (Hildebrand et al. 2010;Arai et al. 1999; Ueda et al. 2000; Kozarsky et al. 2000; Huszar et al. 2000;84 V.I. Zannis et al.Trigatti et al. 1999; Braun et al. 2002, 2003; Karackattu et al. 2005; Zhanget al. 2005).SR-BI deficiency also caused defective maturation of oocytes and red blood cellsdue to accumulation of cholesterol in the plasma membrane of progenitor cells(Trigatti et al. 1999; Holm et al. 2002) and caused infertility in the female but notthe male mice (Trigatti et al. 1999; Yesilaltay et al. 2006a). The infertility could becorrected by restoration of SR-BI gene by adenovirus-mediated gene transfer(Yesilaltay et al. 2006b). Subsequent experiments showed a negative correlationof follicular HDL cholesterol levels in women and embryo fragmentation duringin vitro fertilization (Browne et al. 2009). Taken together these data suggest a roleof HDL in oocyte development and embryogenesis. The SR-BI-mediated selectiveuptake of the CE of HDL by the liver is a complex process and requires thefunctions of a liver-specific protein, PDZK1, that contains four PDZ domains thatcan recognize the C-terminal region of SR-BI. Interaction of PDZK1 with theC-terminal region of SR-BI, posttranscriptionally, regulates localization and stabi-lity of SR-BI (Fenske et al. 2009). Inactivation of hepatic PDZK1 significantlyaffected plasma HDL metabolism and structure and caused occlusive atherosclero-sis in double-deficient mice for apoE and PDKZ (Kocher et al. 2008; Yesilaltayet al. 2009). The detailed mechanism of SR-BI-facilitated selective uptake of theCE of HDL is not yet clear. It has been suggested that HDL binding to hepaticSR-BI allows the entry of cholesteryl esters into a channel that is generated bySR-BI and along which cholesteryl esters move down their concentration gradientinto the cell membrane. During this movement, HDL particles donate their CE tohepatocytes without the simultaneous uptake and degradation of the whole HDLparticle.Recent findings reviewed in Meyer et al. (2013) indicated that SR-BI-indepen-dent cholesterol ester uptake processes may also operate in macrophages. Liver-specific or whole-body ABCA1 deficiency in mice accelerated HDL catabolism inplasma without changing the hepatic expression of SR-BI, suggesting that othermembrane proteins, such as those involved in the hepatic F1-ATPase/P2Y13 path-way (Martinez et al. 2003) (see below for more detail) and CD36 (Brundertet al. 2011), may be involved in the selective cholesteryl ester uptake. In a large-scale human study, several CD36 SNPs were strongly associated with HDL choles-terol levels, thus pointing to a potential role of CD36 in the regulation of humanHDL metabolism (Love-Gregory et al. 2008).Interactions of HDL with SR-BI in endothelial cells trigger signalingmechanisms that involve activation of eNOS and release of nitric oxide that causesvasodilation (Mineo et al. 2003; Yuhanna et al. 2001; Li et al. 2002; Gonget al. 2003).Human subjects have been identified with a P297S substitution in SR-BI.Heterozygote carriers for this mutation had increased HDL levels and decreasedadrenal steroidogenesis and dysfunctional platelets, but did not develop atheroscle-rosis. HDL derived from these subjects had decreased ability to promote cholesterolefflux from macrophages (Vergeer et al. 2011). A recent study has shown theimpact of SR-BI SNPs on female fertility (Yates et al. 2011).HDL Biogenesis, Remodeling, and Catabolism 852.7 Role of Ecto-F1-ATPase/P2Y13 Pathway in Hepatic HDLClearanceBased on Biacore’s surface plasmon resonance studies of hepatic membranes,Martinez and his colleagues (Martinez et al. 2003) demonstrated the presence ofa 50 kDa apoA-I binding protein that was identical to the subunit of the β-chain ofATP synthase (Boyer 1997). The HDL-binding protein was identified as ecto-F1-ATPase that recognizes apoA-I (Fig. 8d). The multi-subunit ATPase complexconsists of two major domains called F0 and F1 (Boyer 1997). The ecto-F1-ATPaseprotein, which resides on cell membranes, hydrolyzes ATP to ADP and phosphateand can be inhibited by the mitochondrial inhibitor protein IF1 (Cabezonet al. 2003). It was recently shown that IF1 is present in the serum, and itsconcentration correlates negatively with HDL-C levels and the risk for coronaryheart disease (Genoux et al. 2013). Binding of lipid-free apoA-I to the high affinityside of ecto-F1-ATPase enhances binding of HDL to the low-affinity binding sites.The apoA-I binding to the ecto-F1-ATPase also increases the production of ADPthat associates with its receptor, purinergic P2Y13 (Jacquet et al. 2005). The ecto-F1-ATPase/P2Y13-mediated HDL uptake pathway is under careful control.Adenylate kinase and niacin are important factors that regulate HDL metabolismand plasma levels via ecto-F1-ATPase (Fabre et al. 2006; Zhang et al. 2008).Inhibitors of ecto-F1-ATPase or adenylate kinase activity that consume ADPgenerated by ecto-F1-ATPase downregulate holo-HDL particle uptake (Genouxet al. 2013). In vivo studies using a P2Y13-deficient mouse model also indicated thatthe P2Y13 ADP receptor may have an important role in HDL-mediated reversecholesterol transport (Fabre et al. 2010). It is possible that induction of hepatic ecto-F1-ATPase/P2Y13 pathway might enhance hepatic HDL endocytosis and turnoverand accelerate cholesterol removal from cholesterol-laden macrophages and othertissues and cells of the body.2.8 Transcytosis of apoA-I and HDL by Endothelial CellsIt has been shown that endothelial cells have the ability to bind and transcytoselipid-free apoA-I in a specific manner. This process depends on ABCA1 and leadsto the generation of a lipidated apoA-I particle that is secreted (Cavelier et al. 2006;Rohrer et al. 2006). Endothelial cells can also transcytose HDL and this processrequired the functions of SR-BI and ABCG1 (Rohrer et al. 2009). ApoA-I mutantswith defective C-terminal apoA-I[Δ(185-243)] and apoA-I[L218A/L219A/V221A/L222A] had 80 % decreased specific binding and 90 % decreased specific transportby aortic endothelial cells. Following lipidation of these mutants, the rHDLparticles formed were transported through endothelial cells by an ABCG1- andSR-BI-dependent process. Amino and combined amino- and carboxy-terminalapoA-I deletion mutants displayed increased nonspecific binding, but the specificbinding or transport remained absent (Ohnsorg et al. 2011). These data support themodel in which apoA-I is initially lipidated by ABCA1 and subsequently processed86 V.I. Zannis et al.by ABCA1-independent mechanisms. Transcytosis of apoA-I and HDL may pro-vide a mechanism for transfer of HDL into the subendothelial space.2.9 The Role of Cubilin in apoA-I and HDL Catabolismby the KidneyIt has been shown that in humans, impaired cubilin and amnionless function resultsin the Imerslunds-Gräsbeck syndrome, which is characterized by intestinal vitaminB12 malabsorption and proteinuria (Fyfe et al. 2004). Cubilin is a 460 kDaendocytic receptor which is co-expressed with megalin, a 600 kDa multi-ligandreceptor belonging to the LDL receptor gene family. It is localized in the apicalmembranes of epithelial cells in the proximal tubules in the kidney cortex(Kozyraki 2001). In addition to co-localization of cubilin with megalin, the trans-membrane protein amnionless is a renal protein that interacts with cubilin andforms a large cubilin/amnionless complex. In this complex, cubilin plays a role asa ligand-binding domain, whereas amnionless is essential for subcellular localiza-tion and endocytosis of cubilin bound to its ligand (Strope et al. 2004; Fyfeet al. 2004). An important function of cubilin is related to its ability to bindapoA-I or HDL (Kozyraki et al. 1999). In the kidney, however, HDL particles aretoo large to cross the glomerular filtration barrier, and therefore megalin andcubilin/amnionless protein receptor system is only exposed to filtered lipid-free orpoorly lipidated apoA-I, thereby affecting the overall HDL metabolism (Kozyrakiet al. 1999; Moestrup and Nielsen 2005). In physiological terms, it is consideredthat the kidney cortex is a major site of catabolism for lipid-free and poorlylipidated apoA-I and that this uptake is a concerted action of glomerular filtration,tubular reabsorption, and intracellular degradation of free apoA-I (Woollett andSpady 1997). Graversen et al. (2008) analyzed urine samples from patients withFanconi syndrome. This is a rare renal proximal tubular reabsorption failure andalso includes dysfunction of cubilin. A high urinary excretion of both apoA-I andapoA-IV but not apoA-II was evident. This study demonstrated that the humankidney is a major site for filtered apoA-I and A-IV but not for HDL particles sinceurinary excretion of all major lipid classes (phospholipids, triglycerides, choles-terol, and cholesterol esters) in Fanconi patients was as low as in control subjects(Graversen et al. 2008). Although the kidney is not considered a central organ inlipoprotein catabolism, it plays an important role in the degradation of lipid-poorapoA-I via the cubilin function.HDL Biogenesis, Remodeling, and Catabolism 873 HDL Subclasses3.1 The Origin and Metabolism of Preb-HDL SubpopulationsSeveral preβ- and α-HDL subpopulations exist in plasma and are generated as aconsequence of the pathway of biogenesis and remodeling of HDL. Thesesubpopulations can be separated based on different fractionation procedures(Fielding and Fielding 1996; Chung et al. 1986; Nichols et al. 1986; Davidsonet al. 1994). The precursor-product relationship between preβ- and α-HDL particlesas well as the precise origin and functions of the preβ-HDL particles is still a matterof investigation.It has been reported earlier that preβ-HDL comprises approximately 5 % of totalplasma apoA-I level. It is heterogeneous in size and contains several species of 5–6 nm in diameter (Fielding and Fielding 1995; Nanjee et al. 2000). The bestcharacterized species are preβ1 and preβ2 (Fielding and Fielding 1995). Theconcentration of preβ1-HDL is increased in large lymph vessels (Asztaloset al. 1993) and in aortic intima (Heideman and Hoff 1982).Preβ-HDL particles can be formed by two different routes. The first is de novosynthesis by the HDL biogenesis pathway (Fig. 8a). The second is generation ofpreβ-HDL particles from α-HDL particles by reactions catalyzed by CETP, PLTP,HL, EL, and apoM discussed earlier (Barrans et al. 1994; Maugeais et al. 2003c;Arai et al. 2000; Huuskonen et al. 2001; Christoffersen et al. 2008b) (Fig. 8a–c).Cell culture studies showed that lipid-free apoA-I added to a culture medium ofCHO cells can recruit phospholipids and cholesterol, initially to form small 73 Åparticles, and subsequently larger apoA-I-containing particles by the action ofLCAT that have a precursor-product relationship (Forte et al. 1993, 1995).Subsequent studies showed that a large proportion of apoA-I is secreted fromHepG-2, CaCo-2, or apoA-I expressing CHO cells in lipid-free monomeric form,with a Stokes radius of 2.6 nm and preα electrophoretic mobility that is unable topromote efflux of phospholipids and cholesterol. It was suggested that in a reactiondependent on ABCA1, the 2.6 nm form was converted into a 3.6 nm monomericapoA-I form with preβ electrophoretic mobility that was able to promote efflux ofphospholipids and cholesterol from cells and thus increase its size (Chauet al. 2006). Expression of apoM in cells transfected with ABCA1 can also increasethe size of preβ-HDL (Mulya et al. 2010). Other studies have shown that some typesof preβ-HDL particles can be formed independently of apoA-I/ABCA1 interactionsin the plasma of humans with Tangier disease and the plasma of apoA-I-deficientmice expressing mutant apoA-I forms (Chroni et al. 2007; Fotakis et al. 2013a;Asztalos et al. 2001). Furthermore, inhibition of ABCA1 in HepG2 cells andmacrophage cultures by glyburide inhibited the formation of α-HDL particles butdid not affect the formation of preβ-HDL particles (Krimbou et al. 2005).The presence of increased concentrations of preβ1-HDL in the vascular bedsuggests that these particles may be generated locally by gradual lipidation of lipid-poor apoA-I (Heideman and Hoff 1982; Smith et al. 1984). Preβ-HDL particles aretypically lipid-poor and therefore they are efficient in promoting ABCA1-mediated88 V.I. Zannis et al.cholesterol efflux. The ABCA1/preβ1-HDL interaction provides phospholipids andcholesterol and thereby converts the preβ-HDL to α-HDL-migrating particles.These particles may be enlarged further by recruitment of phospholipids andcholesterol from cell membranes (Fielding and Fielding 2007). In addition, esterifi-cation of the cholesterol of preβ1-HDL by LCAT contributes to their gradualconversion into spherical HDL without prior formation of discoidal HDL particles(Nakamura et al. 2004b; Fielding and Fielding 2007).3.2 Complexity of HDLGenome-wide association studies demonstrated that new genes and thecorresponding proteins affect plasma HDL levels by unknown mechanisms(Holleboom et al. 2008; Kathiresan et al. 2008b, 2009; Sabatti et al. 2009;Aulchenko et al. 2009; Teslovich et al. 2010; Richards et al. 2009; Willeret al. 2008; Chasman et al. 2009; Waterworth et al. 2010; Laurila et al. 2013). Inparallel, proteomic analysis showed that a large number of plasma proteins canassociate with HDL and this may affect the HDL structure and functions (Fig. 9)(Gordon et al. 2010). The proteins associated with HDL can be classified in sixmajor categories and include proteins involved in lipid, lipoprotein, and HDLbiogenesis and metabolism, acute phase proteins, protease inhibitors, complementregulatory proteins, and a few others (albumin, fibrinogen a chain platelet basicprotein) (Vaisar et al. 2007; Davidson et al. 2009b). Differences were observed inFig. 9 Schematic representation of proteins associated with HDL and factors that may affectHDL levels and functionsHDL Biogenesis, Remodeling, and Catabolism 89the proteomic composition of HDL subpopulations derived from HDL particles ofdifferent sizes (Davidson et al. 2009a). Furthermore the HDL proteome could bealtered by pharmacological treatments (Green et al. 2008).The modulation of the concentration of various proteins associated with HDLhas been studied in more detail under conditions of infection, inflammation, ortissue injury. Under these conditions, acute phase response is triggered that causeshuge alterations in hepatic protein synthesis in response to cytokines that alter HDLprotein composition (Rohrer et al. 2004; Shah et al. 2013). These changes includethe abrupt increase in serum amyloid A, apoJ, and secretory non-pancreatic phos-pholipase A2 and a decrease in apoA-I, paraoxonase-1, LCAT, and PLTP. Thesechanges affect the capacity of HDL to promote cellular cholesterol efflux as well asother HDL functions (Rohrer et al. 2004; Shah et al. 2013).In addition to the variety of proteins, a variety of lipids are also carried by HDL,and some of them are or can be transformed to potent bioactive molecules (Vickersand Remaley 2014). Furthermore, HDL carries and transports fat soluble vitamins,steroid hormones, carotenoids, as well as numerous more polar metabolites such asheneicosanoic acid, pentitol, and oxalic acid which were found to be significantlycorrelated with insulin resistance (Vickers and Remaley 2014). Finally, it has beenreported that HDL also transports small RNAs, including microRNAs, tRNA-derived RNA fragments, and RNase P-derived RNA fragments (Vickerset al. 2011). How all this protein decoration affects HDL metabolism and HDLparticle function remain to be studied.4 Sources of FundingCollaboration among the groups of Drs Vassilis I. Zannis, Dimitris Kardassis, MattiJauhiainen, and Angeliki Chroni is supported by COST Action BM0904. DrVassilis I. Zannis’ research is supported by a grant from the National Institutes ofHealth HL48739. Drs Dimitris Kardassis and Angeliki Chroni’s research issupported by grants of the General Secretariat of Research and Technology ofGreece (Synergasia 09SYN-12-897) and by and the Ministry of Education ofGreece (Thalis MIS 377286). P. Fotakis has been supported by pre-doctoral trainingFellowship HERACLEITUS II by the European Union and Greek national fundsthrough the Operational Program “Education and Lifelong Learning” of theNational Strategic Reference Framework (NSRF). The work was also financiallysupported by the Academy of Finland (grant # 257545 to MJ).Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.90 V.I. Zannis et al.ReferencesActon SL, Scherer PE, Lodish HF, Krieger M (1994) Expression cloning of SR-BI, a CD36-relatedclass B scavenger receptor. J Biol Chem 269:21003–21009Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M (1996) Identification ofscavenger receptor SR-BI as a high density lipoprotein receptor. Science 271:518–520Ahras M, Naing T, McPherson R (2008) Scavenger receptor class B type I localizes to a lateendosomal compartment. J Lipid Res 49:1569–1576Aiello RJ, Brees D, Bourassa PA, Royer L, Lindsey S, Coskran T, Haghpassand M, Francone OL(2002) Increased atherosclerosis in hyperlipidemic mice with inactivation of ABCA1 inmacrophages. Arterioscler Thromb Vasc Biol 22:630–637Albers JJ, Cheung MC (2004) Emerging roles for phospholipid transfer protein in lipid andlipoprotein metabolism. Curr Opin Lipidol 15:255–260Albers JJ, Vuletic S, Cheung MC (2012) Role of plasma phospholipid transfer protein in lipid andlipoprotein metabolism. Biochim Biophys Acta 1821:345–357Amar MJ, Shamburek RD, Vaisman B, Knapper CL, Foger B, Hoyt RF Jr, Santamarina-Fojo S,Brewer HB Jr, Remaley AT (2009) Adenoviral expression of human lecithin-cholesterolacyltransferase in nonhuman primates leads to an antiatherogenic lipoprotein phenotype byincreasing high-density lipoprotein and lowering low-density lipoprotein. Metabolism 58:568–575Arai T, Wang N, Bezouevski M, Welch C, Tall AR (1999) Decreased atherosclerosis in heterozy-gous low density lipoprotein receptor-deficient mice expressing the scavenger receptor BItransgene. J Biol Chem 274:2366–2371Arai T, Tsukada T, Murase T, Matsumoto K (2000) Particle size analysis of high densitylipoproteins in patients with genetic cholesteryl ester transfer protein deficiency. Clin ChimActa 301:103–117Assmann G, von Eckardstein A, Brewer HB (2001) Familial analphalipoproteinemia: Tangierdisease. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic and molecular basisof inherited disease. McGraw-Hill, New York, pp 2937–2960Asztalos BF (2004) High-density lipoprotein metabolism and progression of atherosclerosis: newinsights from the HDL Atherosclerosis Treatment Study. Curr Opin Cardiol 19:385–391Asztalos BF, Sloop CH, Wong L, Roheim PS (1993) Comparison of apo A-I-containingsubpopulations of dog plasma and prenodal peripheral lymph: evidence for alteration insubpopulations in the interstitial space. Biochim Biophys Acta 1169:301–304Asztalos BF, Brousseau ME, McNamara JR, Horvath KV, Roheim PS, Schaefer EJ (2001)Subpopulations of high density lipoproteins in homozygous and heterozygous Tangier disease.Atherosclerosis 156:217–225Aulchenko YS, Ripatti S, Lindqvist I, Boomsma D, Heid IM, Pramstaller PP, Penninx BW,Janssens AC, Wilson JF, Spector T, Martin NG, Pedersen NL, Kyvik KO, Kaprio J,Hofman A, Freimer NB, Jarvelin MR, Gyllensten U, Campbell H, Rudan I, Johansson A,Marroni F, Hayward C, Vitart V, Jonasson I, Pattaro C, Wright A, Hastie N, Pichler I,Hicks AA, Falchi M, Willemsen G, Hottenga JJ, de Geus EJ, Montgomery GW, Whitfield J,Magnusson P, Saharinen J, Perola M, Silander K, Isaacs A, Sijbrands EJ, Uitterlinden AG,Witteman JC, Oostra BA, Elliott P, Ruokonen A, Sabatti C, Gieger C, Meitinger T,Kronenberg F, Doring A, Wichmann HE, Smit JH, McCarthy MI, van Duijn CM, Peltonen L(2009) Loci influencing lipid levels and coronary heart disease risk in 16 European populationcohorts. Nat Genet 41:47–55Axler O, Ahnstrom J, Dahlback B (2007) An ELISA for apolipoprotein M reveals a strongcorrelation to total cholesterol in human plasma. J Lipid Res 48:1772–1780Axler O, Ahnstrom J, Dahlback B (2008) Apolipoprotein M associates to lipoproteins through itsretained signal peptide. FEBS Lett 582:826–828Baldan A, Pei L, Lee R, Tarr P, Tangirala RK, Weinstein MM, Frank J, Li AC, Tontonoz P,Edwards PA (2006) Impaired development of atherosclerosis in hyperlipidemic Ldlr/ andHDL Biogenesis, Remodeling, and Catabolism 91ApoE/ mice transplanted with Abcg1/ bone marrow. Arterioscler Thromb Vasc Biol 26:2301–2307Barrans A, Collet X, Barbaras R, Jaspard B, Manent J, Vieu C, Chap H, Perret B (1994) Hepaticlipase induces the formation of pre-beta 1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2. A study comparing liver perfusion to in vitro incubation with lipases. J Biol Chem269:11572–11577Barter PJ, Brewer HB Jr, Chapman MJ, Hennekens CH, Rader DJ, Tall AR (2003) Cholesterylester transfer protein: a novel target for raising HDL and inhibiting atherosclerosis.Arterioscler Thromb Vasc Biol 23:160–167Belalcazar LM, Merched A, Carr B, Oka K, Chen KH, Pastore L, Beaudet A, Chan L (2003) Long-term stable expression of human apolipoprotein A-I mediated by helper-dependent adenovirusgene transfer inhibits atherosclerosis progression and remodels atherosclerotic plaques in amouse model of familial hypercholesterolemia. Circulation 107:2726–2732Benoit P, Emmanuel F, Caillaud JM, Bassinet L, Castro G, Gallix P, Fruchart JC, Branellec D,Denefle P, Duverger N (1999) Somatic gene transfer of human ApoA-I inhibits atherosclerosisprogression in mouse models. Circulation 99:105–110Biedzka-Sarek M, Metso J, Kateifides A, Meri T, Jokiranta TS, Muszynski A, Radziejewska-Lebrecht J, Zannis V, Skurnik M, Jauhiainen M (2011) Apolipoprotein A-I exerts bactericidalactivity against Yersinia enterocolitica serotype O:3. J Biol Chem 286:38211–38219Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W, Drobnik W, Barlage S,Buchler C, Porsch-Ozcurumez M, Kaminski WE, Hahmann HW, Oette K, Rothe G,Aslanidis C, Lackner KJ, Schmitz G (1999) The gene encoding ATP-binding cassette trans-porter 1 is mutated in Tangier disease. Nat Genet 22:347–351Borhani DW, Rogers DP, Engler JA, Brouillette CG (1997) Crystal structure of truncated humanapolipoprotein A-I suggests a lipid-bound conformation. Proc Natl Acad Sci USA 94:12291–12296Borhani DW, Engler JA, Brouillette CG (1999) Crystallization of truncated human apolipoproteinA-I in a novel conformation. Acta Crystallogr D Biol Crystallogr 55(Pt 9):1578–1583Boyer PD (1997) The ATP synthase–a splendid molecular machine. Annu Rev Biochem 66:717–749Braschi S, Couture N, Gambarotta A, Gauthier BR, Coffill CR, Sparks DL, Maeda N, Schultz JR(1998) Hepatic lipase affects both HDL and ApoB-containing lipoprotein levels in the mouse.Biochim Biophys Acta 1392:276–290Braun A, Trigatti BL, Post MJ, Sato K, Simons M, Edelberg JM, Rosenberg RD, Schrenzel M,Krieger M (2002) Loss of SR-BI expression leads to the early onset of occlusive atheroscleroticcoronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, andpremature death in apolipoprotein E-deficient mice. Circ Res 90:270–276Braun A, Zhang S, Miettinen HE, Ebrahim S, Holm TM, Vasile E, Post MJ, Yoerger DM, PicardMH, Krieger JL, Andrews NC, Simons M, Krieger M (2003) Probucol prevents early coronaryheart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E doubleknockout mouse. Proc Natl Acad Sci USA 100:7283–7288Breckenridge WC, Little JA, Alaupovic P, Wang CS, Kuksis A, Kakis G, Lindgren F, Gardiner G(1982) Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase.Atherosclerosis 45:161–179Brousseau ME, Schaefer EJ, Wolfe ML, Bloedon LT, Digenio AG, Clark RW, Mancuso JP,Rader DJ (2004) Effects of an inhibitor of cholesteryl ester transfer protein on HDL choles-terol. N Engl J Med 350:1505–1515Brown RJ, Lagor WR, Sankaranaravanan S, Yasuda T, Quertermous T, Rothblat GH, Rader DJ(2010) Impact of combined deficiency of hepatic lipase and endothelial lipase on the meta-bolism of both high-density lipoproteins and apolipoprotein B-containing lipoproteins.Circ Res 107:357–36492 V.I. Zannis et al.Browne RW, Bloom MS, Shelly WB, Ocque AJ, Huddleston HG, Fujimoto VY (2009) Follicularfluid high density lipoprotein-associated micronutrient levels are associated with embryofragmentation during IVF. J Assist Reprod Genet 26:557–560Brundert M, Heeren J, Greten H, Rinninger F (2003) Hepatic lipase mediates an increase inselective uptake of HDL-associated cholesteryl esters by cells in culture independent fromSR-BI. J Lipid Res 44:1020–1032Brundert M, Heeren J, Merkel M, Carambia A, Herkel J, Groitl P, Dobner T, Ramakrishnan R,Moore KJ, Rinninger F (2011) Scavenger receptor CD36 mediates uptake of high densitylipoproteins in mice and by cultured cells. J Lipid Res 52:745–758Brunham LR, Kruit JK, Iqbal J, Fievet C, Timmins JM, Pape TD, Coburn BA, Bissada N, Staels B,Groen AK, Hussain MM, Parks JS, Kuipers F, Hayden MR (2006a) Intestinal ABCA1 directlycontributes to HDL biogenesis in vivo. J Clin Invest 116:1052–1062Brunham LR, Singaraja RR, Hayden MR (2006b) Variations on a gene: rare and common variantsin ABCA1 and their impact on HDL cholesterol levels and atherosclerosis. Annu Rev Nutr 26:105–129Brunzell JD, Deeb SS (2001) Familial lipoprotein lipase deficiency, apoC-II deficiency, andhepatic lipase deficiency. In: Scriver CR, Beaudet AL, Valle D, Sly WS (eds) The metabolicand molecular bases of inherited disease. McGraw-Hill, New York, pp 2789–2816Burgess B, Naus K, Chan J, Hirsch-Reinshagen V, Tansley G, Matzke L, Chan B, Wilkinson A,Fan J, Donkin J, Balik D, Tanaka T, Ou G, Dyer R, Innis S, McManus B, Lutjohann D,Wellington C (2008) Overexpression of human ABCG1 does not affect atherosclerosis infat-fed ApoE-deficient mice. Arterioscler Thromb Vasc Biol 28:1731–1737Cabezon E, Montgomery MG, Leslie AG, Walker JE (2003) The structure of bovine F1-ATPase incomplex with its regulatory protein IF1. Nat Struct Biol 10:744–750Calabresi L, Baldassarre D, Castelnuovo S, Conca P, Bocchi L, Candini C, Frigerio B, Amato M,Sirtori CR, Alessandrini P, Arca M, Boscutti G, Cattin L, Gesualdo L, Sampietro T, Vaudo G,Veglia F, Calandra S, Franceschini G (2009a) Functional lecithin: cholesterol acyltransferaseis not required for efficient atheroprotection in humans. Circulation 120:628–635Calabresi L, Favari E, Moleri E, Adorni MP, Pedrelli M, Costa S, Jessup W, Gelissen IC, KovanenPT, Bernini F, Franceschini G (2009b) Functional LCAT is not required for macrophagecholesterol efflux to human serum. Atherosclerosis 204:141–146Cavelier C, Rohrer L, von Eckardstein A (2006) ATP-binding cassette transporter al modulatesapolipoprotein A1 transcytosis through aortic endothelial cells. Circ Res 99:1060–1066Chasman DI, Pare G, Mora S, Hopewell JC, Peloso G, Clarke R, Cupples LA, Hamsten A,Kathiresan S, Malarstig A, Ordovas JM, Ripatti S, Parker AN, Miletich JP, Ridker PM(2009) Forty-three loci associated with plasma lipoprotein size, concentration, and cholesterolcontent in genome-wide analysis. PLoS Genet 5Chau P, Nakamura Y, Fielding CJ, Fielding PE (2006) Mechanism of prebeta-HDL formation andactivation. Biochemistry 45:3981–3987Christoffersen C, Ahnstrom J, Axler O, Christensen EI, Dahlback B, Nielsen LB (2008a) Thesignal peptide anchors apolipoprotein M in plasma lipoproteins and prevents rapid clearance ofapolipoprotein M from plasma. J Biol Chem 283:18765–18772Christoffersen C, Jauhiainen M, Moser M, Porse B, Ehnholm C, Boesl M, Dahlback B, Nielsen LB(2008b) Effect of apolipoprotein M on high density lipoprotein metabolism and atherosclerosisin low density lipoprotein receptor knock-out mice. J Biol Chem 283:1839–1847Chroni A, Liu T, Gorshkova I, Kan HY, Uehara Y, von Eckardstein A, Zannis VI (2003) Thecentral helices of apoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type apoA-I are required forlipid efflux in vitro and high density lipoprotein formation in vivo. J Biol Chem 278:6719–6730Chroni A, Kan HY, Kypreos KE, Gorshkova IN, Shkodrani A, Zannis VI (2004a) Substitutions ofglutamate 110 and 111 in the middle helix 4 of human apolipoprotein A-I (apoA-I) by alanineHDL Biogenesis, Remodeling, and Catabolism 93affect the structure and in vitro functions of apoA-I and induce severe hypertriglyceridemia inapoA-I-deficient mice. Biochemistry 43:10442–10457Chroni A, Liu T, Fitzgerald ML, Freeman MW, Zannis VI (2004b) Cross-linking and lipid effluxproperties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1.Biochemistry 43:2126–2139Chroni A, Duka A, Kan HY, Liu T, Zannis VI (2005a) Point mutations in apolipoprotein a-I mimicthe phenotype observed in patients with classical lecithin:cholesterol acyltransferase defi-ciency. Biochemistry 44:14353–14366Chroni A, Kan HY, Shkodrani A, Liu T, Zannis VI (2005b) Deletions of helices 2 and 3 of humanapoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transferin apoA-I-deficient mice. Biochemistry 44:4108–4117Chroni A, Nieland TJ, Kypreos KE, Krieger M, Zannis VI (2005c) SR-BI mediates cholesterolefflux via its interactions with lipid-bound ApoE. Structural mutations in SR-BI diminishcholesterol efflux. Biochemistry 44:13132–13143Chroni A, Koukos G, Duka A, Zannis VI (2007) The carboxy-terminal region of apoA-I is requiredfor the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo.Biochemistry 46:5697–5708Chung BH, Segrest JP, Ray MJ, Brunzell JD, Hokanson JE, Krauss RM, Beaudrie K, Cone JT(1986) Single vertical spin density gradient ultracentrifugation. Methods Enzymol 128:181–209Croop JM, Tiller GE, Fletcher JA, Lux ML, Raab E, Goldenson D, Son D, Arciniegas S, Wu RL(1997) Isolation and characterization of a mammalian homolog of the Drosophila white gene.Gene 185:77–85Cuchel M, Lund-Katz S, Llera-Moya M, Millar JS, Chang D, Fuki I, Rothblat GH, Phillips MC,Rader DJ (2010) Pathways by which reconstituted high-density lipoprotein mobilizes freecholesterol from whole body and from macrophages. Arterioscler Thromb Vasc Biol 30:526–532Dahlback B, Nielsen LB (2009) Apolipoprotein M affecting lipid metabolism or just catching aride with lipoproteins in the circulation? Cell Mol Life Sci 66:559–564Daniil G, Phedonos AA, Holleboom AG, Motazacker MM, Argyri L, Kuivenhoven JA, Chroni A(2011) Characterization of antioxidant/anti-inflammatory properties and apoA-I-containingsubpopulations of HDL from family subjects with monogenic low HDL disorders.Clin Chim Acta 412:1213–1220Daniil G, Zannis VI, Chroni A (2013) Effect of apoA-I mutations in the capacity of reconstitutedHDL to promote ABCG1-mediated cholesterol efflux. PLoS ONE 8:e67993Davidson WS, Sparks DL, Lund-Katz S, Phillips MC (1994) The molecular basis for the differ-ence in charge between pre-beta- and alpha-migrating high density lipoproteins. J Biol Chem269:8959–8965Davidson WS, Silva RA, Chantepie S, Lagor WR, Chapman MJ, Kontush A (2009) Proteomicanalysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance toantioxidative function. Arterioscler Thromb Vasc Biol 29:870–876de Grooth GJ, Kuivenhoven JA, Stalenhoef AF, de Graaf J, Zwinderman AH, Posma JL, vanTol A, Kastelein JJ (2002) Efficacy and safety of a novel cholesteryl ester transfer proteininhibitor, JTT-705, in humans: a randomized phase II dose-response study. Circulation 105:2159–2165deLemos AS, Wolfe ML, Long CJ, Sivapackianathan R, Rader DJ (2002) Identification of geneticvariants in endothelial lipase in persons with elevated high-density lipoprotein cholesterol.Circulation 106:1321–1326Duka A, Fotakis P, Georgiadou D, Kateifides A, Tzavlaki K, von Eckardstein L, Stratikos E,Kardassis D, Zannis VI (2013) ApoA-IV promotes the biogenesis of apoA-IV-containing HDLparticles with the participation of ABCA1 and LCAT. J Lipid Res 54:107–11594 V.I. Zannis et al.Dullaart RP, Plomgaard P, de Vries R, Dahlback B, Nielsen LB (2009) Plasma apolipoprotein M isreduced in metabolic syndrome but does not predict intima media thickness. Clin Chim Acta406:129–133Eberini I, Calabresi L, Wait R, Tedeschi G, Pirillo A, Puglisi L, Sirtori CR, Gianazza E (2002)Macrophage metalloproteinases degrade high-density-lipoprotein-associated apolipoproteinA-I at both the N- and C-termini. Biochem J 362:627–634Edmondson AC, Brown RJ, Kathiresan S, Cupples LA, Demissie S, Manning AK, Jensen MK,Rimm EB, Wang J, Rodrigues A, Bamba V, Khetarpal SA, Wolfe ML, Derohannessian S,Li M, Reilly MP, Aberle J, Evans D, Hegele RA, Rader DJ (2009) Loss-of-function variants inendothelial lipase are a cause of elevated HDL cholesterol in humans. J Clin Invest 119:1042–1050Edmondson AC, Braund PS, Stylianou IM, Khera AV, Nelson CP,Wolfe ML, Derohannessian SL,Keating BJ, Qu L, He J, Tobin MD, Tomaszewski M, Baumert J, Klopp N, Doring A,Thorand B, Li M, Reilly MP, Koenig W, Samani NJ, Rader DJ (2011) Dense genotyping ofcandidate gene loci identifies variants associated with high-density lipoprotein cholesterol.Circ Cardiovasc Genet 4:145–155Emmanuel F, Caillaud JM, Hennuyer N, Fievet C, Viry I, Houdebine JC, Fruchart JC, Denefle P,Duverger N (1996) Overexpression of human apolipoprotein A-I inhibits atherosclerosisdevelopment in Watanabe rabbits. Circulation 94:632Escola-Gil JC, Chen X, Julve J, Quesada H, Santos D, Metso J, Tous M, Jauhiainen M, Blanco-Vaca F (2013) Hepatic lipase- and endothelial lipase-deficiency in mice promotes macrophage-to-feces RCT and HDL antioxidant properties. Biochim Biophys Acta 1831:691–697Fabre AC, Vantourout P, Champagne E, Terce F, Rolland C, Perret B, Collet X, Barbaras R,Martinez LO (2006) Cell surface adenylate kinase activity regulates the F(1)-ATPase/P2Y(13)-mediated HDL endocytosis pathway on human hepatocytes. Cell Mol Life Sci 63:2829–2837Fabre AC, Malaval C, Ben AA, Verdier C, Pons V, Serhan N, Lichtenstein L, Combes G, Huby T,Briand F, Collet X, Nijstad N, Tietge UJ, Robaye B, Perret B, Boeynaems JM, Martinez LO(2010) P2Y13 receptor is critical for reverse cholesterol transport. Hepatology 52:1477–1483Favari E, Calabresi L, Adorni MP, Jessup W, Simonelli S, Franceschini G, Bernini F (2009) Smalldiscoidal pre-beta1 HDL particles are efficient acceptors of cell cholesterol via ABCA1 andABCG1. Biochemistry 48:11067–11074Fenske SA, Yesilaltay A, Pal R, Daniels K, Barker C, Quinones V, Rigotti A, Krieger M,Kocher O (2009) Normal hepatic cell surface localization of the high density lipoproteinreceptor, scavenger receptor class B, type I, depends on all four PDZ domains of PDZK1.J Biol Chem 284:5797–5806Fielding CJ, Fielding PE (1995) Molecular physiology of reverse cholesterol transport. J Lipid Res36:211–228Fielding CJ, Fielding PE (1996) Two-dimensional nondenaturing electrophoresis of lipoproteins:applications to high-density lipoprotein speciation. Methods Enzymol 263:251–259Fielding CJ, Fielding PE (2007) Reverse cholesterol transport – new roles for preb1-HDL andlecithin: cholesterol acyltransferase. In: Fielding CJ (ed) High-density lipoproteins: from basicbiology to clinical aspects. Wiley, Weinheim, pp 143–162Fielding CJ, Shore VG, Fielding PE (1972) A protein cofactor of lecithin:cholesterolacyltransferase. Biochem Biophys Res Commun 46:1493–1498Fitzgerald ML, Mendez AJ, Moore KJ, Andersson LP, Panjeton HA, Freeman MW (2001)ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence thattranslocates the protein’s first hydrophilic domain to the exoplasmic space. J Biol Chem 276:15137–15145Fitzgerald ML, Morris AL, Rhee JS, Andersson LP, Mendez AJ, Freeman MW (2002) Naturallyoccurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct inter-action with apolipoprotein A-I. J Biol Chem 277:33178–33187HDL Biogenesis, Remodeling, and Catabolism 95Forte TM, Goth-Goldstein R, Nordhausen RW, McCall MR (1993) Apolipoprotein A-I-cellmembrane interaction: extracellular assembly of heterogeneous nascent HDL particles.J Lipid Res 34:317–324Forte TM, Bielicki JK, Goth-Goldstein R, Selmek J, McCall MR (1995) Recruitment of cellphospholipids and cholesterol by apolipoproteins A-II and A-I: formation of nascentapolipoprotein-specific HDL that differ in size, phospholipid composition, and reactivitywith LCAT. J Lipid Res 36:148–157Fotakis P, Kateifides A, Gkolfinopoulou C, Georgiadou D, Beck M, Grundler K, Chroni A,Stratikos E, Kardassis D, Zannis VI (2013a) Role of the hydrophobic and charged residuesin the 218 to 226 region of apoA-I in the biogenesis of HDL. J Lipid Res 54:3281–3292Fotakis P, Tiniakou I, Kateifides AK, Gkolfinopoulou C, Chroni A, Stratikos E, Zannis VI,Kardassis D (2013b) Significance of the hydrophobic residues 225-230 of apoA-I for thebiogenesis of HDL. J Lipid Res 54:3293–3302Francone OL, Royer L, Haghpassand M (1996) Increased prebeta-HDL levels, cholesterol efflux,and LCAT-mediated esterification in mice expressing the human cholesteryl ester transferprotein (CETP) and human apolipoprotein A-I (apoA-I) transgenes. J Lipid Res 37:1268–1277Frikke-Schmidt R, Nordestgaard BG, Jensen GB, Steffensen R, Tybjaerg-Hansen A (2008)Genetic variation in ABCA1 predicts ischemic heart disease in the general population.Arterioscler Thromb Vasc Biol 28:180–186Fyfe JC, Madsen M, Hojrup P, Christensen EI, Tanner SM, de la Chapelle A, He Q, Moestrup SK(2004) The functional cobalamin (vitamin B12)-intrinsic factor receptor is a novel complex ofcubilin and amnionless. Blood 103:1573–1579Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Libby P (1995) Enhanced expressionof vascular matrix metalloproteinases induced in vitro by cytokines and in regions of humanatherosclerotic lesions. Ann N Y Acad Sci 748:501–507Genoux A, Ruidavets JB, Ferrieres J, Combes G, Lichtenstein L, Pons V, Laffargue M,Taraszkiewicz D, Carrie D, Elbaz M, Perret B, Martinez LO (2013) Serum IF1 concentrationis independently associated to HDL levels and to coronary heart disease: the GENES study.J Lipid Res 54:2550–2558Gong M, Wilson M, Kelly T, Su W, Dressman J, Kincer J, Matveev SV, Guo L, Guerin T, Li XA,ZhuW, Uittenbogaard A, Smart EJ (2003) HDL-associated estradiol stimulates endothelial NOsynthase and vasodilation in an SR-BI-dependent manner. J Clin Invest 111:1579–1587Gordon DJ, Probstfield JL, Garrison RJ, Neaton JD, Castelli WP, Knoke JD, Jacobs DR Jr,Bangdiwala S, Tyroler HA (1989) High-density lipoprotein cholesterol and cardiovasculardisease. Four prospective American studies. Circulation 79:8–15Gordon SM, Deng JY, Lu LJ, Davidson WS (2010) Proteomic characterization of human plasmahigh density lipoprotein fractionated by gel filtration chromatography. J Proteome Res 9:5239–5249Gorshkova IN, Atkinson D (2011) Enhanced binding of apolipoprotein A-I variants associatedwith hypertriglyceridemia to triglyceride-rich particles. Biochemistry 50:2040–2047Graversen JH, Castro G, Kandoussi A, Nielsen H, Christensen EI, Norden A, Moestrup SK (2008)A pivotal role of the human kidney in catabolism of HDL protein components apolipoproteinA-I and A-IV but not of A-II. Lipids 43:467–470Green PS, Vaisar T, Pennathur S, Kulstad JJ, Moore AB, Marcovina S, Brunzell J, Knopp RH,Zhao XQ, Heinecke JW (2008) Combined statin and niacin therapy remodels the high-densitylipoprotein proteome. Circulation 118:1259–1267Greene DJ, Skeggs JW, Morton RE (2001) Elevated triglyceride content diminishes the capacity ofhigh density lipoprotein to deliver cholesteryl esters via the scavenger receptor class B type I(SR-BI). J Biol Chem 276:4804–4811Groen AK, Bloks VW, Bandsma RH, Ottenhoff R, Chimini G, Kuipers F (2001) Hepatobiliarycholesterol transport is not impaired in Abca1-null mice lacking HDL. J Clin Invest 108:843–85096 V.I. Zannis et al.Gu X, Trigatti B, Xu S, Acton S, Babitt J, Krieger M (1998) The efficient cellular uptake of highdensity lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellulardomain. J Biol Chem 273:26338–26348Gu X, Kozarsky K, Krieger M (2000a) Scavenger receptor class B, type I-mediated [3H]choles-terol efflux to high and low density lipoproteins is dependent on lipoprotein binding to thereceptor. J Biol Chem 275:29993–30001Gu X, Lawrence R, Krieger M (2000b) Dissociation of the high density lipoprotein and lowdensity lipoprotein binding activities of murine scavenger receptor class B type I (mSR-BI)using retrovirus library-based activity dissection. J Biol Chem 275:9120–9130Guendouzi K, Jaspard B, Barbaras R, Motta C, Vieu C, Marcel Y, Chap H, Perret B, Collet X(1999) Biochemical and physical properties of remnant-HDL2 and of pre beta 1-HDL pro-duced by hepatic lipase. Biochemistry 38:2762–2768Haase CL, Frikke-Schmidt R, Nordestgaard BG, Kateifides AK, Kardassis D, Nielsen LB,Andersen CB, Kober L, Johnsen AH, Grande P, Zannis VI, Tybjaerg-Hansen A (2011)Mutation in APOA1 predicts increased risk of ischaemic heart disease and total mortalitywithout low HDL cholesterol levels. J Intern Med 270:136–146Hailman E, Albers JJ, Wolfbauer G, Tu AY, Wright SD (1996) Neutralization and transfer oflipopolysaccharide by phospholipid transfer protein. J Biol Chem 271:12172–12178Hammad SM, Stefansson S, Twal WO, Drake CJ, Fleming P, Remaley A, Brewer HB Jr, ArgravesWS (1999) Cubilin, the endocytic receptor for intrinsic factor-vitamin B(12) complex,mediates high-density lipoprotein holoparticle endocytosis. Proc Natl Acad Sci USA 96:10158–10163Harder CJ, McPherson R (2007) HDL remodeling by CETP and SR-BI. In: Fielding CJ (ed) High-density lipoproteins. From basic biology to clinical aspects. Wiley, Weinheim, pp 163–182Heideman CL, Hoff HF (1982) Lipoproteins containing apolipoprotein A-I extracted from humanaortas. Biochim Biophys Acta 711:431–444Hildebrand RB, Lammers B, Meurs I, Korporaal SJ, De Haan W, Zhao Y, Kruijt JK, Pratico D,Schimmel AW, Holleboom AG, Hoekstra M, Kuivenhoven JA, van Berkel TJ, Rensen PC, VanEck M (2010) Restoration of high-density lipoprotein levels by cholesteryl ester transferprotein expression in scavenger receptor class B type I (SR-BI) knockout mice does notnormalize pathologies associated with SR-BI deficiency. Arterioscler Thromb Vasc Biol 30:1439–1445Hirano K, Yamashita S, Kuga Y, Sakai N, Nozaki S, Kihara S, Arai T, Yanagi K, Takami S,Menju M et al (1995) Atherosclerotic disease in marked hyperalphalipoproteinemia. Com-bined reduction of cholesteryl ester transfer protein and hepatic triglyceride lipase. ArteriosclerThromb Vasc Biol 15:1849–1856Hirano K, Yamashita S, Nakajima N, Arai T, Maruyama T, Yoshida Y, Ishigami M, Sakai N,Kameda-Takemura K, Matsuzawa Y (1997) Genetic cholesteryl ester transfer protein defi-ciency is extremely frequent in the Omagari area of Japan. Marked hyperalphalipoproteinemiacaused by CETP gene mutation is not associated with longevity. Arterioscler Thromb VascBiol 17:1053–1059Holleboom AG, Vergeer M, Hovingh GK, Kastelein JJ, Kuivenhoven JA (2008) The value of HDLgenetics. Curr Opin Lipidol 19:385–394Holm TM, Braun A, Trigatti BL, Brugnara C, Sakamoto M, Krieger M, Andrews NC (2002)Failure of red blood cell maturation in mice with defects in the high-density lipoproteinreceptor SR-BI. Blood 99:1817–1824Homanics GE, de Silva HV, Osada J, Zhang SH, Wong H, Borensztajn J, Maeda N (1995) Milddyslipidemia in mice following targeted inactivation of the hepatic lipase gene. J Biol Chem270:2974–2980Hopkins GJ, Barter PJ (1980) Transfers of esterified cholesterol and triglyceride between highdensity and very low density lipoproteins: in vitro studies of rabbits and humans. Metabolism29:546–550HDL Biogenesis, Remodeling, and Catabolism 97Hu YW, Zheng L, Wang Q (2010) Characteristics of apolipoprotein M and its relation toatherosclerosis and diabetes. Biochim Biophys Acta 1801:100–105Huang W, Sasaki J, Matsunaga A, Nanimatsu H, Moriyama K, Han H, Kugi M, Koga T,Yamaguchi K, Arakawa K (1998) A novel homozygous missense mutation in the apo A-Igene with apo A-I deficiency. Arterioscler Thromb Vasc Biol 18:389–396Huang R, Silva RA, JeromeWG, Kontush A, Chapman MJ, Curtiss LK, Hodges TJ, Davidson WS(2011) Apolipoprotein A-I structural organization in high-density lipoproteins isolated fromhuman plasma. Nat Struct Mol Biol 18:416–422Huszar D, VarbanML, Rinninger F, Feeley R, Arai T, Fairchild-Huntress V, DonovanMJ, Tall AR(2000) Increased LDL cholesterol and atherosclerosis in LDL receptor-deficient mice withattenuated expression of scavenger receptor B1. Arterioscler Thromb Vasc Biol 20:1068–1073Huuskonen J, Olkkonen VM, Ehnholm C, Metso J, Julkunen I, Jauhiainen M (2000) Phospholipidtransfer is a prerequisite for PLTP-mediated HDL conversion. Biochemistry 39:16092–16098Huuskonen J, Olkkonen VM, Jauhiainen M, Ehnholm C (2001) The impact of phospholipidtransfer protein (PLTP) on HDL metabolism. Atherosclerosis 155:269–281Inazu A, Brown ML, Hesler CB, Agellon LB, Koizumi J, Takata K, Maruhama Y, Mabuchi H,Tall AR (1990) Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation. N Engl J Med 323:1234–1238Ishida T, Choi S, Kundu RK, Hirata K, Rubin EM, Cooper AD, Quertermous T (2003) Endotheliallipase is a major determinant of HDL level. J Clin Invest 111:347–355Ishida T, Choi SY, Kundu RK, Spin J, Yamashita T, Hirata K, Kojima Y, Yokoyama M,Cooper AD, Quertermous T (2004) Endothelial lipase modulates susceptibility to athero-sclerosis in apolipoprotein-E-deficient mice. J Biol Chem 279:45085–45092Jacquet S, Malaval C, Martinez LO, Sak K, Rolland C, Perez C, Nauze M, Champagne E, Terce F,Gachet C, Perret B, Collet X, Boeynaems JM, Barbaras R (2005) The nucleotide receptorP2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis. Cell Mol LifeSci 62:2508–2515Janis MT, Metso J, Lankinen H, Strandin T, Olkkonen VM, Rye KA, Jauhiainen M, Ehnholm C(2005)Apolipoprotein E activates the low-activity form of human phospholipid transfer protein.Biochem Biophys Res Commun 331:333–340Jaye M, Lynch KJ, Krawiec J, Marchadier D, Maugeais C, Doan K, South V, Amin D, Perrone M,Rader DJ (1999) A novel endothelial-derived lipase that modulates HDL metabolism.Nat Genet 21:424–428Ji Y, Jian B, Wang N, Sun Y, Moya ML, Phillips MC, Rothblat GH, Swaney JB, Tall AR (1997)Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux.J Biol Chem 272:20982–20985Ji Y, Wang N, Ramakrishnan R, Sehayek E, Huszar D, Breslow JL, Tall AR (1999) Hepaticscavenger receptor BI promotes rapid clearance of high density lipoprotein free cholesterol andits transport into bile. J Biol Chem 274:33398–33402Jiang XC, Bruce C, Mar J, Lin M, Ji Y, Francone OL, Tall AR (1999) Targeted mutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levels. J ClinInvest 103:907–914Jiang XC, Qin S, Qiao C, Kawano K, Lin M, Skold A, Xiao X, Tall AR (2001) Apolipoprotein Bsecretion and atherosclerosis are decreased in mice with phospholipid-transfer protein defi-ciency. Nat Med 7:847–852Jiao GQ, Yuan ZX, Xue YS, Yang CJ, Lu CB, Lu ZQ, Xiao MD (2007) A prospective evaluationof apolipoprotein M gene T-778C polymorphism in relation to coronary artery disease inHan Chinese. Clin Biochem 40:1108–1112Johannsen TH, Kamstrup PR, Andersen RV, Jensen GB, Sillesen H, Tybjaerg-Hansen A,Nordestgaard BG (2009) Hepatic lipase, genetically elevated high-density lipoprotein, andrisk of ischemic cardiovascular disease. J Clin Endocrinol Metab 94:1264–1273Jonas A (2000) Lecithin cholesterol acyltransferase. Biochim Biophys Acta 1529:245–25698 V.I. Zannis et al.Jones MK, Catte A, Li L, Segrest JP (2009) Dynamics of activation of lecithin:cholesterolacyltransferase by apolipoprotein A-I. Biochemistry 48:11196–11210Kan CC, Yan J, Bittman R (1992) Rates of spontaneous exchange of synthetic radiolabeled sterolsbetween lipid vesicles. Biochemistry 31:1866–1874Karackattu SL, Picard MH, Krieger M (2005) Lymphocytes are not required for the rapid onset ofcoronary heart disease in scavenger receptor class B type I/apolipoprotein E double knockoutmice. Arterioscler Thromb Vasc Biol 25:803–808Karackattu SL, Trigatti B, Krieger M (2006) Hepatic lipase deficiency delays atherosclerosis,myocardial infarction, and cardiac dysfunction and extends lifespan in SR-BI/apolipoprotein Edouble knockout mice. Arterioscler Thromb Vasc Biol 26:548–554Karkkainen M, Oka T, Olkkonen VM, Metso J, Hattori H, Jauhiainen M, Ehnholm C (2002)Isolation and partial characterization of the inactive and active forms of human plasmaphospholipid transfer protein (PLTP). J Biol Chem 277:15413–15418Kateifides AK, Gorshkova IN, Duka A, Chroni A, Kardassis D, Zannis VI (2011) Alteration ofnegatively charged residues in the 89 to 99 domain of apoA-I affects lipid homeostasis andmaturation of HDL. J Lipid Res 52:1363–1372Kathiresan S, Melander O, Guiducci C, Surti A, Burtt NP, Rieder MJ, Cooper GM, Roos C,Voight BF, Havulinna AS, Wahlstrand B, Hedner T, Corella D, Tai ES, Ordovas JM,Berglund G, Vartiainen E, Jousilahti P, Hedblad B, Taskinen MR, Newton-Cheh C,Salomaa V, Peltonen L, Groop L, Altshuler DM, Orho-Melander M (2008a) Six new lociassociated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterolor triglycerides in humans. Nat Genet 40:189–197Kathiresan S, Musunuru K, Orho-Melander M (2008b) Defining the spectrum of alleles thatcontribute to blood lipid concentrations in humans. Curr Opin Lipidol 19:122–127Kathiresan S, Willer CJ, Peloso GM, Demissie S, Musunuru K, Schadt EE, Kaplan L, Bennett D,Li Y, Tanaka T, Voight BF, Bonnycastle LL, Jackson AU, Crawford G, Surti A, Guiducci C,Burtt NP, Parish S, Clarke R, Zelenika D, Kubalanza KA, Morken MA, Scott LJ, StringhamHM, Galan P, Swift AJ, Kuusisto J, Bergman RN, Sundvall J, Laakso M, Ferrucci L, Scheet P,Sanna S, Uda M, Yang Q, Lunetta KL, Dupuis J, de Bakker PI, O’Donnell CJ, Chambers JC,Kooner JS, Hercberg S, Meneton P, Lakatta EG, Scuteri A, Schlessinger D, Tuomilehto J,Collins FS, Groop L, Altshuler D, Collins R, Lathrop GM, Melander O, Salomaa V,Peltonen L, Orho-Melander M, Ordovas JM, Boehnke M, Abecasis GR, Mohlke KL,Cupples LA (2009) Common variants at 30 loci contribute to polygenic dyslipidemia.Nat Genet 41:56–65Kawano K, Qin SC, Lin M, Tall AR, Jiang XC (2000) Cholesteryl ester transfer protein andphospholipid transfer protein have nonoverlapping functions in vivo. J Biol Chem 275:29477–29481Kee P, Rye KA, Taylor JL, Barrett PH, Barter PJ (2002) Metabolism of apoA-I as lipid-free proteinor as component of discoidal and spherical reconstituted HDLs: studies in wild-type andhepatic lipase transgenic rabbits. Arterioscler Thromb Vasc Biol 22:1912–1917Kennedy MA, Barrera GC, Nakamura K, Baldan A, Tarr P, Fishbein MC, Frank J, Francone OL,Edwards PA (2005) ABCG1 has a critical role in mediating cholesterol efflux to HDL andpreventing cellular lipid accumulation. Cell Metab 1:121–131Kielar D, Dietmaier W, Langmann T, Aslanidis C, Probst M, Naruszewicz M, Schmitz G (2001)Rapid quantification of human ABCA1 mRNA in various cell types and tissues by real-timereverse transcription-PCR. Clin Chem 47:2089–2097Kivela AM, Dijkstra MH, Heinonen SE, Gurzeler E, Jauhiainen S, Levonen AL, Yla-Herttuala S(2012) Regulation of endothelial lipase and systemic HDL cholesterol levels by SREBPs andVEGF-A. Atherosclerosis 225:335–340Klucken J, Buchler C, Orso E, Kaminski WE, Porsch-Ozcurumez M, Liebisch G, Kapinsky M,Diederich W, Drobnik W, Dean M, Allikmets R, Schmitz G (2000) ABCG1 (ABC8), thehuman homolog of the Drosophila white gene, is a regulator of macrophage cholesterol andphospholipid transport. Proc Natl Acad Sci USA 97:817–822HDL Biogenesis, Remodeling, and Catabolism 99Kocher O, Yesilaltay A, Shen CH, Zhang S, Daniels K, Pal R, Chen J, Krieger M (2008) Influenceof PDZK1 on lipoprotein metabolism and atherosclerosis. Biochim Biophys Acta 1782:310–316Koizumi J, Mabuchi H, Yoshimura A, Michishita I, Takeda M, Itoh H, Sakai Y, Sakai T, Ueda K,Takeda R (1985) Deficiency of serum cholesteryl-ester transfer activity in patients withfamilial hyperalphalipoproteinaemia. Atherosclerosis 58:175–186Kostner GM, Oettl K, Jauhiainen M, Ehnholm C, Esterbauer H, Dieplinger H (1995) Humanplasma phospholipid transfer protein accelerates exchange/transfer of alpha-tocopherolbetween lipoproteins and cells. Biochem J 305(Pt 2):659–667Koukos G, Chroni A, Duka A, Kardassis D, Zannis VI (2007a) LCAT can rescue the abnormalphenotype produced by the natural ApoA-I mutations (Leu141Arg)Pisa and (Leu159Arg)FIN.Biochemistry 46:10713–10721Koukos G, Chroni A, Duka A, Kardassis D, Zannis VI (2007b) Naturally occurring andbioengineered apoA-I mutations that inhibit the conversion of discoidal to spherical HDL:the abnormal HDL phenotypes can be corrected by treatment with LCAT. Biochem J 406:167–174Kozarsky KF, Donahee MH, Glick JM, Krieger M, Rader DJ (2000) Gene transfer and hepaticoverexpression of the HDL receptor SR-BI reduces atherosclerosis in the cholesterol-fed LDLreceptor-deficient mouse. Arterioscler Thromb Vasc Biol 20:721–727Kozyraki R (2001) Cubilin, a multifunctional epithelial receptor: an overview. J Mol Med (Berl)79:161–167Kozyraki R, Fyfe J, Kristiansen M, Gerdes C, Jacobsen C, Cui S, Christensen EI, Aminoff M, de laChapelle A, Krahe R, Verroust PJ, Moestrup SK (1999) The intrinsic factor-vitamin B12receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein. Nat Med 5:656–661Krauss RM, Levy RI, Fredrickson DS (1974) Selective measurement of two lipase activities inpostheparin plasma from normal subjects and patients with hyperlipoproteinemia. J Clin Invest54:1107–1124Krieger M (1999) Charting the fate of the “good cholesterol”: identification and characterization ofthe high-density lipoprotein receptor SR-BI. Annu Rev Biochem 68:523–558Krieger M (2001) Scavenger receptor class B type I is a multiligand HDL receptor that influencesdiverse physiologic systems. J Clin Invest 108:793–797Krimbou L, Hajj HH, Blain S, Rashid S, Denis M, Marcil M, Genest J (2005) Biogenesis andspeciation of nascent apoA-I-containing particles in various cell lines. J Lipid Res 46:1668–1677Kypreos KE, Zannis VI (2007) Pathway of biogenesis of apolipoprotein E-containing HDL in vivowith the participation of ABCA1 and LCAT. Biochem J 403:359–367Landmesser U, von Eckardstein A, Kastelein J, Deanfield J, Luscher TF (2012) Increasing high-density lipoprotein cholesterol by cholesteryl ester transfer protein-inhibition: a rocky road andlessons learned? The early demise of the dal-HEART programme. Eur Heart J 33:1712–1715Landschulz KT, Pathak RK, Rigotti A, Krieger M, Hobbs HH (1996) Regulation of scavengerreceptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissuesof the rat. J Clin Invest 98:984–995Langmann T, Klucken J, Reil M, Liebisch G, Luciani MF, Chimini G, Kaminski WE, Schmitz G(1999) Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidencefor sterol-dependent regulation in macrophages. Biochem Biophys Res Commun 257:29–33Laurila PP, Surakka I, Sarin AP, Yetukuri L, Hyotylainen T, Soderlund S, Naukkarinen J, Tang J,Kettunen J, Mirel DB, Soronen J, Lehtimaki T, Ruokonen A, Ehnholm C, Eriksson JG,Salomaa V, Jula A, Raitakari OT, Jarvelin MR, Palotie A, Peltonen L, Oresic M,Jauhiainen M, Taskinen MR, Ripatti S (2013) Genomic, transcriptomic, and lipidomicprofiling highlights the role of inflammation in individuals with low high-density lipoproteincholesterol. Arterioscler Thromb Vasc Biol 33:847–857100 V.I. Zannis et al.Leiva A, Verdejo H, Benitez ML, Martinez A, Busso D, Rigotti A (2011) Mechanisms regulatinghepatic SR-BI expression and their impact on HDL metabolism. Atherosclerosis 217:299–307Leren TP, Bakken KS, Daum U, Ose L, Berg K, Assmann G, von Eckardstein A (1997)Heterozygosity for apolipoprotein A-I(R160L)Oslo is associated with low levels of highdensity lipoprotein cholesterol and HDL-subclass LpA-I/A- II but normal levels ofHDL-subclass LpA-I. J Lipid Res 38:121–131Levels JH, Marquart JA, Abraham PR, van den Ende AE, Molhuizen HO, van Deventer SJ,Meijers JC (2005) Lipopolysaccharide is transferred from high-density to low-densitylipoproteins by lipopolysaccharide-binding protein and phospholipid transfer protein.Infect Immun 73:2321–2326Li XA, Titlow WB, Jackson BA, Giltiay N, Nikolova-Karakashian M, Uittenbogaard A, Smart EJ(2002) High density lipoprotein binding to scavenger receptor, Class B, type I activatesendothelial nitric-oxide synthase in a ceramide-dependent manner. J Biol Chem 277:11058–11063LiX,Kypreos K, Zanni EE, ZannisV (2003)Domains of apoE required for binding to apoE receptor2 and to phospholipids: implications for the functions of apoE in the brain. Biochemistry42:10406–10417Li D, Zhang Y, Ma J, Ling W, Xia M (2010) Adenosine monophosphate activated protein kinaseregulates ABCG1-mediated oxysterol efflux from endothelial cells and protects againsthypercholesterolemia-induced endothelial dysfunction. Arterioscler Thromb Vasc Biol 30:1354–1362Liadaki KN, Liu T, Xu S, Ishida BY, Duchateaux PN, Krieger JP, Kane J, Krieger M, Zannis VI(2000) Binding of high density lipoprotein (HDL) and discoidal reconstituted HDL to the HDLreceptor scavenger receptor class B type I. Effect of lipid association and APOA-I mutations onreceptor binding. J Biol Chem 275:21262–21271Lindstedt L, Saarinen J, Kalkkinen N, Welgus H, Kovanen PT (1999) Matrix metalloproteinases-3, -7, and -12, but not -9, reduce high density lipoprotein-induced cholesterol efflux fromhuman macrophage foam cells by truncation of the carboxyl terminus of apolipoprotein A-I.Parallel losses of pre-beta particles and the high affinity component of efflux. J Biol Chem274:22627–22634Liu T, Krieger M, Kan HY, Zannis VI (2002) The effects of mutations in helices 4 and 6 of apoA-Ion scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux suggest that forma-tion of a productive complex between reconstituted high density lipoprotein and SR-BI isrequired for efficient lipid transport. J Biol Chem 277:21576–21584Lorenzi I, von Eckardstein A, Cavelier C, Radosavljevic S, Rohrer L (2008) Apolipoprotein A-Ibut not high-density lipoproteins are internalised by RAWmacrophages: roles of ATP-bindingcassette transporter A1 and scavenger receptor BI. J Mol Med 86:171–183Love-Gregory L, Sherva R, Sun L, Wasson J, Schappe T, Doria A, Rao DC, Hunt SC, Klein S,Neuman RJ, Permutt MA, Abumrad NA (2008) Variants in the CD36 gene associate with themetabolic syndrome and high-density lipoprotein cholesterol. Hum Mol Genet 17:1695–1704Lusa S, Jauhiainen M, Metso J, Somerharju P, Ehnholm C (1996) The mechanism of humanplasma phospholipid transfer protein-induced enlargement of high-density lipoproteinparticles: evidence for particle fusion. Biochem J 313(Pt 1):275–282Ma K, Cilingiroglu M, Otvos JD, Ballantyne CM, Marian AJ, Chan L (2003) Endothelial lipase isa major genetic determinant for high-density lipoprotein concentration, structure, and meta-bolism. Proc Natl Acad Sci USA 100:2748–2753Mardones P, Quinones V, Amigo L, Moreno M, Miquel JF, Schwarz M, Miettinen HE, Trigatti B,Krieger M, VanPatten S, Cohen DE, Rigotti A (2001) Hepatic cholesterol and bile acidmetabolism and intestinal cholesterol absorption in scavenger receptor class B typeI-deficient mice. J Lipid Res 42:170–180Marsh JB, Welty FK, Schaefer EJ (2000) Stable isotope turnover of apolipoproteins of high-density lipoproteins in humans. Curr Opin Lipidol 11:261–266HDL Biogenesis, Remodeling, and Catabolism 101Martinez LO, Jacquet S, Esteve JP, Rolland C, Cabezon E, Champagne E, Pineau T, Georgeaud V,Walker JE, Terce F, Collet X, Perret B, Barbaras R (2003) Ectopic beta-chain of ATP synthaseis an apolipoprotein A-I receptor in hepatic HDL endocytosis. Nature 421:75–79Martinez LO, Perretv B, Barbaras R, Terce F, Collet X (2007) Hepatic and renal HDL receptors.In: Fielding CJ (ed) High-density lipoproteins. From basic biology to clinical aspects. Wiley,Weinheim, pp 307–338Maruyama T, Sakai N, Ishigami M, Hirano K, Arai T, Okada S, Okuda E, Ohya A, Nakajima N,Kadowaki K, Fushimi E, Yamashita S, Matsuzawa Y (2003) Prevalence and phenotypicspectrum of cholesteryl ester transfer protein gene mutations in Japanese hyperalphalipopro-teinemia. Atherosclerosis 166:177–185Matsuura F, Wang N, Chen W, Jiang XC, Tall AR (2006) HDL from CETP-deficient subjectsshows enhanced ability to promote cholesterol efflux from macrophages in an apoE- andABCG1-dependent pathway. J Clin Invest 116:1435–1442Maugeais C, Tietge UJ, Broedl UC, Marchadier D, Cain W, McCoy MG, Lund-Katz S, Glick JM,Rader DJ (2003) Dose-dependent acceleration of high-density lipoprotein catabolism byendothelial lipase. Circulation 108:2121–2126McManus DC, Scott BR, Franklin V, Sparks DL, Marcel YL (2001) Proteolytic degradation andimpaired secretion of an apolipoprotein A-I mutant associated with dominantly inheritedhypoalphalipoproteinemia. J Biol Chem 276:21292–21302McNeish J, Aiello RJ, Guyot D, Turi T, Gabel C, Aldinger C, Hoppe KL, Roach ML, Royer LJ, deWet J, Broccardo C, Chimini G, Francone OL (2000) High density lipoprotein deficiency andfoam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1.Proc Natl Acad Sci USA 97:4245–4250Melchior GW, Castle CK, Murray RW, Blake WL, Dinh DM, Marotti KR (1994) ApolipoproteinA-I metabolism in cholesteryl ester transfer protein transgenic mice. Insights into themechanisms responsible for low plasma high density lipoprotein levels. J Biol Chem 269:8044–8051Meyer JM, Graf GA, van der Westhuyzen DR (2013) New developments in selective cholesterylester uptake. Curr Opin Lipidol 24:386–392Mezdour H, Jones R, Dengremont C, Castro G, Maeda N (1997) Hepatic lipase deficiencyincreases plasma cholesterol but reduces susceptibility to atherosclerosis in apolipoproteinE-deficient mice. J Biol Chem 272:13570–13575Miccoli R, Bertolotto A, Navalesi R, Odoguardi L, Boni A, Wessling J, Funke H, Wiebusch H,Eckardstein A, Assmann G (1996) Compound heterozygosity for a structural apolipoproteinA-I variant, apo A-I(L141R)Pisa, and an apolipoprotein A-I null allele in patients with absenceof HDL cholesterol, corneal opacifications, and coronary heart disease. Circulation 94:1622–1628Miccoli R, Zhu Y, Daum U, Wessling J, Huang Y, Navalesi R, Assmann G, von Eckardstein A(1997) A natural apolipoprotein A-I variant, apoA-I (L141R)Pisa, interferes with the formationof alpha-high density lipoproteins (HDL) but not with the formation of pre beta 1-HDL andinfluences efflux of cholesterol into plasma. J Lipid Res 38:1242Miettinen HE, Gylling H, Miettinen TA, Viikari J, Paulin L, Kontula K (1997a) ApolipoproteinA-IFin. Dominantly inherited hypoalphalipoproteinemia due to a single base substitution in theapolipoprotein A-I gene. Arterioscler Thromb Vasc Biol 17:83–90Miettinen HE, Jauhiainen M, Gylling H, Ehnholm S, Palomaki A, Miettinen TA, Kontula K(1997b) Apolipoprotein A-IFIN (Leu159–>Arg) mutation affects lecithin cholesterolacyltransferase activation and subclass distribution of HDL but not cholesterol efflux fromfibroblasts. Arterioscler Thromb Vasc Biol 17:3021–3032Miller M, Aiello D, Pritchard H, Friel G, Zeller K (1998) Apolipoprotein A-I(Zavalla) (Leu159–> Pro): HDL cholesterol deficiency in a kindred associated with premature coronary arterydisease. Arterioscler Thromb Vasc Biol 18:1242–1247102 V.I. Zannis et al.Mineo C, Yuhanna IS, Quon MJ, Shaul PW (2003) High density lipoprotein-induced endothelialnitric-oxide synthase activation is mediated by Akt and MAP kinases. J Biol Chem 278:9142–9149Moestrup SK, Nielsen LB (2005) The role of the kidney in lipid metabolism. Curr Opin Lipidol 16:301–306Mulya A, Seo J, Brown AL, Gebre AK, Boudyguina E, Shelness GS, Parks JS (2010) Apolipo-protein M expression increases the size of nascent pre beta HDL formed by ATP bindingcassette transporter A1. J Lipid Res 51:514–524Murao K, Terpstra V, Green SR, Kondratenko N, Steinberg D, Quehenberger O (1997) Character-ization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for highdensity lipoprotein and apoptotic thymocytes. J Biol Chem 272:17551–17557Nakamura K, Kennedy MA, Baldan A, Bojanic DD, Lyons K, Edwards PA (2004a) Expressionand regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms thatstimulate cellular cholesterol efflux to high density lipoprotein. J Biol Chem 279:45980–45989Nakamura Y, Kotite L, Gan Y, Spencer TA, Fielding CJ, Fielding PE (2004b) Molecularmechanism of reverse cholesterol transport: reaction of pre-beta-migrating high-density lipo-protein with plasma lecithin/cholesterol acyltransferase. Biochemistry 43:14811–14820Nanjee MN, Cooke CJ, Olszewski WL, Miller NE (2000) Concentrations of electrophoretic andsize subclasses of apolipoprotein A-I-containing particles in human peripheral lymph.Arterioscler Thromb Vasc Biol 20:2148–2155Navab M, Hama SY, Anantharamaiah GM, Hassan K, Hough GP, Watson AD, Reddy ST,Sevanian A, Fonarow GC, Fogelman AM (2000) Normal high density lipoprotein inhibitsthree steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3. J LipidRes 41:1495–1508Neufeld EB, Remaley AT, Demosky SJ, Stonik JA, Cooney AM, Comly M, Dwyer NK, Zhang M,Blanchette-Mackie J, Santamarina-Fojo S, Brewer HB Jr (2001) Cellular localization andtrafficking of the human ABCA1 transporter. J Biol Chem 276:27584–27590Neufeld EB, Demosky SJ Jr, Stonik JA, Combs C, Remaley AT, Duverger N, Santamarina-Fojo S,Brewer HB Jr (2002) The ABCA1 transporter functions on the basolateral surface ofhepatocytes. Biochem Biophys Res Commun 297:974–979Nichols AV, Krauss RM, Musliner TA (1986) Nondenaturing polyacrylamide gradient gel electro-phoresis. Methods Enzymol 128:417–431Nielsen LB, Christoffersen C, Ahnstrom J, Dahlback B (2009) ApoM: gene regulation and effectson HDL metabolism. Trends Endocrinol Metab 20:66–71Nijstad N, Wiersma H, Gautier T, van der Giet M, Maugeais C, Tietge UJ (2009) Scavengerreceptor BI-mediated selective uptake is required for the remodeling of high density lipopro-tein by endothelial lipase. J Biol Chem 284:6093–6100Nishida HI, Nishida T (1997) Phospholipid transfer protein mediates transfer of not onlyphosphatidylcholine but also cholesterol from phosphatidylcholine-cholesterol vesicles tohigh density lipoproteins. J Biol Chem 272:6959–6964Nolte RT, Atkinson D (1992) Conformational analysis of apolipoprotein A-I and E-3 based onprimary sequence and circular dichroism. Biophys J 63:1221–1239Ohnsorg PM, Rohrer L, Perisa D, Kateifides A, Chroni A, Kardassis D, Zannis VI, von EckardsteinA (2011) Carboxyl terminus of apolipoprotein A-I (ApoA-I) is necessary for the transport oflipid-free ApoA-I but not prelipidated ApoA-I particles through aortic endothelial cells. J BiolChem 286:7744–7754Oka T, Kujiraoka T, Ito M, Egashira T, Takahashi S, Nanjee MN, Miller NE, Metso J, OlkkonenVM, Ehnholm C, Jauhiainen M, Hattori H (2000) Distribution of phospholipid transfer proteinin human plasma: presence of two forms of phospholipid transfer protein, one catalyticallyactive and the other inactive. J Lipid Res 41:1651–1657Orso E, Broccardo C, Kaminski WE, Bottcher A, Liebisch G, Drobnik W, Gotz A, Chambenoit O,Diederich W, Langmann T, Spruss T, Luciani MF, Rothe G, Lackner KJ, Chimini G,HDL Biogenesis, Remodeling, and Catabolism 103Schmitz G (2000) Transport of lipids from golgi to plasma membrane is defective in tangierdisease patients and Abc1-deficient mice. Nat Genet 24:192–196Out R, Hoekstra M, Spijkers JA, Kruijt JK, Van Eck M, Bos IS, Twisk J, van Berkel TJ (2004a)Scavenger receptor class B type I is solely responsible for the selective uptake of cholesterylesters from HDL by the liver and the adrenals in mice. J Lipid Res 45:2088–2095Out R, Kruijt JK, Rensen PC, Hildebrand RB, De Vos P, Van Eck M, van Berkel TJ (2004b)Scavenger receptor BI plays a role in facilitating chylomicron metabolism. J Biol Chem 279:18401–18406Out R, Hoekstra M, de Jager SC, Vos PD, van der Westhuyzen DR, Webb NR, Van Eck M,Biessen EA, van Berkel TJ (2005) Adenovirus-mediated hepatic overexpression of scavengerreceptor BI accelerates chylomicron metabolism in C57BL/6J mice. J Lipid Res 46:1172–1181Out R, Hoekstra M, Hildebrand RB, Kruit JK, Meurs I, Li Z, Kuipers F, van Berkel TJ, Van Eck M(2006) Macrophage ABCG1 deletion disrupts lipid homeostasis in alveolar macrophages andmoderately influences atherosclerotic lesion development in LDL receptor-deficient mice.Arterioscler Thromb Vasc Biol 26:2295–2300Out R, Hoekstra M, Meurs I, de Vos P, Kuiper J, Van Eck M, van Berkel TJ (2007) Total bodyABCG1 expression protects against early atherosclerotic lesion development in mice.Arterioscler Thromb Vasc Biol 27:594–599Pagler TA, Rhode S, Neuhofer A, Laggner H, Strobl W, Hinterndorfer C, Volf I, Pavelka M,Eckhardt ER, van der Westhuyzen DR, Schutz GJ, Stangl H (2006) SR-BI-mediated highdensity lipoprotein (HDL) endocytosis leads to HDL resecretion facilitating cholesterol efflux.J Biol Chem 281:11193–11204Parathath S, Sahoo D, Darlington YF, Peng Y, Collins HL, Rothblat GH, Williams DL,Connelly MA (2004) Glycine 420 near the C-terminal transmembrane domain of SR-BI iscritical for proper delivery and metabolism of high density lipoprotein cholesteryl ester. J BiolChem 279:24976–24985Paszty C, Maeda N, Verstuyft J, Rubin EM (1994) Apolipoprotein AI transgene corrects apolipo-protein E deficiency-induced atherosclerosis in mice. J Clin Invest 94:899–903Pisciotta L, Miccoli R, Cantafora A, Calabresi L, Tarugi P, Alessandrini P, Bittolo BG,Franceschini G, Cortese C, Calandra S, Bertolini S (2003) Recurrent mutations of the apolipo-protein A-I gene in three kindreds with severe HDL deficiency. Atherosclerosis 167:335–345Plomgaard P, Dullaart RP, de Vries R, Groen AK, Dahlback B, Nielsen LB (2009) ApolipoproteinM predicts pre-beta-HDL formation: studies in type 2 diabetic and nondiabetic subjects. JIntern Med 266:258–267Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL(1992) Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient micecreated by homologous recombination in ES cells. Cell 71:343–353Plump AS, Masucci-Magoulas L, Bruce C, Bisgaier CL, Breslow JL, Tall AR (1999) Increasedatherosclerosis in ApoE and LDL receptor gene knock-out mice as a result of humancholesteryl ester transfer protein transgene expression. Arterioscler Thromb Vasc Biol 19:1105–1110Ranalletta M, Wang N, Han S, Yvan-Charvet L, Welch C, Tall AR (2006) Decreased atheroscle-rosis in low-density lipoprotein receptor knockout mice transplanted with Abcg1/ bonemarrow. Arterioscler Thromb Vasc Biol 26:2308–2315Rao R, Albers JJ, Wolfbauer G, Pownall HJ (1997) Molecular and macromolecular specificity ofhuman plasma phospholipid transfer protein. Biochemistry 36:3645–3653Reardon CA, Kan HY, Cabana V, Blachowicz L, Lukens JR, Wu Q, Liadaki K, Getz GS,Zannis VI (2001) In vivo studies of HDL assembly and metabolism using adenovirus-mediatedtransfer of ApoA-I mutants in ApoA-I-deficient mice. Biochemistry 40:13670–13680Remaley AT, Stonik JA, Demosky SJ, Neufeld EB, Bocharov AV, Vishnyakova TG,Eggerman TL, Patterson AP, Duverger NJ, Santamarina-Fojo S, Brewer HB Jr (2001) Apolipo-protein specificity for lipid efflux by the human ABCAI transporter. Biochem Biophys ResCommun 280:818–823104 V.I. Zannis et al.Richards JB, Waterworth D, O’Rahilly S, Hivert MF, Loos RJ, Perry JR, Tanaka T, Timpson NJ,Semple RK, Soranzo N, Song K, Rocha N, Grundberg E, Dupuis J, Florez JC, Langenberg C,Prokopenko I, Saxena R, Sladek R, Aulchenko Y, Evans D, Waeber G, Erdmann J,Burnett MS, Sattar N, Devaney J, Willenborg C, Hingorani A, Witteman JC,Vollenweider P, Glaser B, Hengstenberg C, Ferrucci L, Melzer D, Stark K, Deanfield J,Winogradow J, Grassl M, Hall AS, Egan JM, Thompson JR, Ricketts SL, Konig IR,Reinhard W, Grundy S, Wichmann HE, Barter P, Mahley R, Kesaniemi YA, Rader DJ,Reilly MP, Epstein SE, Stewart AF, van Duijn CM, Schunkert H, Burling K, Deloukas P,Pastinen T, Samani NJ, McPherson R, Davey SG, Frayling TM, Wareham NJ, Meigs JB,Mooser V, Spector TD (2009) A genome-wide association study reveals variants in ARL15that influence adiponectin levels. PLoS Genet 5:e1000768Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M (1997) A targeted mutation inthe murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor classB type I reveals its key role in HDL metabolism. Proc Natl Acad Sci USA 94:12610–12615Rohrer L, Hersberger M, von Eckardstein A (2004) High density lipoproteins in the intersection ofdiabetes mellitus, inflammation and cardiovascular disease. Curr Opin Lipidol 15:269–278Rohrer L, Cavelier C, Fuchs S, Schluter MA, Volker W, von Eckardstein A (2006) Binding,internalization and transport of apolipoprotein A-I by vascular endothelial cells. BiochimBiophys Acta 1761:186–194Rohrer L, Ohnsorg PM, Lehner M, Landolt F, Rinninger F, von Eckardstein A (2009) High-densitylipoprotein transport through aortic endothelial cells involves scavenger receptor BI andATP-binding cassette transporter G1. Circ Res 104:1142–1150Rohrl C, Stangl H (2013) HDL endocytosis and resecretion. Biochim Biophys Acta 1831:1626–1633Roosbeek S, Vanloo B, Duverger N, Caster H, Breyne J, De Beun I, Patel H, Vandekerckhove J,Shoulders C, Rosseneu M, Peelman F (2001) Three arginine residues in apolipoprotein A-I arecritical for activation of lecithin:cholesterol acyltransferase. J Lipid Res 42:31–40Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM (1991) Inhibition of early athero-genesis in transgenic mice by human apolipoprotein AI. Nature 353:265–267Ruel IL, Couture P, Cohn JS, Bensadoun A, Marcil M, Lamarche B (2004) Evidence that hepaticlipase deficiency in humans is not associated with proatherogenic changes in HDL compositionand metabolism. J Lipid Res 45:1528–1537Russell RE, Culpitt SV, DeMatos C, Donnelly L, Smith M, Wiggins J, Barnes PJ (2002) Releaseand activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 byalveolar macrophages from patients with chronic obstructive pulmonary disease. Am J RespirCell Mol Biol 26:602–609Rye KA, Jauhiainen M, Barter PJ, Ehnholm C (1998) Triglyceride-enrichment of high densitylipoproteins enhances their remodelling by phospholipid transfer protein. J Lipid Res 39:613–622Sabatti C, Service SK, Hartikainen AL, Pouta A, Ripatti S, Brodsky J, Jones CG, Zaitlen NA,Varilo T, Kaakinen M, Sovio U, Ruokonen A, Laitinen J, Jakkula E, Coin L, Hoggart C,Collins A, Turunen H, Gabriel S, Elliot P, McCarthy MI, Daly MJ, Jarvelin MR, Freimer NB,Peltonen L (2009) Genome-wide association analysis of metabolic traits in a birth cohort froma founder population. Nat Genet 41:35–46Sankaranarayanan S, Oram JF, Asztalos BF, Vaughan AM, Lund-Katz S, Adorni MP, Phillips MC,Rothblat GH (2009) Effects of acceptor composition and mechanism of ABCG1-mediatedcellular free cholesterol efflux. J Lipid Res 50:275–284Santamarina-Fojo S, Hoeg JM, Assmann G, Brewer HB Jr (2001) Lecithin cholesterolacyltransferase deficiency and fish eye disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D(eds) The metabolic and molecular bases of inherited disease. McGraw-Hill, New York,pp 2817–2834Santamarina-Fojo S, Gonzalez-Navarro H, Freeman L, Wagner E, Nong Z (2004) Hepatic lipase,lipoprotein metabolism, and atherogenesis. Arterioscler Thromb Vasc Biol 24:1750–1754HDL Biogenesis, Remodeling, and Catabolism 105Savary S, Denizot F, Luciani M, Mattei M, Chimini G (1996) Molecular cloning of a mammalianABC transporter homologous to Drosophila white gene. Mamm Genome 7:673–676Schaefer EJ, Foster DM, Jenkins LL, Lindgren FT, Berman M, Levy RI, Brewer HB Jr (1979) Thecomposition and metabolism of high density lipoprotein subfractions. Lipids 14:511–522Schaefer EJ, Anderson DW, Zech LA, Lindgren FT, Bronzert TB, Rubalcaba EA, Brewer HB Jr(1981) Metabolism of high density lipoprotein subfractions and constituents in Tangier diseasefollowing the infusion of high density lipoproteins. J Lipid Res 22:217–228Schaefer EJ, Gregg RE, Ghiselli G, Forte TM, Ordovas JM, Zech LA, Brewer HB Jr (1986)Familial apolipoprotein E deficiency. J Clin Invest 78:1206–1219Schou J, Frikke-Schmidt R, Kardassis D, Thymiakou E, Nordestgaard BG, Jensen G, Grande P,Tybjaerg-Hansen A (2012) Genetic variation in ABCG1 and risk of myocardial infarction andischemic heart disease. Arterioscler Thromb Vasc Biol 32:506–515Segrest JP, Jackson RL, Morrisett JD, Gotto AM Jr (1974) A molecular theory of lipid-proteininteractions in the plasma lipoproteins. FEBS Lett 38:247–258Segrest JP, Jones MK, Klon AE, Sheldahl CJ, Hellinger M, De Loof H, Harvey SC (1999) Adetailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein.J Biol Chem 274:31755–31758Setala NL, Holopainen JM, Metso J, Wiedmer SK, Yohannes G, Kinnunen PK, Ehnholm C,Jauhiainen M (2007) Interfacial and lipid transfer properties of human phospholipid transferprotein: implications for the transfer mechanism of phospholipids. Biochemistry 46:1312–1319Shah AS, Tan L, Long JL, Davidson WS (2013) Proteomic diversity of high density lipoproteins:our emerging understanding of its importance in lipid transport and beyond. J Lipid Res 54:2575–2585Shao B, Cavigiolio G, Brot N, Oda MN, Heinecke JW (2008) Methionine oxidation impairsreverse cholesterol transport by apolipoprotein A-I. Proc Natl Acad Sci USA 105:12224–12229Shih DQ, Bussen M, Sehayek E, Ananthanarayanan M, Shneider BL, Suchy FJ, Shefer S,Bollileni JS, Gonzalez FJ, Breslow JL, Stoffel M (2001) Hepatocyte nuclear factor-1alpha isan essential regulator of bile acid and plasma cholesterol metabolism. Nat Genet 27:375–382Siggins S, Rye K-A, Olkkonen VM, Jauhiainen M, Ehnholm C (2007) Human plasma phospho-lipid transfer protein (PLTP) – structural and functional features. In: Fielding CJ (ed) High-density lipoproteins. From basic biology to clinical aspects. Wiley, Weinheim, pp 183–206Silva RA, Huang R, Morris J, Fang J, Gracheva EO, Ren G, Kontush A, Jerome WG, Rye KA,Davidson WS (2008) Structure of apolipoprotein A-I in spherical high density lipoproteins ofdifferent sizes. Proc Natl Acad Sci USA 105:12176–12181Simon JB, Boyer JL (1970) Production of lecithin: cholesterol acyltransferase by the isolatedperfused rat liver. Biochim Biophys Acta 218:549–551Singaraja RR, Brunham LR, Visscher H, Kastelein JJ, Hayden MR (2003) Efflux and athero-sclerosis: the clinical and biochemical impact of variations in the ABCA1 gene. ArteriosclerThromb Vasc Biol 23:1322–1332Singaraja RR, Stahmer B, Brundert M, Merkel M, Heeren J, Bissada N, Kang M, Timmins JM,Ramakrishnan R, Parks JS, Hayden MR, Rinninger F (2006) Hepatic ATP-binding cassettetransporter A1 is a key molecule in high-density lipoprotein cholesteryl ester metabolism inmice. Arterioscler Thromb Vasc Biol 26:1821–1827Singaraja RR, Sivapalaratnam S, Hovingh K, Dube MP, Castro-Perez J, Collins HL, Adelman SJ,Riwanto M, Manz J, Hubbard B, Tietjen I, Wong K, Mitnaul LJ, van Heek M, Lin L,Roddy TA, McEwen J, Dallinge-Thie G, van Vark-van der Zee L, Verwoert G, Winther M,van Duijn C, Hofman A, Trip MD, Marais AD, Asztalos B, Landmesser U, Sijbrands E,Kastelein JJ, HaydenMR (2013) The impact of partial and complete loss-of-function mutationsin endothelial lipase on high-density lipoprotein levels and functionality in humans.Circ Cardiovasc Genet 6:54–62106 V.I. Zannis et al.Smith EB, Ashall C, Walker JE (1984) High density lipoprotein (HDL) subfractions in interstitialfluid from human aortic intima and atherosclerostic lesions. Biochem Soc Trans 12:843–844Smith JD, Waelde C, Horwitz A, Zheng P (2002) Evaluation of the role of phosphatidylserinetranslocase activity in ABCA1-mediated lipid efflux. J Biol Chem 277:17797–17803Sorci-Thomas MG, Thomas MJ (2002) The effects of altered apolipoprotein A-I structure onplasma HDL concentration. Trends Cardiovasc Med 12:121–128Stangl H, Hyatt M, Hobbs HH (1999) Transport of lipids from high and low density lipoproteinsvia scavenger receptor-BI. J Biol Chem 274:32692–32698Stefulj J, Panzenboeck U, Becker T, Hirschmugl B, Schweinzer C, Lang I, Marsche G, Sadjak A,Lang U, Desoye G, Wadsack C (2009) Human endothelial cells of the placental barrierefficiently deliver cholesterol to the fetal circulation via ABCA1 and ABCG1. Circ Res 104:600–608Strope S, Rivi R, Metzger T, Manova K, Lacy E (2004) Mouse amnionless, which is required forprimitive streak assembly, mediates cell-surface localization and endocytic function of cubilinon visceral endoderm and kidney proximal tubules. Development 131:4787–4795Sturek JM, Castle JD, Trace AP, Page LC, Castle AM, Evans-Molina C, Parks JS, Mirmira RG,Hedrick CC (2010) An intracellular role for ABCG1-mediated cholesterol transport in theregulated secretory pathway of mouse pancreatic beta cells. J Clin Invest 120:2575–2589Subbaiah PV, Albers JJ, Chen CH, Bagdade JD (1980) Low density lipoprotein-activated lysoleci-thin acylation by human plasma lecithin-cholesterol acyltransferase. Identity of lysolecithinacyltransferase and lecithin-cholesterol acyltransferase. J Biol Chem 255:9275–9280Takahashi Y, Smith JD (1999) Cholesterol efflux to apolipoprotein AI involves endocytosis andresecretion in a calcium-dependent pathway. Proc Natl Acad Sci USA 96:11358–11363Tall AR, Forester LR, Bongiovanni GL (1983) Facilitation of phosphatidylcholine transfer intohigh density lipoproteins by an apolipoprotein in the density 1.20-1.26 g/ml fraction of plasma.J Lipid Res 24:277–289Tangirala RK, Tsukamoto K, Chun SH, Usher D, Pure E, Rader DJ (1999) Regression of athero-sclerosis induced by liver-directed gene transfer of apolipoprotein A-I in mice. Circulation100:1816–1822Tarling EJ, Edwards PA (2011) ATP binding cassette transporter G1 (ABCG1) is an intracellularsterol transporter. Proc Natl Acad Sci USA 108:19719–19724Terasaka N, Wang N, Yvan-Charvet L, Tall AR (2007) High-density lipoprotein protectsmacrophages from oxidized low-density lipoprotein-induced apoptosis by promoting effluxof 7-ketocholesterol via ABCG1. Proc Natl Acad Sci USA 104:15093–15098Teslovich TM et al (2010) Biological, clinical and population relevance of 95 loci for blood lipids.Nature 466:707–713Thuahnai ST, Lund-Katz S, Williams DL, Phillips MC (2001) Scavenger receptor class B, typeI-mediated uptake of various lipids into cells. Influence of the nature of the donor particleinteraction with the receptor. J Biol Chem 276:43801–43808Timmins JM, Lee JY, Boudyguina E, Kluckman KD, Brunham LR, Mulya A, Gebre AK, CoutinhoJM, Colvin PL, Smith TL, Hayden MR, Maeda N, Parks JS (2005) Targeted inactivation ofhepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism ofapoA-I. J Clin Invest 115:1333–1342Tollefson JH, Ravnik S, Albers JJ (1988) Isolation and characterization of a phospholipid transferprotein (LTP-II) from human plasma. J Lipid Res 29:1593–1602Trigatti B, Rayburn H, Vinals M, Braun A, Miettinen H, Penman M, Hertz M, Schrenzel M,Amigo L, Rigotti A, Krieger M (1999) Influence of the high density lipoprotein receptor SR-BIon reproductive and cardiovascular pathophysiology. Proc Natl Acad Sci USA 96:9322–9327Ueda Y, Royer L, Gong E, Zhang J, Cooper PN, Francone O, Rubin EM (1999) Lower plasmalevels and accelerated clearance of high density lipoprotein (HDL) and non-HDL cholesterol inscavenger receptor class B type I transgenic mice. J Biol Chem 274:7165–7171HDL Biogenesis, Remodeling, and Catabolism 107Ueda Y, Gong E, Royer L, Cooper PN, Francone OL, Rubin EM (2000) Relationship betweenexpression levels and atherogenesis in scavenger receptor class B, type I transgenics. J BiolChem 275:20368–20373Undurti A, Huang Y, Lupica JA, Smith JD, Didonato JA, Hazen SL (2009) Modification of highdensity lipoprotein by myeloperoxidase generates a pro-inflammatory particle. J Biol Chem284:30825–30835Urban S, Zieseniss S, Werder M, Hauser H, Budzinski R, Engelmann B (2000) Scavenger receptorBI transfers major lipoprotein-associated phospholipids into the cells. J Biol Chem 275:33409–33415Vaisar T, Pennathur S, Green PS, Gharib SA, Hoofnagle AN, Cheung MC, Byun J, Vuletic S,Kassim S, Singh P, Chea H, Knopp RH, Brunzell J, Geary R, Chait A, Zhao XQ, Elkon K,Marcovina S, Ridker P, Oram JF, Heinecke JW (2007) Shotgun proteomics implicates proteaseinhibition and complement activation in the antiinflammatory properties of HDL. J Clin Invest117:746–756Vaisman BL, Lambert G, Amar M, Joyce C, Ito T, Shamburek RD, Cain WJ, Fruchart-Najib J,Neufeld ED, Remaley AT, Brewer HB Jr, Santamarina-Fojo S (2001) ABCA1 overexpressionleads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenicmice. J Clin Invest 108:303–309Van Eck M, Bos IS, Kaminski WE, Orso E, Rothe G, Twisk J, Bottcher A, Van Amersfoort ES,Christiansen-Weber TA, Fung-Leung WP, van Berkel TJ, Schmitz G (2002) LeukocyteABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.Proc Natl Acad Sci USA 99:6298–6303Van Eck M, Singaraja RR, Ye D, Hildebrand RB, James ER, Hayden MR, van Berkel TJ (2006)Macrophage ATP-binding cassette transporter A1 overexpression inhibits atheroscleroticlesion progression in low-density lipoprotein receptor knockout mice. Arterioscler ThrombVasc Biol 26:929–934Vaughan AM, Oram JF (2005) ABCG1 redistributes cell cholesterol to domains removable byHDL but not by lipid-depleted apolipoproteins. J Biol Chem 280:30150–30157Venkateswaran A, Repa JJ, Lobaccaro JM, Bronson A, Mangelsdorf DJ, Edwards PA (2000)Human white/murine ABC8 mRNA levels are highly induced in lipid-loaded macrophages. Atranscriptional role for specific oxysterols. J Biol Chem 275:14700–14707Vergeer M, Boekholdt SM, Sandhu MS, Ricketts SL, Wareham NJ, Brown MJ, de Faire U,Leander K, Gigante B, Kavousi M, Hofman A, Uitterlinden AG, van Duijn CM, WittemanJC, Jukema JW, Schadt EE, van der Schoot E, Kastelein JJ, Khaw KT, Dullaart RP, van Tol A,Trip MD, Dallinga-Thie GM (2010a) Genetic variation at the phospholipid transfer proteinlocus affects its activity and high-density lipoprotein size and is a novel marker of cardio-vascular disease susceptibility. Circulation 122:470–477Vergeer M, Cohn DM, Boekholdt SM, Sandhu MS, Prins HM, Ricketts SL, Wareham NJ,Kastelein JJ, Khaw KT, Kamphuisen PW, Dallinga-Thie GM (2010b) Lack of associationbetween common genetic variation in endothelial lipase (LIPG) and the risk for CAD andDVT. Atherosclerosis 211:558–564Vergeer M, Korporaal SJA, Franssen R, Meurs I, Out R, Hovingh GK, Hoekstra M, Sierts JA,Dallinga-Thie GM, Motazacker MM, Holleboom AG, Van Berkel TJC, Kastelein JJP,Van Eck M, Kuivenhoven JA (2011) Genetic variant of the scavenger receptor BI in humans.N Engl J Med 364:136–145Vezeridis AM, Chroni A, Zannis VI (2011) Domains of apoE4 required for the biogenesis of apoE-containing HDL. Ann Med 43:302–311Vickers KC, Remaley AT (2014) HDL and cholesterol: life after the divorce? J Lipid Res 55:4–12Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT (2011) MicroRNAs aretransported in plasma and delivered to recipient cells by high-density lipoproteins. Nat CellBiol 13:423–433108 V.I. Zannis et al.Vikstedt R, Metso J, Hakala J, Olkkonen VM, Ehnholm C, Jauhiainen M (2007a) Cholesterolefflux from macrophage foam cells is enhanced by active phospholipid transfer protein throughgeneration of two types of acceptor particles. Biochemistry 46:11979–11986Vikstedt R, Ye D, Metso J, Hildebrand RB, van Berkel TJ, Ehnholm C, Jauhiainen M, Van EM(2007b) Macrophage phospholipid transfer protein contributes significantly to total plasmaphospholipid transfer activity and its deficiency leads to diminished atherosclerotic lesiondevelopment. Arterioscler Thromb Vasc Biol 27:578–586Wang N, Arai T, Ji Y, Rinninger F, Tall AR (1998) Liver-specific overexpression of scavengerreceptor BI decreases levels of very low density lipoprotein ApoB, low density lipoproteinApoB, and high density lipoprotein in transgenic mice. J Biol Chem 273:32920–32926Wang N, Silver DL, Costet P, Tall AR (2000) Specific binding of ApoA-I, enhanced cholesterolefflux, and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem 275:33053–33058Wang N, Lan D, Chen W, Matsuura F, Tall AR (2004) ATP-binding cassette transporters G1 andG4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci USA101:9774–9779Wang N, Ranalletta M, Matsuura F, Peng F, Tall AR (2006) LXR-induced redistribution ofABCG1 to plasma membrane in macrophages enhances cholesterol mass efflux to HDL.Arterioscler Thromb Vasc Biol 26:1310–1316Wang X, Collins HL, Ranalletta M, Fuki IV, Billheimer JT, Rothblat GH, Tall AR, Rader DJ(2007) Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reversecholesterol transport in vivo. J Clin Invest 117:2216–2224Warden CH, Langner CA, Gordon JI, Taylor BA, McLean JW, Lusis AJ (1989) Tissue-specificexpression, developmental regulation, and chromosomal mapping of the lecithin: cholesterolacyltransferase gene. Evidence for expression in brain and testes as well as liver. J Biol Chem264:21573–21581Waterworth DM, Ricketts SL, Song K, Chen L, Zhao JH, Ripatti S, Aulchenko YS, Zhang W,Yuan X, Lim N, Luan J, Ashford S, Wheeler E, Young EH, Hadley D, Thompson JR,Braund PS, Johnson T, Struchalin M, Surakka I, Luben R, Khaw KT, Rodwell SA, Loos RJ,Boekholdt SM, Inouye M, Deloukas P, Elliott P, Schlessinger D, Sanna S, Scuteri A,Jackson A, Mohlke KL, Tuomilehto J, Roberts R, Stewart A, Kesaniemi YA, Mahley RW,Grundy SM, McArdle W, Cardon L, Waeber G, Vollenweider P, Chambers JC, Boehnke M,Abecasis GR, Salomaa V, Jarvelin MR, Ruokonen A, Barroso I, Epstein SE, Hakonarson HH,Rader DJ, Reilly MP, Witteman JC, Hall AS, Samani NJ, Strachan DP, Barter P, van DuijnCM, Kooner JS, Peltonen L, Wareham NJ, McPherson R, Mooser V, Sandhu MS (2010)Genetic variants influencing circulating lipid levels and risk of coronary artery disease.Arterioscler Thromb Vasc Biol 30:2264–2276Webb NR, de Beer MC, Yu J, Kindy MS, Daugherty A, van der Westhuyzen DR, de Beer FC(2002) Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I, but notapoB, in human apoB transgenic mice. J Lipid Res 43:1421–1428Willer CJ, Sanna S, Jackson AU, Scuteri A, Bonnycastle LL, Clarke R, Heath SC, Timpson NJ,Najjar SS, Stringham HM, Strait J, Duren WL, Maschio A, Busonero F, Mulas A, Albai G,Swift AJ, Morken MA, Narisu N, Bennett D, Parish S, Shen H, Galan P, Meneton P,Hercberg S, Zelenika D, Chen WM, Li Y, Scott LJ, Scheet PA, Sundvall J, Watanabe RM,Nagaraja R, Ebrahim S, Lawlor DA, Ben Shlomo Y, Davey-Smith G, Shuldiner AR, Collins R,Bergman RN, Uda M, Tuomilehto J, Cao A, Collins FS, Lakatta E, Lathrop GM, Boehnke M,Schlessinger D, Mohlke KL, Abecasis GR (2008) Newly identified loci that influence lipidconcentrations and risk of coronary artery disease. Nat Genet 40:161–169Wojcik AJ, Skaflen MD, Srinivasan S, Hedrick CC (2008) A critical role for ABCG1 in macro-phage inflammation and lung homeostasis. J Immunol 180:4273–4282Wolfrum C, Poy MN, Stoffel M (2005) Apolipoprotein M is required for prebeta-HDL formationand cholesterol efflux to HDL and protects against atherosclerosis. Nat Med 11:418–422HDL Biogenesis, Remodeling, and Catabolism 109Woollett LA, Spady DK (1997) Kinetic parameters for high density lipoprotein apoprotein AI andcholesteryl ester transport in the hamster. J Clin Invest 99:1704–1713Wu Z, Gogonea V, Lee X, May RP, Pipich V, Wagner MA, Undurti A, Tallant TC, Baleanu-Gogonea C, Charlton F, Ioffe A, Didonato JA, Rye KA, Hazen SL (2011) The low resolutionstructure of ApoA1 in spherical high density lipoprotein revealed by small angle neutronscattering. J Biol Chem 286:12495–12508Xie Q, Engel T, Schnoor M, Niehaus J, Hofnagel O, Buers I, Cullen P, Seedorf U, Assmann G,Lorkowski S (2006) Cell surface localization of ABCG1 does not require LXR activation.Arterioscler Thromb Vasc Biol 26:e143–e144Xu N, Dahlback B (1999) A novel human apolipoprotein (apoM). J Biol Chem 274:31286–31290Xu N, Nilsson-Ehle P (2007) ApoM – a novel apolipoprotein with antiatherogenic properties.In: Fielding CJ (ed) High-density lipoproteins. From basic biology to clinical aspects. Wiley,Weinheim, pp 89–110Xu S, Laccotripe M, Huang X, Rigotti A, Zannis VI, Krieger M (1997) Apolipoproteins of HDLcan directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediatesselective lipid uptake. J Lipid Res 38:1289–1298Xu N, Nilsson-Ehle P, Ahren B (2006) Suppression of apolipoprotein M expression and secretionin alloxan-diabetic mouse: partial reversal by insulin. Biochem Biophys Res Commun 342:1174–1177Xu WW, Zhang Y, Tang YB, Xu YL, Zhu HZ, Ferro A, Ji Y, Chen Q, Fan LM (2008) A geneticvariant of apolipoprotein M increases susceptibility to coronary artery disease in aChinese population. Clin Exp Pharmacol Physiol 35:546–551Xu Y, Wang W, Zhang L, Qi LP, Li LY, Chen LF, Fang Q, Dang AM, Yan XW (2011) Apolymorphism in the ABCG1 promoter is functionally associated with coronary artery diseasein a Chinese Han population. Atherosclerosis 219:648–654Yamakawa-Kobayashi K, Yanagi H, Fukayama H, Hirano C, Shimakura Y, Yamamoto N,Arinami T, Tsuchiya S, Hamaguchi H (1999) Frequent occurrence of hypoalphalipoproteinemiadue to mutant apolipoprotein A-I gene in the population: a population-based survey. Hum MolGenet 8:331–336Yates M, Kolmakova A, Zhao Y, Rodriguez A (2011) Clinical impact of scavenger receptor classB type I gene polymorphisms on human female fertility. Hum Reprod 26:1910–1916Yesilaltay A, Morales MG, Amigo L, Zanlungo S, Rigotti A, Karackattu SL, Donahee MH,Kozarsky KF, Krieger M (2006) Effects of hepatic expression of the high-density lipoproteinreceptor SR-BI on lipoprotein metabolism and female fertility. Endocrinology 147:1577–1588Yesilaltay A, Daniels K, Pal R, Krieger M, Kocher O (2009) Loss of PDZK1 causes coronaryartery occlusion and myocardial infarction in Paigen diet-fed apolipoprotein E deficient mice.PLoS ONE 4:e8103Yuhanna IS, Zhu Y, Cox BE, Hahner LD, Osborne-Lawrence S, Lu P, Marcel YL, Anderson RG,Mendelsohn ME, Hobbs HH, Shaul PW (2001) High-density lipoprotein binding to scavengerreceptor-BI activates endothelial nitric oxide synthase. Nat Med 7:853–857Yvan-Charvet L, Matsuura F, Wang N, Bamberger MJ, Nguyen T, Rinninger F, Jiang XC,Shear CL, Tall AR (2007) Inhibition of cholesteryl ester transfer protein by torcetrapibmodestly increases macrophage cholesterol efflux to HDL. Arterioscler Thromb Vasc Biol27:1132–1138Yvan-Charvet L, Kling J, Pagler T, Li H, Hubbard B, Fisher T, Sparrow CP, Taggart AK, Tall AR(2010) Cholesterol efflux potential and antiinflammatory properties of high-density lipoproteinafter treatment with niacin or anacetrapib. Arterioscler Thromb Vasc Biol 30:1430–1438Zannis VI, Lees AM, Lees RS, Breslow JL (1982) Abnormal apoprotein A-I isoprotein compo-sition in patients with Tangier disease. J Biol Chem 257:4978–4986Zannis VI, Cole FS, Jackson CL, Kurnit DM, Karathanasis SK (1985) Distribution of apolipo-protein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction ofapolipoprotein E mRNA by cultures of human monocyte-macrophages. Biochemistry 24:4450–4455110 V.I. Zannis et al.Zannis VI, Chroni A, Kypreos KE, Kan HY, Cesar TB, Zanni EE, Kardassis D (2004a) Probing thepathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer.Curr Opin Lipidol 15:151–166Zannis VI, Chroni A, Liu T, Liadaki KN, Laccotripe M (2004) New insights on the roles ofapolipoprotein A-I, the ABCA1 lipid transporter, and the HDL receptor SR-BI in the biogene-sis and the functions of HDL. In: Simionescu M, Sima A, Popov D (eds) Cellular dysfunctionin atherosclerosis and diabetes. Romanian Academy Publishing House, Bucharest, pp 33–72Zannis VI, Chroni A, Krieger M (2006a) Role of apoA-I, ABCA1, LCAT, and SR-BI in thebiogenesis of HDL. J Mol Med 84:276–294Zannis VI, Zanni EE, Papapanagiotou A, Kardassis D, Chroni A (2006b) ApoA-I functions andsynthesis of HDL: insights from mouse models of human HDL metabolism. In: Fielding CJ(ed) High-density lipoproteins. From basic biology to clinical aspects. Wiley, Weinheim,pp 237–265Zhang S, Picard MH, Vasile E, Zhu Y, Raffai RL, Weisgraber KH, Krieger M (2005) Diet-inducedocclusive coronary atherosclerosis, myocardial infarction, cardiac dysfunction and prematuredeath in SR-BI-deficient, hypomorphic apolipoprotein ER61 mice. Circulation 111:3457–3464Zhang Y, Ahmed AM, Tran TL, Lin J, McFarlane N, Boreham DR, Igdoura SA, Truant R,Trigatti BL (2007) The inhibition of endocytosis affects HDL-lipid uptake mediated by thehuman scavenger receptor class B type I. Mol Membr Biol 24:442–454Zhang LH, Kamanna VS, Zhang MC, Kashyap ML (2008) Niacin inhibits surface expression ofATP synthase beta chain in HepG2 cells: implications for raising HDL. J Lipid Res 49:1195–1201Zhang Y, McGillicuddy FC, Hinkle CC, O’Neill S, Glick JM, Rothblat GH, Reilly MP (2010)Adipocyte modulation of high-density lipoprotein cholesterol. Circulation 121:1347–1355Zhong S, Sharp DS, Grove JS, Bruce C, Yano K, Curb JD, Tall AR (1996) Increased coronaryheart disease in Japanese-American men with mutation in the cholesteryl ester transfer proteingene despite increased HDL levels. J Clin Invest 97:2917–2923HDL Biogenesis, Remodeling, and Catabolism 111Regulation of HDL Genes: Transcriptional,Posttranscriptional, and PosttranslationalDimitris Kardassis, Anca Gafencu, Vassilis I. Zannis, and AlbertoDavalosContents1 Regulation of Genes Involved in HDL Metabolism at the Transcriptional Level . . . . . . . 1171.1 General Introduction to Hormone Nuclear Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1181.2 Transcriptional Regulation of the apoA-I Gene in the Liver . . . . . . . . . . . . . . . . . . . . . . 1191.2.1 The Role of the Distal Enhancer in apoA-I Gene Transcription . . . . . . . . . . 1231.2.2 Other Factors Regulating apoA-I Gene Transcription . . . . . . . . . . . . . . . . . . . . . 1241.3 Transcriptional Regulation of the ABCA1 Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1251.3.1 Upregulatory Mechanisms of ABCA1 Gene Expression . . . . . . . . . . . . . . . . . . 1251.3.2 Negative Regulation of ABCA1 Gene Transcription . . . . . . . . . . . . . . . . . . . . . . 1291.4 Transcriptional Regulation of the ABCG1 Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1301.5 Transcriptional Regulation of the Apolipoprotein E Gene . . . . . . . . . . . . . . . . . . . . . . . . 1321.5.1 Proximal Regulatory Binding Sites Involved in the apoE Gene Expression 1341.5.2 Distal Regulatory Binding Sites That Modulate apoE Gene Expressionin Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1351.6 Transcriptional Regulation of the Human apoM Gene in the Liver . . . . . . . . . . . . . . . 1361.7 Transcriptional Regulation of the CETP Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1381.8 Transcriptional Regulation of the PLTP Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1391.9 Transcriptional Regulation of the Bile Acid Transporters ABCG5/ABCG8 . . . . . 1401.10 Transcriptional Regulation of the HDL Receptor SR-BI . . . . . . . . . . . . . . . . . . . . . . . . . . 141D. Kardassis (*)Department of Biochemistry, University of Crete Medical School and Institute of MolecularBiology and Biotechnology, Foundation of Research and Technology of Hellas, Heraklion,Crete 71110, Greecee-mail: kardasis@imbb.forth.grA. GafencuInstitute of Cellular Biology and Pathology “Nicolae Simionescu”, Bucharest, RomaniaV.I. ZannisSection of Molecular Genetics, Boston University Medical Center, Boston, MA 02118, USAA. DavalosLaboratory of Disorders of Lipid Metabolism and Molecular Nutrition, Madrid Institute forAdvanced Studies (IMDEA)-Food, Ctra. de Cantoblanco 8, 28049 Madrid, Spain# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_31132 Posttranscriptional Regulation of HDL Genes by Noncoding RNAs and microRNAs . . 1432.1 miRNAs: Biogenesis and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1442.2 Posttranscriptional Modulation of HDL Metabolism by miRNAs . . . . . . . . . . . . . . . . . . 1462.2.1 Targeting ABCA1 and ABCG1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1462.2.2 Targeting SR-BI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1492.2.3 Targeting Other miRNAs Related to HDL Biogenesis and Function . . . . . . 1503 Posttranslational Mechanisms of HDL Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503.1 ABCA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503.2 ABCG1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1543.3 SR-BI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157AbstractHDL regulation is exerted at multiple levels including regulation at the level oftranscription initiation by transcription factors and signal transduction cascades;regulation at the posttranscriptional level by microRNAs and other noncodingRNAs which bind to the coding or noncoding regions of HDL genes regulatingmRNA stability and translation; as well as regulation at the posttranslationallevel by protein modifications, intracellular trafficking, and degradation. Theabove mechanisms have drastic effects on several HDL-mediated processesincluding HDL biogenesis, remodeling, cholesterol efflux and uptake, as wellas atheroprotective functions on the cells of the arterial wall. The emphasis is onmechanisms that operate in physiologically relevant tissues such as the liver(which accounts for 80 % of the total HDL-C levels in the plasma), themacrophages, the adrenals, and the endothelium. Transcription factors thathave a significant impact on HDL regulation such as hormone nuclear receptorsand hepatocyte nuclear factors are extensively discussed both in terms of genepromoter recognition and regulation but also in terms of their impact on plasmaHDL levels as was revealed by knockout studies. Understanding the differentmodes of regulation of this complex lipoprotein may provide useful insights forthe development of novel HDL-raising therapies that could be used to fightagainst atherosclerosis which is the underlying cause of coronary heart disease.KeywordsHigh-density lipoprotein • Regulation • Transcriptional • Posttranscriptional •Posttranslational • miRNAs • Protein stability • Hormone nuclear receptors •Hepatocyte nuclear factors • apoA-I • ABCA1 • ABCG1 • ABCG5 • ABCG8 •apoE • SR-BI • CETP114 D. Kardassis et al.List of AbbreviationsHMG-CoA 3-Hydroxy-3-methylglutaryl-coenzyme A12/15LO 12/15-LipoxygenaseAP1 Activator protein 1AF1 Activation function 1Ang AngiotensinapoA-I Apolipoprotein A-IapoB Apolipoprotein BapoE Apolipoprotein EapoM Apolipoprotein MARP-1 Apolipoprotein A-I-regulated protein 1DHHC8 Asp-His-His-Cys 8ABCA1 ATP-binding cassette transporter A1ABCG1 ATP-binding cassette transporter G1BIG1 Brefeldin A-inhibited guanine nucleotide-exchange protein 1JNK c-Jun N-terminal kinaseC/EBP CCAAT/enhancer-binding proteinCAV-1 Caveolin 1CXCL Chemokine (C-X-C motif) ligandCOUP-TFI Chicken ovalbumin upstream promoter transcription factor ICEH Cholesterol ester hydrolaseCETP Cholesterol ester transfer protein3C Chromosome conformational captureCLA Conjugated linoleic acidCAR Constitutive androstane receptorCREB Cyclic AMP response element-binding proteinDNase DeoxyribonucleaseDR4 Direct repeat with 4 nucleotides in the spacer regionDBD DNA-binding domainEGR-1 Early growth response protein 1LIPG Endothelial lipaseEE Early endosomeER Endoplasmic reticulumESCRT Endosomal sorting complex required for transportERα and β Estrogen receptors α and βERE Estrogen response elementESRRG Estrogen receptor-related gammaFXR Farnesoid X receptorFXRE Farnesoid X receptor-responsive elementFF FenofibrateFOXA2 Forkhead box A2FOXΟ1 Forkhead box Ο1GF GemfibrozilRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 115GR Glucocorticoid receptorGSK3β Glycogen synthase kinase 3βHNF-4 Hepatocyte nuclear factor-4HDL High-density lipoproteinHDL-C High-density lipoprotein cholesterolHcy HomocysteineHRE Hormone response elementIRAK-1 IL-1 receptor-associated kinase 1IDOL Inducible degrader of the LDLRIFN InterferonIL InterleukinKO KnockoutKLF4 Kruppel-like factor 4LCAT Lecithin-cholesterol acyltransferaseLXR Liver X receptorLXRE Liver X receptor response elementLRH-1 Liver receptor homologue-1LBD Ligand-binding domainLPS LipopolysaccharideLDLR Low-density lipoprotein receptorLAL Lysosomal acid lipaseMCSF Macrophage colony-stimulating factormiRNAs microRNAsMEKK Mitogen-activated protein kinase/ERK kinase kinaseMCP-1 Monocyte chemoattractant protein-1ME MultienhancerMVB Multivesicular bodiesDMHCA N,N-Dimethyl-3β-hydroxycholenamideNHERF Na+/H+ exchanger regulator factorNF-κB Nuclear factor kappa-light-chain-enhancer of activated B cellsNFY Nuclear factor YORP1S Oxysterol-binding protein-related protein 1SPDZK1 PDZ domain-containing adaptor proteinPPAR Peroxisome proliferator-activated receptorsPPRE Peroxisome proliferator-activated receptor-responsive elementPMA Phorbol 12-myristate 13-acetatePI3K phosphatidylinositol 3- kinasePLD Phospholipase DPGC-1 PPARgamma coactivator 1PLTP Phospholipid transfer proteinpri-miRNA Primary long miRNAPREB Prolactin regulatory element-bindingPEST Proline, glutamic acid, serine, threoninePKC Protein kinase CPCA Protocatechuic acid116 D. Kardassis et al.PCSK9 Proprotein convertase subtilisin/kexin type 9RAR Retinoic acid receptorRORα Retinoic acid receptor-related orphan receptor αRXRα Retinoid X receptor αRISC RNA-induced silencing complexSR-BI Scavenger receptor class B type ISPTLC1 Serine palmitoyltransferase 1STAT Signal transducer and activator of transcriptionSHP Small heterodimer partnersiRNAs Small interfering RNAsSP1 Specificity protein 1S1P Sphingosine 1 phosphateSRC-1 Steroid receptor coactivator-1SF-1 Steroidogenic factor-1SREBP Sterol regulatory element-binding proteinSREs Sterol-responsive elementsTRβ Thyroid hormone receptor βTAD Transactivation domainTGFβ1 Transforming growth factor β1TNFα Tumor necrosis factor αTpl2 Tumor progression locus 2UTR Untranslated regionURE Upstream regulatory elementUSF Upstream stimulatory factorsVDR Vitamin D receptorWT Wild typeYY1 Yin Yang 1ZIC Zinc finger of the cerebellumZNF202 Zinc finger protein 2021 Regulation of Genes Involved in HDL Metabolismat the Transcriptional LevelA large body of work generated over the past four decades has revealed thateukaryotic gene transcription is a remarkably intricate biochemical process that istightly regulated at many levels by the ordered assembly of multiprotein transcrip-tion initiation complexes to specific regulatory regions in the promoters of genes(Roeder 1998, 2005; Lemon and Tjian 2000). Despite the progress made, stilllimited knowledge regarding the details exists. It is believed that specificity ingene regulation is determined by the unique order of cis-acting regulatory regionswhich are recognized by sequence-specific DNA-binding transcription factors.Recent advances in gene regulation technologies including the powerful chromatinRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 117immunoprecipitation assay have enabled the monitoring in real time of the orderedassembly and the disassembly of transcription factor complexes on the promotersand the enhancers of genes in response to extracellular or intracellular cues(Christova 2013; Rodriguez-Ubreva and Ballestar 2014). High-throughputsequencing technologies have revolutionized the fields of genomics, epigenomics,and transcriptomics and have provided novel insights into the transcriptionsignatures of human diseases (Churko et al. 2013). Furthermore, using new power-ful methodologies such as chromosome conformation capture (3C) and itsderivatives, we are at a position to monitor dynamic intra- and interchromosomalinteractions that allow the optimal expression of genes at a given time and space(Gavrilov et al. 2009; Wei et al. 2013).Transcription factors may be constitutively active in a cell or work in aninducible mode in response to various ligands and signal transduction pathways.The cross talk between different signaling pathways which orchestrate the cellularresponses can be facilitated by the physical and functional interactions betweentranscription factors, and these interactions can be monitored by various methodsboth in vivo and in vitro. All known transcription factors are modular in nature andcontain a DNA-binding domain and a transcriptional activation domain (Mitchelland Tjian 1989; Lemon and Tjian 2000). In addition, several factors contain adimerization domain that permits them to form homodimers and/or heterodimers. Avariety of nuclear receptors for steroids, thyroids, retinoids, etc. contain a ligandbinding site. Via their transcription activation domains, transcription factors appearto facilitate the recruitment of the proteins of the coactivator complex and the basaltranscription complex to the transcription initiation site of each gene and thusinitiate transcription (Roeder 2005). Importantly, the activity of transcriptionfactors can be modulated by drugs against diseases such as cancer and cardio-vascular disease as exemplified by the drugs that activate or repress the hormonenuclear receptors (Gronemeyer et al. 2004).It is beyond the scope of this chapter to provide a thorough review of thedifferent mechanisms of transcriptional regulation of eukaryotic genes or todescribe extensively the different classes of transcription factors, their structures,and their mode of regulation. We will only focus on those classes of transcriptionfactors that have been shown to play key roles in the regulation of the genesinvolved in lipid and lipoprotein metabolism and more specifically on thoseinvolved in the metabolism of high-density lipoproteins (HDL) such as the hormonenuclear receptors.1.1 General Introduction to Hormone Nuclear ReceptorsHormone nuclear receptors belong to a superfamily of transcription factors that areactivated by steroid hormones (estrogens, androgens, glucocorticoids, etc.),retinoids, thyroids, and products of intermediate metabolism such as bile acids,fatty acids, and cholesterol derivatives, among others (Gronemeyer et al. 2004).Some members of this family do not need ligand binding to regulate transcription118 D. Kardassis et al.and are classified as “orphans” (Blumberg and Evans 1998). Nuclear receptors arestructurally highly conserved. In terms of primary structure, the highest degree ofhomology among family members is in the DNA-binding domain that contains twozinc fingers (Helsen et al. 2012). Nuclear receptors also contain two transactivationdomains (TADs), one N-terminal ligand-independent TAD called activation func-tion 1 (AF1) and a ligand-dependent TAD called AF2 located close to the ligand-binding domain (LBD) (Rochel et al. 2011). Nuclear receptors bind to hormoneresponse elements (HREs) on the promoters of target genes either as homodimers oras heterodimers with the retinoid X receptor (RXR). The HREs consist of directrepeats (DRs), inverted repeats (IRs), or palindromic repeats (PRs) of the consensussequence 50 AG(G/T)TCA 30. The repeats are separated by 1, 2, 3, 4, or5 nucleotides and are designated DR1, DR2, etc. (for the direct repeats); IR1,IR2, etc. (for the inverted repeats); and PR1, PR2, etc. (for the palindromic repeats)as described previously (Kardassis et al. 2007; Helsen et al. 2012). The HRE typeand inter-repeat spacing determine to a large degree the specificity in nuclearreceptor binding, but this rule is not strict at all. For instance, both LXR/RXRand T3R/RXR heterodimers prefer to bind to DR4 HREs, whereas RAR/RXRheterodimers bind to DR5. The direct repeats with one base spacing (DR1 type)appear to be very promiscuous as they bind RXR, COUP-TFI, ARP-1, and HNF-4homodimers and PPAR/RXR, RAR/RXR, COUP-TFI/RXR, and ARP-1/RXRheterodimers (Nakshatri and Bhat-Nakshatri 1998). The elucidation of the three-dimensional structure of the ligand-binding domain of several nuclear receptors byX-ray crystallography in the absence and in the presence of ligands has allowed agood understanding of the modulation of nuclear receptor action by ligands and thedevelopment of very potent agonists and antagonists, some of which have been usedtherapeutically (Bourguet et al. 2000). Chromatin immunoprecipitation studiesusually reveal that nuclear receptors are constitutively nuclear and bound to chro-matin but they are transcriptionally silent in the absence of ligand. The binding ofthe ligand to the LBD causes a major conformational change to this domain whichculminates in the recruitment of nuclear receptor coactivators such as PGC-1 andCBP/p300 and the displacement of corepressors (Chen and Li 1998; Liu and Lin2011). Nuclear receptors can cross talk with other transcription factors in a positiveor a negative manner as exemplified by the negative regulation of Jun or NF-κBtranscription factors by the glucocorticoid receptors during inflammation, a mecha-nism termed trans-repression (Adcock and Caramori 2001).1.2 Transcriptional Regulation of the apoA-I Gene in the LiverThe hypothesis that apolipoprotein (apo) A-I overexpression positively influencesplasma concentrations of HDL cholesterol (HDL-C) has been validated experi-mentally in transgenic mice expressing human apoA-I under homologous or hetero-logous regulatory sequences. These mice have significantly elevated plasma levelsof HDL-C and human apoA-I (Rubin et al. 1991; Kan et al. 2000). These “humanized”apoA-I transgenic mice are valuable tools for the study of apoA-I gene regulationRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 119in vivo. Furthermore, it was demonstrated that the overexpression of apoA-I in apoEKO or LDLR KO mice via transgene- or adenovirus-mediated gene transfer reducedatherosclerosis development confirming the anti-atherogenic role of apoA-Iupregulation (Paszty et al. 1994; Belalcazar et al. 2003; Valenta et al. 2006).In humans, the apoA-I gene is expressed abundantly in the liver and intestine andto a lesser extent in other tissues (Zannis et al. 1985). Early studies had establishedthat the human apoA-I promoter containing 250 bp upstream from the transcriptionstart site of the gene is sufficient to drive liver-specific gene expression both in cellcultures and in mice (Walsh et al. 1989; Tzameli and Zannis 1996; Hu et al. 2010a).This promoter region is rich in nuclear factor binding sites and responds to variousintracellular as well as extracellular ligands (Zannis et al. 2001a; Haas andMooradian 2010). As shown in Fig. 1, prominent role in the regulation of theapoA-I promoter play two hormone response elements (HREs) located at positions210/190 and 132/120 that bind members of the hormone nuclear receptorsuperfamily in a competitive manner (Tzameli and Zannis 1996).One of the nuclear receptors that plays a prominent role in apoA-I gene regu-lation in the liver and the intestine is the hepatocyte nuclear factor-4 (HNF-4).HNF-4 was discovered as a rat liver nuclear protein that binds to the promotersof liver-specific genes such as transthyretin and apolipoprotein C-III (Sladek 1994).In the adult organism, HNF-4 is expressed in the liver, kidney, intestine, andpancreas (Sladek 1994). The total disruption of the HNF-4 gene in mice leads toan embryonic lethal phenotype due to the impairment of endodermal differentiationand gastrulation (Chen et al. 1994). This early developmental arrest was rescued bythe complementation of the HNF-4α/ embryos with a tetraploid embryo-derivedFig. 1 Regulatory elements and transcription factors that control the expression of the apoA-Igene. Arrows and block lines denote activation and repression, respectively. The mechanisms aredescribed in detail in the text. Abbreviations: retinoic acid receptor (RAR); retinoid X receptor(RXR); thyroid receptor β (TRβ); farnesoid X receptor (FXR); hepatocyte nuclear factor (HNF);apoA-I regulatory protein 1 (ARP-1); forkhead box 2 (FOXA2); nuclear factor Y (NFY); CCAAT/enhancer-binding protein (CEBP); liver receptor homologue-1 (LRH-1); small heterodimer part-ner (SHP); specificity protein 1 (Sp1); tumor necrosis factor α (TNFα), interleukin-1 (IL-1);lipopolysaccharide (LPS)120 D. Kardassis et al.wild-type visceral endoderm (Li et al. 2000). The analysis of the rescued miceshowed that the expression of the apoA-I gene as well as of other apolipoproteingenes, shown previously to be regulated by HNF-4 including apoA-II, apoB, apoC-III, and apoC-II, was abolished confirming the cell culture data (Li et al. 2000).Experiments in mice in which the HNF-4 gene was disrupted in the adult liver usingAlb-Cre revealed that HNF-4 is essential not only for the establishment but also forthe maintenance of hepatic differentiation status (Hayhurst et al. 2001). Lipid andlipoprotein analysis of plasma of these mice revealed a dramatic reduction in totalcholesterol, HDL cholesterol, and triglycerides as well as a dramatic increase in theconcentration of bile acids (Hayhurst et al. 2001). Furthermore, FPLC analysisshowed that HDL cholesterol from the HNF-4 Liv KO mice eluted later than thatfrom controls indicative of the presence of smaller HDL populations. Interestingly,the expression of the two essential genes for HDL biogenesis, namely apoA-I andABCA1, was not affected in the livers of the HNF-4 Liv KO, suggesting that thereduction in the plasma HDL levels was the result of altered HDL remodeling ratherthan reduced biosynthesis. In agreement with this, the expression level of the HDLreceptor SR-BI gene was dramatically increased (Hayhurst et al. 2001).Studies in transgenic mice expressing the human apoA-I gene under its ownregulatory sequences and clinical studies in humans have shown that fibrates have apositive effect on apoA-I gene transcription as well as on plasma HDL levels. Theincrease in human apoA-I gene transcription by fibrates is mediated by peroxisomeproliferator-activated receptor α (PPARα) which binds to a PPRE on the proximalapoA-I promoter as a heterodimer with RXRα (Tzameli and Zannis 1996; Staelsand Auwerx 1998). This was confirmed by in vivo experiments performed in micethat express human apoA-I under the control of its own promoter but lack theexpression of PPARα. When these mice were given fenofibrate (FF) or gemfibrozil(GF) for 17 days, an increase in plasma HDL-C levels was observed by FF and to alesser extent by GF only in the mice that express endogenous PPARα (Duezet al. 2005). The fibrate-treated mice had larger HDL particles possibly due to theupregulation of phospholipid transfer protein and downregulation of SR-BI (Duezet al. 2005). Interestingly, the apoA-I gene cannot be upregulated by fibrates inrodents due to a three base pair difference in the PPRE rendering the rodent apoA-IPPRE nonfunctional (Vu-Dac et al. 1994). In line with the above findings, liver-specific inactivation of the PPARα heterodimer partner retinoid X receptor α(RXRα) gene in mice was associated with increased expression of the apoA-Igene (Wan et al. 2000).In a clinical study involving 234 patients with combined hyperlipidemia, bothFF and GF reduced triglycerides and increased HDL-C to a similar extent, but onlyFF treatment increased apoA-I plasma levels, and this was in agreement with theprevious clinical trials (Schaefer et al. 1996; Durrington et al. 1998; Sakaiet al. 2001; Duez et al. 2005).A humanized apoA-I transgenic mouse model expressing human apoA-I underits own regulatory sequences in a mouse apoA-I null background was recently usedto identify by global gene expression profiling candidate genes that affect lipid andlipoprotein metabolism in response to fenofibrate treatment (Sanoudou et al. 2009).Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 121Bioinformatical analysis and stringent selection criteria (twofold change, 0 % falsediscovery rate) identified 267 significantly changed genes. In contrast to the studyby Duez et al. discussed above (Duez et al. 2005), fenofibrates (FF) did notsignificantly alter the levels of hepatic human apoA-I mRNA and plasma apoA-Iprotein. This could be due to differences either in the mouse models used (forinstance, the humanized apoA-I mouse of Sanoudou et al. has 2.1 kb apoA-Ipromoter fragment, whereas the model of Duez et al. has a 5.5 kb apoA-I promoter)or the doses of fibrates (0.2 % w/w in the paper of Duez et al. and 0.03 % in thepaper by Sanoudou et al.). Despite the lack of apoA-I responsiveness, the FFtreatment increased cholesterol levels 1.95-fold mainly due to the increase inHDL-C. The observed changes in HDL were associated with the upregulation ofgenes involved in phospholipid biosynthesis and lipid hydrolysis, as well as in thephospholipid transfer protein. The gene encoding the estrogen receptor-relatedgamma (ESRRG) transcription factor was upregulated 2.36-fold by FF and had asignificant positive correlation with genes of lipid and lipoprotein metabolism andmitochondrial functions, indicating an important role of this orphan receptor inmediating the FF-induced activation of a specific subset of its target genes(Sanoudou et al. 2009).In addition to HNF-4 and PPARα, the two HREs of the proximal human apoA-Ipromoter bind apoA-I regulatory protein 1 (ARP-1) and liver receptor homologue-1(LRH-1) which repress and activate the apoA-I promoter, respectively, as illustratedin Fig. 1 (Ladias and Karathanasis 1991; Delerive et al. 2004). LRH-1 is a member ofthe fushi tarazu subfamily of nuclear receptors that is highly expressed in the liver,intestine, pancreas, and ovary (Fayard et al. 2004). In the liver, LRH-1 plays a keyrole in cholesterol homeostasis, through the control of the expression of genes that areimplicated in bile acid biosynthesis and enterohepatic circulation such as CYP7A1,CYP8B1, and ABCG5/8 (del Castillo-Olivares and Gil 2000; Freeman et al. 2004;Kir et al. 2012; Back et al. 2013), reverse cholesterol transport (SR-BI, apoA-I)(Schoonjans et al. 2002; Delerive et al. 2004), and HDL remodeling (CETP) (Luoet al. 2001). However, mice with targeted inactivation of the LRH-1 gene in the liverare characterized by physiological levels of HDL cholesterol, LDL cholesterol, andtriglycerides but have a profound effect on bile acid composition in the liver whichleads to reduced intestinal reuptake of bile acids and to the enhanced removal oflipids from the body (Mataki et al. 2007). Recent data suggest that LRH-1 functionsin a compensatory safeguard mechanism for adequate induction of bile salt synthesisunder conditions of high bile salt loss (Out et al. 2011).The two HREs of the apoA-I promoter also mediate the response of apoA-I tothyroids, retinoids, and bile acids via heterodimers of RXRα with thyroid hormonereceptor β (TRβ), retinoic acid receptor α (RARα), and farnesoid X receptor α(FXRα), respectively (Rottman et al. 1991; Hargrove et al. 1999). Althoughretinoids activate apoA-I gene expression, thyroids have dual effects on apoA-Ipromoter activity, whereas bile acids inhibit apoA-I gene expression (Tayloret al. 1996; Tzameli and Zannis 1996; Srivastava et al. 2000; Claudelet al. 2002). As shown in Fig. 1, in response to bile acids, FXR downregulatesapoA-I gene transcription by two complementary mechanisms: (a) a direct binding122 D. Kardassis et al.to the apoA-I HRE and (b) an indirect mechanism via the induction of smallheterodimer partner (Bavner et al. 2005) which, in turn, represses the activity ofLRH-1 (Delerive et al. 2004).The nuclear receptor constitutive androstane receptor (CAR) regulates thedetoxification of xenobiotics and endogenous molecules. In mice, the specificCAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) decreasedHDL cholesterol and plasma apoA-I levels in a CAR-specific manner (Massonet al. 2008). In transient transfections, CAR decreased the activity of the humanapoA-I promoter in the presence of TCPOBOP, but the mechanism by which thisrepression is facilitated remains unknown (Masson et al. 2008).Ligands of the vitamin D receptor (VDR) were also shown to affect negativelyapoA-I gene expression in hepatic cells (Wehmeier et al. 2005). In VDR KO mice,serum HDL-C levels were 22 % higher and the mRNA levels of apoA-I were49.2 % higher compared with WT mice. The mechanisms by which VDR ligandsaffect HDL levels remain unclear (Wang et al. 2009).1.2.1 The Role of the Distal Enhancer in apoA-I Gene TranscriptionIn addition to its own promoter, optimal expression of the human apoA-I gene inhepatic and intestinal cells requires the presence of a 200 bp transcriptionalenhancer element located downstream of the apoA-I gene, 500 bp upstream ofthe first exon of the adjacent apoC-III gene. This regulatory region, whichcoordinates the expression of all three genes of the apoA-I/C-III/A-IV cluster,contains two hormone response elements that bind HNF-4 and differentcombinations of ligand-dependent nuclear receptors as well as two binding sitesfor the ubiquitous transcription factor specificity protein 1 (Sp1) (Kardassiset al. 1997; Lavrentiadou et al. 1999). The mutagenesis of the HREs and of theSp1 sites reduced the activity of the apoA-I promoter/C-III enhancer cassette in cellcultures and abolished the binding of the corresponding factors (Kardassiset al. 1997; Lavrentiadou et al. 1999).The contribution of the HREs and the Sp1 binding sites to the tissue-specificexpression of the apoA-I gene in vivo was addressed using transgenic mice bearingthe WT apoA-I/apoC-III gene cluster under the control of their regulatory regionsor the same cluster bearing mutations in different regulatory elements(Georgopoulos et al. 2000; Kan et al. 2000, 2004). It was shown that mutations inone of the two HREs of the enhancer (element I4) abolished the intestinal expres-sion and reduced the hepatic expression of the adjacent apoA-I gene to 20 % of thecontrol. Mutations in the two HREs of the proximal apoA-I promoter reduced thehepatic and intestinal expression of the apoA-I gene to approximately 15 % of thecontrol, whereas combined mutations in all three HREs totally eliminated theintestinal and hepatic expression of the apoA-I gene (Kan et al. 2000). Studies incell cultures established that HNF-4 and Sp1 factors are both required for thesynergy between the apoA-I promoter and the enhancer by physically interactingwith each other and forming transcriptional complexes in order to facilitate therecruitment of the basal transcriptional machinery (Kardassis et al. 2002). Theaforementioned mouse model that expresses human apoA-I under its own promoterRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 123and enhancer (Kan et al. 2000) is very useful for the in vivo characterization of themechanisms that regulate the expression of the apoA-I gene under physiological orpathological conditions as well as for the identification and validation of novelcompounds that are designed to upregulate human apoA-I gene transcription andserve as HDL-raising drugs. This is especially important in light of the differencesbetween the mouse and the human apoA-I promoters. For instance, the mouse genecannot be upregulated by fibrates due to a three base pair difference in the PPREcompared to the human promoter which responds to 0.2 % fibrates but not to 0.03 %fibrates as mentioned above.1.2.2 Other Factors Regulating apoA-I Gene TranscriptionFurther upstream from the two apoA-I HREs, an insulin response core element(IRCE) was identified and shown to bind Sp1 (Murao et al. 1998). Insulin-activatedsignaling pathways including the Ras/raf and the phosphatidylinositol 3-kinase(PI3K) have been shown to posttranslationally modify Sp1, and this leads toincreased apoA-I promoter activity (Mooradian et al. 2004).Early growth response protein 1 (EGR-1) is another transcription factor thatregulates apoA-I expression via the proximal HREs (Kilbourne et al. 1995; Cuiet al. 2002). Mice with experimental nephrotic syndrome are characterized by afivefold increase in the levels of EGR-1, and these changes were associated withhigh plasma apoA-I and HDL-C levels as well as apoA-I gene transcription in theliver (Zaiou et al. 1998). In line with these findings, mice deficient in EGR-1 havereduced plasma HDL-C and apoA-I as well as hepatic apoA-I mRNA levels (Zaiouet al. 1998). EGR-1 was shown to mediate the response of the apoA-I promoter toestrogens (Hargrove et al. 1999).The proximal apoA-I promoter also contains one element that binds the basicleucine zipper (bZip) factor CCAAT/enhancer-binding protein (C/EBP) andnuclear factor Y (NFY) and another element that binds hepatocyte nuclear factor-3β/FOXA2 (Papazafiri et al. 1991; Novak and Bydlowski 1997). Nuclear factorHNF-3β was shown to mediate the response of the apoA-I promoter toglucocorticoids (Hargrove et al. 1999).Several natural compounds with antioxidant, pro-estrogenic, or other activitieswere shown to affect apoA-I and HDL-C levels, and these studies are summarizedin Haas and Mooradian (2010).Pro-inflammatory cytokines including tumor necrosis factor α (TNFα) andinterleukin-1β (IL-1β) were previously shown to inhibit apoA-I gene expressionboth in cell cultures and in animals (Ettinger et al. 1994; Song et al. 1998).Furthermore, plasma levels of HDL-C and apoA-I were shown to be highlyincreased in mice deficient in the p50 subunit of the pro-inflammatory transcriptionfactor NF-κB (Morishima et al. 2003). In agreement with this observation, theactivation of NF-κB by lipopolysaccharide (LPS) caused a reduction in apoA-ImRNA and protein levels in HepG2 cells, whereas the inhibition of NF-κB viaadenovirus-mediated overexpression of IκBα abolished the reduction (Morishimaet al. 2003). This IκBα-induced apoA-I increase was blocked by preincubation withMK886, a selective inhibitor of peroxisome proliferator-activated receptor α, and124 D. Kardassis et al.mutations in the PPARα binding site in the apoA-I promoter abrogated thesechanges (Morishima et al. 2003). In a recent study, it was shown that apoA-Ipromoter activity in HepG2 cells is inhibited by TNFα in a c-Jun-dependent mannerbut no AP1-responsive element within the apoA-I promoter was reported to mediatethis effect (Parseghian et al. 2013). The inhibition of the expression of apoA-I andother HDL genes in hepatocytes during inflammation could also be mediated byHNF-4 which was previously shown to be negatively regulated by the TNFα/NF-κBsignaling pathway by physically interacting with NF-κB (Nikolaidou-Neokosmidou et al. 2006).1.3 Transcriptional Regulation of the ABCA1 GeneThe gene encoding the ATP-binding cassette transporter A1 (ABCA1) is expressedin the liver, small intestine, macrophages, kidney, and various other tissues(Langmann et al. 1999, 2003; Kielar et al. 2001; Wellington et al. 2002). ABCA1is an important regulator of HDL biogenesis in the liver and facilitates the removalof excess cholesterol from macrophages.ABCA1 is particularly abundant in macrophages (Langmann et al. 1999).ABCA1 expression in macrophages has little influence on HDL-C plasma levels(Haghpassand et al. 2001) but is an important factor in the prevention of cholesterolaccumulation in the macrophages found in the atherosclerotic plaque and theirtransformation into foam cells (Aiello et al. 2002). ABCA1 mRNA and protein arevery unstable, having a half-life of 1–2 h in murine macrophages (Wang and Oram2002). Fine-tuning regulatory mechanisms (transcriptional regulation as well asposttranscriptional and posttranslational modifications) are involved to ensure theconstant and inducible ABCA1 expression in macrophages. In this section, we willfocus on the transcriptional regulation of the ABCA1 gene with emphasis onmacrophages. Posttranscriptional and posttranslational regulation of this gene willbe discussed in later sections.The human ABCA1 gene mapped to chromosome 9q31.1 is composed of50 exons, which encode 2261-amino-acid residues (Santamarina-Fojoet al. 2000). The ABCA1 gene promoter contains a TATA box localized 24 bpupstream of the transcription initiation site, essential for promoter activity inmacrophages as well as in hepatocytes (Langmann et al. 2002). The engagementof alternative promoters and transcription initiation sites localized upstream of thefirst exon or inside the first intron of the gene enables the inducible and tissue-specific expression regulation of ABCA1 gene (Huuskonen et al. 2003; Singarajaet al. 2005). In addition, other transcriptional response elements of the promoterinfluence the constitutive and tissue-specific expression of ABCA1 (Fig. 2).1.3.1 Upregulatory Mechanisms of ABCA1 Gene ExpressionThe major transcription factors that upregulate ABCA1 gene expression inmacrophages are the nuclear receptors liver X receptors α and β (LXRα andLXRβ), both expressed by this cell type. LXRs heterodimerize with the retinoicRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 125X receptor (RXRα) to bind to the direct repeats separated by four nucleotides(direct repeat 4; DR4 elements) found at position62/47 of the ABCA1 promoter(Costet et al. 2000). Additional but not well-characterized LXREs are present at theintron 1 promoter of the ABCA1 gene (Singaraja et al. 2001). Since the LXRαpromoter is subject to autoregulation, the LXR-mediated gene expression is auto-amplified (Laffitte et al. 2001a). The natural ligands of LXR are severalhydroxylated derivatives of cholesterol (oxysterols) and include27-hydroxycholesterol (Schwartz et al. 2000; Fu et al. 2001), 22(R)-hydroxycholesterol (Costet et al. 2000), 20(S)-hydroxycholesterol (Schwartzet al. 2000), and desmosterol (Yang et al. 2006). Among these ligands,27-hydroxycholesterol which is endogenously produced by the action ofCYP27A1 enzyme represents the sensor of cholesterol loading in macrophages.In addition to these natural ligands, synthetic LXR ligands such as TO901317 havealso been developed. These synthetic LXR agonists upregulate ABCA1 in culturedmacrophages more efficiently than cholesterol loading via modified LDL particles(Sparrow et al. 2002).LXR tissue-selective gene transcription is dependent on co-regulatory proteins.For example, data showed that the activation of the ABCA1 promoter by LXRα/RXRα heterodimers and their ligands require Sp1 (Thymiakou et al. 2007). Theoverexpression of Sp1 increased ABCA1 mRNA level in HeLa cells and enhancedcellular cholesterol and phospholipid efflux in RAW 246.7 macrophages. Gel shiftexperiments revealed in vitro binding of Sp1 to 85/91 and 151/156 GCFig. 2 Regulatory regions, transcription factors, and signaling molecules (cytokines, growthfactors, metabolites, drugs) that modulate the expression of the ABCA1 gene in macrophagesand other tissues. Arrows and block lines denote activation and repression, respectively. Themechanisms are described in detail in the text. Abbreviations: retinoic acid receptor (RAR);retinoid X receptor (RXR); liver X receptor (LXR); sterol regulatory element-binding protein(SREBP); specificity protein 1 (Sp1); tumor necrosis factor α (TNFα), interleukin (IL); lipopoly-saccharide (LPS); interferon γ (IFNγ); angiotensin II (AngII); nuclear factor kappa beta (NF-κB);upstream stimulatory factor (USF); hypoxia-inducible factor (HIF); transforming growth factor β(TGFβ); peroxisome proliferator-activated receptor (PPAR); Janus kinase (Jak); signal transducerand activator of transcription (STAT)126 D. Kardassis et al.boxes. Moreover, it was shown that Sp3 competed with Sp1 for binding to the latterGC box, acting as a repressor (Langmann et al. 2002). Physical interactionsbetween Sp1 and LXRα require the N-terminal region of LXRα, which includesthe DNA-binding domain and two different domains of Sp1: the transactivationdomain B and the DNA-binding domain (Thymiakou et al. 2007). LXR agonists,such as the natural steroidal ligand 22(R)-hydroxycholesterol and the weak non-steroidal ligand GSK418224, differentially recruit coactivators and corepressorscompared with full LXR agonists, such as the nonsteroidal ligand T0901317 (Alberset al. 2006; Peng et al. 2008, 2011; Phelan et al. 2008). The synthetic oxysterol N,N-dimethyl-3β-hydroxycholenamide (DMHCA) caused a differential induction ofthe ABCA1 and the sterol regulatory element-binding protein (SREBP)-1c genes inhepatic and macrophage cell lines, as well as in mice (Quinet et al. 2004). Incholesterol-loaded or unloaded peritoneal macrophages, DMHCA increasedABCA1 mRNA, whereas SREBP-1c mRNA levels were downregulated (Quinetet al. 2004). Cineole, a small aromatic compound found in teas and herbs, consider-ably stimulated the transactivation potential of LXRα and LXRβ and inducedABCA1 expression in macrophages but significantly reduced the expression ofLXRα- and LXRα-responsive genes in hepatocytes (Jun et al. 2013). AnotherLXR agonist, ATI-111, had a strong effect on ABCA1 expression in macrophagesas well as in the intestine and small effect on ABCA1 expression in the liver.ATI-111 significantly stimulated SREBP-1c mRNA in some tissues but inhibitedthe conversion of SREBP-1c precursor form into its active form (Peng et al. 2011).These findings revealed that LXR agonists have a promising potential for theupregulation of the ABCA1 transporter and the promotion of the cellular lipidefflux capacity of macrophages. Due to the concomitant LXR-mediatedupregulation of two genes involved in the fatty acid biosynthesis, fatty acidsynthase (Joseph et al. 2002a), and SREBP-1c (Yoshikawa et al. 2001), the devel-opment of LXR agonists for therapeutic uses has been limited by their adverseeffects that include hepatic steatosis and hypertriglyceridemia. In order to dissoci-ate the positive effects of LXR agonists on cholesterol homeostasis from theadverse effects on fatty acid metabolism, the next step will be the discovery ofnew LXRβ-selective agonists and the synthesis of novel tissue-specific LXRligands with weaker transcriptional effects on SREBP-1c. SREBP-1a, a differentmember of the SREBP family of proteins, binds to several sites present inside intron1; however, the role of these elements in ABCA1 gene regulation is still unknown(Thymiakou et al., unpublished observations) (Fig. 2).Besides LXR, other nuclear receptors and transcription factors are involved inABCA1 gene regulation in macrophages. Retinoic acid receptor (RAR) activatorssuch as all-trans-retinoic acid and 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (arotinoid acid) were found to increaseABCA1 mRNA and protein levels in macrophages (Costet et al. 2003).Co-transfection experiments showed that the same DR4 promoter element of theABCA1 promoter binds RXR heterodimers in the following order: RARγ/RXRαbound stronger and activated the human ABCA1 promoter, and RARα/RXRαbound weaker, while no RARβ/RXRα binding was detected. However, inRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 127macrophages from RARγ/ mice, arotinoid acid still induced ABCA1 geneexpression and caused marked upregulation of RARα, suggesting that high levelsof RARα can compensate for the absence of RARγ (Costet et al. 2003).Peroxisome proliferator-activated receptors α (PPARα) and γ (PPARγ) are bothexpressed in human macrophages where they exert anti-inflammatory effects. Thehydroxylated derivative of linoleic acid, 13-hydroxy linoleic acid, a natural PPARagonist, and pioglitazone (PPARγ agonist) increased PPAR transcriptional activityand induced ABCA1 gene expression in macrophages (Kammerer et al. 2011;Ozasa et al. 2011). However, data suggest that these effects are indirect and mostprobably are mediated by LXRα (Chinetti et al. 2001). PPARδ activators appearedto induce ABCA1 gene expression and cholesterol efflux moderately and toincrease HDL levels in an obese-monkey model (Oliver et al. 2001).Recent data showed that Clock, a key transcription factor that controls circadianrhythm, is involved in ABCA1 regulation in macrophages (Pan et al. 2013). Thedominant-negative Clock mutant protein (ClockΔ19/Δ19) enhanced plasmacholesterol and atherosclerosis. Mutant ClockΔ19/19/apoE/ mice had macro-phage dysfunction, expressed low levels of ABCA1, and had higher levels ofscavenger receptors. Molecular studies revealed that Clock regulated ABCA1expression in macrophages by modulating the activity of upstream stimulatoryfactor 2 (Pan et al. 2013).Experimental data showed that signaling molecules upregulate ABCA1 expres-sion in macrophages, but the fine-tuning mechanism of modulation and the regu-latory regions involved remain to be elucidated. Transforming growth factor β1(TGFβ1) increased ABCA1 mRNA levels in cholesterol-loaded macrophages(Argmann et al. 2001; Panousis et al. 2001). It was demonstrated that in THP-1macrophage-derived foam cells, the LXRα pathway is involved in TGFβ-mediatedupregulation of ABCA1 expression (Hu et al. 2010b). Interleukin-6 (IL-6) signifi-cantly increased ABCA1 at both the mRNA and protein levels. This effect wasabolished by selective inhibition of the JAK2/STAT3 signaling pathway (Frisdalet al. 2011). ABCA1 mRNA levels were significantly increased by estradioltreatment of macrophages for a short period of time, suggesting a direct activationof the ABCA1 promoter via the estrogen receptor β (Schmitz and Langmann 2005).Membrane-permeable analogues of cyclic adenosine monophosphate (cAMP)induced the ABCA1 mRNA in macrophages and other cells by an unknownmechanism (Lin and Bornfeldt 2002). Inhibitors that are able to block the actionof phosphodiesterase 4 on cAMP have been found to increase ABCA1 mRNA andcellular cholesterol efflux (Lin and Bornfeldt 2002). Verapamil, a calcium channelblocker, enhanced ABCA1 transcription by an LXR-independent process (Suzukiet al. 2004). Toll-like receptor 2 agonist Pam(3)CSK(4) upregulated ABCA1 geneexpression in RAW 264.7 macrophages via the activation of the PKCη/phospho-lipase D2 signaling pathway (Park et al. 2013). S-Allylcysteine, the most abundantorganosulfur compound in aged garlic extract, also elevated ABCA1 content inhuman THP-1 macrophages (Malekpour-Dehkordi et al. 2013). The data showedthat stimulation with CXCL5 that has a protective role in atherosclerosis (Rousselle128 D. Kardassis et al.et al. 2013) induced ABCA1 expression in alternatively activated(M2) macrophages but not in classically activated (M1) macrophages.1.3.2 Negative Regulation of ABCA1 Gene TranscriptionThe thyroid hormone T3 strongly suppressed ABCA1 gene transcription(Huuskonen et al. 2004). It was demonstrated that T3 significantly inhibited theability of oxysterols to activate LXR. Moreover, the TR/RXR heterodimerscompeted with LXR/RXR for the DR4 element in the ABCA1 promoter(Huuskonen et al. 2004). A reciprocal negative cross talk exists between LXRsand STAT1 based on the competition for CREB-binding protein (Pascual-Garciaet al. 2013). This may explain the IFNγ-mediated downregulation of ABCA1 incholesterol-loaded macrophages (Panousis and Zuckerman 2000; Argmannet al. 2001; Ma et al. 2013). Moreover, TNFα downregulates ABCA1 as well asLXRα expression in macrophages via a PKCθ-dependent pathway (Ma et al. 2013).Unsaturated fatty acids decrease the expression of ABCA1 in RAW 264.7macrophages by a mechanism that involves LXR/RXR binding to the promoter(Uehara et al. 2007) and by modulation of the histone acetylation state(Ku et al. 2012). In MCSF-activated human monocytes, linoleic acid decreasesABCA1 gene expression (Mauerer et al. 2009). Geranylgeranyl pyrophosphate, aproduct of the mevalonate pathway that is used for protein isoprenylation,suppresses LXR-induced ABCA1 synthesis in two ways: as an antagonist of theLXR interaction with the steroid receptor coactivator-1 (SRC-1) and as an activatorof Rho GTP-binding proteins (Gan et al. 2001).Among the transcription factors that downregulate ABCA1 expression inmacrophages in an LXR-independent manner is the SCAN domain-containingzinc finger transcription factor ZNF202 which binds to a GnT motif in the region234/215 of the ABCA1 promoter (Porsch-Ozcurumez et al. 2001). High intra-cellular levels of ZNF202 prevented LXR/RXR-mediated induction of the ABCA1promoter in response to oxysterols (Porsch-Ozcurumez et al. 2001).The conserved E-box at position 140 binds transcription factors that modulatethe ABCA1 gene expression. Experimental data indicated that upstream stimu-latory factors (USF) 1 and 2, hepatocyte nuclear factor-1α (HNF-1α), andfos-related antigen (Fra)2 bind to the intact E-box of the human ABCA1 promoterand differentially modulate the gene expression: USF1 and USF2 enhanced andFra2 repressed ABCA1 promoter activity (Langmann et al. 2002; Yang et al. 2002).The same E-box element also binds the sterol regulatory element-binding protein2 (SREBP-2), a key regulator of cholesterol metabolism, which suppresses ABCA1gene transcription in response to cholesterol depletion (73).ABCA1 gene expression was severely decreased in the liver and peritonealmacrophages of diabetic mice (Uehara et al. 2002). This observation was explainedusing in vitro models in which it was revealed that acetoacetate downregulatesABCA1 mRNA and protein in HepG2 hepatocytes and RAW 264.7 macrophages(Uehara et al. 2002) and thus high glucose concentration decreases ABCA1 geneexpression in MCSF-activated monocytes (Mauerer et al. 2009). Data showed thatRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 129the treatment of THP-1 macrophages with 100 nM dexamethasone, a potentsynthetic ligand of the glucocorticoid receptor, decreases the expression of theABCA1 gene (Sporstol et al. 2007).LPS downregulates ABCA1 in macrophages; this inhibition was reverted by thetreatment with betulinic acid acting via the downregulation of miR-33 and suppres-sion of NF-κB pathway (Zhao et al. 2013). IL-18 and IL-12 synergistically decreaseABCA1 levels in THP-1 macrophage-derived foam cells through the IL-18 recep-tor/NF-κB/ZNF202 signaling pathway (Yu et al. 2012). ABCA1 expression wasstrongly suppressed by angiotensin (Ang) II at both mRNA and protein levels in adose-dependent manner in THP-1-derived macrophages, whereas ABCG1 expres-sion was not affected. The effect of Ang II on ABCA1 expression could bemediated by the angiotensin II type 1 (AT1) receptor (Chen et al. 2012a). It wasdemonstrated that clinically relevant concentrations of homocysteine (Hcy)decreased the mRNA and protein expression levels of ABCA1 in macrophages. Itwas revealed that mRNA expression and the activity of DNA methyltransferasewere increased by Hcy, which may explain the higher DNA methylation level ofABCA1 gene in macrophages incubated with Hcy (Liang et al. 2013).1.4 Transcriptional Regulation of the ABCG1 GeneABCG1 mediates cholesterol removal from macrophages to HDL particles, but notto lipid-free apoA-I (Kennedy et al. 2005; Fitzgerald et al. 2010). Although recentdata showed that the combined macrophage deficiency of ABCA1/G1 ispro-atherogenic, probably by promoting plaque inflammation (Westerterpet al. 2013), the data concerning the role of ABCG1 expression in macrophages iscontroversial. Two independent groups reported that LDLR/ mice lackingmacrophage ABCG1 show decreased atherosclerotic lesions (Baldan et al. 2006;Ranalletta et al. 2006), while others reported that the absence of macrophageABCG1 causes a modest increase in atherosclerotic lesions (Out et al. 2006).These contradictory results may be explained by recent data showing that theabsence of ABCG1 leads to increased lesions in early stages of atherosclerosisbut causes retarded lesion progression in more advanced stages of atherosclerosis inLDLR/ mice, suggesting that the influence of ABCG1 deficiency on lesiondevelopment depends on the stage of atherogenesis (Meurs et al. 2012).The human ABCG1 gene has been mapped to chromosome 21q22.3 and encodesfor a 678-amino-acid protein of 75.5 kDa molecular mass (Chen et al. 1996). TheABCG1 gene spans more than 70 kb and includes 15 exons, each containingbetween 30 and 1,081 bp, while the intron size is between 137 bp and more than45 kb. All exon-intron boundaries display the canonical GT/AG sequences.In contrast to the ABCA1 gene, the ABCG1 gene does not contain a canonicalTATA box in the promoter (Langmann et al. 2000).130 D. Kardassis et al.The regulation of ABCG1 expression has similarities with that of ABCA1(Fig. 3). Thus, LXR plays an important role in ABCG1 promoter activation.Experimental data indicated that various LXR ligands upregulate ABCG1 expres-sion. ATI-111, a novel steroidal LXR agonist, induces ABCG1 mRNA expressionin peritoneal macrophages more potently than T0901317 and inhibits its expressionin the liver, suggesting tissue selectivity (Peng et al. 2011). Different naturalcompounds, such as cineole and fucosterol that are LXR activators, are able toincrease ABCG1 levels in macrophages (Hoang et al. 2012; Jun et al. 2013). Recentdata showed that the knockdown of LXRα impaired cholesterol efflux in humanprimary macrophages, while LXRβ silencing had no detectable impact on theexpression of LXR target genes such as ABCA1 and ABCG1 and did not affectcholesterol efflux (Ishibashi et al. 2013). The indirect effects of LXR in ABCG1regulation were recently shown. Adiponectin treatment significantly increasedABCG1 mRNA and protein levels in macrophages from diabetic patients, whereasthe pharmacological or genetic inhibition of LXR abrogated this enhancement;these data demonstrated that the mechanism of adiponectin-mediated upregulationof ABCG1 includes LXRα (Wang et al. 2013b). Similar with ABCA1, TGFβupregulates the expression of ABCG1, while unsaturated fatty acids suppressABCG1 expression via the LXR pathway (Uehara et al. 2007; Hu et al. 2010b).The analysis of potential regulatory elements in the promoter region carried outusing the MatInspector program identified multiple Sp1 sites at positions 184,382, and 566, an AP2 binding site at position 222, a NF-κB site at position338, an E-box motif at position 233, a sterol regulatory element at position660, and the NFY binding site at position 198 in ABCG1 proximal promoter(Langmann et al. 2000). A functional genetic variant of the ABCG1 promoterassociated with an increased risk of myocardial infarction and ischemic heartdisease in the general population was revealed (Schou et al. 2012). This studyFig. 3 Regulatory regions, transcription factors, and signaling molecules (cytokines, growthfactors, metabolites, drugs) that modulate the expression of the ABCG1 gene. Arrows and blocklines denote activation and repression, respectively. The mechanisms are described in detail in thetext. Abbreviations: conjugated linoleic acid (CLA); glycogen synthase kinase 3β (GSK3β); sterolregulatory element-binding protein (SREBP); specificity protein 1 (Sp1)Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 131showed that the ABCG1 expression was decreased by approximately 40 % ing.-376C>T heterozygotes versus noncarriers. This gene polymorphism is includedin a Sp1 binding site located at position382/373 in the ABCG1 promoter. Thus,the presence of the 376 T allele reduced the binding and transactivation of thepromoter by Sp1, leading to a decreased ABCG1 expression (Schou et al. 2012).ZNF202 was identified as a transcriptional repressor of ABCG1 gene whichbinds at position 560 in the ABCG1 promoter (Porsch-Ozcurumez et al. 2001).Recently, microbiotic and dietary factors were shown to regulate the ABCG1expression. Protocatechuic acid (PCA), a gut microbiota metabolite of cyanidin-3formed by 0-β-glucoside, exerts an anti-atherogenic effect partially through theinhibition of miR-10b-mediated downregulation of ABCG1 expression (Wanget al. 2012a). Extra-virgin olive oil intake has been shown to improve the capacityof HDL to mediate cholesterol efflux and increased ABCG1 and ABCA1 expres-sion in human macrophages (Helal et al. 2013). Conjugated linoleic acids (CLAs)are minor components of the diet with many reported biological activities. It wasrevealed that in MCSF-differentiated monocytes, trans-9,trans-11-CLA, but notcis-9,trans-11-CLA and trans-10,cis-12-CLA, activated ABCG1 via SREBP-1c(Ecker et al. 2007). In addition, it was demonstrated that palmitic acid upregulatesthe ABCG1 gene, while high glucose concentration decreased ABCG1 geneexpression in MCSF-activated human monocytes (Mauerer et al. 2009).Among the downregulators of ABCG1, low doses of LPS strongly reduce theexpression of ABCG1 in bone marrow-derived macrophages through IL-1 receptor-associated kinase 1 (IRAK-1)/glycogen synthase kinase 3β (GSK3β)/retinoic acidreceptor α (RARα) signaling pathway (Maitra and Li 2013). HMG-CoA reductaseinhibitors, simvastatin and atorvastatin, decreased ABCG1-mediated cholesterolefflux in human macrophages, despite the fact that the protein expression remainedunaltered (Wang et al. 2013c). In THP-1 monocytes, 100 nM dexamethasone, asynthetic glucocorticoid, inhibited the mRNA expression of ABCG1, although theglucocorticoid receptor expression was very low in this cell line (Sporstolet al. 2007). IL-22, a member of the IL-10 cytokine family secreted primarily byTh17 and Th22 subsets of T lymphocytes, was induced by S100/calgranulin andimpaired cholesterol efflux in macrophages by downregulation of ABCG1 (Chellanet al. 2013).1.5 Transcriptional Regulation of the Apolipoprotein E GeneApolipoprotein E (apoE), a glycoprotein of 35 kDa, plays an important role inplasma cholesterol level regulation and in cholesterol efflux, as documented bystudies in patients and animal models with apoE deficiency or mutated apoE genes(Nakashima et al. 1994; Linton et al. 1995; von Eckardstein 1996; Van Ecket al. 1997; Grainger et al. 2004; Ali et al. 2005; Davignon 2005; Raffaiet al. 2005). ApoE is mainly synthesized by the liver but also by various cells andperipheral tissues (Zannis et al. 2001b). ApoE is a marker for the developmentalstate of macrophages; the culture of mouse bone marrow cells in vitro showed that132 D. Kardassis et al.mature macrophages, but not their monocytic precursors, synthesized apoE (Werband Chin 1983c). At the site of atherosclerotic lesion, apoE is provided byinfiltrated macrophages. Transgenic mice expressing apoE only in macrophagesare protected against atherosclerosis, even though the plasma levels of apoE areexceedingly low and the animals are hypercholesterolemic (Bellosta et al. 1995). Incontrast, transgenic mice with normal levels of apoE in plasma, but not inmacrophages, are more susceptible to atherosclerosis (Fazio et al. 1997). ApoEsecreted by macrophages within the atherosclerotic plaque facilitates the choles-terol efflux to exogenous acceptors (such as HDL), thus assisting the reversecholesterol transport to the liver. The uptake of acetylated LDL or cholesterolester-rich β-VLDL into peritoneal macrophages stimulates apoE synthesis andsecretion (Basu et al. 1981).The human apoE gene is located on chromosome 19 at the 50 end of a clustercontaining also apoC-I, apoC-IV, and apoC-II genes (Myklebost and Rogne 1988;Smit et al. 1988; Allan et al. 1995a, b). The regulation of apolipoprotein E genetranscription is a highly complex process and requires the interaction of transcrip-tion factors with the proximal promoters but also with the distal regulatory regions(Fig. 4).Fig. 4 Regulatory regions, transcription factors, and signaling molecules (cytokines, growthfactors, metabolites, drugs) that modulate the expression of the apoE gene. Arrows and blocklines denote activation and repression, respectively. The mechanisms are described in detail in thetext. Abbreviations: glucocorticoid receptor (GR); signal transducer and activator of transcription(STAT); Jun N-terminal kinase (JNK); retinoid X receptor (RXR); liver X receptor (LXR);specificity protein 1 (Sp1); activator protein 2 (AP2); nuclear factor kappa beta (NF-κB); upstreamregulatory region 3 binding protein (URE3BP); lipopolysaccharide (LPS); multienhancer2 (ME.2); transforming growth factor β (TGFβ)Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 1331.5.1 Proximal Regulatory Binding Sites Involved in the apoE GeneExpressionThe proximal apoE promoter is well conserved in humans and mice, having thesame localization of TATA box and GC box (Rajavashisth et al. 1985; Horiuchiet al. 1989). Multiple positive and negative elements that modulate apoE geneexpression have been detected on the apoE promoter, using different in vitrosystems (Larkin et al. 2000; Zannis et al. 2001b). Smith et al. analyzed the apoEpromoter in both expressing (HepG2) and non-expressing (HeLa) cells (Smithet al. 1988). Within the proximal 50-flanking sequence and the first intron, eightregions were identified which had a positive effect and three regions with a negativeeffect on apoE expression, in both HepG2 and HeLa cells (Smith et al. 1988). Theproximal apoE promoter contains a GC box transcriptional control element at 59/45, a nonspecific enhancer element at 366/246, an upstream regulatory ele-ment (URE1) at 193/124, and a downstream regulatory element at +44/+262(Paik et al. 1988). Within URE1, a sequence spanning 161/141, defined as apositive element for transcription, has the ability to act alone as an enhancerelement (Chang et al. 1990). This element interacts with Sp1 transcription factorthat constitutively binds the GC box motif, suggesting that Sp1 may play animportant role in the basal level of apoE expression, as well as in the activity ofthis enhancer element. Another regulatory element, termed URE3, was identified atposition 101/89 and found to bind a 300 kDa protein from placental nuclearextracts termed URE3 BP (Jo et al. 1995). DNase I footprinting revealed theexistence of two binding sites for recombinant AP2 in the regions from 48/74and from 107/135 of the apoE promoter (Olaisen et al. 1982; Smith et al. 1988;Garcia et al. 1996; Salero et al. 2001, 2003). Gel mobility-shift assays showed thedirect binding of LXRα/RXRα and LXRβ/RXRα to a low-affinity LXRE present inthe region 494/465 of the proximal promoter (Laffitte et al. 2001b). Otherstudies revealed that USF binds to an atypical E-box located in the 101/91region of the apoE promoter (Salero et al. 2003). The same group found that Zic1and Zic2 transcription factors can bind to three binding sites located at 65/54,136/125, and 185/174 in the apoE promoter and stimulate apoE geneexpression (Salero et al. 2001).Bacterial endotoxin and other inflammatory agents decrease apoE production(Werb and Chin 1983a, b; Gafencu et al. 2007). The apoE downregulation inmacrophages impaired the local beneficial effect of apoE during the plaque devel-opment. As a result, despite the fact that macrophages are present in the lesion, theirability to regress atherosclerosis is seriously compromised. We have previouslyreported the mechanisms of apoE downregulation in macrophages exposed toinflammatory conditions, similar to those found at the atherosclerotic site (Gafencuet al. 2007). Tumor progression locus 2 (Tpl2) and mitogen-activated proteinkinase/ERK kinase kinase 1 (MEKK1) were identified as the kinases that areprimarily responsible for the downregulation of apoE promoter activity by LPS.Tpl2 and MEKK1 signaling pathways converge to NF-κB and AP1, acting on theapoE core promoter 55/+73 (Gafencu et al. 2007).134 D. Kardassis et al.1.5.2 Distal Regulatory Binding Sites That Modulate apoE GeneExpression in MacrophagesDespite this complex transcription factor machinery that may be targeted to theapoE promoter, the promoter itself lacks the ability to direct gene transcriptionin vivo in any cells, in the absence of the distal enhancers (Shih et al. 2000). Inmany tissues, cell-specific distal enhancers regulate the expression of genes in theapoE/apoC-I/apoC-I0/apoC-IV/apoC-II gene cluster (Shih et al. 2000). The expres-sion of apoE in macrophages is controlled by two homologous enhancers (95 %identical in sequence), designated as multienhancer 1 (ME.1) and multienhancer2 (ME.2), containing 620 and 619 nucleotides, respectively (Shih et al. 2000).These enhancers are located at 3.3 and 15.9 kb downstream of the apoE gene,respectively. We demonstrated by chromosome conformational capture (3C) andtransient transfections that both ME.1 and ME.2 can interact with the apoE pro-moter only in phorbol 12-myristate 13-acetate (PMA)-differentiated macrophages,but not in undifferentiated monocytes (Trusca et al. 2011). The results showed thatthe interactions take place in antisense orientation of the promoter and ME.1/2. Ourdata obtained using a series of deletion mutants of the promoter or of the ME.2identified the fragment 100/+73 as the minimal region of the apoE promoter thatis activated by the ME.2. We showed that the entire sequence of ME.2 is necessaryfor an optimal interaction with the apoE promoter, but the 50 region of ME.2 is moreimportant than 30 region for enhancing apoE promoter activity (Trusca et al. 2011).The interaction of the apoE promoter with ME.1/2 facilitates the transcriptionalenhancement of the apoE gene by various transcription factors.LXRα and LXRβ and their oxysterol ligands are key regulators of apoE expres-sion in macrophages (Laffitte et al. 2001b; Joseph et al. 2002b; Mak et al. 2002b).The ability of oxysterols and synthetic ligands to regulate apoE expression inperitoneal macrophages as well as in adipose tissue is reduced in LXRα/ orLXRβ/ mice and abolished in double knockouts. However, basal expression ofapoE is not compromised in LXR null mice, suggesting that LXRs mediate lipid-inducible expression rather than tissue-specific expression of this gene (Laffitteet al. 2001b). Data revealed that LXR/RXR binds to a low-affinity LXRE present inthe apoE promoter as well as a high-affinity site conserved in both human ME.1 andME.2 (Laffitte et al. 2001b). Experimental data revealed that the ligand activationof the LXR/RXR heterodimer enhanced the activity of the reporter constructs underthe control of human ME.1 or ME.2 fused to the apoE proximal promoter (Laffitteet al. 2001b). Oxysterol-binding protein-related protein 1S (ORP1S) translocatesfrom the cytoplasm to the nucleus in response to sterol binding and then binds toLXRs, promoting the binding of LXRs to LXREs. Thus, ORP1S mediates theLXR-dependent transcription via the ME.1 and ME.2 of the apoE gene (Leeet al. 2012). An interesting finding was that the induction of apoE gene expressionby LXR agonists is attenuated by inhibitors of JNK and PI3K pathways (Huwaitet al. 2011). A similar inhibition was noticed in the case of TGFβ-induced expres-sion of apoE, which was prevented by pharmacological inhibitors of JNK, p38kinase, and casein kinase 2 (Singh and Ramji 2006).Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 135The synthetic glucocorticoid receptor (GR) ligand, dexamethasone, increasedapoE mRNA levels in mature macrophages up to sixfold over basal levels(Zuckerman et al. 1993). In silico analysis of the ME.1 and ME.2 revealed sometranscription factor binding motifs for the GR (Shih et al. 2000). The presence ofthese GR binding sites in the multienhancers may explain the apoE upregulation byGR ligands, but the biological activity of these GR biding sites remains to berevealed.TRANSFAC analysis of the apoE promoter, ME.1, and ME.2 showed thatSTAT1 transcription factor has a binding site only on the ME.2. Our data showedthat this binding site is biologically active and STAT1 specifically upregulates apoEgene expression via ME.2, in macrophages, but not in hepatocytes. The STAT1binding site was located in the 174/182 region of ME.2 (Trusca et al. 2011).Interestingly, a simultaneous increase in the expression of apoE and STAT1 wasrecorded after monocyte differentiation with PMA treatment (for 4 h). Our modelproposed that after DNA bending, which probably takes place during monocytedifferentiation, STAT1 bound on ME.2 interacts with the transcription initiationcomplex, leading to the activation of apoE expression. In addition, STAT1 caninteract and cooperate with other transcription factors bound on the ME.2 or on theapoE promoter, for the modulation of apoE gene expression. Recently, we haverevealed that STAT1 can interact with RXR and modulate gene expression of theapoC-II gene (Trusca et al. 2012). Since RXRα binds to the ME.2, we can speculatethat apoE expression in macrophages may be modulated by the STAT1-RXRαinteractions, similarly with apoC-II.1.6 Transcriptional Regulation of the Human apoM Genein the LiverApolipoprotein M (apoM) belongs to the lipocalin protein superfamily and differsfrom typical water-soluble apolipoproteins by its tertiary structure (Dahlback andNielsen 2009; Nielsen et al. 2009). ApoM is secreted primarily by the liver andassociates with HDL particles through its retained N-terminal signal peptide (Axleret al. 2008; Christoffersen et al. 2008). The silencing of the endogenous apoM genein mice showed a loss of pre-β-HDL particles and formation of large HDL particles(Wolfrum et al. 2005). In addition to its role in HDL remodeling, it was shown thatapoM is the sole carrier of the bioactive lipid sphingosine 1 phosphate (S1P) inHDL, thus mediating many of the atheroprotective properties of HDL in theendothelium (Christoffersen et al. 2011; Arkensteijn et al. 2013; Christoffersenand Nielsen 2013).The expression of apoM in the liver is primarily controlled by hepatocytenuclear factor-1α (HNF-1α) (Richter et al. 2003). HNF-1α/ mice arecharacterized by the complete absence of apoM from plasma. The plasmaconcentrations of other apolipoproteins in HNF-1α/ mice were either similar(apoA-II, apoB, apoC) or increased (apoA-I, apoE) compared to wild-type mice.This was not due to the absence of apoM since restoration of apoM gene expression136 D. Kardassis et al.in the liver via adenovirus-mediated gene transfer could not rescue the abnormalapolipoprotein profile (Wolfrum et al. 2005). The analysis of the plasma lipoproteinprofile of HNF-1α/ mice showed that similar to the apoM gene-silenced mice,plasma cholesterol was primarily associated with the HDL fraction. In addition, anabnormal large apoE-enriched HDL fraction that was identified as HDLc or HDL1was observed, suggesting that this abnormal lipid profile in HNF-1α/ mice maybe caused by the lack of apoM (Shih et al. 2001). In humans, HNF-1α regulatesapoM gene expression through direct binding to a conserved DNA element locatedin the proximal apoM promoter region between nucleotides 55 and 41 (Richteret al. 2003).ApoM gene expression in the liver is negatively regulated during inflammationor infection via pro-inflammatory cytokines such as TNFα or IL-1β (Feingoldet al. 2008). The HNF-1α binding element in the proximal human apoM promoteris a dual-specificity regulatory element that mediates the activation or repression ofapoM promoter activity by HNF-1 and by activator protein 1 (AP1) proteins (c-Junand JunB), respectively, in hepatic cells (Mosialou et al. 2011). Competitionexperiments showed that the binding of Jun proteins and HNF-1α to the apoMpromoter is mutually exclusive and chromatin immunoprecipitation assaysestablished that AP1 activation leads to the recruitment of c-Jun and JunB proteinsto the proximal apoM promoter with the simultaneous displacement of HNF-1(Mosialou et al. 2011). A similar mechanism of transcriptional repression viadual-specificity AP1-/HNF-1-responsive elements has been demonstrated in thecase of the promoter of the human apolipoprotein A-II gene (Mosialou et al. 2011).AP1 factors were shown to inhibit the promoters of the apolipoprotein C-III(Hadzopoulou-Cladaras et al. 1998) and ABCA1 (Mosialou and Kardassis unpub-lished) genes in hepatic cells suggesting a broader role of AP1 factors in lipoproteinmetabolism in the liver during inflammation.Besides HNF-1α, apoM gene transcription in the liver has been shown to becontrolled positively by liver receptor homologue-1 (LRH-1) and forkhead box A2(FOXA2) transcription factors which bind to distinct sites on the proximal apoMpromoter (Venteclef et al. 2008; Wolfrum et al. 2008). Bile acids suppress apoMexpression in vivo by inhibiting LRH-1 transcriptional activity via the recruitmentof small heterodimer partner (SHP) to the apoM promoter (Venteclef et al. 2008).Insulin, insulin-like growth factor I (IGF-I), and IGF-I potential peptide(IGF-IPP) were all shown to inhibit apoM gene expression in a dose- and time-dependent manner in primary human and murine hepatocytes via a signal transduc-tion pathway that involves the serial activation of phosphatidylinositol 3-kinase(PI3K) and protein kinase B (PKB) and the inactivation of Foxa2 (Xu et al. 2006).In HepG2 cells, glucose and insulin inhibited apoM gene expression in an additivemanner, while in hyperglycemic rats, serum apoM concentrations and hepaticapoM mRNA levels were significantly reduced (Zhang et al. 2007).The human apoM gene is under the control of various orphan- and ligand-dependent nuclear receptors (Mosialou et al. 2010). The overexpression via adeno-virus and silencing via siRNA established that HNF-4 is an important regulator ofapoM gene transcription in hepatic cells (Mosialou et al. 2010). In addition toRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 137HNF-4, homodimers of retinoid X receptor and heterodimers of retinoid X receptorwith receptors for retinoic acid, thyroid hormone, fibrates (peroxisome proliferator-activated receptor), and oxysterols (liver X receptor) were shown to bind withdifferent affinities to the proximal HRE in vitro and in vivo (Mosialouet al. 2010). These findings provide novel insights into the role of apoM in theregulation of HDL by steroid hormones and into the development of novelHDL-based therapies for diseases such as diabetes, obesity, metabolic syndrome,and coronary artery disease that affect a large proportion of the population inWestern countries.1.7 Transcriptional Regulation of the CETP GeneThe gene encoding the cholesterol ester transfer protein (CETP) plays an importantrole in human HDL metabolism because it facilitates the transfer of cholesterylesters from mature spherical HDL particles to VLDL/IDL lipoproteins in exchangeof triglycerides and its activity determines the plasma levels of HDL cholesterol(von Eckardstein et al. 2005; Tall et al. 2008).The CETP gene is expressed mainly in the liver, adipose tissue, and spleen and atlower levels in the small intestine, adrenal, kidneys, and heart (Jiang et al. 1991;Radeau et al. 1995). Atherogenic diets were shown to increase CETP mRNA levelsin rabbits and in human CETP transgenic mice (Quinet et al. 1990; Jianget al. 1992). Both LXRs and SREBPs were shown to bind to regulatory elementson the promoter of the CETP gene and regulate its transcription in response tointracellular cholesterol levels (Gauthier et al. 1999; Luo and Tall 2000). It wasshown recently that synthetic LXR agonists enhanced plasma CETP activity anddecreased HDL-C levels in cynomolgus monkeys and human CETP transgenicmice (Honzumi et al. 2010). The induction of CETP gene expression by the LXRagonist was significantly reduced by knocking down the expression of LXRα butnot LXRβ both ex vivo and in mice (Honzumi et al. 2010). In another study, it wasshown that the LXR agonist T0901317 markedly increased CETP mRNA levelsand CETP production in human differentiated macrophages but not in humanperipheral blood monocytes (Lakomy et al. 2009). In inflammatory mouse andhuman macrophages, LXR-mediated CETP gene upregulation was inhibited andthis inhibition was independent of lipid loading. It was concluded thatLXR-mediated induction of human CETP expression is switched on duringmonocyte-to-macrophage differentiation and is abrogated in inflammatorymacrophages (Lakomy et al. 2009).Other factors that regulate CETP promoter activity include Yin Yang 1 (YY1)that binds to the same element as SREBPs (Gauthier et al. 1999), the LRH-1 thatpotentiates the sterol-mediated induction of the CETP gene by LXRs (Luoet al. 2001), the orphan nuclear receptor ARP-1 (Gaudet and Ginsburg 1995) thatinhibits CETP promoter activity, the retinoic acid receptor (RAR) which regulatesCETP gene expression in response to all-trans-retinoic acid (Jeoung et al. 1999),and the CCAAT/enhancer-binding protein (C/EBP) which is an activator of CETP138 D. Kardassis et al.gene expression (Agellon et al. 1992). Binding sites for the ubiquitous transcriptionfactors SP1 and SP3 have been identified on the CETP promoter at positions 690,623, and 37 and seem to be essential for the basal CETP promoter activity(Le Goff et al. 2003).CETP gene expression was shown recently to be under regulation by bile acidsand their nuclear receptor farnesoid X receptor (FXR) (Gautier et al. 2013). It wasshown that plasma CETP activity and mass was higher in patients with cholestasisthan controls and this was associated with lower HDL-C levels (Gautier et al. 2013).In agreement with this observation, bile acid feeding of APOE3*Leiden miceexpressing the human CETP transgene controlled by its endogenous promoterdecreased HDL-C and increased plasma CETP activity and mass. An FXR responseelement (FXRE) was identified in the first intron of the human CETP gene whichcould be responsible for the upregulation of CETP gene expression in response tobile acids (Gautier et al. 2013). In another study, it was shown that FXRα binds toDR4 LXRE that is present in the proximal CETP promoter and repressesLXR-mediated transactivation of the CETP promoter by a competition mechanism(Park et al. 2008).1.8 Transcriptional Regulation of the PLTP GenePhospholipid transfer protein (PLTP) belongs to the lipopolysaccharide (LPS)binding/lipid transfer gene family that includes the LPS-binding protein (LBP),the neutrophil bactericidal/permeability-increasing protein (BPI), and thecholesteryl ester transfer protein (CETP). PLTP is essential in the transfer of verylow-density lipoprotein phospholipids into HDL (Jiang et al. 2012).PLTP is expressed ubiquitously, but the highest expression levels in humantissues were observed in the ovary, thymus, placenta, and lung (Day et al. 1994).Taking into account the organ size involved, the liver and small intestine appear tobe important sites for the overall PLTP expression. A high-fat, high-cholesterol dietcauses a significant increase in PLTP activity and in mRNA levels. Plasma PLTPactivity and PLTP mRNA levels in the liver and adipose tissues were significantlydecreased following LPS administration (Jiang and Bruce 1995).An FXR-responsive element (FXRE) has been found in the proximal PLTPpromoter that binds FXRα/RXRα heterodimers and mediates the response of thePLTP promoter to bile acids (Urizar et al. 2000). Fibrates were shown to increasePLTP gene expression by activating PPARs which bind to three PPAR-responsiveelements on the PLTP promoter (Tu and Albers 1999; Bouly et al. 2001). Two ofthese PPAR-responsive elements also seem to be responsible for the induction ofPLTP expression by high glucose (Tu and Albers 2001).The human PLTP promoter contains at least two LXR-responsive elements, onein the proximal and one in the distal region, that were shown to mediate PLTP generegulation by oxysterols ex vivo and in vivo (Cao et al. 2002; Mak et al. 2002a;Laffitte et al. 2003). It was recently demonstrated that LXR agonists activatetriglyceride synthesis and PLTP gene transcription by activating SREBP-1cRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 139(Okazaki et al. 2010). In concert with the increase in triglyceride synthesis, theincreased PLTP caused triglyceride incorporation into abnormally large VLDLparticles which were removed from plasma by LDL receptors, whereas in theabsence of LDL receptors, the large VLDLs accumulated and caused massivehypertriglyceridemia (Okazaki et al. 2010).Recently, microarray analysis following alteration of p53 status in severalhuman- and mouse-derived cells identified a group of 341 genes whose expressionwas induced by p53 in the liver-derived cell line HepG2 (Goldstein et al. 2012).Twenty of these genes encode proteins involved in many aspects of lipid homeo-stasis including PLTP (Goldstein et al. 2012).1.9 Transcriptional Regulation of the Bile Acid TransportersABCG5/ABCG8ATP-binding cassette half-transporters G5 and G8 (ABCG5 and ABCG8) playimportant roles in the control of sterol excretion from the liver (Fitzgeraldet al. 2010; Tarling and Edwards 2012; Li et al. 2013; Yu et al. 2014). Mutationsin either of these transporters leads to β-sitosterolemia, an autosomal recessivedisease characterized by premature coronary atherosclerosis and elevated levels ofphytosterols in plasma (Fitzgerald et al. 2010; Tarling and Edwards 2012; Liet al. 2013; Yu et al. 2014). Mice lacking ABCG5 and ABCG8 proteins havedecreased ability to secrete sterols into the bile (Yu et al. 2002a). Theoverexpression of ABCG5 and ABCG8 in the liver increases biliary cholesterolsecretion and decreases dietary cholesterol absorption (Yu et al. 2002b). The humanABCG5 and ABCG8 genes are oriented in a head-to-head configuration, they aretranscribed in opposite directions, and their transcription is coordinated by a short374 bp bidirectional promoter in the intergenic region (Remaley et al. 2002).The bidirectional promoter of ABCG5/ABCG8 genes contains a binding site forLRH-1 at positions 134–142 which is required for the activity of both the ABCG5and ABCG8 promoters (Freeman et al. 2004). Mutating this LRH-1 binding sitereduced promoter activity of the human ABCG5/ABCG8 intergenic region inHepG2 and Caco2 cells. Bile acids such as deoxycholic acid repressed ABCG5and ABCG8 promoters via the FXR-SHP-LXR pathway that was described above(Sumi et al. 2007).Dietary cholesterol feeding was shown to increase duodenal, jejunal, and hepaticexpression levels of ABCG5 and ABCG8 mRNA in wild-type mice (Bergeet al. 2000). The increase in ABCG5 or ABCG8 gene expression by diet wascompromised in mice lacking either LXRα or both LXRα and LXRβ (Repaet al. 2002). Both the RXR-specific agonist LG268 and the LXR-specific agonistT0901317 caused upregulation of ABCG5 and ABCG8 mRNA expression in theliver and intestine of wild-type mice but not in LXRα/β/ mice (Repa et al. 2002).To identify functional LXREs that control the expression of the ABCG5/ABCG8genes in response to oxysterols, a recent study searched for evolutionarilyconserved regions (ECRs) between the human and the mouse genes and identified140 D. Kardassis et al.23 ECRs which were studied by luciferase assays for LXR responsiveness (Backet al. 2013). Two ECRs were found to be responsive to the LXR and binding ofLXRα to these regions was verified (Back et al. 2013).The bidirectional promoter of the ABCG5/G8 genes was shown to bind HNF-4and GATA transcription factors and to be regulated by these factors in a coopera-tive manner and independent of the orientation of the bidirectional promoter (Sumiet al. 2007).It was shown that the expression of both ABCG5 and ABCG8 genes isupregulated in the livers of mice with genetic ablation of the insulin receptorgene (LIRKO mice) both at the mRNA and the protein levels (Biddingeret al. 2008). In agreement with these findings, insulin suppressed the expressionof ABCG5 and ABCG8 genes at subnanomolar concentrations and in a dose-responsive manner in rat hepatoma cells (Biddinger et al. 2008). The observationthat the short intergenic region responded to insulin in both the ABCG5 andABCG8 orientations suggested the presence of an element in the intragenic regionof the ABCG5 and ABCG8 genes that responds to insulin. Using ex vivo andin vivo approaches, it was shown that insulin resistance leads to the activation of theforkhead box 1 (FOXO1) transcription factor which binds to the bidirectionalpromoter and activates the transcription of both genes severalfold (Biddingeret al. 2008).1.10 Transcriptional Regulation of the HDL Receptor SR-BIThe gene encoding the HDL receptor scavenger receptor class B type I (SR-BI) isexpressed at high levels in the liver and steroidogenic tissues. Several transcriptionfactors have been shown to bind to the human or rodent SR-BI promoter and toregulate SR-BI gene transcription in a positive or negative manner.The steroidogenic factor-1 (SF-1) has been shown to regulate both the humanand rat SR-BI promoters and to serve as mediator of the cAMP-dependent regu-lation of the SR-BI gene in response to steroidogenic hormones (Lopez et al. 1999).Liver X receptors α and β and PPARα and γ were shown to bind to distal LXREand PPARE, respectively, on the human and rat SR-BI promoters and regulate theexpression of the human SR-BI gene in response to oxysterols and fibrates (Lopezand McLean 1999; Malerod et al. 2002, 2003), whereas HNF-4 enhances thePPARγ-mediated SR-BI gene transcription (Malerod et al. 2003; Zhanget al. 2011). As discussed above, conditional inactivation of the HNF-4 gene inthe liver of adult mice was associated with a significant increase in hepatic SR-BImRNA levels and a decrease in plasma HDL-C levels, suggesting that HNF-4influences negatively the expression of the HDL receptor (Hayhurst et al. 2001).LRH-1 binds to a proximal response element on the human SR-BI promoter inan overlapping manner with SF-1 and activates the SR-BI promoter (Schoonjanset al. 2002). Retrovirus-mediated overexpression of LRH-1 in hepatic cells inducedSR-BI gene expression, and this was associated with histone H3 acetylation on theSR-BI promoter. In agreement with these findings, the SR-BI mRNA levels wereRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 141decreased in the livers of LRH-1(+/) animals providing evidence that LRH-1regulates SR-BI gene expression in vivo (Schoonjans et al. 2002).Estrogens regulate the activity of the rat SR-BI promoter via estrogen receptorsα and β (ERα and β) which bind to three different estrogen response elements (ERE)on the SR-BI promoter (Lopez et al. 2002; Lopez and McLean 2006). In endothelialcells, 17beta-estradiol (E2) increased the mRNA levels of the human SR-BI geneand the activity of the hSR-BI promoter, and this upregulation was protein kinase C(PKC) dependent since it was blocked by the PKC inhibitor bisindolylmaleimide Iand a dominant-negative mutant of PKC (Fukata et al. 2013).The mRNA levels of the mouse SR-BI gene were decreased in mice lacking theFXR nuclear receptor (FXR/ mice) (Lambert et al. 2003). When WT mice wereplaced on a diet containing 0.4 % of the FXR agonist cholic acid, the hepatic SR-BImRNA and protein levels increased in the wild-type but not in the FXR/ mice,indicating that bile acids positively regulate SR-BI gene expression via FXRs(Lambert et al. 2003). In agreement with these findings, treatment of humanhepatoma HepG2 cells with FXR ligands resulted in the upregulation of SR-BIboth at the mRNA and protein levels via FXR binding to a novel FXRE, a directrepeat 8 at position703/684 of the promoter (Chao et al. 2010). A natural ligandof FXR administered to mice increased hepatic SR-BI expression (Chaoet al. 2010). However, in another study, it was reported that bile acids inhibitSR-BI gene expression in the liver of mice and reduce the SR-BI promoter activity(Malerod et al. 2005). It was proposed that this inhibition was due to theFXR-mediated activation of SHP, which repressed the activity of LRH-1 thatbinds to the proximal SR-BI promoter (Malerod et al. 2005).The zinc finger transcription factor Kruppel-like factor 4 (KLF4) was shown tobind to a putative KLF4 element on the SR-BI promoter at position342/329 andupregulate its activity in peripheral blood mononuclear cells andPMA-differentiated THP-1 cells treated with HDL (Yang et al. 2010).Using high-throughput screening of 6,000 microbial secondary metabolite crudeextracts for the identification of compounds that upregulate the SR-BI promoter inHepG2 cells, several putative SR-BI upregulators were identified: hoxyl-30,5,7-hydroxyl isoflavone; (9R,13S)-7-deoxy-13-dihydrodaunomycinone; pratensein; theisoflavones formononetin, genistein, and daidzein; and the histone deacetylaseinhibitor trichostatin A (Yang et al. 2007, 2009; Bao et al. 2009). Some of thesecompounds were shown to increase the uptake of DiI-labeled HDL and the efflux ofcholesterol to HDL by cells.In steroidogenic tissues, SR-BI supplies the cells with exogenous cholesterol forstorage or for the synthesis of steroid hormones. In these tissues, SR-BI expressionwas shown to be upregulated by adrenocorticotropic hormone (ACTH) (Sunet al. 1999). The suppression of ACTH by the synthetic corticosteroid dexametha-sone (which inhibits the hypothalamic-pituitary axis and decreases ACTH secre-tion) decreased SR-BI levels. However, the mechanism by which ACTH andglucocorticoid regulate the expression of the SR-BI gene in steroidogenic tissuesis unclear. It was shown that the transcription of the human SR-BI gene is subject tofeedback inhibition by glucocorticoids in adrenal and ovarian cells. SR-BI mRNA142 D. Kardassis et al.levels were increased in adrenals from corticosterone-insufficient Crh/ mice,whereas corticosterone replacement by oral administration inhibited SR-BI geneexpression in these mice (Mavridou et al. 2010). The glucocorticoid-mediatedinhibition of SR-BI gene transcription required de novo protein synthesis and theglucocorticoid receptor (GR). No direct binding of GR to the SR-BI promoter couldbe demonstrated in vitro and in vivo, suggesting an indirect mechanism of repres-sion of SR-BI gene transcription by GR in adrenal cells (Mavridou et al. 2010).In the rat ovary, the uptake of cholesterol by the theca-interstitial cells ismediated by SR-BI. It was shown that insulin and the trophic hormone LH/cGHstimulate the expression of SR-BI in theca-interstitial cells and increase intracellu-lar cholesterol, which is subsequently mobilized for androgen biosynthesis(Li et al. 2001; Towns et al. 2005).In the adrenocortical cell line Y-1, adenovirus-mediated overexpression ofprolactin regulatory element-binding (PREB) protein, a transcription factor thatregulates prolactin expression in the anterior pituitary and is induced by cAMP,increased the levels of SR-BI protein and the activity of the SR-BI promoter bybinding to a PREB-responsive cis-element of the SR-BI promoter. Using smallinterfering RNA against PREB in Y-1 cells, the effects of cAMP on SR-BIexpression were attenuated. It was concluded that in the adrenal gland underconditions of cAMP increase, PREB regulates the transcription of the SR-BI gene(Murao et al. 2008).Intracellular sterol levels regulate SR-BI gene expression via SREBP-1a whichbinds to two sterol-responsive elements (SREs) present on the rat SR-BI promoter(Cao et al. 1997; Lopez and McLean 1999). The ubiquitous transcription factorsSP1 and SP3 bind to several GC-rich boxes present on the proximal SR-BIpromoter and have been shown to be important for the basal activity as well asthe SREBP-1a-mediated transactivation of the SR-BI promoter (Shea-Eatonet al. 2001).The expression of the SR-BI gene is also subject to negative regulation bytranscription factors including the orphan nuclear receptor dorsal-sensitive sexadrenal hypoplasia congenital critical region on the X chromosome gene1 (DAX-1), which was shown to repress the rat SR-BI promoter by interferingwith the SF-1- and SREBP-1a-mediated transactivation of the this promoter (Lopezet al. 2001). In addition, YY1 transcription factor binds directly to two sites on theSR-BI promoter and downregulates its activity by interfering with the binding ofSREBP-1a to the SR-BI promoter (Shea-Eaton et al. 2001).2 Posttranscriptional Regulation of HDL Genesby Noncoding RNAs and microRNAsAccording to the Encyclopedia of DNA Elements (ENCODE project), 76 % of thehuman genome is transcribed (Bernstein et al. 2012; Djebali et al. 2012). Whilenovel promoter and enhancer regions have been described (Bernstein et al. 2012;Sanyal et al. 2012), some of the newly described genes encode for noncoding RNAsRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 143including microRNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), circular RNAs (circRNAs), long noncoding RNAs(lncRNAs), trans-acting siRNAs (tasiRNAs), and several other noncoding RNAs.Their abundance and their recognized roles in transcriptional and epigenetic generegulation and their involvement in several diseases suggest the existence of anextensive regulatory network on the basis of RNA signaling (Mattick and Makunin2005). An important class of these endogenous noncoding RNAs is miRNAs.miRNAs are small noncoding RNAs (~22 nt) that have emerged as importantposttranscriptional regulators of different protein-coding genes (Bartel 2009)including those related to HDL metabolism (Davalos and Fernandez-Hernando2013).2.1 miRNAs: Biogenesis and FunctionAlthough originally described as regulators of developmental timing inCaenorhabditis elegans (Lee et al. 1993; Wightman et al. 1993), miRNAs did notreceive special attention until their widespread identification in different species(Lagos-Quintana et al. 2002; Reinhart et al. 2002) and their role in human diseaseswas uncovered (Calin et al. 2002). miRNAs are important posttranscriptionalregulators of gene expression through sequence-specific complementary bindingto the 30 untranslated region (UTR) of the target mRNA (Bartel 2009), even thoughcertain miRNAs can interact with other target mRNA regions including the 50 UTR,coding region, or intron–exon junction and even increase rather than decrease targetmRNA expression (Vasudevan et al. 2007; Orom et al. 2008; Tay et al. 2008;Schnall-Levin et al. 2010). The interaction of a miRNA with its target mRNAresults in the inhibition of translation and/or degradation of mRNAs (Guoet al. 2010; Krol et al. 2010).miRNA biogenesis and function have been intensively studied in recent years(Bartel 2009; Krol et al. 2010). Briefly, in most mammals, these small RNAs aretranscribed by RNA polymerase II into a primary long miRNA (pri-miRNA). Thepri-miRNA is then processed in the nucleus into an ~70 nucleotide precursorhairpin (pre-miRNA) by a multiprotein complex containing different cofactorsand two core components, a ribonuclease III (Drosha) and a double-strandedRNA-binding domain protein (DGCR8/Pasha). Some intronic miRNAs that bypassDrosha-mediated processing are produced by splicing and debranching (miRtrons).Pre-miRNAs are transported to the cytoplasm by Exportin 5 in a Ran-GTP-depen-dent manner (Lund et al. 2004) where they are further cleaved by an RNAse IIIenzyme called Dicer, generating the mature ~22 nt long miRNA/miRNA* duplex.Dicer-independent miRNA biogenesis has also been described (Cifuenteset al. 2010). The duplex miRNA is separated and one of the strands associateswith an argonaute protein (Ago) within the RNA-induced silencing complex(RISC), guiding the complex to the complementary sites within the 30 UTR of thetarget mRNA to induce mRNA repression and/or degradation. Although themiRNA/miRNA* duplex is initially produced in equal amounts, the miRNA*144 D. Kardassis et al.strand (passenger strand) is usually degraded. However, it also contains targetrecognition sites and is thus functional (Yang et al. 2011). Argonaute proteins aremajor players in miRNA-mediated gene regulation (Meister 2013). Althoughseveral aspects of miRNA biogenesis and function are well characterized, thedifferent factors that regulate their decay or turnover are not well known (Krolet al. 2010).Sequencing studies have identified ~2,000 miRNAs encoded in our humangenome, and prediction algorithms suggest that 60 % of human protein-codinggenes have conserved targets for pairing with miRNAs within their 30 UTR(Friedman et al. 2009). Factors that influence miRNA target selection include the“seed” sequence which consists of nucleotides 2–8 at the 50 end of the maturemiRNA (Bartel 2009) and tissue distribution or developmental stage, as certainmiRNAs are highly expressed or even restricted to certain cell types and can onlytarget their mRNA target if they are co-expressed in the same tissue at the sametime (Lagos-Quintana et al. 2002; Small and Olson 2011). In contrast to most plantmiRNAs, which bind with near perfect complementarity to their targets, mostmammalian miRNAs bind with mismatches and bulges and the mode of bindingdetermines the type of posttranscriptional repression (Carthew and Sontheimer2009). Recent experimental data suggest that around 60 % of seed interactionsare noncanonical, containing bulged or mismatched nucleotides which areaccompanied by specific, non-seed base pairing (Helwak et al. 2013). SomemiRNA-mRNA interactions also involve the 30 end of the miRNA (Helwaket al. 2013). Moreover, pseudogenes (Poliseno et al. 2010), long noncodingRNAs (lncRNAs) (Cesana et al. 2011), and circular RNAs (Hansen et al. 2013)that contain miRNA binding sites also influence miRNA activity, acting as com-peting endogenous RNAs (ceRNAs) and thus sequestering miRNAs and preventingthem from binding to their mRNA targets (Salmena et al. 2011).While the primary role of miRNAs seems to be the “fine-tuning” of geneexpression, their capacity to simultaneously bind and repress multiple targetgenes and similarly the possibility of a single mRNA to be targeted by multiplemiRNAs provide a mechanism to synchronize the coordinated regulation of a largenumber of transcripts that govern an entire biological process, thus resulting instrong phenotypic output (Mendell and Olson 2012). The high redundancy amongrelated and non-related miRNAs in regulating gene expression reduces the impor-tance of a particular miRNA under conditions of normal cellular homeostasis.However, compelling evidence suggests that under conditions of stress the functionof miRNAs become especially pronounced (Mendell and Olson 2012) and theirpharmacological modulation represents a novel approach to treat disease bymodulating entire biological pathways as described in Davalos and Chroni (2014).Compelling evidences have shown that miRNAs are present in the systemiccirculation and other human biological fluids associate with extracellularmicrovesicles, exosomes (Valadi et al. 2007; Hunter et al. 2008), Ago2 complex(Arroyo et al. 2011), or HDL (Vickers et al. 2011). Even exogenous miRNAs havebeen described in our circulating plasma (Wang et al. 2012b; Zhang et al. 2012).Although several circulating miRNAs have been implicated as disease biomarkersRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 145in several diseases (Allegra et al. 2012; Kinet et al. 2013) or secreted for intercellu-lar communication (Chen et al. 2012b), the biological significance, the factors thatmodulate their extracellular secretion, and the mechanisms by which they reach thetarget tissue still remain elusive. HDL has been shown to transport and deliverendogenous miRNAs to recipient cells with functional targeting capabilities(Vickers et al. 2011). Although the HDL miRNA signature from normal subjectswas found to be different from hypercholesterolemic subjects (Vickers et al. 2011),the extent by which HDL-bound miRNAs contribute to total circulating miRNAsand thus regulate target cell gene function is not clear (Wagner et al. 2013).In summary, miRNAs play a major role in almost every aspect of cellularfunction by posttranscriptional regulation of gene expression. Whether other non-coding RNAs (that modulate miRNA activity) or circulating or extracellularmiRNAs might modulate HDL metabolism is not known. However, it seemsobvious that by modulating miRNA activity, they might also have a role in HDLmetabolism.2.2 Posttranscriptional Modulation of HDL Metabolismby miRNAsFor normal function, mammalian cells must maintain cellular cholesterol levelswithin tight limits. Thus, complex mechanisms have been evolutionarily developedto control both cellular input from endogenous cholesterol biosynthesis or theuptake from circulating lipoproteins and cellular output by controlling cholesterolefflux. miRNAs have emerged as important regulators of HDL and cholesterolmetabolism, and their functions are summarized in Fig. 5 and described in detailbelow. Although miRNAs have increased the complexity of HDL metabolismregulation, our understanding of every single step in their regulation will providebetter opportunities to develop novel targets for their therapeutic modulation.2.2.1 Targeting ABCA1 and ABCG1ABCA1 is one of the most important proteins directly involved in the elimination ofexcess cholesterol from cells (cholesterol efflux). As described in this chapter,ABCA1 is regulated at the transcriptional, posttranscriptional, and posttranslationallevel. Its regulation is in accordance with the cellular need to handle cholesterollevels within tight limits, as excess of free cholesterol is deleterious to cells.ABCA1 mRNA contains a particularly long 30 UTR (>3.3 kb) as compared toother common genes involved in cholesterol and HDLmetabolism including LCAT(20 bp), apoA-I (55 bp), ApoA-II (112 bp), CETP (178 bp), apoB (301 bp), PDZK1(583 bp), ABCG1 (852 bp), SR-BI (959 bp), PCSK9 (1,269 bp), IDOL (1,496 bp),CAV-1 (1,898 bp), LIPG (2,386 bp), and LDLR (2,513 bp) (Davalos andFernandez-Hernando 2013). This unusually long 30 UTR of ABCA1 clearly raisesthe probability of posttranscriptional regulation by miRNAs (and probably othernoncoding RNAs). Different prediction algorithms indicate that ABCA1 can poten-tially be targeted by ~100 miRs. Some of them have been experimentally validated146 D. Kardassis et al.Fig. 5 Overview of posttranscriptional regulation by miRNAs of proteins involved in cholesterolefflux, RCT, and HDL metabolism. HDL metabolism and the role of individual proteins such asplasma enzymes, membrane transporters, and receptors are described in the text and moreextensively in other chapters. Autophagy, the cell catabolism process through the lysosomalmachinery, has been proposed to participate in cholesterol efflux. The induction of SREBPsinduces the expression of miR-33 family. The induction of LXR induces the expression ofmiR-144 and represses miR-26. The induction of FXR also induces miR-144. miRNAs in blackRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 147for their importance in cholesterol efflux, reverse cholesterol transport, and cardio-vascular disease, whereas other miRNAs still remain to be elucidated. ValidatedmiRNAs that directly target ABCA1 are miR-33 family, miR-758, miR-106b,miR-26, miR-144, miR-10b, miR-128-2, and miR-145 (Fig. 5).miR-33a and miR-33b play a crucial role in controlling cholesterol efflux andHDL function in concert with their host genes, the SREBP transcription factors(Najafi-Shoushtari et al. 2010; Horton et al. 2002; Horie et al. 2010; Marquartet al. 2010; Rayner et al. 2010). While both mature miRNAs only differ in twonucleotides, they are predicted to have largely overlapping sets of target genes inrodents (Rayner et al. 2011b; Horie et al. 2012) and nonhuman primates (Rayneret al. 2011a; Rottiers et al. 2013). The inhibition of the two miR-33 isoforms, eithergenetically or therapeutically, resulted in increased cholesterol efflux, increasedHDL levels, increased reverse cholesterol transport, reduced atherosclerosis, andreduced VLDL triglyceride levels. Although miR-33a and miR-33b target differentgenes involved in lipid homeostasis, fatty acid β-oxidation, insulin signaling, andbiliary transporters (Gerin et al. 2010; Davalos et al. 2011; Rayner et al. 2011a;Allen et al. 2012; Horie et al. 2013), their effects are mediated mainly by ABCA1.The human 30 UTR of ABCA1 has three functional binding sites for miR-33. Whilemost of the studies have been performed in tissues/cells directly related to lipo-protein metabolism and CVD (i.e., hepatocytes, macrophages), it is possible thatfrom these ~100 miRs that potentially target ABCA1 30 UTR, some of them mightbe relevant to other cells and tissues as well.Regarding other tissues, miR-106b (Kim et al. 2012) and miR-758 (Ramirezet al. 2011) were found to regulate neuronal cholesterol excess. While miR-758expression is particularly high in the brain and its expression is mediated by highcholesterol levels, the expression of miR-106 in brain tissues is low. However, theirrole in neuronal cholesterol efflux suggests that these miRNAs may contribute tothe regulation of cholesterol levels in the brain. miR-758 and miR-106 directlytarget the 30 UTR of ABCA1 by binding to two sites and one site, respectively.Moreover, miR-758 has other targets involved in neurological functions (Ramirezet al. 2011). miR-106b also targets the amyloid precursor protein (APP) andincreases the amyloid β (Aβ) peptide secretion and clearance (Kim et al. 2012).HDL metabolism is regulated by the LXRs which control the transcription ofseveral genes related to lipid metabolism in response to hydroxylated products ofcholesterol as described above (Calkin and Tontonoz 2012). Recent data suggestthat LXRs also regulate HDL metabolism posttranscriptionally (Sun et al. 2012; deAguiar Vallim et al. 2013; Ramirez et al. 2013; Vickers and Rader 2013). Theinduction of LXR resulted in the repression of miR-26 and induction of miR-26-a-1,Fig. 5 (continued) labels are validated miRNAs for HDL metabolism. miRNAs in red labels aresuggested miRNAs, but not fully validated, in HDL metabolism. All biological processes may notnecessarily happen in the same cell type. Abbreviations: endoplasmic reticulum (ER); earlyendosome (EE); cholesterol ester hydrolase (CEH); multivesicular bodies (MVB); lysosomalacid lipase (LAL) (Figure courtesy of Dr. Alberto Canfrán-Duque)148 D. Kardassis et al.and miR-144 that directly target the 30 UTR of ABCA1 and thus regulate choles-terol efflux, RCT, and HDL levels. While having one binding site for miR-26-a-1(Sun et al. 2012), the human 30 UTR of ABCA1 has several (up to seven) predictedbinding sites for miR-144 (Ramirez et al. 2013). miR-26-a-1 also targets theADP-ribosylation factor-like 7 (Arl7), which participates in cellular cholesterolefflux (Engel et al. 2004). miR-144 expression is also induced by the nuclearreceptor FXR (Vickers and Rader 2013). By controlling bile acid levels, FXRactivation will not only repress the expression of ABCA1 posttranscriptionallybut will also induce the expression of SR-BI, thereby resulting in an increaseduptake of plasma HDL cholesterol and increased cholesterol biliary excretion viaABCG5/ABCG8 (de Aguiar Vallim et al. 2013).Evolutionarily, the dual effect of LXR activation on miR-144 induction andmR-26-a-1 repression seems obvious, as cells must maintain cellular cholesterollevels within tight limits and these miRNAs might work as buffers against deleteri-ous variation in gene expression programs.Other miRNAs including miR-10b, miR-128-2, and miR-145 also regulatecellular cholesterol efflux by directly targeting the 30 UTR of ABCA1.Protocatechuic acid, an intestinal microbiota metabolite of cyanidin-3-O-glucoside,was found to repress miR-10b and thus regulate cholesterol efflux (Wanget al. 2012a). While in hepatocytes the inhibition of miR145 increases cholesterolefflux, in pancreatic islet beta cells it improves glucose-stimulated insulin secretion(Kang et al. 2013). The proapoptotic miRNA miR-128-2 was also found to regulatecholesterol efflux by targeting (one binding site) the 30 UTR of ABCA1 (Adlakhaet al. 2013). Recent data suggest that this miRNA family governs neuronalexcitability and motor behavior in mice (Tan et al. 2013) and that miR-128-2 might control neuronal cholesterol levels. RXRα is also a direct target ofmiR-128-2 (Adlakha et al. 2013). miR-27a and miR-148 were found to repress anABCA1 30 UTR luciferase reporter construct; however, their physiological role inregulating cholesterol efflux has not been described (Kang et al. 2013).ABCG1, the other member of the ATP-binding cassette transporter family,which also participates in cellular cholesterol efflux to HDL, was found to bedirectly regulated by some of these miRNAs. miR-33, miR-10b, and miR-128-2 regulate cholesterol efflux by posttranscriptionally regulating ABCG1 throughbinding to its 30 UTR (Rayner et al. 2010; Wang et al. 2012a; Adlakha et al. 2013).2.2.2 Targeting SR-BILiver regulation of SR-BI is primarily achieved posttranslationally through a PDZdomain-containing adaptor protein (PDZK1) (Kocher et al. 2003; Kocher andKrieger 2009) as described in detail in a later section of this chapter. Increasingevidence suggests that other posttranscriptional mechanisms, such as targeting bymiRNAs, also regulate SR-BI protein levels. Prediction miRNA analysis (www.targetscan.org) suggests that ~25 miRNA families might target SR-BI, most ofwhich are poorly conserved among mammals and vertebrates. miR-125a andmiR-455 were found to repress the lipoprotein-supported steroidogenesis bydirectly targeting the 30 UTR of SR-BI (Hu et al. 2012). In hepatic cells, whereRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 149SR-BI is expressed at higher levels, the expression of miR-125a was also high andits overexpression resulted in a reduced SR-BI-mediated selective cholesterol esteruptake. In contrast, miR-455 did not show any effect on hepatic SR-BI expression(Hu et al. 2012). miR-185, miR-96, and miR-223 were also found to directly targetthe 30 UTR of SR-BI and to repress HDL cholesterol uptake in hepatic cells (Wanget al. 2013a).2.2.3 Targeting Other miRNAs Related to HDL Biogenesisand FunctionmiR-27a was suggested to regulate apoA-I plasma levels, but the direct interactionto its 30 UTR was not evaluated (Shirasaki et al. 2013). Several other miRNAs wereproposed to regulate genes involved in cholesterol efflux, RCT, or HDL meta-bolism, including ABCG5 (Liu et al. 2012) or endothelial lipase (Kulyte et al. 2013),but either their direct interaction with the 30 UTR of these genes or their physio-logical effects on HDL metabolism were not directly assessed.Autophagy is the catabolic process by which unnecessary or dysfunctionalcellular components are degraded in the lysosome. During starvation, this break-down of cellular components contributes to the maintenance of cellular energylevels. The hydrolysis of cytoplasmic cholesteryl ester of lipid droplets is normallymediated by the action of neutral cholesteryl ester hydrolases. It has been recentlyrecognized that lipid droplets can also be delivered to lysosomes via autophagy,where lysosomal acid lipase can hydrolyze lipid droplet cholesteryl esters togenerate free cholesterol for cholesterol efflux (Ouimet et al. 2011).Several miRNAs have been described that regulate different targets inautophagy (Frankel and Lund 2012). miRNAs that target key pathways in lipid-loaded macrophage autophagy genes and/or cholesterol ester hydrolases might bepromising targets to promote cholesterol efflux (Davalos and Fernandez-Hernando2013). Caveolin, the major protein coat of caveolae, has been proposed to contri-bute to cellular cholesterol efflux (Truong et al. 2010; Kuo et al. 2011). There isaccumulating evidence that several miRNAs including miR-103, miR-107,miR-133a, miR-192, miR-802, and others target caveolin (Nohata et al. 2011;Trajkovski et al. 2011) but their contribution to cholesterol efflux, RCT, andHDL metabolism and their real physiological contribution to HDL function remainto be elucidated.3 Posttranslational Mechanisms of HDL Regulation3.1 ABCA1In addition to the transcriptional and posttranscriptional mechanisms describedabove, the expression of ABCA1 is also regulated at the posttranslational level.Following its synthesis, ABCA1 is inserted into the ER where it undergoes properfolding, N-glycosylation, dimerization, and disulfide bond formation and transportto the plasma membrane via the ER-Golgi system (Fig. 6) (Kang et al. 2010).150 D. Kardassis et al.Transport from the ER to the Golgi is prevented when ABCA1 interacts with theenzyme serine palmitoyltransferase 1 (SPTLC1) which participates in the biosyn-thetic pathway of sphingomyelin, a major phospholipid component of membranes.The ABCA1/SCPTL1 interaction may regulate the levels of intracellular pools ofsphingolipids when the cellular demands for these lipids are high. Pharmacologicalinhibition of SPTLC1 with myriocin, an atypical amino acid and antibiotic derivedfrom thermophilic fungi (Miyake et al. 1995), increases both the ABCA1 levels atthe plasma membrane and the ABCA1-mediated cholesterol efflux (Tamehiroet al. 2008). Mutations in ABCA1 that prevent normal glycosylation cause failureFig. 6 Overview of the mechanisms that regulate the expression of ABCA1, ABCG1, and SR-BIgenes at the posttranslational level. The mechanisms are described in the text. Abbreviations:endoplasmic reticulum (ER); Asp-His-His-Cys 8 (DHHC8); serine palmitoyltransferase1 (SPTLC1); guanine nucleotide-exchange protein 1 (BIG1); endosomal sorting complex requiredfor transport (ESCRT); Pro-GLu-Ser-Thr (PEST); calmodulin (Cam); lymphotoxin (LT); LT-βreceptor (LTβR); phospholipase D (PLD); protein kinase C δ (PKCδ); insulin receptor (IR);phosphatidylinositol 3-kinase (PI3K); 12/15-lipoxygenase (12/15LO); PDZ-containing kidneyprotein 1 (PDZK1); Na+/H+ exchanger regulator factor-3 (NHERF3); 4-hydroxy-2-nonenal(4HNE); monocyte chemoattractant protein-1 (MCP-1)Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 151of ABCA1 to exit the ER (Singaraja et al. 2006). In the Golgi compartment,ABCA1 undergoes palmitoylation at cysteine residues 3, 23, 1110, and 1111 bythe palmitoyltransferase Asp-His-His-Cys 8 (DHHC8) (Singaraja et al. 2009).Inhibiting this enzyme by drugs or preventing ABCA1 palmitoylation by site-specific mutagenesis reduced ABCA1 levels at the plasma membrane anddecreased efflux to apoA-I (Singaraja et al. 2009). The gene encodingcathepsin D, a lysosomal protease, was identified by comparative transcriptomicanalysis to be associated with low HDL-C levels in humans (Haidar et al. 2006).Blocking the activity or expression of cathepsin D reduced ABCA1 expression andprotein abundance as well as apoA-I-mediated lipid efflux by more than 70 % andcaused retention of ABCA1 in lysosomal compartments (Haidar et al. 2006). Veryrecently, BIG1 (brefeldin A-inhibited guanine nucleotide-exchange protein 1) wasfound to modulate ABCA1 trafficking and functions in the liver cells (Linet al. 2013). BIG1 depletion reduced surface ABCA1 on HepG2 cells and inhibitedby 60 % cholesterol efflux, whereas BIG1 overexpression had the opposite effects.RNA interference with BIG1 dramatically decreased the internalization andrecycling of ABCA1. This novel function of BIG1 was dependent on theguanine nucleotide-exchange activity and achieved through the activation ofADP-ribosylation factor 1 (Lin et al. 2013).The transport of ABCA1 to the plasma membrane is also facilitated by certainmembers of the family of small GTPase including Rab8, Rab4A, and Rab4B(Linder et al. 2009) (Jean and Kiger 2012).Calpain, a cysteine protease, cleaves ABCA1 at the PEST (proline, glutamicacid, serine, threonine) sequence (amino acids 1283–1306) that is located in thecytosolic loop of the molecule, and this cleavage takes place at the early endosomes(Fig. 6) (Yokoyama et al. 2012; Miyazaki et al. 2013). The deletion of the PESTsequence increases ABCA1 levels on the plasma membrane and apoA-I binding(Chen et al. 2005). The interaction with apoA-I stabilizes ABCA1 against thisdegradation (Arakawa and Yokoyama 2002; Wang et al. 2003; Arakawaet al. 2004). Importantly, the inhibition of calpain was shown to increase HDLbiogenesis in cultured cells, indicating that the inhibition of proteolytic degradationof ABCA1 could be a promising strategy for increasing HDL (Lu et al. 2008). Thephosphorylation of the PEST sequence of ABCA1 at Thr-1286 and Thr-1305regulates calpain-mediated proteolysis of ABCA1 since the ABCA1-T1286A/T1305A mutant could not be degraded by calpain (Martinez et al. 2003). Theinteraction of ABCA1 with apoA-I results in the dephosphorylation of theABCA1 PEST sequence and inhibition of calpain degradation, leading to anincrease of ABCA1 cell-surface expression (Martinez et al. 2003). Calmodulinwas also shown to interact with ABCA1 in the presence of Ca2+ and to protectfrom calpain-mediated degradation (Iwamoto et al. 2010).In addition to the calpain-mediated proteolytic degradation of ABCA1 describedabove, ABCA1 is subject to ubiquitin-mediated proteolysis. The presence ofubiquitinated ABCA1 in the plasma membrane of several cell lines was recentlydemonstrated (Mizuno et al. 2011). In HuH-7 cells, the degradation of cell-surface-resident ABCA1 was inhibited by the overexpression of a dominant-negative form152 D. Kardassis et al.of ubiquitin. Moreover, the disruption of the endosomal sorting complex requiredfor transport (ESCRT) pathway by the knockdown of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) delayed the degradation of ABCA1(Mizuno et al. 2011). These findings suggested that ubiquitination mediates thelysosomal degradation of plasma membrane ABCA1 and thereby regulates theexpression and cholesterol efflux functions of ABCA1 independently of thecalpain-mediated pathway.Unsaturated fatty acids such as oleate and linoleate were shown to destabilizeABCA1 protein and to inhibit ABCA1-mediated cholesterol efflux in macrophagesby a mechanism that depends on the activity of the enzyme acyl-CoA synthetase1 which is responsible for their activation to their CoA derivatives (Kanteret al. 2012). At the same time, unsaturated fatty acids increase the Ser phosphory-lation of ABCA1 via a phospholipase D (PLD)/protein kinase C δ pathway whichcontributes to ABCA1 destabilization (Fig. 6) (Wang and Oram 2007).Two additional proteins that were identified by two-hybrid screen to physicallyinteract with ABCA1 and to regulate ABCA1 intracellular localization and turnoverby interacting with its PDZ (PSD-95, Dlg, ZO-1) domain are the a1 and b1syntrophins (Munehira et al. 2004). Given the short half-life of ABCA1(t1/2 ¼ 1–2 h) (Wang et al. 2003), interfering with the accessory proteins or theenzymes that posttranslationally modify ABCA1 may prove a valuable strategy toregulate ABCA1-mediated HDL biogenesis in the liver and the intestine or toenhance cholesterol efflux in peripheral cells.Insulin enhances ABCA1 protein degradation in HepG2 cells via PI3K.In addition, it inhibits ABCA1 activity by phosphorylation at Tyr1206 (Nonomuraet al. 2011). The kinase that is responsible for this Tyr phosphorylation of ABCA1is not known, but it was hypothesized that it is the insulin receptor itself.TNFα and lymphotoxin α (LT) are key inflammatory mediators which alsocontribute to the atherogenic process (Schreyer et al. 1996, 2002; Pamiret al. 2012). TNF induces ABCA1 mRNA and protein levels as well as cholesterolefflux from cultured macrophages to apoA-I (Gerbod-Giannone et al. 2006). Theinduction of ABCA1 by TNFα depended primarily on NF-κB (Gerbod-Giannoneet al. 2006). It was also shown that the expression of the two TNF receptors isrequired to mediate full ABCA1 induction by TNFα. In addition, LT increasedABCA1 protein levels by inhibiting protein degradation through the LT-β receptor(LTβR) (Edgel et al. 2010).ABCA1-mediated cholesterol efflux and ABCA1 protein levels were shown tobe decreased by interferon γ (IFNγ) in murine macrophages and macrophage-derived foam cells (Panousis and Zuckerman 2000). This ABCA1 downregulationwas an early event in IFNγ-mediated activation of macrophages (Alfaro Leonet al. 2005) and was dependent on signal transducer and activator of transcription1 (STAT1) since similar effects were not observed in macrophages from STAT1KO mice (Wang et al. 2002).Additional protein kinases were shown to affect the activity and stability ofABCA1 at the posttranslational level including protein kinase A (PKA), proteinkinase C (PKC), Janus kinase 2 (JAK2), and casein kinase (CK2) (Tang et al. 2004).Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 153ABCA1 activity was shown to be regulated by the nuclear receptor LXRβ by amechanism that is distinct from the transcriptional induction of the ABCA1 genecaused by cholesterol accumulation. It was shown that at low cholesterol levels,LXRβ binds to ABCA1 and the ABCA1-LXRβ complex is stably localized at theplasma membrane but is unable to facilitate apoA-I-mediated cholesterol efflux.Exogenously added LXR ligands, which mimic cholesterol accumulation, causeLXRβ to dissociate from ABCA1, thus freeing ABCA1 for apoA-I binding andsubsequent cholesterol efflux (Hozoji et al. 2008; Hozoji-Inada et al. 2011).3.2 ABCG1Similar to ABCA1, the levels of ABCG1 transporter were shown to be subject toregulation at the posttranslational level (Fig. 6) (Tarling and Edwards 2012). Recentstudies showed that the presence of additional 12 amino acids between the WalkerB motif and the first transmembrane domain of ABCG1 can affect ABCG1 proteinstability (Engel et al. 2006). It was shown that stable overexpression of 12/15-lipoxygenase (12/15LO) in macrophages was associated with a 30 % reduction inHDL-mediated cholesterol efflux and reduced ABCG1 protein expression (Nagelinet al. 2008). Treatment of macrophages with the 12/15LO eicosanoid product12SHETE increased serine phosphorylation of ABCG1 and affected the stabilityof the protein (Nagelin et al. 2008). Proteasomal inhibitors blocked thedownregulation of ABCG1 and resulted in the accumulation of phosphorylatedABCG1 (Nagelin et al. 2009). Macrophages that lack 12/15LO had enhancedABCG1 expression, reduced ABCG1 phosphorylation, and increased cholesterolefflux. Conversely, macrophages that overexpress 12/15LO have reduced ABCG1expression, increased transporter phosphorylation, and reduced cholesterol efflux(Nagelin et al. 2009). It was also shown that 12/15LO regulates ABCG1 expressionand function through p38- and JNK2-dependent mechanisms (Nagelin et al. 2009).The activation of adenosine monophosphate-activated protein kinase (AMPK) inhuman aortic endothelial cells resulted in increased levels of ABCG1 protein via aposttranscriptional mechanism that involved increased stability of ABCG1 mRNA(Li et al. 2010a, b). The aminoimidazole carboxamide ribonucleotide (AICAR)-dependent induction of ABCG1 was associated with increased efflux of cellularcholesterol and 7-ketocholesterol to HDL and protected nitric oxide synthaseactivity and vascular reactivity (Li et al. 2010a, b).The protein levels of ABCG1 were shown to be controlled by palmitoylation inboth human embryonic kidney 293 cells and in mouse macrophage, J774(Gu et al. 2013). Five cysteine residues located at positions 26, 150, 311, 390,and 402 in the NH2-terminal cytoplasmic region of ABCG1 were shown to bepalmitoylated. The removal of palmitoylation at Cys311 by mutating the residue toAla (C311A) or Ser significantly decreased ABCG1-mediated cholesterol efflux.On the other hand, the removal of palmitoylation at sites 26, 150, 390, and 402 hadno significant effect (Gu et al. 2013).154 D. Kardassis et al.3.3 SR-BIThe stability of the HDL receptor SR-BI in the hepatocyte plasma membrane isvery important for its functions and is subject to posttranscriptional regulation(Fig. 6). SR-BI stability is primarily controlled by its adapter protein,PDZ-containing kidney protein 1 (PDZK1 or NHERF3 for Na+/H+ exchangerregulator factor-3), since PZK1-knockout mice exhibit a >95 % reduction inhepatic SR-BI protein (but not mRNA) and are characterized by hypercholesterol-emia and the presence of large cholesterol-rich HDL particles in the plasma(Kocher et al. 2003). In adrenal cells where the PDZK1 levels are very low,additional members of the NHERF family such as NHERF1 and NHERF2 (butnot NHERF4) were shown to specifically interact with SR-BI and reduce its proteinlevels (Hu et al. 2013). The data showed that the downregulation of SR-BI by theseNHERF1/2 factors significantly inhibits both HDL-CE uptake and steroid hormoneproduction by adrenal cells. It was concluded that the PDZK1 homologues act asphysiological translational/posttranslational regulators of the functional expressionof SR-BI (Hu et al. 2013).Feeding mice with an atherogenic diet was accompanied by a threefold post-translational downregulation of hepatic SR-BI at the protein level (Niemeieret al. 2009). A similar SR-BI downregulation was observed in transgenic miceoverexpressing SREBP-1a and SREBP-1c on chow diet, and it was associated witha decrease in the expression levels of PDZK1 (Niemeier et al. 2009).The levels of SR-BI protein were shown to be affected in two syndromes: thenephrotic syndrome and the Rett syndrome. It was shown that animals withnephrotic syndrome which is characterized by dyslipidemia, impaired high-densitylipoprotein (HDL)-mediated reverse cholesterol transport, and atherosclerosis(Vaziri et al. 2003) exhibited severe hypercholesterolemia, hypertriglyceridemia,reduced HDL/total cholesterol ratio, significant upregulation of the endocytic HDLreceptor ATP synthase mRNA and protein, and significant reduction of SR-BIprotein despite its normal mRNA abundance. The reduction in SR-BI protein levelsin animals with NS was accompanied by parallel reductions in PDZK1 mRNA andprotein levels (Vaziri et al. 2011). Rett syndrome, a genetic form of infantile autism,is the second most common cause of mental retardation in women (Amiret al. 1999). When compared to healthy subjects, patients with Rett syndromepresent with significant increases in total cholesterol (12 %), LDL cholesterol(15 %), and HDL-C (18 %) (Sticozzi et al. 2013). Skin fibroblasts isolated frompatients with Rett syndrome exhibited low levels of SR-BI as a consequence of itsassociation with 4-hydroxy-2-nonenal (4HNE), a product of lipid peroxidation thatis increased in patients with Rett syndrome, and increased ubiquitination (Sticozziet al. 2013). The role of the proteasome in SR-BI stability was confirmed using theproteasomal inhibitor (MG132). When Rett syndrome fibroblasts were pretreatedwith MG132, the loss of SR-BI was reversed demonstrating that SR-BI is degradedvia the proteasome machinery (Sticozzi et al. 2013).In hepatic cells and fibroblasts, SR-BI protein levels and SR-BI-mediated cho-lesterol transport (export and uptake) were shown to be affected by the Ras/MEK/ERK signaling cascade. This effect was mediated via PPARα-inducibleRegulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 155degradation pathways (Wood et al. 2011). Metabolic labeling experiments inprimary hepatocytes from mice demonstrated that fenofibrate enhances the degra-dation of SR-BI in a post-endoplasmic reticulum compartment (Lan and Silver2005). Moreover, fenofibrate-induced degradation of SR-BI was independent of theproteasome, the calpain protease, or the lysosome (Lan and Silver 2005).In primary macrophages and cell lines derived from female but not from malemice, tamoxifen and 4-hydroxytamoxifen increased SR-BI protein expression viathe estrogen receptor α (Dong et al. 2011). Because SR-BI mRNA expression andpromoter activity were not influenced by tamoxifen and 4-hydroxytamoxifen, it wasdemonstrated that the regulation of SR-BI by these substances takes place at thelevel of protein stability (Dong et al. 2011).The phosphatidylinositol 3-kinase (PI3K) pathway was shown to affect posi-tively the SR-BI-mediated HDL selective cholesterol ester uptake into humanHepG2 cells as this process was compromised in the presence of the PI3K inhibitorswortmannin and LY294002 (Shetty et al. 2006). These inhibitors also blocked thepositive effect of insulin on the SR-BI-dependent selective uptake. HDL cell-surface binding, receptor biotinylation studies, and confocal fluorescence micro-scopy of HepG2 cells expressing green fluorescent protein-tagged SR-BIdemonstrated changes in SR-BI subcellular localization and cell-surface expressionas a result of PI3K activation (Shetty et al. 2006). These data indicate that PI3Kactivation stimulates hepatic SR-BI function posttranslationally by regulating thesubcellular localization of SR-BI. It was recently shown that monocyte chemo-attractant protein-1 (MCP-1), a protein expressed by endothelial cells under inflam-matory conditions, decreases the cell-surface protein expression of ABCA1,ABCG1, and SR-BI in a dose-dependent and time-dependent manner (Huanget al. 2013). It was shown that PI3K activation corrected the MCP-1-induceddecreases in the cell-surface protein expression of the three transporters. MCP-1decreased the lipid uptake by HepG2 cells and the ABCA1-mediated cholesterolefflux to apoA-I, and this was reversed by the activation of PI3K. These datasuggested that MCP-1 impairs RCT activity in HepG2 cells by a PI3K-/AKT-mediated posttranslational regulation of ABCA1, ABCG1, and SR-BI cell-surfaceexpression (Huang et al. 2013).Finally, it was shown that hormones such as triiodothyronine (T3) andthyromimetics and some pharmacological agents such as probucol increase SR-BIlevels posttranscriptionally (Leiva et al. 2011).ConclusionsAs discussed earlier in this chapter as well as in other chapters of this handbook,plasma HDL cholesterol levels are determined by the relative rates of HDLbiogenesis and HDL catabolism which in turn are mediated by several lipid-carrying proteins, plasma enzymes, and membrane transporters. The deregu-lation of the activity or the expression of these proteins by mutations or duringdiseases is associated with non-physiological levels of HDL cholesterol, HDLdysfunction, and predisposition to coronary heart disease. Epidemiologicalstudies as well as experiments in genetically modified animals have providedunequivocal proof that raising HDL cholesterol levels may be therapeutic. Drugs156 D. Kardassis et al.that could specifically increase the rates of HDL biogenesis by inducing thelevels of apoA-I, ABCA1, or LCAT are anticipated to be of great clinical benefit.Other HDL proteins such as ABCG1, ABCG5/G8, CETP, and LPL could alsoprovide useful targets for HDL-based therapies that will interfere with HDLmaturation or catabolism without compromising HDL functionality. The devel-opment of novel HDL-raising drugs that will capitalize on HDL biogenesis willcertainly require a thorough understanding of the mechanisms and the moleculesthat are involved in HDL regulation at the transcriptional, posttranscriptional, orposttranslational level in hepatic cells as well as in other physiologically relevantcells such as the macrophages. For instance, understanding ABCA1 gene regu-lation in the macrophage has led to the elucidation of the important role of LXRsand their ligands in the reverse cholesterol transport and the development ofnovel LXR ligands which however have severe lipogenic effects in the liver andare thus of no clinical value today so the challenge here is to develop morespecific drugs that will avoid lipogenesis. Novel single- or dual-specificityagonists of peroxisome proliferator-activated receptor isoforms are alsoanticipated. Understanding how the levels of HDL proteins and their functional-ity are affected by inflammatory factors will enable us to develop novel drugs torestore HDL levels and functionality and thus reduce the atherosclerotic burdenin patients with chronic inflammatory diseases.In summary, the current challenges in the HDL field are to develop noveltherapies that would raise HDL cholesterol levels without compromising HDLfunctionality and to identify novel HDL-based biomarkers. The advances in thefield of HDL regulation may provide crucial insights and tools to achievethis goal.Acknowledgments This work was supported by the European Cooperation in Science andTechnology (COST) Action BM0904; by grants from the Greek Secretariat for Research andTechnology (SYNERGASIA-09 and THALIS MIS377286 ) to Dimitris Kardassis; by grants fromthe Spanish Instituto de Salud Carlos III (FIS, PI11/00315) to Alberto Dávalos; by a grant of theRomanian National Authority for Scientific Research, CNCS – UEFISCDI, project number PN-II-ID-PCE-2011-3-0591, and the Romanian Academy to Anca Gafencu; and by an NIH grantHL-48739 to Vassilis I. Zannis. We thank Dr. Ovidiu Croitoru for the graphic design.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAdcock IM, Caramori G (2001) Cross-talk between pro-inflammatory transcription factors andglucocorticoids. Immunol Cell Biol 79:376–384Adlakha YK, Khanna S, Singh R, Singh VP, Agrawal A, Saini N (2013) Pro-apoptotic miRNA-128-2 modulates ABCA1, ABCG1 and RXRalpha expression and cholesterol homeostasis.Cell Death Dis 4:e780Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 157Agellon LB, Zhang P, Jiang XC, Mendelsohn L, Tall AR (1992) The CCAAT/enhancer-bindingprotein trans-activates the human cholesteryl ester transfer protein gene promoter. J Biol Chem267:22336–22339Aiello RJ, Brees D, Bourassa PA, Royer L, Lindsey S, Coskran T et al (2002) Increasedatherosclerosis in hyperlipidemic mice with inactivation of ABCA1 in macrophages.Arterioscler Thromb Vasc Biol 22:630–637Albers M, Blume B, Schlueter T, Wright MB, Kober I, Kremoser C et al (2006) A novel principlefor partial agonism of liver X receptor ligands. Competitive recruitment of activators andrepressors. J Biol Chem 281:4920–4930Alfaro Leon ML, Evans GF, Farmen MW, Zuckerman SH (2005) Post-transcriptional regulationof macrophage ABCA1, an early response gene to IFN-gamma. Biochem Biophys ResCommun 333:596–602Ali K, Middleton M, Pure E, Rader DJ (2005) Apolipoprotein E suppresses the type I inflammatoryresponse in vivo. Circ Res 97:922–927Allan CM, Walker D, Segrest JP, Taylor JM (1995a) Identification and characterization of a newhuman gene (APOC4) in the apolipoprotein E, C-I, and C-II gene locus. Genomics 28:291–300Allan CM, Walker D, Taylor JM (1995b) Evolutionary duplication of a hepatic control region inthe human apolipoprotein E gene locus. Identification of a second region that confers high leveland liver-specific expression of the human apolipoprotein E gene in transgenic mice. J BiolChem 270:26278–26281Allegra A, Alonci A, Campo S, Penna G, Petrungaro A, Gerace D et al (2012) CirculatingmicroRNAs: new biomarkers in diagnosis, prognosis and treatment of cancer (review). Int JOncol 41:1897–1912Allen RM, Marquart TJ, Albert CJ, Suchy FJ, Wang DQ, Ananthanarayanan M et al (2012)miR-33 controls the expression of biliary transporters, and mediates statin- and diet-inducedhepatotoxicity. EMBO Mol Med 4:882–895Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY (1999) Rett syndrome iscaused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet23:185–188Arakawa R, Yokoyama S (2002) Helical apolipoproteins stabilize ATP-binding cassette trans-porter A1 by protecting it from thiol protease-mediated degradation. J Biol Chem 277:22426–22429Arakawa R, Hayashi M, Remaley AT, Brewer BH, Yamauchi Y, Yokoyama S (2004) Phosphory-lation and stabilization of ATP binding cassette transporter A1 by synthetic amphiphilic helicalpeptides. J Biol Chem 279:6217–6220Argmann CA, Van Den Diepstraten CH, Sawyez CG, Edwards JY, Hegele RA, Wolfe BMet al (2001) Transforming growth factor-beta1 inhibits macrophage cholesteryl ester accu-mulation induced by native and oxidized VLDL remnants. Arterioscler Thromb Vasc Biol 21:2011–2018Arkensteijn BW, Berbee JF, Rensen PC, Nielsen LB, Christoffersen C (2013) The apolipoproteinm-sphingosine-1-phosphate axis: biological relevance in lipoprotein metabolism, lipiddisorders and atherosclerosis. Int J Mol Sci 14:4419–4431Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF et al (2011) Argonaute2complexes carry a population of circulating microRNAs independent of vesicles in humanplasma. Proc Natl Acad Sci USA 108:5003–5008Axler O, Ahnstrom J, Dahlback B (2008) Apolipoprotein M associates to lipoproteins through itsretained signal peptide. FEBS Lett 582:826–828Back SS, Kim J, Choi D, Lee ES, Choi SY, Han K (2013) Cooperative transcriptional activation ofATP-binding cassette sterol transporters ABCG5 and ABCG8 genes by nuclear receptorsincluding Liver-X-Receptor. BMB Rep 46:322–327Baldan A, Pei L, Lee R, Tarr P, Tangirala RK,Weinstein MM et al (2006) Impaired development ofatherosclerosis in hyperlipidemic Ldlr/ and ApoE/ mice transplanted with Abcg1/bone marrow. Arterioscler Thromb Vasc Biol 26:2301–2307158 D. Kardassis et al.Bao Y, Yang Y, Wang L, Gao L, Jiang W, Wang L et al (2009) Identification of trichostatin A as anovel transcriptional up-regulator of scavenger receptor BI both in HepG2 and RAW 264.7cells. Atherosclerosis 204:127–135Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233Basu SK, Brown MS, Ho YK, Havel RJ, Goldstein JL (1981) Mouse macrophages synthesize andsecrete a protein resembling apolipoprotein E. Proc Natl Acad Sci USA 78:7545–7549Bavner A, Sanyal S, Gustafsson JA, Treuter E (2005) Transcriptional corepression by SHP:molecular mechanisms and physiological consequences. Trends Endocrinol Metab 16:478–488Belalcazar LM, Merched A, Carr B, Oka K, Chen KH, Pastore L et al (2003) Long-term stableexpression of human apolipoprotein A-I mediated by helper-dependent adenovirus genetransfer inhibits atherosclerosis progression and remodels atherosclerotic plaques in a mousemodel of familial hypercholesterolemia. Circulation 107:2726–2732Bellosta S, Mahley RW, Sanan DA, Murata J, Newland DL, Taylor JM et al (1995) Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemicapolipoprotein E-null mice. J Clin Invest 96:2170–2179Berge KE, Tian H, Graf GA, Yu L, Grishin NV, Schultz J et al (2000) Accumulation of dietarycholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science 290:1771–1775Bernstein BE, Birney E, Dunham I, Green ED, Gunter C, Snyder M (2012) An integratedencyclopedia of DNA elements in the human genome. Nature 489:57–74Biddinger SB, Haas JT, Yu BB, Bezy O, Jing E, Zhang W et al (2008) Hepatic insulin resistancedirectly promotes formation of cholesterol gallstones. Nat Med 14:778–782Blumberg B, Evans RM (1998) Orphan nuclear receptors – new ligands and new possibilities.Genes Dev 12:3149–3155Bouly M, Masson D, Gross B, Jiang XC, Fievet C, Castro G et al (2001) Induction of thephospholipid transfer protein gene accounts for the high density lipoprotein enlargement inmice treated with fenofibrate. J Biol Chem 276:25841–25847Bourguet W, Germain P, Gronemeyer H (2000) Nuclear receptor ligand-binding domains: three-dimensional structures, molecular interactions and pharmacological implications. TrendsPharmacol Sci 21:381–388Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E et al (2002) Frequent deletions anddown-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocyticleukemia. Proc Natl Acad Sci USA 99:15524–15529Calkin AC, Tontonoz P (2012) Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR. Nat Rev Mol Cell Biol 13:213–224Cao G, Garcia CK, Wyne KL, Schultz RA, Parker KL, Hobbs HH (1997) Structure and local-ization of the human gene encoding SR-BI/CLA-1. Evidence for transcriptional control bysteroidogenic factor 1. J Biol Chem 272:33068–33076Cao G, Beyer TP, Yang XP, Schmidt RJ, Zhang Y, Bensch WR et al (2002) Phospholipid transferprotein is regulated by liver X receptors in vivo. J Biol Chem 277:39561–39565Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs.Cell 136:642–655Cesana M, Cacchiarelli D, Legnini I, Santini T, Sthandier O, Chinappi M et al (2011) A longnoncoding RNA controls muscle differentiation by functioning as a competing endogenousRNA. Cell 147:358–369Chang DJ, Paik YK, Leren TP, Walker DW, Howlett GJ, Taylor JM (1990) Characterization of ahuman apolipoprotein E gene enhancer element and its associated protein factors. J Biol Chem265:9496–9504Chao F, GongW, Zheng Y, Li Y, Huang G, GaoM et al (2010) Upregulation of scavenger receptorclass B type I expression by activation of FXR in hepatocyte. Atherosclerosis 213:443–448Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 159Chellan B, Yan L, Sontag TJ, Reardon CA, Hofmann Bowman MA (2013) IL-22 is induced byS100/calgranulin and impairs cholesterol efflux in macrophages by down regulating ABCG1.J Lipid Res 55:443–454Chen JD, Li H (1998) Coactivation and corepression in transcriptional regulation by steroid/nuclear hormone receptors. Crit Rev Eukaryot Gene Expr 8:169–190Chen WS, Manova K, Weinstein DC, Duncan SA, Plump AS, Prezioso VR et al (1994) Disruptionof the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectodermand impaired gastrulation of mouse embryos. Genes Dev 8:2466–2477Chen H, Rossier C, Lalioti MD, Lynn A, Chakravarti A, Perrin G et al (1996) Cloning of the cDNAfor a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3.Am J Hum Genet 59:66–75Chen W, Wang N, Tall AR (2005) A PEST deletion mutant of ABCA1 shows impaired internal-ization and defective cholesterol efflux from late endosomes. J Biol Chem 280:29277–29281Chen HY, Xu Z, Chen LF, WangW, Fang Q, Yan XW (2012a) Valsartan and telmisartan abrogateangiotensin II-induced downregulation of ABCA1 expression via AT1 receptor, rather thanAT2 receptor or PPARgamma activation. J Cardiovasc Pharmacol 59:570–575Chen X, Liang H, Zhang J, Zen K, Zhang CY (2012b) Secreted microRNAs: a new form ofintercellular communication. Trends Cell Biol 22:125–132Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Torra IP et al (2001) PPAR-alpha andPPAR-gamma activators induce cholesterol removal from human macrophage foam cellsthrough stimulation of the ABCA1 pathway. Nat Med 7:53–58Christoffersen C, Nielsen LB (2013) Apolipoprotein M: bridging HDL and endothelial function.Curr Opin Lipidol 24:295–300Christoffersen C, Ahnstrom J, Axler O, Christensen EI, Dahlback B, Nielsen LB (2008) The signalpeptide anchors apolipoprotein M in plasma lipoproteins and prevents rapid clearance ofapolipoprotein M from plasma. J Biol Chem 283:18765–18772Christoffersen C, Obinata H, Kumaraswamy SB, Galvani S, Ahnstrom J, Sevvana M et al (2011)Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoproteinM. Proc Natl Acad Sci USA 108:9613–9618Christova R (2013) Detecting DNA-protein interactions in living cells-ChIP approach. AdvProtein Chem Struct Biol 91:101–133Churko JM, Mantalas GL, Snyder MP, Wu JC (2013) Overview of high throughput sequencingtechnologies to elucidate molecular pathways in cardiovascular diseases. Circ Res 112:1613–1623Cifuentes D, Xue H, Taylor DW, Patnode H, Mishima Y, Cheloufi S et al (2010) A novel miRNAprocessing pathway independent of Dicer requires Argonaute2 catalytic activity. Science 328:1694–1698Claudel T, Sturm E, Duez H, Torra IP, Sirvent A, Kosykh V et al (2002) Bile acid-activatednuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR responseelement. J Clin Invest 109:961–971Costet P, Luo Y, Wang N, Tall AR (2000) Sterol-dependent transactivation of the ABC1 promoterby the liver X receptor/retinoid X receptor. J Biol Chem 275:28240–28245Costet P, Lalanne F, Gerbod-Giannone MC, Molina JR, Fu X, Lund EG et al (2003) Retinoic acidreceptor-mediated induction of ABCA1 in macrophages. Mol Cell Biol 23:7756–7766Cui L, Schoene NW, Zhu L, Fanzo JC, Alshatwi A, Lei KY (2002) Zinc depletion reduced Egr-1and HNF-3beta expression and apolipoprotein A-I promoter activity in Hep G2 cells. Am JPhysiol Cell Physiol 283:C623–C630Dahlback B, Nielsen LB (2009) Apolipoprotein M affecting lipid metabolism or just catching aride with lipoproteins in the circulation? Cell Mol Life Sci 66:559–564Davalos A, Chroni A (2014) Antisense oligonucleotides, microRNAs and antibodies. In: vonEckardstein A, Kardassis D (eds) High density lipoproteins – from biological understanding toclinical exploitation. Springer, Heidelberg160 D. Kardassis et al.Davalos A, Fernandez-Hernando C (2013) From evolution to revolution: miRNAs as pharmaco-logical targets for modulating cholesterol efflux and reverse cholesterol transport. PharmacolRes 75:60–72Davalos A, Goedeke L, Smibert P, Ramirez CM, Warrier NP, Andreo U et al (2011) miR-33a/bcontribute to the regulation of fatty acid metabolism and insulin signaling. Proc Natl Acad SciUSA 108:9232–9237Davignon J (2005) Apolipoprotein E and atherosclerosis: beyond lipid effect. Arterioscler ThrombVasc Biol 25:267–269Day JR, Albers JJ, Lofton-Day CE, Gilbert TL, Ching AF, Grant FJ et al (1994) Complete cDNAencoding human phospholipid transfer protein from human endothelial cells. J Biol Chem 269:9388–9391de Aguiar Vallim TQ, Tarling EJ, Kim T, Civelek M, Baldan A, Esau C et al (2013) MicroRNA-144 regulates hepatic ATP binding cassette transporter A1 and plasma high-density lipoproteinafter activation of the nuclear receptor farnesoid X receptor. Circ Res 112:1602–1612del Castillo-Olivares A, Gil G (2000) Alpha 1-fetoprotein transcription factor is required for theexpression of sterol 12alpha-hydroxylase, the specific enzyme for cholic acid synthesis.Potential role in the bile acid-mediated regulation of gene transcription. J Biol Chem 275:17793–17799Delerive P, Galardi CM, Bisi JE, Nicodeme E, Goodwin B (2004) Identification of liver receptorhomolog-1 as a novel regulator of apolipoprotein AI gene transcription. Mol Endocrinol 18:2378–2387Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A et al (2012) Landscape oftranscription in human cells. Nature 489:101–108Dong P, Xie T, Zhou X, Hu W, Chen Y, Duan Y et al (2011) Induction of macrophage scavengerreceptor type BI expression by tamoxifen and 4-hydroxytamoxifen. Atherosclerosis218:435–442Duez H, Lefebvre B, Poulain P, Torra IP, Percevault F, Luc G et al (2005) Regulation of humanapoA-I by gemfibrozil and fenofibrate through selective peroxisome proliferator-activatedreceptor alpha modulation. Arterioscler Thromb Vasc Biol 25:585–591Durrington PN, Mackness MI, Bhatnagar D, Julier K, Prais H, Arrol S et al (1998) Effects of twodifferent fibric acid derivatives on lipoproteins, cholesteryl ester transfer, fibrinogen, plasmi-nogen activator inhibitor and paraoxonase activity in type IIb hyperlipoproteinaemia. Athero-sclerosis 138:217–225Ecker J, Langmann T, Moehle C, Schmitz G (2007) Isomer specific effects of ConjugatedLinoleic Acid on macrophage ABCG1 transcription by a SREBP-1c dependent mechanism.Biochem Biophys Res Commun 352:805–811Edgel KA, Leboeuf RC, Oram JF (2010) Tumor necrosis factor-alpha and lymphotoxin-alphaincrease macrophageABCA1 by gene expression and protein stabilization via different receptors.Atherosclerosis 209:387–392Engel T, Lueken A, Bode G, Hobohm U, Lorkowski S, Schlueter B et al (2004) ADP-ribosylationfactor (ARF)-like 7 (ARL7) is induced by cholesterol loading and participates in apolipo-protein AI-dependent cholesterol export. FEBS Lett 566:241–246Engel T, Bode G, Lueken A, Knop M, Kannenberg F, Nofer JR et al (2006) Expression andfunctional characterization of ABCG1 splice variant ABCG1(666). FEBS Lett 580:4551–4559EttingerWH, VarmaVK, Sorci-ThomasM, Parks JS, Sigmon RC, Smith TK et al (1994) Cytokinesdecrease apolipoprotein accumulation in medium from Hep G2 cells. Arterioscler Thromb14:8–13Fayard E, Auwerx J, Schoonjans K (2004) LRH-1: an orphan nuclear receptor involved indevelopment, metabolism and steroidogenesis. Trends Cell Biol 14:250–260Fazio S, Babaev VR, Murray AB, Hasty AH, Carter KJ, Gleaves LA et al (1997) Increasedatherosclerosis in mice reconstituted with apolipoprotein E null macrophages. Proc Natl AcadSci USA 94:4647–4652Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 161Feingold KR, Shigenaga JK, Chui LG, Moser A, Khovidhunkit W, Grunfeld C (2008) Infectionand inflammation decrease apolipoprotein M expression. Atherosclerosis 199:19–26Fitzgerald ML, Mujawar Z, Tamehiro N (2010) ABC transporters, atherosclerosis and inflammation.Atherosclerosis 211:361–370Frankel LB, Lund AH (2012) MicroRNA regulation of autophagy. Carcinogenesis 33:2018–2025Freeman LA, Kennedy A,Wu J, Bark S, Remaley AT, Santamarina-Fojo S et al (2004) The orphannuclear receptor LRH-1 activates the ABCG5/ABCG8 intergenic promoter. J Lipid Res 45:1197–1206Friedman RC, Farh KK, Burge CB, Bartel DP (2009) Most mammalian mRNAs are conservedtargets of microRNAs. Genome Res 19:92–105Frisdal E, Lesnik P, Olivier M, Robillard P, Chapman MJ, Huby T et al (2011) Interleukin-6protects human macrophages from cellular cholesterol accumulation and attenuates theproinflammatory response. J Biol Chem 286:30926–30936Fu X, Menke JG, Chen Y, Zhou G, MacNaul KL, Wright SD et al (2001) 27-hydroxycholesterol isan endogenous ligand for liver X receptor in cholesterol-loaded cells. J Biol Chem 276:38378–38387Fukata Y, Yu X, Imachi H, Nishiuchi T, Lyu J, Seo K et al (2013) 17β-Estradiol regulates scavengerreceptor class BI gene expression via protein kinase C in vascular endothelial cells.Endocrine 46:644–650Gafencu AV, Robciuc MR, Fuior E, Zannis VI, Kardassis D, Simionescu M (2007) Inflammatorysignaling pathways regulating ApoE gene expression in macrophages. J Biol Chem 282:21776–21785Gan X, Kaplan R, Menke JG, MacNaul K, Chen Y, Sparrow CP et al (2001) Dual mechanisms ofABCA1 regulation by geranylgeranyl pyrophosphate. J Biol Chem 276:48702–48708Garcia MA, Vazquez J, Gimenez C, Valdivieso F, Zafra F (1996) Transcription factor AP-2regulates human apolipoprotein E gene expression in astrocytoma cells. J Neurosci 16:7550–7556Gaudet F, Ginsburg GS (1995) Transcriptional regulation of the cholesteryl ester transfer proteingene by the orphan nuclear hormone receptor apolipoprotein AI regulatory protein-1. J Biol Chem270:29916–29922Gauthier B, Robb M, Gaudet F, Ginsburg GS, McPherson R (1999) Characterization of acholesterol response element (CRE) in the promoter of the cholesteryl ester transfer proteingene: functional role of the transcription factors SREBP-1a, -2, and YY1. J Lipid Res 40:1284–1293Gautier T, de Haan W, Grober J, Ye D, Bahr MJ, Claudel T et al (2013) Farnesoid X receptoractivation increases cholesteryl ester transfer protein expression in humans and transgenicmice.J Lipid Res 54:2195–2205Gavrilov A, Eivazova E, Priozhkova I, Lipinski M, Razin S, Vassetzky Y (2009) Chromosomeconformation capture (from 3C to 5C) and its ChIP-based modification. Methods Mol Biol567:171–188Georgopoulos S, Kan HY, Reardon-Alulis C, Zannis V (2000) The SP1 sites of the human apoCIIIenhancer are essential for the expression of the apoCIII gene and contribute to the hepatic andintestinal expression of the apoA-I gene in transgenic mice. Nucleic Acids Res 28:4919–4929Gerbod-Giannone MC, Li Y, Holleboom A, Han S, Hsu LC, Tabas I et al (2006) TNFalpha inducesABCA1 through NF-kappaB in macrophages and in phagocytes ingesting apoptotic cells.Proc Natl Acad Sci USA 103:3112–3117Gerin I, Clerbaux LA, Haumont O, Lanthier N, Das AK, Burant CF et al (2010) Expression ofmiR-33 from an SREBP2 intron inhibits cholesterol export and fatty acid oxidation. J BiolChem 285:33652–33661Goldstein I, Ezra O, Rivlin N, Molchadsky A, Madar S, Goldfinger N et al (2012) p53, a novelregulator of lipid metabolism pathways. J Hepatol 56:656–662162 D. Kardassis et al.Grainger DJ, Reckless J, McKilligin E (2004) Apolipoprotein E modulates clearance of apoptoticbodies in vitro and in vivo, resulting in a systemic proinflammatory state in apolipoproteinE-deficient mice. J Immunol 173:6366–6375Gronemeyer H, Gustafsson JA, Laudet V (2004) Principles for modulation of the nuclear receptorsuperfamily. Nat Rev Drug Discov 3:950–964Gu HM, Li G, Gao X, Berthiaume LG, Zhang DW (2013) Characterization of palmitoylation ofATP binding cassette transporter G1: effect on protein trafficking and function. BiochimBiophys Acta 1831:1067–1078Guo H, Ingolia NT, Weissman JS, Bartel DP (2010) Mammalian microRNAs predominantly act todecrease target mRNA levels. Nature 466:835–840Haas MJ, Mooradian AD (2010) Therapeutic interventions to enhance apolipoprotein A-I-mediated cardioprotection. Drugs 70:805–821Hadzopoulou-Cladaras M, Lavrentiadou SN, Zannis VI, Kardassis D (1998) Transactivation of theApoCIII promoter by ATF-2 and repression by members of the Jun family. Biochemistry 37:14078–14087Haghpassand M, Bourassa PA, Francone OL, Aiello RJ (2001) Monocyte/macrophage expressionof ABCA1 has minimal contribution to plasma HDL levels. J Clin Invest 108:1315–1320Haidar B, Kiss RS, Sarov-Blat L, Brunet R, Harder C, McPherson R et al (2006) Cathepsin D, alysosomal protease, regulates ABCA1-mediated lipid efflux. J Biol Chem 281:39971–39981Hansen TB, Jensen TI, Clausen BH, Bramsen JB, Finsen B, Damgaard CK et al (2013) NaturalRNA circles function as efficient microRNA sponges. Nature 495:384–388Hargrove GM, Junco A, Wong NC (1999) Hormonal regulation of apolipoprotein AI. J MolEndocrinol 22:103–111Hayhurst GP, Lee YH, Lambert G, Ward JM, Gonzalez FJ (2001) Hepatocyte nuclear factor4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipidhomeostasis. Mol Cell Biol 21:1393–1403Helal O, Berrougui H, Loued S, Khalil A (2013) Extra-virgin olive oil consumption improves thecapacity of HDL to mediate cholesterol efflux and increases ABCA1 and ABCG1 expression inhuman macrophages. Br J Nutr 109:1844–1855Helsen C, Kerkhofs S, Clinckemalie L, Spans L, Laurent M, Boonen S et al (2012) Structural basisfor nuclear hormone receptor DNA binding. Mol Cell Endocrinol 348:411–417Helwak A, Kudla G, Dudnakova T, Tollervey D (2013) Mapping the human miRNA interactomeby CLASH reveals frequent noncanonical binding. Cell 153:654–665Hoang MH, Jia Y, Jun HJ, Lee JH, Lee BY, Lee SJ (2012) Fucosterol is a selective liver X receptormodulator that regulates the expression of key genes in cholesterol homeostasis inmacrophages, hepatocytes, and intestinal cells. J Agric Food Chem 60:11567–11575Honzumi S, Shima A, Hiroshima A, Koieyama T, Ubukata N, Terasaka N (2010) LXRalpharegulates human CETP expression in vitro and in transgenic mice. Atherosclerosis 212:139–145Horie T, Ono K, Horiguchi M, Nishi H, Nakamura T, Nagao K et al (2010) MicroRNA-33 encodedby an intron of sterol regulatory element-binding protein 2 (Srebp2) regulates HDL in vivo.Proc Natl Acad Sci USA 107:17321–17326Horie T, Baba O, Kuwabara Y, Chujo Y, Watanabe S, Kinoshita M et al (2012) MicroRNA-33deficiency reduces the progression of atherosclerotic plaque in ApoE/ mice. J Am HeartAssoc 1:e003376Horie T, Nishino T, Baba O, Kuwabara Y, Nakao T, Nishiga M et al (2013) MicroRNA-33regulates sterol regulatory element-binding protein 1 expression in mice. Nat Commun 4:2883Horiuchi K, Tajima S, Menju M, Yamamoto A (1989) Structure and expression of mouseapolipoprotein E gene. J Biochem 106:98–103Horton JD, Goldstein JL, Brown MS (2002) SREBPs: activators of the complete program ofcholesterol and fatty acid synthesis in the liver. J Clin Invest 109:1125–1131Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 163Hozoji M, Munehira Y, Ikeda Y, Makishima M, Matsuo M, Kioka N et al (2008) Direct interactionof nuclear liver X receptor-beta with ABCA1 modulates cholesterol efflux. J Biol Chem 283:30057–30063Hozoji-Inada M, Munehira Y, Nagao K, Kioka N, Ueda K (2011) Liver X receptor beta (LXRbeta)interacts directly with ATP-binding cassette A1 (ABCA1) to promote high density lipoproteinformation during acute cholesterol accumulation. J Biol Chem 286:20117–20124Hu Y, Ren X, Wang H, Ma Y, Wang L, Shen Y et al (2010a) Liver-specific expression of anexogenous gene controlled by human apolipoprotein A-I promoter. Int J Pharm 398:161–164Hu YW, Wang Q, Ma X, Li XX, Liu XH, Xiao J et al (2010b) TGF-beta1 up-regulates expressionof ABCA1, ABCG1 and SR-BI through liver X receptor alpha signaling pathway in THP-1macrophage-derived foam cells. J Atheroscler Thromb 17:493–502Hu Z, Shen WJ, Kraemer FB, Azhar S (2012) MicroRNAs 125a and 455 repress lipoprotein-supported steroidogenesis by targeting scavenger receptor class B type I in steroidogenic cells.Mol Cell Biol 32:5035–5045Hu Z, Hu J, Zhang Z, Shen WJ, Yun CC, Berlot CH et al (2013) Regulation of expression andfunction of scavenger receptor class B, type I (SR-BI) by Na+/H+ exchanger regulatory factors(NHERFs). J Biol Chem 288:11416–11435Huang CX, Zhang YL, Wang JF, Jiang JY, Bao JL (2013) MCP-1 impacts RCT by repressingABCA1, ABCG1, and SR-BI through PI3K/Akt posttranslational regulation in HepG2 cells.J Lipid Res 54:1231–1240Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, Yu L et al (2008) Detection of microRNAexpression in human peripheral blood microvesicles. PLoS ONE 3:e3694Huuskonen J, Abedin M, Vishnu M, Pullinger CR, Baranzini SE, Kane JP et al (2003)Dynamic regulation of alternative ATP-binding cassette transporter A1 transcripts.Biochem Biophys Res Commun 306:463–468Huuskonen J, Vishnu M, Pullinger CR, Fielding PE, Fielding CJ (2004) Regulation ofATP-binding cassette transporter A1 transcription by thyroid hormone receptor. Biochemistry43:1626–1632Huwait EA, Greenow KR, Singh NN, Ramji DP (2011) A novel role for c-Jun N-terminal kinaseand phosphoinositide 3-kinase in the liver X receptor-mediated induction of macrophage geneexpression. Cell Signal 23:542–549Ishibashi M, Filomenko R, Rebe C, Chevriaux A, Varin A, Derangere V et al (2013) Knock-downof the oxysterol receptor LXRalpha impairs cholesterol efflux in human primary macrophages:lack of compensation by LXRbeta activation. Biochem Pharmacol 86:122–129Iwamoto N, Lu R, Tanaka N, Abe-Dohmae S, Yokoyama S (2010) Calmodulin interacts with ATPbinding cassette transporter A1 to protect from calpain-mediated degradation and upregulateshigh-density lipoprotein generation. Arterioscler Thromb Vasc Biol 30:1446–1452Jean S, Kiger AA (2012) Coordination between RAB GTPase and phosphoinositide regulation andfunctions. Nat Rev Mol Cell Biol 13:463–470Jeoung NH, Jang WG, Nam JI, Pak YK, Park YB (1999) Identification of retinoic acid receptorelement in human cholesteryl ester transfer protein gene. Biochem Biophys Res Commun 258:411–415Jiang XC, Bruce C (1995) Regulation of murine plasma phospholipid transfer protein activity andmRNA levels by lipopolysaccharide and high cholesterol diet. J Biol Chem 270:17133–17138Jiang XC, Moulin P, Quinet E, Goldberg IJ, Yacoub LK, Agellon LB et al (1991) Mammalianadipose tissue and muscle are major sources of lipid transfer protein mRNA. J Biol Chem266:4631–4639Jiang XC, Agellon LB, Walsh A, Breslow JL, Tall A (1992) Dietary cholesterol increasestranscription of the human cholesteryl ester transfer protein gene in transgenic mice. Depen-dence on natural flanking sequences. J Clin Invest 90:1290–1295Jiang XC, Jin W, Hussain MM (2012) The impact of phospholipid transfer protein (PLTP) onlipoprotein metabolism. Nutr Metab (Lond) 9:75164 D. Kardassis et al.Jo DW, Leren TP, Yang ZY, Chung YH, Taylor JM, Paik YK (1995) Characterization of anupstream regulatory element of the human apolipoprotein E gene, and purification of itsbinding protein from the human placenta. J Biochem 117:915–922Joseph SB, Laffitte BA, Patel PH,WatsonMA,MatsukumaKE,Walczak R et al (2002a) Direct andindirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors.J Biol Chem 277:11019–11025Joseph SB, McKilligin E, Pei L, Watson MA, Collins AR, Laffitte BA et al (2002b) Synthetic LXRligand inhibits the development of atherosclerosis in mice. Proc Natl Acad Sci USA 99:7604–7609Jun HJ, Hoang MH, Yeo SK, Jia Y, Lee SJ (2013) Induction of ABCA1 and ABCG1 expression bythe liver X receptor modulator cineole in macrophages. Bioorg Med Chem Lett 23:579–583Kammerer I, Ringseis R, Biemann R, Wen G, Eder K (2011) 13-hydroxy linoleic acid increasesexpression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages. Lipids Health Dis 10:222Kan HY, Georgopoulos S, Zannis V (2000) A hormone response element in the human apolipo-protein CIII (ApoCIII) enhancer is essential for intestinal expression of the ApoA-I andApoCIII genes and contributes to the hepatic expression of the two linked genes in transgenicmice. J Biol Chem 275:30423–30431Kan HY, Georgopoulos S, Zanni M, Shkodrani A, Tzatsos A, Xie HX et al (2004) Contribution ofthe hormone-response elements of the proximal ApoA-I promoter, ApoCIII enhancer, andC/EBP binding site of the proximal ApoA-I promoter to the hepatic and intestinal expression ofthe ApoA-I and ApoCIII genes in transgenic mice. Biochemistry 43:5084–5093KangMH, Singaraja R, Hayden MR (2010) Adenosine-triphosphate-binding cassette transporter-1trafficking and function. Trends Cardiovasc Med 20:41–49Kang MH, Zhang LH, Wijesekara N, de Haan W, Butland S, Bhattacharjee A et al (2013)Regulation of ABCA1 protein expression and function in hepatic and pancreatic islet cellsby miR-145. Arterioscler Thromb Vasc Biol 33:2724–2732Kanter JE, Tang C, Oram JF, Bornfeldt KE (2012) Acyl-CoA synthetase 1 is required for oleateand linoleate mediated inhibition of cholesterol efflux through ATP-binding cassette trans-porter A1 in macrophages. Biochim Biophys Acta 1821:358–364Kardassis D, Tzameli I, Hadzopoulou-Cladaras M, Talianidis I, Zannis V (1997) Distal apolipo-protein C-III regulatory elements F to J act as a general modular enhancer for proximalpromoters that contain hormone response elements. Synergism between hepatic nuclearfactor-4 molecules bound to the proximal promoter and distal enhancer sites. ArteriosclerThromb Vasc Biol 17:222–232Kardassis D, Falvey E, Tsantili P, Hadzopoulou-Cladaras M, Zannis V (2002) Direct physicalinteractions between HNF-4 and Sp1 mediate synergistic transactivation of the apolipoproteinCIII promoter. Biochemistry 41:1217–1228Kardassis D, Drosatos C, Zannis V (2007) Regulation of genes involved in the biogenesis and theremodeling of HDL. In: Fielding CJ (ed) High-density lipoproteins from basic biology toclinical aspects. Wiley-VCH, Weinheim, pp 237–253Kennedy MA, Barrera GC, Nakamura K, Baldan A, Tarr P, Fishbein MC et al (2005) ABCG1 hasa critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumu-lation. Cell Metab 1:121–131Kielar D, Dietmaier W, Langmann T, Aslanidis C, Probst M, Naruszewicz M et al (2001) Rapidquantification of human ABCA1 mRNA in various cell types and tissues by real-time reversetranscription-PCR. Clin Chem 47:2089–2097Kilbourne EJ, Widom R, Harnish DC, Malik S, Karathanasis SK (1995) Involvement of earlygrowth response factor Egr-1 in apolipoprotein AI gene transcription. J Biol Chem 270:7004–7010Kim J, Yoon H, Ramirez CM, Lee SM, Hoe HS, Fernandez-Hernando C (2012) MiR-106b impairscholesterol efflux and increases Aβ levels by repressing ABCA1 expression. Exp Neurol 235:476–483Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 165Kinet V, Halkein J, Dirkx E, Windt LJ (2013) Cardiovascular extracellular microRNAs: emergingdiagnostic markers and mechanisms of cell-to-cell RNA communication. Front Genet 4:214Kir S, Zhang Y, Gerard RD, Kliewer SA, Mangelsdorf DJ (2012) Nuclear receptors HNF4alphaand LRH-1 cooperate in regulating Cyp7a1 in vivo. J Biol Chem 287:41334–41341Kocher O, Krieger M (2009) Role of the adaptor protein PDZK1 in controlling the HDL receptorSR-BI. Curr Opin Lipidol 20:236–241Kocher O, Yesilaltay A, Cirovic C, Pal R, Rigotti A, Krieger M (2003) Targeted disruption of thePDZK1 gene in mice causes tissue-specific depletion of the high density lipoprotein receptorscavenger receptor class B type I and altered lipoprotein metabolism. J Biol Chem 278:52820–52825Krol J, Loedige I, Filipowicz W (2010) The widespread regulation of microRNA biogenesis,function and decay. Nat Rev Genet 11:597–610Ku CS, Park Y, Coleman SL, Lee J (2012) Unsaturated fatty acids repress expression of ATPbinding cassette transporter A1 and G1 in RAW 264.7 macrophages. J Nutr Biochem 23:1271–1276Kulyte A, Lorente-Cebrian S, Gao H, Mejhert N, Agustsson T, Arner P et al (2013) MicroRNAprofiling links miR-378 to enhanced adipocyte lipolysis in human cancer cachexia. Am JPhysiol Endocrinol Metab 306:E267–E274Kuo CY, Lin YC, Yang JJ, Yang VC (2011) Interaction abolishment between mutant caveolin-1(Delta62-100) and ABCA1 reduces HDL-mediated cellular cholesterol efflux. BiochemBiophys Res Commun 414:337–343Ladias JA, Karathanasis SK (1991) Regulation of the apolipoprotein AI gene by ARP-1, a novelmember of the steroid receptor superfamily. Science 251:561–565Laffitte BA, Joseph SB, Walczak R, Pei L, Wilpitz DC, Collins JL et al (2001a) Autoregulation ofthe human liver X receptor alpha promoter. Mol Cell Biol 21:7558–7568Laffitte BA, Repa JJ, Joseph SB, Wilpitz DC, Kast HR, Mangelsdorf DJ et al (2001b) LXRscontrol lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes.Proc Natl Acad Sci USA 98:507–512Laffitte BA, Joseph SB, Chen M, Castrillo A, Repa J, Wilpitz D et al (2003) The phospholipidtransfer protein gene is a liver X receptor target expressed by macrophages in atheroscleroticlesions. Mol Cell Biol 23:2182–2191Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T (2002) Identification oftissue-specific microRNAs from mouse. Curr Biol 12:735–739Lakomy D, Rebe C, Sberna AL, Masson D, Gautier T, Chevriaux A et al (2009) Liver X receptor-mediated induction of cholesteryl ester transfer protein expression is selectively impaired ininflammatory macrophages. Arterioscler Thromb Vasc Biol 29:1923–1929Lambert G, Amar MJ, Guo G, Brewer HB Jr, Gonzalez FJ, Sinal CJ (2003) The farnesoidX-receptor is an essential regulator of cholesterol homeostasis. J Biol Chem 278:2563–2570Lan D, Silver DL (2005) Fenofibrate induces a novel degradation pathway for scavenger receptorB-I independent of PDZK1. J Biol Chem 280:23390–23396Langmann T, Klucken J, Reil M, Liebisch G, Luciani MF, Chimini G et al (1999) Molecularcloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages. Biochem Biophys Res Commun 257:29–33Langmann T, Porsch-Ozcurumez M, Unkelbach U, Klucken J, Schmitz G (2000) Genomicorganization and characterization of the promoter of the human ATP-binding cassettetransporter-G1 (ABCG1) gene. Biochim Biophys Acta 1494:175–180Langmann T, Porsch-Ozcurumez M, Heimerl S, Probst M, Moehle C, Taher M et al (2002)Identification of sterol-independent regulatory elements in the human ATP-binding cassettetransporter A1 promoter: role of Sp1/3, E-box binding factors, and an oncostatin M-responsiveelement. J Biol Chem 277:14443–14450Langmann T, Mauerer R, Zahn A, Moehle C, Probst M, Stremmel W et al (2003) Real-timereverse transcription-PCR expression profiling of the complete human ATP-binding cassettetransporter superfamily in various tissues. Clin Chem 49:230–238166 D. Kardassis et al.Larkin L, Khachigian LM, Jessup W (2000) Regulation of apolipoprotein E production inmacrophages (review). Int J Mol Med 6:253–258Lavrentiadou SN, Hadzopoulou-Cladaras M, Kardassis D, Zannis VI (1999) Binding specificityand modulation of the human ApoCIII promoter activity by heterodimers of ligand-dependentnuclear receptors. Biochemistry 38:964–975Le Goff W, Guerin M, Petit L, Chapman MJ, Thillet J (2003) Regulation of human CETP geneexpression: role of SP1 and SP3 transcription factors at promoter sites 690, 629, and 37.J Lipid Res 44:1322–1331Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes smallRNAs with antisense complementarity to lin-14. Cell 75:843–854Lee S, Wang PY, Jeong Y, Mangelsdorf DJ, Anderson RG, Michaely P (2012) Sterol-dependentnuclear import of ORP1S promotes LXR regulated trans-activation of apoE. Exp Cell Res318:2128–2142Leiva A, Verdejo H, Benitez ML, Martinez A, Busso D, Rigotti A (2011) Mechanisms regulatinghepatic SR-BI expression and their impact on HDL metabolism. Atherosclerosis 217:299–307Lemon B, Tjian R (2000) Orchestrated response: a symphony of transcription factors for genecontrol. Genes Dev 14:2551–2569Li J, Ning G, Duncan SA (2000) Mammalian hepatocyte differentiation requires the transcriptionfactor HNF-4alpha. Genes Dev 14:464–474Li X, Peegel H, Menon KM (2001) Regulation of high density lipoprotein receptor messengerribonucleic acid expression and cholesterol transport in theca-interstitial cells by insulin andhuman chorionic gonadotropin. Endocrinology 142:174–181Li D, Wang D, Wang Y, Ling W, Feng X, Xia M (2010a) Adenosine monophosphate-activatedprotein kinase induces cholesterol efflux from macrophage-derived foam cells and alleviatesatherosclerosis in apolipoprotein E-deficient mice. J Biol Chem 285:33499–33509Li D, Zhang Y, Ma J, Ling W, Xia M (2010b) Adenosine monophosphate activated protein kinaseregulates ABCG1-mediated oxysterol efflux from endothelial cells and protects againsthypercholesterolemia-induced endothelial dysfunction. Arterioscler Thromb Vasc Biol 30:1354–1362Li G, Gu HM, Zhang DW (2013) ATP-binding cassette transporters and cholesterol translocation.IUBMB Life 65:505–512Liang Y, Yang X, Ma L, Cai X, Wang L, Yang C et al (2013) Homocysteine-mediated cholesterolefflux via ABCA1 and ACAT1 DNAmethylation in THP-1 monocyte-derived foam cells. ActaBiochim Biophys Sin (Shanghai) 45:220–228Lin G, Bornfeldt KE (2002) Cyclic AMP-specific phosphodiesterase 4 inhibitors promote ABCA1expression and cholesterol efflux. Biochem Biophys Res Commun 290:663–669Lin S, Zhou C, Neufeld E, Wang YH, Xu SW, Lu L et al (2013) BIG1, a brefeldin A-inhibitedguanine nucleotide-exchange protein modulates ATP-binding cassette transporter A-1 traffick-ing and function. Arterioscler Thromb Vasc Biol 33:e31–e38Linder MD, Mayranpaa MI, Peranen J, Pietila TE, Pietiainen VM, Uronen RL et al (2009) Rab8regulates ABCA1 cell surface expression and facilitates cholesterol efflux in primary humanmacrophages. Arterioscler Thromb Vasc Biol 29:883–888Linton MF, Atkinson JB, Fazio S (1995) Prevention of atherosclerosis in apolipoproteinE-deficient mice by bone marrow transplantation. Science 267:1034–1037Liu C, Lin JD (2011) PGC-1 coactivators in the control of energy metabolism. Acta BiochimBiophys Sin (Shanghai) 43:248–257Liu S, Tetzlaff MT, Cui R, Xu X (2012) miR-200c inhibits melanoma progression and drugresistance through down-regulation of BMI-1. Am J Pathol 181:1823–1835Lopez D, McLean MP (1999) Sterol regulatory element-binding protein-1a binds to cis elementsin the promoter of the rat high density lipoprotein receptor SR-BI gene. Endocrinology 140:5669–5681Lopez D, McLean MP (2006) Estrogen regulation of the scavenger receptor class B gene: anti-atherogenic or steroidogenic, is there a priority? Mol Cell Endocrinol 247:22–33Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 167Lopez D, Sandhoff TW, McLean MP (1999) Steroidogenic factor-1 mediates cyclic 30,5-0-adenosine monophosphate regulation of the high density lipoprotein receptor. Endocrinology140:3034–3044Lopez D, Shea-Eaton W, Sanchez MD, McLean MP (2001) DAX-1 represses the high-densitylipoprotein receptor through interaction with positive regulators sterol regulatory element-binding protein-1a and steroidogenic factor-1. Endocrinology 142:5097–5106Lopez D, Sanchez MD, Shea-Eaton W, McLean MP (2002) Estrogen activates the high-densitylipoprotein receptor gene via binding to estrogen response elements and interaction with sterolregulatory element binding protein-1A. Endocrinology 143:2155–2168Lu R, Arakawa R, Ito-Osumi C, Iwamoto N, Yokoyama S (2008) ApoA-I facilitates ABCA1recycle/accumulation to cell surface by inhibiting its intracellular degradation and increasesHDL generation. Arterioscler Thromb Vasc Biol 28:1820–1824Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U (2004) Nuclear export of microRNAprecursors. Science 303:95–98Luo Y, Tall AR (2000) Sterol upregulation of human CETP expression in vitro and in transgenicmice by an LXR element. J Clin Invest 105:513–520Luo Y, Liang CP, Tall AR (2001) The orphan nuclear receptor LRH-1 potentiates the sterol-mediated induction of the human CETP gene by liver X receptor. J Biol Chem 276:24767–24773Ma AZ, Zhang Q, Song ZY (2013) TNFa alter cholesterol metabolism in human macrophages viaPKC-theta-dependent pathway. BMC Biochem 14:20Maitra U, Li L (2013) Molecular mechanisms responsible for the reduced expression of choles-terol transporters from macrophages by low-dose endotoxin. Arterioscler Thromb Vasc Biol33:24–33Mak PA, Kast-Woelbern HR, Anisfeld AM, Edwards PA (2002a) Identification of PLTP as anLXR target gene and apoE as an FXR target gene reveals overlapping targets for the twonuclear receptors. J Lipid Res 43:2037–2041Mak PA, Laffitte BA, Desrumaux C, Joseph SB, Curtiss LK, Mangelsdorf DJ et al (2002b)Regulated expression of the apolipoprotein E/C-I/C-IV/C-II gene cluster in murine andhuman macrophages. A critical role for nuclear liver X receptors alpha and beta. J BiolChem 277:31900–31908Malekpour-Dehkordi Z, Javadi E, Doosti M, Paknejad M, Nourbakhsh M, Yassa N et al (2013)S-Allylcysteine, a garlic compound, increases ABCA1 expression in human THP-1macrophages. Phytother Res 27:357–361Malerod L, Juvet LK, Hanssen-Bauer A, Eskild W, Berg T (2002) Oxysterol-activated LXRalpha/RXR induces hSR-BI-promoter activity in hepatoma cells and preadipocytes. BiochemBiophys Res Commun 299:916–923Malerod L, Sporstol M, Juvet LK, Mousavi A, Gjoen T, Berg T (2003) Hepatic scavenger receptorclass B, type I is stimulated by peroxisome proliferator-activated receptor gamma and hepato-cyte nuclear factor 4alpha. Biochem Biophys Res Commun 305:557–565Malerod L, Sporstol M, Juvet LK, Mousavi SA, Gjoen T, Berg T et al (2005) Bile acids reduceSR-BI expression in hepatocytes by a pathway involving FXR/RXR, SHP, and LRH-1.Biochem Biophys Res Commun 336:1096–1105Marquart TJ, Allen RM, Ory DS, Baldan A (2010) miR-33 links SREBP-2 induction to repressionof sterol transporters. Proc Natl Acad Sci USA 107:12228–12232Martinez LO, Agerholm-Larsen B, Wang N, Chen W, Tall AR (2003) Phosphorylation of a pestsequence in ABCA1 promotes calpain degradation and is reversed by ApoA-I. J Biol Chem278:37368–37374Masson D, Qatanani M, Sberna AL, Xiao R, Pais de Barros JP, Grober J et al (2008) Activation ofthe constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenicmice. J Lipid Res 49:1682–1691168 D. Kardassis et al.Mataki C, Magnier BC, Houten SM, Annicotte JS, Argmann C, Thomas C et al (2007)Compromised intestinal lipid absorption in mice with a liver-specific deficiency of liverreceptor homolog 1. Mol Cell Biol 27:8330–8339Mattick JS, Makunin IV (2005) Small regulatory RNAs in mammals. HumMol Genet 14(Spec No1):R121–R132Mauerer R, Ebert S, Langmann T (2009) High glucose, unsaturated and saturated fatty acidsdifferentially regulate expression of ATP-binding cassette transporters ABCA1 and ABCG1 inhuman macrophages. Exp Mol Med 41:126–132Mavridou S, Venihaki M, Rassouli O, Tsatsanis C, Kardassis D (2010) Feedback inhibition ofhuman scavenger receptor class B type I gene expression by glucocorticoid in adrenal andovarian cells. Endocrinology 151:3214–3224Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14:447–459Mendell JT, Olson EN (2012) MicroRNAs in stress signaling and human disease. Cell 148:1172–1187Meurs I, Lammers B, Zhao Y, Out R, Hildebrand RB, Hoekstra M et al (2012) The effect ofABCG1 deficiency on atherosclerotic lesion development in LDL receptor knockout micedepends on the stage of atherogenesis. Atherosclerosis 221:41–47Mitchell PJ, Tjian R (1989) Transcriptional regulation in mammalian cells by sequence-specificDNA binding proteins. Science 245:371–378Miyake Y, Kozutsumi Y, Nakamura S, Fujita T, Kawasaki T (1995) Serine palmitoyltransferase isthe primary target of a sphingosine-like immunosuppressant, ISP-1/myriocin. BiochemBiophys Res Commun 211:396–403Miyazaki T, Koya T, Kigawa Y, Oguchi T, Lei XF, Kim-Kaneyama JR et al (2013) Calpain andatherosclerosis. J Atheroscler Thromb 20:228–237Mizuno T, Hayashi H, Naoi S, Sugiyama Y (2011) Ubiquitination is associated with lysosomaldegradation of cell surface-resident ATP-binding cassette transporter A1 (ABCA1) through theendosomal sorting complex required for transport (ESCRT) pathway. Hepatology 54:631–643Mooradian AD, Haas MJ, Wong NC (2004) Transcriptional control of apolipoprotein A-I geneexpression in diabetes. Diabetes 53:513–520Morishima A, Ohkubo N, Maeda N, Miki T, Mitsuda N (2003) NFkappaB regulates plasmaapolipoprotein A-I and high density lipoprotein cholesterol through inhibition of peroxisomeproliferator-activated receptor alpha. J Biol Chem 278:38188–38193Mosialou I, Zannis VI, Kardassis D (2010) Regulation of human apolipoprotein m gene expressionby orphan and ligand-dependent nuclear receptors. J Biol Chem 285:30719–30730Mosialou I, Krasagakis K, Kardassis D (2011) Opposite regulation of the human apolipoprotein Mgene by hepatocyte nuclear factor 1 and Jun transcription factors. J Biol Chem 286:17259–17269Munehira Y, Ohnishi T, Kawamoto S, Furuya A, Shitara K, Imamura M et al (2004) Alpha1-syntrophin modulates turnover of ABCA1. J Biol Chem 279:15091–15095Murao K, Wada Y, Nakamura T, Taylor AH, Mooradian AD, Wong NC (1998) Effects of glucoseand insulin on rat apolipoprotein A-I gene expression. J Biol Chem 273:18959–18965Murao K, Imachi H, Yu X, CaoWM,Muraoka T, Dobashi H et al (2008) The transcriptional factorprolactin regulatory element-binding protein mediates the gene transcription of adrenal scav-enger receptor class B type I via 30,50-cyclic adenosine 50-monophosphate. Endocrinology 149:6103–6112Myklebost O, Rogne S (1988) A physical map of the apolipoprotein gene cluster on humanchromosome 19. Hum Genet 78:244–247Nagelin MH, Srinivasan S, Lee J, Nadler JL, Hedrick CC (2008) 12/15-Lipoxygenase activityincreases the degradation of macrophage ATP-binding cassette transporter G1. ArteriosclerThromb Vasc Biol 28:1811–1819Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 169Nagelin MH, Srinivasan S, Nadler JL, Hedrick CC (2009) Murine 12/15-lipoxygenase regulatesATP-binding cassette transporter G1 protein degradation through p38- and JNK2-dependentpathways. J Biol Chem 284:31303–31314Najafi-Shoushtari SH, Kristo F, Li Y, Shioda T, Cohen DE, Gerszten RE et al (2010) MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis. Science 328:1566–1569Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R (1994) ApoE-deficient mice developlesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb 14:133–140Nakshatri H, Bhat-Nakshatri P (1998) Multiple parameters determine the specificity of transcrip-tional response by nuclear receptors HNF-4, ARP-1, PPAR, RAR and RXR through commonresponse elements. Nucleic Acids Res 26:2491–2499Nielsen LB, Christoffersen C, Ahnstrom J, Dahlback B (2009) ApoM: gene regulation and effectson HDL metabolism. Trends Endocrinol Metab 20:66–71Niemeier A, Kovacs WJ, Strobl W, Stangl H (2009) Atherogenic diet leads to posttranslationaldown-regulation of murine hepatocyte SR-BI expression. Atherosclerosis 202:169–175Nikolaidou-Neokosmidou V, Zannis VI, Kardassis D (2006) Inhibition of hepatocyte nuclearfactor 4 transcriptional activity by the nuclear factor kappaB pathway. Biochem J 398:439–450Nohata N, Hanazawa T, Kikkawa N, Mutallip M, Fujimura L, Yoshino H et al (2011) Caveolin-1mediates tumor cell migration and invasion and its regulation by miR-133a in head and necksquamous cell carcinoma. Int J Oncol 38:209–217Nonomura K, Arai Y, Mitani H, Abe-Dohmae S, Yokoyama S (2011) Insulin down-regulatesspecific activity of ATP-binding cassette transporter A1 for high density lipoprotein biogenesisthrough its specific phosphorylation. Atherosclerosis 216:334–341Novak EM, Bydlowski SP (1997) NFY transcription factor binds to regulatory element AIC andtransactivates the human apolipoprotein A-I promoter in HEPG2 cells. Biochem Biophys ResCommun 231:140–143Okazaki H, Goldstein JL, Brown MS, Liang G (2010) LXR-SREBP-1c-phospholipid transferprotein axis controls very low density lipoprotein (VLDL) particle size. J Biol Chem 285:6801–6810Olaisen B, Teisberg P, Gedde-Dahl T Jr (1982) The locus for apolipoprotein E (apoE) is linked tothe complement component C3 (C3) locus on chromosome 19 in man. Hum Genet 62:233–236Oliver WR Jr, Shenk JL, Snaith MR, Russell CS, Plunket KD, Bodkin NL et al (2001) A selectiveperoxisome proliferator-activated receptor delta agonist promotes reverse cholesterol trans-port. Proc Natl Acad Sci USA 98:5306–5311Orom UA, Nielsen FC, Lund AH (2008) MicroRNA-10a binds the 5’UTR of ribosomal proteinmRNAs and enhances their translation. Mol Cell 30:460–471Ouimet M, Franklin V, Mak E, Liao X, Tabas I, Marcel YL (2011) Autophagy regulatescholesterol efflux from macrophage foam cells via lysosomal acid lipase. Cell Metab 13:655–667Out R, Hoekstra M, Hildebrand RB, Kruit JK, Meurs I, Li Z et al (2006) Macrophage ABCG1deletion disrupts lipid homeostasis in alveolar macrophages and moderately influences athero-sclerotic lesion development in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol26:2295–2300Out C, Hageman J, Bloks VW, Gerrits H, Sollewijn Gelpke MD, Bos T et al (2011) Liver receptorhomolog-1 is critical for adequate up-regulation of Cyp7a1 gene transcription and bile saltsynthesis during bile salt sequestration. Hepatology 53:2075–2085Ozasa H, Ayaori M, Iizuka M, Terao Y, Uto-Kondo H, Yakushiji E et al (2011) Pioglitazoneenhances cholesterol efflux from macrophages by increasing ABCA1/ABCG1 expressions viaPPARgamma/LXRalpha pathway: findings from in vitro and ex vivo studies. Atherosclerosis219:141–150170 D. Kardassis et al.Paik YK, Chang DJ, Reardon CA, Walker MD, Taxman E, Taylor JM (1988) Identification andcharacterization of transcriptional regulatory regions associated with expression of the humanapolipoprotein E gene. J Biol Chem 263:13340–13349Pamir N, McMillen TS, Edgel KA, Kim F, LeBoeuf RC (2012) Deficiency of lymphotoxin-alphadoes not exacerbate high-fat diet-induced obesity but does enhance inflammation in mice. Am JPhysiol Endocrinol Metab 302:E961–E971Pan X, Jiang XC, Hussain MM (2013) Impaired cholesterol metabolism and enhanced atheroscle-rosis in clock mutant mice. Circulation 128:1758–1769Panousis CG, Zuckerman SH (2000) Interferon-gamma induces downregulation of Tangier dis-ease gene (ATP-binding-cassette transporter 1) in macrophage-derived foam cells. ArteriosclerThromb Vasc Biol 20:1565–1571Panousis CG, Evans G, Zuckerman SH (2001) TGF-beta increases cholesterol efflux and ABC-1expression in macrophage-derived foam cells: opposing the effects of IFN-gamma. J Lipid Res42:856–863Papazafiri P, Ogami K, Ramji DP, Nicosia A, Monaci P, Cladaras C et al (1991) Promoterelements and factors involved in hepatic transcription of the human ApoA-I gene positiveand negative regulators bind to overlapping sites. J Biol Chem 266:5790–5797Park SS, Choi H, Kim SJ, Kim OJ, Chae KS, Kim E (2008) FXRalpha down-regulates LXRalphasignaling at the CETP promoter via a common element. Mol Cells 26:409–414Park DW, Lee HK, Lyu JH, Chin H, Kang SW, Kim YJ et al (2013) TLR2 stimulates ABCA1expression via PKC-eta and PLD2 pathway. Biochem Biophys Res Commun 430:933–937Parseghian S, Onstead-Haas LM, Wong NC, Mooradian AD, Haas MJ (2013) Inhibition ofapolipoprotein A-I gene expression by tumor necrosis factor alpha in HepG2 cells: requirementfor c-jun. J Cell Biochem 115:253–260Pascual-Garcia M, Rue L, Leon T, Julve J, Carbo JM, Matalonga J et al (2013) Reciprocal negativecross-talk between liver X receptors (LXRs) and STAT1: effects on IFN-gamma-inducedinflammatory responses and LXR-dependent gene expression. J Immunol 190:6520–6532Paszty C, Maeda N, Verstuyft J, Rubin EM (1994) Apolipoprotein AI transgene corrects apolipo-protein E deficiency-induced atherosclerosis in mice. J Clin Invest 94:899–903Peng D, Hiipakka RA, Dai Q, Guo J, Reardon CA, Getz GS et al (2008) Antiatherosclerotic effectsof a novel synthetic tissue-selective steroidal liver X receptor agonist in low-density lipo-protein receptor-deficient mice. J Pharmacol Exp Ther 327:332–342Peng D, Hiipakka RA, Xie JT, Dai Q, Kokontis JM, Reardon CA et al (2011) A novel potentsynthetic steroidal liver X receptor agonist lowers plasma cholesterol and triglycerides andreduces atherosclerosis in LDLR(/) mice. Br J Pharmacol 162:1792–1804Phelan CA, Weaver JM, Steger DJ, Joshi S, Maslany JT, Collins JL et al (2008) Selective partialagonism of liver X receptor alpha is related to differential corepressor recruitment.Mol Endocrinol 22:2241–2249Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, Pandolfi PP (2010) A coding-independent function of gene and pseudogene mRNAs regulates tumour biology.Nature 465:1033–1038Porsch-Ozcurumez M, Langmann T, Heimerl S, Borsukova H, Kaminski WE, Drobnik Wet al (2001) The zinc finger protein 202 (ZNF202) is a transcriptional repressor of ATP bindingcassette transporter A1 (ABCA1) and ABCG1 gene expression and a modulator of cellularlipid efflux. J Biol Chem 276:12427–12433Quinet EM, Agellon LB, Kroon PA, Marcel YL, Lee YC, Whitlock ME et al (1990) Atherogenicdiet increases cholesteryl ester transfer protein messenger RNA levels in rabbit liver. J ClinInvest 85:357–363Quinet EM, Savio DA, Halpern AR, Chen L, Miller CP, Nambi P (2004) Gene-selective modu-lation by a synthetic oxysterol ligand of the liver X receptor. J Lipid Res 45:1929–1942Radeau T, Lau P, Robb M, McDonnell M, Ailhaud G, McPherson R (1995) Cholesteryl estertransfer protein (CETP) mRNA abundance in human adipose tissue: relationship to cell sizeand membrane cholesterol content. J Lipid Res 36:2552–2561Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 171Raffai RL, Loeb SM, Weisgraber KH (2005) Apolipoprotein E promotes the regression ofatherosclerosis independently of lowering plasma cholesterol levels. Arterioscler ThrombVasc Biol 25:436–441Rajavashisth TB, Kaptein JS, Reue KL, Lusis AJ (1985) Evolution of apolipoprotein E: mousesequence and evidence for an 11-nucleotide ancestral unit. Proc Natl Acad Sci USA 82:8085–8089Ramirez CM, Davalos A, Goedeke L, Salerno AG, Warrier N, Cirera-Salinas D et al (2011)MicroRNA-758 regulates cholesterol efflux through posttranscriptional repression ofATP-binding cassette transporter A1. Arterioscler Thromb Vasc Biol 31:2707–2714Ramirez CM, Rotllan N, Vlassov AV, Davalos A, Li M, Goedeke L et al (2013) Control ofcholesterol metabolism and plasma high-density lipoprotein levels by microRNA-144.Circ Res 112:1592–1601Ranalletta M, Wang N, Han S, Yvan-Charvet L, Welch C, Tall AR (2006) Decreased athero-sclerosis in low-density lipoprotein receptor knockout mice transplanted with Abcg1/ bonemarrow. Arterioscler Thromb Vasc Biol 26:2308–2315Rayner KJ, Suarez Y, Davalos A, Parathath S, Fitzgerald ML, Tamehiro N et al (2010) MiR-33contributes to the regulation of cholesterol homeostasis. Science 328:1570–1573Rayner KJ, Esau CC, Hussain FN, McDaniel AL, Marshall SM, van Gils JM et al (2011a)Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDLtriglycerides. Nature 478:404–407Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S et al (2011b) Antagonism ofmiR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J ClinInvest 121:2921–2931Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants.Genes Dev 16:1616–1626Remaley AT, Bark S, Walts AD, Freeman L, Shulenin S, Annilo T et al (2002) Comparativegenome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster.Biochem Biophys Res Commun 295:276–282Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, Mangelsdorf DJ (2002) Regulation ofATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alphaand beta. J Biol Chem 277:18793–18800Richter S, Shih DQ, Pearson ER, Wolfrum C, Fajans SS, Hattersley AT et al (2003) Regulation ofapolipoprotein M gene expression by MODY3 gene hepatocyte nuclear factor-1alpha:haploinsufficiency is associated with reduced serum apolipoprotein M levels. Diabetes 52:2989–2995Rochel N, Ciesielski F, Godet J, Moman E, Roessle M, Peluso-Iltis C et al (2011) Commonarchitecture of nuclear receptor heterodimers on DNA direct repeat elements with differentspacings. Nat Struct Mol Biol 18:564–570Rodriguez-Ubreva J, Ballestar E (2014) Chromatin immunoprecipitation. Methods Mol Biol 1094:309–318Roeder RG (1998) Role of general and gene-specific cofactors in the regulation of eukaryotictranscription. Cold Spring Harb Symp Quant Biol 63:201–218Roeder RG (2005) Transcriptional regulation and the role of diverse coactivators in animal cells.FEBS Lett 579:909–915Rottiers V, Obad S, Petri A, McGarrah R, Lindholm MW, Black JC et al (2013) Pharmacologicalinhibition of a microRNA family in nonhuman primates by a seed-targeting 8-mer antimiR.Sci Transl Med 5:212ra162Rottman JN, Widom RL, Nadal-Ginard B, Mahdavi V, Karathanasis SK (1991) A retinoic acid-responsive element in the apolipoprotein AI gene distinguishes between two different retinoicacid response pathways. Mol Cell Biol 11:3814–3820Rousselle A, Qadri F, Leukel L, Yilmaz R, Fontaine JF, Sihn G et al (2013) CXCL5 limitsmacrophage foam cell formation in atherosclerosis. J Clin Invest 123:1343–1347172 D. Kardassis et al.Rubin EM, Ishida BY, Clift SM, Krauss RM (1991) Expression of human apolipoprotein A-I intransgenic mice results in reduced plasma levels of murine apolipoprotein A-I and theappearance of two new high density lipoprotein size subclasses. Proc Natl Acad Sci USA 88:434–438Sakai T, Kamanna VS, Kashyap ML (2001) Niacin, but not gemfibrozil, selectively increasesLP-AI, a cardioprotective subfraction of HDL, in patients with low HDL cholesterol.Arterioscler Thromb Vasc Biol 21:1783–1789Salero E, Perez-Sen R, Aruga J, Gimenez C, Zafra F (2001) Transcription factors Zic1 and Zic2bind and transactivate the apolipoprotein E gene promoter. J Biol Chem 276:1881–1888Salero E, Gimenez C, Zafra F (2003) Identification of a non-canonical E-box motif as a regulatoryelement in the proximal promoter region of the apolipoprotein E gene. Biochem J 370:979–986Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP (2011) A ceRNA hypothesis: the Rosetta Stoneof a hidden RNA language? Cell 146:353–358Sanoudou D, Duka A, Drosatos K, Hayes KC, Zannis VI (2009) Role of Esrrg in the fibrate-mediated regulation of lipid metabolism genes in human ApoA-I transgenic mice. Pharmaco-genomics J 10:165–179Santamarina-Fojo S, Peterson K, Knapper C, Qiu Y, Freeman L, Cheng JF et al (2000) Completegenomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-bindingcassette A promoter. Proc Natl Acad Sci USA 97:7987–7992Sanyal A, Lajoie BR, Jain G, Dekker J (2012) The long-range interaction landscape of genepromoters. Nature 489:109–113Schaefer EJ, Lamon-Fava S, Cole T, Sprecher DL, Cilla DD Jr, Balagtas CC et al (1996) Effects ofregular and extended-release gemfibrozil on plasma lipoproteins and apolipoproteins inhypercholesterolemic patients with decreased HDL cholesterol levels. Atherosclerosis 127:113–122Schmitz G, Langmann T (2005) Transcriptional regulatory networks in lipid metabolism controlABCA1 expression. Biochim Biophys Acta 1735:1–19Schnall-Levin M, Zhao Y, Perrimon N, Berger B (2010) Conserved microRNA targeting inDrosophila is as widespread in coding regions as in 30UTRs. Proc Natl Acad Sci USA 107:15751–15756Schoonjans K, Annicotte JS, Huby T, Botrugno OA, Fayard E, Ueda Y et al (2002) Liver receptorhomolog 1 controls the expression of the scavenger receptor class B type I. EMBO Rep 3:1181–1187Schou J, Frikke-Schmidt R, Kardassis D, Thymiakou E, Nordestgaard BG, Jensen G et al (2012)Genetic variation in ABCG1 and risk of myocardial infarction and ischemic heart disease.Arterioscler Thromb Vasc Biol 32:506–515Schreyer SA, Peschon JJ, LeBoeuf RC (1996) Accelerated atherosclerosis in mice lacking tumornecrosis factor receptor p55. J Biol Chem 271:26174–26178Schreyer SA, Vick CM, LeBoeuf RC (2002) Loss of lymphotoxin-alpha but not tumor necrosisfactor-alpha reduces atherosclerosis in mice. J Biol Chem 277:12364–12368Schwartz K, Lawn RM, Wade DP (2000) ABC1 gene expression and ApoA-I-mediated choles-terol efflux are regulated by LXR. Biochem Biophys Res Commun 274:794–802Shea-Eaton W, Lopez D, McLean MP (2001) Yin yang 1 protein negatively regulates high-densitylipoprotein receptor gene transcription by disrupting binding of sterol regulatory elementbinding protein to the sterol regulatory element. Endocrinology 142:49–58Shetty S, Eckhardt ER, Post SR, van der Westhuyzen DR (2006) Phosphatidylinositol-3-kinaseregulates scavenger receptor class B type I subcellular localization and selective lipid uptake inhepatocytes. Arterioscler Thromb Vasc Biol 26:2125–2131Shih SJ, Allan C, Grehan S, Tse E, Moran C, Taylor JM (2000) Duplicated downstream enhancerscontrol expression of the human apolipoprotein E gene in macrophages and adipose tissue.J Biol Chem 275:31567–31572Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 173Shih DQ, Bussen M, Sehayek E, Ananthanarayanan M, Shneider BL, Suchy FJ et al (2001)Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterolmetabolism. Nat Genet 27:375–382Shirasaki T, Honda M, Shimakami T, Horii R, Yamashita T, Sakai Y et al (2013) MicroRNA-27aregulates lipid metabolism and inhibits hepatitis C virus replication in human hepatoma cells.J Virol 87:5270–5286Singaraja RR, Bocher V, James ER, Clee SM, Zhang LH, Leavitt BR et al (2001) Human ABCA1BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor responseelements in intron 1. J Biol Chem 276:33969–33979Singaraja RR, James ER, Crim J, Visscher H, Chatterjee A, Hayden MR (2005) Alternatetranscripts expressed in response to diet reflect tissue-specific regulation of ABCA1. J LipidRes 46:2061–2071Singaraja RR, Visscher H, James ER, Chroni A, Coutinho JM, Brunham LR et al (2006) Specificmutations in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes bothin vivo and in vitro. Circ Res 99:389–397Singaraja RR, Kang MH, Vaid K, Sanders SS, Vilas GL, Arstikaitis P et al (2009) Palmitoylationof ATP-binding cassette transporter A1 is essential for its trafficking and function. Circ Res105:138–147Singh NN, Ramji DP (2006) Transforming growth factor-beta-induced expression of the apolipo-protein E gene requires c-Jun N-terminal kinase, p38 kinase, and casein kinase 2. ArteriosclerThromb Vasc Biol 26:1323–1329Sladek FM (1994) Orphan receptor HNF-4 and liver-specific gene expression. Receptor 4:64Small EM, Olson EN (2011) Pervasive roles of microRNAs in cardiovascular biology. Nature 469:336–342Smit M, van der Kooij-Meijs E, Frants RR, Havekes L, Klasen EC (1988) Apolipoprotein genecluster on chromosome 19. Definite localization of the APOC2 gene and the polymorphic HpaI site associated with type III hyperlipoproteinemia. Hum Genet 78:90–93Smith JD, Melian A, Leff T, Breslow JL (1988) Expression of the human apolipoprotein E gene isregulated by multiple positive and negative elements. J Biol Chem 263:8300–8308Song H, Saito K, Fujigaki S, Noma A, Ishiguro H, Nagatsu T et al (1998) IL-1 beta and TNF-alphasuppress apolipoprotein (apo) E secretion and apo A-I expression in HepG2 cells. Cytokine 10:275–280Sparrow CP, Baffic J, Lam MH, Lund EG, Adams AD, Fu X et al (2002) A potent synthetic LXRagonist is more effective than cholesterol loading at inducing ABCA1 mRNA and stimulatingcholesterol efflux. J Biol Chem 277:10021–10027Sporstol M, Mousavi SA, Eskild W, Roos N, Berg T (2007) ABCA1, ABCG1 and SR-BI:hormonal regulation in primary rat hepatocytes and human cell lines. BMC Mol Biol 8:5Srivastava RA, Srivastava N, Averna M (2000) Dietary cholic acid lowers plasma levels of mouseand human apolipoprotein A-I primarily via a transcriptional mechanism. Eur J Biochem 267:4272–4280Staels B, Auwerx J (1998) Regulation of apo A-I gene expression by fibrates. Atherosclerosis137(Suppl):S19–S23Sticozzi C, Belmonte G, Pecorelli A, Cervellati F, Leoncini S, Signorini C et al (2013) Scavengerreceptor B1 post-translational modifications in Rett syndrome. FEBS Lett 587:2199–2204Sumi K, Tanaka T, Uchida A, Magoori K, Urashima Y, Ohashi R et al (2007) Cooperativeinteraction between hepatocyte nuclear factor 4 alpha and GATA transcription factorsregulates ATP-binding cassette sterol transporters ABCG5 and ABCG8. Mol Cell Biol 27:4248–4260Sun Y, Wang N, Tall AR (1999) Regulation of adrenal scavenger receptor-BI expression byACTH and cellular cholesterol pools. J Lipid Res 40:1799–1805Sun D, Zhang J, Xie J, Wei W, Chen M, Zhao X (2012) MiR-26 controls LXR-dependentcholesterol efflux by targeting ABCA1 and ARL7. FEBS Lett 586:1472–1479174 D. Kardassis et al.Suzuki S, Nishimaki-Mogami T, Tamehiro N, Inoue K, Arakawa R, Abe-Dohmae S et al (2004)Verapamil increases the apolipoprotein-mediated release of cellular cholesterol by induction ofABCA1 expression via Liver X receptor-independent mechanism. Arterioscler Thromb VascBiol 24:519–525Tall AR, Yvan-Charvet L, Terasaka N, Pagler T, Wang N (2008) HDL, ABC transporters, andcholesterol efflux: implications for the treatment of atherosclerosis. Cell Metab 7:365–375Tamehiro N, Zhou S, Okuhira K, Benita Y, Brown CE, Zhuang DZ et al (2008) SPTLC1 bindsABCA1 to negatively regulate trafficking and cholesterol efflux activity of the transporter.Biochemistry 47:6138–6147Tan CL, Plotkin JL, Veno MT, von Schimmelmann M, Feinberg P, Mann S et al (2013)MicroRNA-128 governs neuronal excitability and motor behavior in mice. Science 342:1254–1258Tang C, Vaughan AM, Oram JF (2004) Janus kinase 2 modulates the apolipoprotein interactionswith ABCA1 required for removing cellular cholesterol. J Biol Chem 279:7622–7628Tarling EJ, Edwards PA (2012) Dancing with the sterols: critical roles for ABCG1, ABCA1,miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis.Biochim Biophys Acta 1821:386–395Tay Y, Zhang J, Thomson AM, Lim B, Rigoutsos I (2008) MicroRNAs to Nanog, Oct4 and Sox2coding regions modulate embryonic stem cell differentiation. Nature 455:1124–1128Taylor AH, Wishart P, Lawless DE, Raymond J, Wong NC (1996) Identification of functionalpositive and negative thyroid hormone-responsive elements in the rat apolipoprotein AI pro-moter. Biochemistry 35:8281–8288Thymiakou E, Zannis VI, Kardassis D (2007) Physical and functional interactions between liver Xreceptor/retinoid X receptor and Sp1 modulate the transcriptional induction of the human ATPbinding cassette transporter A1 gene by oxysterols and retinoids. Biochemistry 46:11473–11483Towns R, Azhar S, Peegel H, Menon KM (2005) LH/hCG-stimulated androgen production andselective HDL-cholesterol transport are inhibited by a dominant-negative CREB construct inprimary cultures of rat theca-interstitial cells. Endocrine 27:269–277Trajkovski M, Hausser J, Soutschek J, Bhat B, Akin A, Zavolan M et al (2011) MicroRNAs103 and 107 regulate insulin sensitivity. Nature 474:649–653Truong TQ, Aubin D, Falstrault L, Brodeur MR, Brissette L (2010) SR-BI, CD36, and caveolin-1contribute positively to cholesterol efflux in hepatic cells. Cell Biochem Funct 28:480–489Trusca VG, Fuior EV, Florea IC, Kardassis D, Simionescu M, Gafencu AV (2011) Macrophage-specific up-regulation of apolipoprotein E gene expression by STAT1 is achieved via longrange genomic interactions. J Biol Chem 286:13891–13904Trusca VG, Florea IC, Kardassis D, Gafencu AV (2012) STAT1 interacts with RXRalpha toupregulate ApoCII gene expression in macrophages. PLoS ONE 7:e40463Tu AY, Albers JJ (1999) DNA sequences responsible for reduced promoter activity of humanphospholipid transfer protein by fibrate. Biochem Biophys Res Commun 264:802–807Tu AY, Albers JJ (2001) Glucose regulates the transcription of human genes relevant to HDLmetabolism: responsive elements for peroxisome proliferator-activated receptor are involvedin the regulation of phospholipid transfer protein. Diabetes 50:1851–1856Tzameli I, Zannis VI (1996) Binding specificity and modulation of the ApoA-I promoter activityby homo- and heterodimers of nuclear receptors. J Biol Chem 271:8402–8415Uehara Y, Engel T, Li Z, Goepfert C, Rust S, Zhou X et al (2002) Polyunsaturated fatty acidsand acetoacetate downregulate the expression of the ATP-binding cassette transporter A1.Diabetes 51:2922–2928Uehara Y, Miura S, von Eckardstein A, Abe S, Fujii A, Matsuo Y et al (2007) Unsaturated fattyacids suppress the expression of the ATP-binding cassette transporter G1 (ABCG1) andABCA1 genes via an LXR/RXR responsive element. Atherosclerosis 191:11–21Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 175Urizar NL, Dowhan DH, Moore DD (2000) The farnesoid X-activated receptor mediates bile acidactivation of phospholipid transfer protein gene expression. J Biol Chem 275:39313–39317Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediatedtransfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol 9:654–659Valenta DT, Bulgrien JJ, Banka CL, Curtiss LK (2006) Overexpression of human ApoAI trans-gene provides long-term atheroprotection in LDL receptor-deficient mice. Atherosclerosis 189:255–263Van Eck M, Herijgers N, Yates J, Pearce NJ, Hoogerbrugge PM, Groot PH et al (1997) Bonemarrow transplantation in apolipoprotein E-deficient mice. Effect of ApoE gene dosage onserum lipid concentrations, (beta)VLDL catabolism, and atherosclerosis. Arterioscler ThrombVasc Biol 17:3117–3126Vasudevan S, Tong Y, Steitz JA (2007) Switching from repression to activation: microRNAs canup-regulate translation. Science 318:1931–1934Vaziri ND, Sato T, Liang K (2003) Molecular mechanisms of altered cholesterol metabolism inrats with spontaneous focal glomerulosclerosis. Kidney Int 63:1756–1763Vaziri ND, Gollapudi P, Han S, Farahmand G, Yuan J, Rahimi A et al (2011) Nephrotic syndromecauses upregulation of HDL endocytic receptor and PDZK-1-dependent downregulation ofHDL docking receptor. Nephrol Dial Transplant 26:3118–3123Venteclef N, Haroniti A, Tousaint JJ, Talianidis I, Delerive P (2008) Regulation of anti-atherogenic apolipoprotein M gene expression by the orphan nuclear receptor LRH-1. J BiolChem 283:3694–3701Vickers KC, Rader DJ (2013) Nuclear receptors and microRNA-144 coordinately regulate choles-terol efflux. Circ Res 112:1529–1531Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT (2011) MicroRNAs aretransported in plasma and delivered to recipient cells by high-density lipoproteins. Nat CellBiol 13:423–433von Eckardstein A (1996) Cholesterol efflux from macrophages and other cells. Curr Opin Lipidol7:308–319von Eckardstein A, Hersberger M, Rohrer L (2005) Current understanding of the metabolism andbiological actions of HDL. Curr Opin Clin Nutr Metab Care 8:147–152Vu-Dac N, Schoonjans K, Laine B, Fruchart JC, Auwerx J, Staels B (1994) Negative regulation ofthe human apolipoprotein A-I promoter by fibrates can be attenuated by the interaction of theperoxisome proliferator-activated receptor with its response element. J Biol Chem 269:31012–31018Wagner J, Riwanto M, Besler C, Knau A, Fichtlscherer S, Roxe T et al (2013) Characterization oflevels and cellular transfer of circulating lipoprotein-bound microRNAs. Arterioscler ThrombVasc Biol 33:1392–1400Walsh A, Ito Y, Breslow JL (1989) High levels of human apolipoprotein A-I in transgenic miceresult in increased plasma levels of small high density lipoprotein (HDL) particles comparableto human HDL3. J Biol Chem 264:6488–6494Wan YJ, An D, Cai Y, Repa JJ, Hung-Po Chen T, Flores M et al (2000) Hepatocyte-specificmutation establishes retinoid X receptor alpha as a heterodimeric integrator of multiplephysiological processes in the liver. Mol Cell Biol 20:4436–4444Wang Y, Oram JF (2002) Unsaturated fatty acids inhibit cholesterol efflux from macrophages byincreasing degradation of ATP-binding cassette transporter A1. J Biol Chem 277:5692–5697Wang Y, Oram JF (2007) Unsaturated fatty acids phosphorylate and destabilize ABCA1 through aprotein kinase C delta pathway. J Lipid Res 48:1062–1068Wang XQ, Panousis CG, Alfaro ML, Evans GF, Zuckerman SH (2002) Interferon-gamma-mediated downregulation of cholesterol efflux and ABC1 expression is by the Stat1 pathway.Arterioscler Thromb Vasc Biol 22:e5–e9176 D. Kardassis et al.Wang N, Chen W, Linsel-Nitschke P, Martinez LO, Agerholm-Larsen B, Silver DL et al (2003)A PEST sequence in ABCA1 regulates degradation by calpain protease and stabilization ofABCA1 by apoA-I. J Clin Invest 111:99–107Wang JH, Keisala T, Solakivi T, Minasyan A, Kalueff AV, Tuohimaa P (2009) Serum cholesteroland expression of ApoAI, LXRbeta and SREBP2 in vitamin D receptor knock-out mice.J Steroid Biochem Mol Biol 113:222–226Wang D, Xia M, Yan X, Li D, Wang L, Xu Y et al (2012a) Gut microbiota metabolism ofanthocyanin promotes reverse cholesterol transport in mice via repressing miRNA-10b.Circ Res 111:967–981Wang K, Li H, Yuan Y, Etheridge A, Zhou Y, Huang D et al (2012b) The complex exogenousRNA spectra in human plasma: an interface with human gut biota? PLoS ONE 7:e51009Wang L, Jia XJ, Jiang HJ, Du Y, Yang F, Si SY et al (2013a) MicroRNAs 185, 96, and 223 repressselective high-density lipoprotein cholesterol uptake through posttranscriptional inhibition.Mol Cell Biol 33:1956–1964Wang M, Wang D, Zhang Y, Wang X, Liu Y, Xia M (2013b) Adiponectin increases macrophagescholesterol efflux and suppresses foam cell formation in patients with type 2 diabetes mellitus.Atherosclerosis 229:62–70Wang W, Song W, Wang Y, Chen L, Yan X (2013c) HMG-CoA reductase inhibitors, simvastatinand atorvastatin, downregulate ABCG1-mediated cholesterol efflux in human macrophages.J Cardiovasc Pharmacol 62:90–98Wehmeier K, Beers A, Haas MJ, Wong NC, Steinmeyer A, Zugel U et al (2005) Inhibition ofapolipoprotein AI gene expression by 1, 25-dihydroxyvitamin D3. Biochim Biophys Acta1737:16–26Wei Z, Huang D, Gao F, Chang WH, An W, Coetzee GA et al (2013) Biological implications andregulatory mechanisms of long-range chromosomal interactions. J Biol Chem 288:22369–22377Wellington CL, Walker EK, Suarez A, Kwok A, Bissada N, Singaraja R et al (2002) ABCA1mRNA and protein distribution patterns predict multiple different roles and levels of regu-lation. Lab Invest 82:273–283Werb Z, Chin JR (1983a) Apoprotein E is synthesized and secreted by resident and thioglycollate-elicited macrophages but not by pyran copolymer- or bacillus Calmette-Guerin-activatedmacrophages. J Exp Med 158:1272–1293Werb Z, Chin JR (1983b) Endotoxin suppresses expression of apoprotein E by mousemacrophages in vivo and in culture. A biochemical and genetic study. J Biol Chem 258:10642–10648Werb Z, Chin JR (1983c) Onset of apoprotein E secretion during differentiation of mouse bonemarrow-derived mononuclear phagocytes. J Cell Biol 97:1113–1118Westerterp M, Murphy AJ, Wang M, Pagler TA, Vengrenyuk Y, Kappus MS et al (2013)Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflam-mation and accelerates atherosclerosis in mice. Circ Res 112:1456–1465Wightman B, Ha I, Ruvkun G (1993) Posttranscriptional regulation of the heterochronic genelin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75:855–862Wolfrum C, Poy MN, Stoffel M (2005) Apolipoprotein M is required for prebeta-HDL formationand cholesterol efflux to HDL and protects against atherosclerosis. Nat Med 11:418–422Wolfrum C, Howell JJ, Ndungo E, Stoffel M (2008) Foxa2 activity increases plasma high densitylipoprotein levels by regulating apolipoprotein M. J Biol Chem 283:16940–16949Wood P, Mulay V, Darabi M, Chan KC, Heeren J, Pol A et al (2011) Ras/mitogen-activatedprotein kinase (MAPK) signaling modulates protein stability and cell surface expression ofscavenger receptor SR-BI. J Biol Chem 286:23077–23092Xu N, Ahren B, Jiang J, Nilsson-Ehle P (2006) Down-regulation of apolipoprotein M expression ismediated by phosphatidylinositol 3-kinase in HepG2 cells. Biochim Biophys Acta 1761:256–260Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 177Yang XP, Freeman LA, Knapper CL, Amar MJ, Remaley A, Brewer HB Jr et al (2002) The E-boxmotif in the proximal ABCA1 promoter mediates transcriptional repression of the ABCA1gene. J Lipid Res 43:297–306Yang C, McDonald JG, Patel A, Zhang Y, Umetani M, Xu F et al (2006) Sterol intermediates fromcholesterol biosynthetic pathway as liver X receptor ligands. J Biol Chem 281:27816–27826Yang Y, Zhang Z, Jiang W, Gao L, Zhao G, Zheng Z et al (2007) Identification of novel humanhigh-density lipoprotein receptor Up-regulators using a cell-based high-throughput screeningassay. J Biomol Screen 12:211–219Yang Y, Jiang W, Wang L, Zhang ZB, Si SY, Hong B (2009) Characterization of the isoflavonepratensein as a novel transcriptional up-regulator of scavenger receptor class B type I in HepG2cells. Biol Pharm Bull 32:1289–1294Yang T, Chen C, Zhang B, HuangH,WuG,Wen J et al (2010) Induction of Kruppel-like factor 4 byhigh-density lipoproteins promotes the expression of scavenger receptor class B type I. FEBS J277:3780–3788Yang JS, Phillips MD, Betel D, Mu P, Ventura A, Siepel AC et al (2011) Widespread regulatoryactivity of vertebrate microRNA* species. RNA 17:312–326Yokoyama S, Arakawa R, Wu CA, Iwamoto N, Lu R, Tsujita M et al (2012) Calpain-mediatedABCA1 degradation: post-translational regulation of ABCA1 for HDL biogenesis. BiochimBiophys Acta 1821:547–551Yoshikawa T, Shimano H, Amemiya-Kudo M, Yahagi N, Hasty AH, Matsuzaka T et al (2001)Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatoryelement-binding protein 1c gene promoter. Mol Cell Biol 21:2991–3000Yu L, Hammer RE, Li-Hawkins J, Von Bergmann K, Lutjohann D, Cohen JC et al (2002a)Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterolsecretion. Proc Natl Acad Sci USA 99:16237–16242Yu L, Li-Hawkins J, Hammer RE, Berge KE, Horton JD, Cohen JC et al (2002b) Overexpressionof ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorp-tion of dietary cholesterol. J Clin Invest 110:671–680Yu XH, Jiang HL, Chen WJ, Yin K, Zhao GJ, Mo ZC et al (2012) Interleukin-18 and interleukin-12 together downregulate ATP-binding cassette transporter A1 expression through theinterleukin-18R/nuclear factor-kappaB signaling pathway in THP-1 macrophage-derivedfoam cells. Circ J 76:1780–1791Yu XH, Qian K, Jiang N, Zheng XL, Cayabyab FS, Tang CK (2014) ABCG5/ABCG8 incholesterol excretion and atherosclerosis. Clin Chim Acta 428:82–88Zaiou M, Azrolan N, Hayek T, Wang H, Wu L, Haghpassand M et al (1998) The full induction ofhuman apoprotein A-I gene expression by the experimental nephrotic syndrome in transgenicmice depends on cis-acting elements in the proximal 256 base-pair promoter region and thetrans-acting factor early growth response factor 1. J Clin Invest 101:1699–1707Zannis VI, Cole FS, Jackson CL, Kurnit DM, Karathanasis SK (1985) Distribution of apolipo-protein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction ofapolipoprotein E mRNA by cultures of human monocyte-macrophages. Biochemistry 24:4450–4455Zannis VI, Kan HY, Kritis A, Zanni E, Kardassis D (2001a) Transcriptional regulation of thehuman apolipoprotein genes. Front Biosci 6:D456–D504Zannis VI, Kan HY, Kritis A, Zanni EE, Kardassis D (2001b) Transcriptional regulatorymechanisms of the human apolipoprotein genes in vitro and in vivo. Curr Opin Lipidol 12:181–207Zhang X, Jiang B, Luo G, Nilsson-Ehle P, Xu N (2007) Hyperglycemia down-regulates apolipo-protein M expression in vivo and in vitro. Biochim Biophys Acta 1771:879–882Zhang Y, Shen C, Ai D, Xie X, Zhu Y (2011) Upregulation of scavenger receptor B1 by hepaticnuclear factor 4alpha through a peroxisome proliferator-activated receptor gamma-dependentmechanism in liver. PPAR Res 2011:164925178 D. Kardassis et al.Zhang L, HouD, Chen X, Li D, Zhu L, Zhang Y et al (2012) Exogenous plantMIR168a specificallytargets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res22:107–126Zhao GJ, Tang SL, Lv YC, Ouyang XP, He PP, Yao F et al (2013) Antagonism of betulinic acid onLPS-mediated inhibition of ABCA1 and cholesterol efflux through inhibiting nuclear factor-kappaB signaling pathway and miR-33 expression. PLoS ONE 8:e74782Zuckerman SH, Evans GF, O’Neal L (1993) Exogenous glucocorticoids increase macrophagesecretion of apo E by cholesterol-independent pathways. Atherosclerosis 103:43–54Regulation of HDL Genes: Transcriptional, Posttranscriptional, and. . . 179Cholesterol Efflux and Reverse CholesterolTransportElda Favari, Angelika Chroni, Uwe J.F. Tietge, Ilaria Zanotti,Joan Carles Escolà-Gil, and Franco BerniniContents1 Cholesterol Efflux as the First Step of Reverse Cholesterol Transport (RCT) . . . . . . . . . . . 1831.1 ABCA1-Mediated Lipid Efflux to Lipid-Poor apoA-I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1841.2 Cholesterol Efflux to Lipidated HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1852 HDL Quality and Cholesterol Efflux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1873 RCT in Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1903.1 Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1903.1.1 Methodological Approaches to Quantify RCT In Vivo . . . . . . . . . . . . . . . . . . . . . 1913.1.2 Factors Impacting In Vivo RCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1923.2 Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1963.2.1 CETP Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1963.2.2 Nuclear Receptor Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1973.2.3 Cholesterol Absorption Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1973.2.4 Augmenting or Mimicking apoA-I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1974 Serum Cholesterol Efflux Capacity (CEC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199E. Favari • I. Zanotti • F. Bernini (*)Department of Pharmacy, University of Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italye-mail: fbernini@unipr.itA. ChroniInstitute of Biosciences and Applications, National Center for Scientific Research “Demokritos”,Agia Paraskevi, Athens, GreeceU.J.F. TietgeDepartment of Pediatrics, University of Groningen, University Medical Center Groningen,Groningen, The NetherlandsJ.C. Escolà-GilIIB Sant Pau-CIBER de Diabetes y Enfermedades Metabolicas Asociadas, Barcelona, Spain# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_4181AbstractBoth alterations of lipid/lipoprotein metabolism and inflammatory events con-tribute to the formation of the atherosclerotic plaque, characterized by theaccumulation of abnormal amounts of cholesterol and macrophages in the arterywall. Reverse cholesterol transport (RCT) may counteract the pathogenic eventsleading to the formation and development of atheroma, by promoting the high-density lipoprotein (HDL)-mediated removal of cholesterol from the artery wall.Recent in vivo studies established the inverse relationship between RCT effi-ciency and atherosclerotic cardiovascular diseases (CVD), thus suggesting thatthe promotion of this process may represent a novel strategy to reduce athero-sclerotic plaque burden and subsequent cardiovascular events. HDL plays aprimary role in all stages of RCT: (1) cholesterol efflux, where these lipoproteinsremove excess cholesterol from cells; (2) lipoprotein remodeling, where HDLundergo structural modifications with possible impact on their function; and(3) hepatic lipid uptake, where HDL releases cholesterol to the liver, for the finalexcretion into bile and feces. Although the inverse association between HDLplasma levels and CVD risk has been postulated for years, recently this concepthas been challenged by studies reporting that HDL antiatherogenic functionsmay be independent of their plasma levels. Therefore, assessment of HDLfunction, evaluated as the capacity to promote cell cholesterol efflux may offera better prediction of CVD than HDL levels alone. Consistent with this idea, ithas been recently demonstrated that the evaluation of serum cholesterol effluxcapacity (CEC) is a predictor of atherosclerosis extent in humans.KeywordsHDL • RCT • Cholesterol efflux • CECAbbreviations15-LO-1 15-Lipoxygenase-1ABCA1 ATP-binding cassette transporter A1ABCG1 ATP-binding cassette transporter G1apoB Apolipoprotein BapoE Apolipoprotein ECEC Cholesterol efflux capacityCETP Cholesteryl ester transfer proteinCVD Cardiovascular diseasesEL Endothelial lipaseHDL High-density lipoproteinsLCAT Lecithin-cholesterol acyltransferase182 E. Favari et al.LDL Low-density lipoproteinsLXR Liver X receptorLXR/RXR Liver X receptor/retinoid X receptorMPO MyeloperoxidaseNPC1L1 Niemann-Pick type C like 1PLA2 Phospholipase A2PLTP Phospholipid transfer proteinPPAR Peroxisome proliferator-activated receptorRCT Reverse cholesterol transportSR-BI Scavenger receptor class B type I1 Cholesterol Efflux as the First Step of Reverse CholesterolTransport (RCT)Lipid accumulation within macrophages, leading to the formation of foam cells,represents a main feature of atherogenesis (Wynn et al. 2013). Among the strategiesby which cells may counteract this process, the release of excess cholesterol toextracellular lipid acceptors has been matter of several studies in the recent years.Although cholesterol efflux from macrophages has a minor contribution to thewhole body cholesterol transported by plasma high-density lipoproteins (HDL), itis the most important for the antiatherosclerotic extent. For this reason, the macro-phage RCT concept has been proposed, and it was recently identified as a noveltherapeutic target (Rosenson et al. 2012).Both active and passive mechanisms are responsible for cholesterol efflux, andseveral components, such as cell cholesterol status, lipid transporter activity, andthe nature of extracellular acceptors, have been shown to impact its efficiency(Zanotti et al. 2012).The efflux process involves cholesterol localized on the plasma membrane thatin turn may derive from intracellular sites, such as the late endosomal/lysosomalcompartment and from the Golgi apparatus. Cholesterol localized in the endoplas-mic reticulum originates from endogenous synthesis and can be delivered tointracellular organelles mainly through a nonvesicular pathway involving proteincarriers. Cholesterol in endosomes and lysosomes derives from lipoprotein uptakeand undergoes hydrolyzation by acid hydrolase (Maxfield and Tabas 2005). Thislipoprotein-derived cholesterol is then rapidly released into the cytoplasm anddelivered throughout the cells by Niemann-Pick type C 1 and 2. These proteins,expressed on the membrane of late endosomes and in lysosomes, respectively,facilitate lipid mobilization from these compartments to the plasma membranesvia the trans-Golgi network (Boadu and Francis 2006). In physiological conditionscholesterol content in intracellular locations is low, because most of it (about 80 %)is transferred to the plasma membrane, where it establishes a dynamic equilibriumwith endoplasmic reticulum and the Golgi system pools (Huang et al. 2003). TheCholesterol Efflux and Reverse Cholesterol Transport 183plasma membrane bilayer contains distinct lipid environments in a steady stateequilibrium. Lipid rafts are characterized by a tightly packed, liquid-ordered state,where cholesterol associates with sphingolipids and caveolin, playing a crucial rolein cell signaling (Gargalovic and Dory 2003). Several studies carried out inmacrophages, demonstrated that the major mechanisms of cholesterol efflux areraft-independent (Gargalovic and Dory 2003). The non-raft membranemicrodomains serve as the principal source of cholesterol available for interactionwith extracellular acceptors and subsequent efflux via ATP-binding cassette trans-porter A1 (ABCA1) that is localized in these portions of the membrane (Landryet al. 2006; Vaughan and Oram 2005). Interestingly, RCT can be triggered alsofrom extracellular matrix-associated cholesterol microdomains, whose formation ismediated by ATP-binding cassette transporter G1 (ABCG1) (Freeman et al. 2013).Mechanisms accounting for cholesterol efflux include passive diffusion pro-cesses as well as active pathways mediated by ABCA1, ABCG1, and scavengerreceptor class B type I (SR-BI). Aqueous diffusion mainly involves free cholesterolin the plasma membrane and HDL as lipid acceptors. The nature of this process is amatter of debate: whereas this mechanism appears to be a relevant contributor oflipid removal in foam cell macrophages (Adorni et al. 2007), the involvement of astill unknown transporter is not completely ruled out.1.1 ABCA1-Mediated Lipid Efflux to Lipid-Poor apoA-IABCA1 is a 2,261-amino acid integral membrane protein, member of the largesuperfamily of ABC transporters that use ATP as an energy source to transportlipids across membranes. ABC transporters are characterized by the presence ofnucleotide-binding domains containing two conserved peptide motifs known asWalker A and Walker B, a unique amino acid signature between the two Walkermotifs, which defines the family. ABC transporters are integrated into the mem-brane by domains containing six transmembrane helices. The minimum require-ment for an active ABC transporter is two nucleotide-binding and two 6-helixtransmembrane domains (Oram and Lawn 2001). At the cellular level ABCA1 islocalized both on the plasma membrane and intracellular compartments, the Golgicomplex, and the late endosome/lysosomes, cycling between these loci and pro-moting a flow of intracellular cholesterol from the late endosomes/lysosomes,through the trans-Golgi complex, to the plasma membrane (Boadu and Francis2006). This movement produces an ABCA1-dependent depletion of intracellularpools of cholesterol that affects both the “regulatory” pool of cholesterol and theLDL-derived cholesterol. These activities result in the modulation of a number ofcellular events including: endogenous cholesterol synthesis, LDL receptor expres-sion, cholesteryl ester turnover, and nascent HDL formation (Boadu and Francis2006). The last process is driven by ABCA1-mediated removal of lipids from thecell membrane to extracellular acceptors represented by lipid-free or lipid-poorapolipoproteins. ABCA1 expression on the plasma membrane leads to the genera-tion of non-raft microdomains and enlargement of cholesterol and phospholipid184 E. Favari et al.domains in the outer leaflet, thus facilitating the interaction with apoA-I andsubsequent cholesterol efflux. It is worth noting that these pools of cholesterolcreated by ABCA1 are specific substrates for ABCA1-mediated lipid releasebecause they selectively interact with lipid-free or lipid-poor apolipoproteins(Landry et al. 2006). ABCA1 exerts its function through a floppase activity,which drives cholesterol and phospholipids from the inner leaflet of the membraneto cell surface domains and eventually back to intracellular compartments. Inaddition, ABCA1 promotes vesicular trafficking of cholesterol, phospholipids andABCA1 itself between the plasma membrane and intracellular compartments(Landry et al. 2006). Cholesterol efflux via ABCA1 is related to the protein level,in turn regulated by transcriptional or posttranslational mechanisms. Abca1 geneexpression is primarily induced by the stimulation of liver X receptor/retinoid Xreceptor (LXR/RXR) axis, stimulated by cholesterol accumulation in the cells(Larrede et al. 2009). Recently, the role of microRNAs in the inhibitory controlof Abca1 expression has emerged (Sun et al. 2012; Rayner et al. 2010; Ramirezet al. 2011). The posttranscriptional regulation includes mechanisms that involve(1) the stabilization of ABCA1 protein by apoA-I and (2) the acceleration of itsturnover by calpain-mediated proteolysis or polyunsaturated fatty acids (Oram andVaughan 2006).1.2 Cholesterol Efflux to Lipidated HDLABCA1-mediated generation of nascent HDL particles may in turn promote cho-lesterol efflux via ABCG1 and SR-BI. The former is a half-size ABC protein, wherethe nucleotide-binding domain at the N-terminus is followed by sixtransmembrane-spanning domains. Interestingly, various transcripts of ABCG1have been detected in different cells, possibly arising from alternative splicingevents (Schmitz et al. 2001). Early studies reported many similarities betweenABCG1 and ABCA1, including the cellular localization, the translocation fromintracellular compartments to the plasma membrane, the floppase activity, and theexpression promoted by cholesterol enrichment via LXR. A main feature ofABCG1-mediated efflux is the specific interaction with HDL and low-densitylipoproteins (LDL) thus accounting for the elimination of cholesterol and toxicoxysterols from the macrophages (Vaughan and Oram 2005). It is important to notethat under physiologic conditions ABCA1 and ABCG1 can act in a sequentialfashion, with ABCA1 generating, which then promote lipid release via ABCG1(Gelissen et al. 2006). However, recent data challenge these concepts, suggestingthat, unlike ABCA1, ABCG1 effluxes cellular cholesterol by a process that is notdependent upon interaction with an extracellular protein (Tarling and Edwards2012).Although several cholesterol-responsive ABC transporters, other than ABCA1and ABCG1, have been described in the macrophages, their potential relevance forthe process of foam cell formation and RCT needs further investigations(Fu et al. 2013).Cholesterol Efflux and Reverse Cholesterol Transport 185SR-BI is an 82-kDa integral membrane protein, belonging to the CD36 family,whose physiological role is related to the selective uptake of HDL cholesteryl ester,the process by which the core cholesteryl ester is taken into the cell without theendocytic uptake and degradation of the whole HDL. Since this pathway is themajor route for the delivery of HDL cholesteryl ester to the liver, the role of SR-BIin determining the plasma levels of HDL is of major importance. Importantly,SR-BI has shown to stimulate free cholesterol efflux facilitating the aqueousdiffusion pathway to phospholipid-enriched acceptors. Thus, the expression ofthis receptor establishes a bidirectional flux between cells and HDL, whose neteffect will be related to cell cholesterol status, as well as the composition andconcentration of the acceptor in the extracellular environment. Differently from theABC transporters, SR-BI is localized in caveolae, a subset of lipid rafts, cell surfaceinvaginations enriched in free cholesterol (Rosenson et al. 2012). Despite thesignificant role of SR-BI in cell cholesterol metabolism, its role in cholesterolefflux from macrophages is still unclear. It is important to note that the relativecontribution of a single pathway to cholesterol export is species specific: whereasABCA1-mediated efflux is the predominant mechanism both in human and murinecultured foam cells, the role of ABCG1 is elusive in the former, but not in the latter.Conversely, SR-BI (Cla-1 in humans) plays a pivotal role in human, but not murinecells (Adorni et al. 2007; Sun et al. 2012). Moreover, the antiatheroscleroticproperties of ABCA1 and ABCG1 have recently been challenged by the demon-stration that some efflux-independent activities could be related to deleteriouseffects on macrophage function (Adorni et al. 2011; Olivier et al. 2012).A relevant role in cholesterol efflux from macrophages and RCT has been alsoattributed to apolipoprotein E (apoE) (Zanotti et al. 2011a, b). This 34-kDa proteinis synthesized by many cell types, including macrophages, upon different stimuli,such as differentiation, cytokines, and lipid enrichment. The last process activatesthe LXR pathway, as described for ABCA1 and ABCG1. ApoE synthesis in theendoplasmic reticulum is followed by its movement to the Golgi and trans-Golginetwork and incorporation into vesicular structures, before being transported to theplasma membrane for the final secretion. Secreted apoE can be released into theextracellular medium or alternatively can bind to the cell surface, particularly inassociation with heparan sulfate proteoglycans. Cell surface pools may bereinternalized and subsequently degraded, transported to the Golgi network forposttranslational modifications, or released into the extracellular medium (Kockxet al. 2008). When ApoE is secreted from cholesterol-enriched macrophages, thisprocess can promote cholesterol efflux in the absence of added cholesterolacceptors or in presence of exogenous HDL, causing the generation of nascentHDL particles. There is evidence that apoE can promote cholesterol release by bothABCA1-dependent and independent mechanisms, whereas ABCG1 may contributeby driving cholesterol efflux to apoE-enriched particles (Huang et al. 2001).186 E. Favari et al.2 HDL Quality and Cholesterol EffluxHDL comprises several subclasses that differ in composition and size and exhibit aseries of atheroprotective and other properties, including the ability to effluxcholesterol from various cell types, as well as antioxidative, anti-inflammatory,anticoagulatory, and anti-aggregatory properties (Annema and von Eckardstein2013; Vickers and Remaley 2014). These properties are exerted by the variousprotein and lipid components of HDL (Annema and von Eckardstein 2013; Vickersand Remaley 2014). It is increasingly accepted that the quality rather than quantityof HDL is more relevant for its atheroprotective activity (see chapter “Dysfunctional HDL: From Structure-Function-Relationships to Biomarkers” for moredetails).As anticipated, the ability of HDL to promote cholesterol efflux from lipid-ladenmacrophages is thought to be important for the atheroprotective function of HDL.The capacity of HDL to promote cholesterol efflux from macrophages was shownto have a strong inverse association with both carotid intima–media thickness andthe likelihood of angiographic coronary artery disease, independently of the HDL-Clevel (Khera et al. 2011). As mentioned before, cellular cholesterol efflux ismediated by a number of pathways, with the various HDL subpopulations to displaya varied capacity to promote cholesterol efflux via each of these pathways (Annemaand von Eckardstein 2013; Vickers and Remaley 2014). Therefore, the efficiency ofserum from an individual to accept cellular cholesterol can be affected by thedistribution and composition of HDL particles acting as cholesterol acceptors.Indeed, it has been shown that apolipoprotein B (apoB)-depleted sera from subjectswith similar HDL-C or apoA-I can have higher total macrophage efflux capacitydue to significantly higher ABCA1-mediated efflux, and this efflux is significantlycorrelated with the levels of preβ1-HDL (de la Llera-Moya et al. 2010; Calabresiet al. 2009). Small discoidal preβ1-HDL particles are also efficient acceptors of cellcholesterol via ABCG1 (Favari et al. 2009). In another study, apoB-depleted serafrom patients treated with the cholesteryl ester transfer protein (CETP) inhibitoranacetrapib were shown to have enhanced ability to promote ABCG1-mediatedcholesterol efflux from macrophages, which was associated with increased lecithin-cholesterol acyltransferase (LCAT) and apoE mass in HDL (Yvan-Charvetet al. 2010).The various components of HDL particles can undergo structural or chemicalmodifications during atherogenesis or other pathologic processes, having as a resultadverse effects on HDL functionality, including the cholesterol efflux capacity. Invitro and in vivo studies have shown that enzymatic oxidation, lipolysis, andproteolysis can modify HDL and affect the HDL capacity to promote cellularcholesterol efflux. HDL isolated from humans with established CVD was foundto contain higher levels of protein-bound 3-chlorotyrosine and protein-bound3-nitrotyrosine compared to HDL from controls (Bergt et al. 2004; Pennathuret al. 2004; Zheng et al. 2004). In addition, the levels of both 3-chlorotyrosineand 3-nitrotyrosine were higher in HDL isolated from atherosclerotic lesionscompared with plasma HDL. One pathway that generates such species involvesCholesterol Efflux and Reverse Cholesterol Transport 187myeloperoxidase (MPO), a major constituent of artery wall macrophages (Bergtet al. 2004; Pennathur et al. 2004; Zheng et al. 2004). It was proposed that apoA-I isa selective target for MPO-catalyzed nitration and chlorination in vivo and thatMPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition ofABCA1-dependent cholesterol efflux from macrophages (Zheng et al. 2004).More specifically, it was shown that chlorination of apoA-I impaired the abilityof the protein to promote cholesterol efflux by MPO, while nitration had a muchlesser effect (Bergt et al. 2004; Zheng et al. 2005; Shao et al. 2005) and that acombination of Tyr-192 chlorination and methionine oxidation is necessary fordepriving apoA-I of its ABCA1-dependent cholesterol transport activity (Shaoet al. 2006). More recently it was suggested that oxidative damage to apoA-I byMPO limits the ability of apoA-I to be liberated in a lipid-free form from HDL andthat this impairment of apoA-I exchange reaction may contribute to reducedABCA1-mediated cholesterol efflux (Cavigiolio et al. 2010).Treatment of HDL3 with the reticulocyte-type 15-lipoxygenase-1 (15-LO-1),which has been suggested to play a pathophysiological role in atherosclerosis,induced HDL apolipoprotein cross-linking and reduced cholesterol efflux fromlipid-laden J774 cells (Pirillo et al. 2006). A reduced binding of 15-LO-modifiedHDL3 to SR-BI explained, in part, the observed reduction of cholesterol efflux. Inaddition, the ABCA1-mediated cholesterol efflux was also reduced, as a conse-quence of loss of preβ-particles after HDL3 modification (Pirillo et al. 2006).The capacity of HDL to promote cellular cholesterol efflux from lipid-loadedmouse peritoneal macrophages was significantly decreased after treatment of HDLwith secretory phospholipase A2 (PLA2) group X or V (Zanotti et al. 2011a, b).sPLA2-X and sPLA2-V that have been associated with the pathogenesis of athero-sclerosis affect the capacity of HDL to promote cholesterol efflux by catalyzing thehydrolysis of phosphatidylcholine in HDL without any modification of apoA-I(Ishimoto et al. 2003).Several proteases, including metalloproteinases, cathepsins, chymase, tryptase,kallikrein, neutrophil elastase, and plasmin, that are secreted from various types ofcells of human atherosclerotic arterial intima can proteolytically modify HDLin vitro (Lee-Rueckert et al. 2011). It has been shown that the various proteasesproteolyze apoA-I that is present in preβ-HDL and therefore reduce cholesterolefflux from macrophage foam cells (Favari et al. 2004). A series of studies aimingto elucidate the effect of proteolytic modification of HDL on cholesterol efflux andRCT used mast cells and the neutral serine proteases chymase and tryptase, whichare secreted from mast cells. Treatment of HDL3 with human chymase resulted inrapid depletion of preβ-HDL and a concomitant decrease in the efflux of cholesteroland phospholipids by an ABCA1-dependent pathway, while aqueous or SR-BI-facilitated diffusion of cholesterol was not affected (Favari et al. 2004). Further-more, local activation of mast cells by the specific mast cell degranulating com-pound 48/80 in the mouse peritoneal cavity, which causes acute release of activechymase, resulted in a 90 % reduction of human apoA-I injected into the peritonealcavity. This reduction reflected the reduction in preβ-HDL particles and resulted inattenuation of cholesterol efflux from intraperitoneally co-injected J774188 E. Favari et al.macrophages and a reduced rate of macrophage RCT. In addition, pretreatment ofapoA-I with chymase also fully abolished the stimulatory effect of untreated apoA-Ito promote the transfer of macrophage-derived cholesterol to the intestine(Lee-Rueckert, Silvennoinen et al. 2011).Proteolysis of lipid-free apoA-I or preβ-HDL- or HDL3-associated apoA-I byintima proteases may result in the formation of carboxy-terminal truncated apoA-Ifragments. Specifically, it has been shown recently that lipid-free apoA-I is prefer-entially digested by chymase at the C-terminus rather than the N-terminus and thatthe Phe229 and Tyr192 residues are the main cleavage sites, while the Phe225 residueis a minor cleavage site (Usami et al. 2013). C-terminally truncated apoA-I wasdetected in normal human serum using a specific monoclonal antibody (16-4mAb)recognizing C-terminally truncated apoA-I that has been cleaved after Phe225 bychymase (Usami et al. 2011). In addition, it has been shown that proteolysis ofapoA-I in preβ-HDL by plasmin generated apoA-I fragments lacking theC-terminal region (Kunitake et al. 1990). Such C-terminal truncation of apoA-Ican affect its capacity to promote ABCA1-mediated cholesterol efflux and thebiogenesis of HDL. Previous studies showed that when C-terminal segments thatcontain residues 220–231 are deleted, the apoA-I cannot associate with ABCA1 andhas a diminished capacity to promote ABCA1-mediated phospholipid cholesterolefflux (Favari et al. 2002; Chroni et al. 2003, 2004). In another study, the treatmentof lipid-free human apoA-I by chymase-containing lysate derived from mouseperitoneal mast cells or recombinant human chymase generated apoA-I fragments,lacking the C-terminal site (Usami et al. 2013), which had diminished capacity topromote cholesterol efflux from mouse peritoneal macrophage foam cells(Lee-Rueckert, Silvennoinen et al. 2011). Furthermore, adenovirus-mediated genetransfer in apoA-I-deficient mice showed that the apoA-I mutants that lackC-terminal residues 220–231 fail to form spherical αHDL in vivo (Chroniet al. 2003, 2007).Other proteolysis studies showed that apoA-I in HDL3 by various recombinantmetalloproteinases in vitro or by metalloproteinases secreted from macrophagesgenerated apoA-I fragments also lacking the carboxyl-terminal region (cleavageafter residues 213, 202, 215, 225,199, 191, or 188) (Lindstedt et al. 1999; Eberiniet al. 2002). In addition, chymase, in vitro, cleaves apoA-I in reconstituted HDL atthe C-terminus (after Phe225) (Lee et al. 2003). These C-terminal truncatedlipoprotein-associated apoA-I displayed diminished capacity to promote choles-terol efflux from mouse macrophage foam cells (Lindstedt et al. 1999; Leeet al. 2003). A recent study showed that reconstituted HDL containing theC-terminal deletion mutants apoA-I(Δ(185–243)) or apoA-I(Δ(220–243)) hadstrongly impaired capacity to promote ABCG1-mediated cholesterol efflux (Daniilet al. 2013). In addition, limited proteolysis of rHDL containing wild-type apoA-Iby plasmin resulted in 66 % decrease of ABCG1-dependent cholesterol efflux(Daniil et al. 2013). It is therefore possible that proteolysis of HDL-associatedapoA-I in vivo by proteases, which are present in the human arterial intima, couldyield apoA-I fragments similar to the apoA-I(Δ(185–243)) or apoA-I(Δ(220–243))mutants and thus may impair their capacity to promote ABCG1-mediatedCholesterol Efflux and Reverse Cholesterol Transport 189cholesterol efflux from macrophages. The structure-function relationship seen inthis study between rHDL-associated apoA-I mutants and ABCG1-mediated cho-lesterol efflux closely resembles that seen before in lipid-free apoA-I mutants andABCA1-dependent cholesterol efflux, suggesting that both processes depend on thesame structural determinants of apoA-I.The levels, composition, and the antiatherogenic properties, including thecholesterol efflux capacity, of HDL has been shown to be affected in patientssuffering from chronic inflammatory rheumatic diseases (Onat and Direskeneli2012; Watanabe et al. 2012). Specifically, cholesterol efflux capacity of HDL wasimpaired in rheumatoid arthritis and systemic lupus erythematosus patients withhigh disease activity and was correlated with systemic inflammation, higher plasmaMPO activity, and HDL’s antioxidant capacity (Ronda et al. 2013; Charles-Schoeman et al. 2012). In addition, HDL cholesterol efflux capacity was lower inpatients with psoriasis (Watanabe et al. 2012; Holzer et al. 2013), whileantipsoriatic therapy significantly improved the HDL cholesterol efflux capacity,along with improved serum LCAT activity and without any effect on serum HDLcholesterol levels (Holzer et al. 2013).Overall, modifications of HDL particles have been demonstrated to affect theatheroprotective properties of HDL, including its capacity to promote cellularcholesterol efflux. Elucidation of the conditions and processes that affect thecompositional, structural, and functional intactness of HDL may become an impor-tant tool for the assessment of cardiovascular risk and may provide us with noveltherapeutic approaches. In addition, monitoring of the capacity of HDL to promotecholesterol efflux could be useful to evaluate novel therapeutic approaches for thereduction of cardiovascular risk, as it will be discussed later.3 RCT in Animal Models3.1 PhysiologyThe term RCT summarizes the transport of cholesterol from macrophage foam cellswithin atherosclerotic lesions through the aqueous compartment of the blood forfinal excretion into the feces, which could either occur directly as cholesterol orafter metabolic conversion into bile acids (Annema and Tietge 2012). As detailedabove, cholesterol efflux from lipid-laden macrophages is mediated by thetransporters ABCA1, ABCG1, and SR-BI with HDL as acceptor, and this processis facilitated by apoE. Within the blood HDL can be remodeled in several ways.LCAT esterifies free cholesterol in the particles resulting in larger HDL andincreased plasma HDL-C levels (Annema and Tietge 2012). CETP transfers cho-lesterol out of HDL toward apoB-containing lipoproteins in exchange fortriglycerides, which are then rapidly hydrolyzed by hepatic lipase (HL) (Annemaand Tietge 2012). Thereby CETP and also HL, either alone or in combination,lower plasma HDL-C levels. Other HDL remodeling proteins that increase thecatabolic rate of HDL and lower circulating HDL-C are the phospholipid transfer190 E. Favari et al.protein (PLTP) (Annema and Tietge 2012) and the phospholipases endotheliallipase (EL) (Annema and Tietge 2011) and group IIA secretory phospholipase A2(sPLA2-IIA) (Rosenson and Hurt-Camejo 2012) . Uptake of HDL-C intohepatocytes can be accomplished in two distinct ways, via selective uptakemediated by SR-BI (Annema and Tietge 2012) or via holoparticle uptake in aprocess that has not been fully elucidated yet (Vantourout et al. 2010). However, ithas been shown that via ectopic localization of the mitochondrial F1-beta-ATPaseon the cell membrane, ADP is generated which then stimulates via the ADPreceptor P2Y13 holoparticle uptake of HDL by still elusive receptor mechanisms;thereby, HDL holoparticle uptake is fully dependent on the expression of the ADPreceptor P2Y13 (Vantourout et al. 2010). In hepatocytes, cholesterol is eithersecreted directly into the bile, which is mediated in terms of mass mainly byABCG5/G8 and to a lesser extent by SR-BI, or following conversion into bileacids, for which ABCB11 is the critical transporter into bile (Dikkers and Tietge2010). Cholesterol can be reabsorbed in the intestine by Niemann-Pick type C like1 (NPC1L1) but also (re)secreted by the ABCG5/G8 heterodimer, whereby a lowactivity of NPC1L1 would increase and a low activity of ABCG5/G8 woulddecrease cholesterol excretion into the feces (Annema and Tietge 2012). Bileacids can be reabsorbed in the terminal ileum by ASBT (Dawson 2011). In additionto its role in sterol absorption, the intestine has also been indicated to mediate directsecretion of cholesterol, a process termed transintestinal cholesterol excretion[TICE, current knowledge summarized in Tietge and Groen (2013)]. All differentsteps discussed in this paragraph have the potential to impact on and modulate RCT.3.1.1 Methodological Approaches to Quantify RCT In VivoThe key methodological problem for quantifying RCT specifically frommacrophages, which is most relevant for atherosclerotic disease, is that the macro-phage cholesterol pool is rather small. Initially employed techniques such as massdeterminations of centripetal cholesterol fluxes to the liver or isotope dilutionmethods were therefore not able to accurately allow a conclusion of specificcholesterol fluxes from macrophages (Annema and Tietge 2012). Recently, a nowwidely used and accepted technique had been developed that overcomes suchmethodological drawbacks (Zhang et al. 2003). Thereby, macrophages, eitherprimary or cell lines, are loaded with radiolabeled cholesterol and then injectedintraperitoneally into recipient animals. After injection, the appearance of the traceris determined in plasma at different time points and, most importantly, in the fecesthat are collected continuously. The time course of such experiments is usually24–48 h; in the feces distinguishing between labels in neutral sterols and bile acidsis in our view preferable.Although this method allows tracing of cholesterol from macrophages to feces,there are certain limitations that need to be taken into account when interpretingresults from such studies. One is that in the assay a potential influx of cholesterolfrom the plasma compartment into the macrophage is not taken into account. Asfirst steps to overcome this, two approaches were reported, in which the applicationprocedure of the labeled macrophages is modified in a way that allows reisolation ofCholesterol Efflux and Reverse Cholesterol Transport 191the cells to measure label as well as mass cholesterol content. One of theseapproaches makes use of Matrigel plugs that are implanted subcutaneously(Malik and Smith 2009), while the other another, entrapment of the macrophagesin semipermeable holofibers (Weibel et al. 2011). Another limitation of in vivoRCT assays is that all approaches place the labeled macrophages outside thevascular compartment, which supposedly is a good surrogate but might also notaccurately reflect the situation in an atherosclerotic lesion in terms of oxygentension, pH, or accessibility by the HDL particles. In the following, results obtainedfrom the above-described method will be summarized. It is relevant to point out thatalthough initial steps toward a macrophage RCT assay in humans have beenreported at conferences (Dunbar et al. 2013), current knowledge on the regulationof in vivo RCT is based on studies in animal models that differ in several aspects oftheir lipoprotein metabolism.3.1.2 Factors Impacting In Vivo RCTLooking at the level of the macrophage, available studies consistently indicate thatexpression of ABCA1 and ABCG1 is associated with increased RCT (Wanget al. 2007a, b; Out et al. 2008). However, SR-BI deficiency in macrophages doesnot impact RCT (Wang et al. 2007b; Zhao et al. 2011). On the other hand,macrophage apoE was shown to stimulate RCT (Zanotti et al. 2011a, b).Regarding plasma proteins that have a role in HDL metabolism, for LCATsurprisingly no consistent effects on RCT were observed using several differentmodels and overexpression as well as knockout strategies resulting in the conclu-sion that LCAT only minimally contributes to RCT although it has a major role indetermining plasma HDL-C levels (Tanigawa et al. 2009). In the case of the lipidtransfer proteins, PLTP overexpression resulted in increased RCT (Samynet al. 2009), while for CETP (over)expressing models, either an increase (Tchouaet al. 2008; Tanigawa et al. 2007), dependent on functional expression of the LDLreceptor (Tanigawa et al. 2007), or no effect (Rotllan et al. 2008) was observed.With respect to (phospho)lipases, sPLA2-IIA had no impact on RCT (Annemaet al. 2010). In case of HL and EL, conflicting data have been reported. Whileone group found no effect of knocking out either lipase separate or both incombination (Brown et al. 2010), others reported increased RCT for EL as wellas HL (Escola-Gil et al. 2013). In contrast to apoE expression in macrophages,systemic overexpression of apoE had no effect on RCT either in wild-type or CETPtransgenic mice (Annema et al. 2012).In the case of the hepatic component of RCT, SR-BI expression in hepatocyteshas a clear increasing effect on RCT as indicated in studies using both knockout andoverexpression (Zhao et al. 2011; El Bouhassani et al. 2011; Zhang et al. 2005).Interestingly, it has been shown that in the liver the impact of SR-BI is independentof ABCG5/G8 expression, since double-knockout mice for both transporters had asignificant decrease in RCT compared with ABCG5 knockout mice alone (Dikkerset al. 2013). ABCG5/G8 knockouts on the other hand exhibited no change in RCT(Calpe-Berdiel et al. 2008). In addition, also the consequences of abolishingfunctional HDL holoparticle uptake into hepatocytes on RCT were assessed. In192 E. Favari et al.P2Y13 knockout mice, this pathway is completely absent translating into a signifi-cant reduction in macrophage-to-feces RCT (Fabre et al. 2010; Lichtensteinet al. 2013). These combined studies indicate that both selective uptake andholoparticle uptake of HDL are critical for RCT. Regarding expression ofABCA1 in hepatocytes, liver supposedly contributes around 70 % to total HDLformation (Timmins et al. 2005); the picture is not so clear. While in wild-type andSR-BI knockout mice blocking hepatic ABCA1 with probucol enhanced RCT(Annema et al. 2012; Yamamoto et al. 2011), no such effect was seen in liver-specific ABCA1 knockout mice on a LDLR-deficient background (Bi et al. 2013).With respect to the final step of RCT, sterol excretion from the body, thedifferential contribution of the biliary pathway and the intestine is a subject ofactive study; it might be important to note that no unequivocally establishedconcept has emerged thus far. The classical view on the RCT pathway puts biliarysecretion of RCT-relevant cholesterol central (Annema and Tietge 2012). This viewis supported by studies using either surgical disruption of biliary secretion or Abcb4knockout mice that have a genetic defect in cholesterol secretion into the bilesecondary to their inability to secrete phospholipids and form mixed micelles(Nijstad et al. 2011). In the surgical model RCT was virtually absent, while in thegenetic model, RCT was strongly reduced (Nijstad et al. 2011). Studies inezetimibe-treated mice that express NPC1L1 only in hepatocytes reached a similarconclusion, namely, that functional RCT in this model depended on efficient biliarysterol secretion (Xie et al. 2013). On the other hand, RCT studies in NPC1L1 liver-transgenic mice on a wild-type background as well as a short-term experiment inbile-diverted mice resulted in opposite findings, namely, that RCT can proceedwhen the biliary secretion pathway is impaired (Temel et al. 2010). Further studiesare clearly required to assess the role of the intestine and a possible contribution ofTICE to macrophage RCT. However, currently such experiments are hampered bythe lack of information how TICE is precisely mediated (Tietge and Groen 2013).Commonly accepted on the other hand is the role of intestinal sterol absorption inRCT, and several pharmacological intervention studies were carried out to showthat blocking sterol absorption increases RCT (for details please see Table 1 andtext above).In addition, there are also more complex systemic pathophysiological states thatare clinically associated with an increased atherosclerosis incidence and in whichalso alterations in RCT have been observed in mouse models. One example is aninflammatory response. Consistently, RCT was severely impaired in LPS- (Annemaet al. 2010; McGillicuddy et al. 2009) and zymosan-induced (Malik et al. 2011)models of acute inflammation. Another example is diabetes. In both, an insulin-deficient model of type 1 diabetes (de Boer et al. 2012) as well as in db/db mice(Low et al. 2012), which lack the leptin receptor and serve as model of type2 diabetes, RCT was decreased. Interestingly, the first study indicated that theselective uptake step of HDL into the liver is defective in type 1 diabetic mice(de Boer et al. 2012), while the latter study provided evidence that advancedglycation end products are not likely to be pathophysiologically involved (Lowet al. 2012). Furthermore, acute psychological stress has been shown to increaseCholesterol Efflux and Reverse Cholesterol Transport 193Table1Effectsoftherapeuticagentsoninvivomacrophage-to-fecesRCTTreatmentAgentAnimalmodelMacrophage-to-fecesRCTReferencesCETPinhibitorsTorcetrapibWild-typemice,humanCETP-expressingmice#Tchouaetal.(2008)TorcetrapibHumanCETP/apoB100-expressingmice"Briandetal.(2011)TorcetrapibHamster"Tchouaetal.(2008),Niesoretal.(2010)DalcetrapibHamster"Niesoretal.(2010)AnacetrapibHamster¼or"Niesoretal.(2010),Castro-Perezetal.(2011)AnacetrapibHumanCETP-expressing/LDLR-deficientmice¼Belletal.(2013)CETPantisenseoligonucleotideHumanCETP-expressing/LDLR-deficientmice"Belletal.(2013)NuclearreceptorsSystemicLXRagonistsT0901317andGW3965Wild-typemice,LDLR/apobec-1double-knockoutmice,humanCETP/apoB100-expressingmice"Naiketal.(2006),Zanottietal.(2008),Calpe-Berdieletal.(2008),Yasudaetal.(2010),Nijstadetal.(2011)SystemicLXRagonistGW3965Hamster"Briandetal.(2010)Intestinal-specificLXRagonistGW6340Wild-typemice"Yasudaetal.(2010)PPARβ/δagonistsGW0742Wild-typemice"Briandetal.(2009),Silvennoinenetal.(2012)FenofibrateHumanapoA-Itransgenicmice"Rotllanetal.( 2011)GemfibrozilHumanapoA-Itransgenicmice¼Rotllanetal.(2011)PPARαagonistsGW7647Wild-typemice,LDLR/apobec-1double-knockoutmiceoverexpressinghumanapoA-I"Nakayaetal.(2011)Anti-miR33oligonucleotideLDLR-deficientmice"Rayneretal.(2011)PPARγagonistGW7845Wild-typemice#Tohetal.(2011)FXRagonistGW4064Wild-typemice,SR-BI-deficientmice"Zhangetal.(2010)194 E. Favari et al.CholesterolandbileacidabsorptioninhibitorsEzetimibeWild-typemice"Briandetal.(2009),SehayekandHazen(2008),Silvennoinenetal.(2012),Maugeaisetal.(2013)CholestyramineWild-typemice"Maugeaisetal.(2013)InjectionofreconstitutedHDLandapoA-IformsHumanapoA-IWild-typemice"Lee-Rueckertetal.(2011)ReconstitutedHDLWild-typemice"Maugeaisetal.(2013)MimeticD-4FApoE-deficientmice"Navabetal.(2004)MimeticATI-5261ApoE-deficientmice"Bielickietal.(2010)Mimetic5AWild-typemice"Amaretal.(2010)UpregulationofapoA-IproductionThienotriazolodiazepineRo11-1464HumanapoA-Itransgenicmice"Zanottietal.(2011a,b)"or#denotesthatthemeasuredparameterincreasedordecreased,respectively¼,theparameterremainedunchangedCholesterol Efflux and Reverse Cholesterol Transport 195RCT in mice, mainly by a corticosterone-mediated downregulation of NPC1L1 andsubsequent reduction in intestinal cholesterol absorption (Silvennoinen et al. 2012).These data underline the important role of intestinal NPC1L1 in RCT but arecounterintuitive to the increased atherosclerosis risk attributed to psychologicalstress in clinical settings.Finally, two recent additions of pathways that are linked to RCT have emergedwhich are not so obvious given the classical view on the RCT pathway but areworth mentioning. On the one hand, red blood cells were shown to contribute toRCT in a way that functional RCT was decreased in anemic mice (Hunget al. 2012). On the other hand, lymphatic drainage has been indicated to beinvolved in RCT (Martel et al. 2013; Lim et al. 2013). Hereby, blocking lymphatictransport or lymph vessel regrowth inhibited RCT, while enabling it increased RCT(Martel et al. 2013; Lim et al. 2013). Interestingly, for this route of RCT,transcytosis of HDL through lymphatic endothelium in a SR-BI-dependent fashionwas critical (Lim et al. 2013), findings very similar to the role of SR-BI in HDLtranscytosis through vascular endothelial cells (Rohrer et al. 2009).3.2 PharmacologyRCT-enhancing therapies are currently considered a promising strategy for theprevention and treatment of atherosclerotic CVD. An important number ofRCT-targeted drugs have been used in preclinical animal models (mainly miceand hamsters) to test their effects on in vivo RCT from labeled cholesterolmacrophages to feces. These RCT-targeted drugs can be classified among fourdifferent therapeutic approaches: CETP inhibition, nuclear receptors activation,cholesterol absorption inhibition, and directly augmenting or mimicking apoA-I.Most of these drugs are being used in clinical practice or tested in clinical trials inphases I, II, or III. This section discusses recent findings indicating that some ofthese therapies may be atheroprotective by promoting RCT in vivo (Table 1 showsa summary of available data).3.2.1 CETP InhibitorsCETP inhibition presents a preferential target for raising HDL-C and enhancingRCT. However, available data on the effect of the CETP inhibitors torcetrapib,dalcetrapib, and anacetrapib in macrophage-to-feces RCT have produced divergentresults (Tchoua et al. 2008; Briand et al. 2011; Niesor et al. 2010; Castro-Perezet al. 2011). These controversial effects, together with the disappointing results oftwo large clinical trials using torcetrapib and dalcetrapib, have raised reasonabledoubts regarding the clinical use of CETP inhibitors (Barter and Rye 2012). Thepositive effects of CETP antisense oligonucleotides on macrophage RCT (Bellet al. 2013) open up an alternative pathway to further evaluate whether the inhibi-tion of CETP may improve cardiovascular risk.196 E. Favari et al.3.2.2 Nuclear Receptor ActivationLiver X receptor (LXR) is considered an attractive target for therapeutic strategiesaimed at stimulating RCT since it promotes HDL biogenesis, macrophage choles-terol efflux, and biliary cholesterol excretion. A considerable number of studiestested the effect of systemic LXR agonists T0901317 and GW3965 on macrophage-to-feces RCT and consistently found a higher flux through this pathway (Naiket al. 2006; Zanotti et al. 2008; Calpe-Berdiel et al. 2008; Yasuda et al. 2010;Nijstad et al. 2011; Briand et al. 2010). However, systemic LXR activators havedetrimental consequences for the liver such as the induction of lipogenesis. Asdiscussed in Sect. 3.1.2, multiple evidences indicate that excretion of macrophage-derived cholesterol can be modulated in the last step of RCT pathway which occursin the small intestine. Indeed, intestine-specific LXR activator GW6340 alsoenhances macrophage RCT but avoids the lipogenic toxicity associated with liverLXR activation in mice (Yasuda et al. 2010). Peroxisome proliferator-activatedreceptor (PPAR) α agonists such as GW7647 and fenofibrate promote macrophageRCT (Rotllan et al. 2011; Nakaya et al. 2011). This effect is strongly correlated withthe positive effects on apoA-I levels; more importantly, macrophage PPARα andLXR expression are required for the PPARα-mediated enhancement of macrophageRCT (Nakaya et al. 2011). ABCA1 and G1 are targeted for degradation bymicroRNA (miR)-33, an intronic microRNA located within the SREBF2 gene;anti–miR-33 therapy enhances macrophage RCT (Rayner et al. 2011). In contrast,the PPARγ agonist GW7845 reduces macrophage RCT. The authors hypothesizethat GW7845 redirects macrophage-derived cholesterol to adipose tissue viaSR-BI, thereby reducing its biliary excretion (Toh et al. 2011). Beyond PPAR-LXR activation, the farnesoid X receptor (FXR) agonist GW4064 also promotesmacrophage-to-feces RCT; this effect is related to liver SR-BI upregulation andreduced intestinal cholesterol absorption (Zhang et al. 2010).3.2.3 Cholesterol Absorption InhibitorsInterventions that inhibit cholesterol absorption including ezetimibe administrationand PPAR β/δ activation with GW0742 increase the excretion of macrophage-derived cholesterol in feces by reducing intestinal NPC1L1 activity (Briandet al. 2009; Sehayek and Hazen 2008; Silvennoinen et al. 2012; Maugeaiset al. 2013). The bile acid sequestrant cholestyramine also promotes macrophage-to-feces RCT (Maugeais et al. 2013).3.2.4 Augmenting or Mimicking apoA-IThe use of apoA-I and its different forms for the prevention and treatment ofatherosclerosis is a long-term goal of many laboratories. The full-length apoA-I,reconstituted apoA-I-containing HDL, or apoA-I mimetics D4-F, ATI-5261, and5A have been demonstrated to be effective for enhancing macrophage-to-fecesRCT in different mouse models (Maugeais et al. 2013; Lee-Rueckert and Kovanen2011; Navab et al. 2004; Bielicki et al. 2010; Amar et al. 2010). The upregulation ofliver apoA-I production, as occurred with the thienotriazolodiazepine Ro 11-1464,also enhances macrophage RCT (Zanotti et al. 2011a, b).Cholesterol Efflux and Reverse Cholesterol Transport 1974 Serum Cholesterol Efflux Capacity (CEC)Several epidemiological studies define HDL as the most powerful plasmatic factorwith atheroprotective activity in humans (Di Angelantonio et al. 2009); a 1 mg/dlincrease of plasma HDL-C is associated with a 3–4 % reduction in cardiovascularmortality (Assmann et al. 2002). Some post hoc analyses from randomized con-trolled trials also suggest that raising HDL-C beneficially affects the risk of CVD(Toth et al. 2013). However, the clinical efficacy of raising plasma HDL-C levels toachieve cardiovascular risk reduction has been difficult to prove. Recentlypublished outcome trials involving the addition of niacin or dalcetrapib to standardlow-density lipoprotein cholesterol reduction therapy failed to demonstrate clinicalbenefit despite increases in HDL-C ((AIM-HIGH) trial 2011; Schwartz et al. 2012).Furthermore, genetic variants associated with increased HDL-C, thus conferringlifelong exposure to higher circulating levels, are not consistently associated withimproved vascular outcomes (Voight et al. 2012). These findings have reinforcedthe idea that changes in HDL-C levels are an inadequate surrogate for therapeuticuse. Therefore, an emerging concept is that of the quality of HDL, which areheterogeneous in terms of size, charge, and lipid content (Calabresi et al. 2010)and display functional differences, such as cell cholesterol efflux promotion.Animal studies have suggested that HDL-mediated cholesterol flux through thedifferent RCT steps is a better predictor of the atherosclerotic impact of variousgenetic and pharmacological perturbations than static, mass-based quantification ofcirculating HDL-C (Rader et al. 2009). The efficacy of such HDL function in asingle individual may be estimated by measuring the cholesterol efflux capacity(CEC) using widely standardized techniques that allow distinguishing between thevarious mechanisms involved (Adorni et al. 2007). Several lines of evidencesuggest that CEC is sensitive to HDL composition rather than HDL-C plasmalevels; for instance, it has been shown that the serum capacity to promote choles-terol efflux via ABCA1 strictly depends on the nascent (preβ) HDL plasma levels(de la Llera-Moya et al. 2010; Favari et al. 2004). Subjects with the apoA-IMilanomutation or LCAT deficiency have high levels of circulating particles and efficientserum ability to induce macrophage cholesterol depletion despite very low HDLlevels (Favari et al. 2007; Calabresi et al. 2009). Thus, there is increasing evidencethat the measure of HDL functionality in different populations may be a betterpredictor of coronary artery disease than the measure of absolute HDL-C levels(Khera et al. 2011). As mentioned before, this concept is consistent with studiesrevealing that subjects affected by CVD not only have HDL deficiency but alsomajor rearrangements of their composition (Campos et al. 1995; Sweetnamet al. 1994) and consequently function. The role of serum CEC as an index ofcardiovascular protection has been suggested by a recent study demonstrating thatin two distinct cohorts of subjects, the CEC variable has a stronger predictive powerof the carotid intima-media thickness, an index of subclinical atherosclerosis, thanplasma HDL-C levels (Khera et al. 2011). In addition, in a population of healthysubjects, ABCA1-mediated serum CEC was inversely correlated with pulse wavevelocity, an index of arterial stiffness, independent of HDL-C serum levels (Favari198 E. Favari et al.et al. 2013). A work measuring the flow-mediated dilation, as a parameter ofendothelial function, showed a positive correlation with the ABCA1-mediatedefflux pathway confirming the concept that the functional measures of HDLmight be a better marker for cardiovascular risk rather than HDL cholesterol levels(Vazquez et al. 2012). Recently, Li and colleagues provided data indicating thatindividuals in the top tertile of CEC had a moderately increased risk of a compositecardiovascular end point of incident myocardial infarction, stroke, or death during3 years of follow-up (Li et al. 2013). However, in the same study, CEC wasinversely associated with coronary artery disease. The reasons for this apparentlycontradictory findings require further evaluation, but may be related to thecharacteristics of the population involved in the study (Khera and Rader 2013).Overall, the available data suggest that evaluation of CEC may better correlate withcoronary artery diseases than HDL-C; however, further studies are required todemonstrate its role as predictor of cardiovascular event risk.ConclusionsRCT provides a physiological strategy of protection from atherosclerosis. HDLplays a leading role by promoting the removal of excess cholesterol in the arterialwall through the induction of cellular cholesterol efflux from cells. Cholesterolefflux represents the first and perhaps most important step of RCT. The ability ofHDL to promote cholesterol efflux depends on their quality and the pathologicprocesses that induce their modifications. Many of the available data on thephysiology and pharmacology of RCT depend on studies in animal models thathave demonstrated that the enhancement of RCT is inversely correlated with thedevelopment of atherosclerosis. In humans, the efficiency of the RCT can beevaluated with the surrogate parameter of serum cholesterol efflux capacity(CEC) that indicates the ability of HDL to promote efflux of cholesterol in theindividual patient. Recent clinical data suggest that the evaluation of CEC is astrong predictor of atherosclerosis extent in humans and may represent in thefuture a useful biomarker of cardiovascular risk.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAdorni MP, Zimetti F et al (2007) The roles of different pathways in the release of cholesterol frommacrophages. J Lipid Res 48(11):2453–2462Adorni MP, Favari E et al (2011) Free cholesterol alters macrophage morphology and mobility byan ABCA1 dependent mechanism. Atherosclerosis 215(1):70–76AIM-HIGH (2011) The role of niacin in raising high-density lipoprotein cholesterol to reducecardiovascular events in patients with atherosclerotic cardiovascular disease and optimallytreated low-density lipoprotein cholesterol: baseline characteristics of study participants. TheCholesterol Efflux and Reverse Cholesterol Transport 199Atherothrombosis Intervention in Metabolic syndrome with low HDL/high triglycerides:impact on Global Health outcomes (AIM-HIGH) trial. Am Heart J 161(3):538–543Amar MJ, D’Souza W et al (2010) 5A apolipoprotein mimetic peptide promotes cholesterol effluxand reduces atherosclerosis in mice. J Pharmacol Exp Ther 334(2):634–641Annema W, Tietge UJ (2011) Role of hepatic lipase and endothelial lipase in high-densitylipoprotein-mediated reverse cholesterol transport. Curr Atheroscler Rep 13(3):257–265Annema W, Tietge UJ (2012) Regulation of reverse cholesterol transport - a comprehensiveappraisal of available animal studies. Nutr Metab (Lond) 9(1):25Annema W, von Eckardstein A (2013) High-density lipoproteins. Multifunctional but vulnerableprotections from atherosclerosis. Circ J 77(10):2432–2448Annema W, Nijstad N et al (2010) Myeloperoxidase and serum amyloid A contribute to impairedin vivo reverse cholesterol transport during the acute phase response but not group IIAsecretory phospholipase A(2). J Lipid Res 51(4):743–754Annema W, Dikkers A et al (2012) ApoE promotes hepatic selective uptake but not RCT due toincreased ABCA1-mediated cholesterol efflux to plasma. J Lipid Res 53(5):929–940Assmann G, Cullen P et al (2002) Simple scoring scheme for calculating the risk of acute coronaryevents based on the 10-year follow-up of the prospective cardiovascular Munster (PROCAM)study. Circulation 105(3):310–315Barter PJ, Rye KA (2012) Cholesteryl ester transfer protein inhibition as a strategy to reducecardiovascular risk. J Lipid Res 53(9):1755–1766Bell TA 3rd, Graham MJ et al (2013) Antisense oligonucleotide inhibition of cholesteryl estertransfer protein enhances RCT in hyperlipidemic, CETP transgenic, LDLr-/- mice. J Lipid Res54(10):2647–2657Bergt C, Pennathur S et al (2004) The myeloperoxidase product hypochlorous acid oxidizes HDLin the human artery wall and impairs ABCA1-dependent cholesterol transport. Proc Natl AcadSci U S A 101(35):13032–13037Bi X, Zhu X et al (2013) Liver ABCA1 deletion in LDLrKO mice does not impair macrophagereverse cholesterol transport or exacerbate atherogenesis. Arterioscler Thromb Vasc Biol 33(10):2288–2296Bielicki JK, Zhang H et al (2010) A new HDL mimetic peptide that stimulates cellular cholesterolefflux with high efficiency greatly reduces atherosclerosis in mice. J Lipid Res 51(6):1496–1503Boadu E, Francis GA (2006) The role of vesicular transport in ABCA1-dependent lipid efflux andits connection with NPC pathways. J Mol Med (Berl) 84(4):266–275Briand F, Naik SU et al (2009) Both the peroxisome proliferator-activated receptor delta agonist,GW0742, and ezetimibe promote reverse cholesterol transport in mice by reducing intestinalreabsorption of HDL-derived cholesterol. Clin Transl Sci 2(2):127–133Briand F, Treguier M et al (2010) Liver X receptor activation promotes macrophage-to-fecesreverse cholesterol transport in a dyslipidemic hamster model. J Lipid Res 51(4):763–770Briand F, Thieblemont Q et al (2011) CETP inhibitor torcetrapib promotes reverse cholesteroltransport in obese insulin-resistant CETP-ApoB100 transgenic mice. Clin Transl Sci 4(6):414–420Brown RJ, Lagor WR et al (2010) Impact of combined deficiency of hepatic lipase and endotheliallipase on the metabolism of both high-density lipoproteins and apolipoprotein B-containinglipoproteins. Circ Res 107(3):357–364Calabresi L, Favari E et al (2009) Functional LCAT is not required for macrophage cholesterolefflux to human serum. Atherosclerosis 204(1):141–146Calabresi L, Gomaraschi M et al (2010) High-density lipoprotein quantity or quality for cardio-vascular prevention? Curr Pharm Des 16(13):1494–1503Calpe-Berdiel L, Rotllan N et al (2008) Liver X receptor-mediated activation of reverse choles-terol transport from macrophages to feces in vivo requires ABCG5/G8. J Lipid Res 49(9):1904–1911200 E. Favari et al.Campos H, Roederer GO et al (1995) Predominance of large LDL and reduced HDL2 cholesterolin normolipidemic men with coronary artery disease. Arterioscler Thromb Vasc Biol 15(8):1043–1048Castro-Perez J, Briand F et al (2011) Anacetrapib promotes reverse cholesterol transport and bulkcholesterol excretion in Syrian golden hamsters. J Lipid Res 52(11):1965–1973Cavigiolio G, Geier EG et al (2010) Exchange of apolipoprotein A-I between lipid-associated andlipid-free states: a potential target for oxidative generation of dysfunctional high densitylipoproteins. J Biol Chem 285(24):18847–18857Charles-Schoeman C, Lee YY et al (2012) Cholesterol efflux by high density lipoproteins isimpaired in patients with active rheumatoid arthritis. Ann Rheum Dis 71(7):1157–1162Chroni A, Liu T et al (2003) The central helices of ApoA-I can promote ATP-binding cassettetransporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-typeApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo.J Biol Chem 278(9):6719–6730Chroni A, Liu T et al (2004) Cross-linking and lipid efflux properties of apoA-I mutants suggestdirect association between apoA-I helices and ABCA1. Biochemistry 43(7):2126–2139Chroni A, Koukos G et al (2007) The carboxy-terminal region of apoA-I is required for theABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo. Biochem-istry 46(19):5697–5708Daniil G, Zannis VI et al (2013) Effect of apoA-I mutations in the capacity of reconstituted HDL topromote ABCG1-mediated cholesterol efflux. PLoS ONE 8(6):e67993Dawson PA (2011) Role of the intestinal bile acid transporters in bile acid and drug disposition.Handb Exp Pharmacol 201:169–203de Boer JF, Annema W et al (2012) Type I diabetes mellitus decreases in vivo macrophage-to-feces reverse cholesterol transport despite increased biliary sterol secretion in mice. J Lipid Res53(3):348–357de la Llera-Moya M, Drazul-Schrader D et al (2010) The ability to promote efflux via ABCA1determines the capacity of serum specimens with similar high-density lipoprotein cholesterolto remove cholesterol from macrophages. Arterioscler Thromb Vasc Biol 30(4):796–801Di Angelantonio E, Sarwar N et al (2009) Major lipids, apolipoproteins, and risk of vasculardisease. JAMA 302(18):1993–2000Dikkers A, Tietge UJ (2010) Biliary cholesterol secretion: more than a simple ABC. World JGastroenterol 16(47):5936–5945Dikkers A, Freak de Boer J et al (2013) Scavenger receptor BI and ABCG5/G8 differentiallyimpact biliary sterol secretion and reverse cholesterol transport in mice. Hepatology 58(1):293–303Dunbar RL, Cuchel M, Millar JS, Baer A, Poria R, Freifelder RH, Pryma DA, Billheimer JJ, RaderDJ (2013) Abstract 439: niacin does not accelerate reverse cholesterol transport in man.Arterioscler Thromb Vasc Biol 33:A439Eberini I, Calabresi L et al (2002) Macrophage metalloproteinases degrade high-density-lipopro-tein-associated apolipoprotein A-I at both the N- and C-termini. Biochem J 362(Pt 3):627–634El Bouhassani M, Gilibert S et al (2011) Cholesteryl ester transfer protein expression partiallyattenuates the adverse effects of SR-BI receptor deficiency on cholesterol metabolism andatherosclerosis. J Biol Chem 286(19):17227–17238Escola-Gil JC, Chen X et al (2013) Hepatic lipase- and endothelial lipase-deficiency in micepromotes macrophage-to-feces RCT and HDL antioxidant properties. Biochim Biophys Acta1831(4):691–697Fabre AC, Malaval C et al (2010) P2Y13 receptor is critical for reverse cholesterol transport.Hepatology 52(4):1477–1483Favari E, Bernini F et al (2002) The C-terminal domain of apolipoprotein A-I is involved inABCA1-driven phospholipid and cholesterol efflux. Biochem Biophys Res Commun 299(5):801–805Cholesterol Efflux and Reverse Cholesterol Transport 201Favari E, Lee M et al (2004) Depletion of pre-beta-high density lipoprotein by human chymaseimpairs ATP-binding cassette transporter A1- but not scavenger receptor class B typeI-mediated lipid efflux to high density lipoprotein. J Biol Chem 279(11):9930–9936Favari E, Gomaraschi M et al (2007) A unique protease-sensitive high density lipoprotein particlecontaining the apolipoprotein A-I(Milano) dimer effectively promotes ATP-binding cassetteA1-mediated cell cholesterol efflux. J Biol Chem 282(8):5125–5132Favari E, Calabresi L et al (2009) Small discoidal pre-beta1 HDL particles are efficient acceptorsof cell cholesterol via ABCA1 and ABCG1. Biochemistry 48(46):11067–11074Favari E, Ronda N et al (2013) ABCA1-dependent serum cholesterol efflux capacity inverselycorrelates with pulse wave velocity in healthy subjects. J Lipid Res 54(1):238–243Freeman SR, Jin X et al (2013) ABCG1-mediated generation of extracellular cholesterolmicrodomains. J Lipid Res 55(1):115–127Fu Y, Mukhamedova N et al (2013) ABCA12 regulates ABCA1-dependent cholesterol efflux frommacrophages and the development of atherosclerosis. Cell Metab 18(2):225–238Gargalovic P, Dory L (2003) Caveolins and macrophage lipid metabolism. J Lipid Res 44(1):11–21Gelissen IC, Harris M et al (2006) ABCA1 and ABCG1 synergize to mediate cholesterol export toapoA-I. Arterioscler Thromb Vasc Biol 26(3):534–540Holzer M, Wolf P et al (2013) Anti-psoriatic therapy recovers high-density lipoprotein composi-tion and function. J Invest Dermatol 134(3):635–642Huang ZH, Lin CY et al (2001) Sterol efflux mediated by endogenous macrophage ApoEexpression is independent of ABCA1. Arterioscler Thromb Vasc Biol 21(12):2019–2025Huang ZH, Gu D et al (2003) Expression of scavenger receptor BI facilitates sterol movementbetween the plasma membrane and the endoplasmic reticulum in macrophages. Biochemistry42(13):3949–3955Hung KT, Berisha SZ et al (2012) Red blood cells play a role in reverse cholesterol transport.Arterioscler Thromb Vasc Biol 32(6):1460–1465Ishimoto Y, Yamada K et al (2003) Group V and X secretory phospholipase A(2)s-inducedmodification of high-density lipoprotein linked to the reduction of its antiatherogenicfunctions. Biochim Biophys Acta 1642(3):129–138Khera AV, Rader DJ (2013) Cholesterol efflux capacity: full steam ahead or a bump in the road?Arterioscler Thromb Vasc Biol 33(7):1449–1451Khera AV, Cuchel M et al (2011) Cholesterol efflux capacity, high-density lipoprotein function,and atherosclerosis. N Engl J Med 364(2):127–135Kockx M, Jessup W et al (2008) Regulation of endogenous apolipoprotein E secretion bymacrophages. Arterioscler Thromb Vasc Biol 28(6):1060–1067Kunitake ST, Chen GC et al (1990) Pre-beta high density lipoprotein. Unique disposition ofapolipoprotein A-I increases susceptibility to proteolysis. Arteriosclerosis 10(1):25–30Landry YD, Denis M et al (2006) ATP-binding cassette transporter A1 expression disrupts raftmembrane microdomains through its ATPase-related functions. J Biol Chem 281(47):36091–36101Larrede S, Quinn CM et al (2009) Stimulation of cholesterol efflux by LXR agonists incholesterol-loaded human macrophages is ABCA1-dependent but ABCG1-independent.Arterioscler Thromb Vasc Biol 29(11):1930–1936Lee M, Kovanen PT et al (2003) Apolipoprotein composition and particle size affect HDLdegradation by chymase: effect on cellular cholesterol efflux. J Lipid Res 44(3):539–546Lee-Rueckert M, Kovanen PT (2011) Extracellular modifications of HDL in vivo and theemerging concept of proteolytic inactivation of prebeta-HDL. Curr Opin Lipidol 22(5):394–402Lee-Rueckert M, Silvennoinen R et al (2011) Mast cell activation in vivo impairs the macrophagereverse cholesterol transport pathway in the mouse. Arterioscler Thromb Vasc Biol 31(3):520–527202 E. Favari et al.Li XM, Tang WH et al (2013) Paradoxical association of enhanced cholesterol efflux withincreased incident cardiovascular risks. Arterioscler Thromb Vasc Biol 33(7):1696–1705Lichtenstein L, Serhan N et al (2013) Lack of P2Y13 in mice fed a high cholesterol diet results indecreased hepatic cholesterol content, biliary lipid secretion and reverse cholesterol transport.Nutr Metab (Lond) 10(1):67Lim HY, Thiam CH et al (2013) Lymphatic vessels are essential for the removal of cholesterolfrom peripheral tissues by SR-BI-mediated transport of HDL. Cell Metab 17(5):671–684Lindstedt L, Saarinen J et al (1999) Matrix metalloproteinases-3, -7, and -12, but not -9, reducehigh density lipoprotein-induced cholesterol efflux from human macrophage foam cells bytruncation of the carboxyl terminus of apolipoprotein A-I. Parallel losses of pre-beta particlesand the high affinity component of efflux. J Biol Chem 274(32):22627–22634Low H, Hoang A et al (2012) Advanced glycation end-products (AGEs) and functionality ofreverse cholesterol transport in patients with type 2 diabetes and in mouse models.Diabetologia 55(9):2513–2521Malik P, Smith JP (2009) A novel in vivo assay for reverse cholesterol transport. ArteriosclerThromb Vasc Biol 29:E46Malik P, Berisha SZ et al (2011) Zymosan-mediated inflammation impairs in vivo reversecholesterol transport. J Lipid Res 52(5):951–957Martel C, Li W et al (2013) Lymphatic vasculature mediates macrophage reverse cholesteroltransport in mice. J Clin Invest 123(4):1571–1579Maugeais C, Annema W et al (2013) rHDL administration increases reverse cholesterol transportin mice, but is not additive on top of ezetimibe or cholestyramine treatment. Atherosclerosis229(1):94–101Maxfield FR, Tabas I (2005) Role of cholesterol and lipid organization in disease. Nature 438(7068):612–621McGillicuddy FC, de la Llera Moya M et al (2009) Inflammation impairs reverse cholesteroltransport in vivo. Circulation 119(8):1135–1145Naik SU, Wang X et al (2006) Pharmacological activation of liver X receptors promotes reversecholesterol transport in vivo. Circulation 113(1):90–97Nakaya K, Tohyama J et al (2011) Peroxisome proliferator-activated receptor-alpha activationpromotes macrophage reverse cholesterol transport through a liver X receptor-dependentpathway. Arterioscler Thromb Vasc Biol 31(6):1276–1282Navab M, Anantharamaiah GM et al (2004) Oral D-4 F causes formation of pre-beta high-densitylipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reversecholesterol transport from macrophages in apolipoprotein E-null mice. Circulation 109(25):3215–3220Niesor EJ, Magg C et al (2010) Modulating cholesteryl ester transfer protein activity maintainsefficient pre-beta-HDL formation and increases reverse cholesterol transport. J Lipid Res51(12):3443–3454Nijstad N, Gautier T et al (2011) Biliary sterol secretion is required for functional in vivo reversecholesterol transport in mice. Gastroenterology 140(3):1043–1051Olivier M, Tanck MW et al (2012) Human ATP-binding cassette G1 controls macrophagelipoprotein lipase bioavailability and promotes foam cell formation. Arterioscler ThrombVasc Biol 32(9):2223–2231Onat A, Direskeneli H (2012) Excess cardiovascular risk in inflammatory rheumatic diseases:pathophysiology and targeted therapy. Curr Pharm Des 18(11):1465–1477Oram JF, Lawn RM (2001) ABCA1. The gatekeeper for eliminating excess tissue cholesterol.J Lipid Res 42(8):1173–1179Oram JF, Vaughan AM (2006) ATP-binding cassette cholesterol transporters and cardiovasculardisease. Circ Res 99(10):1031–1043Out R, Jessup W et al (2008) Coexistence of foam cells and hypocholesterolemia in mice lackingthe ABC transporters A1 and G1. Circ Res 102(1):113–120Cholesterol Efflux and Reverse Cholesterol Transport 203Pennathur S, Bergt C et al (2004) Human atherosclerotic intima and blood of patients withestablished coronary artery disease contain high density lipoprotein damaged by reactivenitrogen species. J Biol Chem 279(41):42977–42983Pirillo A, Uboldi P et al (2006) 15-Lipoxygenase-mediated modification of high-densitylipoproteins impairs SR-BI- and ABCA1-dependent cholesterol efflux from macrophages.Biochim Biophys Acta 1761(3):292–300Rader DJ, Alexander ET et al (2009) The role of reverse cholesterol transport in animals andhumans and relationship to atherosclerosis. J Lipid Res 50(Suppl):S189–S194Ramirez CM, Davalos A et al (2011) MicroRNA-758 regulates cholesterol efflux through post-transcriptional repression of ATP-binding cassette transporter A1. Arterioscler Thromb VascBiol 31(11):2707–2714Rayner KJ, Suarez Y et al (2010) MiR-33 contributes to the regulation of cholesterol homeostasis.Science 328(5985):1570–1573Rayner KJ, Sheedy FJ et al (2011) Antagonism of miR-33 in mice promotes reverse cholesteroltransport and regression of atherosclerosis. J Clin Invest 121(7):2921–2931Rohrer L, Ohnsorg PM et al (2009) High-density lipoprotein transport through aortic endothelialcells involves scavenger receptor BI and ATP-binding cassette transporter G1. Circ Res 104(10):1142–1150Ronda N, Favari E et al (2013) Impaired serum cholesterol efflux capacity in rheumatoid arthritisand systemic lupus erythematosus. Ann Rheum Dis 73(3):609–615Rosenson RS, Hurt-Camejo E (2012) Phospholipase A2 enzymes and the risk of atherosclerosis.Eur Heart J 33(23):2899–2909Rosenson RS, Brewer HB Jr et al (2012) Cholesterol efflux and atheroprotection: advancing theconcept of reverse cholesterol transport. Circulation 125(15):1905–1919Rotllan N, Calpe-Berdiel L et al (2008) CETP activity variation in mice does not affect two majorHDL antiatherogenic properties: macrophage-specific reverse cholesterol transport and LDLantioxidant protection. Atherosclerosis 196(2):505–513Rotllan N, Llaverias G et al (2011) Differential effects of gemfibrozil and fenofibrate on reversecholesterol transport from macrophages to feces in vivo. Biochim Biophys Acta 1811(2):104–110Samyn H, Moerland M et al (2009) Elevation of systemic PLTP, but not macrophage-PLTP,impairs macrophage reverse cholesterol transport in transgenic mice. Atherosclerosis 204(2):429–434Schmitz G, Langmann T et al (2001) Role of ABCG1 and other ABCG family members in lipidmetabolism. J Lipid Res 42(10):1513–1520Schwartz GG, Olsson AG et al (2012) Effects of dalcetrapib in patients with a recent acutecoronary syndrome. N Engl J Med 367(22):2089–2099Sehayek E, Hazen SL (2008) Cholesterol absorption from the intestine is a major determinant ofreverse cholesterol transport from peripheral tissue macrophages. Arterioscler Thromb VascBiol 28(7):1296–1297Shao B, Bergt C et al (2005) Tyrosine 192 in apolipoprotein A-I is the major site of nitration andchlorination by myeloperoxidase, but only chlorination markedly impairs ABCA1-dependentcholesterol transport. J Biol Chem 280(7):5983–5993Shao B, Oda MN et al (2006) Myeloperoxidase: an inflammatory enzyme for generating dysfunc-tional high density lipoprotein. Curr Opin Cardiol 21(4):322–328Silvennoinen R, Escola-Gil JC et al (2012) Acute psychological stress accelerates reverse choles-terol transport via corticosterone-dependent inhibition of intestinal cholesterol absorption.Circ Res 111(11):1459–1469Sun D, Zhang J et al (2012) MiR-26 controls LXR-dependent cholesterol efflux by targetingABCA1 and ARL7. FEBS Lett 586(10):1472–1479Sweetnam PM, Bolton CH et al (1994) Associations of the HDL2 and HDL3 cholesterolsubfractions with the development of ischemic heart disease in British men. The Caerphillyand Speedwell collaborative heart disease studies. Circulation 90(2):769–774204 E. Favari et al.Tanigawa H, Billheimer JT et al (2007) Expression of cholesteryl ester transfer protein in micepromotes macrophage reverse cholesterol transport. Circulation 116(11):1267–1273Tanigawa H, Billheimer JT et al (2009) Lecithin: cholesterol acyltransferase expression hasminimal effects on macrophage reverse cholesterol transport in vivo. Circulation 120(2):160–169Tarling EJ, Edwards PA (2012) Dancing with the sterols: critical roles for ABCG1, ABCA1,miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis.Biochim Biophys Acta 1821(3):386–395Tchoua U, D’Souza W et al (2008) The effect of cholesteryl ester transfer protein overexpressionand inhibition on reverse cholesterol transport. Cardiovasc Res 77(4):732–739Temel RE, Sawyer JK et al (2010) Biliary sterol secretion is not required for macrophage reversecholesterol transport. Cell Metab 12(1):96–102Tietge UJ, Groen AK (2013) Role the TICE?: advancing the concept of transintestinal cholesterolexcretion. Arterioscler Thromb Vasc Biol 33(7):1452–1453Timmins JM, Lee JY et al (2005) Targeted inactivation of hepatic Abca1 causes profoundhypoalphalipoproteinemia and kidney hypercatabolism of apoA-I. J Clin Invest 115(5):1333–1342Toh SA, Millar JS et al (2011) PPARgamma activation redirects macrophage cholesterol fromfecal excretion to adipose tissue uptake in mice via SR-BI. Biochem Pharmacol 81(7):934–941Toth PP, Barter PJ et al (2013) High-density lipoproteins: a consensus statement from the nationallipid association. J Clin Lipidol 7(5):484–525Usami Y, Matsuda K et al (2011) Detection of chymase-digested C-terminally truncated apolipo-protein A-I in normal human serum. J Immunol Methods 369(1–2):51–58Usami Y, Kobayashi Y, Kameda T, Miyazaki A, Matsuda K, Sugano M, Kawasaki K, Kurihara Y,Kasama T, Tozuka M (2013) Identification of sites in apolipoprotein A-I susceptible tochymase and carboxypeptidase A digestion. Biosci Rep 33:49–56Vantourout P, Radojkovic C et al (2010) Ecto-F(1)-ATPase: a moonlighting protein complex andan unexpected apoA-I receptor. World J Gastroenterol 16(47):5925–5935Vaughan AM, Oram JF (2005) ABCG1 redistributes cell cholesterol to domains removable byhigh density lipoprotein but not by lipid-depleted apolipoproteins. J Biol Chem 280(34):30150–30157Vazquez E, Sethi AA et al (2012) High-density lipoprotein cholesterol efflux, nitration ofapolipoprotein A-I, and endothelial function in obese women. Am J Cardiol 109(4):527–532Vickers KC, Remaley AT (2014) HDL and cholesterol: life after the divorce? J Lipid Res 55(1):4–12Voight BF, Peloso GM et al (2012) Plasma HDL cholesterol and risk of myocardial infarction: amendelian randomisation study. Lancet 380(9841):572–580Wang MD, Franklin V et al (2007a) In vivo reverse cholesterol transport from macrophageslacking ABCA1 expression is impaired. Arterioscler Thromb Vasc Biol 27(8):1837–1842Wang X, Collins HL et al (2007b) Macrophage ABCA1 and ABCG1, but not SR-BI, promotemacrophage reverse cholesterol transport in vivo. J Clin Invest 117(8):2216–2224Watanabe J, Charles-Schoeman C et al (2012) Proteomic profiling following immunoaffinitycapture of high-density lipoprotein: association of acute-phase proteins and complementfactors with proinflammatory high-density lipoprotein in rheumatoid arthritis. ArthritisRheum 64(6):1828–1837Weibel GL, Hayes S et al (2011) Novel in vivo method for measuring cholesterol mass flux inperipheral macrophages. Arterioscler Thromb Vasc Biol 31(12):2865–2871Wynn TA, Chawla A et al (2013) Macrophage biology in development, homeostasis and disease.Nature 496(7446):445–455Xie P, Jia L et al (2013) Ezetimibe inhibits hepatic Niemann-Pick C1-Like 1 to facilitatemacrophage reverse cholesterol transport in mice. Arterioscler Thromb Vasc Biol 33(5):920–925Cholesterol Efflux and Reverse Cholesterol Transport 205Yamamoto S, Tanigawa H et al (2011) Pharmacologic suppression of hepatic ATP-bindingcassette transporter 1 activity in mice reduces high-density lipoprotein cholesterol levels butpromotes reverse cholesterol transport. Circulation 124(12):1382–1390Yasuda T, Grillot D et al (2010) Tissue-specific liver X receptor activation promotes macrophagereverse cholesterol transport in vivo. Arterioscler Thromb Vasc Biol 30(4):781–786Yvan-Charvet L, Kling J et al (2010) Cholesterol efflux potential and antiinflammatory propertiesof high-density lipoprotein after treatment with niacin or anacetrapib. Arterioscler ThrombVasc Biol 30(7):1430–1438Zanotti I, Poti F et al (2008) The LXR agonist T0901317 promotes the reverse cholesteroltransport from macrophages by increasing plasma efflux potential. J Lipid Res 49(5):954–960Zanotti I, Maugeais C et al (2011a) The thienotriazolodiazepine Ro 11-1464 increases plasmaapoA-I and promotes reverse cholesterol transport in human apoA-I transgenic mice. Br JPharmacol 164(6):1642–1651Zanotti I, Pedrelli M et al (2011b) Macrophage, but not systemic, apolipoprotein E is necessary formacrophage reverse cholesterol transport in vivo. Arterioscler Thromb Vasc Biol 31(1):74–80Zanotti I, Favari E et al (2012) Cellular cholesterol efflux pathways: impact on intracellular lipidtrafficking and methodological considerations. Curr Pharm Biotechnol 13(2):292–302Zhang Y, Zanotti I et al (2003) Overexpression of apolipoprotein A-I promotes reverse transport ofcholesterol from macrophages to feces in vivo. Circulation 108(6):661–663Zhang Y, Da Silva JR et al (2005) Hepatic expression of scavenger receptor class B type I (SR-BI)is a positive regulator of macrophage reverse cholesterol transport in vivo. J Clin Invest 115(10):2870–2874Zhang Y, Yin L et al (2010) Identification of novel pathways that control farnesoid X receptor-mediated hypocholesterolemia. J Biol Chem 285(5):3035–3043Zhao Y, Pennings M et al (2011) Hypocholesterolemia, foam cell accumulation, but no athero-sclerosis in mice lacking ABC-transporter A1 and scavenger receptor BI. Atherosclerosis 218(2):314–322Zheng L, Nukuna B et al (2004) Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease. J ClinInvest 114(4):529–541Zheng L, Settle M et al (2005) Localization of nitration and chlorination sites on apolipoproteinA-I catalyzed by myeloperoxidase in human atheroma and associated oxidative impairment inABCA1-dependent cholesterol efflux from macrophages. J Biol Chem 280(1):38–47206 E. Favari et al.Functionality of HDL: Antioxidationand Detoxifying EffectsHelen Karlsson, Anatol Kontush, and Richard W. JamesContents1 High-Density Lipoproteins and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2091.1 High-Density Lipoproteins: Antioxidative Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2101.2 Mechanisms of Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2101.3 Heterogeneity of Antioxidant Activity of HDL Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . 2132 High-Density Lipoproteins, Paraoxonase-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2142.1 PON1 as an Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2142.2 PON1 and Bacterial Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2163 High-Density Lipoproteins, Environmental Pathogens and Toxins . . . . . . . . . . . . . . . . . . . . . . . 2173.1 Bacterial Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2173.2 Parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2183.3 Hepatitis, Dengue and Other Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2193.4 Metal Oxides, Carbon Nanotubes and PLGA Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . 2193.5 PON1 and Organophosphates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2203.6 Detoxification of Plasma and External Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221H. KarlssonOccupational and Environmental Medicine, Heart Medical Centre, County Council ofOstergotland, Linkoping University, SE-58185 Linkoping, Swedene-mail: helen.m.karlsson@liu.seA. KontushINSERM UMRS 939, University of Pierre and Marie Curie (UPMC) Paris 6, Hôpital de la Pitié,Paris, Francee-mail: anatol.kontush@upmc.frR.W. James (*)Clinical Diabetes Unit, Department of Internal Medicine, University of Geneva, Geneva,Switzerlande-mail: Richard.James@hcuge.ch# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_5207AbstractHigh-density lipoproteins (HDL) are complexes of multiple talents, some ofwhich have only recently been recognised but all of which are under activeinvestigation. Clinical interest initially arose from their amply demonstrated rolein atherosclerotic disease with their consequent designation as a major cardio-vascular disease (CVD) risk factor. However, interest is no longer confined tovascular tissues, with the reports of impacts of the lipoprotein on pancreatic,renal and nervous tissues, amongst other possible targets. The ever-wideningscope of HDL talents also encompasses environmental hazards, including infec-tious agents and environmental toxins. In almost all cases, HDL would appear tohave a beneficial impact on health. It raises the intriguing question of whetherthese various talents emanate from a basic ancestral function to protect the cell.The following chapter will illustrate and review our current understanding ofsome of the functions attributed to HDL. The first section will look at theantioxidative functions of HDL and possible mechanisms that are involved.The second section will focus specifically on paraoxonase-1 (PON1), whichappears to bridge the divide between the two HDL functions discussed herein.This will lead into the final section dealing with HDL as a detoxifying agentprotecting against exposure to environmental pathogens and other toxins.KeywordsHDL • Lipoproteins • Oxidative stress • Paraoxonase • Organophosphates •Bacterial pathogen • Antiviral activity • Nanoparticles • BisphenolAbbreviationsAAPH Azo-initiator (2,20-azobis-(2-amidinopropane) hydrochloride)Apo ApolipoproteinBPA Bisphenol ACEOOH Cholesteryl ester hydroperoxideCETP Cholesterol ester transfer proteinCu CopperCVD Cardiovascular diseaseCys CysteineGSP Glutathione peroxidaseHb HaemoglobinHbr Haptoglobin-related proteinHBV Hepatitis B virusHDL High-density lipoproteinsHETE Hydroxy-eicosatetraenoic acid208 H. Karlsson et al.HODE Hydroxyoctadecadienoic acidHIV Human immunodeficiency virusHSL Homoserine lactonesLCAT Lecithin-cholesterol acyltransferaseLDL Low-density lipoproteinsLOOH Lipid hydroperoxideLPS LipopolysaccharidesLSI Liver somatic indexLTA Lipoteichoic acidMet MethionineMPO MyeloperoxidaseNADPH Nicotinamide adenine dinucleotide phosphateNLF Nasal lavage fluidOP OrganophosphatePAF-AH Platelet-activating factor acetylhydrolasePCB Polychlorinated biphenylPLGA Poly(lactic-co-glycolic acid)PLOOH Phospholipid hydroperoxidePLTP Phospholipid transfer proteinPON1 Paraoxonase-1POP Persistent organic pollutantPOPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholinerHDL Reconstituted HDLSAA Serum amyloid ASRA Serum resistance-associatedSR-BI Scavenger receptor class B-ITLF Trypanolytic factorTLR Toll-like receptorTNF Tumour necrosis factorTyr TyrosineVLDL Low-density lipoproteins1 High-Density Lipoproteins and Oxidative StressThe role of high-density lipoproteins (HDL) in the vascular system has been theprimary focus of clinical interest. It arose from early studies of the influence of thelipoprotein on cholesterol metabolism and atherosclerosis. With increasing under-standing of the complexity of the atherosclerotic process and involvement of otherpathological mechanisms, attention has logically progressed to the impact of HDLon such mechanisms. One process is oxidative stress and there is now persuasiveevidence that the lipoprotein can attenuate its consequences by a number ofmechanisms. These are discussed in this section.Functionality of HDL: Antioxidation and Detoxifying Effects 2091.1 High-Density Lipoproteins: Antioxidative FunctionThe response-to-retention hypothesis of atherosclerosis (Williams and Tabas 1995)postulates that cholesterol-rich lipoproteins, primarily low-density lipoproteins(LDL), are retained in the arterial wall and oxidatively modified under the actionof resident cells (Stocker and Keaney 2004). Oxidation in the arterial intima resultsfrom local oxidative stress, which represents an imbalance between prooxidants andantioxidants in favour of the former. Cellular oxidative systems involved in vivoinclude myeloperoxidase (MPO), NADPH oxidase, nitric oxide synthase andlipoxygenase. They produce a variety of reactive chlorine, nitrogen and oxygenspecies in the form of one-electron (free radical) and two-electron oxidants (Gautand Heinecke 2001).HDL can protect LDL and other lipoproteins from oxidative stress induced byboth one- and two-electron species. It can be observed in vitro on theirco-incubation (Parthasarathy et al. 1990) and in vivo upon HDL supplementation(Klimov et al. 1993). One-electron oxidants modify both lipid and protein moietiesof HDL with formation of lipid and protein radicals. It is followed by accumulationof primary oxidation products, initially lipid hydroperoxides (LOOH), which inturn propagate further oxidation of HDL components to stable termination products(Stocker and Keaney 2004). HDL particles potently protect both lipid and proteinmoieties of LDL from free radical-induced oxidation, inhibiting accumulation ofboth primary and secondary oxidation products (Kontush and Chapman 2010). Thefollowing overview will focus on the impact of HDL on lipid hydroperoxides andother primary products of lipid peroxidation.1.2 Mechanisms of ProtectionHDL potently inhibits accumulation of LOOH in LDL. Removal of LOOH fromLDL (or cells) represents a key step of HDL-mediated protection from oxidativedamage induced by free radicals. Indeed, phospholipid hydroperoxides (PLOOHs)are rapidly transferred from LDL to HDL upon their co-incubation (Zerrad-Saadiet al. 2009). Addition of a hydroperoxyl group to a phospholipid or cholesteryl estermolecule strongly increases hydrophilicity. As a result, LOOH molecules are moresurface active than their non-peroxidised counterparts (Nuchi et al. 2002). Theirexposure to the aqueous phase at the HDL surface facilitates their removal from thelipoprotein. Transfer from LDL to HDL can occur directly between lipoproteinphospholipid monolayers, either spontaneously or mediated by lipid transferproteins, including cholesterol ester transfer protein (CETP). The latter can accel-erate the transfer of both cholesteryl ester hydroperoxides (CEOOHs) and PLOOHs(Christison et al. 1995). Thus, CETP can enhance the antioxidative activity of HDLtowards LDL-derived LOOH. Removal of LOOH molecules from LDL can also bemediated by lipid-free apoA-I. As a consequence, HDL particles constitute a majortransport vehicle of LOOH in human plasma, effectively functioning as a “sink” foroxidised lipids (Bowry et al. 1992). They can accumulate in the particle when its210 H. Karlsson et al.LOOH-inactivating capacity is overwhelmed. Furthermore, HDL represents amajor plasma carrier of F2-isoprostanes, stable final products of lipid peroxidation(Proudfoot et al. 2009). The ability of HDL to remove LOOH from cell membranesof erythrocytes and astrocytes has also been reported (Ferretti et al. 2003; Klimovet al. 2001). Thus, the accumulation of oxidised lipids in HDLmost probably resultsnot only from their transfer from LDL but also from remnant triglyceride-richlipoproteins and/or arterial wall cells, mediated in part by lipid transfer proteins(Christison et al. 1995). Subsequently, LOOHs and their corresponding hydroxidescan be rapidly removed from HDL via scavenger receptor class B-I (SR-BI)-mediated selective uptake by the liver (Christison et al. 1996). This pathway maysignificantly contribute to the removal of toxic, oxidised lipids from the body. AsCEOOH are removed from the circulation more rapidly than the correspondingnonoxidised cholesteryl ester, addition of a hydroperoxyl group may acceleratecholesterol excretion. Following their transfer, HDL-associated LOOH areinactivated by reduction to the corresponding hydroxides in a two-electron redoxreaction with HDL proteins (Garner et al. 1998; Zerrad-Saadi et al. 2009). ApoA-Iplays a central role in redox inactivation of LOOH and may account for a large partof the antioxidation effects of the lipoprotein. ApoA-I Met residues 112 and148 reduce LOOH to redox-inactive lipid hydroxides, thereby terminating chainreactions of lipid peroxidation (Garner et al. 1998; Zerrad-Saadi et al. 2009).Simulation by molecular dynamics shows interaction of Met112 and Met148 withTyr115, creating a microenvironment unique to human apoA-I (Bashtovyyet al. 2011) that may be optimal for such redox reactions. LOOH is also inactivatedby apoA-II, although to a lesser extent. The key role of apoA-I in theHDL-mediated protection of LDL from one-electron oxidants is supported by theobservation that reconstituted HDL (rHDL) containing only purified apoA-I andphospholipid (POPC), but devoid of other protein and lipid components, arecomparable to natural, small, dense HDL3b + 3c subfractions in their capacities todelay lipid peroxidation (Zerrad-Saadi et al. 2009). Concentrations of redox-activeMet residues in apoA-I and of PLOOH suggest a 1:1 reaction stoichiometry (Garneret al. 1998; Zerrad-Saadi et al. 2009). HDL content of apoA-I and oxidative statusof apoA-I Met residues are therefore important determinants of the capacity of HDLto inactivate LOOH and protect LDL from free radical-induced oxidation (Zerrad-Saadi et al. 2009). The redox reaction between HDL Met residues and LOOH isparalleled by the formation of oxidised forms of apoA-I and apoA-II which containmethionine sulfoxides. These methionine sulfoxide moieties can be reduced back tomethionine by methionine sulfoxide reductases. In addition to Met, Cys residuesmay be important for the antioxidative properties of apoA-I, as demonstrated by theelevated inhibitory potency of the N74C mutant of apoA-I towards LDL oxidation(Zhang et al. 2010). His residues may also contribute to the apoA-I-mediatedinhibition of LOOH accumulation in LDL, as a result of their metal-chelatingproperties (Nguyen et al. 2006). Indeed, transition metal ions are well-establishedcatalysers of lipid peroxidation.HDL particles contain other apolipoproteins that may contribute to inhibition ofLOOH accumulation (Davidson et al. 2009; Ostos et al. 2001). Such antioxidativeFunctionality of HDL: Antioxidation and Detoxifying Effects 211properties have been reported for apoA-IV (Ostos et al. 2001) and apoE (Miyataand Smith 1996). In addition, apoM displays antioxidative properties in transgenicmice where possible mechanisms may involve binding of oxidised phospholipids,including LOOH (Elsoe et al. 2012). Enzymatic components potentiallycontributing to antioxidative properties of HDL include paraoxonase-1 (PON1),platelet-activating factor acetylhydrolase (PAF-AH) and lecithin-cholesterolacyltransferase (LCAT), all of which have been proposed to hydrolyse oxidisedphospholipids (Kontush and Chapman 2006). In addition, HDL carries glutathioneperoxidase (GSPx) which can detoxify LOOH by reducing them to thecorresponding hydroxides (Maddipati and Marnett 1987). Finally, the trypanosomelytic factor present in very high-density subpopulations of human HDL exhibits aperoxidase-like activity (Molina Portela et al. 2000) that may provide a minorcontribution to LOOH-inactivating properties of HDL. With respect to PON1,PAF-AH and LCAT, these are weakly reactive towards LOOH (Goyalet al. 1997; Kriska et al. 2007; Teiber et al. 2004). HDL-associated PON1, whichhas been attributed a major antioxidant role in HDL (see Sect. 2.1), does not appearto contribute significantly to the inactivation of LDL-derived PLOOH (Garneret al. 1998; Zerrad-Saadi et al. 2009). Such activity that has been previouslyreported has been ascribed to the presence of detergents or unidentified proteins(Teiber et al. 2004). The major activity of PON1 is that of a calcium-dependentlactonase rather than a peroxidase (Khersonsky and Tawfik 2005); its affinity forLOOH is several orders of magnitude lower than its affinity for lactones (seeSect. 2.1 for further discussion of PON1). It is more probable that PAF-AH, ratherthan PON1, represents the hydrolase for PLOOH in HDL (Kriska et al. 2007).LCAT may equally hydrolyse PLOOH generated during lipoprotein oxidation(Goyal et al. 1997). In addition, it can delay LDL oxidation, acting as a chain-breaking antioxidant, most likely via its Cys residues (McPherson et al. 2007).Inhibition of LCAT does not, however, significantly influence HDL-mediatedinactivation of LDL-derived PLOOH (Zerrad-Saadi et al. 2009), thereby indicatingthat the enzyme is at most a minor factor in PLOOH inactivation. Irrespective of theenzyme involved, PLOOH hydrolysis releases lysophosphatidylcholine and a freefatty acid hydroperoxide. Interestingly, PAF-AH is capable of hydrolysing PLOOHwithin oxidised LDL; in this case, free fatty acid hydroperoxides are transferred toHDL for subsequent two-electron reduction to corresponding hydroxides by apoA-I(Kotosai et al. 2013). As a result of elevated hydrophilicity, free fatty acidhydroperoxides, as compared to PLOOH and CEOOH (Kotosai et al. 2013), arepreferentially transported to HDL and reduced to hydroxides.In contrast to its effects on free radical-induced oxidation, HDL weakly protectsLDL from oxidation by two-electron oxidants, such as hypochlorite, which mainlymodify the protein moiety of LDL. Such antioxidative action appears largelyunspecific, reflecting direct oxidant scavenging by HDL. A rare example ofHDL-mediated protection from two-electron oxidants is inactivation by enzymessuch as PAF-AH and/or PON1 with participation of apoA-I (Ahmed et al. 2001), ofphospholipid core aldehydes generated upon HDL oxidation by peroxynitrite.212 H. Karlsson et al.Finally, HDL lipids can significantly modulate antioxidative activities displayedby the protein components. First, HDL carries small amounts of lipophilicantioxidants, primarily tocopherols, which may provide a minor contribution toits LOOH-inactivating properties (Bowry et al. 1992; Goulinet and Chapman1997). Second, the rigidity of the phospholipid monolayer of HDL particles is akey negative modulator of PLOOH transfer efficiency from LDL and cellmembranes (Vila et al. 2002; Zerrad-Saadi et al. 2009). The surface monolayerrigidity of HDL is primarily determined by the relative content of such lipids(sphingomyelin, free cholesterol, and saturated and monounsaturated fatty acids).Increasing the content of each rigidifies the surface monolayer of HDL particles at agiven protein/lipid ratio (Zerrad-Saadi et al. 2009).1.3 Heterogeneity of Antioxidant Activity of HDL ParticlesApolipoproteins, enzymes and lipids which determine the antioxidative activities ofHDL are non-uniformly distributed across the spectrum of HDL subpopulations.ApoA-I, the major protein component of HDL participating in the reduction ofLOOHs, is enriched relative to apoA-II in small, dense HDL3c and in large, lessdense HDL2b as compared to HDL2a, 3a and 3b subpopulations (Kontushet al. 2007). LCAT, PAF-AH, PON1 (Kontush et al. 2003) and apoA-IV (Bisgaieret al. 1985) are enriched in small, dense HDL. As a consequence, HDL particles areheterogeneous in their capacity to protect LDL from oxidative damage induced byone-electron oxidants. Small, dense, protein-rich HDL particles in particularpotently protect LDL from mild oxidative stress induced by azo-initiator (2,2-0-azobis-(2-amidinopropane) hydrochloride; AAPH) or Cu2+ (Kontush et al. 2003;Yoshikawa et al. 1997). It involves inactivation of LOOHs via a two-step mecha-nism with transfer of LOOHs to HDL (facilitated by enhanced fluidity of the HDLsurface lipid monolayer) and subsequent reduction to redox-inactive hydroxides byMet residues of apoA-I (Christison et al. 1995; Garner et al. 1998; Zerrad-Saadiet al. 2009). Small, dense HDL3 may be superior to large, less dense HDL2 in termsof their capacity to remove oxidised lipids from other lipoproteins and cellularmembranes. Structural defects in the packing of surface lipids, which allow inser-tion of exogenous molecules, become more pronounced with decreasing HDLparticle size and may account for this property (Kumpula et al. 2008). Consistentwith this possibility, small discoid rHDL complexes appear to remove negativelycharged lipids from oxidised LDL to a greater degree than native, spherical HDL(Miyazaki et al. 1994). In addition, a diminished content of sphingomyelin and freecholesterol in small, dense HDL may result in an increased fluidity of the surfacelipid monolayer, thereby facilitating incorporation of oxidised lipids of exogenousorigin (Kontush and Chapman 2010).Inactivation of oxidised lipid molecules following their transfer to HDL mayalso occur more rapidly in small, dense particles. First, reduction of LOOHs tohydroxides is more efficient in HDL3 as compared to HDL2 (Zerrad-Saadiet al. 2009). Relative enrichment of small, dense HDL in apoA-I may underlieFunctionality of HDL: Antioxidation and Detoxifying Effects 213this feature (Kontush et al. 2007). Moreover, the distinctly low lipid content ofsmall, dense HDL can induce conformational changes in apoA-I relative to large,less dense HDL. It results in enhanced exposure to the aqueous phase, as reflected inmodified reactivity towards monoclonal antibodies (Sparks et al. 1995), whichmight in turn facilitate the redox reaction between Met residues of apoA-I andLOOHs. Second, LOOH hydrolysis by HDL-associated hydrolytic enzymesappears to be predominantly associated with small, dense HDL3 (Kontushet al. 2003). Preferential localisation of enzymatic activities of PAF-AH withinHDL3 is probably responsible for such association. Furthermore, enzymaticactivities might be beneficially influenced by the lipidome of small, dense HDL3,which is distinct in displaying a low sphingomyelin to phosphatidylcholine ratio(Kontush et al. 2007). Indeed, sphingomyelin belongs to a class of structural lipidsthat exert a positive impact on surface rigidity and a negative impact on LCATactivity (Subbaiah and Liu 1993).Finally, the unique proteome of HDL3c may have implications for itsantioxidative activity. Several proteins, including apoJ, apoM, serum amyloid A4(SAA4), apoD, apoL-1, PON1 and PON3 and phospholipid transfer protein (PLTP)occur predominantly in this subfraction (Davidson et al. 2009). Indeed, PLTP ispresent on HDL together with apoJ, apoL-I, apoD, apoA-I, apoA-II and severalother proteins, suggesting a direct, functional interaction between them.2 High-Density Lipoproteins, Paraoxonase-1PON1 is an intriguing example of the functional flexibility of HDL components asit bridges the apparent divide between systemic oxidative stress and environmentaltoxicology. Originally identified as an enzyme neutralising environmental toxins, itprogressed to an antioxidant role before revealing its capacity to combat pathogens:the functional complexity and versatility of HDL reflected in a single, peptidecomponent. This section will focus on two aspects of PON1 function. It will expandon the antioxidant capacity of PON1 that was referred to in the preceeding section.Subsequently the ability of PON1 to limit bacterial virulence, notably with respectto Pseudomonas aeruginosa, will be addressed.2.1 PON1 as an AntioxidantEarly, in vitro studies suggested that purified PON1 and HDL-associated PON1could protect LDL from oxidation (Mackness et al. 2003), although theseconclusions were questioned in subsequent reports (James 2006). However, studiesfrom several animal models (James 2006) have provided much stronger support forthe conclusion that PON1 protects against atherosclerotic disease by a process thatinvolves reduction of oxidative stress. Thus PON1 knockout in mice was associatedwith a significant increase in the development of atherosclerotic lesions, whilstHDL from such mice were less able to protect LDL from oxidation (Shih214 H. Karlsson et al.et al. 1998, 2000). The latter was corrected by addition of purified PON1 to theHDL. Conversely, over-expression of PON1 protected against lesion development,with HDL showing a greater capacity to protect against oxidative stress (Twardet al. 2002). A consistent observation from these animal models is that PON1activity is a negative correlate of markers of oxidative stress.Studies in man are also consistent with a protective role of PON1, involving animpact on oxidative stress. In a number of disease states that increase risk ofatherosclerotic disease and where increased oxidative stress is noted, PON1serum activity is significantly reduced. These include diabetes, the metabolicsyndrome, familial hypercholesterolaemia and renal dysfunction (Abbottet al. 1995; James 2006). Enzyme activity was also reduced in coronary diseasepatients, with the extent of coronary lesions correlating inversely with the PON1activity (Graner et al. 2006). The relationship was independent of other risk factors.Other case-control investigations confirm that PON1 is a risk factor for coronarydisease (Soran et al. 2009), although it is not a consistent observation (Troughtonet al. 2008). Whilst the above studies focus on enzyme activity, there have also beena number of studies examining the relationship of PON1 coding regionpolymorphisms to coronary risk. The rationale is that a polymorphism at aminoacid 192 gives rise to two isoforms with strikingly different activities towardsparaoxon, one of the substrates used to analyse PON1 activity. Such studies havegiven somewhat equivocal results, as illustrated in a meta-analysis by Wheeleret al. (2004). However, this data should be interpreted with reserve as the range ofactivities is wide within each isoform. Moreover, the isoforms manifest muchsmaller differences in activities towards a second substrate, phenylacetate, fre-quently used to analyse PON1. It has led to suggestions that PON1 phenotypeshould be used to analyse the relationship of the enzyme to vascular risk (Jarviket al. 2000).More persuasive evidence of a role for PON1 in CVD comes from prospectivestudies. In the Caerphilly prospective study, serum PON1 activity was shown to bean independent determinant of future coronary events (Mackness et al. 2003). Nomeasures of oxidative stress were undertaken. In two prospective studies, Hazenand colleagues (Bhattacharyya et al. 2008; Tang et al. 2012) firmly establishedPON1 as an independent risk factor for vascular disease, in both primary andsecondary disease settings. They also analysed an impressive range of systemicindices of lipid oxidation (HETE, HODE, isoprostane) and showed a strong nega-tive correlation between PON1 activity and serum concentrations of these markers.These studies clearly suggest that it is the ability of PON1 to limit systemicoxidative stress and the generation of oxidised lipids that underlies its links toatherosclerotic disease. Alternative or complementary mechanisms by which PON1could reduce risk have also been proposed (Aviram 2012; Rosenblat et al. 2013).The authors suggest that the enzyme can increase the free radical scavengingcapacity of monocyte/macrophages and lower oxidative stress. The same groupalso proposed that PON1 increased the ability of HDL to remove cholesterol frommacrophages (Rosenblat et al. 2011). The latter is thought to be an important step inreverse cholesterol transport. Data from the same study indicated that PON1 mayFunctionality of HDL: Antioxidation and Detoxifying Effects 215increase the ability of HDL to prevent macrophage-mediated oxidation of LDL. Inanother study, the group proposed that PON1 could reduce the risk of diabetes, apotent CVD risk factor (Koren-Gluzer et al. 2011). The antioxidant function, actingvia sulphydryl groups PON1, was suggested to play a role, but not enzyme activity.Whilst there is persuasive evidence that PON1 prevents systemic lipid oxidation,how and what substrates it may act on are still unclear. As discussed above(Sect. 1.2), LOOHwould appear to be poor substrates for the enzyme. Nevertheless,a feature of the PON1 knockout mice was an accumulation of LOOH in HDL (Shihet al. 1998; Tward et al. 2002). It is possible that short-chain oxidised phospholipidsmay be a better substrate for the enzyme. Increased lipoperoxides as well as lipidoxidation end products were also a feature of reduced PON1 activity in humanstudies (Bhattacharyya et al. 2008; Tang et al. 2012). One possible explanation maybe furnished by a recent, intriguing proposal that PON1 can modulate the activity ofmyeloperoxidase (MPO). The latter is an important source of reactive oxygenspecies and is associated with HDL. Huang et al. (2013) reported that PON1 andMPO form a ternary complex with HDL. In this constellation, PON1 was able tomodulate MPO activity (and conversely MPO inhibited PON1). Confirmation wasobtained in vitro by showing that adding PON1 to active MPO significantly reducedlipid peroxidation by the latter.At present, the question remains open as to how exactly PON1 functions, withpossible explanations being prevention of ROS formation, curtailing propagation ofROS or hydrolysing oxidised lipids.2.2 PON1 and Bacterial PathogensA major advance in our appreciation of the physiological relevance of PON1 wasthe demonstration that its principal enzyme activity is that of a lactonase(Khersonsky and Tawfik 2005). Indeed, lactonase activity would appear to be theinitial, ancestral enzyme activity of the PON gene family. These observationsconsiderably broadened the scope of HDL functions as lactonases are instrumentalin neutralising bacterial virulence factors.Pseudomonas aeruginosa is an opportunist bacterium and a common cause ofdisease in immunocompromised patients. It is also a major contributor to nosoco-mial infections. Bacterial virulence is governed by homoserine lactones (HSL)synthesised and secreted by the bacterium. They are central to the phenomenonof quorum sensing that allows communication between bacteria. The HSLs give ameasure of bacterial density and when a threshold bacteria count is achieved, theyactivate gene programmes that greatly increase the rate of Pseudomonas infection(Juhas et al. 2005). The ability of the PON enzymes to hydrolyse the HSL interruptsbacterial communication and reduces virulence. Whilst the intracellular PON2variant shows the strongest lactonase activity, PON1is also active towards HSL(Ozer et al. 2005) and is able to protect against Pseudomonas infection (Stoltzet al. 2008). Indeed, PON1 is the major source of HSL neutralising activity in serum(Teiber et al. 2008).216 H. Karlsson et al.Discovery of its role in combating bacterial infection and knowledge that theparaoxonases are a highly conserved gene family have led to suggestions that theymay play a role in innate immunity (Shih and Lusis 2009).3 High-Density Lipoproteins, Environmental Pathogensand ToxinsIn recent studies, the molecular composition of the HDL particle has been assignedsignificant, functional importance (Besler et al. 2012), which is of particularrelevance with respect to reported changes in HDL composition in disease. Inter-estingly, alterations in HDL composition have also been observed in man duringendotoxemia (Levels et al. 2011) and in endotoxin-treated mice (Chiba et al. 2011).As suggested above, data indicate that HDL has an important role in neutralisingand detoxifying invading pathogens to prevent systemic inflammatory responses orsepsis, which is the leading cause of death in intensive care units (ICUs) of high-income countries (Levels et al. 2011; Russell 2006). Concurrently, the interactionof HDL with viruses, parasites and environmental toxins has also been a focus ofrecent interest. This is reflected in reviews of the impact of gram-positive and gram-negative bacterial toxins in sepsis (Ramachandran 2013), the role of HDL in innateimmunity (Feingold and Grunfeld 2011) and the immune system (Kaji 2013) andxenobiotic metabolism, disposition and regulation of receptors (Omiecinskiet al. 2011) to which the reader is referred. In this chapter, the role(s) of HDL asa detoxifying agent during exposure to different environmental pathogens or toxinswill be discussed.3.1 Bacterial PathogensMultiple alterations in lipid and lipoprotein metabolism occur during the acutephase response. Within HDL, apoA-I, cholesterol and phospholipid levels decrease,as do certain enzyme activities, e.g. PON1 (Khovidhunkit et al. 2004), whilst freeapoA-I (Cabana et al. 1997) and triglyceride levels increase (Cabana et al. 1996).As a consequence, reverse cholesterol transport decreases, accompanied byincreased cholesterol delivery to immune cells. Such alterations initially protectthe host from the harmful effects of invading pathogens but will contribute toatherogenesis if prolonged (Khovidhunkit et al. 2004).A crucial factor in the detoxifying properties attributed to HDL is the ability toneutralise the invading pathogen. A number of human studies have shown thatcirculating lipoproteins, principally HDL, are able to bind lipopolysaccharides(LPS) from gram-negative bacteria as well as lipoteichoic acid (LTA) from gram-positive bacteria (Khovidhunkit et al. 2004; Levels et al. 2003, 2011). Binding toHDL inhibits LPS interaction with cell surface toll-like receptor 4 (TLR-4)(Underhill and Ozinsky 2002) and LTA interaction with TLR-2 (Flo et al. 2000),receptors known to mediate inflammatory responses (macrophage activation,Functionality of HDL: Antioxidation and Detoxifying Effects 217cytokine release). These neutralising effects of HDL have been confirmed inhumans with low HDL levels who show a more robust inflammatory response toLPS administration (Birjmohun et al. 2007).During the acute phase response, HDL protein and lipid compositions change(Khovidhunkit et al. 2004). Observations indicate that apoA-I can be replaced bythe positive acute phase protein SAA (Artl et al. 2000). SAA is able to influenceHDL-mediated cholesterol metabolism through its inhibitory effects on SR-BI-mediated selective cholesterol uptake (Cai et al. 2005). It has recently beensuggested to facilitate the binding of HDL, isolated from mice injected with LPS,to vascular proteoglycans (Chiba et al. 2011). The relevance of these observations isimportant when taken together with the up to 1,000-fold increase in concentrationsof plasma SAA during acute phase reactions such as inflammation or infection. Inaddition, several isoforms of SAA, still with unknown functions, have recently beendetected in acute phase HDL (Levels et al. 2011).The neutralising effects of HDL during LPS exposure are dependent onphospholipids as well as proteins. ApoA-I is suggested to be the major neutralisingfactor (Massamiri et al. 1997). Thus, direct interaction between LPS and theC-terminus of apoA-I can decrease TNF-α release from macrophages in vitro(Henning et al. 2011). The role of apoA-I as a neutralising agent is furtherstrengthened by the beneficial effects of apoA-I on LPS-induced acute lung injuryand endotoxemia, as shown in mice (Yan et al. 2006).The HDL receptor, SR-BI, is highly expressed in hepatocytes and steroid-producing cells and has an important role regarding the fate of the neutralisedpathogens. In mice, it is suggested to protect against endotoxemia through its rolesin facilitating glucocorticoid production as well as hepatic clearance of LPS. In thisrespect, SR-BI has been suggested to mediate the binding and uptake of LPS as wellas LTA (Vishnyakova et al. 2006). Additionally, an alternatively spliced variant,SR-BII, has been shown to bind directly a variety of bacteria suggesting aconserved role of these receptors in pattern recognition and innate immunity(Webb et al. 1998).3.2 ParasitesSeveral species of African trypanosomes cause fatal disease in livestock, but mostare unable to infect humans due to innate trypanolytic factors (TLFs). It has beenshown that in human serum, TLFs consist of two minor HDL subfractionscharacterised by the presence of haptoglobin-related protein (Hpr) and apoL-I(Vanhamme et al. 2003). The underlying mechanisms of TLFs action duringparasite infection involve their endocytosis by trypanosomes where, subsequently,apoL-I forms membrane pores within the acidic lysosome resulting in iondysregulation that leads to osmotic imbalance, parasite swelling and lysis(Molina-Portela Mdel et al. 2005). Endocytosis of apoL-I is facilitated by acomplex formed by Hpr and haemoglobin that interacts with the haptoglobin-haemoglobin receptor on the trypanosome cell surface (Vanhollebeke218 H. Karlsson et al.et al. 2008). However, Trypanosoma brucei rhodesiense, carried by tsetse flies andcausing sleeping sickness in man, is able to escape lysis by TLFs. It is achievedmainly by the expression of the serum resistance-associated (SRA) protein, whichbinds to and neutralises apoL-I (Vanhamme et al. 2003). It should be noted howeverthat recent population-based observations, focused on individual variations in Hpr,indicate that a more dynamic view of the relative roles of Hpr and Hpr-Hbcomplexes needs to be considered for understanding innate immunity to Africantrypanosomes. Other pathogens including the newly discovered Plasmodium (Imrieet al. 2012) should possibly be considered.3.3 Hepatitis, Dengue and Other VirusesHDL account for part of the broad, non-specific antiviral activity in human serum(Singh et al. 1999). Thus, apoA-I was found to prevent cell penetration by inhibitingfusion of the herpes virus as well as the human immunodeficiency virus (HIV)(Srinivas et al. 1990). Interestingly, HIV infection has also been linked to dysfunc-tional HDL with reduced antioxidant properties, which may be associated toprogression of subclinical atherosclerosis (Kelesidis et al. 2013). In addition,enhanced levels of human apoM, which is mainly associated with HDL, wereobserved during hepatitis B virus (HBV) infection and may reflect feedbacksuppression of HBV replication (Gu et al. 2011). Conversely, HDL may alsopromote virulence by facilitating virus entry via the SR-BI receptor. This wasfirst demonstrated for the hepatitis C viruses (Voisset et al. 2006) and recentlyextended to dengue viruses that directly associate with apoA-I of HDL(Li et al. 2013). It is perhaps unsurprising that SR-BI has recently been proposedas a therapeutic target that warrants further research, based on reports indicating itsinvolvement in the capture and cross-presentation of antigens from viruses, bacteriaand parasites.3.4 Metal Oxides, Carbon Nanotubes and PLGA NanoparticlesNanotechnology is an emerging industry that involves the creation of new materialswith a variety of useful functions. However, concerns are growing that some ofthemmay have toxic effects. This has promoted interest in the role of HDL both as aneutralising agent and carrier of nanosize particles. Thus the biodistribution ofnanoparticles is significantly influenced by their interaction with plasma proteins,notably HDL proteins apoA-I and apoE. These, amongst others, have been found torecognise and interact with carbon nanotubes, a number of metal oxides (Karlssonet al. 2012) and PLGA (poly(lactic-co-glycolic acid)) nanoparticles (Sempfet al. 2013) in in vitro plasma models. There are, as yet, no detailed studiesconfirming HDL neutralisation and clearance of engineered nanoparticles. How-ever, the concept of incorporating lipophilic drugs assembled with apoA-I andphospholipids into soluble, HDL-like nanoparticles for drug delivery is presentlyFunctionality of HDL: Antioxidation and Detoxifying Effects 219widely explored (Marrache and Dhar 2013; Shin et al. 2013). They could serve anumber of therapeutic purposes, for example, targeting tissues expressing highamounts of SR-BI, such as cancer cells (Marrache and Dhar 2013; Shin et al. 2013).3.5 PON1 and OrganophosphatesA crucial function of HDL-associated PON1, in addition to its potential antioxidantrole, is neutralisation of toxic organophosphate (OP) derivatives. Such an activitywas described in human serum many years ago (Aldridge 1953). The activity wasattributed to the enzyme PON1, a name derived from the substrate paraoxon,initially employed in such studies. Its involvement in the detoxification of OPshas since been well documented, encompassing chlorpyrifos, oxon and diazoxon aswell as nerve agents (Costa et al. 2013). Indeed, HDL-associated PON1 has beenshown to be a key determinant in detoxification of OPs (Shih et al. 1998) in mousemodels. Intriguingly, PON1 was shown to be associated with HDL at a time whenits antioxidant activity was unknown (James 2006).A growing area of research is the importance of PON1 in environment toxicol-ogy and its role in susceptibility to OP exposure. Serum concentrations of theenzyme, as well as certain polymorphisms affecting activity, appear important forrisk prediction (Androutsopoulos et al. 2011; Costa et al. 2013). Consequently,children may be more sensitive to environmental OPs since they have reportedlylower PON1 levels than adults (Huen et al. 2009). Finally, its impact on environ-mental toxins, as well as its antioxidant function, has led to suggestions that PON1is linked to neurological disorders (Androutsopoulos et al. 2011), an area thatrequires considerably more research.3.6 Detoxification of Plasma and External FluidsAs discussed in previous sections, HDL and apo A-I have a role in recognition,neutralisation and elimination of xenobiotics. Thus, elevated levels of plasmaapoA-I in female rats have been found after exposure to the plastic chemicalbisphenol A (BPA) (Ronn et al. 2013). The molecular mechanisms behind theincrease in apoA-I may reflect the effects of estrogens, although conflicting datahave been reported (Teeguarden et al. 2013). It could also be related to apoA-I/HDL-controlled clearance mechanisms that involve transport of xenobiotics to theliver. Interestingly, an increase in liver somatic index (LSI) accompanied the apoA-I increase in the exposed rats, indicating less favourable metabolic alterations.Since the structure of BPA is very similar to a number of recently proposedapoA-I enhancing drugs (Du et al. 2012), it may be relevant to investigate thesemechanisms further.Another group of environmental contaminants, present at low levels in mostliving organisms, are persistent organic pollutants (POPs). Due to their hydrophobiccharacter, they can be transported by lipoprotein particles. Interestingly, long-term220 H. Karlsson et al.effects of POPs in human plasma have recently been highlighted in a number ofepidemiological studies (Lind et al. 2012). In a recent study of an exposed Swedishpopulation, significantly higher concentrations of POPs were found amongstindividuals with CVD or cancer compared to controls (Ljunggren et al. 2014).Principal component analyses showed that POP concentrations in HDL were moreassociated with CVD, whilst POP concentrations in LDL/VLDL were moreassociated with cancer. Interestingly, PON-1 activity was negatively correlated tothe sum of polychlorinated biphenyls (PCBs), and covariation between decreasedarylesterase activity, increased PCB concentrations and CVD was found.To expand the knowledge regarding the role of HDL as detoxifier, it may bebeneficial exploring external fluids. Lipids are present in human tears (Rantamakiet al. 2011), and apoA-I has been detected in nasal lavage fluid (NLF) (Ghafouriet al. 2002) and saliva (Ghafouri et al. 2003). Interestingly, in NLF, apoA-I wasshown to increase in hairdressers after 20 min of exposure to persulphates (Karedalet al. 2010), whilst apoA-I in gingival crevicular fluid has been suggested to be anovel periodontal disease marker (Tsuchida et al. 2012). In addition, decreasedlevels of apoA-I have been reported in bronchio-alveolar lavage fluids of patientswith idiopathic pulmonary fibrosis compared to healthy controls (Kim et al. 2010).Together with lipocalin, apoA-I has been suggested as a potential biomarker forchronic obstructive pulmonary disease (Nicholas et al. 2010). The origin of apoA-Iin fluids of the airways has not been extensively studied, but trans-endothelialtransport has been investigated (Ohnsorg et al. 2011). The presence, compositionand possible function of apoA-I containing lipoprotein particles in external fluidsremain to be investigated.Acknowledgements RWJ gratefully acknowledges support received from the Swiss NationalResearch Foundation, the Novartis Consumer Health Foundation and Unitec, University ofGeneva. AK acknowledges financial support from the National Institute for Health and MedicalResearch (INSERM).HK, AK and RWJ are members of the COST Action BM0904.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAbbott CA, Mackness MI, Kumar S, Boulton AJ, Durrington PN (1995) Serum paraoxonaseactivity, concentration, and phenotype distribution in diabetes mellitus and its relationship toserum lipids and lipoproteins. Arterioscler Thromb Vasc Biol 15(11):1812–1818Ahmed Z, Ravandi A, Maguire GF, Emili A, Draganov D, La Du BN, Kuksis A, Connelly PW(2001) Apolipoprotein A-I promotes the formation of phosphatidylcholine core aldehydes thatare hydrolyzed by paraoxonase (PON-1) during high density lipoprotein oxidation with aperoxynitrite donor. J Biol Chem 276(27):24473–24481Aldridge WN (1953) Serum esterases. II. An enzyme hydrolysing diethyl p-nitrophenyl phosphate(E600) and its identity with the A-esterase of mammalian sera. Biochem J 53(1):117–124Functionality of HDL: Antioxidation and Detoxifying Effects 221Androutsopoulos VP, Kanavouras K, Tsatsakis AM (2011) Role of paraoxonase 1 (PON1) inorganophosphate metabolism: implications in neurodegenerative diseases. Toxicol ApplPharmacol 256(3):418–424Artl A, Marsche G, Lestavel S, Sattler W, Malle E (2000) Role of serum amyloid A duringmetabolism of acute-phase HDL by macrophages. Arterioscler Thromb Vasc Biol 20(3):763–772Aviram M (2012) Atherosclerosis: cell biology and lipoproteins – paraoxonases protect againstatherosclerosis and diabetes development. Curr Opin Lipidol 23(2):169–171Bashtovyy D, Jones MK, Anantharamaiah GM, Segrest JP (2011) Sequence conservation ofapolipoprotein A-I affords novel insights into HDL structure-function. J Lipid Res 52(3):435–450Besler C, Luscher TF, Landmesser U (2012) Molecular mechanisms of vascular effects of high-density lipoprotein: alterations in cardiovascular disease. EMBO Mol Med 4(4):251–268Bhattacharyya T, Nicholls SJ, Topol EJ, Zhang R, Yang X, Schmitt D, Fu X, Shao M, BrennanDM, Ellis SG, Brennan ML, Allayee H, Lusis AJ, Hazen SL (2008) Relationship ofparaoxonase 1 (PON1) gene polymorphisms and functional activity with systemic oxidativestress and cardiovascular risk. JAMA 299(11):1265–1276Birjmohun RS, van Leuven SI, Levels JH, van’t Veer C, Kuivenhoven JA, Meijers JC, Levi M,Kastelein JJ, van der Poll T, Stroes ES (2007) High-density lipoprotein attenuates inflamma-tion and coagulation response on endotoxin challenge in humans. Arterioscler Thromb VascBiol 27(5):1153–1158Bisgaier CL, Sachdev OP, Megna L, Glickman RM (1985) Distribution of apolipoprotein A-IV inhuman plasma. J Lipid Res 26(1):11–25Bowry VW, Stanley KK, Stocker R (1992) High density lipoprotein is the major carrier of lipidhydroperoxides in human blood plasma from fasting donors. Proc Natl Acad Sci U S A 89(21):10316–10320Cabana VG, Lukens JR, Rice KS, Hawkins TJ, Getz GS (1996) HDL content and composition inacute phase response in three species: triglyceride enrichment of HDL a factor in its decrease. JLipid Res 37(12):2662–2674Cabana VG, Gidding SS, Getz GS, Chapman J, Shulman ST (1997) Serum amyloid A and highdensity lipoprotein participate in the acute phase response of Kawasaki disease. Pediatr Res 42(5):651–655Cai L, de Beer MC, de Beer FC, van der Westhuyzen DR (2005) Serum amyloid A is a ligand forscavenger receptor class B type I and inhibits high density lipoprotein binding and selectivelipid uptake. J Biol Chem 280(4):2954–2961Chiba T, Chang MY, Wang S, Wight TN, McMillen TS, Oram JF, Vaisar T, Heinecke JW, DeBeer FC, De Beer MC, Chait A (2011) Serum amyloid A facilitates the binding of high-densitylipoprotein from mice injected with lipopolysaccharide to vascular proteoglycans. ArteriosclerThromb Vasc Biol 31(6):1326–1332Christison JK, Rye KA, Stocker R (1995) Exchange of oxidized cholesteryl linoleate betweenLDL and HDL mediated by cholesteryl ester transfer protein. J Lipid Res 36(9):2017–2026Christison J, Karjalainen A, Brauman J, Bygrave F, Stocker R (1996) Rapid reduction and removalof HDL- but not LDL-associated cholesteryl ester hydroperoxides by rat liver perfused in situ.Biochem J 314(Pt 3):739–742Costa LG, Giordano G, Cole TB, Marsillach J, Furlong CE (2013) Paraoxonase 1 (PON1) as agenetic determinant of susceptibility to organophosphate toxicity. Toxicology 307:115–122Davidson WS, Silva RA, Chantepie S, Lagor WR, Chapman MJ, Kontush A (2009) Proteomicanalysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance toantioxidative function. Arterioscler Thromb Vasc Biol 29(6):870–876Du Y, Yang Y, Jiang W, Wang L, Jia XJ, Si SY, Chen XF, Hong B (2012) Substituted benzamidescontaining azaspiro rings as upregulators of apolipoprotein A-I transcription. Molecules 17222 H. Karlsson et al.Elsoe S, Ahnstrom J, Christoffersen C, Hoofnagle AN, Plomgaard P, Heinecke JW, Binder CJ,Bjorkbacka H, Dahlback B, Nielsen LB (2012) Apolipoprotein M binds oxidizedphospholipids and increases the antioxidant effect of HDL. Atherosclerosis 221(1):91–97Feingold KR, Grunfeld C (2011) The role of HDL in innate immunity. J Lipid Res 52(1):1–3Ferretti G, Bacchetti T, Moroni C, Vignini A, Curatola G (2003) Copper-induced oxidativedamage on astrocytes: protective effect exerted by human high density lipoproteins. BiochimBiophys Acta 1635(1):48–54Flo TH, Halaas O, Lien E, Ryan L, Teti G, Golenbock DT, Sundan A, Espevik T (2000) Humantoll-like receptor 2 mediates monocyte activation by Listeria monocytogenes, but not by groupB streptococci or lipopolysaccharide. J Immunol 164(4):2064–2069Garner B, Waldeck AR, Witting PK, Rye KA, Stocker R (1998) Oxidation of high densitylipoproteins. II. Evidence for direct reduction of lipid hydroperoxides by methionine residuesof apolipoproteins AI and AII. J Biol Chem 273(11):6088–6095Gaut JP, Heinecke JW (2001) Mechanisms for oxidizing low-density lipoprotein. Insights frompatterns of oxidation products in the artery wall and from mouse models of atherosclerosis.Trends Cardiovasc Med 11(3–4):103–112. doi:10.1016/S1050-1738(01)00101-3Ghafouri B, Stahlbom B, Tagesson C, Lindahl M (2002) Newly identified proteins in human nasallavage fluid from non-smokers and smokers using two-dimensional gel electrophoresis andpeptide mass fingerprinting. Proteomics 2(1):112–120Ghafouri B, Tagesson C, Lindahl M (2003) Mapping of proteins in human saliva usingtwo-dimensional gel electrophoresis and peptide mass fingerprinting. Proteomics 3(6):1003–1015Goulinet S, Chapman MJ (1997) Plasma LDL and HDL subspecies are heterogenous in particlecontent of tocopherols and oxygenated and hydrocarbon carotenoids. Relevance to oxidativeresistance and atherogenesis. Arterioscler Thromb Vasc Biol 17(4):786–796Goyal J, Wang K, Liu M, Subbaiah PV (1997) Novel function of lecithin-cholesterolacyltransferase. Hydrolysis of oxidized polar phospholipids generated during lipoproteinoxidation. J Biol Chem 272(26):16231–16239Graner M, James RW, Kahri J, Nieminen MS, Syvanne M, Taskinen MR (2006) Association ofparaoxonase-1 activity and concentration with angiographic severity and extent of coronaryartery disease. J Am Coll Cardiol 47(12):2429–2435Gu JG, Zhu CL, Cheng DZ, Xie Y, Liu F, Zhou X (2011) Enchanced levels of apolipoprotein Mduring HBV infection feedback suppresses HBV replication. Lipids Health Dis 10:154Henning MF, Herlax V, Bakas L (2011) Contribution of the C-terminal end of apolipoprotein AI toneutralization of lipopolysaccharide endotoxic effect. Innate Immun 17(3):327–337Huang Y, Wu Z, Riwanto M, Gao S, Levison BS, Gu X, Fu X, Wagner MA, Besler C,Gerstenecker G, Zhang R, Li XM, DiDonato AJ, Gogonea V, Tang WH, Smith JD, PlowEF, Fox PL, Shih DM, Lusis AJ, Fisher EA, DiDonato JA, Landmesser U, Hazen SL (2013)Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex. J Clin Invest123(9):3815–3828Huen K, Harley K, Brooks J, Hubbard A, Bradman A, Eskenazi B, Holland N (2009) Develop-mental changes in PON1 enzyme activity in young children and effects of PON1polymorphisms. Environ Health Perspect 117(10):1632–1638Imrie HJ, Fowkes FJ, Migot-Nabias F, Luty AJ, Deloron P, Hajduk SL, Day KP (2012) Individualvariation in levels of haptoglobin-related protein in children from Gabon. PLoS ONE 7(11):e49816James RW (2006) A long and winding road: defining the biological role and clinical importance ofparaoxonases. Clin Chem Lab Med 44(9):1052–1059. doi:10.1515/CCLM.2006.207Jarvik GP, Rozek LS, Brophy VH, Hatsukami TS, Richter RJ, Schellenberg GD, Furlong CE(2000) Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON1(192) or PON1(55) genotype. Arterioscler Thromb Vasc Biol 20:2441–2447Juhas M, Eberl L, Tummler B (2005) Quorum sensing: the power of cooperation in the world ofPseudomonas. Environ Microbiol 7(4):459–471Functionality of HDL: Antioxidation and Detoxifying Effects 223Kaji H (2013) High-density lipoproteins and the immune system. J Lipids 2013:684903Karedal MH, Mortstedt H, Jeppsson MC, Kronholm Diab K, Nielsen J, Jonsson BA, Lindh CH(2010) Time-dependent proteomic iTRAQ analysis of nasal lavage of hairdressers challengedby persulfate. J Proteome Res 9(11):5620–5628Karlsson H, Ljunggren S, Ahrén M, Ghafouri B, Uvdahl K, Lindahl M, Ljungman A (2012)Two-dimensional gel electrophoresis and mass spectrometry in studies of nanoparticle-proteininteractions. In: Magdeldin S (ed) Gel glectrophoresis – advanced techniques. In Tech, RijekaKelesidis T, Yang OO, Kendall MA, Hodis HN, Currier JS (2013) Dysfunctional HDL andprogression of atherosclerosis in HIV-1-infected and -uninfected adults. Lipids Health Dis12:23Khersonsky O, Tawfik DS (2005) Structure-reactivity studies of serum paraoxonase PON1 suggestthat its native activity is lactonase. Biochemistry 44(16):6371–6382Khovidhunkit W, Kim MS, Memon RA, Shigenaga JK, Moser AH, Feingold KR, Grunfeld C(2004) Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanismsand consequences to the host. J Lipid Res 45(7):1169–1196Kim TH, Lee YH, Kim KH, Lee SH, Cha JY, Shin EK, Jung S, Jang AS, Park SW, Uh ST, KimYH, Park JS, Sin HG, YoumW, Koh ES, Cho SY, Paik YK, Rhim TY, Park CS (2010) Role oflung apolipoprotein A-I in idiopathic pulmonary fibrosis: antiinflammatory and antifibroticeffect on experimental lung injury and fibrosis. Am J Respir Crit Care Med 182(5):633–642Klimov AN, Gurevich VS, Nikiforova AA, Shatilina LV, Kuzmin AA, Plavinsky SL, TeryukovaNP (1993) Antioxidative activity of high density lipoproteins in vivo. Atherosclerosis 100(1):13–18Klimov AN, Kozhevnikova KA, Kuzmin AA, Kuznetsov AS, Belova EV (2001) On the ability ofhigh density lipoproteins to remove phospholipid peroxidation products from erythrocytemembranes. Biochemistry (Mosc) 66(3):300–304Kontush A, Chapman MJ (2006) Functionally defective high-density lipoprotein: a new therapeu-tic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis. Pharmacol Rev58(3):342–374Kontush A, Chapman MJ (2010) Antiatherogenic function of HDL particle subpopulations: focuson antioxidative activities. Curr Opin Lipidol 21(4):312–318Kontush A, Chantepie S, Chapman MJ (2003) Small, dense HDL particles exert potent protectionof atherogenic LDL against oxidative stress. Arterioscler Thromb Vasc Biol 23(10):1881–1888Kontush A, Therond P, Zerrad A, Couturier M, Negre-Salvayre A, de Souza JA, Chantepie S,Chapman MJ (2007) Preferential sphingosine-1-phosphate enrichment and sphingomyelindepletion are key features of small dense HDL3 particles: relevance to antiapoptotic andantioxidative activities. Arterioscler Thromb Vasc Biol 27(8):1843–1849Koren-Gluzer M, Aviram M, Meilin E, Hayek T (2011) The antioxidant HDL-associatedparaoxonase-1 (PON1) attenuates diabetes development and stimulates beta-cell insulinrelease. Atherosclerosis 219(2):510–518Kotosai M, Shimada S, Kanda M, Matsuda N, Sekido K, Shimizu Y, Tokumura A, Nakamura T,Murota K, Kawai Y, Terao J (2013) Plasma HDL reduces nonesterified fatty acidhydroperoxides originating from oxidized LDL: a mechanism for its antioxidant ability. Lipids48(6):569–578Kriska T, Marathe GK, Schmidt JC, McIntyre TM, Girotti AW (2007) Phospholipase action ofplatelet-activating factor acetylhydrolase, but not paraoxonase-1, on long fatty acyl chainphospholipid hydroperoxides. J Biol Chem 282(1):100–108Kumpula LS, Kumpula JM, Taskinen MR, Jauhiainen M, Kaski K, Ala-Korpela M (2008)Reconsideration of hydrophobic lipid distributions in lipoprotein particles. Chem Phys Lipids155(1):57–62Levels JH, Abraham PR, van Barreveld EP, Meijers JC, van Deventer SJ (2003) Distribution andkinetics of lipoprotein-bound lipoteichoic acid. Infect Immun 71(6):3280–3284224 H. Karlsson et al.Levels JH, Geurts P, Karlsson H, Maree R, Ljunggren S, Fornander L, Wehenkel L, Lindahl M,Stroes ES, Kuivenhoven JA, Meijers JC (2011) High-density lipoprotein proteome dynamics inhuman endotoxemia. Proteome Sci 9(1):34Li Y, Kakinami C, Li Q, Yang B, Li H (2013) Human apolipoprotein A-I is associated with denguevirus and enhances virus infection through SR-BI. PLoS ONE 8(7):e70390Lind PM, van Bavel B, Salihovic S, Lind L (2012) Circulating levels of persistent organicpollutants (POPs) and carotid atherosclerosis in the elderly. Environ Health Perspect 120(1):38–43Ljunggren S, Helmfrid I, Salihovic S, van Bavel B, Wingren G, Lindahl M, Karlsson H (2014)Persistent organic pollutants distribution in lipoprotein fractions in relation to cardiovasculardisease and cancer. Environ Int 65:93–99Mackness B, Durrington P, McElduff P, Yarnell J, Azam N, Watt M, Mackness M (2003) Lowparaoxonase activity predicts coronary events in the Caerphilly Prospective Study. Circulation107(22):2775–2779Maddipati KR, Marnett LJ (1987) Characterization of the major hydroperoxide-reducing activityof human plasma. Purification and properties of a selenium-dependent glutathione peroxidase.J Biol Chem 262(36):17398–17403Marrache S, Dhar S (2013) Biodegradable synthetic high-density lipoprotein nanoparticles foratherosclerosis. Proc Natl Acad Sci U S A 110(23):9445–9450Massamiri T, Tobias PS, Curtiss LK (1997) Structural determinants for the interaction oflipopolysaccharide binding protein with purified high density lipoproteins: role of apolipopro-tein A-I. J Lipid Res 38(3):516–525McPherson PA, Young IS, McEneny J (2007) A dual role for lecithin:cholesterol acyltransferase(EC 2.3.1.43) in lipoprotein oxidation. Free Radic Biol Med 43(11):1484–1493Miyata M, Smith JD (1996) Apolipoprotein E allele-specific antioxidant activity and effects oncytotoxicity by oxidative insults and beta-amyloid peptides. Nat Genet 14(1):55–61Miyazaki A, Sakai M, Suginohara Y, Hakamata H, Sakamoto Y, Morikawa W, Horiuchi S (1994)Acetylated low density lipoprotein reduces its ligand activity for the scavenger receptor afterinteraction with reconstituted high density lipoprotein. J Biol Chem 269(7):5264–5269Molina Portela MP, Raper J, Tomlinson S (2000) An investigation into the mechanism oftrypanosome lysis by human serum factors. Mol Biochem Parasitol 110(2):273–282Molina-Portela Mdel P, Lugli EB, Recio-Pinto E, Raper J (2005) Trypanosome lytic factor, asubclass of high-density lipoprotein, forms cation-selective pores in membranes. Mol BiochemParasitol 144(2):218–226Nguyen SD, Jeong TS, Sok DE (2006) Apolipoprotein A-I-mimetic peptides with antioxidantactions. Arch Biochem Biophys 451(1):34–42Nicholas BL, Skipp P, Barton S, Singh D, Bagmane D, Mould R, Angco G, Ward J, Guha-Niyogi B, Wilson S, Howarth P, Davies DE, Rennard S, O’Connor CD, Djukanovic R (2010)Identification of lipocalin and apolipoprotein A1 as biomarkers of chronic obstructive pulmo-nary disease. Am J Respir Crit Care Med 181(10):1049–1060Nuchi CD, Hernandez P, McClements DJ, Decker EA (2002) Ability of lipid hydroperoxides topartition into surfactant micelles and alter lipid oxidation rates in emulsions. J Agric FoodChem 50(19):5445–5449Ohnsorg PM, Rohrer L, Perisa D, Kateifides A, Chroni A, Kardassis D, Zannis VI, von EckardsteinA (2011) Carboxyl terminus of apolipoprotein A-I (ApoA-I) is necessary for the transport oflipid-free ApoA-I but not prelipidated ApoA-I particles through aortic endothelial cells. J BiolChem 286(10):7744–7754Omiecinski CJ, Vanden Heuvel JP, Perdew GH, Peters JM (2011) Xenobiotic metabolism,disposition, and regulation by receptors: from biochemical phenomenon to predictors ofmajor toxicities. Toxicol Sci 120(Suppl 1):S49–S75Ostos MA, Conconi M, Vergnes L, Baroukh N, Ribalta J, Girona J, Caillaud JM, Ochoa A, ZakinMM (2001) Antioxidative and antiatherosclerotic effects of human apolipoprotein A-IV inapolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 21(6):1023–1028Functionality of HDL: Antioxidation and Detoxifying Effects 225Ozer EA, Pezzulo A, Shih DM, Chun C, Furlong C, Lusis AJ, Greenberg EP, Zabner J (2005)Human and murine paraoxonase 1 are host modulators of Pseudomonas aeruginosa quorum-sensing. FEMS Microbiol Lett 253(1):29–37Parthasarathy S, Barnett J, Fong LG (1990) High-density lipoprotein inhibits the oxidativemodification of low-density lipoprotein. Biochim Biophys Acta 1044(2):275–283Proudfoot JM, Barden AE, Loke WM, Croft KD, Puddey IB, Mori TA (2009) HDL is the majorlipoprotein carrier of plasma F2-isoprostanes. J Lipid Res 50(4):716–722Ramachandran G (2013) Gram-positive and gram-negative bacterial toxins in sepsis: a briefreview. Virulence 5(1)Rantamaki AH, Seppanen-Laakso T, Oresic M, Jauhiainen M, Holopainen JM (2011) Human tearfluid lipidome: from composition to function. PLoS ONE 6(5):e19553Ronn M, Lind PM, Karlsson H, Cvek K, Berglund J, Malmberg F, Orberg J, Lind L, Ortiz-Nieto F,Kullberg J (2013) Quantification of total and visceral adipose tissue in fructose-fed rats usingwater-fat separated single echo MRI. Obesity (Silver Spring) 21(9):E388–E395Rosenblat M, Volkova N, AviramM (2011) Injection of paraoxonase 1 (PON1) to mice stimulatestheir HDL and macrophage antiatherogenicity. Biofactors 37(6):462–467Rosenblat M, Elias A, Volkova N, Aviram M (2013) Monocyte-macrophage membrane possessesfree radicals scavenging activity: stimulation by polyphenols or by paraoxonase 1 (PON1).Free Radic Res 47(4):257–267Russell JA (2006) Management of sepsis. N Engl J Med 355(16):1699–1713Sempf K, Arrey T, Gelperina S, Schorge T, Meyer B, Karas M, Kreuter J (2013) Adsorption ofplasma proteins on uncoated PLGA nanoparticles. Eur J Pharm Biopharm 85(1):53–60Shih DM, Lusis AJ (2009) The roles of PON1 and PON2 in cardiovascular disease and innateimmunity. Curr Opin Lipidol 20(4):288–292Shih DM, Gu L, Xia YR, Navab M, Li WF, Hama S, Castellani LW, Furlong CE, Costa LG,Fogelman AM, Lusis AJ (1998) Mice lacking serum paraoxonase are susceptible to organo-phosphate toxicity and atherosclerosis. Nature 394(6690):284–287Shih DM, Xia YR,Wang XP, Miller E, Castellani LW, Subbanagounder G, Cheroutre H, Faull KF,Berliner JA, Witztum JL, Lusis AJ (2000) Combined serum paraoxonase knockout/apolipo-protein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis. J BiolChem 275(23):17527–17535Shin JY, Yang Y, Heo P, Lee JC, Kong B, Cho JY, Yoon K, Shin CS, Seo JH, Kim SG, Kweon DH(2013) pH-responsive high-density lipoprotein-like nanoparticles to release paclitaxel at acidicpH in cancer chemotherapy. Int J Nanomed 7:2805–2816Singh IP, Chopra AK, Coppenhaver DH, Ananatharamaiah GM, Baron S (1999) Lipoproteinsaccount for part of the broad non-specific antiviral activity of human serum. Antiviral Res 42(3):211–218Soran H, Younis NN, Charlton-Menys V, Durrington P (2009) Variation in paraoxonase-1 activityand atherosclerosis. Curr Opin Lipidol 20(4):265–274Sparks DL, Davidson WS, Lund-Katz S, Phillips MC (1995) Effects of the neutral lipid content ofhigh density lipoprotein on apolipoprotein A-I structure and particle stability. J Biol Chem 270(45):26910–26917Srinivas RV, Birkedal B, Owens RJ, Anantharamaiah GM, Segrest JP, Compans RW (1990)Antiviral effects of apolipoprotein A-I and its synthetic amphipathic peptide analogs. Virology176(1):48–57Stocker R, Keaney JF Jr (2004) Role of oxidative modifications in atherosclerosis. Physiol Rev 84(4):1381–1478Stoltz DA, Ozer EA, Taft PJ, Barry M, Liu L, Kiss PJ, Moninger TO, Parsek MR, Zabner J (2008)Drosophila are protected from Pseudomonas aeruginosa lethality by transgenic expression ofparaoxonase-1. J Clin Invest 118(9):3123–3131Subbaiah PV, Liu M (1993) Role of sphingomyelin in the regulation of cholesterol esterification inthe plasma lipoproteins. Inhibition of lecithin-cholesterol acyltransferase reaction. J Biol Chem268(27):20156–20163226 H. Karlsson et al.Tang WH, Hartiala J, Fan Y, Wu Y, Stewart AF, Erdmann J, Kathiresan S, Roberts R,McPherson R, Allayee H, Hazen SL (2012) Clinical and genetic association of serumparaoxonase and arylesterase activities with cardiovascular risk. Arterioscler Thromb VascBiol 32(11):2803–2812Teeguarden J, Hanson-Drury S, Fisher JW, Doerge DR (2013) Are typical human serum BPAconcentrations measurable and sufficient to be estrogenic in the general population? FoodChem Toxicol 62:949–963Teiber JF, Draganov DI, La Du BN (2004) Purified human serum PON1 does not protect LDLagainst oxidation in the in vitro assays initiated with copper or AAPH. J Lipid Res 45(12):2260–2268Teiber JF, Horke S, Haines DC, Chowdhary PK, Xiao J, Kramer GL, Haley RW, Draganov DI(2008) Dominant role of paraoxonases in inactivation of the Pseudomonas aeruginosaquorum-sensing signal N-(3-oxododecanoyl)-L-homoserine lactone. Infect Immun 76(6):2512–2519Troughton JA, Woodside JV, Yarnell JW, Arveiler D, Amouyel P, Ferrieres J, Ducimetiere P,Patterson CC, Luc G (2008) Paraoxonase activity and coronary heart disease risk in healthymiddle-aged males: the PRIME study. Atherosclerosis 197(2):556–563Tsuchida S, Satoh M, Umemura H, Sogawa K, Kawashima Y, Kado S, Sawai S, Nishimura M,Kodera Y, Matsushita K, Nomura F (2012) Proteomic analysis of gingival crevicular fluid fordiscovery of novel periodontal disease markers. Proteomics 12(13):2190–2202Tward A, Xia YR, Wang XP, Shi YS, Park C, Castellani LW, Lusis AJ, Shih DM (2002)Decreased atherosclerotic lesion formation in human serum paraoxonase transgenic mice.Circulation 106(4):484–490Underhill DM, Ozinsky A (2002) Toll-like receptors: key mediators of microbe detection. CurrOpin Immunol 14(1):103–110Vanhamme L, Paturiaux-Hanocq F, Poelvoorde P, Nolan DP, Lins L, Van Den Abbeele J, Pays A,Tebabi P, Van Xong H, Jacquet A, Moguilevsky N, Dieu M, Kane JP, De Baetselier P,Brasseur R, Pays E (2003) Apolipoprotein L-I is the trypanosome lytic factor of humanserum. Nature 422(6927):83–87Vanhollebeke B, De Muylder G, Nielsen MJ, Pays A, Tebabi P, Dieu M, Raes M, Moestrup SK,Pays E (2008) A haptoglobin-hemoglobin receptor conveys innate immunity to Trypanosomabrucei in humans. Science 320(5876):677–681Vila A, Korytowski W, Girotti AW (2002) Spontaneous transfer of phospholipid and cholesterolhydroperoxides between cell membranes and low-density lipoprotein: assessment of reactionkinetics and prooxidant effects. Biochemistry 41(46):13705–13716Vishnyakova TG, Kurlander R, Bocharov AV, Baranova IN, Chen Z, Abu-Asab MS, Tsokos M,Malide D, Basso F, Remaley A, Csako G, Eggerman TL, Patterson AP (2006) CLA-1 and itssplicing variant CLA-2 mediate bacterial adhesion and cytosolic bacterial invasion in mam-malian cells. Proc Natl Acad Sci U S A 103(45):16888–16893Voisset C, Op de Beeck A, Horellou P, Dreux M, Gustot T, Duverlie G, Cosset FL, Vu-Dac N,Dubuisson J (2006) High-density lipoproteins reduce the neutralizing effect of hepatitis C virus(HCV)-infected patient antibodies by promoting HCV entry. J Gen Virol 87(Pt 9):2577–2581Webb NR, Connell PM, Graf GA, Smart EJ, de Villiers WJ, de Beer FC, van der Westhuyzen DR(1998) SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmictail, mediates lipid transfer between high density lipoprotein and cells. J Biol Chem 273(24):15241–15248Wheeler JG, Keavney BD, Watkins H, Collins R, Danesh J (2004) Four paraoxonase genepolymorphisms in 11212 cases of coronary heart disease and 12786 controls: meta-analysisof 43 studies. Lancet 363(9410):689–695Williams KJ, Tabas I (1995) The response-to-retention hypothesis of early atherogenesis.Arterioscler Thromb Vasc Biol 15(5):551–561Yan YJ, Li Y, Lou B, Wu MP (2006) Beneficial effects of ApoA-I on LPS-induced acute lunginjury and endotoxemia in mice. Life Sci 79(2):210–215Functionality of HDL: Antioxidation and Detoxifying Effects 227Yoshikawa M, Sakuma N, Hibino T, Sato T, Fujinami T (1997) HDL3 exerts more powerful anti-oxidative, protective effects against copper-catalyzed LDL oxidation than HDL2. ClinBiochem 30(3):221–225Zerrad-Saadi A, Therond P, Chantepie S, Couturier M, Rye KA, Chapman MJ, Kontush A (2009)HDL3-mediated inactivation of LDL-associated phospholipid hydroperoxides is determinedby the redox status of apolipoprotein A-I and HDL particle surface lipid rigidity: relevance toinflammation and atherogenesis. Arterioscler Thromb Vasc Biol 29(12):2169–2175Zhang X, Zhu X, Chen B (2010) Inhibition of collar-induced carotid atherosclerosis by recombi-nant apoA-I cysteine mutants in apoE-deficient mice. J Lipid Res 51(12):3434–3442228 H. Karlsson et al.Signal Transduction by HDL: Agonists,Receptors, and Signaling CascadesJerzy-Roch NoferContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2312 ApoA-I-Induced Cell Signaling Directly Mediated by ABCA1 . . . . . . . . . . . . . . . . . . . . . . . . . . 2323 ApoA-I-Induced Cell Signaling Indirectly Mediated by β-ATPase and P2Y12/13 ADPReceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2374 ApoA-I- and HDL-Induced Cell Signaling Indirectly Mediated by ABCA1 and/orABCG1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2385 HDL-Induced Cell Signaling Mediated by SR-BI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2396 HDL-Induced Cell Signaling Mediated by S1P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2437 HDL-Induced Cell Signaling: Future Challenges and Opportunities . . . . . . . . . . . . . . . . . . . . . 247References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249AbstractNumerous epidemiologic studies revealed that high-density lipoprotein (HDL) isan important risk factor for coronary heart disease. There are several well-documented HDL functions such as reversed cholesterol transport, inhibitionof inflammation, or inhibition of platelet activation that may account for theatheroprotective effects of this lipoprotein. Mechanistically, these functions arecarried out by a direct interaction of HDL particle or its components withreceptors localized on the cell surface followed by generation of intracellularsignals. Several HDL-associated receptor ligands such as apolipoprotein A-I(apoA-I) or sphingosine-1-phosphate (S1P) have been identified in addition toHDL holoparticles, which interact with surface receptors such as ATP-bindingcassette transporter A1 (ABCA1); S1P receptor types 1, 2, and 3 (S1P1, S1P2,and S1P3); or scavenger receptor type I (SR-BI) and activate intracellularJ.-R. Nofer (*)Center for Laboratory Medicine, University Hospital Münster, Albert-Schweizer-Campus 1,Geb. A1, D-48149 Münster, Germanye-mail: nofer@uni-muenster.de# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_6229signaling cascades encompassing kinases, phospholipases, trimeric and smallG-proteins, and cytoskeletal proteins such as actin or junctional protein such asconnexin43. In addition, depletion of plasma cell cholesterol mediated byABCA1, ATP-binding cassette transporter G1 (ABCG1), or SR-BI wasdemonstrated to indirectly inhibit signaling over proinflammatory orproliferation-stimulating receptors such as Toll-like or growth factor receptors.The present review summarizes the current knowledge regarding theHDL-induced signal transduction and its relevance to athero- andcardioprotective effects as well as other physiological effects exerted by HDL.KeywordsHigh-density lipoprotein (HDL) • Apolipoprotein A-I (apoA-I) • ATP-bindingcassette transporter A1 (ABCA1) • Scavenger receptor type I (SR-BI) •Sphingosine-1-phosphate (S1P) • Signal transductionAbbreviationsABCA1 ATP-binding cassette transporter A1ABCG1 ATP-binding cassette transporter G1AMPK AMP-activated protein kinaseApo ApolipoproteinCAMK Calcium/calmodulin-dependent protein kinasecAMP Cyclic adenosine monophosphateCdc42 Cell division control protein 42CHD Coronary heart diseaseCKD Chronic kidney diseaseCOX-2 Cyclooxygenase-2DAG diacylglycerolDHEA DehydroepiandrosteroneEL Endothelial lipaseeNOS Endothelial nitric oxide synthaseGLUT4 Glucose transporter type 4GM-CSF Granulocyte macrophage colony-stimulating factorHDL High-density lipoproteinHSPC Hematopoietic stem progenitor cellsIL InterleukinJAK2 Janus kinase 2JNK c-Jun N-terminal kinaseLCAT Lecithin:cholesterol acyltransferaseLDL Low-density lipoprotein230 J.-R. NoferLPS LipopolysaccharideMAPK Mitogen-activated protein kinaseMCP1 Monocyte chemotactic protein 1MDCK Madin-Darby canine kidneyPC PhosphatidylcholinePC-PLC PC-specific phospholipase CPC-PLD PC-specific phospholipase DPDZK1 PDZ domain-containing adaptor proteinPGI2 ProstacyclinPHD Prolyl hydroxylasePI3K Phosphatidylinositol 3-kinasePI-PLC Phosphatidylinositol-specific phospholipase CPKA Protein kinase APKC Protein kinase CS1P Sphingosine-1-phosphateSAA Serum amyloid ASDMA Symmetric dimethylarginineSK Sphingosine kinaseSPC SphingosylphosphorylcholineSPM SphingomyelinSR-BI Scavenger receptor type ISTAT3 Signal transducer and activator of transcription 3TGFβ Transforming growth factor-βTLR Toll-like receptorTNFα Tumor necrosis factor αβ-ATPase F1F0 ATP synthase β-subunit1 IntroductionInnumerable epidemiological studies document the inverse relationship betweenplasma levels of high-density lipoprotein (HDL) cholesterol and cardiovascularrisk. The first hypothesis aiming to mechanistically explain this relationship hasbeen formulated by John Glomset (1968), who proposed that HDL particles take upexcess cholesterol in peripheral tissues including atherosclerotically changed arte-rial walls and transport it to the liver for excretion with bile. The first step in thisprocess termed reverse cholesterol transport encompassed direct interactionbetween HDL and cell membrane with the subsequent efflux of cholesterol tolipoprotein particles. Early concepts envisioned cholesterol efflux as a purelyphysicochemical phenomenon with the unidirectional flux of free cholesterolperpetuated by a steady concentration gradient maintained by the activity oflecithin:cholesterol acyltransferase (LCAT) in HDL particles. John F. Oram wasthe first to note that HDL binding to specific binding sites precedes cholesterolSignal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 231efflux and is required for the effective mobilization of free cholesterol from internalpools and its transport to the plasma membrane, where it is available for furthertransfer to lipoprotein particles (Graham and Oram 1987; Slotte et al. 1987). Theseminal discovery of Oram put HDL function in a fully new perspective: ever sincethen HDL has been perceived not only as a cholesterol transporter but also ashormone-like particle exerting its activities through binding to specific receptors.Signal transduction occurs when an extracellular signaling molecule activates asurface receptor, which in turn alters intracellular molecules creating a response,which ultimately modifies cellular functions. The recognition of specific HDLbinding sites raised immediately a question, whether HDL-cell interaction is linkedto signaling events relevant to intracellular cholesterol transport and/or cholesterolefflux. It was Oram again who demonstrated that the exposure of fibroblasts to HDLcauses activation of protein kinase C (PKC), which in turn contributes to transloca-tion of free cholesterol between intracellular membranes and plasmalemma(Mendez et al. 1991). This finding opened a new field of research focusing ondissection of intracellular signaling pathways that are induced by HDL or itsconstituents and mediate cellular responses to lipoprotein particles. In the followingyears, several ligands present in HDL and their receptors expressed on the cellsurface could be identified that mediate one or more physiological effects attributedto this lipoprotein. In addition, the interaction of HDL with the cell membrane wasfound to potently modify signal transduction induced by other ligands such aspathogen-associated molecular patterns (PAMPs) or growth factors. This reviewattempts to summarize the current state of knowledge on HDL-induced signaltransduction and its physiological relevance.2 ApoA-I-Induced Cell Signaling Directly Mediatedby ABCA1ATP-binding cassette transporter A1 (ABCA1) has been originally identified as aprotein defective in familial HDL deficiency (Tangier disease), which ischaracterized on the molecular level by the failure of apoA-I to mobilize cholesterolfrom intracellular stores, to induce cholesterol and phospholipid efflux from cells,and ultimately to initiate the maturation of HDL particles and the HDL-mediatedreversed cholesterol transport (Bodzioch et al. 1999; Brooks-Wilson et al. 1999;Rust et al. 1999). Early models explaining ABCA1-mediated lipid efflux from cellsassumed that this transporter facilitates translocation of cholesterol andphospholipids to the exofacial leaflet of the cell membrane, where they becameaccessible for the passive transfer to apoA-I. Further studies, however, providedseveral pieces of evidence suggesting that apoA-I may directly interact withABCA1 for the effective unloading of cell cholesterol. For instance, cells obtainedfrom patients with Tangier disease displayed reduced number of apoA-I bindingsites, whereas increased apoA-I binding to the cell surface could be observed incells overexpressing full-length cDNA of ABCA1 (Francis et al. 1995; Wanget al. 2000). Moreover, studies utilizing chemical cross-linking indicated that232 J.-R. NoferapoA-I and ABCA1 are in a very close proximity (<7 Å) characteristic of ligand-receptor interaction, while naturally occurring mutations in extracellular ABCA1loops profoundly impaired the cross-linking efficiency (Wang et al. 2000;Fitzgerald et al. 2002, 2004; Chroni et al. 2004). The binding of apoA-I toABCA1 was subsequently found to increase the cell content of ABCA1 proteinbut not the ABCA1 mRNA abundance suggesting that apoA-I-ABCA1 interactionmight be critical to ABCA1 stabilization (Wang et al. 2003). Actually, apoA-I wasfound to promote both the dephosphorylation of the proline-, glutamic acid-, serine-,and threonine-rich (PEST) sequence in the cytoplasmatic domain of ABCA1 and therecruitment of calmodulin to the specific 1-5-8-14 binding motif localized in a closevicinity to the PEST sequence (Wang et al. 2003; Martinez et al. 2003a; Iwamotoet al. 2010). In this manner, apoA-I binding appears to protect ABCA1 againstcalpain-mediated proteolysis. In addition, lipid-free apoA-I was found to retardABCA1 degradation mediated by thiol proteases and thereby to facilitate ABCA1accumulation on the cell surface (Arakawa and Yokoyama 2002).The recognition of the apoA-I-ABCA1 complex formation and its stabilizationas a mechanism protecting against protease-mediated degradation gave rise to thehypothesis that the interaction of apoA-I with ABCA1 may trigger intracellularsignaling cascades that modulate ABCA1 levels and/or ABCA1-mediated lipidtransport activity in a posttranslational manner (see Fig. 1 for schematic presenta-tion of ABCA1-induced signaling). Early studies documented that apoA-I enhancesphosphatidylcholine (PC) turnover in a specific process mediated by a G-protein-dependent activation of PC-specific phospholipases C and D (PC-PLC andPC-PLD) (Walter et al. 1995, 1996). Later investigations attributed the apoA-I-mediated activation of PC-PLC rather to ABCA1-dependent efflux ofsphingomyelin (SPM) and the unspecific depletion of plasma membrane SPMcontent (Yamauchi et al. 2003). In both cases, stimulation of PC-PLC liberatesdiacylglycerol (DAG), which is the primary physiological activator of PKC. Theactivation of both PC-specific phospholipases and PKC seems to be critical forABCA1-mediated lipid transport. Actually, pharmacological interference withPC-PLC and PC-PLD activation reduced apoA-I-induced cholesterol efflux,whereas exogenous application of their products DAG or phosphatidic acid(PA) partially corrected the cholesterol efflux defect in cells obtained from patientswith Tangier disease (Walter et al. 1996; Haidar et al. 2001). Similarly, PKCinhibitors and activators were found, respectively, to block or to enhance apoA-I-induced cholesterol efflux from various cell types including macrophages, smoothmuscle cells, and fibroblasts (Mendez et al. 1991; Li and Yokoyama 1995; Liet al. 1997). More recently, it has been suggested that PKC specifically controlsthe translocation of cholesterol from acylCoA:cholesterol acyltransferase (ACAT)-accessible pools to the as yet poorly defined intracellular cholesterol compartment,where it is available for the ABCA1-mediated release (Yamauchi et al. 2004). Inaddition, PKC isoform alpha (PKCα) was found to increase ABCA1 phosphoryla-tion at as yet unknown serine sites and thereby to enhance it protection againstcalpain-mediated proteolysis (Yamauchi et al. 2003). Apelin, a bioactive peptidemodulating vascular tone and blood pressure and displaying strong antiatherogenicSignal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 233properties, has been recently found to phosphorylate serine residues in ABCA1through the PKCα pathway and to inhibit calpain-mediated proteolysis, therebypromoting cholesterol efflux and reducing foam cell formation (Liu et al. 2013a).ABCA1 is well known to be regulated by cyclic adenosine monophosphate(cAMP) at the transcriptional level, and cAMP responsive elements have beenlocalized in the ABCA1 promoter. The interaction of apoA-I with ABCA1, in turn,was demonstrated to activate adenylate cyclase and to increase cAMP content,while severely reduced apoA-I-induced cAMP production was found in cellsobtained from carriers of ABCA1 mutations (Haidar et al. 2004). Theseobservations strongly suggested that cAMP and its downstream target proteinkinase A (PKA) may represent an important component of apoA-I/ABCA1-inducedsignaling cascade regulating intracellular lipid translocation and/or their removalfrom cells. Actually, PKA phosphorylation sites were identified in the nucleotidebinding domains of ABCA1, and the targeted mutation of one of these sites, S2054,significantly impaired apoA-I-mediated lipid efflux (See et al. 2002). Moreover,PC-PLCPC-PLD AC JAK2Cdc42 Ca2+PKC PKA STAT3JNK CalcineurinActinC h o l e s t e r o l e f f l u xABCA1stabilizationInflammationGαIκBp55p60p55CytokinesChemokinesp60Myd88 TRIFNucleusToll-likereceptorNFκBABCA1ApoA-IPAMPβγFig. 1 ApoA-I-induced cell signaling mediated by ABCA1. The binding of apolipoprotein A-I(apoA-I) to lipid transporter ABCA1 induces the trimeric G-protein-mediated activation ofphosphatidylcholine-specific phospholipases C and/or D (PC-PLC, PC-PLD) and adenylatecyclase (AC) and the ensuing activation of protein kinases C and A (PKC, PKA). Direct interactionof the small G-protein Cdc42 with ABCA1 induces activation of protein kinase JNK and actinpolymerization. Direct interaction of Janus kinase-2 (JAK2) with ABCA1 leads to itsautophosphorylation and activation as well as activation of transcription factor signal transducerand activator of transcription 3 (STAT3). These effects account for cholesterol efflux-inducing,anti-inflammatory, and ABCA1-stabilizing properties of apoA-I. In addition, ABCA1-mediatedcholesterol efflux to apoA-I exerts indirect negative effect on signal transduction mediated byproinflammatory Toll-like receptors and the resulting activation of the transcription factor NF-κBand expression of cyto- and chemokines. PAMP Pathogen-associated molecular patterns234 J.-R. Noferpharmacological modulation of cAMP/PKA signaling pathway exerted profoundeffects on apoA-I-induced cholesterol efflux. For instance, forskolin, an adenylcyclase activator, and 8-Br-cAMP, a stable cAMP analog, substantially increasedboth ABCA1 phosphorylation and cholesterol efflux to apoA-I in wild-type cellsand partly corrected the impaired efflux in cells obtained from patients with Tangierdisease (Haidar et al. 2002). Similar effects were more recently observed inmacrophages treated with Ht31, a PKA-anchoring inhibitor, which robustly ele-vated PKA activity in the cytoplasm, elevated ABCA1-dependent cholesterolexport from cells, and completely reversed foam cell formation (Ma et al. 2011).Conversely, interference with cAMP/PKA signaling accomplished by treating cellswith adenyl cyclase inhibitors, PKA inhibitors, or eicosapentaenoic acid, whichdecreased both cAMP levels and PKA activity, led to concomitant reduction ofABCA1 phosphorylation and ABCA1-mediated cholesterol efflux (Haidaret al. 2002, 2004; Hu et al. 2009). The molecular mechanisms underlying thestimulatory effect of PKA-mediated ABCA1 phosphorylation on the function ofthis transporter remain unclear. In contrast to PKC, PKA neither improves ABCA1stability nor increases apoA-I binding. It seems more likely that phosphorylation ofABCA1 by the cAMP/PKA pathway allows the transporter to assume the confor-mation favoring more effective lipid translocation across the cell membrane (Seeet al. 2002).Janus kinase 2 (JAK2) represents the third kinase, the activity of which isinduced by ApoA-I/ABCA1 interaction. Even short exposure of cells expressingABCA1 to apoA-I produces substantial autophosphorylation of JAK2, which is aprerequisite for further signal transmission to downstream located targets (Tanget al. 2004). Active JAK2 does not seem to directly phosphorylate ABCA1, and it ismore likely that it targets an as yet unknown ABCA1 partner protein. Nevertheless,several experimental approaches clearly documented that JAK2 signaling isrequired for ABCA1 to effectively mediate lipid export from cells. For instance,JAK2-specific inhibitors substantially reduced apoA-I-induced cholesterol efflux,and mutant cells lacking JAK2 exhibited a severely impaired lipid transport (Tanget al. 2004, 2006). In addition, studies exploiting various ABCA1 variants revealedclose interdependency between lipid export and JAK2 activation (Vaughanet al. 2009). In contrast to PKC and PKA, JAK2 activation by apoA-I neitherpromotes ABCA1 stability nor enhances intrinsic lipid translocase activity of thetransporter. However, JAK2 activation seems to be critical for the optimum forma-tion of apoA-I/ABCA1 complex, as JAK2-deficient cells exhibit severely impairedapoA-I binding despite normal ABCA1 protein levels (Tang et al. 2006).JAK2 autophosphorylation is commonly followed by phosphorylation of severaldownstream targets, out of which signal transducer and activator of transcription3 (STAT3) represents its canonical target. As ABCA1 possesses STAT3 dockingsites, this molecule was an obvious candidate for linking JAK2 with ABCA1activation. As a matter of fact, apoA-I induces STAT3 phosphorylation (which istantamount to its activation) in an ABCA1-dependent fashion, and this effect isattenuated in cells expressing ABCA1, in which STAT3 docking sites have beenmutated (Tang et al. 2009). However, neither complete STAT3 deficiency norSignal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 235interruption of ABCA1-STAT3 interaction altered ABCA1 capacity to bind apoA-Iand to efflux cholesterol from cells (Tang et al. 2009). Hence, apoA-I-inducedSTAT3 activation seems dispensable for the ABCA1-mediated lipid export.STAT3 represents an intracellular target of IL-10—a cytokine with well-established anti-inflammatory properties—and activation of STAT3 wasdemonstrated to suppress several aspects of inflammatory macrophage activation.These observations raised the interesting possibility that apoA-I might produceanti-inflammatory effects by activating STAT3 in ABCA1-dependent fashion.Actually, exposure of ABCA1-expressing macrophages to apoA-I suppressedlipopolysaccharide (LPS)-induced production of proinflammatory cytokines suchas interleukin-6 (IL-6), and this effects were abolished after pharmacologicalSTAT3 blockade or knocking down STAT3 expression with siRNA (Tanget al. 2009). Further investigations demonstrated that the suppressing effect ofapoA-I on LPS-induced cytokine production was mediated by mRNA destabiliza-tion brought about by mRNA-destabilizing protein tristetraprolin, the expression ofwhich was induced in cells exposed to apoA-I in an ABCA1- and JAK2/STAT3-dependent manner (Yin et al. 2011). Recently, apoA-I was demonstrated to promotecyclooxygenase-2 (COX-2) activation and prostacyclin (PGI2) production in endo-thelial cells, and these anti-inflammatory effects were also found to depend onABCA1 and JAK2 activation (Liu et al. 2011).Cell division control protein 42 (Cdc42) is a member of a small G-protein familyinvolved in the regulation of cytoskeleton organization and intracellular vesiculartrafficking. Early studies revealed decreased Cdc42 expression and abnormal cyto-skeleton architecture in cells obtained from Tangier patients (Hirano et al. 2000).Moreover, impaired cholesterol efflux observed in aged human fibroblasts as wellas fibroblasts from Werner syndrome characterized by the early onset of senescentphenotypes including premature atherosclerotic cardiovascular disease could becorrected by forced expression of wild-type Cdc42 (Tsukamoto et al. 2002; Zhanget al. 2005). Expression of constitutively active Cdc42 variant in normal Madin-Darby canine kidney (MDCK) cells potentiated apoA-I-induced cholesterol efflux,whereas expression of dominant negative Cdc42 variants in normal MDCK cells orfibroblasts exerted opposite effects (Hirano et al. 2000; Nofer et al. 2003). ApoA-I-induced cholesterol efflux was also reduced in cells pretreated with Clostridiumdifficile toxin, which irreversibly modifies and disables Cdc42 (Nofer et al. 2003).Both Cdc42 and ABCA1 were found to co-segregate into lubrol-insoluble, triton-soluble lipid rafts (Drobnik et al. 2002). In addition, Cdc42 could be immunopre-cipitated with wild-type ABCA1, but not with C-terminally truncated variant of thistransporter indicating that both proteins interact with each other and suggesting thatC-terminus of ABCA1 harbors a potential docking site for Cdc42 (Tsukamotoet al. 2001; Nofer et al. 2006). Exposure of cells expressing wild-type ABCA, butnot cells obtained from Tangier patients or expressing C-terminally truncatedvariant of ABCA1 to apoA-I, induced Cdc42 activation and subsequent actinpolymerization (Nofer et al. 2006). Taken together, these findings suggest thatapoA-I signals through ABCA1 to activate Cdc42 and thereby to promote choles-terol efflux. How exactly Cdc42 activation promotes lipid export from cells remains236 J.-R. Noferto be elucidated. One possibility is that Cdc42 modulates intracellular vesiculartraffic and thereby helps to supply cholesterol and/or phospholipids to an intracel-lular compartment, where they are available for the ABCA1-mediated release. Theretardation of intracellular lipid transport in cells with reduced Cdc42 levels and itsintensification after enforced expression of Cdc42 appears to support this concept(Tsukamoto et al. 2002). Alternatively, components of the signaling machinerylocalized downstream to Cdc42 might accelerate intracellular lipid transfer or theABCA1-dependent lipid translocation across the cell membrane. Actually, apoA-Iwas demonstrated to produce consecutive activation of two serine/threoninekinases, namely, p21-activated kinase 1 (PAK1) and c-Jun N-terminal kinase(JNK) in ABCA1- and Cdc42-dependent manner (Nofer et al. 2003). The potentialinvolvement of these two kinases in the processes of lipid export from cells hasbeen not clarified to date.Mobilization of intracellular Ca(2+) from intracellular stores or Ca(2+) influxfrom extracellular space represents an integral component of diverse intracellularsignaling pathways. In one single report, evidence has been provided that apoA-Iprovokes extracellular Ca(2+) influx in an ABCA1-dependent manner and thatinhibition of calcineurin, the downstream target of Ca(2+) influx, with cyclosporineA or FK506 completely abolished apoA-I lipidation and also interfered with JAK2phosphorylation (Karwatsky et al. 2010). However, other authors failed to observeCa(2+) mobilization in fibroblasts or smooth muscle cells after exposure to apoA-Ior other apolipoproteins (apoA-II, apoC-III) (Nofer et al. 2000 and unpublishedobservations). Further studies are necessary, therefore, to fully understand thecontribution of Ca(2+) mobilization to ABCA1-mediated signaling and lipidexport.3 ApoA-I-Induced Cell Signaling Indirectly Mediatedby b-ATPase and P2Y12/13 ADP ReceptorF1F0 ATP synthase is the terminal enzyme of the oxidative phosphorylationpathway responsible for the lion part of ATP synthesis in humans and other livingbeing except in Archaea and present in mitochondria, chloroplasts, and prokaryoticmembranes. Unexpectedly, the F1F0 ATP synthase (with F1 domain facing out-side) has been found on the surface of several cells including hepatocytes,adipocytes, and endothelial cells, and its β-chain (β-ATPase) has been characterizedas a receptor for apoA-I (Martinez et al. 2003b; Fabre et al. 2006; Radojkovicet al. 2009; Howard et al. 2011). The binding of apoA-I to β-ATPase induces ATPhydrolysis and promotes extracellular ADP generation, which in turn stimulatesintracellular signaling by activating purinergic receptors, which belong to thefamily of G-protein-coupled receptors stimulated by extracellular nucleotides(Martinez et al. 2003b; Fabre et al. 2006). In hepatocytes, the β-ATPase/P2Y13-induced signaling was reported to activate the small G-protein RhoA and thedownstream-located Rho-associated protein kinase (ROCK-I) and thereby to pro-mote HDL endocytosis by as yet poorly defined mechanism (Fabre et al. 2006;Signal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 237Malaval et al. 2009). In endothelial cells, binding of apoA-I to β-ATPase and thesubsequent generation of extracellular ADP stimulated endothelial proliferationand survival, but components of intracellular signaling cascade responsible forthese effects remained uncharacterized (Radojkovic et al. 2009). In a recentstudy, the activation of β-ATPase/P2Y12 was found to precede HDL binding andcell association as well as ABCG1- and SR-BI-dependent internalization, andtranscytosis in endothelial cells (Cavelier et al. 2012). The identity of intracellularsignaling pathway linking P2Y12 activation to HDL internalization has not beenaddressed in this study.4 ApoA-I- and HDL-Induced Cell Signaling IndirectlyMediated by ABCA1 and/or ABCG1ABCA1 is unique among ABC transporters in that it requires the direct binding ofacceptors for the transported substances. ATP-binding cassette transporter G1(ABCG1) was also found to be a crucial mediator of lipid export. Studies byWang et al. demonstrated that ABCG1 promotes efflux to a variety of acceptors,including HDL, low-density lipoprotein (LDL), phospholipid vesicles, and cyclo-dextrin (Wang et al. 2004, 2006). In major contrast to ABCA1, ABCG1 does notengage into direct interaction with lipid acceptors and for this reason cannot serveas a triggering element of intracellular second messenger cascade. However, recentstudies document that ABCG1 (and to lesser extent ABCA1) profoundly impacts onsignaling processes taking place in contiguity. Several studies showed that ABCA1regulates cholesterol distribution between raft and non-raft membrane fractions andpointed to increased lipid raft formation in macrophages and other cells deficient inABCG1 and ABCA1 (Landry et al. 2006; Sano et al. 2007; Koseki et al. 2007; Zhuet al. 2008a, Yvan-Charvet et al. 2008). Lipid rafts provide a molecular platformsecuring efficient signal transduction through several receptor families includingproinflammatory Toll-like receptors (TLRs). Not surprisingly, therefore, bothABCA1-deficient cells and ABCG1-deficient cells, and in particular cells lackingboth ABC transporters, exhibited increased cell surface expression of TLR4 andsensitivity to LPS stimulation (Koseki et al. 2007; Zhu et al. 2008a, Yvan-Charvetet al. 2008; Mogilenko et al. 2012). Also the NF-κB activation-dependent cytokineand chemokine release in response to stimulation with agonists of other TLRreceptors (TLR2, TLR3) or other proinflammatory receptors (CD40) revealed tobe increased in macrophages deficient in ABCA1 and/or ABCG1 (Sun et al. 2009;Yin et al. 2012). Such cells were also found to be prone to apoptosis duringefferocytosis (phagocytosis of apoptotic cells) as compared to wild-type cells(Yvan-Charvet et al. 2010a). The enhanced apoptotic response observed inABCA1- and ABCG1-deficient macrophages was dependent on an excessiveassembly of NADPH oxidase/NOX2 complexes within lipid rafts followed byoxidative burst and ensuing sustained JNK activation, which turned on the apopto-tic cell death program. It is very likely that similar mechanisms consisting inamplification of transmembrane signaling under conditions of decreased ABCG1238 J.-R. Noferand/or ABCA1 activity and the resulting accumulation of lipid rafts may substan-tially contribute to the enhancement of several proinflammatory processes. Forinstance, cholesterol accumulation within lipid rafts was suggested to enhancemonocyte adhesion and neutrophil activation, and both processes could be reversedby cholesterol removal from cells carried out either with help of synthetic choles-terol acceptors (e.g., cyclodextrin) or through induction of ABCA1-dependentcholesterol efflux (Murphy et al. 2008, 2011). In addition, ABCG1- and ABCA1-dependent modulation of cell membrane cholesterol content was demonstrated tocritically regulate cellular signaling related to proliferatory responses. For instance,mice deficient in ABCG1- and ABCA1 developed severe monocytosis andneutrophilia, which was dependent on uncontrolled proliferation and expansion ofhematopoietic stem progenitor cells (HSPC) (Yvan-Charvet et al. 2010b). Themechanistic analysis revealed increased plasma membrane lipid raft formation inthese cells accompanied by elevated cell surface presence of interleukin IL-3/granulocyte macrophage colony-stimulating factor (GM-CSF) receptor. Additionof IL-3 or GM-CSF to ABCA1/G1-deficient bone marrow led to increased prolif-eration of HSPCs, reflecting activation of signaling pathways downstream of theIL-3/GM-CSF receptor. Similarly, defects in cholesterol efflux pathways inmacrophages and dendritic cells in spleens of ABCA1/G1-deficient mice ledto increased production of IL-23 and activation of a proinflammatory andproatherogenic signaling axis involving IL-23/IL-17 (Westerterp et al. 2012).Other studies showed increased lipid raft content in plasma membrane andenhanced proliferation in response to anti-CD3 antibody stimulation in T cellsobtained from ABCG1-deficient animals (Bensinger et al. 2008). Similar increasein lymph node T-cell proliferation has been noted after disruption of cholesterolefflux pathway by deletion of apoA-I on a hypercholesterolemic background(Wilhelm et al. 2009).5 HDL-Induced Cell Signaling Mediated by SR-BIScavenger receptor type I (SR-BI) is a cell surface glycoprotein originally identifiedby its homology to the scavenger receptor CD36 and capacity to bind acetylatedLDL. Later studies in vitro revealed that SR-BI serves as the binding partner for avariety of ligands including native and oxidized LDL, very-low-densitylipoproteins (VLDL), Lp(a), modified serum proteins such as maleylated albumin,lipid vesicles containing negatively charged phospholipids, apoptotic cells, andHDL. Despite this promiscuous binding behavior SR-BI is believed to primarilyfunction as a physiologically relevant HDL receptor because of its tissue expressionpattern reflecting sites of HDL cholesterol uptake and because of the profoundimpact of complete or tissue-specific SR-BI deficiency on HDL metabolism. Theclassic cellular function of SR-BI is to mediate the selective uptake of lipids fromHDL and to transfer them into cells. Cholesteryl esters represent the primarysubstrate for SR-BI, but this receptor also facilitates transfer of various other lipidspecies including unesterified cholesterol, phospholipids, triglycerides, and lipid-Signal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 239soluble vitamins. SR-BI also mediates the efflux of cholesterol from cells to HDL,which in contrast to ABCA1- and ABCG1-mediated efflux is a passive, concentra-tion gradient-dependent, and energy-independent process. Importantly, SR-BI-mediated lipid transfer from and to HDL occurs in the absence of consecutiveinternalization and degradation of HDL particle, which is a precedent condition foreffective signal transduction (for recent reviews, see Hoekstra et al. 2010; Mineoand Shaul 2012).Binding of HDL to SR-BI has been demonstrated to trigger several intracellularsignaling events (see Fig. 2). The recruitment of non-receptor tyrosine kinase src tothe C-terminal cytoplasmic tail of SR-BI followed by src autophosphorylation andphosphorylation and activation of AMP-activated protein kinase (AMPK) mediatedthrough calcium/calmodulin-dependent protein kinase (CAMK) and/or serine/thre-onine liver kinase B1 (LKB1) appear to constitute initial steps in SR-BI signalingHDLN CAkt MAPKsrcLKB1CAMKAMPKeNOSNOPCOX-2PGI2GLUT4GlucoseTransportRac-1LamellipodiaSiah 1/2HIF-1aPDZK1(CLAMP)Vasodilation ReedothelializationInsulinsensitivityIkBp55p60p55CytokinesChemokinesCholesterolEffluxp60Myd88 TRIFHypoxia -angiogenesisNucleusToll-likereceptorNFkBPAMPFig. 2 HDL-induced cell signaling mediated by SR-BI. The binding of HDL to scavengerreceptor type 1 (SR-BI) induces activation of protein kinases src, liver kinase B1 (LKB1), andcalcium calmodulin-dependent protein kinase (CAMK) in a process dependent on an adapterprotein PDZK1 (CLAMP). The resulting activation of AMP-activated protein kinase (AMPK) andprotein kinase Akt as well as mitogen-activated protein kinase (MAPK) transduces signal todownstream effectors including endothelial nitric oxide synthase (eNOS) generating NO,cyclooxygenase-2 (COX-2) producing prostacyclin (PGI2), glucose transporter GLUT4 mediatingcellular glucose uptake, ubiquitin ligases Siah 1 and 2 stabilizing hypoxia-inducible factor-1α(HIF-1α), and small G-protein Rac-1 regulating formation of lamellipodia. These effects accountfor vasodilatory, insulin-sensitizing, proangiogenic, and endothelial regenerating properties ofHDL. In addition, SR-BI-mediated cholesterol efflux to HDL exerts indirect negative effects onsignal transduction mediated by proinflammatory Toll-like receptors and the resulting activationof the transcription factor NF-κB and expression of cyto- and chemokines. Pathogen-associatedmolecular patterns (PAMP)240 J.-R. Nofer(Mineo et al. 2003; Kimura et al. 2010). Detailed studies revealed that phosphor-ylations of src (though not its interaction with SR-BI) and AMPK are criticallydependent on the presence of the PDZ domain-containing adaptor protein, PDZK1(also termed CLAMP), which interacts with extreme C-terminal residues of SR-BIand is essential for its stability in the plasma membrane (Kimura et al. 2010;Assanasen et al. 2005; Zhu et al. 2008a, b). Src and AMPK activations lead to theactivation of phosphatidylinositol 3-kinase (PI3K), which in turn induces parallelactivation of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK)(Kimura et al. 2010; Assanasen et al. 2005; Zhu et al. 2008b). The latter eventseems to be required for the subsequent stimulation of the activity of smallG-protein Rac1, which promotes formation of lamellipodia and induces cellshape change (Seetharam et al. 2006). It remains currently unclear, whether SR-BI-mediated lipid transfer is required for the effective initiation of signaling byHDL. The C-terminal transmembrane domain of SR-BI contains a cholesterol-binding site, which interacts with plasma cholesterol, as could be documented inphotocholesterol binding experiments (Assanasen et al. 2005). Studies utilizingSR-BI/CD36 chimeras showed that only transfer of both C-terminal domains(cytoplasmatic and transmembrane) endows CD36 with signaling properties ofSR-BI, whereas the transfer of the C-terminal cytoplasmatic domain only is insuf-ficient (Assanasen et al. 2005). In addition, introduction of mutations into thetransmembrane C-terminal domain severely impeded SR-BI-mediated signal trans-duction (Saddar et al. 2013). Other studies pointed to the enhancement of SR-BIsignaling in the presence of extracellular cholesterol acceptors such ascyclodextrins or reconstituted HDL particles containing 2 molecules of apoA-Ibut not cholesterol (Assanasen et al. 2005). Furthermore, HDL enriched in phos-phatidylcholine, which increases its cholesterol acceptor capacity, induces signaltransduction at concentrations that are otherwise insufficient to invoke signaling.Collectively, these observations suggest that SR-BI-mediated cholesterol effluxexerts at least potentiating effects on HDL-triggered signaling transduced by thisreceptor. Recently, Al-Jarallah and Trigatti hypothesized that SR-BI-mediatedremoval of cholesterol from caveolae might suppress the activity of high molecularweight phosphatase complex, which would in turn help to maintain protein kinasetargets such as MAPK in the phosphorylated state and to prolong their activation(Al-Jarallah and Trigatti 2010). Whether this mechanism indeed contributes toHDL-induced signaling mediated by SR-BI remains to be clarified.The concerted activation of both Akt and MAPK appears to constitute thecenterpiece of SR-BI-triggered signaling cascades. Both kinases are required forphosphorylation and full activation of endothelial nitric oxide synthase (eNOS),nitric oxide generation, and vasorelaxation, which are observed in endothelial cellsor isolated aortas in the presence of HDL (Yuhanna et al. 2001; Mineo et al. 2003;Assanasen et al. 2005). In addition, PI3K/Akt/eNOS signaling was found to beinvolved in the HDL-induced COX-2 expression and PGI2 release in endothelialcells (Zhang et al. 2012). The proliferation of bone-derived mesenchymal stem cellsin response to HDL was likewise mediated via its binding to scavenger receptor-Btype I and activation of PI3K/Akt/MAPK pathways (Xu et al. 2012). Activation ofSignal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 241MAPK but not Akt with subsequent induction of Rac1-GTPase was demonstratedto promote HDL-induced endothelial cell migration, which is critical for carotidartery reendothelialization after perivascular electric injury (Seetharam et al. 2006).Conversely, activation of Akt but not MAPK was necessary to sustain the migrationand the proliferation of breast cancer cells (Danilo et al. 2013). In adipocytes, SR-BI-mediated activations of Akt and AMPK were found to promote glucose uptakeand translocation of the glucose transporter GLUT4 to the plasma membranesuggesting that SR-BI signaling might contribute to insulin-sensitizing effects ofHDL (Zhang et al. 2011). Finally, augmentation of hypoxia-induced angiogenesis,which is observed in the presence of HDL, was demonstrated to depend on SR-BI-mediated activation of Akt linked to increased expression of E3 ubiquitin ligasesSiah1 and Siah2 and accelerated degradation of prolyl hydroxylases (PHD1-PHD3)combined with stabilization of hypoxia-inducible factor 1α (HIF-1α) (Tanet al. 2013). Recent studies provide evidence that similar to ABCG1 also SR-BImay indirectly affect proinflammatory signaling by reducing membrane cholesterolcontent. Actually, macrophages obtained from SR-BI-deficient animals werecharacterized by increased inflammatory responses to Toll-like receptor agonists(Guo et al. 2009; Cai et al. 2012). Feng et al. found that SR-BI deficiency enhancedlymphocyte proliferation, caused imbalanced interferon-γ (IFNγ) and IL-4 produc-tion in lymphocytes, and led to elevated inflammatory cytokine production inmacrophages (Feng et al. 2011). Umemoto et al. demonstrated that HDL-induceddisruption of lipid raft in adipocytes, which is accompanied by reduced activationof NADPH oxidase and production of monocyte chemotactic protein 1 (MCP1),was reversed by silencing both ABCG1 and SR-BI expression (Umemotoet al. 2013).It is well established that HDL inhibits the activation of platelets by strongagonists such as thrombin and collagen (for review, see Nofer and van Eck 2011).The presence of SR-BI has been identified on platelet surface (Imachi et al. 2003).Recent investigations demonstrated that HDL binding to platelets was reduced in aconcentration-dependent fashion by SR-BI ligands such as negatively chargedliposomes, which in addition potently inhibited thrombin-induced platelet aggre-gation, granule secretion, fibrinogen binding, and mobilization of intracellular Ca2+(Nofer et al. 2011). Furthermore, both native HDL and other SR-BI ligands failed toinhibit thrombin-induced activation of platelets obtained fromSR-BI-deficientmice.These findings together with the results of Imachi et al. (2003), who observed aninverse relationship between the levels of SR-BI expression and the platelet aggre-gation in response to ADP, corroborate the hypothesis that SR-BI is a true functionalHDL receptor on platelets. The likely consequence of the HDL binding to SR-BI onplatelets is the induction of intracellular second messenger cascade. It has beenpreviously demonstrated that incubation of platelets with HDL leads to the release ofDAG from the plasma membrane phosphatidylcholine and to subsequent activationof PKC, which in turn stimulates the Na+/H+ antiport, promotes the alkalization ofthe cytoplasmic compartment, and inhibits the release of calcium ions from intra-cellular storage sites (Nofer et al. 1996). In addition, activation of PKC inhibits theactivity of phosphatidylinositol-specific phospholipase C (PI-PLC), which is one of242 J.-R. Noferthe most important signal transduction mediators of agonists such as thrombin andcollagen (Nofer et al. 1998). It is conceivable that this signaling cascade is launchedby interaction of HDL with SR-BI on platelet surface.6 HDL-Induced Cell Signaling Mediated by S1PEarly studies on HDL-induced signal transduction focused on apoA-I or otherapolipoproteins as potential binding partners for HDL receptor responsible forinitiating the cascade of intracellular signaling events. However, it has soon becomeclear that neither apoA-I nor other proteins encountered in HDL particles canexclusively account for HDL signaling activities. Studies utilizing fibroblastsdemonstrated that PI-PLC activation, which is observed in cells exposed to HDLand accompanied by increased phosphatidylinositol bisphosphate (PIP2) turnoverand the mobilization of intracellular calcium, is not emulated by purified apoA-I orapoA-I-containing proteoliposomes, but occurs in the presence of lipids extractedfrom HDL holoparticles (Nofer et al. 2000). Likewise, certain cellular responsestriggered by HDL in fibroblasts of smooth muscle cells such as cell proliferation orproliferation-linked activation of MAPK could be observed in the presence of HDLlipids orHDLparticles, inwhich proteins were covalentlymodified or enzymaticallydegraded, but not in the presence of proteins isolated from HDL (Deeg et al. 1997;Sachinidis et al. 1999; Nofer et al. 2001a). The intense search for lipid speciesaccounting for the signal-inducing capacity of HDL revealed that these lipoproteinsserve as a carrier for bioactive lysosphingolipids such as sphingosine-1-phosphate(S1P), sphingosylphosphorylcholine (SPC), and lysosulfatide (LSF) and that signal-ing events observed in cells exposed to native HDL or isolated lysosphingolipidslargely overlap (Sachinidis et al. 1999; Nofer et al. 2000, 2004; Kimura et al. 2001).S1P is the most prominent and best characterized member of the lysosphingolipidfamily. It is produced by phosphorylation of sphingosine by sphingosine kinases1 and 2 (SK1 and SK2) in response to a variety of stimuli and degraded tophosphoethanolamine and hexadecanal by S1P-lyase or to sphingosine and organicphosphate by S1P phosphatase. S1P interacts with five related G-protein-coupledreceptors termed S1P1–5, which regulate a wide spectrum of cellular functions,including proliferation and survival, cytoskeletal rearrangements, and cell motility,and exert potent cytoprotective effects. In the vasculature, S1P receptors wereidentified on endothelial and smooth muscle cells, and S1P provides essential contri-bution to the new vessel formation and the maintenance of vascular barrier integrity.In addition, S1P was demonstrated to interfere with proliferation, migration, andactivation of monocytes/macrophages and to regulate their recruitment to sites ofinflammation (for recent reviews, see Maceyka et al. 2012; Levkau 2013).Erythrocytes and platelets are major sources of S1P in plasma, where it is preferen-tially (up to 60 %) associated with HDL subfraction rich in apolipoprotein M (apoM)(Dahm et al. 2006; Hänel et al. 2007; Christoffersen et al. 2011). Recent studiesdemonstrate that apoM specifically binds S1P and stimulates its biosynthesis forsecretion (Liu et al. 2013b). There is substantial evidence suggesting that S1P maySignal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 243directly contribute to atheroprotective effects attributed to HDL. For instance, S1Pclosely correlates with HDL in a concentration range, in which HDL most effectivelyprotects against atherosclerosis, and decreased HDL-bound S1P levels were noted inpatients with coronary artery disease and myocardial infarction (Karuna et al. 2011;Sattler et al. 2010; Argraves et al. 2011). In addition, S1P was found to emulatein vitro several atheroprotective effects of HDL, which themselves could beattenuated by interrupting signaling through S1P receptors (Levkau et al. 2013).Protein kinases MAPK, AMPK, and Akt are critical for the S1P-dependentsignaling cascade, which unfolds in endothelial cells in the presence of HDL (seeFig. 3 for schematic presentation of S1P-mediated signaling). Kimura andAktHDL S1P1/S1P3eNOSNOVasodilationSR-BIN CA-IS1PMigrationRac1BADBcl-XLPPCaspase 9/3ApoptosisN-BkFE-selectinVCAM-1Adhesionp38MAPKCOX-2PGI2TGFbGa bgSmad2/3MMP9PLA2InflammationRhoAS1P2Rac1Smad2/3NADPHoxidaseROSGa bgCardiomyocyteProtectionEndothelialbarrierNF-kBTLR2CytokinessrcSTAT3AMPKFAKFig. 3 HDL-induced cell signaling mediated by S1P. The binding of HDL to scavenger receptortype I (SR-BI) may provide a molecular platform for efficient receptor stimulation by sphingosine-1-phosphate (S1P) present in HDL molecule. Activation of AMP-activated protein kinase(AMPK) and protein kinase Akt via S1P1 and/or S1P3 receptors leads to the stimulation of focaladhesion kinase (FAK), the inactivation of proapoptotic protein BAD and the inhibition ofcaspases 9 and 3, the inhibition of the transcription factor NF-κB and the expression of adhesivemolecules E-selectin and vascular cell adhesion molecule 1 (VCAM-1), as well as the activation ofendothelial nitric oxide synthase (eNOS) and the generation of NO. Activation of the mitogen-activated protein kinase p38MAPK results in activation of cyclooxygenase-2 (COX-2) andproduction of prostacyclin (PGI2). Increased expression of transforming growth factor β (TGFβ)contributes to the increased activation of transcription factors Smad 2 and 3, which suppressproduction of proinflammatory matrix metalloproteinases such as MMP9 as well as phospholipaseA2 (PLA2). Inhibition of small G-protein Rac1 suppresses activity of NADPH oxidase andgeneration of reactive oxygen species. Inhibition of Toll-like receptor 2 (TLR2) reduces NF-κBactivation and production of proinflammatory cytokines. These effects account for endothelialbarrier-enhancing, anti-inflammatory, antiapoptotic, anti-adhesive, and vasodilatory properties ofHDL. Activation of protein kinase src and transcription factor signal transducer and activator oftranscription 3 (STAT3) via S1P2 protects against cardiomyocyte apoptosis. Activation of thesmall G-protein RhoA via S1P2 leads to inhibition of the small G-protein Rac1, which regulatessmooth muscle cell migration244 J.-R. Nofercolleagues demonstrated that activation of MAPK is essential for endothelialproliferation and survival mediated by HDL-associated lysosphingolipids (Kimuraet al. 2001). Using antisense oligonucleotides and siRNA strategy combined withapplication of pharmacological G-protein inhibitors, they further show that S1Preceptor types 1 and 3 and inhibitory G-protein (Gi) contribute to HDL-mediatedinduction of endothelial migration, whereas the augmentation of endothelial sur-vival in endothelial cells exposed to HDL is exclusively dependent on S1P1. Miuraet al. demonstrated that HDL promotes endothelial cell tube formation via activa-tion of MAPK and suggested that this effect is mediated by S1P present in HDLparticles (Miura et al. 2003). Later these authors showed that reconstituted HDLparticles enriched in S1P are particularly effective in stimulating angiogenicresponse (Matsuo et al. 2007). HDL-like lipoproteins are also present in follicularfluid and induce angiogenesis in the S1P- and MAPK-dependent manner (von Otteet al. 2006). The activation of AMPK, which itself was entirely dependent on theparallel activation of CAMK, was localized upstream to Akt in the signaling sequel(Kimura et al. 2010). The stimulation of Akt by HDL-associated lysosphingolipidswas—depending on the experimental system used—mediated by either S1P1 orS1P3 and controlled an array of important antiatherogenic activities attributed toHDL. Nofer et al. (2001b) found that activation of Akt by HDL and its associatedlysosphingolipids disabled a proapoptotic protein BAD and thereby inhibited thecollapse of mitochondrial potential, the cytochrome C release from mitochondria,and the ensuing activation of apoptosis-executing caspases 3 and 9. Noferet al. (2004) and Kimura et al. (2006) demonstrated that the S1P content in HDLat least partially accounts for the HDL-stimulated activation of eNOS, generation ofNO, and vasorelaxation. Stimulation of endothelial cells with statins such aspitavastatin or simvaststin increased the expression of S1P1 and potentiated thestimulatory effect of HDL on eNOS activation (Igarashi et al. 2007; Kimuraet al. 2008). Likewise, upregulated S1P1 and S1P3 expression and increased Aktand eNOS activation were found in fenofibrate-treated mice, which werecharacterized by elevated plasma levels of HDL and S1P (Krishna et al. 2012).Kimura et al. (2006) reported that signaling through S1P1 receptor partly accountsfor the inhibitory effects exerted by HDL on tumor necrosis factor α (TNFα)-induced NF-κB activation as well as endothelial expression of adhesins such asvascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Argraves et al. (2008) found that HDL increased endothelial barrierintegrity as measured by electric cell substrate impedance sensing in a process thatwas entirely dependent on S1P, S1P1 expression, and Akt activation. The favorableeffect of S1P on endothelial barrier was sustained for considerably longer time withHDL-associated rather than albumin-associated S1P, and this was related to theinhibitory effect of HDL on S1P1 degradation (Wilkerson et al. 2012). Norataet al. reported that HDL potently stimulates endothelial expression of longpentraxin 3—a key component of innate immunity—as well as transforminggrowth factor-β (TGFβ), a compound with potent anti-inflammatory and immuno-modulatory activities, in Akt-dependent manner. They attributed these effects to thepresence of S1P and SPC in HDL particles and S1P1 and S1P3 receptors onSignal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 245endothelial cells (Norata et al. 2005, 2008). Increased activation of Smad 2/3, atranscription factor controlled by TGFβ, found in the latter study suggests thatHDL-associated S1P may transactivate TGFβ signaling pathways in an autocrinefashion. Most recently, Tatematsu et al. reported that endothelial lipase (EL) is acritical determinant of HDL-stimulated S1P-dependent signaling (Tatematsuet al. 2013). Actually, HDL-induced endothelial cell migration and angiogenicresponses as well as Akt activation were markedly decreased in EL-deficientendothelial cells, but restored in the presence of exogenous S1P. Basing on appli-cation of pharmacological inhibitors, Tatematsu and colleagues concluded that theeffects of HDL on endothelial cell migration and Akt activation are mediatedby S1P1.As pointed out above, HDL is strongly mitogenic for smooth muscle cells, andHDL-associated lysosphingolipids appear to account for this effect. On the otherhand, HDL was identified as a potent inhibitor of smooth muscle cell migration,which is a hallmark of the development of advanced atherosclerotic plaque andrestenosis (Tamama et al. 2005; Damirin et al. 2007). The inhibitory effect of HDLwas likely mediated by its S1P content, as it was abolished in the presence of S1P2-specific siRNA and following desensitization of S1P receptors and much enhancedin cells overexpressing S1P2 receptor. Further effects exerted by HDL on smoothmuscle cell through its S1P content include modulation of the activity of severalvasoactive factors. Chrisman et al. found that HDL-associated S1P desensitizesguanylyl cyclase B, a receptor for C-type natriuretic peptide (CNP), and therebyinhibits CNP-induced cyclic guanosine monophosphate (cGMP) accumulation invascular smooth muscle cells (Chrisman et al. 2003). Gonzalez-Diez and colleaguesdemonstrated induction of COX-2 expression and PGI2 production in vascularsmooth muscle cells exposed to HDL, which was mediated through S1P receptors2 and 3 and protein kinase p38MAPK (González-Dı́ez et al. 2008). They have alsoshown that simvastatin potentiates the HDL- and S1P-induced COX-2 expressionby upregulating cellular levels of S1P3. Finally, HDL-associated lysosphingolipidsS1P and SPC were found to suppress the proinflammatory activation of smoothmuscle cells brought about by stimulation with thrombin and characterized by theincreased release of inflammatory cyto- and chemokines such as MCP1 (Tölleet al. 2008). The detailed examination of signaling pathways accounting for inhibi-tory effects exerted by HDL-associated lysosphingolipids revealed that they wererelated to the inhibition of Rac1-mediated activation of NADPH oxidase andgeneration of reactive oxygen species. S1P3 was identified to mediate anti-inflammatory effects of HDL, S1P, and SPC in smooth muscle cells.Cardiomyocytes express several S1P receptors, and HDL was found to exertprotective effects on these cells through its S1P content. HDL and S1P showedpotent capacity to protect cardiomyocytes against hypoxia-reoxygenation- ordoxorubicin-induced apoptosis, and these effects were attributed to the inductionof protein kinases Src, MAPK, and Akt and the transcription factor STAT3(Frias et al. 2009, 2010; Tao et al. 2010). However, investigations concerning theidentity of receptor-mediating protective effects of HDL-associated S1P wereinconclusive, and equally supportive evidence for the involvement of either S1P2246 J.-R. Noferor S1P1 and S1P3 has been provided. Recently, short-term treatment ofcardiomyocytes with HDL or S1P was found to induce a PKC-dependent phos-phorylation of connexin43, which is a gap junction protein present in ventricularcardiomyocytes and involved in cardioprotection by modulating ischemicpreconditioning (Morel et al. 2012). In addition, HDL and S1P improved gapjunctional communication, but only incrementally affected conduction velocitiesin cardiomyocytes. The physiological relevance of these effects has been clearlydocumented using an ex vivo approach, in which both HDL and S1P protectedperfused hearts against ischemia/reperfusion-induced cell death.Several other cell types were found to be responsive to stimulation withHDL-associated lysosphingolipids, but the physiological relevance of these effectsremains obscure. For instance, HDL and S1P were demonstrated to stimulateprostate cancer cell migration and invasion through S1P receptors 2 and 3 andactivation of MAPK and STAT3, to stimulate plasminogen activator inhibitor-1(PAI-1) release from adipocytes through S1P2 and activation of PKC and Rhokinase, and to promote release of fibroblast growth factor-2 (FGF2), a potentneurotrophic factor, from astroglial cells through activation of PI-PLC andMAPK (Malchinkhuu et al. 2003; Lee et al. 2010; Sekine et al. 2011). HDLparticles isolated from body fluids other than plasma were also found to containS1P and to stimulate intracellular signaling through their S1P content. For instance,HDL-like particles obtained from cerebrospinal fluid stimulated astrocyte migra-tion and oligodendrocyte retraction in a manner sensitive to inhibitors of S1Preceptors 1 and 3 (Sato et al. 2007). Follicular fluid-derived HDL was found topromote human granulosa lutein cell migration through S1P3 and activation ofsmall G-protein Rac1 (Becker et al. 2011). Despite the prominent role played bymonocytes and macrophages in the pathogenesis of atherosclerosis, very little isknown about the modulatory effects exerted by S1P cargo of HDL particles on thesecells. In one study, both HDL and S1P were found to blunt monocyte activationinduced by agonists of TLR2, but the attribution of this effect to theHDL-associated S1P was not unequivocally demonstrated (Dueñas et al. 2008).7 HDL-Induced Cell Signaling: Future Challengesand OpportunitiesHDL constitutes a large and heterogenous macromolecule carrying more than50 proteins, several amphipathic peptides, miRNAs, and hundreds of lipid speciesrepresenting almost all known lipid classes. Several proteins and lipids identified inHDL—among others, apolipoprotein H, haptoglobin, and steroid hormones—arerecognized receptor ligands and inductors of intracellular signaling events. How-ever, the rigid evidence demonstrating that these proteins or lipids contribute to theHDL-induced signaling governing diverse functional responses to this lipoproteinhas been not provided to date or is at best equivocal. For instance, several studiesdocumented that HDL carries estradiol in the form of fatty acyl esters produced in areaction catalyzed by LCAT. HDL-derived estradiol esters are internalized via SR-Signal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 247BI-mediated mechanisms and subsequently hydrolyzed intracellularly to unfoldtheir effects. However, the specific signaling events and/or physiological effectsclearly attributable to the estradiol content in HDL particles have not been unequiv-ocally defined. In one study, HDL-associated estradiol was found to stimulateeNOS and vasodilation in an SR-BI- and Akt-dependent manner, but these resultswere questioned by others (Gong et al. 2003; Nofer et al. 2004). In other study,dehydroepiandrosterone (DHEA) fatty acyl esters once incorporated in HDL werefound to induce stronger vasodilatory response, and this effect appeared also to bemediated by SR-BI (Paatela et al. 2011). However, no clear signaling link betweenthe HDL-associated DHEA and vasodilation has been established in this study.These two examples clearly show that the assignment of ligands present in HDL tothe appending signaling machinery and to various functions exerted by theselipoproteins in physiology will be a major challenge in the future.The composition of HDL particles is variable and influenced both by normalmetabolism and in various states of pathology. For instance, HDL particles isolatedfrom subjects suffering from acute or chronic inflammatory diseases were found tolose proteins and enzymes with established or presumed antiatherogenic functionsuch as apoA-I or LCAT and to concomitantly acquire proinflammatory orprooxidative factors such as serum amyloid A (SAA), ceruloplasmin, lipoprotein-specific phospholipase A2, or myeloperoxidase. Not surprisingly, such inflamma-tory HDL particles are severely impeded in their capacity to trigger certain signal-ing pathways, but at the same time may acquire new ligands and thereby alter theirsignaling properties. For instance, the relatively high content of apolipoprotein J(apoJ) in HDL particles obtained from healthy subjects endowed them with impor-tant antiatherogenic property to promote endothelial survival by stimulation of theexpression of antiapoptotic protein Bcl-XL (Riwanto et al. 2013). By contrast, HDLparticles isolated from patients with stable coronary heart disease (CHD) or acutecoronary syndromes were found to be poor in apoJ, but instead enriched in apoC-Ior apoC-III (McNeal et al. 2013; Riwanto et al. 2013). Furthermore, the increasedcontent of these apolipoproteins turned HDL particles into potent apoptosisinducers in vascular smooth muscle and endothelial cells by virtue of stimulatingthe expression of proapoptotic protein tBid. In addition to CHD, dysfunctional HDLis also encountered in chronic conditions such as chronic kidney disease (CKD).Abnormal HDL composition characterized primarily by increased serum amyloid Aand apoC-III content as well as compromised HDL functionality has been repeat-edly reported in uremic patients (Holzer et al. 2011; Weichhart et al. 2012). In onerecent study, SAA-enriched HDL encountered in CKD was found to induce pro-duction of a proinflammatory chemokine MCP1 in smooth muscle cells by interac-tion with formyl peptide receptor 2 (FPR2) (Tölle et al. 2012). In other studyconducted in CKD patients, accumulation of symmetric dimethylarginine(SDMA) in HDL was found to turn this lipoprotein into a noxious particle reducingNO bioavailability, evoking endothelial dysfunction, and subsequently increasingarterial blood pressure. The deleterious effects of SDMA-modified HDL wereattributed to the noncanonical activation of TLR2 (Speer et al. 2013). Thisexemplifies another challenge facing HDL research in the future, namely,248 J.-R. Noferidentification of ligands, receptors, and signaling cascades attributable to HDL invarious states of pathology.Notwithstanding the physiological or pathological character of HDL-inducedsignal transduction, its elucidation may open new opportunities for the develop-ment of antiatherogenic, anti-inflammatory, or antithrombotic drugs. Several inter-ventional studies conducted recently and aiming at cardiovascular risk reductionthrough simple elevation of plasma HDL cholesterol fall through. This epic failureemphasizes the necessity to develop more sophisticated strategies based on targetedexploitation of selected antiatherogenic or anti-inflammatory activities exerted byHDL particles. The proper understanding of HDL-induced signaling may help tospecifically enhance beneficial physiological or to disable harmful pathologicaleffects of HDL and thereby to design more efficacious therapeutic approaches forcardiovascular and other inflammatory diseases.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAl-Jarallah A, Trigatti BL (2010) A role for the scavenger receptor, class B type I in high densitylipoprotein dependent activation of cellular signaling pathways. Biochim Biophys Acta1801:1239–1248Arakawa R, Yokoyama S (2002) Helical apolipoproteins stabilize ATP-binding cassette trans-porter A1 by protecting it from thiol protease-mediated degradation. J Biol Chem 277:22426–22429Argraves KM, Gazzolo PJ, Groh EM et al (2008) High density lipoprotein-associated sphingosine1-phosphate promotes endothelial barrier function. J Biol Chem 283:25074–25081Argraves KM, Sethi AA, Gazzolo PJ et al (2011) S1P, dihydro-S1P and C24:1-ceramide levels inthe HDL-containing fraction of serum inversely correlate with occurrence of ischemic heartdisease. Lipids Health Dis 10:70Assanasen C, Mineo C, Seetharam D et al (2005) Cholesterol binding, efflux, and aPDZ-interacting domain of scavenger receptor-BI mediate HDL-initiated signaling. J ClinInvest 115:969–977Becker S, von Otte S, Robenek H et al (2011) Follicular fluid high-density lipoprotein-associatedsphingosine 1-phosphate (S1P) promotes human granulosa lutein cell migration via S1Preceptor type 3 and small G-protein RAC1. Biol Reprod 84:604–612Bensinger SJ, Bradley MN, Joseph SB et al (2008) LXR signaling couples sterol metabolism toproliferation in the acquired immune response. Cell 134:97–111Bodzioch M, Orso E, Klucken J et al (1999) The gene encoding ATP-binding cassette transporter1 is mutated in Tangier disease. Nat Genet 22:347–351Brooks-Wilson A, Marcil M, Clee SM et al (1999) Mutations in ABC1 in Tangier disease andfamilial high-density lipoprotein deficiency. Nat Genet 22:336–345Cai L, Wang Z, Meyer JM et al (2012) Macrophage SR-BI regulates LPS-inducedpro-inflammatory signaling in mice and isolated macrophages. J Lipid Res 53:1472–1481Cavelier C, Ohnsorg PM, Rohrer L et al (2012) The β-chain of cell surface F(0)F(1) ATPasemodulates apoA-I and HDL transcytosis through aortic endothelial cells. Arterioscler ThrombVasc Biol 32:131–139Signal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 249Chrisman TD, Perkins DT, Garbers DL (2003) Identification of a potent serum factor that causesdesensitization of the receptor for C-Type natriuretic peptide. Cell Commun Signal 1:4Christoffersen C, Obinata H, Kumaraswamy SB et al (2011) Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M. Proc Natl Acad Sci USA108:9613–9618Chroni A, Liu T, Fitzgerald ML, Freeman MW et al (2004) Cross-linking and lipid effluxproperties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1.Biochemistry 43:2126–2139Dahm F, Nocito A, Bielawska A et al (2006) Distribution and dynamic changes of sphingolipids inblood in response to platelet activation. J Thromb Haemost 4:2704–2709Damirin A, Tomura H, Komachi M et al (2007) Role of lipoprotein-associated lysophospholipidsin migratory activity of coronary artery smooth muscle cells. Am J Physiol Heart Circ Physiol292:H2513–H2522Danilo C, Gutierrez-Pajares JL et al (2013) Scavenger receptor class B type I regulates cellularcholesterol metabolism and cell signaling associated with breast cancer development. BreastCancer Res 15:R87Deeg MA, Bowen RF, Oram JF et al (1997) High density lipoproteins stimulate mitogen-activatedprotein kinases in human skin fibroblasts. Arterioscler Thromb Vasc Biol 17:1667–1674Drobnik W, Borsukova H, Böttcher A et al (2002) Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains. Traffic3:268–278Dueñas AI, Aceves M, Fernández-Pisonero I et al (2008) Selective attenuation of Toll-likereceptor 2 signalling may explain the atheroprotective effect of sphingosine 1-phosphate.Cardiovasc Res 79:537–544Fabre AC, Vantourout P, Champagne E et al (2006) Cell surface adenylate kinase activityregulates the F(1)-ATPase/P2Y (13)-mediated HDL endocytosis pathway on humanhepatocytes. Cell Mol Life Sci 63:2829–2837Feng H, Guo L, Wang D et al (2011) Deficiency of scavenger receptor BI leads to impairedlymphocyte homeostasis and autoimmune disorders in mice. Arterioscler Thromb Vasc Biol31:2543–2551Fitzgerald ML, Morris AL, Rhee JS et al (2002) Naturally occurring mutations in the largestextracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I. J BiolChem 277:33178–33187Fitzgerald ML, Morris AL, Chroni A et al (2004) ABCA1 and amphipathic apolipoproteins formhigh-affinity molecular complexes required for cholesterol efflux. J Lipid Res 45:287–294Francis GA, Knopp RH, Oram JF (1995) Defective removal of cellular cholesterol andphospholipids by apolipoprotein A-I in Tangier disease. J Clin Invest 96:78–87Frias MA, James RW, Gerber-Wicht C et al (2009) Native and reconstituted HDL activate Stat3 inventricular cardiomyocytes via ERK1/2: role of sphingosine-1-phosphate. Cardiovasc Res82:313–323Frias MA, Lang U, Gerber-Wicht C et al (2010) Native and reconstituted HDL protectcardiomyocytes from doxorubicin-induced apoptosis. Cardiovasc Res 85:118–126Glomset JA (1968) The plasma lecithin: cholesterol acyltransferase reaction. J Lipid Res 9:155–167Gong M, Wilson M, Kelly T et al (2003) HDL-associated estradiol stimulates endothelial NOsynthase and vasodilation in an SR-BI-dependent manner. J Clin Invest 111:1579–1587González-Dı́ez M, Rodrı́guez C, Badimon L et al (2008) Prostacyclin induction by high-densitylipoprotein (HDL) in vascular smooth muscle cells depends on sphingosine 1-phosphatereceptors: effect of simvastatin. Thromb Haemost 100:119–126Graham DL, Oram JF (1987) Identification and characterization of a high density lipoprotein-binding protein in cell membranes by ligand blotting. J Biol Chem 262:7439–7442Guo L, Song Z, Li M, Li XA et al (2009) Scavenger receptor BI protects against septic deaththrough Its role in modulating inflammatory response. J Biol Chem 284:19826–19834250 J.-R. NoferHaidar B, Mott S, Boucher B et al (2001) Cellular cholesterol efflux is modulated by phospholipid-derived signaling molecules in familial HDL deficiency/Tangier disease fibroblasts. J LipidRes 42:249–257Haidar B, Denis M, Krimbou L et al (2002) cAMP induces ABCA1 phosphorylation activity andpromotes cholesterol efflux from fibroblasts. J Lipid Res 43:2087–2094Haidar B, Denis M, Marcil M et al (2004) Apolipoprotein A-I activates cellular cAMP signalingthrough the ABCA1 transporter. J Biol Chem 279:9963–9969Hänel P, Andréani P, Gräler MH (2007) Erythrocytes store and release sphingosine 1-phosphate inblood. FASEB J 21:1202–1209Hirano K, Matsuura F, Tsukamoto K et al (2000) Decreased expression of a member of the RhoGTPase family, Cdc42Hs, in cells from Tangier disease – the small G protein may play a role incholesterol efflux. FEBS Lett 484:275–279Hoekstra M, Van Berkel TJ, Van Eck M (2010) Scavenger receptor BI: a multi-purpose player incholesterol and steroid metabolism. World J Gastroenterol 16:5916–5924Holzer M, Birner-Gruenberger R, Stojakovic T et al (2011) Uremia alters HDL composition andfunction. J Am Soc Nephrol 22:1631–1641Howard AD, Verghese PB, Arrese EL et al (2011) The β-subunit of ATP synthase is involved incellular uptake and resecretion of apoA-I but does not control apoA-I-induced lipid efflux inadipocytes. Mol Cell Biochem 348:155–164Hu YW, Ma X, Li XX et al (2009) Eicosapentaenoic acid reduces ABCA1 serine phosphorylationand impairs ABCA1-dependent cholesterol efflux through cyclic AMP/protein kinase Asignaling pathway in THP-1 macrophage-derived foam cells. Atherosclerosis 204:e35–e43Igarashi J, Miyoshi M, Hashimoto T et al (2007) Statins induce S1P1 receptors and enhanceendothelial nitric oxide production in response to high-density lipoproteins. Br J Pharmacol150:470–479Imachi H, Murao K, Cao W et al (2003) Expression of human scavenger receptor B1 on and inhuman platelets. Arterioscler Thromb Vasc Biol 23:898–904Iwamoto N, Lu R, Tanaka N, Abe-Dohmae S et al (2010) Calmodulin interacts with ATP bindingcassette transporter A1 to protect from calpain-mediated degradation and upregulates high-density lipoprotein generation. Arterioscler Thromb Vasc Biol 30:1446–1452Karuna R, Park R, Othman A et al (2011) Plasma levels of sphingosine-1-phosphate and apolipo-protein M in patients with monogenic disorders of HDL metabolism. Atherosclerosis 219:855–863Karwatsky J, Ma L, Dong F et al (2010) Cholesterol efflux to apoA-I in ABCA1-expressing cells isregulated by Ca2 + -dependent calcineurin signaling. J Lipid Res 51:1144–1156Kimura T, Sato K, Kuwabara A et al (2001) Sphingosine 1-phosphate may be a major componentof plasma lipoproteins responsible for the cytoprotective actions in human umbilical veinendothelial cells. J Biol Chem 276:31780–31785Kimura T, Tomura H, Mogi C et al (2006) Role of scavenger receptor class B type I andsphingosine 1-phosphate receptors in high density lipoprotein-induced inhibition of adhesionmolecule expression in endothelial cells. J Biol Chem 281:37457–37467Kimura T, Mogi C, Tomura H et al (2008) Induction of scavenger receptor class B type I is criticalfor simvastatin enhancement of high-density lipoprotein-induced anti-inflammatory actions inendothelial cells. J Immunol 181:7332–7340Kimura T, Tomura H, Sato K et al (2010) Mechanism and role of high density lipoprotein-inducedactivation of AMP-activated protein kinase in endothelial cells. J Biol Chem 285:4387–4397Koseki M, Hirano K, Masuda D et al (2007) Increased lipid rafts and acceleratedlipopolysaccharide-induced tumor necrosis factor-alpha secretion in Abca1-deficientmacrophages. J Lipid Res 48:299–306Krishna SM, Seto SW, Moxon JV et al (2012) Fenofibrate increases high-density lipoprotein andsphingosine 1 phosphate concentrations limiting abdominal aortic aneurysm progression in amouse model. Am J Pathol 181:706–718Signal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 251Landry YD, Denis M, Nandi S et al (2006) ATP-binding cassette transporter A1 expressiondisrupts raft membrane microdomains through its ATPase-related functions. J Biol Chem281:36091–36101Lee MH, Hammad SM, Semler AJ et al (2010) HDL3, but not HDL2, stimulates plasminogenactivator inhibitor-1 release from adipocytes: the role of sphingosine-1-phosphate. J Lipid Res51:2619–2628Levkau B (2013) Cardiovascular effects of sphingosine-1-phosphate (S1P). Handb Exp Pharmacol216:147–170Li Q, Yokoyama S (1995) Independent regulation of cholesterol incorporation into freeapolipoprotein-mediated cellular lipid efflux in rat vascular smooth muscle cells. J BiolChem 270:26216–26223Li Q, Tsujita M, Yokoyama S (1997) Selective down-regulation by protein kinase C inhibitors ofapolipoprotein-mediated cellular cholesterol efflux in macrophages. Biochemistry 36:12045–12052Liu D, Ji L, Tong X et al (2011) Human apolipoprotein A-I induces cyclooxygenase-2 expressionand prostaglandin I-2 release in endothelial cells through ATP-binding cassette transporter A1.Am J Physiol Cell Physiol 301:C739–C748Liu M, Seo J, Allegood J, Bi X et al (2013a) Hepatic ApoM overexpression stimulates formation oflarger, ApoM/S1P-enriched plasma HDL. J Biol Chem 289:2801–2814Liu XY, Lu Q, Ouyang XP et al (2013b) Apelin-13 increases expression of ATP-binding cassettetransporter A1 via activating protein kinase C α signaling in THP-1 macrophage-derived foamcells. Atherosclerosis 226:398–407Ma L, Dong F, Denis M et al (2011) Ht31, a protein kinase A anchoring inhibitor, induces robustcholesterol efflux and reverses macrophage foam cell formation through ATP-binding cassettetransporter A1. J Biol Chem 286:3370–3378Maceyka M, Harikumar KB, Milstien S et al (2012) Sphingosine-1-phosphate signaling and itsrole in disease. Trends Cell Biol 22:50–60Malaval C, Laffargue M, Barbaras R et al (2009) RhoA/ROCK I signalling downstream of theP2Y13 ADP-receptor controls HDL endocytosis in human hepatocytes. Cell Signal 21:120–127Malchinkhuu E, Sato K, Muraki T et al (2003) Assessment of the role of sphingosine 1-phosphateand its receptors in high-density lipoprotein-induced stimulation of astroglial cell function.Biochem J 370:817–827Martinez LO, Agerholm-Larsen B, Wang N et al (2003a) Phosphorylation of a pest sequence inABCA1 promotes calpain degradation and is reversed by ApoA-I. J Biol Chem 278:37368–37374Martinez LO, Jacquet S, Esteve JP et al (2003b) Ectopic beta-chain of ATP synthase is anapolipoprotein A-I receptor in hepatic HDL endocytosis. Nature 421:75–79Matsuo Y, Miura S, Kawamura A et al (2007) Newly developed reconstituted high-densitylipoprotein containing sphingosine-1-phosphate induces endothelial tube formation. Athero-sclerosis 194:159–168McNeal CJ, Chatterjee S, Hou J et al (2013) Human HDL containing a novel apoC-I isoforminduces smooth muscle cell apoptosis. Cardiovasc Res 98:83–93Mendez AJ, Oram JF, Bierman EL (1991) Protein kinase C as a mediator of high densitylipoprotein receptor-dependent efflux of intracellular cholesterol. J Biol Chem 266:10104–10111Mineo C, Shaul PW (2012) Functions of scavenger receptor class B, type I in atherosclerosis. CurrOpin Lipidol 23:487–493Mineo C, Yuhanna IS, Quon MJ et al (2003) High density lipoprotein induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J Biol Chem 278:9142–9149Miura S, Fujino M, Matsuo Y et al (2003) High density lipoprotein-induced angiogenesis requiresthe activation of Ras/MAP kinase in human coronary artery endothelial cells. ArteriosclerThromb Vasc Biol 23:802–808252 J.-R. NoferMogilenko DA, Orlov SV, Trulioff AS et al (2012) Endogenous apolipoprotein A-I stabilizesATP-binding cassette transporter A1 and modulates Toll-like receptor 4 signaling in humanmacrophages. FASEB J 26:2019–2030Morel S, Frias MA, Rosker C et al (2012) The natural cardioprotective particle HDL modulatesconnexin43 gap junction channels. Cardiovasc Res 93:41–49Murphy AJ, Woollard KJ, Hoang A et al (2008) High-density lipoprotein reduces the humanmonocyte inflammatory response. Arterioscler Thromb Vasc Biol 28:2071–2077Murphy AJ, Woollard KJ, Suhartoyo A et al (2011) Neutrophil activation is attenuated by high-density lipoprotein and apolipoprotein A-I in in vitro and in vivo models of inflammation.Arterioscler Thromb Vasc Biol 31:1333–1341Nofer JR, van Eck M (2011) HDL scavenger receptor class B type I and platelet function. CurrOpin Lipidol 22:277–282Nofer J-R, Tepel M, Kehrel B et al (1996) High density lipoproteins enhance the Na+/H+ antiportin human platelets. Thromb Haemost 75:635–641Nofer JR, Walter M, Kehrel B et al (1998) HDL3-mediated inhibition of thrombin-induced plateletaggregation and fibrinogen binding occurs via decreased production of phosphoinositide-derived second messengers 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. ArteriosclerThromb Vasc Biol 18:861–869Nofer JR, Fobker M, Höbbel G et al (2000) Activation of phosphatidylinositol-specific phospholi-pase C by HDL-associated lysosphingolipid. Involvement in mitogenesis but not in cholesterolefflux. Biochemistry 39:15199–15207Nofer JR, Junker R, Pulawski E et al (2001a) High density lipoproteins induce cell cycle entry invascular smooth muscle cells via mitogen activated protein kinase-dependent pathway.Thromb Haemost 85:730–735Nofer JR, Levkau B, Wolinska I et al (2001b) Suppression of endothelial cell apoptosis by highdensity lipoproteins (HDL) and HDL-associated lysosphingolipids. J Biol Chem 276:34480–34485Nofer JR, Feuerborn R, Levkau B et al (2003) Involvement of Cdc42 signaling in apoA-I-inducedcholesterol efflux. J Biol Chem 278:53055–53062Nofer JR, van der Giet M, Tölle M et al (2004) HDL induces NO-dependent vasorelaxation via thelysophospholipid receptor S1P3. J Clin Invest 113:569–581Nofer JR, Remaley AT, Feuerborn R et al (2006) Apolipoprotein A-I activates Cdc42 signalingthrough the ABCA1 transporter. J Lipid Res 47:794–803Nofer JR, Brodde M, Korporaal SJ et al (2011) Native high density lipoproteins inhibit thrombin-induced platelet activation via scavenger receptor B1 (SR-B1). Atherosclerosis 215:374–382Norata GD, Callegari E, Marchesi M et al (2005) High-density lipoproteins induce transforminggrowth factor-beta2 expression in endothelial cells. Circulation 111:2805–2811Norata GD, Marchesi P, Pirillo A et al (2008) Long pentraxin 3, a key component of innateimmunity, is modulated by high density lipoproteins in endothelial cells. Arterioscler ThrombVasc Biol 28:925–931Paatela H, Vihma V, Jauhiainen M et al (2011) Dehydroepiandrosterone fatty acyl esters in highdensity lipoprotein: interaction with human vascular endothelial cells and vascular responsesex vivo. Steroids 76:376–380Radojkovic C, Genoux A, Pons V et al (2009) Stimulation of cell surface F1-ATPase activity byapolipoprotein A-I inhibits endothelial cell apoptosis and promotes proliferation. ArteriosclerThromb Vasc Biol 29:1125–1130Riwanto M, Rohrer L, Roschitzki B et al (2013) Altered activation of endothelial anti- andproapoptotic pathways by high-density lipoprotein from patients with coronary artery disease:role of high-density lipoprotein-proteome remodeling. Circulation 127:891–904Rust S, Rosier M, Funke H et al (1999) Tangier disease is caused by mutations in the geneencoding ATP-binding cassette transporter 1. Nat Genet 22:352–355Sachinidis A, Kettenhofen R, Seewald S et al (1999) Evidence that lipoproteins are carriers ofbioactive factors. Arterioscler Thromb Vasc Biol 19:2412–2421Signal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 253Saddar S, Carriere V, Lee WR et al (2013) Scavenger receptor class B type I is a plasma membranecholesterol sensor. Circ Res 112:140–151Sano O, Kobayashi A, Nagao K et al (2007) Sphingomyelin-dependence of cholesterol effluxmediated by ABCG1. J Lipid Res 48:2377–2384Sato K, Malchinkhuu E, Horiuchi Y et al (2007) HDL-like lipoproteins in cerebrospinal fluidaffect neural cell activity through lipoprotein-associated sphingosine 1-phosphate. BiochemBiophys Res Commun 359:649–654Sattler KJ, Elbasan S, Keul P et al (2010) Sphingosine 1-phosphate levels in plasma and HDL arealtered in coronary artery disease. Basic Res Cardiol 105:821–832See RH, Caday-Malcolm RA, Singaraja RR et al (2002) Protein kinase A site-specific phosphory-lation regulates ATP-binding cassette A1 (ABCA1)-mediated phospholipid efflux. J BiolChem 277:41835–41842Seetharam D, Mineo C, Gormley AK et al (2006) High-density lipoprotein promotes endothelialcell migration and reendothelialization via scavenger receptor-B type I. Circ Res 98:63–72Sekine Y, Suzuki K, Remaley AT (2011) HDL and sphingosine-1-phosphate activate stat3 inprostate cancer DU145 cells via ERK1/2 and S1P receptors, and promote cell migration andinvasion. Prostate 71:690–699Slotte JP, Oram JF, Bierman EL (1987) Binding of high density lipoproteins to cell receptorspromotes translocation of cholesterol from intracellular membranes to the cell surface. J BiolChem 262:12904–12907Speer T, Rohrer L, Blyszczuk P et al (2013) Abnormal high-density lipoprotein induces endothe-lial dysfunction via activation of toll-like receptor-2. Immunity 38:754–768Sun Y, Ishibashi M, Seimon T et al (2009) Free cholesterol accumulation in macrophagemembranes activates Toll-like receptors and p38 mitogen-activated protein kinase and inducescathepsin K. Circ Res 104:455–465Tamama K, Tomura H, Sato K et al (2005) High density lipoprotein inhibits migration of vascularsmooth muscle cells through its sphingosine 1-phosphate component. Atherosclerosis 178:19–23Tan JT, Prosser HC, Vanags LZ et al (2013) High-density lipoproteins augment hypoxia-inducedangiogenesis via regulation of post-translational modulation of hypoxia-inducible factor 1α.FASEB J 28:206–217Tang C, Vaughan AM, Oram JF (2004) Janus kinase 2 modulates the apolipoprotein interactionswith ABCA1 required for removing cellular cholesterol. J Biol Chem 279:7622–7628Tang C, Vaughan AM, Anantharamaiah GM et al (2006) Janus kinase 2 modulates the lipid-removing but not protein-stabilizing interactions of amphipathic helices with ABCA1. J LipidRes 47:107–114Tang C, Liu Y, Kessler PS, Vaughan AM, Oram JF (2009) The macrophage cholesterol exporterABCA1 functions as an anti-inflammatory receptor. J Biol Chem 284:32336–32343Tao R, Hoover HE, Honbo N et al (2010) High-density lipoprotein determines adult mousecardiomyocyte fate after hypoxia-reoxygenation through lipoprotein-associated sphingosine1-phosphate. Am J Physiol Heart Circ Physiol 298:H1022–H1028Tatematsu S, Francis SA, Natarajan P et al (2013) Endothelial lipase is a critical determinant ofhigh-density lipoprotein-stimulated sphingosine 1-phosphate-dependent signaling in vascularendothelium. Arterioscler Thromb Vasc Biol 33:1788–1794Tölle M, Pawlak A, Schuchardt M et al (2008) HDL-associated lysosphingolipids inhibit NAD(P)H oxidase-dependent monocyte chemoattractant protein-1 production. Arterioscler ThrombVasc Biol 28:1542–1548Tölle M, Huang T, Schuchardt M et al (2012) High-density lipoprotein loses its anti-inflammatorycapacity by accumulation of pro-inflammatory-serum amyloid A. Cardiovasc Res 94:154–162Tsukamoto K, Hirano K, Tsujii K et al (2001) ATP-binding cassette transporter-1 inducesrearrangement of actin cytoskeletons possibly through Cdc42/N-WASP. Biochem BiophysRes Commun 287:757–765254 J.-R. NoferTsukamoto K, Hirano K, Yamashita S et al (2002) Retarded intracellular lipid transport associatedwith reduced expression of Cdc42, a member of Rho-GTPases, in human aged skin fibroblasts:a possible function of Cdc42 in mediating intracellular lipid transport. Arterioscler ThrombVasc Biol 22:1899–1904Umemoto T, Han CY, Mitra P et al (2013) Apolipoprotein AI and high-density lipoprotein haveanti-inflammatory effects on adipocytes via cholesterol transporters: ATP-binding cassetteA-1, ATP-binding cassette G-1, and scavenger receptor B-1. Circ Res 112:1345–1354Vaughan AM, Tang C, Oram JF (2009) ABCA1 mutants reveal an interdependency between lipidexport function, apoA-I binding activity, and Janus kinase 2 activation. J Lipid Res 50:285–292von Otte S, Paletta JR, Becker S et al (2006) Follicular fluid high density lipoprotein-associatedsphingosine 1-phosphate is a novel mediator of ovarian angiogenesis. J Biol Chem 281:5398–5405Walter M, Reinecke H, Nofer JR et al (1995) HDL3 stimulates multiple signaling pathways inhuman skin fibroblasts. Arterioscler Thromb Vasc Biol 15:1975–1986Walter M, Reinecke H, Gerdes U et al (1996) Defective regulation of phosphatidylcholine-specificphospholipases C and D in a kindred with Tangier disease. Evidence for the involvement ofphosphatidylcholine breakdown in HDL-mediated cholesterol efflux mechanisms. J ClinInvest 98:2315–2323Wang N, Silver DL, Costet P, Tall AR (2000) Specific binding of ApoA-I, enhanced cholesterolefflux, and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem275:33053–33058Wang N, Chen W, Linsel-Nitschke P et al (2003) A PEST sequence in ABCA1 regulatesdegradation by calpain protease and stabilization of ABCA1 by apoA-I. J Clin Invest111:99–107Wang N, Lan D, Chen W et al (2004) ATP-binding cassette transporters G1 and G4 mediatecellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci USA 101:9774–9779Wang N, Ranalletta M, Matsuura F et al (2006) LXR-induced redistribution of ABCG1 to plasmamembrane in macrophages enhances cholesterol mass efflux to HDL. Arterioscler ThrombVasc Biol 26:1310–1316Weichhart T, Kopecky C, Kubicek M et al (2012) Serum amyloid A in uremic HDL promotesinflammation. J Am Soc Nephrol 23:934–947Westerterp M, Gourion-Arsiquaud S, Murphy AJ et al (2012) Regulation of hematopoietic stemand progenitor cell mobilization by cholesterol efflux pathways. Cell Stem Cell 11:195–206Wilhelm AJ, Zabalawi M, Grayson JM et al (2009) Apolipoprotein A-I and its role in lymphocytecholesterol homeostasis and autoimmunity. Arterioscler Thromb Vasc Biol 29:843–849Wilkerson BA, Grass GD, Wing SB et al (2012) Sphingosine 1-phosphate (S1P) carrier-dependentregulation of endothelial barrier: high density lipoprotein (HDL)-S1P prolongs endothelialbarrier enhancement as compared with albumin-S1P via effects on levels, trafficking, andsignaling of S1P1. J Biol Chem 287:44645–44653Xu J, Qian J, Xie X et al (2012) High density lipoprotein cholesterol promotes the proliferation ofbone-derived mesenchymal stem cells via binding scavenger receptor-B type I and activationof PI3K/Akt, MAPK/ERK1/2 pathways. Mol Cell Biochem 371:55–64Yamauchi Y, Hayashi M, Abe-Dohmae S et al (2003) Apolipoprotein A-I activates protein kinaseC alpha signaling to phosphorylate and stabilize ATP binding cassette transporter A1 for thehigh density lipoprotein assembly. J Biol Chem 278:47890–47897Yamauchi Y, Chang CC, Hayashi M et al (2004) Intracellular cholesterol mobilization involved inthe ABCA1/apolipoprotein-mediated assembly of high density lipoprotein in fibroblasts. JLipid Res 45:1943–1951Yin K, Deng X, Mo ZC et al (2011) Tristetraprolin-dependent post-transcriptional regulation ofinflammatory cytokine mRNA expression by apolipoprotein A-I: role of ATP-binding mem-brane cassette transporter A1 and signal transducer and activator of transcription 3. J BiolChem 286:13834–13845Signal Transduction by HDL: Agonists, Receptors, and Signaling Cascades 255Yin K, Chen WJ, Zhou ZG et al (2012) Apolipoprotein A-I inhibits CD40 proinflammatorysignaling via ATP-binding cassette transporter A1-mediated modulation of lipid raft inmacrophages. J Atheroscler Thromb 19:823–836Yuhanna IS, Zhu Y, Cox BE et al (2001) High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase. Nat Med 7:853–857Yvan-Charvet L, Welch C, Pagler TA et al (2008) Increased inflammatory gene expression inABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling viatoll-like receptors, and neutrophil infiltration of atherosclerotic lesions. Circulation 118:1837–1847Yvan-Charvet L, Pagler TA, Seimon TA et al (2010a) ABCA1 and ABCG1 protect againstoxidative stress-induced macrophage apoptosis during efferocytosis. Circ Res 106:1861–1869Yvan-Charvet L, Pagler T, Gautier EL et al (2010b) ATP-binding cassette transporters and HDLsuppress hematopoietic stem cell proliferation. Science 328:1689–1693Zhang Z, Hirano K, Tsukamoto K et al (2005) Defective cholesterol efflux in Werner syndromefibroblasts and its phenotypic correction by Cdc42, a RhoGTPase. Exp Gerontol 40:286–294Zhang Q, Zhang Y, Feng H et al (2011) High density lipoprotein (HDL) promotes glucose uptakein adipocytes and glycogen synthesis in muscle cells. PLoS ONE 6:e23556Zhang QH, Zu XY, Cao RX et al (2012) An involvement of SR-B1 mediated PI3K-Akt-eNOSsignaling in HDL-induced cyclooxygenase 2 expression and prostacyclin production in endo-thelial cells. Biochem Biophys Res Commun 420:17–23Zhu X, Lee JY, Timmins JM et al (2008a) Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages. J BiolChem 283:22930–22941Zhu W, Saddar S, Seetharam D et al (2008b) The scavenger receptor class B type I adaptor proteinPDZK1 maintains endothelial monolayer integrity. Circ Res 102:480–487256 J.-R. NoferPart IIPathology of HDLEpidemiology: Disease AssociationsandModulators of HDL-Related BiomarkersMarkku J. SavolainenContents1 Protective Role of HDL: Evidence from Epidemiological Studies . . . . . . . . . . . . . . . . . . . . . 2612 HDL Cholesterol as a Risk Factor for Atherosclerosis and Its Complications . . . . . . . . . 2623 HDL Cholesterol as a Risk Factor for Other Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2624 Total HDL-C in Various Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2635 Total HDL-C Modulated by Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2646 HDL-C in Diseases and Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2667 High HDL Levels Do Not Add to the Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2678 Effect of HDL on Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2679 Time Trends in Total HDL-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26810 Are There Other Biomarkers than the Total HDL-C? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26811 HDL Fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26812 HDL Particle Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26913 HDL Particle Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26914 HDL Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26915 HDL Apolipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27016 HDL Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27017 HDL Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27118 Pleiotropy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27219 Future Approaches of Epidemiological Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274AbstractEpidemiological studies have shown an inverse association between high-density lipoprotein cholesterol (HDL-C) levels and risk of ischemic heart dis-ease. In addition, a low level of HDL-C has been shown to be a risk factor forother diseases not related to atherosclerosis. However, recent studies have notM.J. Savolainen (*)Department of Internal Medicine, Institute of Clinical Medicine, University of Oulu, Kajaanintie50, P.O.Box 5000, 90014 Oulu, Finlande-mail: markku.savolainen@oulu.fi# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_7259supported a causal effect of HDL-C in the development of atherosclerosis.Furthermore, new drugs markedly elevating HDL-C levels have been disap-pointing with respect to clinical endpoints. Earlier, most studies have focusedalmost exclusively on the total HDL-C without regard to the chemical composi-tion or multiple subclasses of HDL particles. Recently, there have been efforts todissect the HDL fraction into as many well-defined subfractions and individualmolecules of HDL particles as possible. On the other hand, the focus is shiftingfrom the structure and composition to the function of HDL particles. Biomarkersand mechanisms that could potentially explain the beneficial characteristics ofHDL particles unrelated to their cholesterol content have been sought withsophisticated methods such as proteomics, lipidomics, metabonomics, and func-tion studies including efflux capacity. These new approaches have been used inorder to resolve the complex effects of diseases, conditions, environmentalfactors, and genes in relation to the protective role of HDL but high-throughputmethods are still needed for large-scale epidemiological studies.KeywordsHigh-density lipoproteins • Cholesterol • Atherosclerosis • Coronary heartdisease • Apolipoproteins • Cholesterol efflux • Diabetes • Obesity • Cancer •Proteomics • Lipidomics • MetabonomicsAbbreviationsApoA-I Apolipoprotein A-IApoA-II Apolipoprotein A-IIApoC-III Apolipoprotein C-IIICHD Coronary heart diseaseCETP Cholesteryl ester transfer proteinCVD Cardiovascular diseaseEPA Eicosapentaenoic acidDHA Docosahexaenoic acidGI Glycemic indexGL Glycemic loadHDL High-density lipoproteinsHDL-C High-density lipoprotein cholesterolHDL2 High-density lipoprotein fraction 2LDL Low-density lipoproteinLDL-C Low-density lipoprotein cholesterolNMR Nuclear magnetic resonancePON1 Paraoxonase 1S1P Sphingosine-1-phosphateT2DM Type 2 diabetes mellitus260 M.J. SavolainenLow levels of high-density lipoprotein (HDL) particles in the plasma of patientswith coronary heart disease (CHD) were observed already in the early 1950s (Barret al. 1951; Nikkilä 1953). At that time the lipoprotein fraction was called alphalipoprotein as electrophoresis methods were used. Twenty years later the samerelationship was confirmed in large epidemiological studies using ultracentrifuga-tion or precipitation methods. The new interest for HDL research was thenstimulated by the reverse cholesterol transport hypothesis (Glomset et al. 1966)and a Lancet review written by Miller and Miller (1975). These authors proposedthat low plasma HDL concentration accelerates the development of atherosclerosisby impairing the clearance of cholesterol from the arterial wall.1 Protective Role of HDL: Evidence from EpidemiologicalStudiesThe protective role of HDL has been well established in several epidemiologicalstudies. An increment of 2.5 % corresponding to 1 mg/dl or 0.04 mmol/l isassociated with a 2 and 3 % reduction in CHD risk in men and women, respectively(Gordon et al. 1989; Jacobs et al. 1990). So far, it is mainly the total cholesterolconcentration in HDL particles that has been determined using standardizedmethods applicable also to routine clinical work. However, cholesterol representsonly a small fraction, approximately 15 %, of the HDL particle mass. Furthermore,the proportion of free cholesterol to esterified cholesterol varies between lipopro-tein particles.The relationship between HDL-C and CHD is complex and HDL-C may not bean appropriate indicator of the impact of this lipoprotein fraction on cardiovascularrisk. This has been demonstrated, e.g., by the fact that carotid intima mediathickness is not increased in apoA-I(Milano) mutation carriers with very low levelsof HDL-C (Sirtori et al. 2001). Moreover, recently, drugs that increase HDLcholesterol (HDL-C) level have failed to reduce cardiovascular risk, and Mendelianrandomization studies have failed to show a causal relationship between HDL-Cand cardiovascular diseases (van Capelleveen et al. 2013).Despite this controversy, the hard evidence for low HDL-C level as a risk factorof atherosclerosis will be described in the first part of this presentation. It isnoteworthy that the cholesterol concentration in HDL fraction is not necessarilyassociated with the antiatherogenic properties of HDL. Therefore, later in thischapter, other potential HDL-related biomarkers for the prevention of atheroscle-rosis by HDL will be presented. These include not only the other major lipid andapolipoprotein components of HDL but also minor bioactive lipid moleculesresiding in the HDL particles. Furthermore, the physicochemical characteristicsof various subfractions of HDL as well as the large number of molecules circulatingmore or less firmly bound to HDL particles may contribute to the antiatherogenicpotential of HDL via antioxidative and anti-inflammatory effects or cholesteroltransport capacity.Epidemiology: Disease Associations and Modulators of HDL-Related Biomarkers 2612 HDL Cholesterol as a Risk Factor for Atherosclerosisand Its ComplicationsUntil recently, the clinical evaluation of HDL as a risk factor has focused almostexclusively on the total HDL-C without regard to the chemical composition ormultiple subclasses of HDL particles. A large body of epidemiological research hasshown a solid inverse and independent relationship between HDL-C and the risk ofcardiovascular disease (Toth et al. 2013).Gofman et al. first reported an inverse association between HDL-C levels and therisk of ischemic heart disease (Gofman et al. 1966). This was shown in case–controlstudies from Framingham and Livermore cohorts with 10–12 years of follow-up.Later, the inverse relationship has been observed also in several larger studies in theUSA (the Honolulu Heart Program, Rhoads et al. 1976, and the Framingham HeartStudy, Gordon et al. 1977), in Norway (the Tromsø Heart Study, Miller et al. 1977),in Germany (the Prospective Cardiovascular Münster Study, Assmann et al. 1996),and in Israel (the Israeli Ischemic Heart Disease Study, Goldbourt et al. 1997).Recent meta-analyses have corroborated the relationship between HDL-C andatherosclerosis and its complications (Chirovsky et al. 2009; Boekholdtet al. 2013; Touboul et al. 2014). The association is independent of triglyceridelevels and other risk factors (Goldbourt et al. 1997). Many CHD risk algorithmshave included HDL-C as a factor to improve the prediction of CHD events (Cooperet al. 2005; Halcox et al. 2013; Hippisley-Cox et al. 2013; Tehrani et al. 2013).Recently, the picture has become less clear. Mendelian randomization studieshave not supported a causal effect of HDL-C in the atherosclerotic disease process(Voight et al. 2012; Holmes et al. 2014). Moreover, statin trials have shown thatHDL-C is predictive among patients treated with statin even at low LDL levels(Barter et al. 2007), whereas it is not predictive among patients taking placebo(Ridker et al. 2010; Mora et al. 2012). Further research is needed to clarify the roleof HDL-C as a risk factor. It is possible that other characteristics of HDL particlesmay be more important in this respect than the total cholesterol concentration.In patients with CHD, the protective role of HDL-C is controversial (Silbernagelet al. 2013). Some studies have shown that low HDL-C is associated with athero-sclerotic progression in myocardial infarction survivors (Johansson et al. 1991;Duffy et al. 2012; Liosis et al. 2013), provides additional prognostic value also inpatients with acute coronary syndrome (Correia et al. 2009), and reduces the riskafter coronary interventions (Sattler et al. 2009), whereas some studies have shownthat HDL-C has no protective role in the secondary prevention of CHD after bypassoperation (Angeloni et al. 2013).3 HDL Cholesterol as a Risk Factor for Other DiseasesThe low level of HDL-C has been shown to be a risk factor for other diseases notrelated to atherosclerosis. Recent reports have shown that HDL-C may be abiomarker for diseases like psoriasis (Holzer et al. 2012), rheumatoid arthritis262 M.J. Savolainen(Raterman et al. 2013), and liver fibrosis in hepatitis C patients (Gangadharanet al. 2012). Alterations in HDL composition have been observed also in hemodial-ysis patients (Mangé et al. 2012).Special interest has been recently focused on HDL in cancer patients. Severalepidemiological studies have shown that low HDL-C level may be a risk and/orprognostic factor of biliary tract cancers (Andreotti et al. 2008), prostate cancer(Mondul et al. 2011; Kotani et al. 2013), colon cancer (van Duijnhoven et al. 2011),breast cancer (Furberg et al. 2005, Melvin et al. 2012) and gastric cancer (Tamuraet al. 2012), but not that of endometrial cancer (Cust et al. 2007; Espositoet al. 2014) or rectal cancer (van Duijnhoven et al. 2011). Confounding factorsrelated to obesity and metabolic syndrome may be more strongly associated withthe latter cancer types (Kotani et al. 2013).The association with low HDL-C levels is shared among many types of cancer,and it is mainly linked to obesity and inflammation, suggesting a common pathway(Melvin et al. 2013; Vı́lchez et al. 2014). The mechanism of cancer protection is notknown. Lipoprotein particles may carry cancerogenic molecules but at least onesmall study has shown that persistent organogenic pollutants in HDL particles aremore associated with CVD than cancer (Ljunggren et al. 2014).The potential importance of HDL particles in cancer protection has recently ledto attempts to develop cancer treatments by HDL-mimetic synthetic nanoparticles(Zheng et al. 2013; Yang et al. 2013). These interesting approaches need to befollowed closely in the future.4 Total HDL-C in Various PopulationsRacial differences. Several studies have shown differences in HDL-C betweenvarious ethnic groups (Thelle et al. 1982; Heiss et al. 1984; Haffner et al. 1986;Saha 1987). The differences may in part be due to genetic factors but the role ofbehavioral and anthropometric variables seem to affect the HDL-C (Thelleet al. 1982; Haffner et al. 1986). Environmental factors such as diet, smoking,and alcohol use may confound the differences observed between ethnic groups.In the USA, the African-American population has higher HDL-C thanCaucasians after adjustment for weight, smoking, and alcohol consumption thanCaucasians (Heiss et al. 1984), while people originating from India have lowerHDL-C than Europeans or many other populations (Saha 1987; de Munteret al. 2011). Differences in HDL-C and other lipoproteins between various ethnicgroups can be observed already in children (van Vliet et al. 2011). Even at an earlyage, the HDL-C levels are confounded by other cardiovascular risk factors includ-ing blood pressure, overweight, and obesity.Gender differences. Women have higher HDL-C levels than men (Heisset al. 1980; Seidell et al. 1991), and significantly increased CHD risk is defined atlevels below 50 mg/dl (1.29 mmol/l) and 40 mg/dl (1.03 mmol/l), respectively. Thereason for the gender difference may mainly be sex hormones but the fat distribu-tion seems to play a central role since adjustment for waist/thigh ratio almost totallyEpidemiology: Disease Associations and Modulators of HDL-Related Biomarkers 263removed the gender difference in HDL cholesterol (Seidell et al. 1991). In womenHDL-C levels decline after menopause (Heiss et al. 1980) but the sex differenceremains significant even in the seventh decade of life (Ostlund et al. 1990).Age-related differences. Lipoprotein levels are very low at birth and thenincrease during childhood. HDL-C levels decrease in males during puberty andearly adulthood and thereafter remain lower than those in women (Kreisberg andKasim 1987; Walter 2009). HDL-C levels decline with age and low HDL choles-terol remains a powerful risk predictor into old age (Kreisberg and Kasim 1987;Walter 2009). A selection bias by HDL-lowering genetic variation may explainwhy HDL deficiency is rare among very old people (Kervinen et al. 1994; Baggioet al. 1998).5 Total HDL-C Modulated by Environmental FactorsAlcohol consumption in moderation is associated with a reduced risk for cardiovas-cular diseases. Alcohol consumption increases the concentration of HDL-C, possi-bly secondary to an inhibition of CETP (cholesteryl ester transfer protein)(Savolainen et al. 1990; Hannuksela et al. 1992). A common polymorphism inthe CETP gene (TaqIB2) modifies the relationship of alcohol intake with HDL-Csuggesting a gene-environment interaction on the risk of CHD (Jensen et al. 2008).However, it has not been definitely shown whether the HDL-C elevation associatedwith alcohol consumption is cardioprotective.The quantity of alcohol needed to increase HDL-C by 0.1 mmol/l (3.87 mg/dl) isabout 30 g/d (Brien et al. 2011). Therefore, alcohol drinking cannot berecommended as a method to raise low HDL-C levels. The alcohol-inducedincrease in HDL-C occurs commonly without significant changes in other lipidsalthough alcohol may increase triglyceride levels especially in subjects with ele-vated triglyceride concentration.The death rate from CHD among moderate alcohol users is lower than in totalabstainers or heavy drinkers (Rimm et al. 1999; Brien et al. 2011; Bergmannet al. 2013). Even very low alcohol consumption, e.g., a couple of drinks perweek, seems to protect from CHD even though it does not have any significanteffect on HDL-C. To explain this fact, it has been suggested that ethanol metabo-lism may produce specific bioactive lipids, e.g., phosphatidylethanol, that couldserve as a memory molecule in the body (Liisanantti et al. 2004). If HDL particlesof alcohol drinkers contain this bioactive lipid, it could circulate for several dayseven without daily alcohol drinking, and when HDL enters into the endothelial cell,it could then exert its positive effects on the vascular endothelium (Liisanantti andSavolainen 2005). However, the concentration of phosphatidylethanol in HDL maybe in the low nanomolar range which makes the analysis challenging and mayhamper its determination of epidemiological studies.Smoking reduces HDL-C level and smoking cessation is associated with anincrease in the plasma concentration of HDL-C (Maeda et al. 2003). The mecha-nism by which smoking reduces HDL-C is not known. It is noteworthy that in many264 M.J. Savolainencases smoking is associated with alcohol drinking and therefore smoking couldattenuate the alcohol-induced increase in HDL-C.Physical activity is associated with high HDL-C (Marti 1991). Low level ofphysical activity is very common in the developed countries, and therefore, increas-ing the level of exercise might be more beneficial for HDL-C than any otherpreventive measure.Education and socioeconomic status. HDL-C levels increase with income andeducational attainment after controlling diet, exercise, and other risk factors forelevated cholesterol (Muennig et al. 2007). The mechanisms are not clear. It hasbeen suggested that stress differences by social class may play a role (Muenniget al. 2007). However, several lifestyle factors including leisure-time physicalactivity, smoking, alcohol drinking, and dietary habits correlate with the socioeco-nomic status, classified as the degree of educational level (Schröder et al. 2004).Dietary carbohydrates affect the lipoprotein profile. The epidemiological stud-ies can be divided into three categories. First, the type of carbohydrate modulatesthe impact of carbohydrates on plasma lipoproteins. The intake of refinedcarbohydrates has increased in Western societies, and they have more deleteriouseffects on abdominal obesity and consequently on insulin resistance and hepaticlipogenesis in comparison with complex carbohydrates or starches (Ma et al. 2012;Stanhope et al. 2009). The end result of high intake of refined carbohydrates is a lowHDL-C level (Heiss et al. 1980; Sonestedt et al. 2012).Second, trials focusing on dietary carbohydrate restriction have shown modula-tion of atherogenic dyslipidemia. The effect on HDL-C is modest but commonlygreater than that on total cholesterol, and thus, the atherogenic burden is improved.Results from epidemiological studies are difficult to interpret due to differences indiets.The third type of studies involves the replacement of carbohydrate with differentfats in order to maintain isocaloric intake of macronutrients. A classic meta-analysis of 60 trials (Mensink et al. 2003) showed that replacement of 10 % ofenergy from carbohydrate with saturated fat, monounsaturated fat, and polyunsatu-rated fat increased HDL-C by 4.7, 3.4, and 2.8 mg/dl (0.12, 0.09, and 0.07 mmol/l),respectively. However, LDL cholesterol increased by 13 mg/dl (0.34 mmol/l) withsaturated fat substitution and decreased by 3.3 mg/dl (0.09 mmol/l) with polyunsat-urated fat substitution while the substitution with monounsaturated fat had no effecton LDL-C. Thus, the atherogenicity of the plasma lipoprotein profile improved withthe unsaturated fat substitution of carbohydrate.The effects of dietary carbohydrates on HDL-C and CHD risk have beenanalyzed also on the basis of their glycemic index (GI, the effect on blood glucoselevel) or glycemic load (GL, including carbohydrate content and intake of foods inaddition to GI). High GL and GI were associated with significant increased risk ofCVDs, specifically for women. Several cross-sectional studies have reportedinverse associations of low GI and GL diets with HDL-C, but meta-analyses(Kelly et al. 2004; Goff et al. 2013) have not found any effect on HDL-C.Fatty acids and especially omega-3 fatty acids have been the focus of manyepidemiological studies. The most important omega-3 fatty acids in this respect areEpidemiology: Disease Associations and Modulators of HDL-Related Biomarkers 265eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that increaseHDL-C by 1–3 % (Balk et al. 2006).6 HDL-C in Diseases and ConditionsType 2 diabetes. Patients with type 2 diabetes may have various types ofdyslipidemias that usually are accompanied with a low concentration of HDL-C(Chehade et al. 2013; Morgantini et al. 2014). Low HDL-C contributes to diabetes-related CHD risk more in women than in men, the diabetes-related hazard ratio for amajor CHD event being 3 times higher in women after adjustment for othercardiovascular risk factors (Juutilainen et al. 2004). On the other hand, lipidcomposition of HDL particles is associated with the development of metabolicsyndrome (Abbasi et al. 2013; Onat et al. 2013).Dyslipidemias. Traditionally, dyslipidemia has been characterized by an eleva-tion in plasma triglycerides and total or LDL cholesterol and reduction in HDL-C.This is commonly referred to as the atherogenic triad. LDL particles are small anddense (Mooradian 2009). Genetic studies have indicated linkage of apoB gene topeak LDL size and plasma triglycerides, HDL-C, and apoB levels.Obesity. Overweight and obese subjects usually have low HDL-C concentrations(Seidell et al. 1991). The underlying cause may be insulin resistance that promotesfree fatty acid flux to the liver, stimulates hepatic lipogenesis, and finally enhancesthe secretion of triglyceride-rich apoB-containing lipoproteins from the liver. Theexcess triglyceride-rich particles also enhance the CETP-mediated exchange ofcholesteryl esters from HDL to apoB-containing lipoprotein particles and simulta-neous transfer of triglycerides into HDL (Mann et al. 1991; Liinamaa et al. 1997).This may further reduce HDL-C levels.Weight reduction is an important modulator of the lipoprotein profile and long-term weight reduction increases HDL-C levels especially in subjects with type2 diabetes. It is noteworthy that the effect of weight reduction on HDL-C isdifferent in the initial weight loss period compared with the weight maintenancephase. A meta-analysis has shown that HDL-C is increased by 0.35 mg/dl(0.009 mmol/l) per kilogram weight lost during the stable weight reduction. Duringactive weight loss, however, HDL-C is reduced by 0.27 mg/dl (0.007 mmol/l) forevery kilogram of weight lost (Browning et al. 2011). Theoretically, an increase inHDL-C of 2.45 mg/dl (0.06 mmol/l) could be expected during the stable weightmaintenance stage after a weight reduction of 7 % in an individual with an initialweight of 100 kg. Furthermore, this would translate into 7.4 % reduction in CHDrisk in women.266 M.J. Savolainen7 High HDL Levels Do Not Add to the ProtectionWhile low levels of HDL-C are associated with increased CHD risk, high HDL-Clevels are not uniformly atheroprotective. At higher concentrations the curve ofCHD risk gradually tapers off as depicted in Fig. 1 which is based on the resultsfrom the Framingham Heart Study (Gordon et al. 1977), and there is no additionaleffect of higher HDL-C levels compared with average HDL-C concentrations.8 Effect of HDL on StrokeIn contrast to the role of HDL-C as a major risk factor of CHD, the role of HDL-C inthe pathogenesis of ischemic stroke is less clear. Epidemiological studies of carotidintima media thickness and stroke protection have provided conflicting results.Even in the studies reporting positive results, the effect of HDL-C on protectionof stroke is modest (a 10 mg/dl or 0.38 mmol/l increase in HDL-C reduces strokerisk by 11–15 %) compared with its protective effect on CHD. The discordantresults in prospective cohort studies as well as in case-control studies may be due tothe heterogeneity of stroke, since dyslipidemia including low HDL-C levels maynot be involved in the pathogenesis of some subtypes such as lacunar andcardioembolic strokes (Amarenco et al. 2008).Fig. 1 Potential differences in the functions of HDL particles. HDLaa depicts a theoreticalsubclass of particles comprising the bulk of plasma HDL-C concentration but without anysignificant role in reverse cholesterol transport or pleiotropic effects of HDL, whereas HDLbbmay be a smaller fraction but more active in reverse cholesterol transport. HDLcc could be a smallsubclass without any significant contribution to the HDL-C quantity. These particles may, how-ever, have many pleiotropic effects on cytokines, growth factors, etc. [Modified from Hannukselaet al. (2004)]Epidemiology: Disease Associations and Modulators of HDL-Related Biomarkers 2679 Time Trends in Total HDL-CDuring the last decades, favorable trends in HDL-C levels have occurred in USadults and youths aged 6–19 years despite changes in physical activity, obesity, anddiabetes (Carroll et al. 2012; Kit et al. 2012). Similar trends have been observedalso in Europe and India (Muntoni et al. 2009; Gupta et al. 2012).10 Are There Other Biomarkers than the Total HDL-C?The cholesterol in HDL (i.e., HDL-C) may reflect the rate of reverse cholesteroltransport from the peripheral tissues to liver. However, it may be that the bulk ofHDL cholesterol does not necessarily represent the particles that are involved in thereverse cholesterol transport or any other potentially antiatherogenic activity ofHDL (Fig. 1).HDL particles comprise a heterogeneous fraction of lipoprotein which vary bythe apolipoprotein and lipid content and consequently also in density, shape, size,and charge. Various subfractions can be analyzed, e.g., by ultracentrifugation(density), electron microscopy (shape), nondenaturing gel electrophoresis, gelfiltration or nuclear magnetic resonance (NMR) spectroscopy (size), NMR orimmunoaffinity (composition), and 2-dimensional electrophoresis (charge).11 HDL FractionsAnalytic ultracentrifugation was first used to separate subclasses of HDL (DeLallaet al. 1954). Later, the sequential ultracentrifugation and various precipitationmethods enabled the analysis of larger series in epidemiological studies.Sequential ultracentrifugation methods have enabled separation of two majorHDL subfractions, namely, HDL2 and HDL3, while the isolation of furthersubclasses has required more tedious methods such as gradient ultracentrifugation.Nuclear magnetic resonance (NMR) spectroscopy has simplified the differentiationinto five subclasses. However, the nomenclature has varied and it is generally quitedifficult to compare HDL subclasses separated by different methods. Recently, aunified nomenclature for a simplified differentiation into 5 subclasses according toparticle size (very small, small, medium, large, and very large HDL) has beenproposed (Rosenson et al. 2011).Controversial results have been obtained in studies aimed at distinguishingcardiovascular differences between HDL subclasses separated by density (Superkoet al. 2012; Pirillo et al. 2013). A majority of the studies, however, have foundHDL2-C to be more predictive of CHD risk than total HDL-C or HDL3-C(Johansson et al. 1991; Drexel et al. 1992; Lamarche et al. 1997). A recent study268 M.J. Savolainenwith 29-year follow-up of the Gofman’s Livermore cohort showed that HDL2 andHDL3 are independently related to CHD risk (Williams and Feldman 2011).12 HDL Particle SizeFurther characterization of HDL subfractions by nondenaturing polyacrylamidegels has identified three HDL3 and two HDL2 subclasses. Very large HDL particles(HDL2b subfraction with diameter between 9.7 and 12.9 nm) are stronglycorrelated with the total HDL-C concentration and most strongly inversely relatedto CHD risk in normotriglyceridemic subjects (Johansson et al. 1991). On the otherhand, an increased concentration small HDL particles (HDL3b subfraction, 7.8–8.2 nm) is associated with an atherogenic lipoprotein profile characterized by lowHDL2b levels, high plasma triglyceride concentration, and increased level of small,dense LDL particles (Berneis and Krauss 2002). Low concentration of HDL2bsubclass has also been shown in patients with T2DM (Xian et al. 2009).13 HDL Particle NumberThe complexity of HDL metabolism leads to the formation of multiple HDLsubpopulations with varying density, size, charge, and chemical composition.Two individuals with the same HDL-C concentration may have HDL particles ofdifferent size distribution, and consequently, the particle number is different.Recently, NMR methods have enabled high-throughput determination of HDLparticle concentration. Plasma levels of large particles and low particle numberare consistently associated with low CHD prevalence (Mora et al. 2012).14 HDL LipidsSome of the effects of HDL on cellular functions are mediated by sphingomyelin-1-phosphate (S1P) that is a bioactive lipid in HDL. Alterations in S1P content in HDLparticles could explain the dysfunction of HDL since the HDL-associated S1Pseems to be responsible for many of the pleiotropic effects of HDL by activatingspecial S1P receptors. In epidemiological studies low levels of S1P in HDL hasbeen associated with coronary heart disease (Argraves et al. 2011). Sphingomyelincontent in HDL particles has been associated with coronary heart disease inpostmenopausal women (Horter et al. 2002). Sphingomyelin has also beenassociated with kidney disease in patients with type 1 diabetes (Mäkinenet al. 2012).Epidemiology: Disease Associations and Modulators of HDL-Related Biomarkers 26915 HDL ApolipoproteinsThe structure of HDL particles is very complex, apoA-I and apoA-II being themajor protein components. ApoA-I accounts for about two-thirds of the proteincontent of HDL and is also functionally important since it is the acceptor in theefflux of phospholipids and free cholesterol from peripheral cells. The measurementof apolipoprotein levels is more expensive and time-consuming than that of lipidconcentrations. To circumvent this, data from large epidemiological cohorts wasrecently used for the development of computer software that enables accurateestimation of apolipoprotein levels on the basis of lipid measurements (Raitakariet al. 2013).High plasma levels of apoA-I are protective against atherosclerosis in somestudies (Arsenault et al. 2011; Emerging Risk Factor Collaboration 2012). It hasbeen proposed that apoA-I concentration could even be a better predictor ofatherosclerosis development than HDL-C. A recent study reported that adjustmentfor apoA-I changes the direction of the association between HDL-C and the severityof atherosclerotic lesions as determined with coronary artery calcium score (Sunget al. 2013). Among patients treated with statin therapy, apoA-I levels are stronglyassociated with a reduced cardiovascular risk, even among those achieving verylow LDL-C level (Boekholdt et al. 2013).ApoA-II is the second major apolipoprotein in HDL particles. It is present inmost but not all particles and the clinical significance of apoA-II-containing andapoA-II-free particles is controversial (Rosenson et al. 2011).Some studies have reported enrichment with apoC-III in the HDL of patientswith CHD (Vaisar et al. 2010; Kavo et al. 2012; Jensen et al. 2012). Interestingly,Jensen and coworkers found that HDL particles without apoC-III were inverselyassociated with the risk of CHD while apoC-III-containing HDL particles weredirectly associated with increased risk of CHD (Jensen et al. 2012). HDL withapoC-III comprised about 13 % of the total HDL-C.16 HDL ProteomicsSeveral proteins circulate attached to HDL particles although most of them in muchlower numbers than apolipoproteins mentioned above. The protein content of HDLparticles has been characterized recently using various methods commonlydescribed as proteomics. It has been shown that HDL2 fraction of CHD patientscarries a distinct protein cargo (Vaisar et al. 2010; Gordon et al. 2010; Kavoet al. 2012; Alwaili et al. 2012).Proteomics as a new promising approach for detecting biomarkers andmechanisms could potentially explain the beneficial characteristics of HDLparticles unrelated to their cholesterol content. To this end, novel sophisticatedmethods have been recently developed (Gordon et al. 2010; Mazur et al. 2010;270 M.J. SavolainenBurillo et al. 2013; Hoofnagle et al. 2012; Mazur and Cardasis 2013; Riwantoet al. 2013). High-throughput methods are urgently needed for large-scale epidemi-ological studies.17 HDL FunctionCholesterol efflux is the first step of reverse cholesterol transport. Excess cellularcholesterol from peripheral tissues is effluxed to extracellular HDL-based acceptorparticles through the action of active transporters and passive diffusion. Recently, itwas reported that the cholesterol efflux measured from cultured macrophagesenriched with free cholesterol to apoB-depleted serum as cholesterol acceptorwas inversely associated with the history of CHD independent of HDL-C levels(Khera et al. 2011). However, more recently, enhanced efflux has been associatedwith high cardiovascular risk (Li et al. 2013). Further studies are urgently needed toresolve the issue with conflicting results.Antioxidative properties of HDL are mainly attributed to apoA-I althoughparaoxonase 1 (PON1) may also play an important role. Antioxidative activity ofHDL subfractions increases with increment in density as follows:HDL2b20 cM wide including the gene encodinghepatic lipase (LIPC), which has important functions in HDL metabolism. How-ever, the same investigators suspected that variations in LIPC might not fullyexplain this linkage peak and sought additional gene(s) that might contribute tothe peak. This is how they identified GLCE encoding glucuronic acid epimerase, aheparan sulfate proteoglycan biosynthetic enzyme (Hodoglugil et al. 2011). So far,no other studies on the role of this enzyme in HDL metabolism have beenconducted while the gene was not found associated with HDL cholesterol levelsin the latest meta-analyses GWA study (Willer et al. 2013).3.1.6 Beta-Chain of ATP SynthaseIn 2003, beta-chain of ATP synthase (encoded by ATPB5), a principal proteincomplex of the mitochondrial inner membrane, was surprisingly identified as ahigh-affinity HDL receptor for apoA-I receptor with a role in hepatic HDL endocy-tosis (Martinez et al. 2003). A subsequent study demonstrated a major role ofcytoskeleton reorganization in F(1)-ATPase/P2Y(13)-dependent HDL endocytosisunder the control of the small GTPase RhoA and its effector ROCK I (Malavalet al. 2009). Others showed that binding of HDL to this receptor triggers thegeneration of ADP, which via the activation of the purinergic receptor P2Y13stimulates the uptake and transport of HDL and initially lipid-free apoA-I byBeyond the Genetics of HDL: Why Is HDL Cholesterol Inversely Related to. . . 293endothelial cells (Cavelier et al. 2012). So far, there is no genetic support for theinvolvement of beta-chain of ATP synthase and/or P2Y(13), but even mildmutations could have in the case of ATPB5 lethal consequences. Most recently, itwas reported that there may be a role for beta-chain of ATP synthase in regulatingHDL cholesterol levels as mitochondrial inhibitor factor 1, which inhibits ATPB5and can be measured in serum, is associated with HDL cholesterol levels (Genouxet al. 2013).3.2 HDL Conversion and Remodeling3.2.1 Angptl Family of ProteinsIn the year 2000, ANGPTL4 was identified as a peroxisome proliferator-activatedreceptor α response gene (Kersten et al. 2000) while ANGPTL3 is since 2002 knownfor its effects on glucose and lipid metabolism through a hypolipidemic mousestrain (Koishi et al. 2002). Both Angptl3 and Angptl4 are known to inhibit LPL,thereby having an indirect effect on HDL cholesterol concentration in plasma, butAngptl3 may also exert direct effects on HDL remodeling through inhibition ofendothelial lipase (Shimamura et al. 2007). In GWA studies published in 2008 and2010, ANGPTL3 was shown to be associated primarily with triglycerides(Kathiresan et al. 2008; Teslovich et al. 2010) while more recently it was shownthat human ANGPTL3 deficiency also causes marked reduction in HDL cholesterol(Musunuru et al. 2010). In 2010, ANGPTL4was reported to be associated with HDLcholesterol as primary trait (Teslovich et al. 2010) while later on ANGPTL4 variantswere found associated with lower triglycerides and elevated HDL cholesterol(Romeo et al. 2007). Another study showed that mutant alleles of ANGPTL3 andANGPTL4 that were associated with low plasma triglyceride levels interfered eitherwith the synthesis or secretion of the protein or with the ability of the ANGPTLprotein to inhibit LPL (Romeo et al. 2009). More recently, ANGPTL8 (also knownas betatrophin) was shown to also affect HDL cholesterol through affecting theactivity of Angpl3 (Quagliarini et al. 2012). In addition, low-frequency variants inANGPTL8 were shown to affect HDL cholesterol levels (Peloso et al. 2014). For arecent review on the current eight Angptl proteins, please see Santulli (2014).3.2.2 Tribbles Homolog 1Variation at the TRIB1 gene locus has been reported to be associated withtriglycerides (as lead trait), HDL cholesterol, total cholesterol, and LDL cholesterol(Kathiresan et al. 2008; Teslovich et al. 2010). It is one of the few genes subse-quently studied in depth in mouse models, in vitro experiments, and further humangenetic association studies. Viral-mediated overexpression in the liver of micesimultaneously reduced plasma triglycerides and cholesterol of all majorlipoproteins (including HDL). On the other hand, TRIB1 knockout mice showedelevated levels of triglycerides (Burkhardt et al. 2010) without statistically signifi-cant effects on HDL cholesterol. These effects were related to an on VLDLproduction but it is not known what mechanisms are responsible. Further294 J.A. Kuivenhoven and A.K. Groenepidemiological studies confirmed that TRIB1 is associated with HDL cholesterolbut interestingly without affecting apoA-I concentration (Varbo et al. 2011). Morerecently it was shown that Trib1 is important to adipose tissue maintenance andsuppression of metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages (Satoh et al. 2013). In addition, a TRIB1 single-nucleotide polymorphism was found associated with nonalcoholic fatty liver dis-ease in humans while the same investigators showed that TRIB1 expression affectshepatic lipogenesis and glycogenesis through multiple molecular interactions(including a molecular interaction with MLXIPL or CHREBP, a hepatic lipogenicmaster regulator) (Ishizuka et al. 2014). In addition to a role in lipid metabolism,there is a substantial literature on the role of tribbles in cancer (Liang et al. 2013).3.2.3 Tetratricopeptide Repeat Domain/Glycogen-Targeting PP1Subunit G(L)TTC39B was discovered through GWA studies in 2009 (Kathiresan et al. 2009).When knocked down in mice (using a viral-mediated strategy), it was shown toincrease HDL cholesterol levels (Teslovich et al. 2010). However, no studies havesince revealed insight into how tetratricopeptide repeat domain 39B may affectlipid metabolism while another GWA study could not replicate the effect ofTTC39B variation on plasma lipid levels (Dumitrescu et al. 2011).PPP1R3B was also found to be associated with HDL cholesterol as primary trait(Teslovich et al. 2010), and in the same paper, viral-mediated overexpression wasshown to reduce HDL cholesterol levels in mice. The gene encoding for glycogen-targeting PP1 (protein phosphatase 1) subunit G(L) has also been associated withtype 2 diabetes and maturity-onset diabetes of the young but sequence variants atthe PPP1R3B locus were not found to be related to diabetes in mostly Caucasianfamilies (Dunn et al. 2006). Other studies proposed a role for this gene in inflam-mation (Dehghan et al. 2011), Alzheimer’s disease (Kamboh et al. 2012), andhepatic steatosis (Palmer et al. 2013). As for TTC39B, there are no mechanisticstudies that have revealed insight into how PPP1R3B gene products may affectlipid metabolism.3.2.4 ppGalNAc-T2Variation at the GALNT2 locus was also shown to be associated with HDL choles-terol (Teslovich et al. 2010). Viral-mediated overexpression and knockdown wereshown to decrease and increase HDL cholesterol levels in mice, respectively. In alater study a rare functional GALNT2 variant, identified in two index cases with veryhigh HDL cholesterol, was reported to affect HDLmetabolism indirectly via an effecton the lipolysis of triglycerides (Holleboom et al. 2011a, b). This was suggested to bein part due to attenuated glycosylation of apoC-III which impaired its capacity toinhibit LPL in the catabolism of triglycerides. Other investigators reported thatGALNT2 regulates plasma lipid levels through the glycosylation of ANGTPL3,another inhibitor of the LPL reaction (Schjoldager et al. 2010). Recently,ppGalNAc-T2 was identified as a novel regulator of insulin signaling (MarucciBeyond the Genetics of HDL: Why Is HDL Cholesterol Inversely Related to. . . 295et al. 2013) while there appears to be a role for GALNT2 in hypertension too(Pendergrass et al. 2013; McDonough et al. 2013).3.2.5 Glucokinase (Hexokinase 4) RegulatorGCKR (also known as GKRP) encodes for a regulatory protein that inhibitsglucokinase in the liver and pancreatic islet cells by binding non-covalently toform an inactive complex with the enzyme. This gene is considered a susceptibilitygene candidate for a form of maturity-onset diabetes of the young. GCKR waspicked up in both GWA studies with a focus on plasma lipids but also with type2 diabetes (van de Bunt and Gloyn 2010). Varbo et al. showed that a GCKR SNPwas associated with increased triglycerides, decreased HDL cholesterol, andremarkably increased apoA-I (Varbo et al. 2011). Several genetic associationstudies have shown that GCKR variants are associated with triglycerides, insulinresistance (Shen et al. 2013), type 2 diabetes fasting plasma glucose (Li et al. 2013a,b), hyperglycemia (Stancakova et al. 2012), and nonalcoholic fatty liver disease(Lin et al. 2014). In 2013, the crystal structure was resolved (Pautsch et al. 2013)while two potent small-molecule GK–GKRP disruptors were recently reported tonormalize blood glucose levels in several rodent models of diabetes (Lloydet al. 2013).Conclusions and PerspectivesThe harvest of screening the most recent published literature for novel HDLinfluencing genes is extensive. However, it also shows that there are only veryfew mechanistic clues on how “the new kids on the block” (primarily identifiedthrough GWA studies) affect HDL cholesterol or HDL function.It is interesting that none of the genes encoding for the proteins and enzymesthat have been discussed in the section on the de novo synthesis of HDL arelisted in the current largest GWA studies. This may be surprising in light of theevidence for the roles of especially bone morphogenetic protein-1, procollagenC-proteinase enhancer 2, apoM, and CT alpha in HDL metabolism. It shows thatGWA strategies, like any approach, come with limitations. Thus, prioritizingresearch interest on the basis of the top ranking GWA hits is not necessarily themost promising route to go.In the section on (new) insights in HDL conversion and remodeling, it is clearthat the factors discussed are only indirectly associated with HDL cholesterollevels through effects on triglyceride and/or glucose metabolism. It may benoted in this regard that HDL cholesterol was proposed to be the lead trait forANGPTL4, TTC39B, PPP1R3B, and GALNT2, in GWA studies which highlightsa need for more comprehensive studies with focus on the mechanistic relationsbetween HDL cholesterol, triglycerides, and glucose metabolism. Such studiesmay need the use of genome-scale metabolic maps.Finally, this review underlines an urgent need of mechanistic studies. Butwhere to start? Answering this question is today’s challenge as evidence-basedprioritization tools and high-throughput functional studies are yet to come.296 J.A. Kuivenhoven and A.K. GroenAcknowledgments I wish to apologize to colleagues whose work may not be cited in this review:It remains an attempt to cover the existing literature. This work is supported by Fondation LeDucq(Transatlantic Network, 2009–2014), the Netherlands CardioVascular Research Initiative(CVON2012-17; Genius), and the European Union (COST BM0904; Resolve: FP7-305707;TransCard: FP7-603091–2).Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAung LH et al (2013) Association of the apolipoprotein M gene polymorphisms and serum lipidlevels. Mol Biol Rep 40(2):1843–1853Burkhardt R et al (2010) Trib1 is a lipid- and myocardial infarction-associated gene that regulateshepatic lipogenesis and VLDL production in mice. J Clin Invest 120(12):4410–4414Candini C et al (2010) Identification and characterization of novel loss of function mutations inATP-binding cassette transporter A1 in patients with low plasma high-density lipoproteincholesterol. Atherosclerosis 213(2):492–498Cavelier C et al (2012) The beta-chain of cell surface F(0)F(1) ATPase modulates apoA-I andHDL transcytosis through aortic endothelial cells. Arterioscler Thromb Vasc Biol 32(1):131–139ChapmanMJ et al (2011) Triglyceride-rich lipoproteins and high-density lipoprotein cholesterol inpatients at high risk of cardiovascular disease: evidence and guidance for management. EurHeart J 32(11):1345–1361Chau P et al (2007) Bone morphogenetic protein-1 (BMP-1) cleaves human proapolipoprotein A1and regulates its activation for lipid binding. Biochemistry 46(28):8445–8450Christoffersen C, Nielsen LB (2013) Apolipoprotein M: bridging HDL and endothelial function.Curr Opin Lipidol 24(4):295–300Cohen JC et al (2004) Multiple rare alleles contribute to low plasma levels of HDL cholesterol.Science 305(5685):869–872Dehghan A et al (2011) Meta-analysis of genome-wide association studies in >80,000 subjectsidentifies multiple loci for C-reactive protein levels. Circulation 123(7):731–738Dumitrescu L et al (2011) Genetic determinants of lipid traits in diverse populations from thepopulation architecture using genomics and epidemiology (PAGE) study. PLoS Genet 7(6):e1002138Dunn JS et al (2006) Examination of PPP1R3B as a candidate gene for the type 2 diabetes andMODY loci on chromosome 8p23. Ann Hum Genet 70(Pt 5):587–593Feig JE et al (2014) High-density lipoprotein and atherosclerosis regression: evidence frompreclinical and clinical studies. Circ Res 114(1):205–213Francone OL et al (2011) Disruption of the murine procollagen C-proteinase enhancer 2 genecauses accumulation of proapoA-I and increased HDL levels. J Lipid Res 52(11):1974–1983Frikke-Schmidt R (2011) Genetic variation in ABCA1 and risk of cardiovascular disease. Athero-sclerosis 218(2):281–282Genoux A et al (2013) Serum IF1 concentration is independently associated to HDL levels and tocoronary heart disease: the GENES study. J Lipid Res 54(9):2550–2558Ginsberg HN, Reyes-Soffer G (2013) Niacin: a long history, but a questionable future. Curr OpinLipidol 24(6):475–479Goode EL et al (2007) Heritability of longitudinal measures of body mass index and lipid andlipoprotein levels in aging twins. Twin Res Hum Genet 10(5):703–711Beyond the Genetics of HDL: Why Is HDL Cholesterol Inversely Related to. . . 297Gordts SC et al (2013) Pleiotropic effects of HDL: towards new therapeutic areas forHDL-targeted interventions. Curr Mol Med 14(4):481–503Haase CL et al (2012) Population-based resequencing of APOA1 in 10,330 individuals: spectrumof genetic variation, phenotype, and comparison with extreme phenotype approach. PLoSGenet 8(11):e1003063Hinds DA et al (2004) Application of pooled genotyping to scan candidate regions for associationwith HDL cholesterol levels. Hum Genomics 1(6):421–434Hodoglugil U et al (2011) Glucuronic acid epimerase is associated with plasma triglyceride andhigh-density lipoprotein cholesterol levels in Turks. Ann Hum Genet 75(3):398–417Holleboom AG et al (2011a) Heterozygosity for a loss-of-function mutation in GALNT2 improvesplasma triglyceride clearance in man. Cell Metab 14(6):811–818Holleboom AG et al (2011b) High prevalence of mutations in LCAT in patients with low HDLcholesterol levels in The Netherlands: identification and characterization of eight novelmutations. Hum Mutat 32(11):1290–1298Ishizuka Y et al (2014) TRIB1 down-regulates hepatic lipogenesis and glycogenesis via multiplemolecular interactions. J Mol Endocrinol 52(2):145–158Jacobs RL et al (2004) Targeted deletion of hepatic CTP:phosphocholine cytidylyltransferasealpha in mice decreases plasma high density and very low density lipoproteins. J Biol Chem279(45):47402–47410Jacobs RL et al (2008) Hepatic CTP:phosphocholine cytidylyltransferase-alpha is a criticalpredictor of plasma high density lipoprotein and very low density lipoprotein. J Biol Chem283(4):2147–2155Johansen CT et al (2010) Excess of rare variants in genes identified by genome-wide associationstudy of hypertriglyceridemia. Nat Genet 42(8):684–687Kamboh MI et al (2012) Genome-wide association study of Alzheimer’s disease. Transl Psychia-try 2:e117Kathiresan S et al (2008) Six new loci associated with blood low-density lipoprotein cholesterol,high-density lipoprotein cholesterol or triglycerides in humans. Nat Genet 40(2):189–197Kathiresan S et al (2009) Common variants at 30 loci contribute to polygenic dyslipidemia. NatGenet 41(1):56–65Kersten S et al (2000) Characterization of the fasting-induced adipose factor FIAF, a novelperoxisome proliferator-activated receptor target gene. J Biol Chem 275(37):28488–28493Khera AV et al (2011) Cholesterol efflux capacity, high-density lipoprotein function, and athero-sclerosis. N Engl J Med 364(2):127–135Kiss RS et al (2007) Genetic etiology of isolated low HDL syndrome: incidence and heterogeneityof efflux defects. Arterioscler Thromb Vasc Biol 27(5):1139–1145Koishi R et al (2002) Angptl3 regulates lipid metabolism in mice. Nat Genet 30(2):151–157LagorWR et al (2009) Overexpression of apolipoprotein F reduces HDL cholesterol levels in vivo.Arterioscler Thromb Vasc Biol 29(1):40–46Lagor WR et al (2012) The effects of apolipoprotein F deficiency on high density lipoproteincholesterol metabolism in mice. PLoS ONE 7(2):e31616Li H et al (2013a) Association of glucokinase regulatory protein polymorphism with type 2 diabe-tes and fasting plasma glucose: a meta-analysis. Mol Biol Rep 40(6):3935–3942Li XM et al (2013b) Paradoxical association of enhanced cholesterol efflux with increased incidentcardiovascular risks. Arterioscler Thromb Vasc Biol 33(7):1696–1705Liang KL et al (2013) Tribbles in acute leukemia. Blood 121(21):4265–4270Lin YC et al (2014) Genetic variants in GCKR and PNPLA3 confer susceptibility to nonalcoholicfatty liver disease in obese individuals. Am J Clin Nutr 99(4):869–874Lloyd DJ et al (2013) Antidiabetic effects of glucokinase regulatory protein small-moleculedisruptors. Nature 504(7480):437–440Luscher TF et al (2014) High-density lipoprotein: vascular protective effects, dysfunction, andpotential as therapeutic target. Circ Res 114(1):171–182298 J.A. Kuivenhoven and A.K. GroenMalaval C et al (2009) RhoA/ROCK I signalling downstream of the P2Y13 ADP-receptor controlsHDL endocytosis in human hepatocytes. Cell Signal 21(1):120–127Martinez LO et al (2003) Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor inhepatic HDL endocytosis. Nature 421(6918):75–79Marucci A et al (2013) Role of GALNT2 in the modulation of ENPP1 expression, and insulinsignaling and action: GALNT2: a novel modulator of insulin signaling. Biochim Biophys Acta1833(6):1388–1395McDonough CW et al (2013) Atenolol induced HDL-C change in the pharmacogenomic evalua-tion of antihypertensive responses (PEAR) study. PLoS ONE 8(10):e76984Morton RE et al (2008) Lipid transfer inhibitor protein (apolipoprotein F) concentration innormolipidemic and hyperlipidemic subjects. J Lipid Res 49(1):127–135Motazacker MM et al (2013) Evidence of a polygenic origin of extreme high-density lipoproteincholesterol levels. Arterioscler Thromb Vasc Biol 33(7):1521–1528Musunuru K et al (2010) Exome sequencing, ANGPTL3 mutations, and familial combinedhypolipidemia. N Engl J Med 363(23):2220–2227Oldoni F et al (2014) Mendelian disorders of high-density lipoprotein metabolism. Circ Res 114(1):124–142Palmer ND et al (2013) Characterization of European ancestry nonalcoholic fatty liver disease-associated variants in individuals of African and Hispanic descent. Hepatology 58(3):966–975Park YJ et al (2013) The APOM polymorphism as a novel risk factor for dyslipidaemia inrheumatoid arthritis: a possible shared link between disease susceptibility and dyslipidaemia.Clin Exp Rheumatol 31(2):180–188Pautsch A et al (2013) Crystal structure of glucokinase regulatory protein. Biochemistry 52(20):3523–3531Peloso GM et al (2014) Association of low-frequency and rare coding-sequence variants withblood lipids and coronary heart disease in 56,000 whites and blacks. Am J Hum Genet 94(2):223–232Pendergrass SA et al (2013) Phenome-wide association study (PheWAS) for detection of pleiot-ropy within the population architecture using genomics and epidemiology (PAGE) network.PLoS Genet 9(1):e1003087Plomgaard P et al (2009) Apolipoprotein M predicts pre-beta-HDL formation: studies in type2 diabetic and nondiabetic subjects. J Intern Med 266(3):258–267Quagliarini F et al (2012) Atypical angiopoietin-like protein that regulates ANGPTL3. Proc NatlAcad Sci USA 109(48):19751–19756Rader DJ, Degoma EM (2014) Future of cholesteryl ester transfer protein inhibitors. Annu RevMed 65:385–403Riwanto M, Landmesser U (2013) High density lipoproteins and endothelial functions: mechanis-tic insights and alterations in cardiovascular disease. J Lipid Res 54(12):3227–3243Romeo S et al (2007) Population-based resequencing of ANGPTL4 uncovers variations thatreduce triglycerides and increase HDL. Nat Genet 39(4):513–516Romeo S et al (2009) Rare loss-of-function mutations in ANGPTL family members contribute toplasma triglyceride levels in humans. J Clin Invest 119(1):70–79Rye KA, Barter PJ (2014) Cardioprotective functions of HDLs. J Lipid Res 55(2):168–179Santulli G (2014) Angiopoietin-like proteins: a comprehensive look. Front Endocrinol (Lausanne)5:4Satoh T et al (2013) Critical role of Trib1 in differentiation of tissue-resident M2-likemacrophages. Nature 495(7442):524–528Schjoldager KT et al (2010) O-glycosylation modulates proprotein convertase activation ofangiopoietin-like protein 3: possible role of polypeptide GalNAc-transferase-2 in regulationof concentrations of plasma lipids. J Biol Chem 285(47):36293–36303Shen Y et al (2013) GCKR variants increase triglycerides while protecting from insulin resistancein Chinese children. PLoS ONE 8(1):e55350Beyond the Genetics of HDL: Why Is HDL Cholesterol Inversely Related to. . . 299Shimamura M et al (2007) Angiopoietin-like protein3 regulates plasma HDL cholesterol throughsuppression of endothelial lipase. Arterioscler Thromb Vasc Biol 27(2):366–372Stancakova A et al (2012) Hyperglycemia and a common variant of GCKR are associated with thelevels of eight amino acids in 9,369 Finnish men. Diabetes 61(7):1895–1902Talmud PJ et al (2013) Use of low-density lipoprotein cholesterol gene score to distinguishpatients with polygenic and monogenic familial hypercholesterolaemia: a case-control study.Lancet 381(9874):1293–1301Teslovich TM et al (2010) Biological, clinical and population relevance of 95 loci for blood lipids.Nature 466(7307):707–713Tietjen I et al (2012) Segregation of LIPG, CETP, and GALNT2 mutations in Caucasian familieswith extremely high HDL cholesterol. PLoS ONE 7(8):e37437van Capelleveen JC et al (2014) Novel therapies focused on the high-density lipoprotein particle.Circ Res 114(1):193–204van de Bunt M, Gloyn AL (2010) From genetic association to molecular mechanism. Curr DiabRep 10(6):452–466Varbo A et al (2011) TRIB1 and GCKR polymorphisms, lipid levels, and risk of ischemic heartdisease in the general population. Arterioscler Thromb Vasc Biol 31(2):451–457Vergeer M et al (2010) The HDL hypothesis: does high-density lipoprotein protect from athero-sclerosis? J Lipid Res 51(8):2058–2073Voight BF et al (2012) Plasma HDL cholesterol and risk of myocardial infarction: a mendelianrandomisation study. Lancet 380(9841):572–580Willer CJ et al (2013) Discovery and refinement of loci associated with lipid levels. Nat Genet 45(11):1274–1283Wolfrum C et al (2005) Apolipoprotein M is required for prebeta-HDL formation and cholesterolefflux to HDL and protects against atherosclerosis. Nat Med 11(4):418–422Xu N, Dahlback B (1999) A novel human apolipoprotein (apoM). J Biol Chem 274(44):31286–31290Zhu J et al (2009) Regulation of apoAI processing by procollagen C-proteinase enhancer-2 andbone morphogenetic protein-1. J Lipid Res 50(7):1330–1339300 J.A. Kuivenhoven and A.K. GroenMouse Models of Disturbed HDLMetabolismMenno Hoekstra and Miranda Van EckContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3032 Apolipoprotein A-I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3033 ATP-Binding Cassette Transporter A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3054 ATP-Binding Cassette Transporter G1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3095 Lecithin-Cholesterol Acyltransferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3116 Phospholipid Transfer Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3127 Scavenger Receptor BI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3158 Insights from Intercrossing of the Different Knockout Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3199 Conclusions from the Gene Knockout Mouse Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32010 Cholesterol Ester Transfer Protein Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325AbstractHigh-density lipoprotein (HDL) is considered to be an anti-atherogenic lipopro-tein moiety. Generation of genetically modified (total body and tissue-specificknockout) mouse models has significantly contributed to our understanding ofHDL function. Here we will review data from knockout mouse studies on theimportance of HDL’s major alipoprotein apoA-I, the ABC transporters A1 andG1, lecithin:cholesterol acyltransferase, phospholipid transfer protein, and scav-enger receptor BI for HDL’s metabolism and its protection against atherosclero-sis in mice. The initial generation and maturation of HDL particles as well as theselective delivery of its cholesterol to the liver are essential parameters in the lifecycle of HDL. Detrimental atherosclerosis effects observed in response to HDLdeficiency in mice cannot be solely attributed to the low HDL levels per se, asM. Hoekstra (*) • M. Van EckDivision of Biopharmaceutics, Gorlaeus Laboratories, Leiden Academic Centre for DrugResearch, Leiden University, Einsteinweg 55, 2333CC Leiden, The Netherlandse-mail: hoekstra@lacdr.leidenuniv.nl; m.eck@lacdr.leidenuniv.nl# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_9301the low HDL levels are in most models paralleled by changes in non-HDL-cholesterol levels. However, the cholesterol efflux function of HDL is of criticalimportance to overcome foam cell formation and the development of atheroscle-rotic lesions in mice. Although HDL is predominantly studied for itsatheroprotective action, the mouse data also suggest an essential role for HDLas cholesterol donor for steroidogenic tissues, including the adrenals and ovaries.Furthermore, it appears that a relevant interaction exists between HDL-mediatedcellular cholesterol efflux and the susceptibility to inflammation, which(1) provides strong support for the novel concept that inflammation and metabo-lism are intertwining biological processes and (2) identifies the efflux function ofHDL as putative therapeutic target also in other inflammatory diseases thanatherosclerosis.KeywordsKnockout mice • High-density lipoprotein • Reverse cholesterol transport • Ath-erosclerosis • Steroidogenesis • Inflammation • ABC transporters • Scavengerreceptor BI • Apolipoprotein • Lecithin-cholesterol acyltransferase • Phospho-lipid transfer proteinAbbreviationsABCA1 ATP-binding cassette transporter A1ABCG1 ATP-binding cassette transporter G1apoA-I Apolipoprotein A-IapoA-II Apolipoprotein A-IIapoA-IV Apolipoprotein A-IVapoB Apolipoprotein BapoE Apolipoprotein ECETP Cholesterol ester transfer proteinHDL High-density lipoproteinLCAT Lecithin-cholesterol acyltransferaseLDL Low-density lipoproteinLDLr Low-density lipoprotein receptorLPS LipopolysaccharidePLTP Phospholipid transfer proteinSR-BI Scavenger receptor BIVLDL Very-low-density lipoprotein302 M. Hoekstra and M. Van Eck1 IntroductionHigh levels of low-density lipoprotein (LDL) cholesterol are associated with anincreased risk for cardiovascular disease. In contrast, plasma levels of cholesterolassociated with high-density lipoprotein (HDL) are inversely correlated with therisk of cardiovascular disease (Gordon et al. 1977). Since standard treatment withstatins to lower plasma levels of LDL cholesterol only reduces the risk of cardio-vascular disease by ~30 % (Cholesterol Treatment Trialists’ (CTT) Collaborationet al. 2010; Cholesterol Treatment Trialists’ (CTT) Collaborators et al. 2012),raising plasma levels of HDL cholesterol has been considered a potential additionaltherapeutic strategy to overcome disease.The term HDL refers to a class of alpha-migrating protein/lipid complexes thatdiffer in size (5–12 nm), shape, and lipidation pattern. In order to be able toeffectively modulate plasma HDL-cholesterol levels, it is of critical importanceto understand the anti-atherogenic potential and metabolism of the different HDLparticles. Due to the large biological/genetic variation and lifestyle of people, it isdifficult to gain clear insight into the relation between the flux of cholesterolthrough the HDL pathway and the development of atherosclerosis—the primaryunderlying cause of cardiovascular disease—in the general human population.Small animal models, in particular mice, have been proven valuable tools toincrease the understanding of the complexity of HDL metabolism and theconsequences of interfering in specific pathways or atherosclerosis susceptibility.Here we will review data from several genetically modified (total body and tissue-specific knockout) mouse models to (1) show the importance of specific geneproducts in the life cycle of HDL and their contribution to HDL’s primary function,reverse cholesterol transport, i.e., the flux of cholesterol from peripheral cells backto the liver for subsequent excretion, and (2) highlight the potential of modulationof HDL metabolism as an approach to lower atherosclerotic disease burden.2 Apolipoprotein A-IApolipoprotein A-I (apoA-I) is produced by the liver (Zannis et al. 1983) andintestine (Gordon et al. 1982) and represents the primary apolipoprotein constituentof HDL particles (Scanu et al. 1969). Several mutations in the apoA-I gene havebeen causally linked to HDL deficiency in humans (Tilly-Kiesi et al. 1995; Lerenet al. 1997; Matsunaga et al. 1999; Hovingh et al. 2004; Dastani et al. 2006; Wadaet al. 2009; Berge and Leren 2010; Lee et al. 2013).The groups of Nobuyo Maeda (Williamson et al. 1992) and Jan Breslow (Plumpet al. 1997) both inactivated the apoA-I gene in mice using dedicated homologousrecombination strategies, leading to the absence of detectable amounts of apoA-Iprotein in plasma. In accordance with an important function for apoA-I in theformation and stability of HDL particles, genetic disruption of apoA-I in mice isassociated with an 83 % decrease in the level of HDL cholesterol, contributing to a68 % decrease in plasma total cholesterol levels (Williamson et al. 1992). TheMouse Models of Disturbed HDL Metabolism 303limited amount of HDL cholesterol present in apoA-I knockout mice is carried byparticles that are enriched in triglycerides (Plump et al. 1997). However, HDLparticle size and shape are similar to that of wild-type controls. ApoA-I-deficientHDL particles display increased levels of other apolipoprotein subspecies, includ-ing apolipoprotein A-II (apoA-II), apolipoprotein E (apoE), and apolipoproteinA-IV (apoA-IV) (Li et al. 1993; Plump et al. 1997; Moore et al. 2003). Homozy-gous apoA-I deficiency in mice not only lowers plasma levels of HDL cholesterol,but also decreases levels of cholesterol associated with apoB-containing very-low-density lipoproteins (VLDL) (Plump et al. 1997). The cholesterol ester tounesterified cholesterol ratio is lower in plasma of apoA-I-deficient mice, whichcan be attributed to a reduced endogenous cholesterol esterification rate (Parkset al. 1995). Furthermore, the cholesterol ester fatty acid composition of plasma ismarkedly different between the different genotypes, with apoA-I knockout micedisplaying a higher degree of fatty acid saturation as compared to wild-type mice(Parks et al. 1995). ApoA-I, in addition to its structural role, thus also serves as anessential activator of the enzyme lecithin-cholesterol acyltransferase (LCAT) thatesterifies cholesterol.ApoA-I deficiency in mice does, in general, not affect tissue cholesterol levels(Plump et al. 1997). In the liver, decreased delivery of cholesterol esters tohepatocytes is compensated by a concomitant decrease in bile acid formation asjudged from a decrease in hepatic mRNA expression levels of cholesterol 7alpha-hydroxylase (CYP7A1) in apoA-I knockout mice (Plump et al. 1997). In contrast,the adrenals are not able to cope with the diminished plasma cholesterol flux due tothe lack of apoA-I. Cortical cells located in the zona glomerulosa and zonafasciculata from the adrenals of wild-type mice are filled with large lipid dropletscontaining cholesterol esters, while adrenals from apoA-I-deficient micecompletely lack cholesterol esters and display a parallel decrease in microvillarchannel width (Plump et al. 1996). Importantly, apoA-I-deficient mice show adiminished adrenal glucocorticoid output in response to steroidogenic triggers(Plump et al. 1996), suggesting that a lack of apoA-I-containing HDL particlesimpairs normal adrenal function in mice. Although the cholesterol ester content ofthe testis is similar between apoA-I knockout and wild-type mice, interstitial andtheca cells within the ovaries of apoA-I knockout mice are similarly deprived ofcholesterol esters (Plump et al. 1996). Currently no evidence is present for analtered female fertility, despite the clear ovarian cholesterol depletion phenotype.Combined, these findings indicate that apoA-I-containing HDL particles act asimportant cholesterol donors for several, but not all, steroidogenic tissues.Human apoA-I mutation carriers may develop premature atherosclerosis(Hovingh et al. 2004; Dastani et al. 2006). The formation of atherosclerotic lesionsin normolipidemic mice can be induced by feeding them a cholic acid-containingdiet enriched in cholesterol and fat that increases levels of pro-atherogenic apoB-containing lipoproteins (Paigen et al. 1987; Ishida et al. 1991). Strikingly, deletionof apoA-I in wild-type mice does not alter the incidence or extent of atherogenicdiet-induced lesion formation (Li et al. 1993). This may be explained by the factthat, under cholic acid-containing diet feeding conditions, levels of anti-atherogenic304 M. Hoekstra and M. Van EckHDL as well as levels of pro-atherogenic VLDL/LDL are lower in apoA-I knockoutmice as compared to wild-type controls (Li et al. 1993). To further determine theimpact of apoA-I deficiency on atherosclerosis susceptibility, apoA-I knockoutmice have been crossbred with genetically hyperlipidemic low-density lipoproteinreceptor (LDLr) knockout mice that spontaneously develop atherosclerotic lesionson a chow diet, albeit at a slow rate (Ishibashi et al. 1994). Chow diet-fed apoA-I xLDLr double knockout (DKO) mice as compared to LDLr single knockout controlsexhibit an increase in the plasma non-HDL-cholesterol to HDL-cholesterol ratio.This can be attributed to a marked increase in VLDL-cholesterol levels and adecrease in plasma HDL-cholesterol levels (Moore et al. 2003; Zabalawiet al. 2003). In accordance with the more atherogenic lipoprotein profile (higherVLDL cholesterol and lower HDL cholesterol), atherosclerotic lesion burden isincreased in chow diet-fed apoA-I x LDLr DKO mice, as measured by en faceanalysis of the aorta, irrespective of the age of the mice (Moore et al. 2003).Feeding a Western-type high fat/high cholesterol diet induces a rapid progressionof atherosclerotic lesion development in LDLr knockout mice (Ishibashiet al. 1994). In great contrast with the chow diet findings, the extent of atheroscle-rotic lesion formation was not significantly different between LDLr KO and apoA-Ix LDLr DKO mice upon feeding an atherogenic diet containing 10 % saturated fatfrom palm oil and 0.1 % cholesterol for 16 weeks (Zabalawi et al. 2003). However,it should be noted that apoA-I x LDLr DKO mice show a much lower diet-inducedincrease in VLDL-cholesterol levels. Identical aortic cholesterol contents are thusfound in the context of a ~70 % lower plasma total cholesterol level (Zabalawiet al. 2003). It therefore appears that ablation of apoA-I function and the concomi-tant decrease in plasma HDL-cholesterol levels do predispose for atheroscleroticlesion development in mice, but only after correcting for apoA-I genotype-associated changes in VLDL-cholesterol levels.ApoA-I x LDLr DKO mice develop severe (fatal) skin lesions containing lipid-filled macrophages upon feeding the high cholesterol/high fat diet (Zabalawiet al. 2003). In addition, B-cells, T-cells, and dendritic cells within lymph nodesof apoA-I x LDLr DKO mice are enriched in cholesterol esters (Wilhelmet al. 2009). This is associated with an autoimmune phenotype characterized byrelatively high autoantibody titers in plasma upon feeding an atherogenic highfat/high cholesterol diet (Wilhelm et al. 2009). ApoA-I-containing HDL particlesthus also play an essential role in maintaining cholesterol homeostasis in the skin aswell as in cells from the lymphoid system.3 ATP-Binding Cassette Transporter A1The cholesterol used to transform pre-beta1HDL into discoidal alpha-migratingHDL particles can be supplied through passive diffusion driven by a concentrationgradient. However, it has become clear that energy-dependent flux of cholesteroland phospholipid across the membrane facilitated by members of the ATP-bindingMouse Models of Disturbed HDL Metabolism 305cassette (ABC) family of transporters contributes significantly to the lipidation ofapoA-I.Human carriers of a mutation in the full size ABC transporter ABCA1 genesuffer from familial HDL deficiency (Brooks-Wilson et al. 1999; Bodziochet al. 1999; Hong et al. 2002). In accordance with a prominent role for ABCA1 inthe control of HDL biogenesis, total body ABCA1-deficient mice are characterizedby an almost complete lack of HDL cholesterol (>90 % decrease) in the context ofsimilar levels of triglyceride-rich lipoproteins (Christiansen-Weber et al. 2000;Orsó et al. 2000; Calpe-Berdiel et al. 2005; Brunham et al. 2006). Mature alpha-migrating HDL particles are virtually absent in ABCA1 knockout mice. Theremaining HDL cholesterol is carried by pre-beta HDL particles that are relativelyenriched in triglycerides and exhibit an altered phospholipid species distribution(Orsó et al. 2000; Francone et al. 2003). ABCA1 knockout mice display a dimin-ished LCAT activity (Francone et al. 2003), which highlights that lipidation ofapoA-I via ABCA1 is required for LCAT activity and maturation of HDL.Adrenocortical cells (Orsó et al. 2000) and testical Sertoli cells (Christiansen-Weber et al. 2000) of mice lacking ABCA1 show distinct accumulation of choles-terol esters. This can possibly be attributed to the observed increase in the activityof the cholesterol synthesis gene HMG-CoA reductase (Drobnik et al. 2001). How-ever, ABCA1 may also act as a local regulator of steroidogenic tissue cholesterolhomeostasis. In further support of a crucial role of ABCA1 in the control of normalsteroidogenesis and development, pregnant ABCA1-deficient females exhibitlower plasma progesterone and estrogen levels (Christiansen-Weber et al. 2000).Furthermore, placentas of ABCA1 knockout mice often show malformations,which translates into a diminished birth and survival rate of ABCA1 knockoutpups (Christiansen-Weber et al. 2000).Livers and intestines of ABCA1 knockout mice are depleted of cholesterol esters(Orsó et al. 2000). Within the liver of wild-type mice, relatively high mRNA andprotein expression levels of ABCA1 are found in hepatocytes and tissuemacrophages (Kupffer cells) (Lawn et al. 2001; Hoekstra et al. 2003), while inthe intestine ABCA1 expression seems to be mostly restricted to macrophages inthe lamina propria of villi (Lawn et al. 2001). Dedicated gene targeting strategieshave been employed to delineate the contribution of the different cell compartmentsto plasma HDL-cholesterol levels and total body cholesterol homeostasis. Condi-tional disruption of ABCA1 function specifically in parenchymal liver cells withthe use of the Cre/Flox gene targeting system impairs the apoA-I-mediated efflux ofcholesterol and phospholipid from hepatocytes without altering the efflux of cho-lesterol from macrophages to apoA-I (Timmins et al. 2005). PlasmaHDL-cholesterol levels are ~80 % lower as a result of liver-specific ABCA1deficiency (Timmins et al. 2005). Liver-specific deletion of ABCA1 is alsoassociated with hypercatabolism of apoA-I by the kidneys resulting in an increasedturnover of HDL particles and a marked decrease in steady-state plasma apoA-Ilevels (Timmins et al. 2005; Singaraja et al. 2006). Although no data on the effect ofgeneral ABCA1 deficiency in mice on apoA-I flux have been described, it can beassumed total body ABCA1 knockout mice similarly show an increased catabolism306 M. Hoekstra and M. Van Eckand clearance of apoA-I by the kidneys. ABCA1 deficiency in hepatocytes isassociated with a rise in plasma levels and size of triglyceride-rich VLDL particles,which can be attributed to an increase in hepatic VLDL secretion and a paralleldecrease in lipoprotein lipase (LPL)-mediated catabolism of triglycerides (Sahooet al. 2004; Chung et al. 2010). ABCA1-mediated transport of cholesterol fromhepatocytes to the plasma compartment thus not only controls HDL biogenesis, butalso impacts on the synthesis and catabolism of non-HDL particles. Deletion ofABCA1 specifically in intestinal epithelial cells is associated with an accumulationof cholesterol esters in the intestine, a lower plasma apoA-I concentration, and a~30 % decrease in plasma HDL-cholesterol levels (Brunham et al. 2006). Incontrast to total body and hepatocyte-specific knockout mice, intestinal-specificABCA1 knockout mice do not show major changes in non-HDL-cholesterol andplasma triglyceride levels (Brunham et al. 2006), which further highlights the roleof hepatocyte ABCA1 in the control of plasma triglyceride levels. Importantly,mice genetically lacking ABCA1 in both the liver and intestine show a greaterdecrease in plasma HDL-cholesterol levels as compared to their single tissueknockout controls, almost reaching similarly low levels as those found in totalbody ABCA1 knockout mice (Brunham et al. 2006). Hepatocytes and the intestinalepithelium can thus be considered the primary cellular sources of the cholesterolthat is used for lipidation of apoA-I and generation of HDL.Loss-of-function mutations in the ABCA1 gene could not be associated withincreased coronary heart disease in the general population (Frikke-Schmidtet al. 2008a). However, several studies have suggested that premature atheroscle-rosis and cardiovascular disease are a common finding in human carriers offunctional mutations in the ABCA1 gene (Huang et al. 2001; Hong et al. 2002;Frikke-Schmidt et al. 2008b). In contrast, total body ABCA1 deficiency does notalter the susceptibility to atherosclerosis in hyperlipidemic apoE knockout and LDLreceptor (LDLr) knockout mice fed either regular chow or an atherogenic highfat/high cholesterol diet (Aiello et al. 2002). However, deletion of ABCA1 functionin the context of genetic hyperlipidemia is not only associated with HDL deficiencybut also with a significant decrease in the plasma concentration of cholesterolcarried by pro-atherogenic VLDL/LDL (Aiello et al. 2002). As is also the casefor apoA-I, normal ABCA1 function in mice thus only protects against atheroscle-rosis after correcting for ABCA1 genotype-associated confounding differences inatherogenic lipoprotein levels. The atheroprotective effect of ABCA1 may beattributed to its general impact on plasma HDL-cholesterol levels or specificallydue to its potential role in the efflux of cholesterol from macrophages. Lipid-filledfoam cells can be found in all macrophage-rich tissues of hyperlipidemic total bodyABCA1 knockout mice (Aiello et al. 2002), which already hints to an importantin vivo role for ABCA1 in macrophage cholesterol efflux. The capacity of ABCA1knockout mice to facilitate reverse cholesterol transport, the transport from periph-eral cells—i.e., macrophages—back to the liver for subsequent excretion, has beenvalidated by measuring the recovery in the plasma compartment and feces ofradiolabeled cholesterol from intraperitoneal administered macrophages. In linewith the hypothesis that ABCA1-mediated cholesterol efflux is an importantMouse Models of Disturbed HDL Metabolism 307determinant of reverse cholesterol transport, a lower amount of radiolabeled cho-lesterol is excreted under the condition of both total body and macrophage-specificABCA1 deficiency (Calpe-Berdiel et al. 2005; Wang et al. 2007).Aiello et al. (2002) and Van Eck et al. (2002) utilized bone marrow transplanta-tion to explore the specific effect of ABCA1-dependent macrophage cholesterolefflux on atherosclerosis susceptibility. In accordance with a negligible impact ofmacrophage ABCA1 on total HDL biogenesis, bone marrow ABCA1 deficiencydoes not alter plasma HDL-cholesterol levels (Haghpassand et al. 2001; Aielloet al. 2002; Van Eck et al. 2002). Atherosclerotic lesion development in apoEknockout mice is markedly enhanced—at multiple sites—in response to disruptionof ABCA1 expression in bone marrow (Aiello et al. 2002). Transplantation ofABCA1 knockout bone marrow into lethally irradiated LDLr knockout micesimilarly stimulates atherosclerotic lesion development (Van Eck et al. 2002).ABCA1-mediated efflux of cholesterol from leukocytes is thus an important pro-tective mechanism to inhibit macrophage foam cell formation and atherosclerosisin mice. Remarkably, macrophage-specific ABCA1 deficiency does not affect theextent of atherosclerosis in LDLr knockout mice (Brunham et al. 2009). It cantherefore be concluded that ABCA1’s function in macrophages does not conferprotection against atherosclerosis, but that rather an efficient efflux of cholesterolfrom lymphocytes or other immune cells is essential to overcome disease.Relative HDL deficiency in liver-specific ABCA1 knockout mice on ahyperlipidemic LDLr knockout background is not associated with any enhancedsusceptibility for the initial development of macrophage-rich atheroscleroticlesions, while the development of more advanced (collagen-containing) plaquesis inhibited (Bi et al. 2013). In accordance with a prominent role for macrophageABCA1 in reverse cholesterol transport, no change is observed in the flux ofcholesterol from macrophages to the feces in LDLr knockout mice lackingABCA1 function only in hepatocytes (Bi et al. 2013). Strikingly, apoE knockoutmice that contain hepatocyte-specific ABCA1 deficiency do show an increasedsusceptibility to atherosclerosis, despite lowered plasma VLDL/LDL-cholesterollevels (Brunham et al. 2009). A critical review of these data has suggested that thediscrepancy in the atherosclerosis findings from liver-specific ABCA1 knockoutmice may be attributed to differences in inflammatory status of the twohyperlipidemic mouse models (Van Eck and Van Berkel 2013), which makesproper interpretation of the findings difficult. However, due to the inconsistencyof the aforementioned results, it seems clear that the decrease in plasmaHDL-cholesterol levels due to deletion of ABCA1 function should not be consid-ered the driving force for the increase in atherosclerosis in total body ABCA1knockout mice.A consistent decrease in plasma VLDL-cholesterol levels is observed in LDLrknockout mice that have been transplanted with ABCA1-deficient bone marrowupon feeding an atherogenic Western-type diet (Van Eck et al. 2002; Lammerset al. 2011, 2012). This finding suggests that modulation of the leukocyte choles-terol efflux rate can directly impact on the metabolism of VLDL particles in vivo.However, to date, the mechanism underlying this effect remains to be resolved. It is308 M. Hoekstra and M. Van Eckbecoming more and more evident that immunology and metabolism are not just twoseparate disciplines, but that they intervene and impact each other at multiple levels(Mathis and Shoelson 2011). ABCA1-deficient macrophages display a highersensitivity to the bacterial membrane component lipopolysaccharide (LPS) (Zhuet al. 2008; Yvan-Charvet et al. 2008) and myeloid cell-specific ABCA1 knockoutmice are protected against bacterial infection (Zhu et al. 2012). Furthermore,ABCA1 deficiency in bone marrow is associated with an increase in blood leuko-cyte counts (Van Eck et al. 2002). Given the apparent link between ABCA1 andimmune function in vivo, future research should be aimed at uncovering to whatextent ABCA1 contributes to the interplay between inflammation and cholesterolmetabolism.4 ATP-Binding Cassette Transporter G1ABCG1, formerly known as the ABC8/white gene, belongs to the family of ABChalf transporters that need to dimerize to execute their function. ABCG1 expressionis mostly restricted to macrophage-rich tissues, including liver, spleen, and lung(Klucken et al. 2000), where it acts as an intracellular sterol transporter (Tarling andEdwards 2011). ABCG1 mRNA expression can actually be used as a measure forthe hepatic macrophage content in rodent livers, despite detectable levels ofABCG1 expression in hepatocytes (Hoekstra et al. 2003; Ye et al. 2008; Liet al. 2012). The capacity to efflux cholesterol to HDL is diminished in ABCG1-deficient macrophages, while the apoA-I-mediated cholesterol efflux rate is similaras compared to wild-type macrophages (Out et al. 2006). ABCG1 and ABCA1 thusinteract with different HDL subspecies (mature HDL vs. pre-beta HDL) to facilitatetheir cholesterol efflux function. To date, no association between ABCG1mutations and HDL-cholesterol levels has been described in humans, suggestingthat ABCG1 does not significantly contribute to HDL biogenesis. In accordance,under basal chow diet feeding conditions, ABCG1 knockout mice do not exhibit achange in plasma total or HDL-cholesterol levels (Out et al. 2007; Wiersmaet al. 2009).Chow-fed ABCG1-deficient mice exhibit a severe pulmonary lipidosis pheno-type, starting from the age of ~6 months, which is characterized by accumulation ofphospholipids, cholesterol crystals, and inflammatory infiltrates in sub-pleural areasof the lung (Baldán et al. 2006a, 2008; Wojcik et al. 2008). The distinct lungphenotype in total body ABCG1 knockout mice can be specifically attributed to thelack of ABCG1 in bone marrow-derived cells, presumably macrophages. Lethallyirradiated normolipidemic mice transplanted with ABCG1-deficient bone marrowalso show inflammatory cell infiltrates in their lungs, while normalization ofmacrophage ABCG1 function prevents the occurrence of pulmonary lipidosis intotal body ABCG1 knockout mice (Wojcik et al. 2008). Alveolar macrophagesfrom ABCG1 knockout mice have a foamy appearance and display compensatoryupregulation of the cholesterol efflux gene ABCA1 (Baldán et al. 2006a). In vitrocultured macrophages lacking ABCG1 are more susceptible to surfactant-inducedMouse Models of Disturbed HDL Metabolism 309cholesterol accumulation (Baldán et al. 2006a). ABCG1-mediated cholesterolefflux to HDL is thus essential for the protection against specifically surfactant-induced foam cell formation under normolipidemic conditions, since no accumula-tion of lipid has been noted in other macrophage-rich tissues in chow-fed ABCG1-deficient mice (Out et al. 2008a). The pulmonary lipidosis phenotype is exacerbatedupon challenging the mice with a cholic acid-containing atherogenic diet (Outet al. 2007). Under atherogenic diet feeding conditions, ABCG1 knockout micealso show marked lipid accumulation in cells within and surrounding the germinalcenters of the spleen (Out et al. 2007). This finding highlights that ABCG1 is notsolely involved in maintaining cholesterol homeostasis locally within the lungs.Variations in the human ABCG1 gene have been linked to an altered risk foratherosclerotic disease that cannot be explained by changes in lipoprotein levels(Furuyama et al. 2009). In accordance with a role for murine ABCG1 in theprotection against atherosclerosis, a higher extent of lesion formation, in the contextof similar plasma lipoprotein levels, is detected in ABCG1 knockout mice ascompared to wild-type littermate controls upon feeding atherogenic diet (Outet al. 2007). The bone marrow transplantation technique has been applied toelucidate the specific contribution of macrophages in the atheroprotective effectof ABCG1. In line with the prominent role of macrophage ABCG1 in the preven-tion of pulmonary lipidosis, extensive lipid accumulation is observed also in lungsof hyperlipidemic mice transplanted with ABCG1 knockout bone marrow (Outet al. 2006; Baldán et al. 2006b). However, contrasting effects on atherosclerosisoutcome have been noted upon bone marrow-specific ABCG1 deletion. Macro-phage ABCG1 appears to protect against atherosclerosis in LDLr knockout mice asevident from the initial studies by Out et al. (2006). In marked contrast, similarstudies by Baldán et al. in LDLr and apoE knockout mice (Baldán et al. 2006b) andRanalletta et al. in LDLr-deficient mice (Ranalletta et al. 2006) have identifiedmacrophage ABCG1 as being a pro-atherogenic factor. Importantly, followingcritical review of all the present data and additional bone marrow transplantationstudies, Meurs et al. have been able to clarify the evident discrepancy in the findingsregarding the role of macrophage ABCG1 in atherosclerosis. The effect of disrup-tion of ABCG1 function in macrophages on atherogenesis appears to be highlydependent on the stage of lesion development (Meurs et al. 2012). DiminishedABCG1-mediated efflux of cholesterol to HDL stimulates the formation of foamcells and accelerates lesion development in initial plaques that primarily containmacrophages. During later stages of the disease, macrophage ABCG1 deficiencyinhibits plaque progression as a result of activation of compensatoryatheroprotective mechanisms, i.e., hypersecretion of apoE from macrophages(Ranalletta et al. 2006), and/or an increased apoptosis rate of lipid-laden macro-phage foam cells (Baldán et al. 2006b).310 M. Hoekstra and M. Van Eck5 Lecithin-Cholesterol AcyltransferaseDiscoidal small HDL particles are able to carry a relatively limited load of choles-terol. In order for nascent HDL species to become fully enriched in cholesterol, theunesterified cholesterol acquired upon cellular efflux has to be esterified forsubsequent storage in the core of more mature spherical HDL particles. Thehepatocyte-derived enzyme lecithin-cholesterol acyltransferase (LCAT) is consid-ered to be the sole mediator of cholesterol esterification in HDL in humans, sincefamilial LCAT deficiency is characterized by a >95 % decrease in plasmaHDL-cholesterol ester levels (Glomset et al. 1970). As such, homozygous carriersof loss-of-function mutations in LCAT present with HDL deficiency, while half ofnormal HDL-cholesterol values are observed in heterozygous carriers(Santamarina-Fojo et al. 2000; Hovingh et al. 2005). Two different strains ofLCAT knockout mice have been generated through genetic deletion of respectivelyexon 1 or exons 2–5. In accordance with a major role for LCAT in the maturation ofHDL, both types of LCAT-deficient mice show severe HDL-cholesterol deficiency(>90 % decreased; Ng et al. 1997; Sakai et al. 1997). This coincides with asignificant decrease in plasma apoA-I levels (Ng et al. 1997), probably as a resultof apoA-I hypercatabolism by the kidneys. The remaining plasma HDL cholesterolin LCAT knockout mice is contained in its unesterified form (Ng et al. 1997; Sakaiet al. 1997).Besides the clear HDL deficiency phenotype, LCAT knockout mice displaysignificant changes in the metabolism of triglyceride-rich lipoproteins. Homozy-gous LCAT-deficient mice show hypertriglyceridemia and an increase in plasmaVLDL-cholesterol levels on a chow diet (Sakai et al. 1997; Lambert et al. 2001; Nget al. 2002). This phenotype is preserved when mice are fed a Western-type dietenriched in fat (Li et al. 2007). Furthermore, extensive triglyceride deposition canbe observed within livers of LCAT knockout mice upon Western-type diet feeding(Li et al. 2007). Mechanistic studies in LCAT x LDLr double knockout mice haveindicated that the hypertriglyceridemia can be attributed to an increase in secretionof triglyceride-rich VLDL particles and a decrease in the LPL-mediated lipolysisrate (Ng et al. 2004). As evidenced by lower fasting glucose and insulin levels,LCAT knockout mice exhibit an improved glucose tolerance and insulin sensitivitywhen crossbred onto the hyperlipidemic LDLr knockout background(Ng et al. 2004; Li et al. 2007). LCAT-mediated cholesterol ester formation thusnot only contributes to the formation of mature HDL particles, but also (indirectly)impacts on fatty acid and glucose metabolism.A genetic defect in LCAT does, in general, not affect the tissue cholesterolbalance in mice (Ng et al. 1997). However, adrenals of LCAT knockout mice showgross morphological changes associated with neutral lipid depletion, i.e., they havea more red/brownish color instead of the normal white appearance after perfusion(Ng et al. 1997). Specifically adrenocortical cells within the zona fasciculata, butnot zona glomerulosa, are deprived from neutral lipid stores (Hoekstra et al. 2013a).This results in an overall ~80 % decrease in adrenal cholesterol ester content(Ng et al. 1997). Probably as a compensatory response to overcome cholesterolMouse Models of Disturbed HDL Metabolism 311insufficiency, marked increases in the expression of genes involved in the de novosynthesis and extracellular acquisition of cholesterol can be detected in LCATknockout adrenals (Ng et al. 1997). Importantly, in support of the notion thatHDL act as cholesterol donors for the synthesis of glucocorticoids by the adrenals,LCAT knockout mice display a 40–50 % reduction in the maximal glucocorticoidoutput (Hoekstra et al. 2013a). A minor decrease in tissue cholesterol content is alsonoted in ovaries, but not testis, in response to LCAT deficiency in mice (Tomimotoet al. 2001), which does not translate into apparent changes in female fertility.LCAT knockout mice on a normolipidemic background are virtually protectedagainst cholic acid containing diet-induced formation of atherosclerotic lesions(Lambert et al. 2001). Chow diet-fed LCAT x apoE double knockout mice alsodisplay a decrease in atherosclerotic lesion formation, as compared with apoEsingle knockout controls (Lambert et al. 2001; Ng et al. 2002). Ablation of LCATfunction is associated with marked HDL deficiency under all hyperlipidemicconditions. Strikingly, mixed results on the effect of LCAT deficiency on athero-sclerosis susceptibility, however, have been noted in hyperlipidemic mice uponfeeding diets enriched in fat. LCAT x LDLr double knockout mice display a loweratherosclerotic lesion burden upon feeding a cholic acid-containing diet (Lambertet al. 2001). In contrast, LCAT deficiency stimulates the formation of atheroscle-rotic lesions feeding in LDLr knockout mice upon feeding a Western-type dietdevoid of cholic acid, as judged by the aortic cholesterol content (Furbeeet al. 2002; Lee et al. 2004). Furthermore, LCAT x apoE double knockout micealso exhibit an increase in their aortic cholesterol content upon feeding theWestern-type high fat diet (Furbee et al. 2002). Importantly, lower plasma levelsof non-HDL cholesterol are found in the hyperlipidemic LCAT knockout miceunder cholic acid-containing diet feeding conditions (Lambert et al. 2001), whilenon-HDL-cholesterol levels are actually increased in response to LCAT deficiencyupon Western-type diet feeding (Furbee et al. 2002; Lee et al. 2004). It thus seemsthat the diverse effects of LCAT deficiency on atherosclerosis outcome in thedifferent mouse models cannot be attributed to the HDL deficiency, but are rathera consequence of the genotype-associated changes in plasma levels of apoB-containing lipoproteins (Kunnen and Van Eck 2012).6 Phospholipid Transfer ProteinTransfer of cholesterol esters generated by LCAT to the core of the HDL particlesand the subsequent maturation of the HDL particles require the action of phospho-lipid transfer protein (PLTP), which supplies phospholipids allowing surfaceexpansion of the shell of the particles. The phospholipids are liberated during thelipolysis of triglycerides in the core of apoB-containing lipoproteins by the action oflipoprotein lipase (Tall et al. 1985). In addition, PLTP facilitates the fusion ofHDL3 particles to enlarged particles (Lusa et al. 1996). During this process lipid-poor apoA-I particles are released that can act as substrate for ABCA1-mediatedcholesterol efflux from macrophages. In humans, common variants of PLTP have312 M. Hoekstra and M. Van Eckbeen identified that are associated with alterations in serum HDL cholesterol andthe accumulation of small HDL particles (Albers et al. 2012). Furthermore, PLTPexpression and activity is regulated during several diseases, including sepsis,multiple sclerosis, cancer, and cardiovascular disease (Albers et al. 2012), but itis largely unknown if the regulation of PLTP is a causative factor or simply aconsequence of the processes underlying the diseases. The generation of PLTPknockout mice by the group of Alan Tall 15 years ago has greatly contributed to thegeneral understanding of the role of PLTP in HDL metabolism. PLTP wasinactivated in mice by replacing exon 2 containing the translation initiationcodon, the signal peptide, and the first 16 amino acids of mature PLTP with aneomycin-resistant gene (Jiang et al. 1999). The plasma transfer activity of themajor phospholipids, including phosphatidylcholine, phosphatidylethanolamine,phosphatidylinositol, and sphingomyelin into HDL, was completely blocked,while also the transfer of free cholesterol was impaired in PLTP knockout mice.Deletion of PLTP in mice led to markedly decreased levels of HDL phospholipidsand cholesterol (65–70 %), illustrating the importance of transfer of surfacephospholipids from apoB-containing lipoproteins by PLTP for maintaining HDLlevels (Jiang et al. 1999). Furthermore, similarly as described for LCAT knockoutmice the decrease in HDL cholesterol coincided with a significant decrease inplasma apoA-I levels. Hepatocytes isolated from PLTP knockout mice synthesizednormal amounts of apoA-I, albeit with reduced amounts of phosphatidylcholine(Siggins et al. 2007), indicating that the reduced plasma apoA-I levels were not dueto impaired production. Qin et al. showed by in vivo turnover studies usingautologous HDL that the reduced apoA-I levels are likely the consequence ofincreased catabolism of HDL in the PLTP knockout mice (Qin et al. 2000).On regular chow diet, PLTP-deficient mice absorb less cholesterol in the intes-tine (Liu et al. 2007), while hepatic phospholipids are increased and triglyceridesare reduced in mice lacking PLTP (Siggins et al. 2007).Feeding the mice a high saturated fat diet, containing 20 % hydrogenatedcoconut oil and 0.15 % cholesterol, led to the accumulation of surface componentsof apoB-containing lipoproteins as evidenced by a massive increase in VLDL andLDL phospholipids and cholesterol in the absence of changes in apoB (Jianget al. 1999). Subsequent studies showed that the animals accumulated phospholipidand free cholesterol-rich lamellar particles containing apoA-IV and apoE (Qinet al. 2000). The accumulation of the lamellar particles specifically in coconutoil-fed PLTP knockout mice has been attributed to markedly reduced removal ofthese particles via scavenger receptor BI (SR-BI) by parenchymal liver cells(Kawano et al. 2002) and impaired secretion of biliary cholesterol and phospholipid(Yeang et al. 2010). Interestingly, these particles did not accumulate in the circula-tion when the mice were fed regular chow diet or a Western diet, containing 20 %milk fat and 0.15 % cholesterol (Kawano et al. 2002). In contrast, while feeding aWestern diet, containing milk fat and 0.15 % cholesterol, PLTP deficiency wasassociated with an attenuated diet-induced hypercholesterolemia due to twofoldlower concentrations of cholesterol transported by apoB-containing lipoproteinsand HDL. Hepatic and intestinal lipid levels were not affected under theseMouse Models of Disturbed HDL Metabolism 313conditions, but cholesterol absorption in the intestine was reduced (Shellyet al. 2008).Both on chow and while feeding the milk fat Western diet, PLTP knockout micedisplay reduced systemic inflammation as evidenced by lower levels of interleukin6 and reduced expression of intercellular adhesion molecule 1 (ICAM1) andvascular adhesion molecule 1 (VCAM1) in aorta, indicating that mice lackingPLTP might have a reduced atherosclerosis susceptibility (Shelly et al. 2008).To study the effect of PLTP deficiency on atherosclerotic lesion development,PLTP knockout mice were crossbred with human apoB transgenic mice, apoEknockout mice, and LDLr knockout mice (Jiang et al. 2001). Both in the apoBtransgenic and the apoE knockout background, PLTP deficiency led, on top of thereduction in HDL lipids, to substantially lower VLDL lipid levels. Interestingly,this effect on VLDL lipids was not found in the LDLr knockout background,indicating the involvement of the LDLr in the lowering of VLDL lipids. In line,apoB production was reduced in the apoB transgenic and apoE knockout back-ground, but not in the LDLr knockout background (Jiang et al. 2001). The contentof the antioxidant vitamin E was significantly increased in all three backgrounds inthe absence of PLTP, while autoantibodies to oxidized LDL were largely decreased(Jiang et al. 2002). HDL isolated from PLTP knockout mice crossbred to both theapoB transgenic and the LDLr knockout background displayed improved anti-inflammatory properties and reduced the ability of LDL to induce monocytechemotaxis (Yan et al. 2004). After 6 months of feeding apoB transgenic miceWestern diet and after feeding apoE knockout mice chow for 3 months, atheroscle-rotic lesion size was reduced fivefold in mice lacking PLTP. In contrast, in LDLrknockout mice PLTP deficiency led to a twofold reduction in atherosclerotic lesionsize after 8 weeks Western diet, whereas no significant effects were observed after12 weeks Western diet feeding (Jiang et al. 2001). Thus, while PLTP deficiency canreduce early atherosclerotic lesion development in mice lacking the LDLr, thepro-atherogenic effects of PLTP should be primarily attributed to its effects onthe production of apoB-containing lipoproteins in mice fed regular chow or aWestern diet. Notably, when apoE knockout mice lacking PLTP were challengedwith a coconut oil-enriched, high fat diet for 7 weeks plasma levels of freecholesterol were 23 % higher due to the accumulation of the lamellar free choles-terol and phospholipid-rich particles. Under these conditions no effects on athero-sclerotic lesion development were found (Yeang et al. 2010).PLTP is widely distributed with expression in placenta > pancreas > lung >kidney > heart > liver > skeletal muscle > brain (Day et al. 1994). In addition,PLTP is found in endothelial cells (Day et al. 1994) and in smooth muscle cell andmacrophage foam cells in atherosclerotic lesions (Desrumaux et al. 2003; Laffitteet al. 2003; O’Brien et al. 2003). Interestingly, Vikstedt and colleagues showed thatthe expression of PLTP is sixfold higher in Kupffer cells, macrophages of the liveras compared to hepatocytes (Vikstedt et al. 2007). Mice with a hepatocyte-specificdeletion of PLTP were generated by injection of PLTP-flox/flippase animals withadenovirus-associated virus expressing Cre-recombinase under control of the thy-roxine binding globulin promoter (Yazdanyar et al. 2013). PLTP activity was314 M. Hoekstra and M. Van Eckreduced by approximately 25 % in these animals with a hepatocyte-specific deletionof PLTP, leading to a 20 % reduction of HDL cholesterol. In addition, non-HDLcholesterol was 29 % lower due to an impaired production of apoB-containinglipoproteins (Yazdanyar et al. 2013). Conversely, liver-specific expression of PLTPin a PLTP knockout background promoted the secretion of apoB-containinglipoproteins by the liver (Yazdanyar and Jiang 2012). The contribution of macro-phage PLTP to plasma PLTP activity was determined by bone marrow transplanta-tion studies in LDLr knockout mice. Selective deletion of PLTP in bone marrow-derived cells led to a decrease in plasma PLTP activity on chow and Western diet(Vikstedt et al. 2007). Vikstedt et al. showed that after 9 weeksWestern diet feedingatherosclerotic lesion size was 29 % smaller in LDLr knockout mice transplantedwith PLTP-deficient bone marrow, which coincided with decreased serum VLDL-cholesterol and phospholipid levels, while HDL phospholipid and apoA-I wereincreased (Vikstedt et al. 2007). In contrast, Valenta et al. also found lower levels ofcholesterol in apoB-containing lipoproteins upon disruption of PLTP in bonemarrow-derived cells of LDLr knockout mice, but surprisingly atheroscleroticlesion development was increased (Valenta et al. 2006). No effect on atherosclero-sis was observed upon transplantation of PLTP-deficient bone marrow into LDLrknockout mice overexpressing human apoA-I (Valenta et al. 2006). The studies byValenta et al. suggest that locally in the arterial wall PLTP produced bymacrophages can be anti-atherogenic. A possible explanation is that PLTP produc-tion by macrophages promotes ABCA1-mediated cholesterol efflux, as evidencedby decreased efflux to apoA-I from PLTP-deficient macrophages (Lee-Rueckertet al. 2006). In agreement, macrophages isolated from PLTP knockout mice wereshown to accumulate more cholesterol upon incubation with native or acetylatedLDL (Ogier et al. 2007). Importantly, the higher levels of lipid-poor apoA-I inLDLr knockout mice overexpressing human apoA-I can overcome thepro-atherogenic effects of deletion of PLTP in macrophages, probably bystimulating the cholesterol efflux capacity of the macrophages.In summary, the effects of PLTP on atherosclerotic lesion development aredetermined by a balance between systemic effects influencing the levels of anti-atherogenic HDL and pro-atherogenic apoB-containing lipoproteins and the anti-oxidant vitamin E and local effects of PLTP produced by macrophages in thearterial wall influencing macrophage apoE production and ABCA1-mediated cho-lesterol efflux.7 Scavenger Receptor BIDuring the final step of the reverse cholesterol transport process, mature HDLparticles deliver their cholesterol load to the liver for subsequent excretion via thebile. The transmembrane glycoprotein scavenger receptor BI (SR-BI) is consideredto be the sole mediator of selective uptake of cholesterol esters from HDL byhepatocytes in mice (Out et al. 2004). Several genetic variations at the SR-BIlocus—either translating in an altered SR-BI protein expression orMouse Models of Disturbed HDL Metabolism 315functionality—have been associated with a change in plasma HDL-cholesterollevels (Hsu et al. 2003; West et al. 2009; Vergeer et al. 2011; Brunhamet al. 2011; Chadwick and Sahoo 2012), which highlights the crucial role forSR-BI in human HDL metabolism. To uncover the role of SR-BI in lipoproteinmetabolism, the group of Monty Krieger developed a mouse model carrying atargeted mutation in the SR-BI gene that leads to complete absence of functionalSR-BI protein in all tissues (Rigotti et al. 1997). In addition, a strain of SR-BIattmice has been generated by the group of Dennis Huszar in which the SR-BIpromoter is mutated, resulting in a lowered protein expression of SR-BI in liver,testis, and adrenals (Varban et al. 1998). Furthermore, hypomorphic SR-BI knock-out mice have been constructed that do not suffer from complete SR-BI deficiency,but show a restricted expression pattern of the protein in liver, kidney, aorta, andsteroidogenic tissues (Huby et al. 2006).Total body SR-BI knockout mice are characterized by a diet-independent hyper-cholesterolemia, which can be primarily attributed to a marked rise in plasmaHDL-cholesterol levels (Rigotti et al. 1997; Van Eck et al. 2003, 2008). Thefractional catabolic rate, plasma decay, and subsequent uptake ofHDL-cholesterol esters by the liver are diminished in SR-BI knockout mice (Outet al. 2004). SR-BIatt mice similarly show a diminished fractional catabolic rateand hepatic selective uptake of HDL-associated cholesterol, also leading to a highersteady-state level of HDL cholesterol in the plasma of these mice (Varbanet al. 1998; Ji et al. 1999). In accordance with a role for hepatic SR-BI in theregulation of plasma HDL-cholesterol levels, both total body and hepatocyte-specific restriction of SR-BI expression in hypomorphic SR-BI knockout mice isalso associated with an increase in plasma HDL-cholesterol levels (Hubyet al. 2006). Although SR-BI is thought to primarily mediate the uptakeHDL-cholesterol esters, in particular free cholesterol levels are higher in plasmain the different SR-BI-deficient mice (Van Eck et al. 2003; Huby et al. 2006; ElBouhassani et al. 2011). The increase in plasma free cholesterol to cholesterol esterratio underlies anemia and thrombocytopenia phenotypes in SR-BI knockout mice(Holm et al. 2002; Meurs et al. 2005; Dole et al. 2008; Korporaal et al. 2011) andcan be attributed to a reduction in the LCAT activity (El Bouhassani et al. 2011). Asjudged by the unchanged plasma apoA-I levels in total body SR-BI knockout mice,only the size and cholesterol content but not the absolute number of HDL particlesis increased in response to SR-BI deficiency (Rigotti et al. 1997). The enlargedHDL particles observed in plasma of SR-BI knockout mice are highly enriched inapoE. In contrast, apoA-II cannot be detected in the HDL fraction of SR-BI-deficient mice, which suggests a generally altered apolipoprotein composition(Rigotti et al. 1997). In line with the notion that SR-BI facilitates HDL-mediatedreverse cholesterol transport, the recovery of radiolabeled cholesterol in feces uponinjection of either peritoneal or J774 macrophages is lower in SR-BI knockout miceas compared to their wild-type littermate controls, despite an increased appearanceof radioactivity in the plasma (HDL) compartment (Zhang et al. 2005a; Zhaoet al. 2011).316 M. Hoekstra and M. Van EckSR-BI knockout mice also display a significant increase in non-HDL-cholesterollevels (Rigotti et al. 1997; Van Eck et al. 2003, 2008). Hepatic VLDL secretion andLPL-mediated lipolysis rates are not affected by SR-BI deficiency (Van Ecket al. 2008). SR-BI has been shown to be directly involved in the selective uptakeof cholesterol esters from LDL and VLDL particles by hepatocytes (Rhaindset al. 2003; Van Eck et al. 2008). It can therefore be anticipated that the increasein the level of cholesterol associated with apoB-containing lipoproteins in SR-BIknockout mice is directly resulting from a diminished clearance by the liver.Two variations in the SR-BI gene have been linked to changes in fertility inhumans (Yates et al. 2011). Without dietary addition of the lipid-lowering drugprobucol, SR-BI knockout female mice are virtually sterile, with a ~90 % reductionin fertility and pup yield as compared to wild-type controls (Miettinen et al. 2001).The development of SR-BI knockout embryos is impaired in ex vivo cultures(Trigatti et al. 1999). Furthermore, developmental abnormalities are also observedin SR-BI knockout embryos in vivo (Santander et al. 2013). Although steroidogenictissues, i.e., ovaries and adrenals, from SR-BI knockout mice exhibit an apparentdecrease in their cholesterol ester stores (Rigotti et al. 1997; Trigatti et al. 1999),levels of the steroid hormone progesterone as well as the number of oocytes andcyclic activity are unaffected by murine SR-BI deficiency (Trigatti et al. 1999). Thelack of pups generated from SR-BI-deficient breedings can thus rather be attributedto embryonic defects than to infertility of the mothers.Human carriers of functional mutations in the SR-BI gene exhibit a decrease intheir adrenal steroidogenesis rate (Vergeer et al. 2011). Similarly, depletion ofadrenocortical cholesterol stores due to SR-BI deficiency translates into a signifi-cant change of the adrenal steroid function in mice. Basal glucocorticoid levels inplasma are similar between SR-BI knockout and wild-type mice (Hoekstraet al. 2008). However, total body SR-BI deficiency is associated with primaryglucocorticoid insufficiency. Total body SR-BI knockout mice display increasedplasma ACTH levels and are unable to increase their adrenal glucocorticoid outputin response to established steroidogenic triggers (Cai et al. 2008; Hoekstraet al. 2008, 2009). It appears that SR-BI-mediated uptake of HDL-cholesterol estersby the adrenals is necessary for a proper steroid output, since the glucocorticoidfunction is also impaired in adrenocortical cell-specific SR-BI knockout mice(Hoekstra et al. 2013b). In further support, hepatocyte-specific SR-BI deficiencydoes not lower adrenal cholesterol levels or impair the adrenal corticosteroneresponse to fasting (El Bouhassani et al. 2011).No atherosclerotic lesion development is normally observed in normolipidemicwild-type mice upon feeding a regular chow diet or a high cholesterol high fatWestern-type diet that does not contain cholic acid. No atherosclerotic plaques canalso be detected in the aortic root of chow-fed SR-BI knockout mice (Zhaoet al. 2011). In contrast, SR-BI knockout mice with increased HDL-cholesterollevels do readily develop macrophage-rich fatty streak lesions after 20 weeks ofWestern-type diet feeding (Van Eck et al. 2003). SR-BI knockout mice display anincrease in non-HDL cholesterol under these feeding conditions (Van Ecket al. 2003). However, the measured levels of apoB-containing lipoproteins uponMouse Models of Disturbed HDL Metabolism 317Western-type diet feeding are still considered to be too low to stimulate theformation of atherosclerotic lesions. This suggests that other SR-BI-relatedmechanisms may underlie the susceptibility for Western-type diet-induced athero-genesis in SR-BI knockout mice. Follow-up studies in liver-specific hypomorphicSR-BI knockout mice have indicated that the SR-BI-mediated atheroprotectionoriginates from both hepatic and peripheral cell sources (Huby et al. 2006; ElBouhassani et al. 2011). A similarly increased susceptibility for atherosclerosisupon ablation of total body SR-BI function has been noted in response to feeding acholic acid-containing atherogenic diet (Zhang et al. 2005b). In accordance with arole for SR-BI in the clearance of apoB-containing lipoproteins, high fat diet-fedSR-BIatt x LDLr knockout mice exhibit higher plasma LDL-cholesterol levels andan increased extent of atherosclerosis as compared to LDLr knockout controls(Huszar et al. 2000). In contrast, total body SR-BI deficiency in high fat diet-fedLDLr knockout mice is associated with lower VLDL/LDL levels, but a similarlyenhanced formation of atherosclerotic lesions in the context of a higher plasmaHDL-cholesterol concentration (Covey et al. 2003). Loss of total body SR-BIfunction in chow diet-fed hyperlipidemic apoE knockout mice is associated withsevere cardiac dysfunction, i.e., a reduced contractility and ejection fraction, andocclusion of the coronary arteries, leading to premature death of these animals atthe age of 6–8 weeks (Braun et al. 2002). Occlusive coronary atherosclerosis and anincreased extent of atherosclerosis in the aortic root are also seen upon geneticdeletion of SR-BI in atherogenic diet-fed hypomorphic APOER61h/h mice (Zhanget al. 2005b). An optimal total body SR-BI function thus protects against athero-sclerosis and myocardial infarction, through (1) its role in hepatic uptake oflipoprotein-derived cholesterol and (2) via its action extrahepatic/peripheral cells.In rodents, high expression of SR-BI is not only found in liver and steroidogenictissues (Acton et al. 1996), but also in tissue macrophages such as liver Kupffercells (Fluiter et al. 1998; Malerød et al. 2002; Hoekstra et al. 2003), which suggeststhat SR-BI can also contribute to macrophage cholesterol homeostasis locallywithin atherosclerotic lesions. Studies using isolated SR-BI-deficient peritonealmacrophages have suggested that SR-BI may facilitate cholesterol efflux to matureHDL (Van Eck et al. 2004). However, macrophage SR-BI does not contribute toreverse cholesterol in vivo, based upon radiolabeled cholesterol recovery studies(Wang et al. 2007). Bone marrow transplantation studies have been executed todelineate the specific role of macrophage SR-BI in the protection against athero-sclerosis. Transplantation of SR-BI knockout bone marrow into LDLr knockoutmice stimulates atherogenesis without altering plasma lipoprotein cholesterol levels(Covey et al. 2003; Van Eck et al. 2004). Inactivation of macrophage SR-BI alsopromotes the development of atherosclerotic plaques in the context of unchangedlipid levels in apoE knockout mice (Zhang et al. 2003). In addition, the cardiachypertrophy and dysfunction and coronary atherosclerosis development areattenuated upon restoration of macrophage SR-BI function in apoE x SR-BI doubleknockout mice (Pei et al. 2013). Strikingly, in marked contrast to the findings fromhyperlipidemic mice, disruption of macrophage SR-BI expression innormolipidemic C57BL/6 mice results in a lower susceptibility for atherosclerosis318 M. Hoekstra and M. Van Eckupon feeding a cholic acid-containing atherogenic diet (Van Eck et al. 2004).C57Bl/6 mice fed a cholic acid-containing atherogenic diet develop small initialatherosclerotic lesions, suggesting that SR-BI might be pro-atherogenic in this earlystage of lesion development. In line, early atherosclerotic lesion development inLDLr knockout mice challenged with Western diet for only 4 weeks was reducedupon disruption of SR-BI in bone marrow-derived cells. These combined studiessuggest that macrophage SR-BI plays a dual role in foam cell formation andatherogenesis, which may be dependent on the stage of atherosclerotic lesiondevelopment.8 Insights from Intercrossing of the Different Knockout MiceSince some of the aforementioned proteins execute both different and overlappingfunctions in the formation of HDL particles and reverse cholesterol transport, it isconceivable that they may act synergistically and/or be able to compensate for eachother’s loss. To uncover interactions between specific gene products several doubleknockout mice have been generated via intercrossing. In line with a differential rolein HDL formation and maturation, studies in mice lacking both ABCA1 and LCATfunction have suggested that these two proteins act synergistically. Hepatic choles-terol accumulation is higher, while peripheral tissue cholesterol levels are lower inABCA1 x LCAT double knockout mice as compared to the respective singleknockout mice, despite an even higher HDL fractional catabolic rate (Hossainet al. 2009). In parallel, probucol-induced inhibition of ABCA1 function furtherlowers adrenal cholesterol levels in LCAT knockout mice (Tomimoto et al. 2001).The ABCA1-mediated formation of HDL particles rather than the SR-BI-mediatedcholesterol uptake is the driving force in the maintenance of plasmaHDL-cholesterol levels, as HDL-cholesterol levels in ABCA1 x SR-BI doubleknockout mice are still close to zero (Zhao et al. 2011). In contrast, ablation ofeither ABCA1 or SR-BI function is equally effective in lowering the rate of in vivomacrophage reverse cholesterol transport (Zhao et al. 2011). The mild pulmonarylipidosis phenotype of ABCA1 knockout mice is severely aggravated in response todeletion of SR-BI function, which suggests that macrophage SR-BI is able topartially compensate for the inability of ABCA1 to mediate cholesterol efflux(Zhao et al. 2011). In accordance, specific disruption of both ABCA1 and SR-BIin bone marrow-derived cells of LDLr knockout mice leads to an added increase in(tissue) macrophage foam cell formation and Western-type diet-induced athero-sclerotic lesion development, in the context of lower plasma cholesterol levels,when compared to both single knockout bone marrow transplanted controls (Zhaoet al. 2010). Importantly, ABCG1 seems to play a more essential role in thecompensatory response to macrophage ABCA1 deficiency. Mass efflux of choles-terol from macrophages to both apoA-I and HDL is virtually absent as a result ofcombined deletion of ABCA1 and ABCG1 (Out et al. 2008a). Already on a chowdiet ABCA1 x ABCG1 double knockout mice display marked accumulation ofcholesterol not only within the lungs (reminiscent of the ABCG1 knockoutMouse Models of Disturbed HDL Metabolism 319phenotype), but also in primary and secondary lymphoid organs and macrophage-rich areas of the liver and intestine (Out et al. 2008a). Furthermore, the doubledeletion exacerbates high fat diet-induced lymphocytosis (Yvan-Charvetet al. 2008). Transplantation of bone marrow from ABCA1 x ABCG1 doubleknockout mice into LDLr knockout mice is associated with resistance toWestern-type diet-induced hyperlipidemia (Out et al. 2008b). However, despitethe rather low plasma cholesterol levels under Western-type diet feedingconditions, ABCA1 x ABCG1 double knockout bone marrow recipient miceshow significant plaque development and exhibit distinct lipid deposition inmacrophage-rich tissues (Out et al. 2008b). This highlights the necessity for thesetransporters to overcome macrophage foam cell formation and atherosclerosis.Subsequent bone marrow transplantations into mice with a heterozygous mutationin the LDLr gene (LDLr+/) have validated the increased susceptibility to macro-phage cholesterol accumulation and atherosclerosis in response to the combineddeficiency in macrophage ABCA1 and ABCG1 upon challenge with a cholate-containing diet (Yvan-Charvet et al. 2007). Furthermore, these studies havesuggested that the combined deficiency of these ABC transporters in bone marrowalso impacts on the inflammatory function and susceptibility to apoptosis ofmacrophages as well as the cyclic activity of hematopoietic stem cells (Yvan-Charvet et al. 2008, 2010a).9 Conclusions from the Gene Knockout Mouse StudiesGeneration of the different knockout mice has significantly contributed to ourunderstanding of HDL’s metabolism and function (see Fig. 1 for overview). Ithas become clear that the initial generation and maturation of HDL particles as wellas the selective uptake of its cholesterol by the liver are important parameters in thelife cycle of HDL. In line with the proposed atheroprotective function of HDL,murine HDL deficiency consistently predisposes to the development of atheroscle-rotic lesions. However, it should be noted that many of the detrimental atheroscle-rosis effects observed in response to HDL deficiency in mice cannot be solelyattributed to the low HDL levels per se, as the low HDL levels are in most modelsparalleled by changes in non-HDL-cholesterol levels. Although in the humansetting, low HDL-cholesterol levels predict increased cardiovascular risk at allplasma non-HDL-cholesterol levels, the mouse data firmly establish that HDLand non-HDL metabolism are heavily intertwined.Based upon the bone marrow transplantation studies in hyperlipidemic mice, itappears that the cholesterol efflux function of HDL is of critical importance toovercome foam cell formation and the development of atherosclerotic lesions. Wetherefore consider it therapeutically highly relevant to increase the expressionand/or activity of the cholesterol transporters in macrophages. In this regard, specialfocus should be given to macrophage ABCA1 as this transporter seems to be thedriving force in the atheroprotection.320 M. Hoekstra and M. Van EckAlthough HDL is predominantly studied for its atheroprotective action, it can beappreciated that the mouse data suggest an essential role for HDL as cholesteroldonor for steroidogenic tissues, including the adrenals and ovaries. Furthermore, itappears that a relevant interaction exists between HDL-mediated cellular choles-terol efflux and the susceptibility to inflammation, which (1) provides strongsupport for the novel concept that inflammation and metabolism are intertwiningbiological processes and (2) identifies the efflux function of HDL as putativetherapeutic target also in other inflammatory diseases than atherosclerosis, includ-ing sepsis and diabetes.10 Cholesterol Ester Transfer Protein Transgenic MiceDespite the fact that a great deal regarding the function of specific gene productshas been learned from the murine transgenic studies it should be acknowledged thatone should take care when directly translating the data to the human situation.Humans, in contrast to mice, naturally express cholesterol ester transfer protein(CETP) that plays a major role in total body cholesterol homeostasis as it is able toexchange cholesterol esters and triglycerides between cholesterol-rich HDL andApoA-I knockout- Plasma HDL-C ↓↓↓- Adrenal cholesterol ↓; adrenal funcon ↓- Ovarian cholesterol ↓; female ferlity =- Atherosclerosis ↑ (LDLr KO on chow); plasma VLDL-C ↑ - Atherosclerosis = (LDLr KO on Western diet); plasma VLDL-C ↓- Development of skin lesions containing lipid-filled macrophages- Cholesterol in B and T cells of lymph nodes ↑ABCG1 knockout- Plasma HDL-C =- Adrenal funcon ?- Female ferlity =- Pulmonary lipidosis- Inial atherosclerosis↑- Advanced atherosclerosis ↓- Similar effects in general KO’sand bone marrow deleonABCA1 knockout- Plasma HDL-C ↓↓↓- Adrenal cortex cholesterol esters ↑- Female ferlity ↓; steroid hormones ↓ ; placenta malformaons- Atherosclerosis =; plasma VLDL-C ↓- Bone marrow deleon: atherosclerosis ↑, blood leukocytes ↑- Macrophage/Neutrophil deleon: atherosclerosis =- Liver deleon: atherosclerosis = (LDLr KO), atherosclerosis ↑ (apoE KO)LCAT knockout- Plasma HDL-C ↓↓↓ - Adrenal cholesterol ↓; adrenal funcon ↓ - Female ferlity =- Plasma TG ↑ and VLDL-C ↑- Atherosclerosis =↓↑ (depending on thegenec background, diet and effects onplasma VLDL-C)PLTP knockout- Plasma HDL-C ↓↓- Adrenal funcon ?- Female ferlity =- PL and FC-rich lamellar parcles ↑ (saturated fat-rich diet)- Atherosclerosis ↓= (depending on the genec background, dietand effects on plasma VLDL-C)- Bone marrow deleon: atherosclerosis ↓↑; plasma VLDL-C ↓SR-BI knockout- Plasma HDL-C ↑↑- Plasma VLDL-C ↑- Adrenal cholesterol ↓; adrenal funcon ↓ - Female ferlity ↓; abnormal embryo development- Atherosclerosis ↑- Severe cardiac dysfuncon and premature death in apoE KO’s- Bone marrow deleon: inial lesions ↓, advanced lesions ↑Fig. 1 Major phenotypic changes in mouse models of disturbed HDLmetabolism. An overview isgiven of the effects of deletion of apolipoprotein A-I (apoA-I), the ABC transporters A1 (ABCA1)and G1 (ABCG1), lecithin-cholesterol acyltransferase (LCAT), phospholipid transfer protein(PLTP), and scavenger receptor BI (SR-BI) for HDL’s metabolism, adrenal function, femalefertility, and its protection against atherosclerosis in mice. ¼ no effect, # decreased, " increased,C cholesterol, PL phospholipids, TG triglycerides, KO knockoutMouse Models of Disturbed HDL Metabolism 321triglyceride-rich VLDL/LDL particles. In agreement with a major role for CETP inthe modulation of plasma HDL-cholesterol levels, genetic CETP deficiency isassociated with marked hyperalphalipoproteinemia, i.e., relatively highHDL-cholesterol levels, in humans (Rhyne et al. 2006; Calabresi et al. 2009). Toovercome this apparent discrepancy between mice and humans in studies dealingwith lipoprotein metabolism, the group of Alan Tall generated a mouse line(NFR-CETP) expressing the human CETP transgene under the control of its naturalflanking regulatory elements (Jiang et al. 1992). Initial cloning studies (Draynaet al. 1987) indicated that CETP is primarily derived from liver, small intestine,adrenals, and spleen in humans. In accordance, relatively high CETP mRNA levelsare found in liver, spleen, and small intestine of human NFR-CETP transgenic mice(Jiang et al. 1992). Of note, the Tall group also generated a transgenic mouse lineexpressing CETP under control of the methallothionein-I (MT) gene (Jianget al. 1992). In MT-CETP transgenic mice, additional high expression of CETPcan be detected in kidney, adipose tissue, and heart. Importantly, the expression ofCETP in MT-CETP transgenic is not responsive to high cholesterol/high fat dietfeeding, which contrasts the findings from NFR-CETP transgenic mice (Jianget al. 1992) and species naturally expressing CETP, i.e., monkeys and rabbits(Quinet et al. 1990; Pape et al. 1991). Given the apparent limitations ofMT-CETP transgenic mice, in our opinion the NFR-CETP mouse model shouldpreferentially be used in studies aimed at translation to the human situation.Therefore, findings from NFR-CETP transgenic mice will only be reviewed inthe next part.Chow-fed CETP transgenic mice exhibit a ~15 % lower plasma total cholesterollevel as compared to non-transgenic controls (Masucci-Magoulas et al. 1996;Harada et al. 2007). In contrast, plasma triglyceride levels are marginally increasedin CETP carrying mice upon feeding chow diet (Zhou et al. 2006; Haradaet al. 2007) due to a slightly diminished clearance of triglyceride-rich lipoproteins(Salerno et al. 2009). The decrease in total cholesterol in CETP transgenic mice canprimarily be attributed to lower levels of cholesterol associated with HDL (Zhouet al. 2006). The fractional catabolic rate and uptake of HDL-cholesterol esters bythe liver, adrenals, and adipose tissue are increased in CETP transgenic mice, whichis associated with an increase in hepatic cholesterol stores (Harada et al. 2007). As aresult, CETP transgenic mice exhibit a rise in the plasma total cholesterol overHDL-cholesterol ratio (Clee et al. 1997). The decrease in HDL-cholesterol levelscannot be attributed to a change in LCAT levels. Intriguingly, the presence of CETPactually enhances the saturation level of the LCAT-mediated cholesterol esterifica-tion reaction (Oliveira et al. 1997). This suggests that CETP generates particles thatcan serve as optimal substrates for LCAT.Despite the marked increase in plasma HDL-cholesterol levels associated withcomplete CETP deficiency, variable effects on atherosclerosis and cardiovasculardisease risk in response to genetic variations in the CETP gene have been noted inthe human setting (Zhong et al. 1996; Agerholm-Larsen et al. 2000; Curbet al. 2004; Zheng et al. 2004; Rhyne et al. 2006). To provide clear insight intothe contribution of CETP to atherosclerotic lesion development, NFR-CETP322 M. Hoekstra and M. Van Ecktransgenic mice have been bred onto several different atherosclerosis-susceptiblemouse backgrounds. CETP expression in heterozygous LDLr knockout mice on acholic acid-containing atherogenic diet does not significantly alter plasma HDL- ornon-HDL-cholesterol levels. As a result, CETP does not impact on the susceptibil-ity to atherosclerosis in heterozygous LDLr knockout mice (Casquero et al. 2006).In contrast, CETP executes a clear pro-atherogenic effect in hyperlipidemic apoEknockout and homozygous LDLr knockout mice (Plump et al. 1999). CETP lowersplasma HDL-cholesterol levels in chow-fed apoE knockout mice without changinglevels of cholesterol associated with non-HDL particles. In addition, triglyceridelevels are increased in the plasma of CETP-expressing apoE knockout mice. CETPexpression does not alter plasma HDL-cholesterol levels in LDLr knockout micechallenged with a Western-type high fat/high cholesterol diet. However, levels ofcholesterol associated with pro-atherogenic apoB-containing lipoproteins are mark-edly increased in response to CETP expression in LDLr knockout mice. It thusappears that the increased atherosclerotic lesion development in apoE and LDLrknockout mice upon CETP expression can be directly related to an increasedatherogenic index of the plasma compartment (higher non-HDL/HDL ratio) inboth types of hyperlipidemic mice. In further support, a marked increase in plasmanon-HDL-cholesterol levels and atherosclerotic lesion development—in the con-text of a marginal but significant decrease in HDL-cholesterol levels—was notedwhen we expressed CETP only in bone marrow-derived cells of LDLr knockoutmice through bone marrow transplantation (Van Eck et al. 2007).A clear drawback of using LDLr and apoE knockout mice for studies regardinglipoprotein metabolism is that they do not exhibit a normal clearance of apoE-containing VLDL/LDL particles. To determine the effect of CETP expression onlipoprotein metabolism and atherosclerosis development in a more relevant“humanized” setting, CETP transgenic mice have also been crossbred withhyperlipidemic apoE*3-Leiden mice. Transgenic apoE*3-Leiden mice express amutation of the human apoE3 gene resulting in a slightly attenuated clearance ofapoB-containing particles via the LDLr pathway (van den Maagdenberget al. 1993). As a result, these mice (1) exhibit a lipoprotein cholesterol distributionprofile that is highly similar to that found in humans (van den Maagdenberget al. 1993; van Vlijmen et al. 1994), (2) are susceptible to atherosclerotic lesiondevelopment when fed a high fat/high cholesterol Western-type diet devoid ofcholic acid (van Vlijmen et al. 1994), and (3) show a similar plasma lipid responseas human subjects to several drug interventions (van der Hoogt et al. 2007; de Haanet al. 2008a; van der Hoorn et al. 2008). ApoE*3-Leiden x CETP double transgenicmice as compared to apoE*3-Leiden controls display a significant increase inplasma total cholesterol levels on both chow and under Western-type diet feedingconditions (Westerterp et al. 2006). Under these dietary conditions, VLDL-cholesterol levels are increased twofold, while HDL-cholesterol levels as well asplasma apoA-I levels are 25 % lower in apoE*3-Leiden x CETP transgenic mice(Westerterp et al. 2006). Importantly, apoE*3-Leiden x CETP transgenic miceexhibit increased atherosclerotic lesion development in the aortic root as comparedMouse Models of Disturbed HDL Metabolism 323to apoE*3-Leiden controls (Westerterp et al. 2006). Based upon these combinedfindings, CETP should clearly be considered a pro-atherogenic protein moiety.In the last decade, therapeutic strategies have focused on inhibiting CETPactivity through the use of a variety of CETP-binding drugs, i.e., torcetrapib, thatform an inactive complex between CETP and HDL. Studies in apoE*3-Leiden xCETP transgenic mice have suggested that the HDL increase associated withtorcetrapib treatment does not add to the beneficial impact of statin-induced lipid-lowering on atherosclerosis (de Haan et al. 2008b). Importantly, the ILLUMINATEand dal-OUTCOMES trials also failed to show the beneficial effect of CETPinhibition in statin-treated cardiovascular disease patients (Barter et al. 2007;Schwartz et al. 2012). Combination treatment with torcetrapib and statin increasesthe macrophage content in atherosclerotic lesions in apoE*3-Leiden x CETPtransgenic mice as compared to statin treatment alone (de Haan et al. 2008b). Ahigher lesional macrophage content is generally associated with an increasedsusceptibility for plaque rupture and future cardiovascular events in the humansituation (Redgrave et al. 2006; Marnane et al. 2014). An effect of CETP inhibitionon plaque stability may therefore possibly have contributed to the negative outcomeof the aforementioned torcetrapib and dalcetrapib trials.Concluding RemarkThe use of dedicated knockout and transgenic mouse models has significantlyincreased our knowledge on the different functions and metabolism of HDL.However, as evident from the recent failures of the CETP inhibition-basedclinical trials, more research is clearly needed to better understand how we canuse HDL as target in the treatment of patients at risk of cardiovascular diseaseand other immune-related disorders. The generation of “humanized” mousemodels such as the apoE*3-Leiden x CETP transgenic mice will hopefully aidin the development of novel therapeutic approaches beneficially impacting onHDL functionality and atherosclerosis outcome also in the clinical setting.Acknowledgments This work was supported by the European COST Action BM0904 “HDL–From Biological Understanding to Clinical Exploitation,” the Netherlands Heart Foundation(Established Investigator Grant 2007 T056 to M. Van Eck and Senior Postdoc Grants 2008 T70and 2012 T80 to M. Hoekstra), and VICI Grant 91813603 from the Netherlands Organization forScientific Research.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.324 M. Hoekstra and M. Van EckReferencesActon S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M (1996) Identification ofscavenger receptor SR-BI as a high density lipoprotein receptor. Science 271:518–520Agerholm-Larsen B, Tybjaerg-Hansen A, Schnohr P, Steffensen R, Nordestgaard BG (2000)Common cholesteryl ester transfer protein mutations, decreased HDL cholesterol, and possibledecreased risk of ischemic heart disease: the Copenhagen City heart study. Circulation102:2197–2203Aiello RJ, Brees D, Bourassa PA, Royer L, Lindsey S, Coskran T, Haghpassand M, Francone OL(2002) Increased atherosclerosis in hyperlipidemic mice with inactivation of ABCA1 inmacrophages. Arterioscler Thromb Vasc Biol 22:630–637Albers JJ, Vuletic S, Cheung MC (2012) Role of plasma phospholipid transfer protein in lipid andlipoprotein metabolism. Biochim Biophys Acta 1821:345–357Baigent C, Blackwell L, Emberson J, Holland LE, Reith C, Bhala N, Peto R, Barnes EH, Keech A,Simes J, Collins R, Cholesterol Treatment Trialists’ (CTT) Collaboration (2010) Efficacy andsafety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000participants in 26 randomised trials. Lancet 376:1670–1681Baldán A, Tarr P, Vales CS, Frank J, Shimotake TK, Hawgood S, Edwards PA (2006a) Deletion ofthe transmembrane transporter ABCG1 results in progressive pulmonary lipidosis. J Biol Chem281:29401–29410Baldán A, Pei L, Lee R, Tarr P, Tangirala RK, Weinstein MM, Frank J, Li AC, Tontonoz P,Edwards PA (2006b) Impaired development of atherosclerosis in hyperlipidemic Ldlr-/- andApoE-/- mice transplanted with Abcg1-/- bone marrow. Arterioscler Thromb Vasc Biol26:2301–2307Baldán A, Gomes AV, Ping P, Edwards PA (2008) Loss of ABCG1 results in chronic pulmonaryinflammation. J Immunol 180:3560–3568Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJ, Komajda M, Lopez-Sendon J,Mosca L, Tardif JC, Waters DD, Shear CL, Revkin JH, Buhr KA, Fisher MR, Tall AR,Brewer B, Investigators ILLUMINATE (2007) Effects of torcetrapib in patients at high riskfor coronary events. N Engl J Med 357:2109–2122Berge KE, Leren TP (2010) Mutations in APOA-I and ABCA1 in Norwegians with low levels ofHDL cholesterol. Clin Chim Acta 411:2019–2023Bi X, Zhu X, Duong M, Boudyguina EY, Wilson MD, Gebre AK, Parks JS (2013) Liver ABCA1deletion in LDLrKO mice does not impair macrophage reverse cholesterol transport orexacerbate atherogenesis. Arterioscler Thromb Vasc Biol 33:2288–2296Bodzioch M, Orsó E, Klucken J, Langmann T, Böttcher A, Diederich W, Drobnik W, Barlage S,Büchler C, Porsch-Ozcürümez M, Kaminski WE, Hahmann HW, Oette K, Rothe G,Aslanidis C, Lackner KJ, Schmitz G (1999) The gene encoding ATP-binding cassette trans-porter 1 is mutated in Tangier disease. Nat Genet 22:347–351Braun A, Trigatti BL, Post MJ, Sato K, Simons M, Edelberg JM, Rosenberg RD, Schrenzel M,Krieger M (2002) Loss of SR-BI expression leads to the early onset of occlusive atheroscleroticcoronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, andpremature death in apolipoprotein E-deficient mice. Circ Res 90:270–276Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, Yu L, Brewer C,Collins JA, Molhuizen HO, Loubser O, Ouelette BF, Fichter K, Ashbourne-Excoffon KJ,Sensen CW, Scherer S, Mott S, Denis M, Martindale D, Frohlich J, Morgan K, Koop B,Pimstone S, Kastelein JJ, Genest J Jr, Hayden MR (1999) Mutations in ABC1 in Tangierdisease and familial high-density lipoprotein deficiency. Nat Genet 22:336–345Brunham LR, Kruit JK, Iqbal J, Fievet C, Timmins JM, Pape TD, Coburn BA, Bissada N, Staels B,Groen AK, Hussain MM, Parks JS, Kuipers F, Hayden MR (2006) Intestinal ABCA1 directlycontributes to HDL biogenesis in vivo. J Clin Invest 116:1052–1062Mouse Models of Disturbed HDL Metabolism 325Brunham LR, Singaraja RR, Duong M, Timmins JM, Fievet C, Bissada N, Kang MH, Samra A,Fruchart JC, McManus B, Staels B, Parks JS, Hayden MR (2009) Tissue-specific roles ofABCA1 influence susceptibility to atherosclerosis. Arterioscler Thromb Vasc Biol 29:548–554Brunham LR, Tietjen I, Bochem AE, Singaraja RR, Franchini PL, Radomski C, Mattice M,Legendre A, Hovingh GK, Kastelein JJ, Hayden MR (2011) Novel mutations in scavengerreceptor BI associated with high HDL cholesterol in humans. Clin Genet 79:575–581Cai L, Ji A, de Beer FC, Tannock LR, van der Westhuyzen DR (2008) SR-BI protects againstendotoxemia in mice through its roles in glucocorticoid production and hepatic clearance. JClin Invest 118:364–375Calabresi L, Nilsson P, Pinotti E, Gomaraschi M, Favari E, Adorni MP, Bernini F, Sirtori CR,Calandra S, Franceschini G, Tarugi P (2009) A novel homozygous mutation in CETP gene as acause of CETP deficiency in a Caucasian kindred. Atherosclerosis 205:506–511Calpe-Berdiel L, Rotllan N, Palomer X, Ribas V, Blanco-Vaca F, Escolà-Gil JC (2005) Directevidence in vivo of impaired macrophage-specific reverse cholesterol transport inATP-binding cassette transporter A1-deficient mice. Biochim Biophys Acta 1738:6–9Casquero AC, Berti JA, Salerno AG, Bighetti EJ, Cazita PM, Ketelhuth DF, Gidlund M, OliveiraHC (2006) Atherosclerosis is enhanced by testosterone deficiency and attenuated by CETPexpression in transgenic mice. J Lipid Res 47:1526–1534Chadwick AC, Sahoo D (2012) Functional characterization of newly-discovered mutations inhuman SR-BI. PLoS ONE 7:e45660Christiansen-Weber TA, Voland JR, Wu Y, Ngo K, Roland BL, Nguyen S, Peterson PA, Fung-Leung WP (2000) Functional loss of ABCA1 in mice causes severe placental malformation,aberrant lipid distribution, and kidney glomerulonephritis as well as high-density lipoproteincholesterol deficiency. Am J Pathol 157:1017–1029Chung S, Timmins JM, Duong M, Degirolamo C, Rong S, Sawyer JK, Singaraja RR, Hayden MR,Maeda N, Rudel LL, Shelness GS, Parks JS (2010) Targeted deletion of hepatocyte ABCA1leads to very low density lipoprotein triglyceride overproduction and low density lipoproteinhypercatabolism. J Biol Chem 285:12197–12209Clee SM, Zhang H, Bissada N, Miao L, Ehrenborg E, Benlian P, Shen GX, Angel A, LeBoeuf RC,Hayden MR (1997) Relationship between lipoprotein lipase and high density lipoproteincholesterol in mice: modulation by cholesteryl ester transfer protein and dietary status. JLipid Res 38:2079–2089Covey SD, Krieger M, Wang W, Penman M, Trigatti BL (2003) Scavenger receptor class B typeI-mediated protection against atherosclerosis in LDL receptor-negative mice involves itsexpression in bone marrow-derived cells. Arterioscler Thromb Vasc Biol 23:1589–1594Curb JD, Abbott RD, Rodriguez BL, Masaki K, Chen R, Sharp DS, Tall AR (2004) A prospectivestudy of HDL-C and cholesteryl ester transfer protein gene mutations and the risk of coronaryheart disease in the elderly. J Lipid Res 45:948–953Dastani Z, Dangoisse C, Boucher B, Desbiens K, Krimbou L, Dufour R, Hegele RA, Pajukanta P,Engert JC, Genest J, Marcil M (2006) A novel nonsense apolipoprotein A-I mutation (apoA-I(E136X)) causes low HDL cholesterol in French Canadians. Atherosclerosis 185:127–136Day JR, Albers JJ, Lofton-Day CE, Gilbert TL, Ching AF, Grant FJ, O’Hara PJ, Marcovina SM,Adolphson JL (1994) Complete cDNA encoding human phospholipid transfer protein fromhuman endothelial cells. J Biol Chem 269:9388–9391de Haan W, van der Hoogt CC, Westerterp M, Hoekstra M, Dallinga-Thie GM, Princen HM,Romijn JA, Jukema JW, Havekes LM, Rensen PC (2008a) Atorvastatin increases HDLcholesterol by reducing CETP expression in cholesterol-fed APOE*3-Leiden.CETP mice.Atherosclerosis 197:57–63de Haan W, de Vries-van der Weij J, van der Hoorn JW, Gautier T, van der Hoogt CC,Westerterp M, Romijn JA, Jukema JW, Havekes LM, Princen HM, Rensen PC (2008b)Torcetrapib does not reduce atherosclerosis beyond atorvastatin and induces moreproinflammatory lesions than atorvastatin. Circulation 117:2515–2522326 M. Hoekstra and M. Van EckDesrumaux CM, Mak PA, Boisvert WA, Masson D, Stupack D, Jauhiainen M, Ehnholm C, CurtissLK (2003) Phospholipid transfer protein is present in human atherosclerotic lesions and isexpressed by macrophages and foam cells. J Lipid Res 44:1453–1461Dole VS, Matuskova J, Vasile E, Yesilaltay A, Bergmeier W, Bernimoulin M, Wagner DD,Krieger M (2008) Thrombocytopenia and platelet abnormalities in high-density lipoproteinreceptor-deficient mice. Arterioscler Thromb Vasc Biol 28:1111–1116Drayna D, Jarnagin AS, McLean J, Henzel W, Kohr W, Fielding C, Lawn R (1987) Cloning andsequencing of human cholesteryl ester transfer protein cDNA. Nature 327:632–634Drobnik W, Lindenthal B, Lieser B, Ritter M, Christiansen Weber T, Liebisch G, Giesa U, Igel M,Borsukova H, Büchler C, Fung-Leung WP, Von Bergmann K, Schmitz G (2001) ATP-bindingcassette transporter A1 (ABCA1) affects total body sterol metabolism. Gastroenterology120:1203–1211El Bouhassani M, Gilibert S, Moreau M, Saint-Charles F, Tréguier M, Poti F, Chapman MJ, LeGoff W, Lesnik P, Huby T (2011) Cholesteryl ester transfer protein expression partiallyattenuates the adverse effects of SR-BI receptor deficiency on cholesterol metabolism andatherosclerosis. J Biol Chem 286:17227–17238Fluiter K, van der Westhuijzen DR, van Berkel TJ (1998) In vivo regulation of scavenger receptorBI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchy-mal and Kupffer cells. J Biol Chem 273:8434–8438Francone OL, Subbaiah PV, van Tol A, Royer L, Haghpassand M (2003) Abnormal phospholipidcomposition impairs HDL biogenesis and maturation in mice lacking Abca1. Biochemistry42:8569–8578Frikke-Schmidt R, Nordestgaard BG, Stene MC, Sethi AA, Remaley AT, Schnohr P, Grande P,Tybjaerg-Hansen A (2008a) Association of loss-of-function mutations in the ABCA1 genewith high-density lipoprotein cholesterol levels and risk of ischemic heart disease. JAMA299:2524–2532Frikke-Schmidt R, Nordestgaard BG, Jensen GB, Steffensen R, Tybjaerg-Hansen A (2008b)Genetic variation in ABCA1 predicts ischemic heart disease in the general population.Arterioscler Thromb Vasc Biol 28:180–186Furbee JW Jr, Sawyer JK, Parks JS (2002) Lecithin:cholesterol acyltransferase deficiencyincreases atherosclerosis in the low density lipoprotein receptor and apolipoprotein E knockoutmice. J Biol Chem 277:3511–3519Furuyama S, Uehara Y, Zhang B, Baba Y, Abe S, Iwamoto T, Miura S, Saku K (2009) Genotypiceffect of ABCG1 gene promoter257T>G polymorphism on coronary artery disease severityin Japanese men. J Atheroscler Thromb 16:194–200Glomset JA, Norum KR, King W (1970) Plasma lipoproteins in familial lecithin: cholesterolacyltransferase deficiency: lipid composition and reactivity in vitro. J Clin Invest49:1827–1837Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR (1977) High density lipoproteinas a protective factor against coronary heart disease. The Framingham study. Am J Med62:707–714Gordon JI, Smith DP, Andy R, Alpers DH, Schonfeld G, Strauss AW (1982) The primarytranslation product of rat intestinal apolipoprotein A-I mRNA is an unusual preproprotein. JBiol Chem 257:971–978Haghpassand M, Bourassa PA, Francone OL, Aiello RJ (2001) Monocyte/macrophage expressionof ABCA1 has minimal contribution to plasma HDL levels. J Clin Invest 108:1315–1320Harada LM, Amigo L, Cazita PM, Salerno AG, Rigotti AA, Quintão EC, Oliveira HC (2007)CETP expression enhances liver HDL-cholesteryl ester uptake but does not alter VLDL andbiliary lipid secretion. Atherosclerosis 191:313–318Hoekstra M, Kruijt JK, Van Eck M, Van Berkel TJ (2003) Specific gene expression ofATP-binding cassette transporters and nuclear hormone receptors in rat liver parenchymal,endothelial, and Kupffer cells. J Biol Chem 278:25448–25453Mouse Models of Disturbed HDL Metabolism 327Hoekstra M, Meurs I, Koenders M, Out R, Hildebrand RB, Kruijt JK, Van Eck M, Van Berkel TJ(2008) Absence of HDL cholesteryl ester uptake in mice via SR-BI impairs an adequateadrenal glucocorticoid-mediated stress response to fasting. J Lipid Res 49:738–745Hoekstra M, Ye D, Hildebrand RB, Zhao Y, Lammers B, Stitzinger M, Kuiper J, Van Berkel TJ,Van Eck M (2009) Scavenger receptor class B type I-mediated uptake of serum cholesterol isessential for optimal adrenal glucocorticoid production. J Lipid Res 50:1039–1046Hoekstra M, Korporaal SJ, van der Sluis RJ, Hirsch-Reinshagen V, Bochem AE, Wellington CL,Van Berkel TJ, Kuivenhoven JA, Van Eck M (2013a) LCAT deficiency in mice is associatedwith a diminished adrenal glucocorticoid function. J Lipid Res 54:358–364Hoekstra M, van der Sluis RJ, Van Eck M, Van Berkel TJ (2013b) Adrenal-specific scavengerreceptor BI deficiency induces glucocorticoid insufficiency and lowers plasma very-low-density and low-density lipoprotein levels in mice. Arterioscler Thromb Vasc Biol 33:e39–e46Holm TM, Braun A, Trigatti BL, Brugnara C, Sakamoto M, Krieger M, Andrews NC (2002)Failure of red blood cell maturation in mice with defects in the high-density lipoproteinreceptor SR-BI. Blood 99:1817–1824Hong SH, Rhyne J, Zeller K, Miller M (2002) ABCA1(Alabama): a novel variant associated withHDL deficiency and premature coronary artery disease. Atherosclerosis 164:245–250Hossain MA, Tsujita M, Akita N, Kobayashi F, Yokoyama S (2009) Cholesterol homeostasis inABCA1/LCAT double-deficient mouse. Biochim Biophys Acta 1791:1197–1205Hovingh GK, Brownlie A, Bisoendial RJ, Dube MP, Levels JH, Petersen W, Dullaart RP, StroesES, Zwinderman AH, de Groot E, Hayden MR, Kuivenhoven JA, Kastelein JJ (2004) A novelapoA-I mutation (L178P) leads to endothelial dysfunction, increased arterial wall thickness,and premature coronary artery disease. J Am Coll Cardiol 44:1429–1435Hovingh GK, Hutten BA, Holleboom AG, Petersen W, Rol P, Stalenhoef A, Zwinderman AH, deGroot E, Kastelein JJ, Kuivenhoven JA (2005) Compromised LCAT function is associatedwith increased atherosclerosis. Circulation 112:879–884Hsu LA, Ko YL, Wu S, Teng MS, Peng TY, Chen CF, Chen CF, Lee YS (2003) Associationbetween a novel 11-base pair deletion mutation in the promoter region of the scavengerreceptor class B type I gene and plasma HDL cholesterol levels in Taiwanese Chinese.Arterioscler Thromb Vasc Biol 23:1869–1874Huang W, Moriyama K, Koga T, Hua H, Ageta M, Kawabata S, Mawatari K, Imamura T, Eto T,Kawamura M, Teramoto T, Sasaki J (2001) Novel mutations in ABCA1 gene in Japanesepatients with Tangier disease and familial high density lipoprotein deficiency with coronaryheart disease. Biochim Biophys Acta 1537:71–78Huby T, Doucet C, Dachet C, Ouzilleau B, Ueda Y, Afzal V, Rubin E, Chapman MJ, Lesnik P(2006) Knockdown expression and hepatic deficiency reveal an atheroprotective role for SR-BIin liver and peripheral tissues. J Clin Invest 116:2767–2776Huszar D, Varban ML, Rinninger F, Feeley R, Arai T, Fairchild-Huntress V, Donovan MJ, TallAR (2000) Increased LDL cholesterol and atherosclerosis in LDL receptor-deficient mice withattenuated expression of scavenger receptor B1. Arterioscler Thromb Vasc Biol 20:1068–1073Ishibashi S, Goldstein JL, Brown MS, Herz J, Burns DK (1994) Massive xanthomatosis andatherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice. J Clin Invest93:1885–1893Ishida BY, Blanche PJ, Nichols AV, Yashar M, Paigen B (1991) Effects of atherogenic dietconsumption on lipoproteins in mouse strains C57BL/6 and C3H. J Lipid Res 32:559–568Ji Y, Wang N, Ramakrishnan R, Sehayek E, Huszar D, Breslow JL, Tall AR (1999) Hepaticscavenger receptor BI promotes rapid clearance of high density lipoprotein free cholesterol andits transport into bile. J Biol Chem 274:33398–33402Jiang XC, Agellon LB, Walsh A, Breslow JL, Tall A (1992) Dietary cholesterol increasestranscription of the human cholesteryl ester transfer protein gene in transgenic mice. Depen-dence on natural flanking sequences. J Clin Invest 90:1290–1295328 M. Hoekstra and M. Van EckJiang XC, Bruce C, Mar J, Lin M, Ji Y, Francone OL, Tall AR (1999) Targeted mutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levels. J ClinInvest 103:907–914Jiang XC, Qin S, Qiao C, Kawano K, Lin M, Skold A, Xiao X, Tall AR (2001) Apolipoprotein Bsecretion and atherosclerosis are decreased in mice with phospholipid-transfer protein defi-ciency. Nat Med 7:847–852Jiang XC, Tall AR, Qin S, Lin M, Schneider M, Lalanne F, Deckert V, Desrumaux C, Athias A,Witztum JL, Lagrost L (2002) Phospholipid transfer protein deficiency protects circulatinglipoproteins from oxidation due to the enhanced accumulation of vitamin E. J Biol Chem277:31850–31856Kawano K, Qin S, Vieu C, Collet X, Jiang XC (2002) Role of hepatic lipase and scavengerreceptor BI in clearing phospholipid/free cholesterol-rich lipoproteins in PLTP-deficient mice.Biochim Biophys Acta 1583:133–140Klucken J, Büchler C, Orsó E, Kaminski WE, Porsch-Ozcürümez M, Liebisch G, Kapinsky M,Diederich W, Drobnik W, Dean M, Allikmets R, Schmitz G (2000) ABCG1 (ABC8), thehuman homolog of the Drosophila white gene, is a regulator of macrophage cholesterol andphospholipid transport. Proc Natl Acad Sci USA 97:817–822Korporaal SJ, Meurs I, Hauer AD, Hildebrand RB, Hoekstra M, Cate HT, Praticò D, AkkermanJW, Van Berkel TJ, Kuiper J, Van Eck M (2011) Deletion of the high-density lipoproteinreceptor scavenger receptor BI in mice modulates thrombosis susceptibility and indirectlyaffects platelet function by elevation of plasma free cholesterol. Arterioscler Thromb Vasc Biol31:34–42Kunnen S, Van Eck M (2012) Lecithin:cholesterol acyltransferase: old friend or foe in atheroscle-rosis? J Lipid Res 53:1783–1799Laffitte BA, Joseph SB, Chen M, Castrillo A, Repa J, Wilpitz D, Mangelsdorf D, Tontonoz P(2003) The phospholipid transfer protein gene is a liver X receptor target expressed bymacrophages in atherosclerotic lesions. Mol Cell Biol 23:2182–2191Lambert G, Sakai N, Vaisman BL, Neufeld EB, Marteyn B, Chan CC, Paigen B, Lupia E,Thomas A, Striker LJ, Blanchette-Mackie J, Csako G, Brady JN, Costello R, Striker GE,Remaley AT, Brewer HB Jr, Santamarina-Fojo S (2001) Analysis of glomerulosclerosis andatherosclerosis in lecithin cholesterol acyltransferase-deficient mice. J Biol Chem276:15090–15098Lammers B, Zhao Y, Hoekstra M, Hildebrand RB, Ye D, Meurs I, Van Berkel TJ, Van Eck M(2011) Augmented atherogenesis in LDL receptor deficient mice lacking both macrophageABCA1 and ApoE. PLoS ONE 6:e26095Lammers B, Zhao Y, Foks AC, Hildebrand RB, Kuiper J, Van Berkel TJ, Van Eck M (2012)Leukocyte ABCA1 remains atheroprotective in splenectomized LDL receptor knockout mice.PLoS ONE 7:e48080Lawn RM, Wade DP, Couse TL, Wilcox JN (2001) Localization of human ATP-binding cassettetransporter 1 (ABC1) in normal and atherosclerotic tissues. Arterioscler Thromb Vasc Biol21:378–385Lee RG, Kelley KL, Sawyer JK, Farese RV Jr, Parks JS, Rudel LL (2004) Plasma cholesterylesters provided by lecithin: cholesterol acyltransferase and acyl-coenzyme a:cholesterolacyltransferase 2 have opposite atherosclerotic potential. Circ Res 95:998–1004Lee EY, Klementowicz PT, Hegele RA, Asztalos BF, Schaefer EJ (2013) HDL deficiency due to anew insertion mutation (ApoA-INashua) and review of the literature. J Clin Lipidol 7:169–173Lee-Rueckert M, Vikstedt R, Metso J, Ehnholm C, Kovanen PT, Jauhiainen M (2006) Absence ofendogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterolfrom macrophage foam cells. J Lipid Res 47:1725–1732Leren TP, Bakken KS, Daum U, Ose L, Berg K, Assmann G, von Eckardstein A (1997)Heterozygosity for apolipoprotein A-I(R160L)Oslo is associated with low levels of highdensity lipoprotein cholesterol and HDL-subclass LpA-I/A-II but normal levels ofHDL-subclass LpA-I. J Lipid Res 38:121–131Mouse Models of Disturbed HDL Metabolism 329Li H, Reddick RL, Maeda N (1993) Lack of apoA-I is not associated with increased susceptibilityto atherosclerosis in mice. Arterioscler Thromb 13:1814–1821Li L, Naples M, Song H, Yuan R, Ye F, Shafi S, Adeli K, Ng DS (2007) LCAT-null mice developimproved hepatic insulin sensitivity through altered regulation of transcription factors andsuppressors of cytokine signaling. Am J Physiol Endocrinol Metab 293:E587–E594Li Z, Wang Y, van der Sluis RJ, van der Hoorn JW, Princen HM, Van Eck M, Van Berkel TJ,Rensen PC, Hoekstra M (2012) Niacin reduces plasma CETP levels by diminishing livermacrophage content in CETP transgenic mice. Biochem Pharmacol 84:821–829Liu R, Iqbal J, Yeang C, Wang DQ, Hussain MM, Jiang XC (2007) Phospholipid transfer protein-deficient mice absorb less cholesterol. Arterioscler Thromb Vasc Biol 27:2014–2021Lusa S, Jauhiainen M, Metso J, Somerharju P, Ehnholm C (1996) The mechanism of humanplasma phospholipid transfer protein-induced enlargement of high-density lipoproteinparticles: evidence for particle fusion. Biochem J 313:275–282Malerød L, Juvet K, Gjøen T, Berg T (2002) The expression of scavenger receptor class B, type I(SR-BI) and caveolin-1 in parenchymal and nonparenchymal liver cells. Cell Tissue Res307:173–180Marnane M, Prendeville S, McDonnell C, Noone I, Barry M, Crowe M, Mulligan N, Kelly PJ(2014) Plaque inflammation and unstable morphology are associated with early stroke recur-rence in symptomatic carotid stenosis. Stroke 45:801–806Masucci-Magoulas L, Plump A, Jiang XC, Walsh A, Breslow JL, Tall AR (1996) Profoundinduction of hepatic cholesteryl ester transfer protein transgene expression in apolipoproteinE and low density lipoprotein receptor gene knockout mice. A novel mechanism signalschanges in plasma cholesterol levels. J Clin Invest 97:154–161Mathis D, Shoelson SE (2011) Immunometabolism: an emerging frontier. Nat Rev Immunol 11:81Matsunaga A, Sasaki J, Han H, Huang W, Kugi M, Koga T, Ichiki S, Shinkawa T, Arakawa K(1999) Compound heterozygosity for an apolipoprotein A1 gene promoter mutation and astructural nonsense mutation with apolipoprotein A1 deficiency. Arterioscler Thromb VascBiol 19:348–355Meurs I, Hoekstra M, van Wanrooij EJ, Hildebrand RB, Kuiper J, Kuipers F, Hardeman MR, VanBerkel TJ, Van Eck M (2005) HDL cholesterol levels are an important factor for determiningthe lifespan of erythrocytes. Exp Hematol 33:1309–1319Meurs I, Lammers B, Zhao Y, Out R, Hildebrand RB, Hoekstra M, Van Berkel TJ, Van Eck M(2012) The effect of ABCG1 deficiency on atherosclerotic lesion development in LDL receptorknockout mice depends on the stage of atherogenesis. Atherosclerosis 221:41–47Miettinen HE, Rayburn H, Krieger M (2001) Abnormal lipoprotein metabolism and reversiblefemale infertility in HDL receptor (SR-BI)-deficient mice. J Clin Invest 108:1717–1722Mihaylova B, Emberson J, Blackwell L, Keech A, Simes J, Barnes EH, Voysey M, Gray A,Collins R, Baigent C, Cholesterol Treatment Trialists’ (CTT) Collaborators (2012) The effectsof lowering LDL cholesterol with statin therapy in people at low risk of vascular disease: meta-analysis of individual data from 27 randomised trials. Lancet 380:581–590Moore RE, Kawashiri MA, Kitajima K, Secreto A, Millar JS, Pratico D, Rader DJ (2003)Apolipoprotein A-I deficiency results in markedly increased atherosclerosis in mice lackingthe LDL receptor. Arterioscler Thromb Vasc Biol 23:1914–1920Ng DS, Francone OL, Forte TM, Zhang J, Haghpassand M, Rubin EM (1997) Disruption of themurine lecithin: cholesterol acyltransferase gene causes impairment of adrenal lipid deliveryand up-regulation of scavenger receptor class B type I. J Biol Chem 272:15777–15781Ng DS, Maguire GF, Wylie J, Ravandi A, XuanW, Ahmed Z, Eskandarian M, Kuksis A, ConnellyPW (2002) Oxidative stress is markedly elevated in lecithin: cholesterol acyltransferase-deficient mice and is paradoxically reversed in the apolipoprotein E knockout background inassociation with a reduction in atherosclerosis. J Biol Chem 277:11715–11720Ng DS, Xie C, Maguire GF, Zhu X, Ugwu F, Lam E, Connelly PW (2004) Hypertriglyceridemia inlecithin-cholesterol acyltransferase-deficient mice is associated with hepatic overproduction of330 M. Hoekstra and M. Van Ecktriglycerides, increased lipogenesis, and improved glucose tolerance. J Biol Chem279:7636–7642O’Brien KD, Vuletic S, McDonald TO, Wolfbauer G, Lewis K, Tu AY, Marcovina S, Wight TN,Chait A, Albers JJ (2003) Cell-associated and extracellular phospholipid transfer protein inhuman coronary atherosclerosis. Circulation 108:270–274Ogier N, Klein A, Deckert V, Athias A, Bessède G, Le Guern N, Lagrost L, Desrumaux C (2007)Cholesterol accumulation is increased in macrophages of phospholipid transfer protein-deficient mice: normalization by dietary alpha-tocopherol supplementation. ArteriosclerThromb Vasc Biol 27:2407–2412Oliveira HC, Ma L, Milne R, Marcovina SM, Inazu A, Mabuchi H, Tall AR (1997) Cholesterylester transfer protein activity enhances plasma cholesteryl ester formation. Studies in CETPtransgenic mice and human genetic CETP deficiency. Arterioscler Thromb Vasc Biol17:1045–1052Orsó E, Broccardo C, Kaminski WE, Böttcher A, Liebisch G, Drobnik W, Götz A, Chambenoit O,Diederich W, Langmann T, Spruss T, Luciani MF, Rothe G, Lackner KJ, Chimini G, SchmitzG (2000) Transport of lipids from golgi to plasma membrane is defective in tangier diseasepatients and Abc1-deficient mice. Nat Genet 24:192–196Out R, Hoekstra M, Spijkers JA, Kruijt JK, van Eck M, Bos IS, Twisk J, Van Berkel TJ (2004)Scavenger receptor class B type I is solely responsible for the selective uptake of cholesterylesters from HDL by the liver and the adrenals in mice. J Lipid Res 45:2088–2095Out R, Hoekstra M, Hildebrand RB, Kruit JK, Meurs I, Li Z, Kuipers F, Van Berkel TJ, Van EckM(2006) Macrophage ABCG1 deletion disrupts lipid homeostasis in alveolar macrophages andmoderately influences atherosclerotic lesion development in LDL receptor-deficient mice.Arterioscler Thromb Vasc Biol 26:2295–2300Out R, Hoekstra M, Meurs I, de Vos P, Kuiper J, Van Eck M, Van Berkel TJ (2007) Total bodyABCG1 expression protects against early atherosclerotic lesion development in mice.Arterioscler Thromb Vasc Biol 27:594–599Out R, Jessup W, Le Goff W, Hoekstra M, Gelissen IC, Zhao Y, Kritharides L, Chimini G,Kuiper J, Chapman MJ, Huby T, Van Berkel TJ, Van Eck M (2008a) Coexistence of foam cellsand hypocholesterolemia in mice lacking the ABC transporters A1 and G1. Circ Res102:113–120Out R, Hoekstra M, Habets K, Meurs I, de Waard V, Hildebrand RB, Wang Y, Chimini G,Kuiper J, Van Berkel TJ, Van Eck M (2008b) Combined deletion of macrophage ABCA1 andABCG1 leads to massive lipid accumulation in tissue macrophages and distinct atherosclerosisat relatively low plasma cholesterol levels. Arterioscler Thromb Vasc Biol 28:258–264Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA (1987) Quantitative assessment ofatherosclerotic lesions in mice. Atherosclerosis 68:231–240Pape ME, Rehberg EF, Marotti KR, Melchior GW (1991) Molecular cloning, sequence, andexpression of cynomolgus monkey cholesteryl ester transfer protein. Inverse correlationbetween hepatic cholesteryl ester transfer protein mRNA levels and plasma high densitylipoprotein levels. Arterioscler Thromb 11:1759–1771Parks JS, Li H, Gebre AK, Smith TL, Maeda N (1995) Effect of apolipoprotein A-I deficiency onlecithin: cholesterol acyltransferase activation in mouse plasma. J Lipid Res 36:349–355Pei Y, Chen X, Aboutouk D, Fuller MT, Dadoo O, Yu P, White EJ, Igdoura SA, Trigatti BL (2013)SR-BI in bone marrow derived cells protects mice from diet induced coronary artery athero-sclerosis and myocardial infarction. PLoS ONE 8:e72492Plump AS, Erickson SK, WengW, Partin JS, Breslow JL, Williams DL (1996) Apolipoprotein A-Iis required for cholesteryl ester accumulation in steroidogenic cells and for normal adrenalsteroid production. J Clin Invest 97:2660–2671Plump AS, Azrolan N, Odaka H, Wu L, Jiang X, Tall A, Eisenberg S, Breslow JL (1997) ApoA-Iknockout mice: characterization of HDL metabolism in homozygotes and identification of apost-RNA mechanism of apoA-I up-regulation in heterozygotes. J Lipid Res 38:1033–1047Mouse Models of Disturbed HDL Metabolism 331Plump AS, Masucci-Magoulas L, Bruce C, Bisgaier CL, Breslow JL, Tall AR (1999) Increasedatherosclerosis in ApoE and LDL receptor gene knock-out mice as a result of humancholesteryl ester transfer protein transgene expression. Arterioscler Thromb Vasc Biol19:1105–1110Qin S, Kawano K, Bruce C, Lin M, Bisgaier C, Tall AR, Jiang X (2000) Phospholipid transferprotein gene knock-out mice have low high density lipoprotein levels, due to hypercatabolism,and accumulate apoA-IV-rich lamellar lipoproteins. J Lipid Res 41:269–276Quinet EM, Agellon LB, Kroon PA, Marcel YL, Lee YC, Whitlock ME, Tall AR (1990)Atherogenic diet increases cholesteryl ester transfer protein messenger RNA levels in rabbitliver. J Clin Invest 85:357–363Ranalletta M, Wang N, Han S, Yvan-Charvet L, Welch C, Tall AR (2006) Decreased atheroscle-rosis in low-density lipoprotein receptor knockout mice transplanted with Abcg1-/- bonemarrow. Arterioscler Thromb Vasc Biol 26:2308–2315Redgrave JN, Lovett JK, Gallagher PJ, Rothwell PM (2006) Histological assessment of 526 symp-tomatic carotid plaques in relation to the nature and timing of ischemic symptoms: the Oxfordplaque study. Circulation 113:2320–2328Rhainds D, Brodeur M, Lapointe J, Charpentier D, Falstrault L, Brissette L (2003) The role ofhuman and mouse hepatic scavenger receptor class B type I (SR-BI) in the selective uptake oflow-density lipoprotein-cholesteryl esters. Biochemistry 42:7527–7538Rhyne J, Ryan MJ, White C, Chimonas T, Miller M (2006) The two novel CETP mutationsGln87X and Gln165X in a compound heterozygous state are associated with markedhyperalphalipoproteinemia and absence of significant coronary artery disease. J Mol Med(Berl) 84:647–650Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M (1997) A targeted mutation inthe murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor classB type I reveals its key role in HDL metabolism. Proc Natl Acad Sci USA 94:12610–12615Sahoo D, Trischuk TC, Chan T, Drover VA, Ho S, Chimini G, Agellon LB, Agnihotri R, FrancisGA, Lehner R (2004) ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDLparticle formation and decreases VLDL secretion from murine hepatocytes. J Lipid Res45:1122–1131Sakai N, Vaisman BL, Koch CA, Hoyt RF Jr, Meyn SM, Talley GD, Paiz JA, Brewer HB Jr,Santamarina-Fojo S (1997) Targeted disruption of the mouse lecithin: cholesterolacyltransferase (LCAT) gene. Generation of a new animal model for human LCAT deficiency.J Biol Chem 272:7506–7510Salerno AG, Patrı́cio PR, Berti JA, Oliveira HC (2009) Cholesteryl ester transfer protein (CETP)increases postprandial triglyceridaemia and delays triacylglycerol plasma clearance in trans-genic mice. Biochem J 419:629–634Santamarina-Fojo S, Lambert G, Hoeg JM, Brewer HB Jr (2000) Lecithin-cholesterolacyltransferase: role in lipoprotein metabolism, reverse cholesterol transport and atherosclero-sis. Curr Opin Lipidol 11:267–275Santander NG, Contreras-Duarte S, Awad MF, Lizama C, Passalacqua I, Rigotti A, Busso D(2013) Developmental abnormalities in mouse embryos lacking the HDL receptor SR-BI. HumMol Genet 22:1086–1096Scanu A, Toth J, Edelstein C, Koga S, Stiller E (1969) Fractionation of human serum high densitylipoprotein in urea solutions. Evidence for polypeptide heterogeneity. Biochemistry8:3309–3316Schwartz GG, Olsson AG, Abt M, Ballantyne CM, Barter PJ, Brumm J, Chaitman BR, Holme IM,Kallend D, Leiter LA, Leitersdorf E, McMurray JJ, Mundl H, Nicholls SJ, Shah PK, Tardif JC,Wright RS, dal-OUTCOMES Investigators (2012) Effects of dalcetrapib in patients with arecent acute coronary syndrome. N Engl J Med 367:2089–2099Shelly L, Royer L, Sand T, Jensen H, Luo Y (2008) Phospholipid transfer protein deficiencyameliorates diet-induced hypercholesterolemia and inflammation in mice. J Lipid Res49:773–781332 M. Hoekstra and M. Van EckSiggins S, Bykov I, Hermansson M, Somerharju P, Lindros K, Miettinen TA, Jauhiainen M,Olkkonen VM, Ehnholm C (2007) Altered hepatic lipid status and apolipoprotein A-I metabo-lism in mice lacking phospholipid transfer protein. Atherosclerosis 190:114–123Singaraja RR, Stahmer B, Brundert M, Merkel M, Heeren J, Bissada N, Kang M, Timmins JM,Ramakrishnan R, Parks JS, Hayden MR, Rinninger F (2006) Hepatic ATP-binding cassettetransporter A1 is a key molecule in high-density lipoprotein cholesteryl ester metabolism inmice. Arterioscler Thromb Vasc Biol 26:1821–1827Tall AR, Krumholz S, Olivecrona T, Deckelbaum RJ (1985) Plasma phospholipid transfer proteinenhances transfer and exchange of phospholipids between very low density lipoproteins andhigh density lipoproteins during lipolysis. J Lipid Res 26:842–851Tarling EJ, Edwards PA (2011) ATP binding cassette transporter G1 (ABCG1) is an intracellularsterol transporter. Proc Natl Acad Sci USA 108:19719–19724Tilly-Kiesi M, Zhang Q, Ehnholm S, Kahri J, Lahdenperä S, Ehnholm C, Taskinen MR (1995)ApoA-IHelsinki (Lys107–> 0) associated with reduced HDL cholesterol and LpA-I:A-IIdeficiency. Arterioscler Thromb Vasc Biol 15:1294–1306Timmins JM, Lee JY, Boudyguina E, Kluckman KD, Brunham LR, Mulya A, Gebre AK, CoutinhoJM, Colvin PL, Smith TL, Hayden MR, Maeda N, Parks JS (2005) Targeted inactivation ofhepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism ofapoA-I. J Clin Invest 115:1333–1342Tomimoto S, Tsujita M, Okazaki M, Usui S, Tada T, Fukutomi T, Ito S, Itoh M, Yokoyama S(2001) Effect of probucol in lecithin-cholesterol acyltransferase-deficient mice: inhibition of2 independent cellular cholesterol-releasing pathways in vivo. Arterioscler Thromb Vasc Biol21:394–400Trigatti B, Rayburn H, Viñals M, Braun A, Miettinen H, Penman M, Hertz M, Schrenzel M,Amigo L, Rigotti A, Krieger M (1999) Influence of the high density lipoprotein receptor SR-BIon reproductive and cardiovascular pathophysiology. Proc Natl Acad Sci USA 96:9322–9327Valenta DT, Ogier N, Bradshaw G, Black AS, Bonnet DJ, Lagrost L, Curtiss LK, Desrumaux CM(2006) Atheroprotective potential of macrophage-derived phospholipid transfer protein inlow-density lipoprotein receptor-deficient mice is overcome by apolipoprotein AIoverexpression. Arterioscler Thromb Vasc Biol 26:1572–1578van den Maagdenberg AM, Hofker MH, Krimpenfort PJ, de Bruijn I, van Vlijmen B, van derBoom H, Havekes LM, Frants RR (1993) Transgenic mice carrying the apolipoproteinE3-Leiden gene exhibit hyperlipoproteinemia. J Biol Chem 268:10540–10545van der Hoogt CC, de Haan W, Westerterp M, Hoekstra M, Dallinga-Thie GM, Romijn JA,Princen HM, Jukema JW, Havekes LM, Rensen PC (2007) Fenofibrate increasesHDL-cholesterol by reducing cholesteryl ester transfer protein expression. J Lipid Res48:1763–1771van der Hoorn JW, de Haan W, Berbée JF, Havekes LM, Jukema JW, Rensen PC, Princen HM(2008) Niacin increases HDL by reducing hepatic expression and plasma levels of cholesterylester transfer protein in APOE*3Leiden.CETP mice. Arterioscler Thromb Vasc Biol28:2016–2022Van Eck M, Van Berkel TJ (2013) ATP-binding cassette transporter A1 in lipoprotein metabolismand atherosclerosis: a new piece of the complex puzzle. Arterioscler Thromb Vasc Biol33:2281–2283Van Eck M, Bos IS, Kaminski WE, Orsó E, Rothe G, Twisk J, Böttcher A, Van Amersfoort ES,Christiansen-Weber TA, Fung-Leung WP, Van Berkel TJ, Schmitz G (2002) LeukocyteABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.Proc Natl Acad Sci USA 99:6298–6303Van Eck M, Twisk J, Hoekstra M, Van Rij BT, Van der Lans CA, Bos IS, Kruijt JK, Kuipers F,Van Berkel TJ (2003) Differential effects of scavenger receptor BI deficiency on lipidmetabolism in cells of the arterial wall and in the liver. J Biol Chem 278:23699–23705Mouse Models of Disturbed HDL Metabolism 333Van Eck M, Bos IS, Hildebrand RB, Van Rij BT, Van Berkel TJ (2004) Dual role for scavengerreceptor class B, type I on bone marrow-derived cells in atherosclerotic lesion development.Am J Pathol 165:785–794Van Eck M, Ye D, Hildebrand RB, Kar Kruijt J, de Haan W, Hoekstra M, Rensen PC, Ehnholm C,Jauhiainen M, Van Berkel TJ (2007) Important role for bone marrow-derived cholesteryl estertransfer protein in lipoprotein cholesterol redistribution and atherosclerotic lesion developmentin LDL receptor knockout mice. Circ Res 100:678–685Van Eck M, Hoekstra M, Out R, Bos IS, Kruijt JK, Hildebrand RB, Van Berkel TJ (2008)Scavenger receptor BI facilitates the metabolism of VLDL lipoproteins in vivo. J Lipid Res49:136–146van Vlijmen BJ, van den Maagdenberg AM, Gijbels MJ, van der Boom H, HogenEsch H, FrantsRR, Hofker MH, Havekes LM (1994) Diet-induced hyperlipoproteinemia and atherosclerosisin apolipoprotein E3-Leiden transgenic mice. J Clin Invest 93:1403–1410Varban ML, Rinninger F, Wang N, Fairchild-Huntress V, Dunmore JH, Fang Q, Gosselin ML,Dixon KL, Deeds JD, Acton SL, Tall AR, Huszar D (1998) Targeted mutation reveals a centralrole for SR-BI in hepatic selective uptake of high density lipoprotein cholesterol. Proc NatlAcad Sci USA 95:4619–4624Vergeer M, Korporaal SJ, Franssen R, Meurs I, Out R, Hovingh GK, Hoekstra M, Sierts JA,Dallinga-Thie GM, Motazacker MM, Holleboom AG, Van Berkel TJ, Kastelein JJ, VanEck M, Kuivenhoven JA (2011) Genetic variant of the scavenger receptor BI in humans. NEngl J Med 364:136–1345Vikstedt R, Ye D, Metso J, Hildebrand RB, Van Berkel TJ, Ehnholm C, Jauhiainen M, Van Eck M(2007) Macrophage phospholipid transfer protein contributes significantly to total plasmaphospholipid transfer activity and its deficiency leads to diminished atherosclerotic lesiondevelopment. Arterioscler Thromb Vasc Biol 27:578–586Wada M, Iso T, Asztalos BF, Takama N, Nakajima T, Seta Y, Kaneko K, Taniguchi Y,Kobayashi H, Nakajima K, Schaefer EJ, Kurabayashi M (2009) Marked high density lipopro-tein deficiency due to apolipoprotein A-I Tomioka (codon 138 deletion). Atherosclerosis207:157–161Wang X, Collins HL, Ranalletta M, Fuki IV, Billheimer JT, Rothblat GH, Tall AR, Rader DJ(2007) Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reversecholesterol transport in vivo. J Clin Invest 117:2216–2224West M, Greason E, Kolmakova A, Jahangiri A, Asztalos B, Pollin TI, Rodriguez A (2009)Scavenger receptor class B type I protein as an independent predictor of high-density lipopro-tein cholesterol levels in subjects with hyperalphalipoproteinemia. J Clin Endocrinol Metab94:1451–1457Westerterp M, van der Hoogt CC, de Haan W, Offerman EH, Dallinga-Thie GM, Jukema JW,Havekes LM, Rensen PC (2006) Cholesteryl ester transfer protein decreases high-densitylipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice. ArteriosclerThromb Vasc Biol 26:2552–2559Wiersma H, Nijstad N, de Boer JF, Out R, Hogewerf W, Van Berkel TJ, Kuipers F, Tietge UJ(2009) Lack of Abcg1 results in decreased plasma HDL cholesterol levels and increased biliarycholesterol secretion in mice fed a high cholesterol diet. Atherosclerosis 206:141–147Wilhelm AJ, Zabalawi M, Grayson JM, Weant AE, Major AS, Owen J, Bharadwaj M, Walzem R,Chan L, Oka K, Thomas MJ, Sorci-Thomas MG (2009) Apolipoprotein A-I and its role inlymphocyte cholesterol homeostasis and autoimmunity. Arterioscler Thromb Vasc Biol29:843–849Williamson R, Lee D, Hagaman J, Maeda N (1992) Marked reduction of high density lipoproteincholesterol in mice genetically modified to lack apolipoprotein A-I. Proc Natl Acad Sci USA89:7134–7138Wojcik AJ, Skaflen MD, Srinivasan S, Hedrick CC (2008) A critical role for ABCG1 in macro-phage inflammation and lung homeostasis. J Immunol 180:4273–4282334 M. Hoekstra and M. Van EckYan D, Navab M, Bruce C, Fogelman AM, Jiang XC (2004) PLTP deficiency improves the anti-inflammatory properties of HDL and reduces the ability of LDL to induce monocyte chemo-tactic activity. J Lipid Res 45:1852–1858Yates M, Kolmakova A, Zhao Y, Rodriguez A (2011) Clinical impact of scavenger receptor classB type I gene polymorphisms on human female fertility. Hum Reprod 26:1910–1916Yazdanyar A, Jiang XC (2012) Liver phospholipid transfer protein (PLTP) expression with aPLTP-null background promotes very low-density lipoprotein production in mice. Hepatology56:576–584Yazdanyar A, Quan W, Jin W, Jiang XC (2013) Liver-specific phospholipid transfer proteindeficiency reduces high-density lipoprotein and non-high-density lipoprotein production inmice. Arterioscler Thromb Vasc Biol 33:2058–2064Ye D, Hoekstra M, Out R, Meurs I, Kruijt JK, Hildebrand RB, Van Berkel TJ, Van Eck M (2008)Hepatic cell-specific ATP-binding cassette (ABC) transporter profiling identifies putativenovel candidates for lipid homeostasis in mice. Atherosclerosis 196:650–658Yeang C, Qin S, Chen K, Wang DQ, Jiang XC (2010) Diet-induced lipid accumulation inphospholipid transfer protein-deficient mice: its atherogenicity and potential mechanism. JLipid Res 51:2993–3002Yvan-Charvet L, Ranalletta M, Wang N, Han S, Terasaka N, Li R, Welch C, Tall AR (2007)Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation andaccelerates atherosclerosis in mice. J Clin Invest 117:3900–3908Yvan-Charvet L, Welch C, Pagler TA, Ranalletta M, Lamkanfi M, Han S, Ishibashi M, Li R,Wang N, Tall AR (2008) Increased inflammatory gene expression in ABC transporter-deficientmacrophages: free cholesterol accumulation, increased signaling via toll-like receptors, andneutrophil infiltration of atherosclerotic lesions. Circulation 118:1837–1847Yvan-Charvet L, Pagler TA, Seimon TA, Thorp E, Welch CL, Witztum JL, Tabas I, Tall AR(2010a) ABCA1 and ABCG1 protect against oxidative stress-induced macrophage apoptosisduring efferocytosis. Circ Res 106:1861–1869Yvan-Charvet L, Pagler T, Gautier EL, Avagyan S, Siry RL, Han S, Welch CL, Wang N,Randolph GJ, Snoeck HW, Tall AR (2010b) ATP-binding cassette transporters and HDLsuppress hematopoietic stem cell proliferation. Science 328:1689–1693Zabalawi M, Bhat S, Loughlin T, Thomas MJ, Alexander E, Cline M, Bullock B, Willingham M,Sorci-Thomas MG (2003) Induction of fatal inflammation in LDL receptor and ApoA-Idouble-knockout mice fed dietary fat and cholesterol. Am J Pathol 163:1201–1213Zannis VI, Karathanasis SK, Keutmann HT, Goldberger G, Breslow JL (1983) Intracellular andextracellular processing of human apolipoprotein A-I: secreted apolipoprotein A-I isoprotein2 is a propeptide. Proc Natl Acad Sci USA 80:2574–2578Zhang W, Yancey PG, Su YR, Babaev VR, Zhang Y, Fazio S, Linton MF (2003) Inactivation ofmacrophage scavenger receptor class B type I promotes atherosclerotic lesion development inapolipoprotein E-deficient mice. Circulation 108:2258–2263Zhang Y, Da Silva JR, Reilly M, Billheimer JT, Rothblat GH, Rader DJ (2005a) Hepaticexpression of scavenger receptor class B type I (SR-BI) is a positive regulator of macrophagereverse cholesterol transport in vivo. J Clin Invest 115:2870–2874Zhang S, Picard MH, Vasile E, Zhu Y, Raffai RL, Weisgraber KH, Krieger M (2005b) Diet-induced occlusive coronary atherosclerosis, myocardial infarction, cardiac dysfunction, andpremature death in scavenger receptor class B type I-deficient, hypomorphic apolipoproteinER61 mice. Circulation 111:3457–3464Zhao Y, Pennings M, Hildebrand RB, Ye D, Calpe-Berdiel L, Out R, Kjerrulf M, Hurt-Camejo E,Groen AK, Hoekstra M, Jessup W, Chimini G, Van Berkel TJ, Van Eck M (2010) Enhancedfoam cell formation, atherosclerotic lesion development, and inflammation by combineddeletion of ABCA1 and SR-BI in Bone marrow-derived cells in LDL receptor knockoutmice on western-type diet. Circ Res 107:e20–e31Zhao Y, Pennings M, Vrins CL, Calpe-Berdiel L, Hoekstra M, Kruijt JK, Ottenhoff R, HildebrandRB, van der Sluis R, Jessup W, Le Goff W, Chapman MJ, Huby T, Groen AK, Van Berkel TJ,Mouse Models of Disturbed HDL Metabolism 335Van Eck M (2011) Hypocholesterolemia, foam cell accumulation, but no atherosclerosis inmice lacking ABC-transporter A1 and scavenger receptor BI. Atherosclerosis 218:314–322Zheng KQ, Zhang SZ, Zhang L, Huang DJ, Liao LC, Hou YP (2004) A novel missense mutation(L296 Q) in cholesteryl ester transfer protein gene related to coronary heart disease. ActaBiochim Biophys Sin (Shanghai) 36:33–36Zhong S, Sharp DS, Grove JS, Bruce C, Yano K, Curb JD, Tall AR (1996) Increased coronaryheart disease in Japanese-American men with mutation in the cholesteryl ester transfer proteingene despite increased HDL levels. J Clin Invest 97(12):2917–2923Zhou H, Li Z, Silver DL, Jiang XC (2006) Cholesteryl ester transfer protein (CETP) expressionenhances HDL cholesteryl ester liver delivery, which is independent of scavenger receptor BI,LDL receptor related protein and possibly LDL receptor. Biochim Biophys Acta1761:1482–1488Zhu X, Lee JY, Timmins JM, Brown JM, Boudyguina E, Mulya A, Gebre AK, Willingham MC,Hiltbold EM, Mishra N, Maeda N, Parks JS (2008) Increased cellular free cholesterol inmacrophage-specific Abca1 knock-out mice enhances pro-inflammatory response ofmacrophages. J Biol Chem 283:22930–22941Zhu X, Westcott MM, Bi X, Liu M, Gowdy KM, Seo J, Cao Q, Gebre AK, Fessler MB, HiltboldEM, Parks JS (2012) Myeloid cell-specific ABCA1 deletion protects mice from bacterialinfection. Circ Res 111:1398–1409336 M. Hoekstra and M. Van EckDysfunctional HDL: From Structure-Function-Relationships to BiomarkersMeliana Riwanto, Lucia Rohrer, Arnold von Eckardstein,and Ulf LandmesserContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3402 HDL and Reverse Cholesterol Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3412.1 Mechanisms Under Physiological Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3412.2 Alterations of the Cholesterol Efflux Capacity of HDL in Cardiovascular Disease 3423 Effects of HDL on LDL Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3433.1 Mechanisms Under Physiological Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3433.2 Impairment of the Anti-Oxidative Effects of HDL in Patients After Surgery andWith Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3454 Effects of HDL on Endothelial Nitric Oxide Bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3464.1 Mechanisms Under Physiological Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3464.2 Impaired HDL Capacity to Stimulate NO Production in Patients withCardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3475 Endothelial Anti-Inflammatory Effects of HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3485.1 Mechanisms Under Physiological Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3485.2 Impaired Endothelial Anti-Inflammatory Effects of HDL in Patients with CAD,Diabetes, or Chronic Kidney Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3506 Effects of HDL on Endothelial Cell Apoptotic Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3516.1 Mechanisms Under Physiological Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3516.2 Impairment of the Endothelial Anti-Apoptotic Effects of HDL in Patients withCardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3527 HDL Protein Cargo and Prognostic Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355M. Riwanto • U. Landmesser (*)Cardiology, University Heart Center, University Hospital Zurich and Center of MolecularCardiology, University of Zurich, Rämistrasse 100, CH 8091 Zurich, Switzerlande-mail: ulf.landmesser@usz.chL. Rohrer • A. von EckardsteinInstitute of Clinical Chemistry, University Hospital Zurich, Zurich, Switzerland# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_10337AbstractReduced plasma levels of HDL-C are associated with an increased risk of CADand myocardial infarction, as shown in various prospective population studies.However, recent clinical trials on lipid-modifying drugs that increase plasmalevels of HDL-C have not shown significant clinical benefit. Notably, in somerecent clinical studies, there is no clear association of higher HDL-C levels witha reduced risk of cardiovascular events observed in patients with existing CAD.These observations have prompted researchers to shift from a cholesterol-centricview of HDL towards assessing the function and composition of HDL particles.Of importance, experimental and translational studies have furtherdemonstrated various potential antiatherogenic effects of HDL. HDL has beenproposed to promote macrophage reverse cholesterol transport and to protectendothelial cell functions by prevention of oxidation of LDL and its adverseendothelial effects. Furthermore, HDL from healthy subjects can directly stimu-late endothelial cell production of nitric oxide and exert anti-inflammatory andantiapoptotic effects. Of note, increasing evidence suggests that the vasculareffects of HDL can be highly heterogeneous and HDL may lose important anti-atherosclerotic properties and turn dysfunctional in patients with chronic inflam-matory disorders. A greater understanding of mechanisms of action of HDL andits altered vascular effects is therefore critical within the context ofHDL-targeted therapies.KeywordsHDL dysfunction • Endothelial function • Atherosclerosis • CADAbbreviationsABCA1 ATP-binding cassette transporter A1ABCG1 ATP-binding cassette transporter G1ACS Acute coronary syndromeapoA-I Apolipoprotein A1apoA-II Apolipoprotein A2apoB Apolipoprotein BapoA-IV Apolipoprotein A-IVapoC-III Apolipoprotein C-IIIapoE Apolipoprotein EapoJ Apolipoprotein JBad Bcl-2-associated death promoterBcl-xL B-cell lymphoma-extra largeBid BH3 interacting domain death agonist338 M. Riwanto et al.CAD Coronary artery diseasecaspase Cysteine-aspartic proteasesCETP Cholesteryl ester transfer proteinCHF Chronic heart failureCKD Chronic kidney diseaseeNOS Endothelial nitric oxide synthaseHDL High-density lipoproteinHDL-C High-density lipoprotein cholesterolHPLC High-performance liquid chromatographyICAM-1 Intercellular Adhesion Molecule 1LCAT Lecithin cholesterol acyltransferaseMS/MS Tandem mass spectrometryLDL Low density lipoproteinL-NAME L-NG-Nitroarginine Methyl EsterLOX-1 Lectin-type oxidized LDL receptor 1LpPLA2 Lipoprotein-associated phospholipase A2MALDI-TOF-MS Matrix-assisted laser desorption/ionization-time-of-flight massspectrometryMAPK Mitogen-activated protein kinaseMCP Monocyte chemoattractant proteinMDA MalondialdehydeMI Myocardial infarctionMPO MyeloperoxidaseNF-kB Nuclear factor kappa-light-chain-enhancer of activated B cellsNO Nitric oxideoxLDL Oxidized low density lipoproteinPAF-AH Platelet-activating factor acetylhydrolasePI3K Phosphoinositide-3-kinasePLTP Phospholipid transport proteinPON1 Paraoxonase 1POPC 1-palmitoyl-2-oleoyl-phosphatidylcholineRCT Reverse cholesterol transportrHDL Reconstituted high-density lipoproteinS1P Sphingosine-1-phosphateSAA Serum amyloid ASNP Single nucleotide polymorphismsPLA2-IIa Secretory phospholipase A2-IIaSR-BI Scavenger receptor type B1TNF-α Tumor necrosis factor-alphaVCAM-1 Vascular cell adhesion molecule 1VLDL Very low density lipoproteinDysfunctional HDL: From Structure-Function-Relationships to Biomarkers 3391 IntroductionIt was shown in multiple large prospective studies of cardiovascular risk factors thatreduced plasma levels of HDL-cholesterol (HDL-C) are associated with anincreased risk of coronary artery disease (CAD) (Castelli et al. 1986; Cullenet al. 1997; Di Angelantonio et al. 2009; Gordon et al. 1977; Sharrett et al. 2001).Multiple biological functions of HDL have been identified, whereby HDL mayexert antiatherogenic effects (Annema and von Eckardstein 2013; Barteret al. 2004; Mineo et al. 2006; Rader 2006; Riwanto and Landmesser 2013), e.g.,HDL from healthy subjects has been shown to directly promote endothelialantiapoptotic, anti-inflammatory, and antithrombotic effects (Mineo et al. 2006;Nofer et al. 2004; Rye and Barter 2008; Tall et al. 2008; Yuhanna et al. 2001).Accordingly, interventions to improve HDL-C levels and/or HDL function arebeing intensely evaluated as a potential therapeutic strategy to reduce cardiovascu-lar risk. However, increasing evidence suggests that the endothelial and vasculareffects of HDL are highly heterogeneous and vasoprotective properties of HDL areimpaired in patients with diabetes, CAD, or chronic kidney dysfunction (Besleret al. 2011; Khera et al. 2011; Riwanto et al. 2013; Sorrentino et al. 2010).Intriguingly, several recent clinical trials testing the effects of HDL-C-raisingtherapies have failed to demonstrate cardiovascular risk reduction in patients withCAD. The Investigation of Lipid Level Management to Understand its Impact inAtherosclerotic Events (ILLUMINATE) trial testing the impact of the CETPinhibitor torcetrapib on clinical outcome showed increased risk of mortality andmorbidity in patients at high risk for coronary events, despite a substantial increaseof HDL-C levels (Barter et al. 2007). Dalcetrapib, another CETP inhibitor, mod-estly increased HDL-C levels, but the phase 3 trial dal-OUTCOMES study wasterminated before completion to a lack of efficacy (Schwartz et al. 2012). Morerecently, the HPS2-THRIVE trial results showed that adding extended-releaseniacin/laropiprant, another HDL-cholesterol-raising agent, to statins did not reducethe risk of cardiovascular event (Haynes et al. 2013). Taken together, theseobservations strongly suggest that plasma HDL-C levels per se are not an optimaltherapeutic target.Of note, accumulating evidence suggests that the vascular effects of HDL can behighly heterogeneous and may turn dysfunctional. HDL loses potential anti-atherosclerotic properties in patients with chronic inflammatory disorders, such asthe antiphospholipid syndrome (Charakida et al. 2009), systemic lupuserythematosus and rheumatoid arthritis (McMahon et al. 2006), scleroderma(Weihrauch et al. 2007), metabolic syndrome (de Souza et al. 2008), diabetes(Persegol et al. 2006; Sorrentino et al. 2010), and CAD (Ansell et al. 2003; Besleret al. 2011; Riwanto et al. 2013). In a study of 189 patients with chronic kidneydisease on hemodialysis, an impaired anti-inflammatory capacity of HDL wascorrelated with a poor clinical outcome (Kalantar-Zadeh et al. 2007). Furthermore,HDL isolated from subjects with type 1 or type 2 diabetes mellitus or abdominalobesity had reduced capacity to reverse the inhibition of aortic ring endothelium-dependent relaxation by oxLDL as compared to HDL from healthy control subjects340 M. Riwanto et al.(Persegol et al. 2006, 2007). Importantly, the heterogeneity of the vascular effectsof HDL may be attributed to changes in the HDL-associated proteome andlipidome, i.e., changes in the amount and type of proteins and lipids bound to theHDL particle and also posttranslational modifications. In particular, HDL is knownto be susceptible to modification in vitro by a variety of oxidants, such as metalions, peroxyl and hydroxyl radicals, aldehydes, various myeloperoxidase (MPO)-generated oxidants, lipoxygenase, phospholipase A2, elastase, nonenzymaticglycation, and homocysteinylation (Ferretti et al. 2006). These oxidativemodifications may contribute to the generation of dysfunctionalproinflammatory HDL.2 HDL and Reverse Cholesterol Transport2.1 Mechanisms Under Physiological ConditionsOne of the antiatherogenic effects of HDL has been attributed to its function inmacrophage reverse cholesterol transport (RCT), i.e., the removal of excess choles-terol from lipid-laden macrophage foam cells in the atherosclerotic plaque and itstransport to the liver for excretion in the bile (Rader 2006). The first step ofmacrophage RCT involves the hydrolysis of cytoplasmic cholesteryl ester intofree cholesterol followed by the efflux of the free cholesterol to mature HDL orextracellular lipid-poor apoA-I. Macrophage cholesterol efflux is mediated byactive transport systems, which include the ATP-binding cassette transportersABCA1 and ABCG1 (Rader 2006; Tall et al. 2008). Studies in macrophagesfrom ABCA1-knockout or ABCA1-overexpressing mice have shown thatABCA1 primarily mediates the cholesterol efflux to lipid-poor apoA-I (Bortnicket al. 2000; Haghpassand et al. 2001). In contrast, ABCG1 largely mediatescholesterol efflux from macrophages to mature HDL (Tall et al. 2008).A study by Wang et al. quantitatively assessed the roles of SR-BI, ABCA1, andABCG1 in macrophage RCT in mice in vivo (Wang et al. 2007a). Using primarymacrophages lacking SR-BI, the authors demonstrated that SR-BI did not promotemacrophage RCT in vivo after intraperitoneal injection. In contrast, both ABCA1and ABCG1 contributed to macrophage RCT in vivo. The study also demonstratedthat transplantation of bone marrow from ABCA1/ABCG1-deficient miceaccelerated atherosclerotic lesion formation in LDL receptor-deficient mice(Wang et al. 2007a). Interestingly, Yvan-Charvet et al. also demonstrated thatSR-BI failed to stimulate net cholesterol efflux from HEK293 cells to plasmaHDL and inhibited ABCG1-mediated cholesterol efflux, which was at least inpart due to the increased uptake of HDL cholesteryl esters into these cells (Yvan-Charvet et al. 2008).HDL-associated cholesterol is subsequently esterified by LCAT which transfersa fatty acyl residue from phospholipids to the 3-beta-hydroxy group of cholesterol.The final steps of RCT involve the uptake of HDL-C by the liver and its excretion inthe bile. Cholesterol esters and free cholesterol in HDL can either be directly takenDysfunctional HDL: From Structure-Function-Relationships to Biomarkers 341up by the liver via SR-BI or transferred to apoB containing lipoproteins in a processmediated by CETP (Cuchel et al. 2009). In the latter pathway, cholesterol esters aretaken up by the liver via the LDL receptor and then hydrolyzed to free cholesterolwhich can be excreted directly into the bile or following conversion to bile acid.2.2 Alterations of the Cholesterol Efflux Capacity of HDLin Cardiovascular DiseaseA case-control study by Khera et al. showed that the cholesterol efflux capacity ofapoB-depleted serum was inversely related to carotid IMT in healthy volunteersand to the likelihood of angiographic CAD, even after adjustment for HDL-C andapoA-I levels (Khera et al. 2011). In a separate study, enhanced cholesterol effluxactivity from ABCA1-stimulated macrophages was associated with reduced risk ofprevalent CAD in unadjusted models in two case-control cohorts: an angiographiccohort comprising stable subjects undergoing elective diagnostic coronary angiog-raphy and an outpatient cohort (Li et al. 2013). However, the inverse risk relation-ship remained significant after adjustment for traditional CAD risk factors onlywithin the outpatient cohort (Li et al. 2013). Surprisingly, higher cholesterol effluxactivity was associated with an increase in prospective 3-year risk of myocardialinfarction/stroke and major adverse cardiovascular events. The authors furtherobserved that the HDL fraction (1.063< d< 1.21) contained only a minority(40 %) of [(14)C] cholesterol released, with the majority found within thelipoprotein particle-depleted fraction, where 60 % was recovered after apoA-Iimmunoprecipitation. This is in agreement with previous findings on plasmas frompatients with apoA-I deficiency, such as Tangier disease and LCAT deficiency,whereby the cholesterol efflux capacity amounted to at least 40 and 20 % of totaland apoB-depleted plasmas from healthy controls, respectively (von Eckardsteinet al. 1995a). However, in these studies the considerable residual cholesterol effluxcapacity of HDL-free or HDL-poor plasma was explained by the abundance ofapoA-I-free HDL particles containing apoA-IV, apoA-II, or apoE rather than byalbumin (Huang et al. 1994, 1995; von Eckardstein et al. 1995a, b).Importantly, impaired cholesterol efflux capacity of HDL has been associatedwith structural changes of the HDL components. Two groups independentlydemonstrated that oxidative modification of apoA-I, particularly by oxidation ofmethionine, tyrosine, or tryptophan residues through the myeloperoxidase pathway,dramatically reduced the ability of apoA-I to promote cholesterol efflux through theABCA1 pathway (Bergt et al. 2004; Huang et al. 2014; Shao et al. 2006, 2010).Both groups also demonstrated that apoA-I in patients with cardiovascular diseasewas oxidatively modified and defective in promoting cholesterol efflux frommacrophages via ABCA1 (Bergt et al. 2004; Shao et al. 2012; Zheng et al. 2004).Of note, the cholesterol efflux capacity as determined by the assay protocol ofKhera et al. which is widely used after its prominent publications is potentiallyconfounded by several HDL-independent factors: the 4-h long incubation with cellsraises the possibility that serum components deregulate the activity of ABCA1 and342 M. Riwanto et al.ABCG1. For example, free fatty acids and cytokines are known to alter theexpression of ABCA1 and ABCG1 on both the transcriptional and posttranslationallevel. The use of serum instead of plasma implies the activation of thrombin andother proteases which are known to degrade prebeta-HDL (Eckardstein 2012).3 Effects of HDL on LDL Oxidation3.1 Mechanisms Under Physiological ConditionsThe antioxidative properties of HDL were first reported by Bowry (Bowryet al. 1992). It has been suggested that HDL limits the formation of oxidizedLDL (oxLDL) and oxidation of LDL has been proposed for decades to be a keyevent in atherogenesis (Heinecke 1998; Witztum and Steinberg 1991). LDLaccumulates in the subendothelial space where it is oxidized by different pathwayssuch as lipoxygenase, myeloperoxidase, or NADPH oxidase pathways (Diazet al. 1997). OxLDL induces the expression of various proinflammatory cytokines,chemokines, and adhesion molecules (Navab et al. 1996). In addition, it promotesmonocyte chemotaxis, their differentiation into macrophages, and the subsequentoxLDL uptake that convert them into foam cells, a hallmark of atheroscleroticplaques (Chisolm et al. 1999).HDL is a major carrier of lipid oxidation products and specifically apoA-I, themain protein constituent of HDL, binds to and removes lipid hydroperoxides ofLDL in vitro and in vivo (Navab et al. 2000b). In addition, apoA-I may directlyreduce cholesteryl ester hydroperoxides and phosphatidylcholine hydroperoxidesby oxidation of specific Met residues in the protein (Garner et al. 1998). Interest-ingly, the resulting oxidized HDL is more rapidly and selectively removed byhepatocytes than native HDL (Garner et al. 1998). Treatment of human arterywall cells with apoA-I but not apoA-II, or by addition of native HDL, apoA-Ipeptide mimetics, or paraoxonase, prevents the cells from oxidizing LDL in vitro(Navab et al. 2000a). The antioxidative function of apoA-I was corroborated by thefinding that recombinant HDL containing only apoA-I and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) was as effective as native HDL in preventing LDLoxidation. In addition, in vivo studies have demonstrated that apoA-I can act as anantioxidative, anti-inflammatory, and anti-atherosclerotic agent (Nichollset al. 2005a, b; Paszty et al. 1994). This further underlines a key antioxidant rolefor the HDL major protein apoA-I (Zerrad-Saadi et al. 2009).Several other HDL-associated apolipoproteins have also been shown to contrib-ute to its antioxidant effects. ApoE has been demonstrated to show isoform-dependent antioxidant activity (Miyata and Smith 1996). ApoE2 stimulates endo-thelial NO release and exerts anti-inflammatory effects (Sacre et al. 2003). Incontrast, apoE4 has been suggested to be proinflammatory (Ophir et al. 2005).Another HDL-associated protein, apoA-IV, showed anti-atherosclerotic, anti-inflammatory, and antioxidant actions in vivo (Ostos et al. 2001; RecaldeDysfunctional HDL: From Structure-Function-Relationships to Biomarkers 343et al. 2004; Vowinkel et al. 2004). In addition, apoJ, also called clusterin, has beenreported to block LDL oxidation by artery wall cells (Navab et al. 1997).Furthermore, ApoA-II-enriched HDL isolated from transgenic human apoA-IImice protected VLDL from oxidation more efficiently than control HDL (Boisferet al. 2002). In contrast, in other studies, dyslipidemic mice overexpressing humanapoA-II showed accelerated atherosclerosis and reduced antioxidative activity ofHDL, which may be attributed to the displacement of apoA-I and PON1 by apoA-IIin HDL particles (Ribas et al. 2004; Rotllan et al. 2005). Notably, in apparentlyhealthy subjects from the prospective EPIC-Norfolk (European Prospective Inves-tigation into Cancer and Nutrition-Norfolk) cohort, apoA-II was found to beassociated with a decreased risk of future CAD in a nested case-control study(Birjmohun et al. 2007).Importantly, HDL carries antioxidant enzymes that have the capacity to preventlipid oxidation or degrade lipid hydroperoxides such as serum paraoxonase/arylesterase 1 (PON1), lecithin/cholesterol acyltransferase (LCAT), and platelet-activating factor acetylhydrolase (PAF-AH). Specifically PON1 has been suggestedto be an important regulator of the potential antiatherogenic capacity of HDL (Shihet al. 1998; Tward et al. 2002). It is secreted from the liver and associates with HDLin the plasma. Many studies revealed that PON1 degrades oxidizedproinflammatory lipids, measured as a reduction in lipid peroxides (Macknesset al. 1991; Shih et al. 2000; Watson et al. 1995). Higher PON1 activity has beenreported to be associated with a lower incidence of major cardiovascular events inhuman. Conversely, reduced activity of PON1 has been associated with pathologi-cal conditions such as diabetes, chronic renal failure rheumatoid arthritis, andvarious dementia (Soran et al. 2009).LCAT is another HDL-associated enzyme with antioxidative properties. Theantioxidant role of LCAT is thought to be its capacity to hydrolyze oxidized acylchains from phosphatidylcholine-based oxidized phospholipids and oxidized freefatty acids (Goyal et al. 1997; Subramanian et al. 1999). Genetics and biochemicalcharacterizations of HDLs from patients with low HDL disorders demonstrated thatHDL-LCAT activity was reduced in all LCAT mutation carriers as well as inpatients with HDL deficiency due to mutation in APOA1 or ABCA1 (Soranet al. 2009; von Eckardstein et al. 1995a). In vivo study in mice deficient forLDL receptor and leptin revealed that LCAT overexpression decreasedautoantibodies to oxLDL (Mertens et al. 2003). Besides LCAT and PON1,PAF-AH, another HDL-associated enzyme, nowadays also known as lipoprotein-associated phospholipase A2 (LpPLA2), is able to hydrolyze oxidizedphospholipids (Marathe et al. 2003; Noto et al. 2003). The local expression ofPAF-AH in arteries of non-hyperlipidemic rabbits reduced the accumulation ofoxidatively modified LDL without changing plasma levels of PAF-AH and reducedthe expression of endothelial cell adhesion molecules (Arakawa et al. 2005).PAF-AH deficiency caused by a missense mutation in the gene has been indicatedto be an independent risk factor for CAD in Japanese men (McIntyre et al. 2009). Incontrast to this both activity and mass concentration of LpPLA2 in total serum hasbeen associated with increased rather than decreased cardiovascular risk(Lp et al. 2010). However, recently HDL-associated LpPLA2 was found to be344 M. Riwanto et al.inversely related to risk of coronary events in stable CAD patients (Rallidiset al. 2012). Hence, the lipoprotein distribution of LpPLA2 may determine thepro- or antiatherogenic role of PAF-AH/LpPLA2. Furthermore, plasma levels ofPAF-AH are also shown to be an independent risk marker of CAD (Garzaet al. 2007). However, a study by Holleboom et al. showed that the reduction inLCAT and PAF-AH activites due to LCAT mutations was not associated withincreased plasma lipid peroxidation (Holleboom et al. 2012).3.2 Impairment of the Anti-Oxidative Effects of HDL in PatientsAfter Surgery and With Cardiovascular DiseaseIn the acute phase response to surgery, van Lenten et al. demonstrated that theserum amyloid A (SAA) levels in HDL was increased and the activities of PON1and PAF-AH were reduced (Van Lenten et al. 1995). Concomitant with thesechanges the anti-inflammatory capacity of HDL was lost in both humans and rabbits(Van Lenten et al. 1995). Isolated HDL from patients before and immediately aftersurgery were compared for the effects of HDL on LDL-induced monocyte transmi-gration and lipid hydroperoxide formation (Van Lenten et al. 1995). Prior tosurgery, HDL completely inhibited the LDL-induced increase in monocyte trans-migration and lipid hydroperoxide formation. In marked contrast, the “acute phase”HDL obtained from the same patients 2–3 days after surgery revealed a significantLDL-induced monocyte transmigration and was less effective in inhibiting lipidhydroperoxide formation. In other words, HDL in the same patient had beentransformed from anti-inflammatory towards proinflammatory particles (VanLenten et al. 1995). Similarly findings were reported in mice infected with influenzaA or Chlamydia pneumonia. HDL isolated from virus-infected wild-type mice hadimpaired capacity to block LDL oxidation as well as LDL-induced monocytechemotactic activity in human artery wall cell co-cultures (Van Lentenet al. 2001). The mice infected with C. pneumoniae had decreased PON1 activityand reduced HDL capacity to prevent LDL oxidation at days 2 and 3 postinfection(Campbell et al. 2010). Of note, studies by Navab et al. showed that HDL isolatedfrom patients with CAD failed to prevent LDL oxidation by human artery wall cells(Navab et al. 2000a) and had impaired capacity to inhibit LDL-induced monocytechemotactiy activity (Navab et al. 2001b). Similar results were obtained with HDLisolated from mice genetically predisposed to diet-induced atherosclerosis; itbecame proinflammatory when the mice were fed an atherogenic diet (Navabet al. 2001a). A subsequent small study by Ansell et al. suggested that the capacityof HDL to alter LDL-induced monocyte chemotactic activity in patients with CADwas somewhat improved after 6 weeks of simvastatin therapy (Ansell et al. 2003).However, HDL from patients with CAD on statin therapy remainedproinflammatory when compared to HDL from healthy subjects, despite a signifi-cant improvement in plasma lipid levels.Interestingly, impaired antioxidant properties and increased oxidized fatty acidscontent were found in HDL from patients with type 2 diabetes (MorgantiniDysfunctional HDL: From Structure-Function-Relationships to Biomarkers 345et al. 2011). The authors speculated that the elevated levels of oxidized fatty acidsin HDL from these patients may account for the impaired antioxidant properties ofthe lipoprotein (Morgantini et al. 2011). Moreover, in a different study, the abilityof HDL to prevent LDL oxidation was found to be reduced in patients with acutecoronary syndrome but not in patients with stable coronary artery syndrome (Patelet al. 2011). The antioxidative and cholesterol efflux capacities of HDL were alsofound to be reduced in ischemic cardiomyopathy (Patel et al. 2013).4 Effects of HDL on Endothelial Nitric Oxide Bioavailability4.1 Mechanisms Under Physiological ConditionsEndothelial nitric oxide plays a crucial role in the regulation of vascular tone,platelet aggregation, and angiogenesis. Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) has been shown to exert a variety of atheroprotectiveeffects in the vasculature, such as anti-inflammatory and antithrombotic effects(Landmesser et al. 2004). Consequently, reduced endothelial NO bioavailabilityhas been proposed to promote initiation and progression of atherosclerosis(Landmesser et al. 2004).It was shown that HDL can directly stimulate eNOS-mediated NO production(Yuhanna et al. 2001). There have been several studies that consistentlydemonstrated the capacity of HDL to modulate eNOS expression and to stimulateendothelial NO production in vitro and in vivo (Besler et al. 2011; Kuvinet al. 2002; Mineo et al. 2003; Nofer et al. 2004; Ramet et al. 2003; Sorrentinoet al. 2010). Furthermore, administration of reconstituted HDL (rHDL) has beenshown to improve endothelial function in patients with hypercholesterolemia or insubjects with isolated low HDL due to heterozygous loss-of-function mutations inthe ABCA1 gene locus (Bisoendial et al. 2003; Spieker et al. 2002). Severalmechanisms have been proposed to account for the endothelial NO-stimulatingcapacity of HDL. Early studies have demonstrated that HDL prevents oxLDL-mediated eNOS displacements from caveolae and restores enzyme stimulation(Uittenbogaard et al. 2000). Yuhanna et al. demonstrated that HDL binding toendothelial SR-BI is required for eNOS activation (Yuhanna et al. 2001). Thisbinding activates the phosphatidylinositol-3-kinase/Akt signaling pathway and theMAP kinase/extracellular signal-regulated kinase pathway (Mineo et al. 2003).Activation of endothelial Akt by HDL stimulates phosphorylation of eNOS atserine residue 1177 (Mineo et al. 2003; Nofer et al. 2004), which is known to bean important regulatory mechanism leading to eNOS activation (Dimmeleret al. 1999).There are many different components of HDL known to play a role in theendothelial NO-stimulating capacity. The potential interaction of apoA-I witheNOS in cultured endothelial cells has been previously reported (Rametet al. 2003). However, lipid-free apoA-I failed to activate eNOS despite being theligand for SR-BI, suggesting that other HDL components may be important or are346 M. Riwanto et al.required to support the conformation of apoA-I to allow its interaction with SR-BIand to stimulate eNOS (de Beer et al. 2001). In isolated endothelial cell plasmamembranes, anti-apoA-I antibody blocks eNOS activation by HDL in vitro(Yuhanna et al. 2001). In contrast anti-apoA-II antibody further enhances eNOSstimulation by HDL (Yuhanna et al. 2001). Furthermore, lysophospholipids mayplay a role in eNOS activation as demonstrated in several studies. A study by Noferet al. suggested that HDL-associated sphingolipids such as sphingosylphosphor-ylcholine, sphingosine-1-phosphate, and lysosulfatide caused eNOS-dependentrelaxation of precontracted aortic rings from mice by binding to thelysophospholipid receptor S1P3 expressed in endothelial cells (Nofer et al. 2004).However, the vasodilatory response of HDL was not completely abolished in S1P3-deficient mice (Nofer et al. 2004).Recently, we demonstrated that the HDL-associated enzyme PON1 isparticipating in HDL’s capacity to stimulate endothelial NO production and toexert NO-dependent endothelial atheroprotective effects (Besler et al. 2011). Inhi-bition of PON1 in HDL from healthy subjects impaired the capacity of HDL tostimulate endothelial NO production in human aortic endothelial cells. In additionHDL isolated from PON1-deficient mice failed to stimulate NO production inmouse aortic endothelial cells (Besler et al. 2011). Furthermore, inhibition ofeNOS-mediated NO production by the pharmacological inhibitor L-NAMEprevented the inhibitory effects of HDL from healthy subjects on nuclear factorκB (NF-κB) activity, vascular cell adhesion molecule (VCAM)-1 expression, andendothelial monocyte adhesion. These clearly indicate that the capacity of HDL tostimulate endothelial NO production is important for these endothelial anti-inflammatory effects of HDL (Besler et al. 2011).4.2 Impaired HDL Capacity to Stimulate NO Production inPatients with Cardiovascular DiseaseWe and others have recently shown that HDL isolated from patients with stableCAD, diabetes, chronic kidney diseases (CKD), or acute coronary artery syndrome(ACS) displays altered endothelial effects when compared to HDL from healthysubjects. Of note, HDL from patients with diabetes in contrast to HDL from healthysubjects failed to stimulate endothelial cell NO production and to promote endo-thelial repair in a carotid artery injury model in mice (Sorrentino et al. 2010).Moreover, HDL from patients with either stable CAD or an ACS, in contrast toHDL from age- and gender-matched healthy subjects, inhibited endothelial cell NOproduction and lost the capacity to limit endothelial inflammatory activation as wellas to promote endothelial repair in vivo (Besler et al. 2011). Interestingly, thecapacity to stimulate endothelial NO production could be improved upon exercisetraining (Adams et al. 2013).HDL from patients with CAD and chronic heart failure (CHF) showed anelevated malondialdehyde (MDA) content as compared to HDL from healthysubjects, which may contribute to the impaired endothelial NO production (BeslerDysfunctional HDL: From Structure-Function-Relationships to Biomarkers 347et al. 2011). The MDA-lysine adducts in HDL was determined to act via lectin-typeoxidized LDL receptor 1 (LOX-1) activation of protein kinase C-βII, which blocksAkt-activating phosphorylation at Ser473 and eNOS-activating phosphorylation atSer1177. Furthermore, inactivation of PON1 in HDL from healthy subjects resultedin attenuated NO production, in greater protein kinase C-βII activation, decreasedactivating eNOS-Ser1177 phosphorylation, and increased inactivating eNOS-Thr495 phosphorylation. Furthermore, HDL isolated from PON1-deficient micefailed to stimulate endothelial cell NO production (Besler et al. 2011). Theseobservations likely suggest that alterations of HDL-associated PON1 may severelyaffect endothelial effects of HDL.Very recently, we have also shown that MPO and PON1 reciprocally modulateeach other’s function in vivo (Huang et al. 2013). MPO may promote site-specificoxidative modification of PON1, therefore limiting its activity. In addition, HDLisolated from patients with an ACS carries enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity (Huanget al. 2013).Several groups have reported an inverse relationship between PON1 serumactivity and cardiovascular events (Bhattacharyya et al. 2008; Regieliet al. 2009). Interestingly, analysis of SNPs for PON1 identified in genome wideassociation studies did not reveal a significant association between SNPs,associated with mildly reduced PON1 activity, and the risk of cardiovascular events(Tang et al. 2012). The interpretation of PON1 activity is difficult because it is notknown to which extent the paraoxonase and arylesterase activities represent bio-logically relevant functions. Furthermore, we and others observed important post-translational modifications of the proteins, which could explain alterations ofbiological properties of the enzymes (Aviram et al. 1999; Besler et al. 2011;Huang et al. 2013).5 Endothelial Anti-Inflammatory Effects of HDL5.1 Mechanisms Under Physiological ConditionsAtherosclerosis is a chronic inflammatory disorder. The cellular inflammatoryresponse is triggered by modified LDL accumulated in the subendothelium andthe induction of proinflammatory cytokines and adhesion protein expression. As aconsequence monocytes/macrophages infiltrate and accumulate in the arterial walland express scavenger receptors and toll-like receptors to sequester cholesterol. Thecholesterol-loaded macrophages together with T lymphocytes produce a wide arrayof proinflammatory cytokines and accelerate plague progression (Hansson 2005).In vitro, HDL has been shown to inhibit the expression of monocytechemoattractant protein (MCP)-1, an important proinflammatory chemokine inendothelial cells (Mackness et al. 2004; Navab et al. 1991). Furthermore, thepotential anti-inflammatory effects of HDL have been demonstrated by severalin vivo studies. Infusion of native HDL as well as reconstituted HDL containing348 M. Riwanto et al.apoA-I or apoA-I Milano suppressed cytokine and chemokine expression in animalmodels of inflammation (Calabresi et al. 1997; Cockerill et al. 1995). ReducedVCAM-1 expression and decreased monocyte/macrophage infiltration werereported after carotid artery cuff injury in apoE-deficient mice (Dimayugaet al. 1999). In the streptozotocin-induced diabetic cardiomyopathy rat model,human apoA-I gene transfer increased HDL-C plasma levels and blocked thediabetes-induced myocardial mRNA expression of VCAM-1 and ICAM-1 (VanLinthout et al. 2008). In contrast, overexpression of human apoA-I in apoE-deficient mice did not result in any change of endothelial VCAM-1 expressionand monocyte adherence in early atherosclerotic lesions at the aortic branch sites,despite overall reduction in aortic atherosclerotic lesion formation (Danskyet al. 1999). These studies support the concept that the anti-inflammatory capacityof HDL is heterogeneous, and is dependent on the pathophysiological conditions. Invitro studies showed variations in the capacity of HDL isolated from differenthuman subjects to reduce TNF-α stimulated endothelial VCAM-1 expression(Ashby et al. 1998; Van Lenten et al. 1995). In human studies, the administrationof reconstituted HDL increased the anti-inflammatory capacity of HDL frompatients with type-2 diabetes (Patel et al. 2009).One of the proposed mechanisms for the anti-inflammatory effect of HDL is theremoval of cholesterol from cell membranes of macrophages and also endothelialcells. The cholesterol depletion and consequently the lipid rafts disruption maydownregulate signaling pathways and interfere with the antigen presentation or theexpression of toll-like receptor (Anderson et al. 2000; Norata and Catapano 2012;Wang et al. 2012). Additional studies suggest that HDL and apoA-I are able toinhibit the capacity of antigen-presenting cells to stimulate T-cell activation. Thisinhibition was attributed to cholesterol efflux through ABCA1, an ATP-bindingtransporter (Tang et al. 2009; Zhu et al. 2010).The anti-inflammatory capacity of HDL has been mainly attributed to apoA-I,the major protein constituent of HDL. In an in vivo study, apoA-I infusion in rabbitsin acute vascular inflammation model reduced neutrophil infiltration and endothe-lial cell inflammatory activation (Puranik et al. 2008). In addition, apoA-1 mimeticpeptides have been shown to reduce vascular inflammation in type I diabetic ratsand improve insulin sensitivity in obese mice (Peterson et al. 2007, 2008). It hasalso been demonstrated that treatment with lipid-free apoA-I and rHDL treatmentreduced the expression of chemokines and chemokine receptors in vitro and in vivoby modulating NF-κB and peroxisome proliferator-activated receptor γ (Bursillet al. 2010). Interestingly, apoA-I has also been shown to inhibit palmitate-inducedNF-κB activation by reducing Toll-like receptor-4 recruitment into lipid rafts(Cheng et al. 2012). More recently, De Nardo et al. using a systems biologyapproach identified the transcription factor ATF3 as an HDL-inducible gene tosuppress Toll-like receptor-induced proinflammatory cytokines. Mice deficient inATF3 and apoE revealed enhanced atherosclerotic lesions, and administration ofrHDL into apoE-deficient mice resulted in ATF3 upregulation. The authors usedrHDL in their study, suggesting that apoA-I is a key mechanistic player, and ofDysfunctional HDL: From Structure-Function-Relationships to Biomarkers 349note, the effects seem to be unrelated to cholesterol transport (De Nardoet al. 2014).The lipid moiety of HDL has also been proposed to be important for the anti-inflammatory effects of HDL. In vitro studies using reconstituted HDL containingonly apoA-I with a few phospholipid molecules suggested that inhibitory effects ofHDL on endothelial cell adhesion molecule expression are also, at least in part,dependent on HDL-associated phospholipid species (Baker et al. 2000). The inhi-bition of cytokine-induced expression of VCAM-1 by reconstituted HDL variedwhen different phosphatidylcholine species were used. This suggests that the lipidcomposition of HDL influences its anti-inflammatory capacity and is likely animportant determinant of HDL functionality (Baker et al. 2000; Barteret al. 2004). In addition there are multiple reports suggesting the involvement ofsphingosine-1-phosphate (S1P), a biologically active sphingolipid that plays keyfunctions in the immune, inflammatory, and cardiovascular systems. S1P carried byHDL is believed to regulate arterial tone, vascular permeability, and tissue perfu-sion. During inflammation it induces endothelial adhesion molecules and recruitsinflammatory cells, and furthermore it activates a negative feedback loop thatconsecutively decreases vascular leakage by improving the endothelial barrierfunction and preventing cytokine-induced leukocyte adhesion (Garcia et al. 2001;Lucke and Levkau 2010; Takeya et al. 2003).5.2 Impaired Endothelial Anti-Inflammatory Effects of HDL inPatients with CAD, Diabetes, or Chronic Kidney DysfunctionHDL isolated from patients with inflammatory disease such as CAD, diabetes, orchronic kidney disease display reduced eNOS activation and impaired endothelialrepair capacity in a carotid artery injury mouse model. There have been variousmechanisms proposed to account for the impaired endothelial anti-inflammatoryeffects of HDL. Reduced HDL apoA-I levels in inflammatory states has beenrelated to accelerated HDL catabolism and apoA-I substitution in HDL particlesby serum amyloid A (SAA) (Esteve et al. 2005; Khovidhunkit et al. 2004). In acutephase, SAA is able to replace apoA-I in HDL, resulting in reduced plasma levels ofapoA-I (Parks and Rudel 1985). ApoA-I can be completely replaced by SAA inrabbits and mice, in a subset of small, dense HDL particles and therefore function-ing as a structural apolipoprotein (Cabana et al. 1999). HDL isolated from patientswith chronic kidney disease is enriched in SAA, which correlated with its reducedanti-inflammatory capacity to inhibit monocyte chemoattractant protein-1 forma-tion in vascular smooth muscle cells (Tolle et al. 2012).Moreover, HDL carries potential cardioprotective molecules that are signifi-cantly altered in compositions in patients with CAD or ACS (Riwanto et al. 2013;Vaisar et al. 2007). Oxidative modifications of HDL, in particular the amino acidresidues in apoA-I, have been shown to contribute to the generation of dysfunc-tional HDL (Bergt et al. 2004; Shao et al. 2006, 2012; Zheng et al. 2004). In vitrostudy has demonstrated that MPO-catalyzed oxidative modification of HDL or350 M. Riwanto et al.apoA-I converts HDL into a proinflammatory particle which promotes NF-κBactivation and endothelial VCAM-1 expression (Undurti et al. 2009). Furthermore,glycation of HDL and apoA-I, a process that is known to occur in diabetes in vivo(Curtiss and Witztum 1985), has been shown to impair the anti-inflammatorycapacity of HDL (Nobecourt et al. 2010). Infusion of glycated lipid-free apoA-Ifailed to decrease adhesion molecule expression following vascular injury(Nobecourt et al. 2010). Moreover, glycation of HDL has also been shown toinhibit the HDL capacity to inhibit oxLDL-induced monocyte adhesion to humanaortic endothelial cells in vitro (Hedrick et al. 2000).6 Effects of HDL on Endothelial Cell Apoptotic Pathways6.1 Mechanisms Under Physiological ConditionsEndothelial cell dysfunction and injury has been associated with the pathogenesisof atherosclerosis (Landmesser et al. 2004; Nabel and Braunwald 2012; Ross 1999).Studies in pigs have shown that atherosclerotic lesion-prone regions arecharacterized by a high endothelial cell turnover (Caplan and Schwartz 1973),which has been suggested to be due to increased endothelial cell apoptosis (Caplanand Schwartz 1973). Several studies have indicated an important contribution ofendothelial cell apoptosis to the pathophysiology of CAD (Burke et al. 1997; Rossiget al. 2001). Furthermore, it is evident that apoptosis of endothelial cells and smoothmuscle cells is detrimental to plaque stability (Bombeli et al. 1997; Kockx andHerman 2000). In the normal arterial wall, endothelial cells may protect themselvesagainst apoptosis using NO-dependent mechanisms. The situation is completelydifferent in atherosclerotic plaques, in a high oxidative stress environment wheremacrophages produce high amounts of nitric oxide or peroxynitrite that couldinduce apoptotic cell death (Kockx and Herman 2000). In the aorta the capacityof HDL to quench endothelial cell apoptosis may therefore represent anantiatherogenic property of HDL (de Souza et al. 2010; Nofer et al. 2001; Sucet al. 1997; Sugano et al. 2000).HDL has been shown to attenuate endothelial cell apoptosis induced by differentstimuli such as TNF-α, oxLDL, and growth factor deprivation (de Souza et al. 2010;Nofer et al. 2001; Suc et al. 1997; Sugano et al. 2000). It has been suggested thatHDL may inhibit both death-receptor and mitochondrial-mediated apoptoticpathways. Importantly, both the protein and lipid moieties have been reported tocontribute to the antiapoptotic capacity of HDL. ApoA-I has been shown to inhibitendothelial cell apoptosis induced by oxLDL, VLDL, and TNF-α (Speidelet al. 1990; Suc et al. 1997; Sugano et al. 2000). In a more detailed study, HDLsubpopulations enriched with apoA-I account for approximately 70 % of theantiapoptotic activity of HDL in a cell culture model with human microvascularendothelial cells that were treated with mildly oxidized LDL. Using rHDLconsisting of apoA-I, cholesterol and phospholipids potently inhibited oxLDL-induced apoptosis in these cells (de Souza et al. 2010). This suggests that apoA-IDysfunctional HDL: From Structure-Function-Relationships to Biomarkers 351plays an important role for the antiapoptotic capacity of HDL in oxLDL-stimulatedendothelial cells.HDL-associated lysosphingolipids have been identified to inhibit endothelialcell apoptosis induced by growth factor deprivation (Kimura et al. 2001, 2003;Nofer et al. 2001). The antiapoptotic capacity of the lipid moiety of HDL wasfurther supported by the findings that the ratio of sphingosine-1-phosphate andsphingomyelin in small dense HDL3 particles was increased and correlated posi-tively with the characteristics of these HDL subpopulations to inhibit endothelialcell apoptosis (Kontush et al. 2007).Several mechanisms have been suggested for the endothelial antiapoptoticeffects of HDL, depending on the stimulus of apoptosis. Endothelial cell deathtriggered by oxLDL causes a delayed but sustained increase in intracellular calciumwhich is shown to be prevented by HDL (Suc et al. 1997). HDL inhibits tumornecrosis factor-α-induced endothelial cell death by association as well as suppres-sion of caspase-3, which is a key factor of all primary apoptotic pathways (Suganoet al. 2000). Nofer et al. suggested that sphingosine-1-phosphate (S1P) carried byHDL prevents the growth factor deprived activation of the intrinsic pathway to celldeath (Nofer et al. 2001). Of note, lysophospholipids have been shown to enhanceendothelial cell survival, but the effects were inhibited by S1P receptor knockdown,as well as by the presence of phosphoinositide 3 (PI3) kinase and Erk pathwayantagonists (Kimura et al. 2003).In addition, HDL activates Akt and causes phosphorylation of the Akt targetBcl-2-associated death promoter (BAD), preventing it from binding to theantiapoptotic protein Bcl-xL (Nofer et al. 2001). HDL also causes phosphoinositide3 (PI3) kinase-mediated upregulation of the antiapoptotic Bcl-2 family proteinBcl-xL expression (Riwanto et al. 2013). Interestingly, HDL retained itsantiapoptotic activity after knockdown of eNOS using specific RNA interferenceor pharmacological inhibition using L-NAME (Riwanto et al. 2013), indicating thatHDL may exert its antiapoptotic activity independently of eNOS activation.6.2 Impairment of the Endothelial Anti-Apoptotic Effects of HDLin Patients with Cardiovascular DiseaseRecently, we reported that HDL isolated from patients with stable CAD or ACS, incontrast to HDL from healthy subjects, failed to inhibit endothelial cell apoptosisin vitro and in apoE-deficient-mice in vivo. Using proteomic analysis of HDL, weobserved reduced clusterin and increased apo C-III content in HDL isolated frompatients with CAD. Both, supplementing HDL from healthy subjects with apoC-IIIor pretreatment of healthy HDL with an antibody against clusterin, lead to theactivation of proapoptotic signaling pathways in endothelial cells (Riwantoet al. 2013). HDL isolated from these patients stimulated endothelial proapoptoticpathways, in particular p38-MAPK-mediated activation of the proapoptotic Bcl-2-protein tBid. Our study further suggests that differences in the proteome of HDLfrom patients with CAD, in particular reduced HDL-associated clusterin andincreased HDL-associated apoC-III, play an important role for altered activation352 M. Riwanto et al.of endothelial anti- and proapoptotic signaling pathways (Riwanto et al. 2013). Inline with this, a nested case-control study found that cholesterol levels in apoC-III-containing HDL particles show a positive association with cardiovascular risk,whereas both total HDL-C and cholesterol in apoC-III-free HDL particles showthe expected inverse associations (Jensen et al. 2012). In addition, Undurtiet al. demonstrated that MPO-catalyzed oxidation of HDL impaired its capacityto inhibit endothelial apoptosis in vitro (Undurti et al. 2009).7 HDL Protein Cargo and Prognostic BiomarkersAs highlighted above, alterations in HDL functions are likely, at least in part,attributed to changes in the composition of HDL particles. HDL particles are highlyheterogenous in their structure and compositions. They undergo continuousremodeling through apolipoprotein exchanges with other circulating lipoproteinsand tissues (Lund-Katz et al. 2003; Saito et al. 2004).An early study has shown that acute phase response causes significant changes inthe HDL-associated proteins and apolipoprotein composition, i.e., enrichment inC-reactive protein (CRP), secretory phospholipase A2-IIa (sPLA2-IIa), serum amy-loid A (SAA), and cholesterol ester transfer protein (CETP) (Coetzee et al. 1986). Ithas been suggested that such remodeling may compromise the antiatherogenicfunctions of HDL (Jahangiri et al. 2009). These observations have promptedexplorative studies into HDL composition. Evaluations of the HDL proteomehave been reported with one- or two-dimensional electrophoresis combined withhigh performance liquid chromatography (HPLC) and tandem mass spectrometry(MS/MS) or matrix-assisted laser desorption/ionization-time flight mass spectrom-etry (MALDI-TOF-MS) (Heller et al. 2007; Karlsson et al. 2005). More recentstudies have used shotgun proteomics, through label-free MS techniques, such asspectral counting and XIC inspection, to analyze HDL protein compositions(Riwanto et al. 2013; Vaisar et al. 2007). Overall, the identification techniquescurrently available for the analysis of the HDL proteome are semi-quantitative.Given the complexity of HDL and its multitude of biological functions, it isconceivable to expect that sets of functionally associated proteins can provideinformation about their participation in the spectrum of atheroprotective actionsattributed to HDL. This has further led to studies comparing the HDL proteome ofhealthy subjects with that of particles from patients with dyslipidemias or CAD(Green et al. 2008; Heller et al. 2007; Riwanto et al. 2013; Vaisar et al. 2007). In thestudy by Vaisar et al., HDL was shown to carry apolipoproteins and proteins withfunctions in lipid metabolism, the acute phase response, complement regulation,and blood coagulation (Vaisar et al. 2007). The same group further compared theproteome of HDL3 from 6 CAD patients before and after 1-year treatment withcombined statin/niacin therapy (Green et al. 2008). In this study, HDL3 of CADpatients is significantly enriched in apoE and apoCII and carry less apoJ andphospholipid transport protein (PLTP) as compared to HDL from control subjects.Treatment with niacin/statin decreases HDL3 apoE and raises apoJ and PLTP levelsDysfunctional HDL: From Structure-Function-Relationships to Biomarkers 353(Green et al. 2008). In our recent study, we have observed that reduced clusterin andincreased apoC-III content in HDL isolated from patients with CAD lead toactivation of proapoptotic signaling pathways in endothelial cells (Riwantoet al. 2013).Of note, oxidative modifications of HDL-associated proteins have also beendemonstrated to contribute to the functional impairment of HDL. In particular,MPO-mediated oxidation of apoA-I has been linked to the generation of dysfunc-tional forms of HDL (Huang et al. 2014; Shao et al. 2010; Undurti et al. 2009; Wanget al. 2007b; Zheng et al. 2004). Recently, we have shown that MPO and PON1interact and reciprocally modulate each other’s function in vivo, i.e., PON1 par-tially inhibits MPO activity, while MPO oxidizes and inactivates PON1 (Huanget al. 2013). Furthermore, HDL isolated from patients with an ACS carriesenhanced chlorotyrosine content, site-specific PON1 methionine oxidation, andreduced PON1 activity (Huang et al. 2013).Taken together, these observations indicate that the HDL proteomic compositionmay be altered in different disease states which likely have an impact on HDLfunction. Unraveling the complexities of the HDL through proteomics studies maytherefore allow for better understanding of the HDL composition and function.However, proteomics studies of HDL have been limited in size and number due tothe labor-intensive nature of the approach. Furthermore, given the diverse concen-tration range of the proteins in the HDL particles, some proteomics approaches mayfail to reflect the actual amount or alteration of low abundant proteins due to signalsuppression in the presence of highly abundant proteins. Targeted proteomics usingselected reaction monitoring is a powerful tandem mass spectrometry method thatcan be used to monitor target peptides within a complex protein digest (Picotti andAebersold 2012; Picotti et al. 2010). The approach will enable absolute quantifica-tion of the individual protein contained within HDL particles.Conclusion and PerspectivesOver the past two decades, many studies have emerged showing variousantiatherogenic effects of HDL, including the capacity of HDL to mediatereverse cholesterol transport, and antioxidative, anti-inflammatory, andantiapoptotic effects (Riwanto and Landmesser 2013). Growing evidence hasshown that HDL particles are highly heterogeneous and the vasoprotectiveeffects of HDL are altered in patients with CAD, diabetes, and chronic kidneydysfunction, i.e., patients with a high cardiovascular risk profile. Of note, thesefindings have more recently been supported by observations in cohort studies,suggesting that in patients with advanced CAD, higher plasma levels of HDL-Care no longer associated with reduced risk of cardiovascular events (Angeloniet al. 2013; Schwartz et al. 2012). Importantly, the increase in HDL-C observedwith some lipid-modifying drugs has not been uniformly associated with clinicalbenefit (Barter et al. 2007; Schwartz et al. 2012). These findings have clearlyindicated that the association between HDL and cardiovascular disease is farmore complex than previously thought and is likely mediated by different HDL354 M. Riwanto et al.functional properties, independent of the cholesterol component of the HDLparticle (Heinecke 2011).It is increasingly evident that the functional heterogeneity of HDL may beattributed to the complexity of the HDL particles, containing proteins and lipidsthat can be modified or altered in their composition. Of note, rapid advances inthe MS proteome technology have allowed tremendous leaps in characterizingthe HDL proteome. Studies investigating the lipidome of HDL are still some-what limited by the available technologies. Importantly, several studies haveassociated changes in specific HDL protein either due to altered composition ormodification to the loss of function of HDL (Besler et al. 2011; Huanget al. 2013; Riwanto et al. 2013; Shao et al. 2006, 2012; Zheng et al. 2004).Nevertheless, it remains to be determined if the alterations are secondary tochanges that occur during the disease progression or if the alteration has indeed acausal effect on disease etiology. In this context it is also important to emphasizethe problem of confounding between different diseases: for example, manydiabetic patients have (sub)clinical chronic kidney disease and/or CAD. As aconsequence one must also expect considerable overlap of HDL modificationswhich were initially identified in patients with either diabetes, CAD, or CKD. Itis hence rather unlikely that each disease has its specific footprint of HDLdysfunction and alterations in proteome and lipidome.Taken together, it is becoming clear that plasma HDL-C levels are not anappropriate marker of vascular effects of high-density lipoproteins and thereforedo not represent a reliable therapeutic target. Importantly, HDL-targeted treat-ment approaches need to take into account the altered vascular effects of HDL incardiovascular disease. Therefore, targeting HDL-mediated antiatherogenicmechanisms, rather than plasma HDL-C levels, may represent a more promisingand interesting therapeutic target. Studies looking into the specific mechanismsleading to a loss of antiatherogenic effects of HDL are of particular importance,in order to develop therapeutic approaches that aim to restore the vasoprotectiveproperties of HDL.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAdams V, Besler C, Fischer T, Riwanto M, Noack F, Hollriegel R, Oberbach A, Jehmlich N,Volker U, Winzer EB, Lenk K, Hambrecht R, Schuler G, Linke A, Landmesser U, Erbs S(2013) Exercise training in patients with chronic heart failure promotes restoration of high-density lipoprotein functional properties. Circ Res 113:1345–1355Anderson HA, Hiltbold EM, Roche PA (2000) Concentration of MHC class II molecules in lipidrafts facilitates antigen presentation. Nat Immunol 1:156–162Dysfunctional HDL: From Structure-Function-Relationships to Biomarkers 355Angeloni E, Paneni F, Landmesser U, Benedetto U, Melina G, Luscher TF, Volpe M, Sinatra R,Cosentino F (2013) Lack of protective role of HDL-C in patients with coronary artery diseaseundergoing elective coronary artery bypass grafting. Eur Heart J 34(46):3557–3562Annema W, von Eckardstein A (2013) High-density lipoproteins. Multifunctional but vulnerableprotections from atherosclerosis. Circ J 77:2432–2448Ansell BJ, Navab M, Hama S, Kamranpour N, Fonarow G, Hough G, Rahmani S, Mottahedeh R,Dave R, Reddy ST, Fogelman AM (2003) Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish patients from control subjects better than high-density lipopro-tein cholesterol levels and are favorably affected by simvastatin treatment. Circulation108:2751–2756Arakawa H, Qian JY, Baatar D, Karasawa K, Asada Y, Sasaguri Y, Miller ER, Witztum JL, UenoH (2005) Local expression of platelet-activating factor-acetylhydrolase reduces accumulationof oxidized lipoproteins and inhibits inflammation, shear stress-induced thrombosis, andneointima formation in balloon-injured carotid arteries in nonhyperlipidemic rabbits. Circula-tion 111:3302–3309Ashby DT, Rye KA, Clay MA, Vadas MA, Gamble JR, Barter PJ (1998) Factors influencing theability of HDL to inhibit expression of vascular cell adhesion molecule-1 in endothelial cells.Arterioscler Thromb Vasc Biol 18:1450–1455Aviram M, Rosenblat M, Billecke S, Erogul J, Sorenson R, Bisgaier CL, Newton RS, La Du B(1999) Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoproteinand preserved by antioxidants. Free Radic Biol Med 26:892–904Baker PW, Rye KA, Gamble JR, Vadas MA, Barter PJ (2000) Phospholipid composition ofreconstituted high density lipoproteins influences their ability to inhibit endothelial celladhesion molecule expression. J Lipid Res 41:1261–1267Barter PJ, Nicholls S, Rye KA, Anantharamaiah GM, Navab M, Fogelman AM (2004)Antiinflammatory properties of HDL. Circ Res 95:764–772Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJ, Komajda M, Lopez-Sendon J,Mosca L, Tardif JC, Waters DD, Shear CL, Revkin JH, Buhr KA, Fisher MR, Tall AR, BrewerB (2007) Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med357:2109–2122Bergt C, Pennathur S, Fu X, Byun J, O’Brien K, McDonald TO, Singh P, Anantharamaiah GM,Chait A, Brunzell J, Geary RL, Oram JF, Heinecke JW (2004) The myeloperoxidase producthypochlorous acid oxidizes HDL in the human artery wall and impairs ABCA1-dependentcholesterol transport. Proc Natl Acad Sci USA 101:13032–13037Besler C, Heinrich K, Rohrer L, Doerries C, Riwanto M, Shih DM, Chroni A, Yonekawa K,Stein S, Schaefer N, Mueller M, Akhmedov A, Daniil G, Manes C, Templin C, Wyss C,Maier W, Tanner FC, Matter CM, Corti R, Furlong C, Lusis AJ, von Eckardstein A, FogelmanAM, Luscher TF, Landmesser U (2011) Mechanisms underlying adverse effects of HDL oneNOS-activating pathways in patients with coronary artery disease. J Clin Invest121:2693–2708Bhattacharyya T, Nicholls SJ, Topol EJ, Zhang R, Yang X, Schmitt D, Fu X, Shao M, BrennanDM, Ellis SG, Brennan ML, Allayee H, Lusis AJ, Hazen SL (2008) Relationship ofparaoxonase 1 (PON1) gene polymorphisms and functional activity with systemic oxidativestress and cardiovascular risk. JAMA 299:1265–1276Birjmohun RS, Dallinga-Thie GM, Kuivenhoven JA, Stroes ES, Otvos JD, Wareham NJ, Luben R,Kastelein JJ, Khaw KT, Boekholdt SM (2007) Apolipoprotein A-II is inversely associated withrisk of future coronary artery disease. Circulation 116:2029–2035Bisoendial RJ, Hovingh GK, Levels JH, Lerch PG, Andresen I, Hayden MR, Kastelein JJ, StroesES (2003) Restoration of endothelial function by increasing high-density lipoprotein insubjects with isolated low high-density lipoprotein. Circulation 107:2944–2948Boisfer E, Stengel D, Pastier D, Laplaud PM, Dousset N, Ninio E, Kalopissis AD (2002)Antioxidant properties of HDL in transgenic mice overexpressing human apolipoproteinA-II. J Lipid Res 43:732–741356 M. Riwanto et al.Bombeli T, Karsan A, Tait JF, Harlan JM (1997) Apoptotic vascular endothelial cells becomeprocoagulant. Blood 89:2429–2442Bortnick AE, Rothblat GH, Stoudt G, Hoppe KL, Royer LJ, McNeish J, Francone OL (2000) Thecorrelation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various celllines. J Biol Chem 275:28634–28640Bowry VW, Stanley KK, Stocker R (1992) High density lipoprotein is the major carrier of lipidhydroperoxides in human blood plasma from fasting donors. Proc Natl Acad Sci USA89:10316–10320Burke AP, Farb A, Malcom GT, Liang YH, Smialek J, Virmani R (1997) Coronary risk factors andplaque morphology in men with coronary disease who died suddenly. N Engl J Med336:1276–1282Bursill CA, Castro ML, Beattie DT, Nakhla S, van der Vorst E, Heather AK, Barter PJ, Rye KA(2010) High-density lipoproteins suppress chemokines and chemokine receptors in vitro andin vivo. Arterioscler Thromb Vasc Biol 30:1773–1778Cabana VG, Reardon CA, Wei B, Lukens JR, Getz GS (1999) SAA-only HDL formed during theacute phase response in apoA-I+/+and apoA-I-/- mice. J Lipid Res 40:1090–1103Calabresi L, Franceschini G, Sirtori CR, De Palma A, Saresella M, Ferrante P, Taramelli D (1997)Inhibition of VCAM-1 expression in endothelial cells by reconstituted high densitylipoproteins. Biochem Biophys Res Commun 238:61–65Campbell LA, Yaraei K, Van Lenten B, Chait A, Blessing E, Kuo CC, Nosaka T, Ricks J,Rosenfeld ME (2010) The acute phase reactant response to respiratory infection with Chla-mydia pneumoniae: implications for the pathogenesis of atherosclerosis. Microbes Infect12:598–606Caplan BA, Schwartz CJ (1973) Increased endothelial cell turnover in areas of in vivo Evans Blueuptake in the pig aorta. Atherosclerosis 17:401–417Castelli WP, Garrison RJ, Wilson PW, Abbott RD, Kalousdian S, Kannel WB (1986) Incidence ofcoronary heart disease and lipoprotein cholesterol levels. The Framingham study. JAMA256:2835–2838Charakida M, Besler C, Batuca JR, Sangle S, Marques S, Sousa M,Wang G, Tousoulis D, DelgadoAlves J, Loukogeorgakis SP, Mackworth-Young C, D’Cruz D, Luscher T, Landmesser U,Deanfield JE (2009) Vascular abnormalities, paraoxonase activity, and dysfunctional HDL inprimary antiphospholipid syndrome. JAMA 302:1210–1217Cheng AM, Handa P, Tateya S, Schwartz J, Tang C, Mitra P, Oram JF, Chait A, Kim F (2012)Apolipoprotein A-I attenuates palmitate-mediated NF-kappaB activation by reducing Toll-likereceptor-4 recruitment into lipid rafts. PLoS ONE 7:e33917Chisolm GM 3rd, Hazen SL, Fox PL, Cathcart MK (1999) The oxidation of lipoproteins bymonocytes-macrophages. Biochemical and biological mechanisms. J Biol Chem274:25959–25962Cockerill GW, Rye KA, Gamble JR, Vadas MA, Barter PJ (1995) High-density lipoproteins inhibitcytokine-induced expression of endothelial cell adhesion molecules. Arterioscler Thromb VascBiol 15:1987–1994Coetzee GA, Strachan AF, van der Westhuyzen DR, Hoppe HC, Jeenah MS, de Beer FC (1986)Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipopro-tein composition. J Biol Chem 261:9644–9651Cuchel M, Lund-Katz S, de la Llera-Moya M, Millar JS, Chang D, Fuki I, Rothblat GH, PhillipsMC, Rader DJ (2009) Pathways by which reconstituted high-density lipoprotein mobilizes freecholesterol from whole body and from macrophages. Arterioscler Thromb Vasc Biol30:526–532Cullen P, Schulte H, Assmann G (1997) The Munster heart study (PROCAM): total mortality inmiddle-aged men is increased at low total and LDL cholesterol concentrations in smokers butnot in nonsmokers. Circulation 96:2128–2136Curtiss LK, Witztum JL (1985) Plasma apolipoproteins AI, AII, B, CI, and E are glucosylated inhyperglycemic diabetic subjects. Diabetes 34:452–461Dysfunctional HDL: From Structure-Function-Relationships to Biomarkers 357Dansky HM, Charlton SA, Barlow CB, Tamminen M, Smith JD, Frank JS, Breslow JL (1999) ApoA-I inhibits foam cell formation in Apo E-deficient mice after monocyte adherence to endo-thelium. J Clin Invest 104:31–39de Beer MC, Durbin DM, Cai L, Jonas A, de Beer FC, van der Westhuyzen DR (2001) Apolipo-protein A-I conformation markedly influences HDL interaction with scavenger receptor BI. JLipid Res 42:309–313De Nardo D, Labzin LI, Kono H, Seki R, Schmidt SV, Beyer M, Xu D, Zimmer S, Lahrmann C,Schildberg FA, Vogelhuber J, Kraut M, Ulas T, Kerksiek A, Krebs W, Bode N, Grebe A,Fitzgerald ML, Hernandez NJ, Williams BR, Knolle P, Kneilling M, Rocken M, Lutjohann D,Wright SD, Schultze JL, Latz E (2014) High-density lipoprotein mediates anti-inflammatoryreprogramming of macrophages via the transcriptional regulator ATF3. Nat Immunol15:152–160de Souza JA, Vindis C, Hansel B, Negre-Salvayre A, Therond P, Serrano CV Jr, Chantepie S,Salvayre R, Bruckert E, Chapman MJ, Kontush A (2008) Metabolic syndrome features small,apolipoprotein A-I-poor, triglyceride-rich HDL3 particles with defective anti-apoptotic activ-ity. Atherosclerosis 197:84–94de Souza JA, Vindis C, Negre-Salvayre A, Rye KA, Couturier M, Therond P, Chantepie S,Salvayre R, Chapman MJ, Kontush A (2010) Small, dense HDL 3 particles attenuate apoptosisin endothelial cells: pivotal role of apolipoprotein A-I. J Cell Mol Med 14:608–620Di Angelantonio E, Sarwar N, Perry P, Kaptoge S, Ray KK, Thompson A, Wood AM,Lewington S, Sattar N, Packard CJ, Collins R, Thompson SG, Danesh J (2009) Major lipids,apolipoproteins, and risk of vascular disease. JAMA 302:1993–2000Diaz MN, Frei B, Vita JA, Keaney JF Jr (1997) Antioxidants and atherosclerotic heart disease. NEngl J Med 337:408–416Dimayuga P, Zhu J, Oguchi S, Chyu KY, Xu XO, Yano J, Shah PK, Nilsson J, Cercek B (1999)Reconstituted HDL containing human apolipoprotein A-1 reduces VCAM-1 expression andneointima formation following periadventitial cuff-induced carotid injury in apoE null mice.Biochem Biophys Res Commun 264:465–468Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM (1999) Activation of nitricoxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399:601–605Eckardstein A (2012) Tachometer for reverse cholesterol transport? J Am Heart Assoc 1:e003723Esteve E, Ricart W, Fernandez-Real JM (2005) Dyslipidemia and inflammation: an evolutionaryconserved mechanism. Clin Nutr 24:16–31Ferretti G, Bacchetti T, Negre-Salvayre A, Salvayre R, Dousset N, Curatola G (2006) Structuralmodifications of HDL and functional consequences. Atherosclerosis 184:1–7Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, English D(2001) Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependentcytoskeletal rearrangement. J Clin Invest 108:689–701Garner B, Waldeck AR, Witting PK, Rye KA, Stocker R (1998) Oxidation of high densitylipoproteins. II. Evidence for direct reduction of lipid hydroperoxides by methionine residuesof apolipoproteins AI and AII. J Biol Chem 273:6088–6095Garza CA, Montori VM, McConnell JP, Somers VK, Kullo IJ, Lopez-Jimenez F (2007) Associa-tion between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematicreview. Mayo Clin Proc 82:159–165Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR (1977) High density lipoproteinas a protective factor against coronary heart disease. The Framingham study. Am J Med62:707–714Goyal J, Wang K, Liu M, Subbaiah PV (1997) Novel function of lecithin-cholesterolacyltransferase. Hydrolysis of oxidized polar phospholipids generated during lipoproteinoxidation. J Biol Chem 272:16231–16239Green PS, Vaisar T, Pennathur S, Kulstad JJ, Moore AB, Marcovina S, Brunzell J, Knopp RH,Zhao XQ, Heinecke JW (2008) Combined statin and niacin therapy remodels the high-densitylipoprotein proteome. Circulation 118:1259–1267358 M. Riwanto et al.Haghpassand M, Bourassa PA, Francone OL, Aiello RJ (2001) Monocyte/macrophage expressionof ABCA1 has minimal contribution to plasma HDL levels. J Clin Invest 108:1315–1320Hansson GK (2005) Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med352:1685–1695Haynes R, Jiang L, Hopewell JC, Li J, Chen F, Parish S, Landray MJ, Collins R, Armitage J,Collins R, Armitage J, Baigent C, Chen Z, Landray M, Chen Y, Jiang L, Pedersen TR,Landray M, Bowman L, Chen F, Hill M, Haynes R, Knott C, Rahimi K, Tobert J, Sleight P,Simpson D, Parish S, Baxter A, Lay M, Bray C, Wincott E, Leijenhorst G, Skattebol A,Moen G, Mitchel Y, Kuznetsova O, Macmahon S, Kjekshus J, Hill C, Lam TH, Sandercock P,Peto R, Hopewell JC (2013) HPS2-THRIVE randomized placebo-controlled trial in 25 673high-risk patients of ER niacin/laropiprant: trial design, pre-specified muscle and liveroutcomes, and reasons for stopping study treatment. Eur Heart J 34:1279–1291Hedrick CC, Thorpe SR, Fu MX, Harper CM, Yoo J, Kim SM, Wong H, Peters AL (2000)Glycation impairs high-density lipoprotein function. Diabetologia 43:312–320Heinecke JW (1998) Oxidants and antioxidants in the pathogenesis of atherosclerosis:implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis 141:1–15Heinecke J (2011) HDL and cardiovascular-disease risk–time for a new approach? N Engl J Med364:170–171Heller M, Schlappritzi E, Stalder D, Nuoffer JM, Haeberli A (2007) Compositional proteinanalysis of high density lipoproteins in hypercholesterolemia by shotgun LC-MS/MS andprobabilistic peptide scoring. Mol Cell Proteomics 6:1059–1072Holleboom AG, Daniil G, Fu X, Zhang R, Hovingh GK, Schimmel AW, Kastelein JJ, Stroes ES,Witztum JL, Hutten BA, Tsimikas S, Hazen SL, Chroni A, Kuivenhoven JA (2012) Lipidoxidation in carriers of lecithin:cholesterol acyltransferase gene mutations. ArteriosclerThromb Vasc Biol 32:3066–3075Huang Y, von Eckardstein A, Wu S, Maeda N, Assmann G (1994) A plasma lipoprotein containingonly apolipoprotein E and with gamma mobility on electrophoresis releases cholesterol fromcells. Proc Natl Acad Sci USA 91:1834–1838Huang Y, von Eckardstein A, Wu S, Assmann G (1995) Cholesterol efflux, cholesterol esterifica-tion, and cholesteryl ester transfer by LpA-I and LpA-I/A-II in native plasma. ArteriosclerThromb Vasc Biol 15:1412–1418Huang Y, Wu Z, Riwanto M, Gao S, Levison BS, Gu X, Fu X, Wagner MA, Besler C,Gerstenecker G, Zhang R, Li XM, DiDonato AJ, Gogonea V, Tang WH, Smith JD, PlowEF, Fox PL, Shih DM, Lusis AJ, Fisher EA, DiDonato JA, Landmesser U, Hazen SL (2013)Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex. J Clin Invest123:3815–3828Huang Y, DiDonato JA, Levison BS, Schmitt D, Li L, Wu Y, Buffa J, Kim T, Gerstenecker GS,Gu X, Kadiyala CS, Wang Z, Culley MK, Hazen JE, Didonato AJ, Fu X, Berisha SZ, Peng D,Nguyen TT, Liang S, Chuang CC, Cho L, Plow EF, Fox PL, Gogonea V, Tang WH, Parks JS,Fisher EA, Smith JD, Hazen SL (2014) An abundant dysfunctional apolipoprotein A1 inhuman atheroma. Nat Med 20:193–203Jahangiri A, de Beer MC, Noffsinger V, Tannock LR, Ramaiah C, Webb NR, van der WesthuyzenDR, de Beer FC (2009) HDL remodeling during the acute phase response. Arterioscler ThrombVasc Biol 29:261–267Jensen MK, Rimm EB, Furtado JD, Sacks FM (2012) Apolipoprotein C-III as a potentialmodulator of the association between HDL-cholesterol and incident coronary heart disease. JAm Heart Assoc 1Kalantar-Zadeh K, Kopple JD, Kamranpour N, Fogelman AM, Navab M (2007)HDL-inflammatory index correlates with poor outcome in hemodialysis patients. Kidney Int72:1149–1156Karlsson H, Leanderson P, Tagesson C, Lindahl M (2005) Lipoproteomics II: mapping of proteinsin high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry.Proteomics 5:1431–1445Dysfunctional HDL: From Structure-Function-Relationships to Biomarkers 359Khera AV, Cuchel M, de la Llera-Moya M, Rodrigues A, Burke MF, Jafri K, French BC, PhillipsJA, Mucksavage ML, Wilensky RL, Mohler ER, Rothblat GH, Rader DJ (2011) Cholesterolefflux capacity, high-density lipoprotein function, and atherosclerosis. N Engl J Med364:127–135Khovidhunkit W, Kim MS, Memon RA, Shigenaga JK, Moser AH, Feingold KR, Grunfeld C(2004) Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanismsand consequences to the host. J Lipid Res 45:1169–1196Kimura T, Sato K, Kuwabara A, Tomura H, Ishiwara M, Kobayashi I, Ui M, Okajima F (2001)Sphingosine 1-phosphate may be a major component of plasma lipoproteins responsible for thecytoprotective actions in human umbilical vein endothelial cells. J Biol Chem276:31780–31785Kimura T, Sato K, Malchinkhuu E, Tomura H, Tamama K, Kuwabara A, Murakami M, Okajima F(2003) High-density lipoprotein stimulates endothelial cell migration and survival throughsphingosine 1-phosphate and its receptors. Arterioscler Thromb Vasc Biol 23:1283–1288Kockx MM, Herman AG (2000) Apoptosis in atherosclerosis: beneficial or detrimental?Cardiovasc Res 45:736–746Kontush A, Therond P, Zerrad A, Couturier M, Negre-Salvayre A, de Souza JA, Chantepie S,Chapman MJ (2007) Preferential sphingosine-1-phosphate enrichment and sphingomyelindepletion are key features of small dense HDL3 particles: relevance to antiapoptotic andantioxidative activities. Arterioscler Thromb Vasc Biol 27:1843–1849Kuvin JT, Ramet ME, Patel AR, Pandian NG, Mendelsohn ME, Karas RH (2002) A novelmechanism for the beneficial vascular effects of high-density lipoprotein cholesterol: enhancedvasorelaxation and increased endothelial nitric oxide synthase expression. Am Heart J144:165–172Landmesser U, Hornig B, Drexler H (2004) Endothelial function: a critical determinant inatherosclerosis? Circulation 109:II27–II33Li XM, Tang WH, Mosior MK, Huang Y, Wu Y, Matter W, Gao V, Schmitt D, Didonato JA,Fisher EA, Smith JD, Hazen SL (2013) Paradoxical association of enhanced cholesterol effluxwith increased incident cardiovascular risks. Arterioscler Thromb Vasc Biol 33:1696–1705Lp PLASC, Thompson A, Gao P, Orfei L, Watson S, Di Angelantonio E, Kaptoge S, Ballantyne C,Cannon CP, Criqui M, Cushman M, Hofman A, Packard C, Thompson SG, Collins R, Danesh J(2010) Lipoprotein-associated phospholipase A(2) and risk of coronary disease, stroke, andmortality: collaborative analysis of 32 prospective studies. Lancet 375:1536–1544Lucke S, Levkau B (2010) Endothelial functions of sphingosine-1-phosphate. Cell PhysiolBiochem 26:87–96Lund-Katz S, Liu L, Thuahnai ST, Phillips MC (2003) High density lipoprotein structure. FrontBiosci 8:d1044–d1054Mackness MI, Arrol S, Durrington PN (1991) Paraoxonase prevents accumulation oflipoperoxides in low-density lipoprotein. FEBS Lett 286:152–154Mackness B, Hine D, Liu Y, Mastorikou M, Mackness M (2004) Paraoxonase-1 inhibits oxidisedLDL-induced MCP-1 production by endothelial cells. Biochem Biophys Res Commun318:680–683Marathe GK, Zimmerman GA, McIntyre TM (2003) Platelet-activating factor acetylhydrolase,and not paraoxonase-1, is the oxidized phospholipid hydrolase of high density lipoproteinparticles. J Biol Chem 278:3937–3947McIntyre TM, Prescott SM, Stafforini DM (2009) The emerging roles of PAF acetylhydrolase. JLipid Res 50(Suppl):S255–S259McMahon M, Grossman J, FitzGerald J, Dahlin-Lee E, Wallace DJ, Thong BY, Badsha H,Kalunian K, Charles C, Navab M, Fogelman AM, Hahn BH (2006) Proinflammatory high-density lipoprotein as a biomarker for atherosclerosis in patients with systemic lupuserythematosus and rheumatoid arthritis. Arthritis Rheum 54:2541–2549Mertens A, Verhamme P, Bielicki JK, Phillips MC, Quarck R, Verreth W, Stengel D, Ninio E,Navab M, Mackness B, Mackness M, Holvoet P (2003) Increased low-density lipoproteinoxidation and impaired high-density lipoprotein antioxidant defense are associated with360 M. Riwanto et al.increased macrophage homing and atherosclerosis in dyslipidemic obese mice: LCAT genetransfer decreases atherosclerosis. Circulation 107:1640–1646Mineo C, Yuhanna IS, Quon MJ, Shaul PW (2003) High density lipoprotein-induced endothelialnitric-oxide synthase activation is mediated by Akt and MAP kinases. J Biol Chem278:9142–9149Mineo C, Deguchi H, Griffin JH, Shaul PW (2006) Endothelial and antithrombotic actions ofHDL. Circ Res 98:1352–1364Miyata M, Smith JD (1996) Apolipoprotein E allele-specific antioxidant activity and effects oncytotoxicity by oxidative insults and beta-amyloid peptides. Nat Genet 14:55–61Morgantini C, Natali A, Boldrini B, Imaizumi S, Navab M, Fogelman AM, Ferrannini E, ReddyST (2011) Anti-inflammatory and antioxidant properties of HDLs are impaired in type2 diabetes. Diabetes 60:2617–2623Nabel EG, Braunwald E (2012) A tale of coronary artery disease and myocardial infarction. NEngl J Med 366:54–63Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, Valente AJ, Berliner JA,Drinkwater DC, Laks H et al (1991) Monocyte transmigration induced by modification oflow density lipoprotein in cocultures of human aortic wall cells is due to induction of monocytechemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest88:2039–2046Navab M, Berliner JA, Watson AD, Hama SY, Territo MC, Lusis AJ, Shih DM, Van Lenten BJ,Frank JS, Demer LL, Edwards PA, Fogelman AM (1996) The Yin and Yang of oxidation in thedevelopment of the fatty streak. A review based on the 1994 George Lyman Duff MemorialLecture. Arterioscler Thromb Vasc Biol 16:831–842Navab M, Hama-Levy S, Van Lenten BJ, Fonarow GC, Cardinez CJ, Castellani LW, Brennan ML,Lusis AJ, Fogelman AM, La Du BN (1997) Mildly oxidized LDL induces an increasedapolipoprotein J/paraoxonase ratio. J Clin Invest 99:2005–2019Navab M, Hama SY, Anantharamaiah GM, Hassan K, Hough GP, Watson AD, Reddy ST,Sevanian A, Fonarow GC, Fogelman AM (2000a) Normal high density lipoprotein inhibitsthree steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3. J LipidRes 41:1495–1508Navab M, Hama SY, Cooke CJ, Anantharamaiah GM, Chaddha M, Jin L, Subbanagounder G,Faull KF, Reddy ST, Miller NE, Fogelman AM (2000b) Normal high density lipoproteininhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1. J LipidRes 41:1481–1494Navab M, Berliner JA, Subbanagounder G, Hama S, Lusis AJ, Castellani LW, Reddy S, Shih D,Shi W, Watson AD, Van Lenten BJ, Vora D, Fogelman AM (2001a) HDL and the inflamma-tory response induced by LDL-derived oxidized phospholipids. Arterioscler Thromb Vasc Biol21:481–488Navab M, Hama SY, Hough GP, Subbanagounder G, Reddy ST, Fogelman AM (2001b) A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of orinactivating oxidized phospholipids. J Lipid Res 42:1308–1317Nicholls SJ, Cutri B, Worthley SG, Kee P, Rye KA, Bao S, Barter PJ (2005a) Impact of short-termadministration of high-density lipoproteins and atorvastatin on atherosclerosis in rabbits.Arterioscler Thromb Vasc Biol 25:2416–2421Nicholls SJ, Dusting GJ, Cutri B, Bao S, Drummond GR, Rye KA, Barter PJ (2005b)Reconstituted high-density lipoproteins inhibit the acute pro-oxidant and proinflammatoryvascular changes induced by a periarterial collar in normocholesterolemic rabbits. Circulation111:1543–1550Nobecourt E, Tabet F, Lambert G, Puranik R, Bao S, Yan L, Davies MJ, Brown BE, Jenkins AJ,Dusting GJ, Bonnet DJ, Curtiss LK, Barter PJ, Rye KA (2010) Nonenzymatic glycationimpairs the antiinflammatory properties of apolipoprotein A-I. Arterioscler Thromb VascBiol 30:766–772Dysfunctional HDL: From Structure-Function-Relationships to Biomarkers 361Nofer JR, Levkau B, Wolinska I, Junker R, Fobker M, von Eckardstein A, Seedorf U, Assmann G(2001) Suppression of endothelial cell apoptosis by high density lipoproteins (HDL) andHDL-associated lysosphingolipids. J Biol Chem 276:34480–34485Nofer JR, van der Giet M, Tolle M, Wolinska I, von Wnuck LK, Baba HA, Tietge UJ, Godecke A,Ishii I, Kleuser B, Schafers M, Fobker M, Zidek W, Assmann G, Chun J, Levkau B (2004)HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J ClinInvest 113:569–581Norata GD, Catapano AL (2012) HDL and adaptive immunity: a tale of lipid rafts. Atherosclerosis225:34–35Noto H, Hara M, Karasawa K, Iso ON, Satoh H, Togo M, Hashimoto Y, Yamada Y, Kosaka T,Kawamura M, Kimura S, Tsukamoto K (2003) Human plasma platelet-activating factoracetylhydrolase binds to all the murine lipoproteins, conferring protection against oxidativestress. Arterioscler Thromb Vasc Biol 23:829–835Ophir G, Amariglio N, Jacob-Hirsch J, Elkon R, Rechavi G, Michaelson DM (2005) Apolipopro-tein E4 enhances brain inflammation by modulation of the NF-kappaB signaling cascade.Neurobiol Dis 20:709–718Ostos MA, Conconi M, Vergnes L, Baroukh N, Ribalta J, Girona J, Caillaud JM, Ochoa A, ZakinMM (2001) Antioxidative and antiatherosclerotic effects of human apolipoprotein A-IV inapolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 21:1023–1028Parks JS, Rudel LL (1985) Alteration of high density lipoprotein subfraction distribution withinduction of serum amyloid A protein (SAA) in the nonhuman primate. J Lipid Res 26:82–91Paszty C, Maeda N, Verstuyft J, Rubin EM (1994) Apolipoprotein AI transgene corrects apolipo-protein E deficiency-induced atherosclerosis in mice. J Clin Invest 94:899–903Patel S, Drew BG, Nakhla S, Duffy SJ, Murphy AJ, Barter PJ, Rye KA, Chin-Dusting J, Hoang A,Sviridov D, Celermajer DS, Kingwell BA (2009) Reconstituted high-density lipoproteinincreases plasma high-density lipoprotein anti-inflammatory properties and cholesterol effluxcapacity in patients with type 2 diabetes. J Am Coll Cardiol 53:962–971Patel PJ, Khera AV, Jafri K, Wilensky RL, Rader DJ (2011) The anti-oxidative capacity of high-density lipoprotein is reduced in acute coronary syndrome but not in stable coronary arterydisease. J Am Coll Cardiol 58:2068–2075Patel PJ, Khera AV, Wilensky RL, Rader DJ (2013) Anti-oxidative and cholesterol effluxcapacities of high-density lipoprotein are reduced in ischaemic cardiomyopathy. Eur J HeartFail 15(11):1215–1219Persegol L, Verges B, Foissac M, Gambert P, Duvillard L (2006) Inability of HDL from type2 diabetic patients to counteract the inhibitory effect of oxidised LDL on endothelium-dependent vasorelaxation. Diabetologia 49:1380–1386Persegol L, Verges B, Gambert P, Duvillard L (2007) Inability of HDL from abdominally obesesubjects to counteract the inhibitory effect of oxidized LDL on vasorelaxation. J Lipid Res48:1396–1401Peterson SJ, Husney D, Kruger AL, Olszanecki R, Ricci F, Rodella LF, Stacchiotti A, Rezzani R,McClung JA, Aronow WS, Ikehara S, Abraham NG (2007) Long-term treatment with theapolipoprotein A1 mimetic peptide increases antioxidants and vascular repair in type I diabeticrats. J Pharmacol Exp Ther 322:514–520Peterson SJ, Drummond G, Kim DH, Li M, Kruger AL, Ikehara S, Abraham NG (2008) L-4 Ftreatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity inobese mice. J Lipid Res 49:1658–1669Picotti P, Aebersold R (2012) Selected reaction monitoring-based proteomics: workflows, poten-tial, pitfalls and future directions. Nat Methods 9:555–566Picotti P, Rinner O, Stallmach R, Dautel F, Farrah T, Domon B, Wenschuh H, Aebersold R (2010)High-throughput generation of selected reaction-monitoring assays for proteins and proteomes.Nat Methods 7:43–46Puranik R, Bao S, Nobecourt E, Nicholls SJ, Dusting GJ, Barter PJ, Celermajer DS, Rye KA(2008) Low dose apolipoprotein A-I rescues carotid arteries from inflammation in vivo.Atherosclerosis 196:240–247362 M. Riwanto et al.Rader DJ (2006) Molecular regulation of HDL metabolism and function: implications for noveltherapies. J Clin Invest 116:3090–3100Rallidis LS, Tellis CC, Lekakis J, Rizos I, Varounis C, Charalampopoulos A, Zolindaki M,Dagres N, Anastasiou-Nana M, Tselepis AD (2012) Lipoprotein-associated phospholipase A(2) bound on high-density lipoprotein is associated with lower risk for cardiac death in stablecoronary artery disease patients: a 3-year follow-up. J Am Coll Cardiol 60:2053–2060Ramet ME, Ramet M, Lu Q, Nickerson M, Savolainen MJ, Malzone A, Karas RH (2003) High-density lipoprotein increases the abundance of eNOS protein in human vascular endothelialcells by increasing its half-life. J Am Coll Cardiol 41:2288–2297Recalde D, Ostos MA, Badell E, Garcia-Otin AL, Pidoux J, Castro G, Zakin MM, Scott-Algara D(2004) Human apolipoprotein A-IV reduces secretion of proinflammatory cytokines andatherosclerotic effects of a chronic infection mimicked by lipopolysaccharide. ArteriosclerThromb Vasc Biol 24:756–761Regieli JJ, Jukema JW, Doevendans PA, Zwinderman AH, Kastelein JJ, Grobbee DE, van derGraaf Y (2009) Paraoxonase variants relate to 10-year risk in coronary artery disease: impact ofa high-density lipoprotein-bound antioxidant in secondary prevention. J Am Coll Cardiol54:1238–1245Ribas V, Sanchez-Quesada JL, Anton R, Camacho M, Julve J, Escola-Gil JC, Vila L, Ordonez-Llanos J, Blanco-Vaca F (2004) Human apolipoprotein A-II enrichment displaces paraoxonasefrom HDL and impairs its antioxidant properties: a new mechanism linking HDL proteincomposition and antiatherogenic potential. Circ Res 95:789–797Riwanto M, Landmesser U (2013) High density lipoproteins and endothelial functions: mechanis-tic insights and alterations in cardiovascular disease. J Lipid Res 54:3227–3243Riwanto M, Rohrer L, Roschitzki B, Besler C, Mocharla P, Mueller M, Perisa D, Heinrich K,Altwegg L, von Eckardstein A, Luscher TF, Landmesser U (2013) Altered activation ofendothelial anti- and pro-apoptotic pathways by high-density lipoprotein from patients withcoronary artery disease: role of HDL-proteome remodeling. Circulation 127(8):891–904Ross R (1999) Atherosclerosis – an inflammatory disease. N Engl J Med 340:115–126Rossig L, Dimmeler S, Zeiher AM (2001) Apoptosis in the vascular wall and atherosclerosis.Basic Res Cardiol 96:11–22Rotllan N, Ribas V, Calpe-Berdiel L, Martin-Campos JM, Blanco-Vaca F, Escola-Gil JC (2005)Overexpression of human apolipoprotein A-II in transgenic mice does not impair macrophage-specific reverse cholesterol transport in vivo. Arterioscler Thromb Vasc Biol 25:e128–e132Rye KA, Barter PJ (2008) Antiinflammatory actions of HDL: a new insight. Arterioscler ThrombVasc Biol 28:1890–1891Sacre SM, Stannard AK, Owen JS (2003) Apolipoprotein E (apoE) isoforms differentially inducenitric oxide production in endothelial cells. FEBS Lett 540:181–187Saito H, Lund-Katz S, Phillips MC (2004) Contributions of domain structure and lipid interactionto the functionality of exchangeable human apolipoproteins. Prog Lipid Res 43:350–380Schwartz GG, Olsson AG, Abt M, Ballantyne CM, Barter PJ, Brumm J, Chaitman BR, Holme IM,Kallend D, Leiter LA, Leitersdorf E, McMurray JJ, Mundl H, Nicholls SJ, Shah PK, Tardif JC,Wright RS (2012) Effects of dalcetrapib in patients with a recent acute coronary syndrome. NEngl J Med 367:2089–2099Shao B, Oda MN, Bergt C, Fu X, Green PS, Brot N, Oram JF, Heinecke JW (2006)Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidationand site-specific tyrosine chlorination of apolipoprotein A-I. J Biol Chem 281:9001–9004Shao B, Tang C, Heinecke JW, Oram JF (2010) Oxidation of apolipoprotein A-I bymyeloperoxidase impairs the initial interactions with ABCA1 required for signaling andcholesterol export. J Lipid Res 51:1849–1858Shao B, Pennathur S, Heinecke JW (2012) Myeloperoxidase targets apolipoprotein A-I, the majorhigh density lipoprotein protein, for site-specific oxidation in human atherosclerotic lesions. JBiol Chem 287:6375–6386Sharrett AR, Ballantyne CM, Coady SA, Heiss G, Sorlie PD, Catellier D, Patsch W (2001)Coronary heart disease prediction from lipoprotein cholesterol levels, triglycerides,Dysfunctional HDL: From Structure-Function-Relationships to Biomarkers 363lipoprotein(a), apolipoproteins A-I and B, and HDL density subfractions: the atherosclerosisrisk in communities (ARIC) study. Circulation 104:1108–1113Shih DM, Gu L, Xia YR, Navab M, Li WF, Hama S, Castellani LW, Furlong CE, Costa LG,Fogelman AM, Lusis AJ (1998) Mice lacking serum paraoxonase are susceptible to organo-phosphate toxicity and atherosclerosis. Nature 394:284–287Shih DM, Xia YR,Wang XP, Miller E, Castellani LW, Subbanagounder G, Cheroutre H, Faull KF,Berliner JA, Witztum JL, Lusis AJ (2000) Combined serum paraoxonase knockout/apolipo-protein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis. J BiolChem 275:17527–17535Soran H, Younis NN, Charlton-Menys V, Durrington P (2009) Variation in paraoxonase-1 activityand atherosclerosis. Curr Opin Lipidol 20:265–274Sorrentino SA, Besler C, Rohrer L, Meyer M, Heinrich K, Bahlmann FH, Mueller M, Horvath T,Doerries C, Heinemann M, Flemmer S, Markowski A, Manes C, Bahr MJ, Haller H, vonEckardstein A, Drexler H, Landmesser U (2010) Endothelial-vasoprotective effects of high-density lipoprotein are impaired in patients with type 2 diabetes mellitus but are improved afterextended-release niacin therapy. Circulation 121:110–122Speidel MT, Booyse FM, Abrams A, Moore MA, Chung BH (1990) Lipolyzed hypertrigly-ceridemic serum and triglyceride-rich lipoprotein cause lipid accumulation in and are cytotoxicto cultured human endothelial cells. High density lipoproteins inhibit this cytotoxicity. ThrombRes 58:251–264Spieker LE, Sudano I, Hurlimann D, Lerch PG, Lang MG, Binggeli C, Corti R, Ruschitzka F,Luscher TF, Noll G (2002) High-density lipoprotein restores endothelial function in hypercho-lesterolemic men. Circulation 105:1399–1402Subramanian VS, Goyal J, Miwa M, Sugatami J, Akiyama M, Liu M, Subbaiah PV (1999) Role oflecithin-cholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma:studies with platelet-activating factor-acetyl hydrolase-deficient plasma. Biochim BiophysActa 1439:95–109Suc I, Escargueil-Blanc I, Troly M, Salvayre R, Negre-Salvayre A (1997) HDL and ApoA preventcell death of endothelial cells induced by oxidized LDL. Arterioscler Thromb Vasc Biol17:2158–2166Sugano M, Tsuchida K, Makino N (2000) High-density lipoproteins protect endothelial cells fromtumor necrosis factor-alpha-induced apoptosis. Biochem Biophys Res Commun 272:872–876Takeya H, Gabazza EC, Aoki S, Ueno H, Suzuki K (2003) Synergistic effect of sphingosine1-phosphate on thrombin-induced tissue factor expression in endothelial cells. Blood102:1693–1700Tall AR, Yvan-Charvet L, Terasaka N, Pagler T, Wang N (2008) HDL, ABC transporters, andcholesterol efflux: implications for the treatment of atherosclerosis. Cell Metab 7:365–375Tang C, Liu Y, Kessler PS, Vaughan AM, Oram JF (2009) The macrophage cholesterol exporterABCA1 functions as an anti-inflammatory receptor. J Biol Chem 284:32336–32343Tang WH, Hartiala J, Fan Y, Wu Y, Stewart AF, Erdmann J, Kathiresan S, Roberts R,McPherson R, Allayee H, Hazen SL (2012) Clinical and genetic association of serumparaoxonase and arylesterase activities with cardiovascular risk. Arterioscler Thromb VascBiol 32:2803–2812Tolle M, Huang T, Schuchardt M, Jankowski V, Prufer N, Jankowski J, Tietge UJ, Zidek W, vander Giet M (2012) High-density lipoprotein loses its anti-inflammatory capacity by accumula-tion of pro-inflammatory-serum amyloid A. Cardiovasc Res 94:154–162Tward A, Xia YR, Wang XP, Shi YS, Park C, Castellani LW, Lusis AJ, Shih DM (2002)Decreased atherosclerotic lesion formation in human serum paraoxonase transgenic mice.Circulation 106:484–490Uittenbogaard A, Shaul PW, Yuhanna IS, Blair A, Smart EJ (2000) High density lipoproteinprevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxidesynthase localization and activation in caveolae. J Biol Chem 275:11278–11283Undurti A, Huang Y, Lupica JA, Smith JD, DiDonato JA, Hazen SL (2009) Modification of highdensity lipoprotein by myeloperoxidase generates a pro-inflammatory particle. J Biol Chem284:30825–30835364 M. Riwanto et al.Vaisar T, Pennathur S, Green PS, Gharib SA, Hoofnagle AN, Cheung MC, Byun J, Vuletic S,Kassim S, Singh P, Chea H, Knopp RH, Brunzell J, Geary R, Chait A, Zhao XQ, Elkon K,Marcovina S, Ridker P, Oram JF, Heinecke JW (2007) Shotgun proteomics implicates proteaseinhibition and complement activation in the antiinflammatory properties of HDL. J Clin Invest117:746–756Van Lenten BJ, Hama SY, de Beer FC, Stafforini DM, McIntyre TM, Prescott SM, La Du BN,Fogelman AM, Navab M (1995) Anti-inflammatory HDL becomes pro-inflammatory duringthe acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wallcell cocultures. J Clin Invest 96:2758–2767Van Lenten BJ, Wagner AC, Nayak DP, Hama S, Navab M, Fogelman AM (2001) High-densitylipoprotein loses its anti-inflammatory properties during acute influenza a infection. Circula-tion 103:2283–2288Van Linthout S, Spillmann F, Riad A, Trimpert C, Lievens J, Meloni M, Escher F, Filenberg E,Demir O, Li J, Shakibaei M, Schimke I, Staudt A, Felix SB, Schultheiss HP, De Geest B,Tschope C (2008) Human apolipoprotein A-I gene transfer reduces the development ofexperimental diabetic cardiomyopathy. Circulation 117:1563–1573von Eckardstein A, Huang Y, Wu S, Funke H, Noseda G, Assmann G (1995a) Reverse cholesteroltransport in plasma of patients with different forms of familial HDL deficiency. ArteriosclerThromb Vasc Biol 15:691–703von Eckardstein A, Huang Y, Wu S, Sarmadi AS, Schwarz S, Steinmetz A, Assmann G (1995b)Lipoproteins containing apolipoprotein A-IV but not apolipoprotein A-I take up and esterifycell-derived cholesterol in plasma. Arterioscler Thromb Vasc Biol 15:1755–1763Vowinkel T, Mori M, Krieglstein CF, Russell J, Saijo F, Bharwani S, Turnage RH, Davidson WS,Tso P, Granger DN, Kalogeris TJ (2004) Apolipoprotein A-IV inhibits experimental colitis. JClin Invest 114:260–269Wang X, Collins HL, Ranalletta M, Fuki IV, Billheimer JT, Rothblat GH, Tall AR, Rader DJ(2007a) Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reversecholesterol transport in vivo. J Clin Invest 117:2216–2224Wang Z, Nicholls SJ, Rodriguez ER, Kummu O, Horkko S, Barnard J, Reynolds WF, Topol EJ,DiDonato JA, Hazen SL (2007b) Protein carbamylation links inflammation, smoking, uremiaand atherogenesis. Nat Med 13:1176–1184Wang SH, Yuan SG, Peng DQ, Zhao SP (2012) HDL and ApoA-I inhibit antigen presentation-mediated T cell activation by disrupting lipid rafts in antigen presenting cells. Atherosclerosis225:105–114Watson AD, Berliner JA, Hama SY, La Du BN, Faull KF, Fogelman AM, Navab M (1995)Protective effect of high density lipoprotein associated paraoxonase. Inhibition of thebiological activity of minimally oxidized low density lipoprotein. J Clin Invest 96:2882–2891Weihrauch D, Xu H, Shi Y, Wang J, Brien J, Jones DW, Kaul S, Komorowski RA, Csuka ME,Oldham KT, Pritchard KA (2007) Effects of D-4 F on vasodilation, oxidative stress,angiostatin, myocardial inflammation, and angiogenic potential in tight-skin mice. Am JPhysiol Heart Circ Physiol 293:H1432–H1441Witztum JL, Steinberg D (1991) Role of oxidized low density lipoprotein in atherogenesis. J ClinInvest 88:1785–1792Yuhanna IS, Zhu Y, Cox BE, Hahner LD, Osborne-Lawrence S, Lu P, Marcel YL, Anderson RG,Mendelsohn ME, Hobbs HH, Shaul PW (2001) High-density lipoprotein binding to scavengerreceptor-BI activates endothelial nitric oxide synthase. Nat Med 7:853–857Yvan-Charvet L, Pagler TA, Wang N, Senokuchi T, Brundert M, Li H, Rinninger F, Tall AR(2008) SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL. JLipid Res 49:107–114Zerrad-Saadi A, Therond P, Chantepie S, Couturier M, Rye KA, Chapman MJ, Kontush A (2009)HDL3-mediated inactivation of LDL-associated phospholipid hydroperoxides is determinedby the redox status of apolipoprotein A-I and HDL particle surface lipid rigidity: relevance toinflammation and atherogenesis. Arterioscler Thromb Vasc Biol 29:2169–2175Dysfunctional HDL: From Structure-Function-Relationships to Biomarkers 365Zheng L, Nukuna B, Brennan ML, Sun M, Goormastic M, Settle M, Schmitt D, Fu X, Thomson L,Fox PL, Ischiropoulos H, Smith JD, Kinter M, Hazen SL (2004) Apolipoprotein A-I is aselective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjectswith cardiovascular disease. J Clin Invest 114:529–541Zhu X, Owen JS, Wilson MD, Li H, Griffiths GL, Thomas MJ, Hiltbold EM, Fessler MB, Parks JS(2010) Macrophage ABCA1 reduces MyD88-dependent toll-like receptor trafficking to lipidrafts by reduction of lipid raft cholesterol. J Lipid Res 51:3196–3206366 M. Riwanto et al.Part IIIPossible Indications and Target Mechanismsof HDL TherapyHDL and Atherothrombotic VascularDiseaseWijtske Annema, Arnold von Eckardstein, and Petri T. KovanenContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3712 Interactions of HDLs with the Endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3732.1 Preservation of Endothelial Integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3732.2 Preservation of Endothelial Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3753 Anti-inflammatory Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3763.1 Suppression of Myelopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3763.2 Suppression of Monocyte Extravasation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3773.3 Interference with Macrophage Differentiation and Activation . . . . . . . . . . . . . . . . . . . . . 3794 Effects of HDLs on Vascular Lipid and Lipoprotein Homeostasis . . . . . . . . . . . . . . . . . . . . . . . 3794.1 From LDL Retention to Atheroma Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3794.1.1 Initial Lipid Accumulation in Atherosclerosis-Prone Arterial Intima: ASign of LDL Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3794.1.2 Formation of a Fatty Streak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3814.1.3 Development of an Atheroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3824.2 Inhibition of LDL Modification by HDLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3834.3 Macrophage Cholesterol Efflux and Reverse Cholesterol Transport . . . . . . . . . . . . . . . 3854.3.1 Transendothelial HDL Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3854.3.2 Cholesterol Efflux from Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3864.3.3 Exit from the Arterial Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3874.3.4 Delivery of Cholesterol to the Liver and Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . 388W. Annema (*)Institute of Clinical Chemistry, University Hospital Zurich, Zurich, Switzerlande-mail: wijtske.annema@usz.chA. von EckardsteinInstitute of Clinical Chemistry, University Hospital Zurich, Zurich, SwitzerlandCenter of integrative Human Physiology, University of Zurich, Zurich, Switzerlande-mail: arnold.voneckardstein@usz.chP.T. KovanenWihuri Research Institute, Helsinki, Finlande-mail: petri.kovanen@wri.fi# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_113695 Atherothrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3885.1 Effects of HDLs on Smooth Muscle Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3905.2 Antithrombotic Effects of HDLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3915.2.1 HDLs and Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3915.2.2 HDLs and Coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3926 Myocardial Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394AbstractHigh-density lipoproteins (HDLs) exert many beneficial effects which may helpto protect against the development or progression of atherosclerosis or evenfacilitate lesion regression. These activities include promoting cellular choles-terol efflux, protecting low-density lipoproteins (LDLs) from modification,preserving endothelial function, as well as anti-inflammatory and antithromboticeffects. However, questions remain about the relative importance of theseactivities for atheroprotection. Furthermore, the many molecules (both lipidsand proteins) associated with HDLs exert both distinct and overlappingactivities, which may be compromised by inflammatory conditions, resultingin either loss of function or even gain of dysfunction. This complexity of HDLfunctionality has so far precluded elucidation of distinct structure–functionrelationships for HDL or its components. A better understanding of HDLmetabolism and structure–function relationships is therefore crucial to exploitHDLs and its associated components and cellular pathways as potential targetsfor anti-atherosclerotic therapies and diagnostic markers.KeywordsAtherosclerosis • Cholesterol efflux • Endothelial function • Foam cells • High-density lipoproteins • Inflammation • Macrophages • Oxidation • Thrombosis •Smooth muscle cellsList of AbbreviationsAAPH 2,20-Azobis(2-amidinopropane) dihydrochlorideApoA-I Apolipoprotein A-IABCA1 ATP-binding cassette transporter A1ABCG1 ATP-binding cassette transporter G1ATF3 Activating transcription factor 3β-ATPase β-Chain of F0F1 ATPaseBSEP Bile salt export pumpCETP Cholesteryl ester transfer proteinCYP7A1 Cholesterol 7α-hydroxylaseeNOS Endothelial nitric oxide synthaseEPC Endothelial progenitor cellHDL High-density lipoproteinHO-1 Heme oxygenase-1370 W. Annema et al.IL-1β Interleukin-1βLCAT Lecithin-cholesterol acyltransferaseLDL Low-density lipoproteinLp-PLA2 Lipoprotein-associated phospholipase A2MAP Mitogen-activated proteinMCP-1 Monocyte chemotactic protein-1MDA MalondialdehydeMMP Matrix metalloproteinaseNF-κβ Nuclear factor-κβNO Nitric oxidePAF-AH Platelet-activating factor acetylhydrolasePDZK1 PDZ domain-containing protein 1PI3K Phosphatidylinositol 3-kinasePON1 Paraoxonase 1rHDL Reconstituted high-density lipoproteinROS Reactive oxygen speciesS1P Sphingosine-1-phosphateSMC Smooth muscle cellSR-BI Scavenger receptor BISTAT3 Signal transducer and activator of transcription 3TCFA Thin-cap fibroatheromaTLR Toll-like receptorTNFα Tumor necrosis factor αVCAM-1 Vascular cell adhesion molecule 1VSMC Vascular smooth muscle cell1 IntroductionAtherothrombotic diseases, such as coronary heart disease, stroke, and peripheralarterial disease, are the world’s leading cause of death. The first-line therapy forprevention and treatment of coronary heart disease is low-density lipoprotein(LDL) lowering by statin therapy. Although statin drugs are very effective inreducing plasma LDL cholesterol concentrations, the risk of major cardiovascularevents is only decreased by 15–40 % (Baigent et al. 2005; Ridker et al. 2010).Additional therapeutic approaches are needed to minimize this considerableremaining cardiovascular risk in statin-treated patients. It has been postulated thatpart of this residual risk may be explained by low circulating levels of high-densitylipoproteins (HDLs). Clinical and large population-based epidemiological studieshave consistently shown that low HDL cholesterol is independently associated withan increased risk of atherothrombotic diseases (Di Angelantonio et al. 2009; Barteret al. 2007a). However, this long-standing inverse relationship between HDL andatherosclerotic cardiovascular disease has been challenged by recent findings.HDL and Atherothrombotic Vascular Disease 371Indeed, high plasma concentrations of HDL cholesterol might not be beneficial forcertain subgroups of patients. Thus, in high-risk postinfarction patientscharacterized by high total cholesterol and C-reactive protein, elevated levels ofHDL cholesterol were paradoxically found to be related to poor clinical outcome(Corsetti et al. 2006). Similarly, the inverse relationship between HDL cholesterollevels and major cardiovascular events was found lost in patients with establishedcoronary artery disease undergoing diagnostic angiography or coronary arterybypass grafting (Angeloni et al. 2013; Silbernagel et al. 2013). Moreover, severallarge randomized clinical trials assessing the effects of new therapies targetingHDL metabolism, such as cholesteryl ester transfer protein (CETP) inhibitors andniacin, reported disappointing results. The Investigation of Lipid Level Manage-ment to Understand its Impact in Atherosclerotic Events (ILLUMINATE) trialrevealed that the combination of atorvastatin with the CETP inhibitor torcetrapibincreased HDL cholesterol levels by 72.1 % relative to atorvastatin alone (Barteret al. 2007b). This raise in HDL cholesterol upon torcetrapib administration did,however, not lower the risk of the major cardiovascular outcome (Barteret al. 2007b). On the contrary, patients in the atorvastatin/torcetrapib group unex-pectedly experienced significantly more adverse events (Barter et al. 2007b). Morerecently, another phase 3 clinical trial involving patients with a recent acutecoronary syndrome, named dal-OUTCOMES, was designed to evaluate the effectsof the CETP inhibitor dalcetrapib (Schwartz et al. 2012). Addition of dalcetrapib tostatin therapy raised HDL cholesterol levels by 31–40 % but failed to reduce therisk of recurrent cardiovascular events (Schwartz et al. 2012). Niacin is anothereffective agent for raising HDL cholesterol and also lowering LDL cholesterol,triglycerides, and lipoprotein(a). Data from the Atherothrombosis Intervention inMetabolic Syndrome with Low HDL/High Triglycerides: Impact on Global HealthOutcomes (AIM-HIGH) trial, which included patients with established cardiovas-cular disease, showed that adding extended-release niacin to statin therapy was noteffective in reducing the risk of cardiovascular events during a follow-up of36 months despite a 25 % increase in HDL cholesterol (Boden et al. 2011).Genetic studies also questioned a role for HDLs in atherothrombotic diseases.Lifelong reductions of plasma HDL cholesterol levels due to heterozygosity for aloss-of-function mutation in ABCA1 did not affect the risk for ischemic heartdisease (Frikke-Schmidt et al. 2008). Likewise, genetic variation in the apolipopro-tein A-I (apoA-I) gene, APOA1, affecting apoA-I and HDL cholesterol levels didnot predict ischemic heart disease or myocardial infarction in the general popula-tion (Haase et al. 2010). A more recent Mendelian randomization analysisdemonstrated that there is no significant association between risk of myocardialinfarction and a loss-of-function SNP in the gene coding for endothelial lipase(LIPG Asn396Ser), which results in elevated levels of HDL cholesterol (Voightet al. 2012). Hence, there is ongoing discussion whether HDLs are causallyinvolved in the development of atherothrombotic diseases or are merely an innocentbystander. On the other hand, there is considerable evidence from in vitro andin vivo studies that HDL particles isolated from human plasma as well as artificially372 W. Annema et al.reconstituted HDLs (rHDLs) exert multiple atheroprotective effects (Annema andvon Eckardstein 2013).The above-outlined controversies have shifted the interest of the cardiovascularresearch field to HDL functionality, meaning that the functional quality of theparticles might be more important than the absolute amount of HDL cholesterol.Currently, the available HDL-targeted therapies for atherothrombotic diseases raiseplasma HDL cholesterol levels per se. Nonetheless, plasma concentrations of HDLcholesterol do not necessarily reflect the functional capacity of HDL particles. Inaddition, there is evidence that the anti-atherogenic functions of HDLs are impairedin patients with cardiovascular disease (Annema and von Eckardstein 2013), andthese dysfunctional HDLs might even promote atherothrombotic diseases. There-fore, a better understanding of the molecular mechanisms behind the protectivefunctions of HDLs in the context of atherothrombotic diseases is fundamental forsuccessful exploitation of HDLs for the treatment of coronary heart disease.2 Interactions of HDLs with the Endothelium2.1 Preservation of Endothelial IntegrityPerturbations in the function of endothelial cells lining the lumen of blood vessels arean important hallmark of the earliest stages of the atherosclerotic process. HDLs mayimprove or help reverse endothelial dysfunction by several endothelial-vasoprotectivemeans. Several in vitro studies indicate that HDLs are able to counteract endothelialcell death. Treatment of endothelial cells with HDLs caused a marked attenuation ofapoptosis in response to pro-atherogenic signals such as oxidized LDL (Suc et al. 1997;de Souza et al. 2010; Kimura et al. 2001), tumor necrosis factor α (TNFα) (Suganoet al. 2000; Riwanto et al. 2013), and growth factor deprivation (Kimura et al. 2001;Nofer et al. 2001b; Riwanto et al. 2013). Although the underlying mechanism has notbeen fully elucidated, it has been suggested that HDLs regulate endothelial cell survivalby interfering with critical steps in apoptotic pathways. Several studies reported thatHDLs block the activation of the mitochondrial pathway of apoptosis by preventingdepolarization of the mitochondrial transmembrane and subsequent mitochondrialrelease of cytochrome c and apoptosis-inducing factor into the cytoplasm (Noferet al. 2001b; de Souza et al. 2010). As a consequence, HDLs inhibit the cytochromec-initiated caspase activation cascade (Sugano et al. 2000; Nofer et al. 2001b; de Souzaet al. 2010). Moreover, levels of the apoptosis-inducing protein truncated Bid arereduced in endothelial cells cultured in the presence of HDLs, whereas endothelialexpression of the antiapoptotic protein Bcl-xL is enhanced (Riwanto et al. 2013). Thecapacity of HDLs to attenuate apoptosis in endothelial cells requires induction ofphosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS)signaling (Nofer et al. 2001b; Riwanto et al. 2013) and was attributed to apoA-I(Radojkovic et al. 2009) as well as HDL-associated lysophospholipids (Kimuraet al. 2001; Nofer et al. 2001b). More recently, a lower clusterin (or apoJ) and increasedapoC-III content in the HDLs of patients with stable coronary artery disease or acuteHDL and Atherothrombotic Vascular Disease 373coronary syndrome was found to be associated with reduced antiapoptotic activities inhuman endothelial cells (Riwanto et al. 2013).Disruption of vascular endothelium integrity is another central event promotingatherosclerotic plaque formation. Sphingosine-1-phosphate (S1P), a bioactivesphingolipid metabolite, has been identified as an important endothelial barrierfunction-enhancing agonist (Garcia et al. 2001). In plasma S1P preferentiallyassociates with HDL particles via binding to apoM (Christoffersen et al. 2011). Invitro studies provided evidence that delivery of S1P carried by apoM-containingHDLs to S1P1 receptors on endothelial cells leads to activation of Akt and eNOS,which in turn promotes endothelial barrier integrity (Argraves et al. 2008;Wilkerson et al. 2012). Interestingly, the lung vascular barrier function, as deter-mined by Evan’s blue extravasation, is decreased in mice that lack S1P in theirHDL fraction due to deficiency of apoM (Christoffersen et al. 2011). These dataunderline a significant role of HDL-associated S1P in endothelial barrier protectionin vivo. In this context it is important to highlight the submicromolar concentrationof S1P in HDLs which amounts to only 5–10 % of HDL particle concentration but isin the range of the affinity constants of the G-protein-coupled S1P receptors(Annema and von Eckardstein 2013; Karuna et al. 2011). Moreover, erythrocytesrelease S1P in response to HDLs (Lucke and Levkau 2010) so that in vivo the localconcentration of S1P may be considerably higher than in isolated HDLs.Acceleration of reendothelialization after vascular injury is another mechanismproposed to contribute to the positive influence of HDLs on endothelial function. Inthis respect, HDLs might be specifically important in driving endothelial cellmigration during repair. Incubation of cultured endothelial cells with HDLsstimulated cell migration in response to wounding (Seetharam et al. 2006). More-over, hepatic expression of human apoA-I rescued the impaired carotid arteryreendothelialization following perivascular electric injury in apoA-I knockoutmice (Seetharam et al. 2006). The promotion of endothelial cell migration byHDLs has been demonstrated to involve scavenger receptor BI (SR-BI)-mediatedactivation of Rac-directed formation of actin-based lamellipodia (Seetharamet al. 2006). An SR-BI-dependent mechanism for the promigratory actions ofHDL was confirmed by experiments showing that the capacity of HDLs to stimulateendothelial cell migration was reduced by transfection of endothelial cells withsiRNA specific to PDZ domain-containing protein 1 (PDZK1), an SR-BI adaptorprotein that controls steady-state SR-BI levels, and that the reendothelializationcapacity of injured vessels is lost in mice in which the pdzk1 gene has beendisrupted (Zhu et al. 2008). Other studies suggested that HDL-induced endothelialcell migration is mediated by stimulation of Akt/eNOS phosphorylation throughHDL-bound S1P in an S1P1-dependent manner and that this process is facilitatedby endothelial lipase (Tatematsu et al. 2013).Besides endothelial cell migration, also recruitment and incorporation of bonemarrow-derived endothelial progenitor cells (EPCs) to sites of vessel injurycontributes to the repair of the damaged vessel wall. HDLs activate the PI3K/Aktpathway in EPCs resulting in enhanced EPC proliferation, migration, and survival(Sumi et al. 2007; Petoumenos et al. 2009; Zhang et al. 2010). Moreover,374 W. Annema et al.preincubation of EPCs with HDL augmented their adhesive capacity to endothelialcells and potentiated their transendothelial migration (Petoumenos et al. 2009).Mice that were injected with rHDLs had an increased number of circulating EPCsand showed improved reendothelialization after carotid artery injury (Petoumenoset al. 2009). Additional mouse studies indicated that infusion of rHDLs enhancedbone marrow progenitor cell mobilization, neovascularization, and functionalrecovery after hind limb ischemia in wild-type mice (Sumi et al. 2007). In contrast,no beneficial effects of rHDL treatment on ischemia-induced angiogenesis wereobserved in eNOS knockout mice (Sumi et al. 2007), suggesting an essential rolefor eNOS in mediating HDL-stimulated EPC recruitment and the subsequentfunctional effects on the ischemic limb. There is some evidence that HDLs alsoaffect circulating EPCs in humans. In patients with coronary artery disease, higherHDL cholesterol levels were significantly correlated with higher circulating EPCnumbers (Petoumenos et al. 2009). These findings are corroborated by data from astudy in patients with type 2 diabetes mellitus which found that rHDL infusion at adose of 80 mg/kg body weight markedly increased circulating EPC levels comparedto baseline (van Oostrom et al. 2007).2.2 Preservation of Endothelial FunctionOther reports on HDL functionality in endothelial cells established that HDLs havedirect effects on vascular endothelium-relaxing properties. HDLs induceendothelium-derived vasorelaxation in aortic segments ex vivo in a dose-dependentmanner (Nofer et al. 2004). Mechanistically, binding of HDL to SR-BI and thelysophospholipid receptor S1P3 on endothelial cells leads to PI3K-stimulatedactivation of Akt and mitogen-activated protein (MAP) kinase pathways, and Aktin turn phosphorylates eNOS at Ser-1177 to increase endothelial nitric oxide(NO) production and ultimately endothelium-dependent vasorelaxation (Noferet al. 2004; Mineo et al. 2003; Yuhanna et al. 2001). The stimulatory effect ofHDLs on NO-dependent vasorelaxation could be mimicked by threeHDL-associated lysophospholipids, i.e., sphingosylphosphorylcholine, S1P, andlysosulfatide (Nofer et al. 2004). In addition to eNOS activation, modulation of Aktand MAP signaling by HDLs has been reported to delay the degradation of eNOSmRNA in endothelial cells, thereby upregulating eNOS protein levels (Rametet al. 2003). Dietary oxysterols, in particular 7-oxysterols, impair endothelialvasorelaxant function by disrupting the active dimeric form of eNOS and by inducingproduction of reactive oxygen species (ROS) (Terasaka et al. 2010). HDLs cancounteract the negative effects of 7-oxysterols on endothelial eNOS function bypromoting efflux of 7-oxysterols from endothelial cells via ATP-binding cassettetransporter G1 (ABCG1) (Terasaka et al. 2010). The same researchers revealed thatHDL-mediated removal of cholesterol and oxysterols from endothelial cells throughABCG1 could reverse the cholesterol loading-induced inactivation of eNOS resultingfrom its increased interaction with caveolin (Terasaka et al. 2010). Similar to NO,there is evidence to suggest that the release of the endothelium-derived vasodilatorHDL and Atherothrombotic Vascular Disease 375prostacyclin can be influenced by HDLs. In human umbilical vein endothelial cells,HDLs elevated cyclooxygenase-2 protein expression and thereby induced prostacy-clin synthesis via a pathway that involved PKC, ERK1/2, and sphingosine kinaseSphK-2 (Xiong et al. 2014). The human relevance of these findings is underscored bystudies demonstrating that infusion of rHDLs in hypercholesterolemic patients led tonormalization of endothelium-dependent vasodilation in response to acetylcholine(Spieker et al. 2002). Moreover, heterozygosity for a loss-of-function mutation inABCA1 has been reported to be associated with low levels of HDL cholesterol as wellas an impaired endothelium-dependent forearm vasodilatory response, a phenotypewhich could be restored by intravenous administration of apoA-I/phosphatidylcholine discs (Bisoendial et al. 2003).3 Anti-inflammatory Effects3.1 Suppression of MyelopoiesisVascular inflammation, one of the main forces driving the formation of atheroscle-rotic plaques, is accompanied by the rolling, firm adhesion, and subsequent migra-tion of monocytes across the vascular endothelium. A first critical determinant formonocyte transmigration is the number of monocytes present in the circulation.HDL-mediated cholesterol efflux pathways appear to play a prime role in theproliferation of myeloid cells in the bone marrow, cell mobilization from thebone marrow, and subsequent myeloid differentiation, at least in mice. Mice thatlack both ABCA1 and ABCG1 have increased numbers of myeloid progenitorcells in bone marrow, blood, spleen, and liver, suggestive of aberrant myeloproli-feration, myeloid progenitor cell mobilization, and extramedullary hematopoiesis(Yvan-Charvet et al. 2010; Westerterp et al. 2012). As a consequence monocytecounts in blood are elevated in ABCA1 x ABCG1 double knockout mice (Yvan-Charvet et al. 2010). In addition, the myeloproliferative phenotype and theenhanced mobilization of hematopoietic progenitor cells associated with bonemarrow deficiency of ABCA1 and ABCG1 were reversed in mice carrying ahuman apoA-I transgene, and in high cholesterol diet-fed LDL receptor knockoutmice, this was associated with less severe atherosclerosis (Yvan-Charvetet al. 2010; Westerterp et al. 2012). Correspondingly, raising HDL cholesterollevels in other murine models of myeloproliferative disorders also efficientlyreduced the appearance of hematopoietic progenitor cells in the circulation(Westerterp et al. 2012). Direct proof for a link between HDL and myeloproli-feration was provided by experiments showing that incubation of myeloid progeni-tor cells with HDLs ex vivo suppressed cell expansion (Yvan-Charvet et al. 2010).Taken together, these findings indicate that HDLs may beneficially impact thenumber of circulating monocytes. In humans there is some evidence for a correla-tion between HDL cholesterol levels and monocyte numbers in patients withfamilial hypercholesterolemia (Tolani et al. 2013). However, only few and incon-sistent data on the association between monocyte counts and risk of atherosclerotic376 W. Annema et al.vascular disease in humans has been reported. In addition, it will be important toconsider the heterogeneity of monocytes for any association with coronary heartdisease (Zawada et al. 2012).3.2 Suppression of Monocyte ExtravasationThe arrest of circulating monocytes on inflamed endothelial monolayers is triggeredby chemokines and their corresponding receptors, which both have beendemonstrated to be subject to regulation by HDLs. Infusions of lipid-free apoA-Iin apoE-deficient mice that were fed a high-fat diet reduced the relative abundanceof cells stained positive for the chemokine receptors CCR2 and CX3CR1 inatherosclerotic plaques in the aortic sinus and decreased the circulating levels ofthe chemokines CCL2 and CCL5 (Bursill et al. 2010). Moreover, in vitro rHDLswere found to downregulate cell surface expression of chemokine receptors onmonocytes and to render human monocytes and endothelial cells partly resistant tophytohemagglutinin- or cytokine-induced expression of chemokines (Bursillet al. 2010; Spirig et al. 2013). Although it was shown that HDLs counterbalancethe upregulation of chemokines in response to pro-inflammatory cytokines byreducing nuclear factor-κβ (NF-κβ) pathway activation, how HDLs modulatemonocyte expression of chemokine receptors on the molecular level remains tobe established (Bursill et al. 2010). In addition to these effects on monocytes andendothelial cells, HDLs were also able to limit the production of CCL2 in vascularsmooth muscle cells (VSMCs) exposed to thrombin (Tolle et al. 2008). The studyidentified HDL as a negative regulator of thrombin-induced activation of Rac1-dependent NADPH oxidases and subsequent ROS generation. This process ismediated by binding of HDL to SR-BI on VSMCs, and moreover lysosphingolipidspresent in HDL particles can act on local S1P3 receptor to inhibit CCL2 expression(Tolle et al. 2008).Once monocytes are attracted by chemokines to inflamed endothelium,monocyte–endothelial cell interactions are mediated by several cell adhesionmolecules. Experiments in vitro provided compelling evidence that cytokine-induced adhesion molecule expression on the endothelial cell surface is diminishedin the presence of either native HDLs (Cockerill et al. 1995; Ashby et al. 1998) orrHDLs (Baker et al. 1999; Clay et al. 2001). Notably, the effects of HDLs onendothelial expression of adhesion molecules not only occurred in cultured cells butalso in the in vivo setting under inflammatory conditions. For example, a singleinjection of discoidal rHDL particles in pigs prevented upregulation of E-selectinexpression in response to intradermal interleukin-1α (Cockerill et al. 2001). More-over, daily treatment of normocholesterolemic rabbits with rHDLs or lipid-freeapoA-I markedly attenuated periarterial collar-induced endothelial expression ofvascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule1 as well as concomitant neutrophil infiltration into the vessel wall and generationof ROS (Nicholls et al. 2005). The positive effects of lipid-free apoA-I onpro-inflammatory vascular changes were still observed when administered as aHDL and Atherothrombotic Vascular Disease 377single low dose 3 h after insertion of the non-occlusive periarterial collar (Puraniket al. 2008), indicating that infusion of apoA-I may also be able to rescuepreestablished vascular inflammation. In patients suffering from claudication, treat-ment with rHDLs led to a significant reduction in the percentage of VCAM-1positive cells in femoral artery lesions (Shaw et al. 2008). Diminution of adhesionmolecule expression on endothelial cells by HDLs may involve the activation ofmultiple receptors and signaling cascades. There are data supporting that HDLsalter adhesion molecule expression as a consequence of perturbation of cellularsphingolipid metabolism and the NF-κβ signaling pathway. HDL3 was found toimpede TNFα-induced upregulation of VCAM-1 and E-selectin expression inhuman umbilical vein endothelial cells by antagonizing activation of sphingosinekinase and subsequent intracellular formation of S1P (Xia et al. 1999). Further-more, it has been documented that in cultured endothelial cells, incubation withHDLs prevented NF-κβ activation, movement of p65 to the nucleus, and expressionof NF-κβ target genes in response to TNFα (Xia et al. 1999; Park et al. 2003;Kimura et al. 2006; McGrath et al. 2009). The basal molecular mechanisms ofthe beneficial effects of HDL on endothelial inflammation are dependent on anincrease in the expression of 3β-hydroxysteroid-Δ24 reductase that results inactivation of PI3K and downstream activation of eNOS and heme oxygenase-1(HO-1) (McGrath et al. 2009; Wu et al. 2013). Specific knockdown of SR-BI orS1P1 receptor by treatment of human endothelial cells with small interfering RNAsagainst SR-BI and PDZK1 or S1P1, respectively, markedly reduced the ability ofHDLs to inhibit VCAM-1 expression and NF-κβ activation (Kimura et al. 2006; Wuet al. 2013), suggesting that both receptors may be critical for the anti-inflammatoryendothelial actions of HDLs. Corresponding with the possible involvement of theS1P1 receptor, the inhibitory activity of HDLs on the expression of endothelial celladhesion molecules has been proposed to be a function of biologically activelysosphingolipids carried by HDL particles (Nofer et al. 2003; Kimuraet al. 2006). Besides the lipid component, the anti-inflammatory properties ofHDLs could also be related to the microRNA content of HDL particles. Recently,it has been reported that transfer of microRNA-223 by HDLs to human aorticendothelial cells significantly repressed expression of intracellular adhesion mole-cule 1 (Tabet et al. 2014).Monocytes bind to adhesion molecules on activated endothelial cells throughmonocytic cell surface receptors, such as CD11b. Ex vivo incubation of stimulatedhuman peripheral blood monocytes with apoA-I in the lipid-free or HDL-associatedstate caused a reduction in cell surface expression of CD11b (Murphy et al. 2008).These results might be directly translatable to the human in vivo situation, asevidenced by the observation that infusion of rHDLs in patients with atherosclerosisdisease in the femoral artery led to a significantly lower expression of CD11b oncirculating monocytes (Shaw et al. 2008). The fact that cellular depletion ofcholesterol by cyclodextrin mimicked the protective effects of apoA-I on theexpression of monocyte CD11b and that inhibition of CD11b expression is lost inthe presence of an antibody against ABCA1 or in monocytes derived from Tangierdisease patients is indicative of a role for ABCA1-mediated cholesterol efflux in themechanism of monocyte deactivation by apoA-I (Murphy et al. 2008).378 W. Annema et al.3.3 Interference with Macrophage Differentiation and ActivationMonocytes that have entered the arterial intima subsequently differentiate intoresident macrophages of at least two major subtypes that differentially produce avariety of pro- and anti-inflammatory mediators. In the subendothelial space, themacrophages become activated and comprise key targets for the anti-inflammatoryaction of HDLs. Incubation with HDLs primed isolated murine bone marrowmacrophages into alternative M2 macrophages with anti-inflammatory features(Sanson et al. 2013). Moreover, HDLs were potent inhibitors of interferon-gamma-mediated expression of pro-inflammatory M1 markers in isolated mousemacrophages, and, interestingly, experiments performed with macrophages fromSTAT6-deficient mice demonstrated that STAT6 plays an essential role in theHDL-mediated shift of the macrophage phenotype to M2 (Sanson et al. 2013).However, other investigators could not replicate these observations, when usingprimary human monocyte-derived macrophages (Colin et al. 2014). A recent studyprovided novel mechanistic insights into the anti-inflammatory effects of HDLs inmacrophages. Thus, administration of native HDL, rHDL, or apoA-I to micechallenged with a toll-like receptor (TLR) agonist protected from liver damageand reduced serum levels of pro-inflammatory cytokines, which was supposed to bedue the ability of HDLs to reduce cytokine production in activated macrophages(De Nardo et al. 2014). The anti-inflammatory action of HDL particles onmacrophages was explained by their ability to induce the expression of activatingtranscription factor 3 (ATF3), which functions as a repressor of pro-inflammatorytarget genes (De Nardo et al. 2014).During the past decades, a considerable amount of data has been publishedrevealing that natural regulatory T cells, which dampen inflammatory responses,may help decrease the atherosclerotic disease burden. The findings of a recentmurine study imply that apoA-I could promote the expansion of regulatory T cells.Subcutaneous injection of human lipid-free apoA-I in LDL receptor/apoA-I doubleknockout mice fed an atherogenic diet restored the regulatory T-cell population inthe lymph nodes (Wilhelm et al. 2010). In these animals the raise in the number ofregulatory T cells in the lymph nodes was accompanied by a reversal of theinflammatory and autoimmune phenotype.4 Effects of HDLs on Vascular Lipid and LipoproteinHomeostasis4.1 From LDL Retention to Atheroma Formation4.1.1 Initial Lipid Accumulation in Atherosclerosis-Prone ArterialIntima: A Sign of LDL RetentionThe intima is disproportionally thick at the outer curvatures of the proximalbranches of coronary and carotid arteries, where turbulent flow conditions prevailand the pulse wave amplitude is relatively high (Wentzel et al. 2012). TheHDL and Atherothrombotic Vascular Disease 379thickening is a physiological response to these conditions and ensues when thevascular smooth muscle cells (VSMCs) divide and secrete various components ofthe extracellular matrix at the stressed sites. One major factor explaining therelationship between the thickness of the intima and its susceptibility to atheroscle-rotic lesion development is the lack of a capillary delivery system and lack of alymphatic drainage system in this tissue (Hulten and Levin 2009; Eliskaet al. 2006). As a consequence, the inflowing apoB100-containing lipoproteinparticles (notably LDL and also VLDL remnants) are only slowly conveyed bythe intimal fluid from the subendothelial superficial layer into the deeper intimallayers and ultimately to the medial layer of the arterial wall where lymphaticcapillaries are present. Indeed, in cholesterol-fed rabbits, intimal LDL concentra-tion initially increases in atherosclerosis-susceptible sites, where their intimalresidence time of the LDL particles is long, the transit time being calculated to behours (Schwenke and Carew 1989a, b).Since progressive accumulation of LDL cholesterol is a key feature of athero-genesis, we can deduce that some intimal LDL particles never leave the site. Theconstant inflow without compensatory outflow of LDL particles results in a dra-matic increase in their concentration in the intimal fluid to reach values equaling oreven exceeding (by twofold) those in the corresponding human blood plasma(Smith 1990). Thus, the concentration of LDL in the arterial intimal fluid is10–20 times higher than in the extracellular fluids of other extrahepatic tissues, inwhich its concentration is about 1/10th of that in the corresponding plasma, sorendering the intima a truly “hypercholesterolemic” site.During their lengthy passage across the thick avascular tissue, the LDL particlestend to bind to the subendothelial extracellular proteoglycan matrix, a phenomenonknown as retention of LDL (Williams and Tabas 1995). The proteoglycans form atight network that is negatively charged. To this network the LDL particles bind viaionic interactions, when positively charged lysine and arginine residues located inthe proteoglycan binding site of apoB100 interact with the negatively chargedsulfate groups of the glycosaminoglycan components of the proteoglycans (Camejoet al. 1998). The prolonged residence time of LDL particles in the intima, whetherthe particles are in solution or trapped in the matrix, enhances their exposure to avariety of local LDL-modifying factors. Thus, during their long extracellularresidence time, the LDL particles are modified, for example, by oxygen radicalssecreted by the resident cells of the intima, notably by endothelial cells, and also bySMCs. It is of particular relevance that the endothelial cells become activated tosecrete pro-oxidative compounds at sites with turbulent flow, i.e., at theatherogenesis-prone sites of the arterial tree (Nigro et al. 2011). Moreover, thesubendothelially located LDL particles are not protected by the powerful antioxi-dant systems present in the circulating blood plasma, and they also graduallybecome depleted of their own antioxidant molecules capable of protecting themfrom free radical attack and oxidation (Esterbauer et al. 1991). In addition to theoxidative processes, various hydrolytic (both proteolytic and lipolytic) enzymesmay also modify the LDL particles in the intima (Tabas 1999). Importantly, theoxidative and hydrolytic changes of LDL particles render them unstable, trigger380 W. Annema et al.their aggregation and fusion into lipid droplets, and markedly strengthen theirbinding to arterial proteoglycans (Oorni et al. 2000). These in vitro observationshave their close counterparts in various animal models, and also in the humancarotid arteries, in which tiny proteoglycan-, collagen-, and elastin-associated lipiddeposits, detectable only by aid of special electron microscopic techniques, arelocated in the subendothelial extracellular space even before any microscopic signsof atherosclerosis are visible (Nievelstein-Post et al. 1994; Tamminen et al. 1999;Pasquinelli et al. 1989).4.1.2 Formation of a Fatty StreakAs stated above, atherosclerosis typically develops at certain predilection sites as aresponse to the hemodynamic stress and other types of injury affecting the endo-thelial cells. Importantly, in such stressed areas with disturbed flow conditions,endothelial inflammatory signaling pathways become activated and a localpro-inflammatory environment is created (Nigro et al. 2011). We can considerthis local scenario as a singular starting condition to the development of atheroscle-rosis. Thus, the flow-mediated preconditioning of the intima renders it a suitablesoil for the retention, modification, and accumulation of LDL particles. Because thesubendothelially located modified LDL particles and the products released fromthem are pro-inflammatory, they are likely to have profound local cell-activatingeffects and to worsen the inflammation by inducing local generation of variousLDL-modifying enzymes and agents and so creating a positive feedback loop(Pentikainen et al. 2000; Tabas et al. 2007). Moreover, synthesis and secretion ofpro-inflammatory cytokines and chemokines, such as the monocyte chemotacticprotein-1 (MCP-1; CCL2), are induced (Libby 2002; Zernecke and Weber 2010).Indeed, monocytes preferentially adhere to the endothelium covering inflamedtissue sites, i.e., regarding atherogenesis, they tend to enter the intimal areascontaining oxidized or otherwise modified LDL particles (Schmitt et al. 2014a, b).The extracellularly located modified LDL particles, whether free floating in theintimal fluid or whether matrix bound, are ingested by intimal macrophages and, tosome extent, also by intimal SMCs (Witztum 2005; Wang et al. 1996). On theirsurfaces, the macrophages express scavenger receptors, which recognize oxidizedand otherwise modified LDL particles (Krieger and Herz 1994). The macrophagesmay also phagocytose modified LDL particles, provided the particles are in aggre-gate form or have been converted into droplets via fusion. Since uptake of LDLcholesterol by macrophages is not negatively feedback regulated by the inflowingcholesterol, uptake of LDL cholesterol, be it scavenger receptor dependent ormediated by other mechanisms, may continue until the cells are filled with choles-terol (Brown and Goldstein 1983). Of great interest, also unmodified native LDLscan be ingested by macrophages at a rate which is linear to their concentration in theextracellular fluid (Kruth 2011). Since the concentration of LDL in the intimal fluidis exceptionally high (see above), such micropinocytotic uptake of LDLs bymacrophages may actually be of great significance in the atherosclerosis-pronearterial intima.HDL and Atherothrombotic Vascular Disease 381In the macrophages, the excess of intracellular cholesterol is esterified with fattyacids and stored in their cytoplasm as cholesteryl ester droplets (Brown andGoldstein 1983). Macrophages full of such droplets look foamy under the micro-scope and are called foam cells. When numerous foam cells form clusters, theyhave yellow appearance visible to the naked eye and the site appears as a fattystreak (Stary et al. 1994). Importantly, when fatty streaks develop in human carotidarteries, the subendothelial perifibrous lipid droplets disappear (Pasquinelliet al. 1989). This switch conceivably reflects uptake of the perifibrous droplets bythe subendothelial macrophages. Thus, in a fatty streak the LDL-derived choles-terol is located mostly intracellularly. Taken together, an important role ofsubendothelial macrophages in the arterial intima is to clear unmodified or modifiedLDL particles from their surrounding and to store their cholesterol as cholesterylesters. Indeed, foam cells in the arterial intima are the most defining feature ofatherosclerosis.HDLs directly bind unesterified cholesterol. As the consequence, HDL canmobilize unesterified cholesterol both from cells and extracellular locations (Free-man et al. 2014). Regarding cholesteryl esters, the extracellularly stored esters,present either in the core of the matrix-bound LDL particles or in the core of theperifibrous lipid droplets derived from modified LDL particles, are likely to fullyresist transfer to HDLs. In sharp contrast, intracellular cholesterol stored ascholesteryl esters in the cytoplasmic lipid droplets of macrophage foam cells issensitive to transfer to HDLs (Rosenson et al. 2012). Thus, regarding the return ofLDL cholesterol from the arterial intima back to the circulating blood, it appears tobe of fundamental importance that the LDL particles are first taken up andmetabolized by intimal macrophages. Accordingly, this macrophage-dependentpreparative step appears to be mandatory for the initiation of reverse cholesteroltransport in the arterial intima. However, since the macrophages accumulateLDL-derived cholesterol, an imbalance between cholesterol influx and cholesterolefflux exists in the atherosclerosis-prone arterial intima. One reason for the relativeinability of HDLs to effectively remove cholesterol from the intimal macrophagesis their functional modification including proteolysis, oxidation, and lipolysis tovarious extents (Lee-Rueckert and Kovanen 2011; DiDonato et al. 2013).4.1.3 Development of an AtheromaConversion of a fatty streak into an atheroma is the next phase in atherogenesis(Stary et al. 1995). It is characterized by extracellular accumulation of cholesterolin the deep layers of the intima, i.e., below the superficial foam cell layers.However, in sharp contrast to the superficial layers of the intima, where atherogen-esis initiates and where macrophages swiftly arrive and efficiently remove the lipidsso preventing their extracellular accumulation, in the deep layers, the macrophagesarrive late (Nakashima et al. 2008). Accordingly, once formed, the deep extracellu-lar lipids are not rapidly scavenged by macrophages. Rather, the continuousformation of lipid droplets without their concomitant removal leads to their pro-gressive deposition, which ultimately leads to the appearance of “lipid lakes”. Thelakes consist of huge numbers of such droplets, and together the lakes form a lipid382 W. Annema et al.core (Guyton and Klemp 1996). The presence of a lipid core is the hallmark of anatheroma.During the process of lipid core formation, macrophages start to migrate fromthe superficial layer into the deep intimal area where they ingest the extracellularlipid droplets in the periphery of the lipid core and themselves become convertedinto foam cells. Since the deep intimal region is distant from the circulating blood, iteasily becomes hypoxic (Hulten and Levin 2009). Although survival mechanismsare activated in macrophages trapped in such hypoxic areas (Ramkhelawonet al. 2013), promotion of lipid accumulation, ATP depletion, and immobilizationof the macrophage foam cells is likely to ultimately lead to their death. Of note,death of a foam cell is bound to liberation of its cytoplasmic cholesteryl esterdroplets into the extracellular space, where the droplets mix with the preexistingextracellular lipid droplets. Moreover, the nonviable macrophages (foam cells) failto effectively remove the cellular debris derived from the dying or dead foam cells(Thorp et al. 2011), so leaving the formed debris deposited among the lipids of thegrowing core. A lipid core also containing remnants derived from dead cells iscalled a “necrotic lipid core”.In summary, regarding cholesterol metabolism in the arterial intima, the super-ficial and the deep layers appear to be fundamentally different. Mechanistically, inthe superficial compartment of the arterial intima, viable macrophages ingest andstore LDL cholesterol as cholesteryl esters, and transfer it to HDLs for ultimatereturn to the circulation, continual hydrolysis of the cytoplasmic cholesteryl estersbeing the critical factor for the transfer of cholesterol from LDL to HDL. Incontrast, in the deep intimal compartment, macrophages only inefficiently removeLDL cholesterol, and, provided a macrophage is converted into a foam cell, it maydie and release its cholesteryl ester cargo into the extracellular space, where thecholesteryl esters are not available for removal by HDLs. Thus, in the absence of anefficient transfer of cholesterol from LDL to HDL, the necrotic lipid core growsprogressively and may convert an atheroma into a clinically significant atheroscle-rotic lesion.4.2 Inhibition of LDL Modification by HDLsResident macrophages in the subendothelial space take up oxidized LDLs viascavenger receptors and become macrophage foam cells, the hallmark cell type ofatherosclerotic lesions. HDLs have been proven to help to reduce the formation oflipid-laden macrophage foam cells by rendering LDL particles resistant to oxida-tion. Preexposure of LDL particles to apoA-I or mature HDL particles completelyprevented the oxidative modification of LDLs by cultured human aortic endothelialcells and SMCs (Navab et al. 2000). Moreover, the human artery wall cells were nolonger able to oxidize LDLs isolated from mice and humans that received a singletreatment with human apoA-I. Likewise, HDLs have been shown to possess potentantioxidative activity in chemical in vitro models for oxidative modifications ofHDL and Atherothrombotic Vascular Disease 383LDLs, using, for example, copper ions (Parthasarathy et al. 1990) or the more mildoxidizing agent 2,20-azobis(2-amidinopropane) dihydrochloride (AAPH) (Kontushet al. 2003). At least part of the ability of HDLs to inhibit LDL oxidation wasattributed to the capacity of apoA-I to remove seeding molecules present in freshlyisolated LDLs (Navab et al. 2000). Notably, the reduction of lipid hydroperoxidesto their corresponding hydroxides by HDLs was associated with the selectiveoxidation of Met residues in apoA-I to their Met sulfoxide form (Garneret al. 1998), suggesting that Met residues in apoA-I may act as endogenousantioxidants within the HDL particle.In addition to apoA-I, antioxidant properties of HDLs are conferred byparaoxonase 1 (PON1), an antioxidant enzyme transported in the circulation byHDL particles. The inhibitory effect of HDLs on LDL oxidation by human arterywall cells or copper could be mimicked by incubation of LDLs with purified PON1(Navab et al. 2000; Liu et al. 2008). After treatment of oxidized phospholipids withPON1, oxygenated polyunsaturated fatty acids were no longer detectable (Watsonet al. 1995a), indicating that PON1 was able to destroy these multi-oxygenatedbiologically active molecules which are abundantly formed during the atherogenicoxidation of LDL particles. Studies in transgenic and knockout mice on the role ofPON1 in the pathogenesis of atherosclerosis have supported the protective effectsof PON1 on LDL oxidation. The lack of PON1 in the HDL fraction of PON1knockout mice resulted in an inability of these HDL particles to prevent theformation of oxidized LDLs, and accordingly pon1-null mice were more suscepti-ble to atherosclerosis than their wild-type littermates (Shih et al. 1998). Expressionof human PON1 in transgenic mice led to the opposite phenotype. On theatherosclerosis-prone apoE/ background, mice transgenic for human PON1developed significantly less atherosclerotic plaques in the aorta than the controlmice. Moreover, in vitro PON1-enriched HDLs from PON1 transgenic/apoEknockout were more potent inhibitors of copper-catalyzed oxidation of LDLparticles (Tward et al. 2002).Another antioxidant enzyme that has been related to the cardioprotectiveantioxidative function of HDL particles is lipoprotein-associated phospholipaseA2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase(PAF-AH). In human plasma a small proportion of the circulating Lp-PLA2 enzymeactivity is found in HDL particles (Tellis and Tselepis 2009). Inhibition of theenzymatic activity of Lp-PLA2 rendered HDLs unable to prevent monocyte bindinginduced by oxidized LDLs in endothelial cells, and supplementation of the dys-functional HDL particles with purified Lp-PLA2 restored the normal protectivefunction (Watson et al. 1995b). In mice, adenovirus-mediated expression of humanLp-PLA2 leads to a reduction in the autoantibody titers against oxidized LDLsand malondialdehyde (MDA)-modified LDLs (Quarck et al. 2001; Notoet al. 2003). Despite the fact that the antioxidative potential of HDL-boundLp-PLA2 has been well documented, several lines of evidence suggest that thepresence of Lp-PLA2 in LDL particles may actually stimulate atherogenesis. Thepro-atherogenic actions of Lp-PLA2 in LDLs are thought to arise from oxidized freefatty acids and lysophospholipids, two inflammatory mediators which are released384 W. Annema et al.after the hydrolysis of oxidized phospholipids by Lp-PLA2 (Tellis and Tselepis2009). LDLs are the preferred lipoprotein carriers for Lp-PLA2 in the circulation,which would explain why previous prospective cohort studies paradoxically linkedhigh plasma Lp-PLA2 levels with adverse cardiovascular outcomes (Brilakiset al. 2005; Koenig et al. 2004; Sabatine et al. 2007). In support of the hypothesisthat Lp-PLA2 is atheroprotective only when incorporated in HDL particles, Rallidiset al. found that elevated total plasma Lp-PLA2 mass and activity increased the riskfor future cardiac death in patients with stable coronary artery disease, whileLp-PLA2 mass and activity in apoB-depleted plasma showed a significant associa-tion with future cardiac death in the opposite direction (Rallidis et al. 2012).The protective effect of HDLs on LDL oxidation has been tentatively ascribed tothe antioxidant activities of lecithin-cholesterol acyltransferase (LCAT). Similar toHDLs, purified LCAT has been shown to diminish the formation of both lipidhydroperoxides and conjugated dienes in copper-oxidized LDLs (Vohl et al. 1999).Moreover, adenovirus-mediated transfer of the human LCAT gene into mice withcombined leptin and LDL receptor deficiency decreased circulating levels ofautoantibodies binding to MDA-LDL by 40 % and accordingly limited the accu-mulation of oxidized LDLs in the arterial wall (Mertens et al. 2003).4.3 Macrophage Cholesterol Efflux and Reverse CholesterolTransportThe uptake of modified LDLs in arterial macrophages leads to the cellular accumu-lation of cholesterol and oxidized lipids. This process causes the macrophages totransform into lipid-laden macrophage foam cells which can no longer escape thearterial wall (Potteaux et al. 2011). The excessive cellular cholesterol in macro-phage foam cells can be efficiently removed by the potent cholesterol acceptorslipid-free apoA-I and HDLs to be transported back to the liver by a process termedreverse cholesterol transport.4.3.1 Transendothelial HDL TransportIn order to reach these resident macrophage foam cells in the innermost layer of thearterial wall, apoA-I and HDLs first need to passage across the endothelial celllayer. There is cell culture evidence that lipid-free apoA-I binds to specific saturablehigh-affinity binding sites on aortic endothelial cells, which is followed by inter-nalization, transcytosis, and delivery of lipidated apoA-I to the basolateral side(Rohrer et al. 2006). A similar phenomenon was observed when cultivated aorticendothelial cells were treated with mature HDLs (Rohrer et al. 2009). However, themechanisms involved in regulating this endothelial transport of apoA-I and HDLsare not fully understood. The ABCA1 transporter is thought to play major role inthe apical-to-basolateral transport of apoA-I in aortic endothelial cells (Cavelieret al. 2006). Induction of ABCA1 expression by a combination of oxysterol and9-cis-retinoic acid increased the binding and internalization of apoA-I in endothe-lial cells, whereas knockdown of endothelial ABCA1 by RNA interference reducedHDL and Atherothrombotic Vascular Disease 385the cellular uptake and transcytosis of apoA-I (Cavelier et al. 2006). Conversely,siRNA-mediated gene silencing experiments revealed that the cell surface bindingand transport of HDLs through aortic endothelial cells are highly dependent onSR-BI and ABCG1, but not on ABCA1 (Rohrer et al. 2009). Subsequent researchhas demonstrated expression of the ectopic β-chain of F0F1 ATPase (β-ATPase) onthe endothelial cell surface (Cavelier et al. 2012). The results of this study furthersuggest that the binding of lipid-free apoA-I to endothelial cell surface β-ATPasefacilitates the uptake and transport of lipidated apoA-I and mature HDLs byenhancing β-ATPase-mediated hydrolysis of extracellular ATP into ADP andinducing consecutive activation of the purinergic P2Y12 receptor (Cavelieret al. 2012). More recently, transendothelial transport of HDL particles was foundto be modulated by endothelial lipase both through its enzymatic lipolytic activityand its ability to bridge the binding of lipoproteins to the endothelial surface(Robert et al. 2013). Initiation of an atherosclerosis-related inflammatory pheno-type after stimulation of vascular endothelial cells with interleukin-6 significantlyincreased the binding, cell association, and transport of HDLs, which was linkedmechanistically to the increase in endothelial lipase expression in response tointerleukin-6 (Robert et al. 2013).4.3.2 Cholesterol Efflux from MacrophagesOnce apoA-I and HDLs have reached macrophage foam cells in the atheromatousvessel wall, macrophage cholesterol efflux can be elicited via several differentpathways. It is well documented that lipid-free apoA-I and pre-beta HDL particlesare able to remove cholesterol and phospholipids from macrophage foam cells viathe ABCA1 transporter (Wang et al. 2000; Wang and Tall 2003). It has beenpostulated that apoA-I-dependent cholesterol efflux from cells is mediated eitherby a direct protein–protein interaction between apoA-I and ABCA1 or indirectly byABCA1-induced changes in the membrane cholesterol distribution (Wang and Tall2003). The ability of ABCA1 to mobilize cellular lipids to apoA-I is essential forthe initial lipidation of apoA-I. The ABCG1 transporter is responsible for a majorpart of the macrophage cholesterol efflux towards mature HDL particles (Wanget al. 2004). Since cellular removal of cholesterol via ABCG1 was not accompaniedby specific binding of HDLs to the plasma membrane (Wang et al. 2004), themolecular basis of efflux of cholesterol mediated by ABCG1, like ABCA1, is notfully understood and warrants further investigation. Another cell surface receptorinvolved in macrophage cholesterol efflux elicited by mature HDLs is SR-BI.Binding of HDL to macrophage SR-BI facilitates a bidirectional exchange ofunesterified cholesterol and other lipids between the cell membrane and HDLacceptor particles according to the cholesterol concentration gradient (de LaLlera-Moya et al. 2001). This cholesterol concentration gradient between cellsand HDL not only allows cholesterol transfer via SR-BI but also passive diffusionof cholesterol molecules to nearby HDL particles by receptor-independent pro-cesses (von Eckardstein et al. 2001; Yancey et al. 2003). The HDL-associatedenzyme LCAT catalyzes the esterification of cholesterol in HDL particles(Calabresi and Franceschini 2010). The generated cholesteryl esters leave the386 W. Annema et al.particle surface and form the core of the maturing HDL particle; hence, a localgradient of unesterified cholesterol is created that maintains a continuous flow ofunesterified cholesterol from macrophages towards HDLs (Calabresi andFranceschini 2010).4.3.3 Exit from the Arterial WallThere is little reported data on the route used by HDLs to leave the arterial intima.Recent lines of evidence support a role for the lymphatic vasculature in thetransport of HDLs from the interstitial space back to the bloodstream. Followinginjection of fluorescently labeled HDLs in the mouse footpad, HDLs were subse-quently found in the draining lymph node and its afferent and efferent lymphaticvessels (Lim et al. 2013). Moreover, the movement of cholesterol frommacrophages in the footpad to the lymph and plasma was substantially reduced inmice with surgical interruption of the afferent lymphatic vessels or in Chy mutantmice that lack dermal lymphatic capillaries (Lim et al. 2013; Martel et al. 2013).The importance of the lymphatic system in the reentry of cholesterol originatingfrom macrophages via HDLs in the circulation was further confirmed by additionalexperiments showing that the appearance of macrophage-derived cholesterol in theplasma was impaired after implantation of [3H]-cholesterol-labeled macrophages inthe tail skin of apoA-I transgenic mice with microsurgical ablation of the majorlymphatic conduits in the tail (Martel et al. 2013). To provide more direct proof forthe involvement of the lymphatic vasculature in the transport of cholesterol fromthe atherosclerotic plaque, aortic segments with advanced atherosclerotic lesionswere loaded with D6-cholesterol and transplanted in apoE knockout mice (Martelet al. 2013). Inhibition of regrowth of a functional lymphatic vasculature in theaortic donor wall by an antibody blocking the function of vascular endothelialgrowth factor receptor 3 markedly suppressed the apoE-induced removal ofD6-cholesterol from the transplanted aorta (Martel et al. 2013), hence emphasizingthe pivotal role of the lymphatic system for the egress of cholesterol from the aorticwall. The entry of HDL particles into lymphatic vessels appears to be primarilydependent on SR-BI. Experiments in cultured cells demonstrate that the internali-zation and transcytosis of HDL particles by lymphatic endothelial cells are dimin-ished by an SR-BI blocking antibody or a selective SR-BI inhibitor (Limet al. 2013). In agreement, lymphatic transport of HDLs is compromised in SR-BI-null mice and in mice treated with an antibody against SR-BI (Lim et al. 2013).Although not formally tested, it is also possible that, in order to reach the circulationagain, HDL particles undergo transcytosis through the luminal endothelial cellbarrier or transfer cholesterol to endothelial cells for delivery to circulatingHDLs. The importance of a well-regulated homeostasis between entry and exit ofHDL particles into and from the arterial wall, respectively, is indicated by theenrichment of modified and dysfunctional HDLs and apoA-I in atheroscleroticlesions (DiDonato et al. 2013, 2014; Huang et al. 2014).HDL and Atherothrombotic Vascular Disease 3874.3.4 Delivery of Cholesterol to the Liver and IntestineAfter having left the arterial wall and entered the circulation, the HDL particlestransport the cholesterol either directly or indirectly via apoB-containinglipoproteins to the liver for either secretion into bile or for de novo assembly oflipoproteins. The first direct way by which HDL can deliver cholesterol to the liveris via SR-BI, a cell surface receptor that binds HDLs and mediates the selectiveuptake of HDL-associated cholesteryl esters and the subsequent resecretion ofcholesteryl ester-poor HDLs (Acton et al. 1996). Secondly, HDL particles can betaken up from the circulation by the liver via HDL holoparticle endocytosis (i.e.,uptake of the whole HDL particle). The binding of apoA-I to the ectopic β-chain ofF0F1 ATPase on hepatocytes triggers extracellular production of ADP, which inturn acts on the P2Y13 receptor to stimulate HDL holoparticle endocytosis(Martinez et al. 2003; Jacquet et al. 2005). In humans, the major fraction of thecholesteryl esters in HDL particles are shuttled via the action of CETP to apoB-containing lipoproteins (Charles and Kane 2012). These cholesteryl estersoriginating from HDLs are internalized as part of apoB-containing lipoproteinsby hepatic LDL receptors. Following hepatic uptake, HDL-derived cholesterol is, atleast in part, targeted for fecal excretion via the biliary route (Lewis and Rader2005; Nijstad et al. 2011). Hepatic cholesterol can either be secreted directly in thefree form into the bile by the ABCG5/G8 heterodimer or alternatively be convertedby cholesterol 7α-hydroxylase (CYP7A1) and other enzymes into bile acids forhepatic excretion via the bile salt export pump (BSEP) (Dikkers and Tietge 2010).Secretion into bile enables cholesterol to move from the hepatocyte to the intestinallumen for final excretion via the stool. This major HDL-driven pathway to elimi-nate atherogenic cholesterol from the body is also known as reverse cholesteroltransport (Annema and Tietge 2012). Although reverse cholesterol transport fromplaque macrophages to feces only represents a very small proportion of the totalreverse cholesterol transport flux from peripheral tissues, it is key component of thereverse transport potentially capable of initiating and maintaining the regression ofatherosclerosis (Lee-Rueckert et al. 2013).5 AtherothrombosisThe most important mechanism of acute coronary syndromes, i.e., unstable angina,acute myocardial infarction, and sudden cardiac death, is rupture or erosion of acoronary atheroma (Libby 2013b). The risk of atheromatous rupture or erosionappears to critically depend on both the cellular and extracellular compositions ofthe atheroma. The superficial layer of an atheroma separating a necrotic lipid corefrom the circulating blood is called the “fibrous cap”. Although a cap usually containsfoam cells, its critical components are VSMCs, which synthesize and secrete collagenand other components of the extracellular matrix. It is current understanding that thecap is thinning as the necrotic lipid core is growing. Whether the thinning is aresponse to the enlargement of the necrotic core, or whether thinning is an indepen-dent process and just allows the core to expand, has not been established.388 W. Annema et al.As expected, a lesion with a thick fibrous cap and a small lipid core is stable,whereas a lesion with a thin fibrous cap and a large lipid core is unstable and proneto rupture. Indeed, it is now well recognized that acute coronary syndromes mostcommonly result from rupture of a thin-cap fibroatheroma (TCFA), which ischaracterized by a large necrotic core with an overlying thin fibrous cap measuringless than 65 μm (Sakakura et al. 2013; Fujii et al. 2013).Provided a stable lesion with a thick fibrous cap causes significant stenosis of thearterial lumen, and turbulent flow conditions are created, some endothelial cellsmay detach, that is, the plaque surface becomes eroded (Libby 1995; Farbet al. 1996). Apoptosis of endothelial cells also leads to endothelial denudation,i.e., to plaque erosion when, for example, subendothelial mast cells degrade thebasement membrane of endothelial cells (Durand et al. 2004; Mayranpaaet al. 2006). The exposed subendothelial structures are prothrombotic and aplatelet-rich thrombus is formed locally. Since the activated platelets secreteplatelet-derived growth factor which stimulates SMCs to divide and generateextracellular matrix, a neointima is formed at the site of erosion. This is onemechanism by which the fibrotic cap of a stenosing plaque becomes thicker andthe plaque grows into the lumen and so aggravates the local stenosis.When an unstable TCFA ruptures, the flowing blood becomes exposed to thenecrotic lipid core. The various components of the core, notably tissue factorreleased by macrophages, apoptotic bodies, remnants of dead cells, and extracellu-larly located lipids, all are contributing to the strong thrombogenic response usuallyassociated with a plaque rupture (Fuster et al. 2005; Corti et al. 2003). Thus, theplatelet-rich arterial thrombus growing at the site of plaque rupture easily becomesocclusive, even if the plaque only had caused a mild stenosis which was hemody-namically nonsignificant.In view of the importance of the thickness of collagen-rich matrix of the fibrouscap in determining plaque stability and instability, it is essential to understand thefactors that may regulate its thickness. Regarding such regulation, two relatedobservations made in advanced human atherosclerotic plaques are of prime impor-tance. First, irrespective of the structure of an atherosclerotic plaque, the actual siteof atheromatous ulceration with ensuing local formation of a platelet-rich thrombusis characterized by an inflammatory process (van der Wal et al. 1994). Thus,atheromatous coronary and carotid ulcerations have invariably shown the presenceof inflammatory cell infiltrates, composed of macrophages, T cells, and mast cells(Kovanen et al. 1995; Bui et al. 2009). When compared with the evolving athero-sclerotic plaques, which contain inflammatory cell infiltrates, composed of varioussubsets of macrophages and T cells and also of mast cells and neutrophils(Woollard 2013), the vulnerable plaques appear to be equally, if not even moreimmunologically, active. The second critical aspect of a vulnerable atheroscleroticplaque is the fact that SMCs are dying in their fibrous caps (Bennett 1999).Fundamentally, the various activities of SMCs tend to thicken the cap, whilethose of the inflammatory cells tend to oppose them. The net production of matrixcomponents depends on the number of the matrix-producing SMCs and the abilityof the individual cells to produce such components. The number of SMCs dependsHDL and Atherothrombotic Vascular Disease 389on the balance of cell replication and cell death. In human atherosclerotic plaques,the infiltrating lymphocytes are of the Th1 subpopulation of CD4+ cells and thepro-inflammatory Th1-derived cytokines, notably interferon-gamma, dominate(Hansson 2005). This cytokine is able to inhibit the capacity of SMCs to producecollagen (Libby 1995) and together with TNFα and interleukin-1β (IL-1β) contrib-ute to their apoptotic death. Thus, activation of Th1 cells, together with IL-1-β-producing macrophages and TNFα-producing mast cells within the cap of anatheroma, could lower the local rate of collagen production by lowering the numberof SMCs and by inhibiting the rate of synthesis in those SMCs that survive(Kaartinen et al. 1996). Importantly, the fibrous caps often contain cholesterolcrystals, which have the potential to activate the NLRP3 inflammasome inmacrophages and so trigger release of IL-1β from the macrophages (Rajamakiet al. 2010; Duewell et al. 2010). Since it is unlikely that HDLs are capable ofremoving crystalline cholesterol from the intima, the crystals, once formed, maypermanently stimulate cap macrophages to secrete IL-1β and so contribute to theongoing cap thinning.The various components of the extracellular matrix are degraded by members ofthe superfamily of matrix metalloproteinases (MMPs) primarily produced by themacrophages of the caps (Newby 2005). For the MMPs to become active, they needto be proteolytically activated. Among the proteolytic enzymes capable ofactivating plaque MMPs are plasmin, chymase, and tryptase, the two latter onesbeing neutral proteases secreted by activated mast cells in atherosclerotic lesions(Johnson et al. 1998). Once activated, interstitial collagenase, MMP-1, can degradethe otherwise protease-resistant fibrillar collagen fibers into fragments, and anothermember of the family, MMP-9, can break collagen fragments further. Importantly,stromelysin, MMP-3, has a broad spectrum and can degrade other components ofthe extracellular matrix as well, including proteoglycans and elastin (Libby 2013a).Cathepsins are another group of enzymes capable of effectively degrading thevarious components of the fibrous cap (Fonovic and Turk 2014). Thus, the presenceof inflammatory cells in the caps of advanced plaques tends to render the plaqueunstable and prone to rupture by both decreasing the production and increasing thedegradation of the extracellular matrix of the fibrous cap.5.1 Effects of HDLs on Smooth Muscle CellsVSCMs can indirectly influence the development of atherosclerosis by the secretionof growth factors and vasoactive agents, and the protective effects of HDLs onatherosclerotic lesion formation might, at least partly, be related to their impact onthe secretory function of VSMCs. Exposure of aortic SMCs to mildly oxidized LDLsenhanced the production and release of platelet-derived growth factor and basicfibroblast growth factor, while addition of HDLs abolished the stimulatory effect ofoxidized LDLs on growth factor secretion by these VSMCs (Cucina et al. 1998,2006). Conversely, treatment of rabbit SMCs in culture with HDLs potentiated thesynthesis of the vasodilator prostacyclin via a mechanism involving MAP kinase390 W. Annema et al.kinase signaling and increased cyclooxygenase-2 expression (Vinals et al. 1997,1999). VSMCs are also considered to protect the atherosclerotic plaque againstdestabilization and rupture. In this respect, HDLs regulate both the proliferation andmigration of VSMCs. Nofer and colleagues showed that HDLs accelerate prolifer-ation of VSMCs by driving the cell cycle forward in the G1 phase via cyclinD1-induced phosphorylation of pRb (Nofer et al. 2001a). Elevated cyclin D1expression in VSMCs in response to treatment with HDLs in turn relies on activa-tion of the Raf/MEK/ERK signal transduction pathway (Nofer et al. 2001a). On theother hand, the platelet-derived growth factor-mediated migration of VCMCs islimited by HDLs through the interaction of HDL-bound S1P with the S1P2 receptor(Tamama et al. 2005). Finally, HDLs were found to suppress inflammatoryresponses in VSMCs. Oxidized LDLs, via formation of ROS, induced NF-κβactivation in cultured VSMCs, while their pretreatment with HDLs inhibited theintracellular rise in ROS and the subsequent upregulation of NF-κβ elicited by theoxidized LDLs (Robbesyn et al. 2003).5.2 Antithrombotic Effects of HDLs5.2.1 HDLs and PlateletsThe majority of acute coronary syndromes are caused by the rupture of an athero-sclerotic plaque in a coronary artery and subsequent formation of an occlusivecoronary thrombus. There appears to be a relationship between plasma HDLs andthe thrombogenic potential of blood, as evidenced by the fact that the total throm-bus area in an ex vivo flow chamber model for coronary thrombosis was lower inhuman subjects with higher HDL cholesterol levels (Naqvi et al. 1999). HDLs haveseveral favorable effects on platelets that might explain its interference withthrombus formation in the coronary vasculature. First, it has been reported thatHDLs can block the activation and aggregation of platelets. In a small clinical trail,13 patients with type 2 diabetes mellitus were randomized to a single infusion ofrHDLs (CSL-111) or placebo (Calkin et al. 2009). Study results established that a40 % increase in HDL cholesterol levels in patients with type 2 diabetes mellituswas associated with a 50–75 % reduction in platelet aggregation (Calkinet al. 2009). A similar decrease in the aggregation of platelets was observed whenisolated human platelets were incubated with rHDLs or native HDLs in vitro(Calkin et al. 2009; Nofer et al. 1998; Badrnya et al. 2013). Native HDLs andrHDLs not only modulate platelet aggregation but also prevent platelet degranula-tion and spreading as well as adhesion of platelets to fibrinogen (Calkin et al. 2009;Nofer et al. 1998; Badrnya et al. 2013). In addition, rHDLs limited theincorporation of platelets in growing thrombi under flow conditions in vitro (Calkinet al. 2009). The ability of HDLs to block platelet aggregation was proposed to bedue to a reduction in the availability of the second messengers 1,2-diacylglyceroland inositol 1,4,5-trisphosphate, a decreased mobilization of intracellular calcium,activation of protein kinase C, and elevated Na+/H+ exchange (Nofer et al. 1996;1998). The role of SR-BI for the anti-aggregatory effect of HDLs is controversial.HDL and Atherothrombotic Vascular Disease 391One study reported that inhibition of platelet reactivity by HDLs results fromcholesterol depletion in lipid rafts but that this is independent of ABCG1 andSR-BI (Calkin et al. 2009). In contrast, Brodde et al. found that SR-BI is thereceptor responsible for binding of HDL to platelets and that HDLs are no longerable to attenuate thrombin-induced adhesion of platelets to fibrinogen when usingplatelets isolated from SR-BI knockout mice (Brodde et al. 2011). The inhibitoryactivity of HDLs on platelets is mimicked by soybean phosphocholine (Calkinet al. 2009) and phosphatidylserine-containing liposomes (Brodde et al. 2011),underlining the importance of the phospholipid component in this antithromboticfunction of HDLs. As a final point, HDLs may regulate the platelet coagulantactivity by reducing the synthesis of platelet-activating factor in endothelial cells(Sugatani et al. 1996).Another step of the arterial atherothrombotic process in which HDLs mayinterfere is the production of platelets from bone marrow megakaryocyte progenitorcells. In vitro experiments have revealed that rHDLs promote the removal ofcholesterol from megakaryocyte progenitor cells in an ABCG4-dependent fashion,thereby decreasing cellular proliferation (Murphy et al. 2013). Consistent withthese observations, intravenous administration of rHDLs resulted in a decrease inthe amount of bone marrow megakaryocyte progenitor cells and concomitantlyreduced circulating platelets in Ldlr/ mice transplanted with wild-type bonemarrow, while rHDLs lost their effects on bone marrow megakaryocyte progenitorcells and platelet numbers when injected into Ldlr/ mice that were transplantedwith ABCG4-deficient bone marrow (Murphy et al. 2013). A small placebo-controlled clinical study demonstrated that a single injection of rHDLs significantlyreduced platelet counts in patients with peripheral vascular disease (Murphyet al. 2013). The importance of deregulation of cholesterol homeostasis in mega-karyocyte progenitor cells for atherothrombosis was supported by evidence thatselective disruption of ABCG4 in bone marrow cells of LDL receptor knockoutmice accelerated the progression of atherosclerotic lesion formation and wasassociated with marked thrombocytosis and an increased tendency to developarterial thrombosis (Murphy et al. 2013).5.2.2 HDLs and CoagulationVarious publications have described an impact of HDLs on the extrinsic coagula-tion pathway that might result in a decreased tendency for thrombosis. Inhibition ofthe catalytic activity of the tissue factor–factor VIIA complex by HDLs has beenshown to impede the activation of the procoagulant factor X (Carson 1981). Besidesdirect inactivation of tissue factor and factor Va, HDLs are able to modulate thetissue factor-activated coagulation cascade by downregulating tissue factor expres-sion in thrombin-stimulated endothelial cells through suppression of RhoA andinduction of PI3K (Viswambharan et al. 2004). In addition, incorporation of anionicphospholipids into apoA-I-containing rHDL particles resulted in the loss of theirprocoagulant properties, revealing that, by scavenging anionic phospholipids,apoA-I may control blood coagulation (Oslakovic et al. 2009). HDLs have alsobeen shown to promote the ability of the anticoagulant activated protein C and its392 W. Annema et al.cofactor protein S to cause inactivation of factor Va through Arg306 cleavage, and innormal healthy subjects, plasma levels of apoA-I were positively correlated withthe anticoagulant potency of activated protein C and protein S in a prothrombin-time clotting assay (Griffin et al. 1999). Of note, these findings were laterquestioned by another study revealing that the previously observed capacity ofthe HDL fraction to boost the activated protein C system was not an actual propertyof HDLs, but instead was due to co-isolation of anionic phospholipid membraneswith the HDL fraction during ultracentrifugation (Oslakovic et al. 2010).6 Myocardial InjuryThe main pathophysiological manifestation of coronary heart disease is myocardialdamage due to ischemia–reperfusion. It has been postulated that HDLs can provideprotection against myocardial injury following ischemia–reperfusion. In an in vivomurine ischemia–reperfusion model, it was found that the infarcted area wasdecreased when mice were treated 30 min before ischemia–reperfusion with eitherhuman HDLs or S1P (Theilmeier et al. 2006). In this regard, HDL or S1P injectionmitigated the inflammatory response to ischemia–reperfusion as manifested by alower accumulation of leukocytes in the infarcted area (Theilmeier et al. 2006).Furthermore, cardiomyocytes were better able to resist ischemia–reperfusion-induced apoptosis in the presence of HDLs or S1P (Theilmeier et al. 2006), poten-tially due to a higher tolerance of HDL-treated cardiomyocytes to hypoxia-inducedopening of the mitochondrial permeability transition pore (Frias et al. 2013). S1P3was identified as the receptor responsible for the beneficial effect of HDLs and S1Pon reperfusion injury (Theilmeier et al. 2006). The finding that HDLs amelioratemyocardial ischemia–reperfusion damage has subsequently been confirmed andextended by others. Ex vivo exposure of isolated hearts from wild-type mice toHDLs provided dose-dependent cardioprotection from reperfusion injury (Friaset al. 2013). On the other hand, HDL-mediated protection of isolated hearts orcardiomyocytes against hypoxia was lost in mice deficient in tumor necrosis factorand in mice with cardiomyocyte-restricted knockout of signal transducer andactivator of transcription 3 (STAT3) (Frias et al. 2013), suggesting a role for thesurvivor activating factor enhancement pathway which is initiated by tumor necro-sis factor and involves activation of STAT3. In another study, HDL cholesterollevels were raised in LDL receptor-deficient mice using adenovirus-mediated genetransfer of human apoA-I (Gordts et al. 2013). After induction of myocardialinfarction by permanent ligation of the left anterior descending coronary artery,mice that were injected with an adenovirus expressing human apoA-I exhibited ahigher survival rate, reduced infarct expansion, attenuated dilatation of the leftventricle, as well as improved systolic and diastolic cardiac function when com-pared with controls (Gordts et al. 2013).HDL and Atherothrombotic Vascular Disease 393ConclusionsHDL particles exert many effects which may help either to protect arteries fromthe development, progression, and complication of atherosclerosis or evenfacilitate repair and regression of lesions. Nevertheless, HDL has not yet beensuccessfully exploited for preventive or curative therapy of cardiovasculardiseases. One potential reason for this shortfall is the structural and functionalcomplexity of HDL particles which is further increased in several inflammatoryconditions including CAD itself and which is not discerned by the biomarkerHDL cholesterol. Moreover, the relative importance of the many physiologicaland pathological activities of normal and dysfunctional HDL, respectively, forthe pathogenesis of atherosclerosis is unknown. The elucidation of structure–function relationships of HDL-associated molecules is essential to exploit HDLfor the development of anti-atherogenic drugs as well as of diagnosticbiomarkers for the identification, personalized treatment stratification, and mon-itoring of patients at increased cardiovascular risk. In addition to the metabolismof HDL or its anti-atherogenic molecules, also the cellular pathways positivelyregulated by HDL are interesting targets for anti-atherosclerotic therapies.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesActon S, Rigotti A, Landschulz KT et al (1996) Identification of scavenger receptor SR-BI as ahigh density lipoprotein receptor. Science 271(5248):518–520Angeloni E, Paneni F, Landmesser U et al (2013) Lack of protective role of HDL-C in patientswith coronary artery disease undergoing elective coronary artery bypass grafting. Eur Heart J34(46):3557–3562Annema W, Tietge UJ (2012) Regulation of reverse cholesterol transport - a comprehensiveappraisal of available animal studies. Nutr Metab (Lond) 9(1):25Annema W, von Eckardstein A (2013) High-density lipoproteins. Multifunctional but vulnerableprotections from atherosclerosis. Circ J 77(10):2432–2448Argraves KM, Gazzolo PJ, Groh EM et al (2008) High density lipoprotein-associated sphingosine1-phosphate promotes endothelial barrier function. J Biol Chem 283(36):25074–25081Ashby DT, Rye KA, Clay MA et al (1998) Factors influencing the ability of HDL to inhibitexpression of vascular cell adhesion molecule-1 in endothelial cells. Arterioscler Thromb VascBiol 18(9):1450–1455Badrnya S, Assinger A, Volf I (2013) Native high density lipoproteins (HDL) interfere withplatelet activation induced by oxidized low density lipoproteins (OxLDL). Int J Mol Sci 14(5):10107–10121Baigent C, Keech A, Kearney PM et al (2005) Efficacy and safety of cholesterol-loweringtreatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trialsof statins. Lancet 366(9493):1267–1278Baker PW, Rye KA, Gamble JR et al (1999) Ability of reconstituted high density lipoproteins toinhibit cytokine-induced expression of vascular cell adhesion molecule-1 in human umbilicalvein endothelial cells. J Lipid Res 40(2):345–353394 W. Annema et al.Barter P, Gotto AM, LaRosa JC et al (2007a) HDL cholesterol, very low levels of LDL cholesterol,and cardiovascular events. N Engl J Med 357(13):1301–1310Barter PJ, Caulfield M, Eriksson M et al (2007b) Effects of torcetrapib in patients at high risk forcoronary events. N Engl J Med 357(21):2109–2122Bennett MR (1999) Apoptosis of vascular smooth muscle cells in vascular remodelling andatherosclerotic plaque rupture. Cardiovasc Res 41(2):361–368Bisoendial RJ, Hovingh GK, Levels JH et al (2003) Restoration of endothelial function byincreasing high-density lipoprotein in subjects with isolated low high-density lipoprotein.Circulation 107(23):2944–2948Boden WE, Probstfield JL, Anderson T et al (2011) Niacin in patients with low HDL cholesterollevels receiving intensive statin therapy. N Engl J Med 365(24):2255–2267Brilakis ES, McConnell JP, Lennon RJ et al (2005) Association of lipoprotein-associated phospho-lipase A2 levels with coronary artery disease risk factors, angiographic coronary artery disease,and major adverse events at follow-up. Eur Heart J 26(2):137–144Brodde MF, Korporaal SJ, Herminghaus G et al (2011) Native high-density lipoproteins inhibitplatelet activation via scavenger receptor BI: role of negatively charged phospholipids. Athero-sclerosis 215(2):374–382Brown MS, Goldstein JL (1983) Lipoprotein metabolism in the macrophage: implications forcholesterol deposition in atherosclerosis. Annu Rev Biochem 52:223–261Bui QT, Prempeh M, Wilensky RL (2009) Atherosclerotic plaque development. Int J BiochemCell Biol 41(11):2109–2113Bursill CA, Castro ML, Beattie DT et al (2010) High-density lipoproteins suppress chemokinesand chemokine receptors in vitro and in vivo. Arterioscler Thromb Vasc Biol 30(9):1773–1778Calabresi L, Franceschini G (2010) Lecithin:cholesterol acyltransferase, high-density lipoproteins,and atheroprotection in humans. Trends Cardiovasc Med 20(2):50–53Calkin AC, Drew BG, Ono A et al (2009) Reconstituted high-density lipoprotein attenuatesplatelet function in individuals with type 2 diabetes mellitus by promoting cholesterol efflux.Circulation 120(21):2095–2104Camejo G, Hurt-Camejo E, Wiklund O et al (1998) Association of apo B lipoproteins with arterialproteoglycans: pathological significance and molecular basis. Atherosclerosis 139(2):205–222Carson SD (1981) Plasma high density lipoproteins inhibit the activation of coagulation factor Xby factor VIIa and tissue factor. FEBS Lett 132(1):37–40Cavelier C, Rohrer L, von Eckardstein A (2006) ATP-Binding cassette transporter A1 modulatesapolipoprotein A-I transcytosis through aortic endothelial cells. Circ Res 99(10):1060–1066Cavelier C, Ohnsorg PM, Rohrer L et al (2012) The beta-chain of cell surface F(0)F(1) ATPasemodulates apoA-I and HDL transcytosis through aortic endothelial cells. Arterioscler ThrombVasc Biol 32(1):131–139Charles MA, Kane JP (2012) New molecular insights into CETP structure and function: a review.J Lipid Res 53(8):1451–1458Christoffersen C, Obinata H, Kumaraswamy SB et al (2011) Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M. Proc Natl Acad Sci USA 108(23):9613–9618Clay MA, Pyle DH, Rye KA et al (2001) Time sequence of the inhibition of endothelial adhesionmolecule expression by reconstituted high density lipoproteins. Atherosclerosis 157(1):23–29Cockerill GW, Rye KA, Gamble JR et al (1995) High-density lipoproteins inhibit cytokine-induced expression of endothelial cell adhesion molecules. Arterioscler Thromb Vasc Biol15(11):1987–1994Cockerill GW, Huehns TY, Weerasinghe A et al (2001) Elevation of plasma high-densitylipoprotein concentration reduces interleukin-1-induced expression of E-selectin in anin vivo model of acute inflammation. Circulation 103(1):108–112Colin S, Fanchon M, Belloy L et al (2014) HDL does not influence the polarization of humanmonocytes toward an alternative phenotype. Int J Cardiol 172(1):179–184HDL and Atherothrombotic Vascular Disease 395Corsetti JP, Zareba W, Moss AJ et al (2006) Elevated HDL is a risk factor for recurrent coronaryevents in a subgroup of non-diabetic postinfarction patients with hypercholesterolemia andinflammation. Atherosclerosis 187(1):191–197Corti R, Fuster V, Badimon JJ (2003) Pathogenetic concepts of acute coronary syndromes. J AmColl Cardiol 41(4 Suppl S):7S–14SCucina A, Pagliei S, Borrelli V et al (1998) Oxidised LDL (OxLDL) induces production of plateletderived growth factor AA (PDGF AA) from aortic smooth muscle cells. Eur J Vasc EndovascSurg 16(3):197–202Cucina A, Scavo MP, Muzzioli L et al (2006) High density lipoproteins downregulate basicfibroblast growth factor production and release in minimally oxidated-LDL treated smoothmuscle cells. Atherosclerosis 189(2):303–309de La Llera-Moya M, Connelly MA, Drazul D et al (2001) Scavenger receptor class B type Iaffects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL. J LipidRes 42(12):1969–1978De Nardo D, Labzin LI, Kono H et al (2014) High-density lipoprotein mediates anti-inflammatoryreprogramming of macrophages via the transcriptional regulator ATF3. Nat Immunol 15(2):152–160de Souza JA, Vindis C, Negre-Salvayre A et al (2010) Small, dense HDL 3 particles attenuateapoptosis in endothelial cells: pivotal role of apolipoprotein A-I. J Cell Mol Med 14(3):608–620Di Angelantonio E, Sarwar N, Perry P et al (2009) Major lipids, apolipoproteins, and risk ofvascular disease. JAMA 302(18):1993–2000DiDonato JA, Huang Y, Aulak KS et al (2013) Function and distribution of apolipoprotein A1 inthe artery wall are markedly distinct from those in plasma. Circulation 128(15):1644–1655DiDonato JA, Aulak K, Huang Y et al (2014) Site-specific nitration of apolipoprotein A-I attyrosine 166 is both abundant within human atherosclerotic plaque and dysfunctional. J BiolChem 289(15):10276–10292Dikkers A, Tietge UJ (2010) Biliary cholesterol secretion: more than a simple ABC. World JGastroenterol 16(47):5936–5945Duewell P, Kono H, Rayner KJ et al (2010) NLRP3 inflammasomes are required for atherogenesisand activated by cholesterol crystals. Nature 464(7293):1357–1361Durand E, Scoazec A, Lafont A et al (2004) In vivo induction of endothelial apoptosis leads tovessel thrombosis and endothelial denudation: a clue to the understanding of the mechanismsof thrombotic plaque erosion. Circulation 109(21):2503–2506Eliska O, Eliskova M, Miller AJ (2006) The absence of lymphatics in normal and atheroscleroticcoronary arteries in man: a morphologic study. Lymphology 39(2):76–83Esterbauer H, Puhl H, Dieber-Rotheneder M et al (1991) Effect of antioxidants on oxidativemodification of LDL. Ann Med 23(5):573–581Farb A, Burke AP, Tang AL et al (1996) Coronary plaque erosion without rupture into a lipid core.A frequent cause of coronary thrombosis in sudden coronary death. Circulation 93(7):1354–1363Fonovic M, Turk B (2014) Cysteine cathepsins and extracellular matrix degradation. BiochimBiophys Acta 1840(8):2560–2570Freeman SR, Jin X, Anzinger JJ et al (2014) ABCG1-mediated generation of extracellularcholesterol microdomains. J Lipid Res 55(1):115–127Frias MA, Pedretti S, Hacking D et al (2013) HDL protects against ischemia reperfusion injury bypreserving mitochondrial integrity. Atherosclerosis 228(1):110–116Frikke-Schmidt R, Nordestgaard BG, Stene MC et al (2008) Association of loss-of-functionmutations in the ABCA1 gene with high-density lipoprotein cholesterol levels and risk ofischemic heart disease. JAMA 299(21):2524–2532Fujii K, Hao H, Ohyanagi M et al (2013) Intracoronary imaging for detecting vulnerable plaque.Circ J 77(3):588–595396 W. Annema et al.Fuster V, Moreno PR, Fayad ZA et al (2005) Atherothrombosis and high-risk plaque: part I:evolving concepts. J Am Coll Cardiol 46(6):937–954Garcia JG, Liu F, Verin AD et al (2001) Sphingosine 1-phosphate promotes endothelial cell barrierintegrity by Edg-dependent cytoskeletal rearrangement. J Clin Invest 108(5):689–701Garner B, Waldeck AR, Witting PK et al (1998) Oxidation of high density lipoproteins.II. Evidence for direct reduction of lipid hydroperoxides by methionine residues ofapolipoproteins AI and AII. J Biol Chem 273(11):6088–6095Gordts SC, Muthuramu I, Nefyodova E et al (2013) Beneficial effects of selective HDL-raisinggene transfer on survival, cardiac remodelling and cardiac function after myocardial infarctionin mice. Gene Ther 20(11):1053–1061Griffin JH, Kojima K, Banka CL et al (1999) High-density lipoprotein enhancement of anticoagu-lant activities of plasma protein S and activated protein C. J Clin Invest 103(2):219–227Guyton JR, Klemp KF (1996) Development of the lipid-rich core in human atherosclerosis.Arterioscler Thromb Vasc Biol 16(1):4–11Haase CL, Tybjaerg-Hansen A, Grande P et al (2010) Genetically elevated apolipoprotein A-I,high-density lipoprotein cholesterol levels, and risk of ischemic heart disease. J ClinEndocrinol Metab 95(12):E500–E510Hansson GK (2005) Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 352(16):1685–1695Huang Y, DiDonato JA, Levison BS et al (2014) An abundant dysfunctional apolipoprotein A1 inhuman atheroma. Nat Med 20(2):193–203Hulten LM, Levin M (2009) The role of hypoxia in atherosclerosis. Curr Opin Lipidol 20(5):409–414Jacquet S, Malaval C, Martinez LO et al (2005) The nucleotide receptor P2Y13 is a key regulatorof hepatic high-density lipoprotein (HDL) endocytosis. Cell Mol Life Sci 62(21):2508–2515Johnson JL, Jackson CL, Angelini GD et al (1998) Activation of matrix-degrading metallopro-teinases by mast cell proteases in atherosclerotic plaques. Arterioscler Thromb Vasc Biol 18(11):1707–1715Kaartinen M, Penttila A, Kovanen PT (1996) Mast cells in rupture-prone areas of human coronaryatheromas produce and store TNF-alpha. Circulation 94(11):2787–2792Karuna R, Park R, Othman A et al (2011) Plasma levels of sphingosine-1-phosphate and apolipo-protein M in patients with monogenic disorders of HDL metabolism. Atherosclerosis 219(2):855–863Kimura T, Sato K, Kuwabara A et al (2001) Sphingosine 1-phosphate may be a major componentof plasma lipoproteins responsible for the cytoprotective actions in human umbilical veinendothelial cells. J Biol Chem 276(34):31780–31785Kimura T, Tomura H, Mogi C et al (2006) Role of scavenger receptor class B type I andsphingosine 1-phosphate receptors in high density lipoprotein-induced inhibition of adhesionmolecule expression in endothelial cells. J Biol Chem 281(49):37457–37467Koenig W, Khuseyinova N, Lowel H et al (2004) Lipoprotein-associated phospholipase A2 addsto risk prediction of incident coronary events by C-reactive protein in apparently healthymiddle-aged men from the general population: results from the 14-year follow-up of a largecohort from southern Germany. Circulation 110(14):1903–1908Kontush A, Chantepie S, Chapman MJ (2003) Small, dense HDL particles exert potent protectionof atherogenic LDL against oxidative stress. Arterioscler Thromb Vasc Biol 23(10):1881–1888Kovanen PT, Kaartinen M, Paavonen T (1995) Infiltrates of activated mast cells at the site ofcoronary atheromatous erosion or rupture in myocardial infarction. Circulation 92(5):1084–1088Krieger M, Herz J (1994) Structures and functions of multiligand lipoprotein receptors: macro-phage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem 63:601–637Kruth HS (2011) Receptor-independent fluid-phase pinocytosis mechanisms for induction of foamcell formation with native low-density lipoprotein particles. Curr Opin Lipidol 22(5):386–393HDL and Atherothrombotic Vascular Disease 397Lee-Rueckert M, Kovanen PT (2011) Extracellular modifications of HDL in vivo and theemerging concept of proteolytic inactivation of prebeta-HDL. Curr Opin Lipidol 22(5):394–402Lee-Rueckert M, Blanco-Vaca F, Kovanen PT et al (2013) The role of the gut in reversecholesterol transport–focus on the enterocyte. Prog Lipid Res 52(3):317–328Lewis GF, Rader DJ (2005) New insights into the regulation of HDL metabolism and reversecholesterol transport. Circ Res 96(12):1221–1232Libby P (1995) Molecular bases of the acute coronary syndromes. Circulation 91(11):2844–2850Libby P (2002) Inflammation in atherosclerosis. Nature 420(6917):868–874Libby P (2013a) Collagenases and cracks in the plaque. J Clin Invest 123(8):3201–3203Libby P (2013b) Mechanisms of acute coronary syndromes and their implications for therapy.N Engl J Med 368(21):2004–2013Lim HY, Thiam CH, Yeo KP et al (2013) Lymphatic vessels are essential for the removal ofcholesterol from peripheral tissues by SR-BI-mediated transport of HDL. Cell Metab 17(5):671–684Liu Y, Mackness B, Mackness M (2008) Comparison of the ability of paraoxonases 1 and 3 toattenuate the in vitro oxidation of low-density lipoprotein and reduce macrophage oxidativestress. Free Radic Biol Med 45(6):743–748Lucke S, Levkau B (2010) Endothelial functions of sphingosine-1-phosphate. Cell PhysiolBiochem 26(1):87–96Martel C, Li W, Fulp B et al (2013) Lymphatic vasculature mediates macrophage reversecholesterol transport in mice. J Clin Invest 123(4):1571–1579Martinez LO, Jacquet S, Esteve JP et al (2003) Ectopic beta-chain of ATP synthase is anapolipoprotein A-I receptor in hepatic HDL endocytosis. Nature 421(6918):75–79Mayranpaa MI, Heikkila HM, Lindstedt KA et al (2006) Desquamation of human coronary arteryendothelium by human mast cell proteases: implications for plaque erosion. Coron Artery Dis17(7):611–621McGrath KC, Li XH, Puranik R et al (2009) Role of 3beta-hydroxysteroid-delta 24 reductase inmediating antiinflammatory effects of high-density lipoproteins in endothelial cells.Arterioscler Thromb Vasc Biol 29(6):877–882Mertens A, Verhamme P, Bielicki JK et al (2003) Increased low-density lipoprotein oxidation andimpaired high-density lipoprotein antioxidant defense are associated with increased macro-phage homing and atherosclerosis in dyslipidemic obese mice: LCAT gene transfer decreasesatherosclerosis. Circulation 107(12):1640–1646Mineo C, Yuhanna IS, Quon MJ et al (2003) High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J Biol Chem 278(11):9142–9149Murphy AJ, Woollard KJ, Hoang A et al (2008) High-density lipoprotein reduces the humanmonocyte inflammatory response. Arterioscler Thromb Vasc Biol 28(11):2071–2077Murphy AJ, Bijl N, Yvan-Charvet L et al (2013) Cholesterol efflux in megakaryocyte progenitorssuppresses platelet production and thrombocytosis. Nat Med 19(5):586–594Nakashima Y, Wight TN, Sueishi K (2008) Early atherosclerosis in humans: role of diffuse intimalthickening and extracellular matrix proteoglycans. Cardiovasc Res 79(1):14–23Naqvi TZ, Shah PK, Ivey PA et al (1999) Evidence that high-density lipoprotein cholesterol is anindependent predictor of acute platelet-dependent thrombus formation. Am J Cardiol 84(9):1011–1017Navab M, Hama SY, Cooke CJ et al (2000) Normal high density lipoprotein inhibits three steps inthe formation of mildly oxidized low density lipoprotein: step 1. J Lipid Res 41(9):1481–1494Newby AC (2005) Dual role of matrix metalloproteinases (matrixins) in intimal thickening andatherosclerotic plaque rupture. Physiol Rev 85(1):1–31Nicholls SJ, Dusting GJ, Cutri B et al (2005) Reconstituted high-density lipoproteins inhibit theacute pro-oxidant and proinflammatory vascular changes induced by a periarterial collar innormocholesterolemic rabbits. Circulation 111(12):1543–1550398 W. Annema et al.Nievelstein-Post P, Mottino G, Fogelman A et al (1994) An ultrastructural study of lipoproteinaccumulation in cardiac valves of the rabbit. Arterioscler Thromb 14(7):1151–1161Nigro P, Abe J, Berk BC (2011) Flow shear stress and atherosclerosis: a matter of site specificity.Antioxid Redox Signal 15(5):1405–1414Nijstad N, Gautier T, Briand F et al (2011) Biliary sterol secretion is required for functional in vivoreverse cholesterol transport in mice. Gastroenterology 140(3):1043–1051Nofer JR, Tepel M, Kehrel B et al (1996) High density lipoproteins enhance the Na+/H+ antiport inhuman platelets. Thromb Haemost 75(4):635–641Nofer JR, Walter M, Kehrel B et al (1998) HDL3-mediated inhibition of thrombin-induced plateletaggregation and fibrinogen binding occurs via decreased production of phosphoinositide-derived second messengers 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. ArteriosclerThromb Vasc Biol 18(6):861–869Nofer JR, Junker R, Pulawski E et al (2001a) High density lipoproteins induce cell cycle entry invascular smooth muscle cells via mitogen activated protein kinase-dependent pathway.Thromb Haemost 85(4):730–735Nofer JR, Levkau B, Wolinska I et al (2001b) Suppression of endothelial cell apoptosis by highdensity lipoproteins (HDL) and HDL-associated lysosphingolipids. J Biol Chem 276(37):34480–34485Nofer JR, Geigenmuller S, Gopfert C et al (2003) High density lipoprotein-associated lysosphin-golipids reduce E-selectin expression in human endothelial cells. Biochem Biophys ResCommun 310(1):98–103Nofer JR, van der Giet M, Tolle M et al (2004) HDL induces NO-dependent vasorelaxation via thelysophospholipid receptor S1P3. J Clin Invest 113(4):569–581Noto H, Hara M, Karasawa K et al (2003) Human plasma platelet-activating factor acetylhydrolasebinds to all the murine lipoproteins, conferring protection against oxidative stress. ArteriosclerThromb Vasc Biol 23(5):829–835Oorni K, Pentikainen MO, Ala-Korpela M et al (2000) Aggregation, fusion, and vesicle formationof modified low density lipoprotein particles: molecular mechanisms and effects on matrixinteractions. J Lipid Res 41(11):1703–1714Oslakovic C, Krisinger MJ, Andersson A et al (2009) Anionic phospholipids lose theirprocoagulant properties when incorporated into high density lipoproteins. J Biol Chem 284(9):5896–5904Oslakovic C, Norstrom E, Dahlback B (2010) Reevaluation of the role of HDL in the anticoagulantactivated protein C system in humans. J Clin Invest 120(5):1396–1399Park SH, Park JH, Kang JS et al (2003) Involvement of transcription factors in plasma HDLprotection against TNF-alpha-induced vascular cell adhesion molecule-1 expression. Int JBiochem Cell Biol 35(2):168–182Parthasarathy S, Barnett J, Fong LG (1990) High-density lipoprotein inhibits the oxidativemodification of low-density lipoprotein. Biochim Biophys Acta 1044(2):275–283Pasquinelli G, Preda P, Vici M et al (1989) Electron microscopy of lipid deposits in humanatherosclerosis. Scanning Microsc 3(4):1151–1159Pentikainen MO, Oorni K, Ala-Korpela M et al (2000) Modified LDL - trigger of atherosclerosisand inflammation in the arterial intima. J Intern Med 247(3):359–370Petoumenos V, Nickenig G, Werner N (2009) High-density lipoprotein exerts vasculoprotectionvia endothelial progenitor cells. J Cell Mol Med 13(11–12):4623–4635Potteaux S, Gautier EL, Hutchison SB et al (2011) Suppressed monocyte recruitment drivesmacrophage removal from atherosclerotic plaques of Apoe-/- mice during disease regression.J Clin Invest 121(5):2025–2036Puranik R, Bao S, Nobecourt E et al (2008) Low dose apolipoprotein A-I rescues carotid arteriesfrom inflammation in vivo. Atherosclerosis 196(1):240–247Quarck R, De Geest B, Stengel D et al (2001) Adenovirus-mediated gene transfer of humanplatelet-activating factor-acetylhydrolase prevents injury-induced neointima formation andHDL and Atherothrombotic Vascular Disease 399reduces spontaneous atherosclerosis in apolipoprotein E-deficient mice. Circulation 103(20):2495–2500Radojkovic C, Genoux A, Pons V et al (2009) Stimulation of cell surface F1-ATPase activity byapolipoprotein A-I inhibits endothelial cell apoptosis and promotes proliferation. ArteriosclerThromb Vasc Biol 29(7):1125–1130Rajamaki K, Lappalainen J, Oorni K et al (2010) Cholesterol crystals activate the NLRP3inflammasome in human macrophages: a novel link between cholesterol metabolism andinflammation. PLoS ONE 5(7):e11765Rallidis LS, Tellis CC, Lekakis J et al (2012) Lipoprotein-associated phospholipase A(2) bound onhigh-density lipoprotein is associated with lower risk for cardiac death in stable coronary arterydisease patients: a 3-year follow-up. J Am Coll Cardiol 60(20):2053–2060Ramet ME, Ramet M, Lu Q et al (2003) High-density lipoprotein increases the abundance ofeNOS protein in human vascular endothelial cells by increasing its half-life. J Am Coll Cardiol41(12):2288–2297Ramkhelawon B, Yang Y, van Gils JM et al (2013) Hypoxia induces netrin-1 and Unc5b inatherosclerotic plaques: mechanism for macrophage retention and survival. ArteriosclerThromb Vasc Biol 33(6):1180–1188Ridker PM, Genest J, Boekholdt SM et al (2010) HDL cholesterol and residual risk of firstcardiovascular events after treatment with potent statin therapy: an analysis from the JUPITERtrial. Lancet 376(9738):333–339Riwanto M, Rohrer L, Roschitzki B et al (2013) Altered activation of endothelial anti- andproapoptotic pathways by high-density lipoprotein from patients with coronary artery disease:role of high-density lipoprotein-proteome remodeling. Circulation 127(8):891–904Robbesyn F, Garcia V, Auge N et al (2003) HDL counterbalance the proinflammatory effect ofoxidized LDL by inhibiting intracellular reactive oxygen species rise, proteasome activation,and subsequent NF-kappaB activation in smooth muscle cells. FASEB J 17(6):743–745Robert J, Lehner M, Frank S et al (2013) Interleukin 6 stimulates endothelial binding and transportof high-density lipoprotein through induction of endothelial lipase. Arterioscler Thromb VascBiol 33(12):2699–2706Rohrer L, Cavelier C, Fuchs S et al (2006) Binding, internalization and transport of apolipoproteinA-I by vascular endothelial cells. Biochim Biophys Acta 1761(2):186–194Rohrer L, Ohnsorg PM, Lehner M et al (2009) High-density lipoprotein transport through aorticendothelial cells involves scavenger receptor BI and ATP-binding cassette transporter G1. CircRes 104(10):1142–1150Rosenson RS, Brewer HB Jr, Davidson WS et al (2012) Cholesterol efflux and atheroprotection:advancing the concept of reverse cholesterol transport. Circulation 125(15):1905–1919Sabatine MS, Morrow DA, O’Donoghue M et al (2007) Prognostic utility of lipoprotein-associatedphospholipase A2 for cardiovascular outcomes in patients with stable coronary artery disease.Arterioscler Thromb Vasc Biol 27(11):2463–2469Sakakura K, Nakano M, Otsuka F et al (2013) Pathophysiology of atherosclerosis plaque progres-sion. Heart Lung Circ 22(6):399–411Sanson M, Distel E, Fisher EA (2013) HDL induces the expression of the M2 macrophage markersarginase 1 and Fizz-1 in a STAT6-dependent process. PLoS ONE 8(8):e74676Schmitt MM, Fraemohs L, Hackeng TM et al (2014a) Atherogenic mononuclear cell recruitment isfacilitated by oxidized lipoprotein-induced endothelial junctional adhesion molecule-Aredistribution. Atherosclerosis 234(2):254–264Schmitt MM, Megens RT, Zernecke A et al (2014b) Endothelial junctional adhesion molecule-aguides monocytes into flow-dependent predilection sites of atherosclerosis. Circulation 129(1):66–76Schwartz GG, Olsson AG, Abt M et al (2012) Effects of dalcetrapib in patients with a recent acutecoronary syndrome. N Engl J Med 367(22):2089–2099400 W. Annema et al.Schwenke DC, Carew TE (1989a) Initiation of atherosclerotic lesions in cholesterol-fed rabbits.I. Focal increases in arterial LDL concentration precede development of fatty streak lesions.Arteriosclerosis 9(6):895–907Schwenke DC, Carew TE (1989b) Initiation of atherosclerotic lesions in cholesterol-fed rabbits.II. Selective retention of LDL vs. selective increases in LDL permeability in susceptible sitesof arteries. Arteriosclerosis 9(6):908–918Seetharam D, Mineo C, Gormley AK et al (2006) High-density lipoprotein promotes endothelialcell migration and reendothelialization via scavenger receptor-B type I. Circ Res 98(1):63–72Shaw JA, Bobik A, Murphy A et al (2008) Infusion of reconstituted high-density lipoprotein leadsto acute changes in human atherosclerotic plaque. Circ Res 103(10):1084–1091Shih DM, Gu L, Xia YR et al (1998) Mice lacking serum paraoxonase are susceptible toorganophosphate toxicity and atherosclerosis. Nature 394(6690):284–287Silbernagel G, Schottker B, Appelbaum S et al (2013) High-density lipoprotein cholesterol,coronary artery disease, and cardiovascular mortality. Eur Heart J 34(46):3563–3571Smith EB (1990) Transport, interactions and retention of plasma proteins in the intima: the barrierfunction of the internal elastic lamina. Eur Heart J 11(Suppl E):72–81Spieker LE, Sudano I, Hurlimann D et al (2002) High-density lipoprotein restores endothelialfunction in hypercholesterolemic men. Circulation 105(12):1399–1402Spirig R, Schaub A, Kropf A et al (2013) Reconstituted high-density lipoprotein modulatesactivation of human leukocytes. PLoS ONE 8(8):e71235Stary HC, Chandler AB, Glagov S et al (1994) A definition of initial, fatty streak, and intermediatelesions of atherosclerosis. A report from the committee on vascular lesions of the council onarteriosclerosis, American heart association. Arterioscler Thromb 14(5):840–856Stary HC, Chandler AB, Dinsmore RE et al (1995) A definition of advanced types of athero-sclerotic lesions and a histological classification of atherosclerosis. A report from the commit-tee on vascular lesions of the council on arteriosclerosis, American heart association.Circulation 92(5):1355–1374Suc I, Escargueil-Blanc I, Troly M et al (1997) HDL and ApoA prevent cell death of endothelialcells induced by oxidized LDL. Arterioscler Thromb Vasc Biol 17(10):2158–2166Sugano M, Tsuchida K, Makino N (2000) High-density lipoproteins protect endothelial cells fromtumor necrosis factor-alpha-induced apoptosis. Biochem Biophys Res Commun 272(3):872–876Sugatani J, Miwa M, Komiyama Y et al (1996) High-density lipoprotein inhibits the synthesis ofplatelet-activating factor in human vascular endothelial cells. J Lipid Mediat Cell Signal 13(1):73–88Sumi M, Sata M, Miura S et al (2007) Reconstituted high-density lipoprotein stimulates differen-tiation of endothelial progenitor cells and enhances ischemia-induced angiogenesis.Arterioscler Thromb Vasc Biol 27(4):813–818Tabas I (1999) Nonoxidative modifications of lipoproteins in atherogenesis. Annu Rev Nutr 19:123–139Tabas I, Williams KJ, Boren J (2007) Subendothelial lipoprotein retention as the initiating processin atherosclerosis: update and therapeutic implications. Circulation 116(16):1832–1844Tabet F, Vickers KC, Cuesta Torres LF et al (2014) HDL-transferred microRNA-223 regulatesICAM-1 expression in endothelial cells. Nat Commun 5:3292Tamama K, Tomura H, Sato K et al (2005) High-density lipoprotein inhibits migration of vascularsmooth muscle cells through its sphingosine 1-phosphate component. Atherosclerosis 178(1):19–23Tamminen M, Mottino G, Qiao JH et al (1999) Ultrastructure of early lipid accumulation in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 19(4):847–853Tatematsu S, Francis SA, Natarajan P et al (2013) Endothelial lipase is a critical determinant ofhigh-density lipoprotein-stimulated sphingosine 1-phosphate-dependent signaling in vascularendothelium. Arterioscler Thromb Vasc Biol 33(8):1788–1794HDL and Atherothrombotic Vascular Disease 401Tellis CC, Tselepis AD (2009) The role of lipoprotein-associated phospholipase A2 in atheroscle-rosis may depend on its lipoprotein carrier in plasma. Biochim Biophys Acta 1791(5):327–338Terasaka N, Westerterp M, Koetsveld J et al (2010) ATP-binding cassette transporter G1 and high-density lipoprotein promote endothelial NO synthesis through a decrease in the interaction ofcaveolin-1 and endothelial NO synthase. Arterioscler Thromb Vasc Biol 30(11):2219–2225Theilmeier G, Schmidt C, Herrmann J et al (2006) High-density lipoproteins and their constituent,sphingosine-1-phosphate, directly protect the heart against ischemia/reperfusion injury in vivovia the S1P3 lysophospholipid receptor. Circulation 114(13):1403–1409Thorp E, Subramanian M, Tabas I (2011) The role of macrophages and dendritic cells in theclearance of apoptotic cells in advanced atherosclerosis. Eur J Immunol 41(9):2515–2518Tolani S, Pagler TA, Murphy AJ et al (2013) Hypercholesterolemia and reduced HDL-C promotehematopoietic stem cell proliferation and monocytosis: studies in mice and FH children.Atherosclerosis 229(1):79–85Tolle M, Pawlak A, Schuchardt M et al (2008) HDL-associated lysosphingolipids inhibit NAD(P)H oxidase-dependent monocyte chemoattractant protein-1 production. Arterioscler ThrombVasc Biol 28(8):1542–1548Tward A, Xia YR, Wang XP et al (2002) Decreased atherosclerotic lesion formation in humanserum paraoxonase transgenic mice. Circulation 106(4):484–490van der Wal AC, Becker AE, van der Loos CM et al (1994) Site of intimal rupture or erosion ofthrombosed coronary atherosclerotic plaques is characterized by an inflammatory processirrespective of the dominant plaque morphology. Circulation 89(1):36–44van Oostrom O, Nieuwdorp M, Westerweel PE et al (2007) Reconstituted HDL increasescirculating endothelial progenitor cells in patients with type 2 diabetes. Arterioscler ThrombVasc Biol 27(8):1864–1865Vinals M, Martinez-Gonzalez J, Badimon JJ et al (1997) HDL-induced prostacyclin release insmooth muscle cells is dependent on cyclooxygenase-2 (Cox-2). Arterioscler Thromb VascBiol 17(12):3481–3488Vinals M, Martinez-Gonzalez J, Badimon L (1999) Regulatory effects of HDL on smooth musclecell prostacyclin release. Arterioscler Thromb Vasc Biol 19(10):2405–2411Viswambharan H, Ming XF, Zhu S et al (2004) Reconstituted high-density lipoprotein inhibitsthrombin-induced endothelial tissue factor expression through inhibition of RhoA and stimu-lation of phosphatidylinositol 3-kinase but not Akt/endothelial nitric oxide synthase. Circ Res94(7):918–925Vohl MC, Neville TA, Kumarathasan R et al (1999) A novel lecithin-cholesterol acyltransferaseantioxidant activity prevents the formation of oxidized lipids during lipoprotein oxidation.Biochemistry 38(19):5976–5981Voight BF, Peloso GM, Orho-Melander M et al (2012) Plasma HDL cholesterol and risk ofmyocardial infarction: a mendelian randomisation study. Lancet 380(9841):572–580von Eckardstein A, Nofer JR, Assmann G (2001) High density lipoproteins and arteriosclerosis.Role of cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol 21(1):13–27Wang N, Tall AR (2003) Regulation and mechanisms of ATP-binding cassette transporterA1-mediated cellular cholesterol efflux. Arterioscler Thromb Vasc Biol 23(7):1178–1184Wang Y, Lindstedt KA, Kovanen PT (1996) Phagocytosis of mast cell granule remnant-boundLDL by smooth muscle cells of synthetic phenotype: a scavenger receptor-mediated processthat effectively stimulates cytoplasmic cholesteryl ester synthesis. J Lipid Res 37(10):2155–2166Wang N, Silver DL, Costet P et al (2000) Specific binding of ApoA-I, enhanced cholesterol efflux,and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem 275(42):33053–33058Wang N, Lan D, Chen W et al (2004) ATP-binding cassette transporters G1 and G4 mediatecellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci USA 101(26):9774–9779402 W. Annema et al.Watson AD, Berliner JA, Hama SY et al (1995a) Protective effect of high density lipoproteinassociated paraoxonase. Inhibition of the biological activity of minimally oxidized low densitylipoprotein. J Clin Invest 96(6):2882–2891Watson AD, Navab M, Hama SY et al (1995b) Effect of platelet activating factor-acetylhydrolaseon the formation and action of minimally oxidized low density lipoprotein. J Clin Invest 95(2):774–782Wentzel JJ, Chatzizisis YS, Gijsen FJ et al (2012) Endothelial shear stress in the evolution ofcoronary atherosclerotic plaque and vascular remodelling: current understanding andremaining questions. Cardiovasc Res 96(2):234–243Westerterp M, Gourion-Arsiquaud S, Murphy AJ et al (2012) Regulation of hematopoietic stemand progenitor cell mobilization by cholesterol efflux pathways. Cell Stem Cell 11(2):195–206Wilhelm AJ, Zabalawi M, Owen JS et al (2010) Apolipoprotein A-I modulates regulatory T cellsin autoimmune LDLr-/-, ApoA-I-/- mice. J Biol Chem 285(46):36158–36169Wilkerson BA, Grass GD, Wing SB et al (2012) Sphingosine 1-phosphate (S1P) carrier-dependentregulation of endothelial barrier: high density lipoprotein (HDL)-S1P prolongs endothelialbarrier enhancement as compared with albumin-S1P via effects on levels, trafficking, andsignaling of S1P1. J Biol Chem 287(53):44645–44653Williams KJ, Tabas I (1995) The response-to-retention hypothesis of early atherogenesis.Arterioscler Thromb Vasc Biol 15(5):551–561Witztum JL (2005) You are right too! J Clin Invest 115(8):2072–2075Woollard KJ (2013) Immunological aspects of atherosclerosis. Clin Sci (Lond) 125(5):221–235Wu BJ, Chen K, Shrestha S et al (2013) High-density lipoproteins inhibit vascular endothelialinflammation by increasing 3beta-hydroxysteroid-Delta24 reductase expression and inducingheme oxygenase-1. Circ Res 112(2):278–288Xia P, Vadas MA, Rye KA et al (1999) High density lipoproteins (HDL) interrupt the sphingosinekinase signaling pathway. A possible mechanism for protection against atherosclerosis byHDL. J Biol Chem 274(46):33143–33147Xiong SL, Liu X, Yi GH (2014) High-density lipoprotein induces cyclooxygenase-2 expressionand prostaglandin I-2 release in endothelial cells through sphingosine kinase-2. Mol CellBiochem 389(1–2):197–207Yancey PG, Bortnick AE, Kellner-Weibel G et al (2003) Importance of different pathways ofcellular cholesterol efflux. Arterioscler Thromb Vasc Biol 23(5):712–719Yuhanna IS, Zhu Y, Cox BE et al (2001) High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase. Nat Med 7(7):853–857Yvan-Charvet L, Pagler T, Gautier EL et al (2010) ATP-binding cassette transporters and HDLsuppress hematopoietic stem cell proliferation. Science 328(5986):1689–1693Zawada AM, Rogacev KS, Schirmer SH et al (2012) Monocyte heterogeneity in human cardio-vascular disease. Immunobiology 217(12):1273–1284Zernecke A, Weber C (2010) Chemokines in the vascular inflammatory response of atherosclero-sis. Cardiovasc Res 86(2):192–201Zhang Q, Yin H, Liu P et al (2010) Essential role of HDL on endothelial progenitor cellproliferation with PI3K/Akt/cyclin D1 as the signal pathway. Exp Biol Med (Maywood) 235(9):1082–1092Zhu W, Saddar S, Seetharam D et al (2008) The scavenger receptor class B type I adaptor proteinPDZK1 maintains endothelial monolayer integrity. Circ Res 102(4):480–487HDL and Atherothrombotic Vascular Disease 403HDLs, Diabetes, and Metabolic SyndromePeter Vollenweider, Arnold von Eckardstein, and ChristianWidmannContents1 Type 2 Diabetes Mellitus and the Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4062 Low HDL Cholesterol Levels in Diabetes and Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . 4082.1 Insulin Resistance, Hypertriglyceridemia, and DecreasedHDL Cholesterol Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4082.2 FFAs and Decreased HDL Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4102.3 HDL Metabolism, Subclasses, and Catabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4103 Does HDL Play a Causal Role in the Pathogenesis of Diabetes? . . . . . . . . . . . . . . . . . . . . . . . . . 4113.1 Effects of HDL on Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4123.2 Effects of HDL on Insulin Sensitivity and Glucose Utilizationby Skeletal Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4133.3 The Beneficial Effects of HDL in Pancreatic Beta Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4143.3.1 Beneficial Effects of HDL on Insulin Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4143.3.2 Effects of HDL on Pancreatic Beta Cell Survival and ER Stress . . . . . . . . . . 415Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417AbstractThe prevalence of type 2 diabetes mellitus and of the metabolic syndrome isrising worldwide and reaching epidemic proportions. These pathologies areassociated with significant morbidity and mortality, in particular with an excessof cardiovascular deaths. Type 2 diabetes mellitus and the cluster of pathologiesP. VollenweiderDepartment of Internal Medicine, University Hospital Center (CHUV) and University ofLausanne, Lausanne, SwitzerlandA. von EckardsteinInstitute of Clinical Chemistry, University Hospital Zurich, Zurich, SwitzerlandC. Widmann (*)Department of Physiology, University of Lausanne, Bugnon 7, 1005 Lausanne, Switzerlande-mail: christian.widmann@unil.ch# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_12405including insulin resistance, central obesity, high blood pressure, and hypertri-glyceridemia that constitute the metabolic syndrome are associated with lowlevels of HDL cholesterol and the presence of dysfunctional HDLs. We herereview the epidemiological evidence and the potential underlying mechanismsof this association. We first discuss the well-established association of type2 diabetes mellitus and insulin resistance with alterations of lipid metabolismand how these alterations may lead to low levels of HDL cholesterol and theoccurrence of dysfunctional HDLs. We then present and discuss the evidenceshowing that HDL modulates insulin sensitivity, insulin-independent glucoseuptake, insulin secretion, and beta cell survival. A dysfunction in these actionscould play a direct role in the pathogenesis of type 2 diabetes mellitus.KeywordsHDLs • Diabetes • Metabolic syndrome1 Type 2 Diabetes Mellitus and the Metabolic SyndromeDiabetes mellitus (DM) is characterized by increased fasting and/or postprandialglucose levels. The disease had been known for many centuries (Laios et al. 2012).Its etiology1 however started to be unraveled in the late nineteenth century/earlytwentieth century. During this period, it was eventually discovered that a compoundin pancreatic extracts (later identified to be insulin) could cure young diabeticpatients (Banting et al. 1922). From the early work of H. P. Himsworth in 1936, aconcept emerged that diabetes could be divided into what he termed “insulinsensitive” and “insulin insensitive” [the pioneering work of Himsworth is nicelyportrayed in a historical perspective written by Gerald Reaven (Reaven 2005)]. Butit is only once a reliable radioimmunoassay became available to measurecirculating insulin levels (Bray 1996) that it was realized that some patientssuffered from a disease associated with low to undetectable insulin levels andprone to ketoacidosis (type 1 DM [T1DM]), while others, the vast majority, hadhigh circulating insulin levels despite increased glucose levels (type 2 DM[T2DM]) (Inzucchi 2012). Patients with T2DM are often overweight and obese,and their diabetes develops later in life. These patients are characterized as havinginsulin resistance (IR), i.e., a decreased response of insulin-sensitive tissues to theeffects of the hormone, in particular its ability to induce glucose uptake. Insulinresistance is usually associated with compensatory hyperinsulinemia in an attemptto maintain normoglycemia. Insulin resistance is a hallmark of T2DM as well as ofoverweight and obesity. Insulin resistance precedes the development of T2DM, andit is once that the beta cell can no longer compensate to the insulin resistance thatthe full-blown disease with hyperglycemia develops (Tabak et al. 2009). Gerald1 The study of causes or origins (of a disease).406 P. Vollenweider et al.Reaven, in his seminal Banting Lecture in 1988 (Reaven 1988), summarized hiswork that put insulin resistance not only at the center of diabetes research butproposed that insulin resistance may directly contribute to the increased risk ofcoronary artery disease associated with T2DM or obesity. In this chapter, hesuggested that IR and hyperinsulinemia were, together with increased VLDL/triglyceride levels, low HDL cholesterol levels, and hypertension, directlyassociated with and responsible for the development of diabetes. He termed “syn-drome2 X” the clustering of these features. Syndrome X was also named “insulinresistance syndrome” but is now generally called “metabolic syndrome.” Thissyndrome corresponds to a cluster of increased abdominal obesity (a good proxyof insulin resistance), increased blood glucose levels, high blood pressure andelevated triglycerides, and low HDL cholesterol levels. Several definitions of theTable 1 Definitions of the metabolic syndromeNCEP ATP III WHO (1998) IDF (2005)Criteria Three of thefollowingPresence of DM, IGF, IGT,or insulin resistance andtwo of the followingCentral obesity(ethnicity specificity)and two of thefollowingAnthropometricdataWaistcircumference>102 cm (men)>88 cm (women)WHR> 0.9 (men)WHR> 0.85 (women)and/orBMI> 30 kg/m2Lipids TG 1.7 mmol/land/orHDL-C< 1.03 mmol/l(men)or< 1.29 mmol/l(women)or treatment forthese specific lipidabnormalitiesTG 1.7 mmol/l and/orHDL-C< 0.9 mmol/l(men) or <1.0 mmol/l(women)TG 1.7 mmol/land/orHDL-C< 1.03 mmol/l (men)or< 1.29 mmol/l(women)or treatment for thesespecific lipidabnormalitiesBlood pressure 130/85 mmHgor treatment140/90 mmHg 130/85 mmHgr treatmentBlood glucose FPG 5.6 mmol/lor treatmentFPG 5.6 mmol/l ortreatmentOthers Urinary albuminexcretion 20 μg/min oralbumin/creatinineratio 30 mg/gBMI body mass index, DM diabetes mellitus, FPG fasting plasma glucose, HDL-C high-densitylipoprotein cholesterol, IDF International Diabetes Federation, IFG impaired fasting glucose, IGTimpaired glucose tolerance, NCEP National Cholesterol Education Program, TG triglycerides,WHO World Health Organization, WHR waist-hip ratio2 A group of symptoms that collectively indicate or characterize a disease.HDLs, Diabetes, and Metabolic Syndrome 407metabolic syndrome have been proposed with the National Cholesterol EducationProgram Adult Treatment Panel III (NCEP ATP III) definition being the one mostwidely used. TheWorld Health Organization (WHO) and the International DiabetesFederation (IDF) have suggested other definitions. Table 1 summarizes thesevarious definitions. These current definitions, albeit arbitrary in the cutoffs thatwere chosen for the different variables included, are intended to provide a usefultool for clinicians to identify individuals at increased risk for the development ofT2DM, atherosclerotic cardiovascular disease, and cardiovascular death. T2DMand the metabolic syndrome are characterized not only by insulin resistance butalso by a dyslipidemia that includes high triglyceride and low HDL cholesterollevels. With the presence of small dense LDLs in T2DM, these lipid abnormalitiessubstantially contribute to the increased cardiovascular morbidity and mortalityassociated with these conditions (Grundy 2012). Several epidemiological studieshave clearly shown that these lipid abnormalities are present before the develop-ment of T2DM. Furthermore, longitudinal studies have provided evidence thatthese abnormalities represent an independent risk factor for T2DM (vonEckardstein et al. 2000). Hence, the concept has emerged that lipid abnormalitiesassociated with IR and the metabolic syndrome are not only bystanders of themetabolic dysfunctions but contribute directly to the progression to type DM.2 Low HDL Cholesterol Levels in Diabetes and MetabolicSyndromeT2DM and the metabolic syndrome are characterized by the presence of anatherogenic dyslipidemia typically including low HDL cholesterol, hypertrigly-ceridemia, and increased small dense LDL blood concentrations. Individuals withlow HDL cholesterol are often insulin resistant and at increased risk of developingT2DM (Li et al. 2014; von Eckardstein and Sibler 2011). T2DM, the metabolicsyndrome, and IR are characterized by hyperinsulinemia and increased free fattyacids (FFA). Both insulin resistance and FFAs appear to contribute to the patho-genesis associated with decreased HDL levels (Rohrer et al. 2004).2.1 Insulin Resistance, Hypertriglyceridemia, and DecreasedHDL Cholesterol LevelsInsulin has pleiotropic effects on lipid metabolism, in particular on the metabolismof HDLs and the TG-rich VLDLs. In rats, insulin can acutely decrease the hepaticproduction of ApoB100 (Chirieac et al. 2000), the apolipoprotein required forVLDL assembly, by favoring ApoB100 degradation. In the Hep2G liver cell line,insulin also decreases the levels of microsomal TG transfer protein that is essentialfor VLDL synthesis and secretion (Lin et al. 1995). Insulin could therefore nega-tively affect the hepatic production of VLDL. However, the situation is morecomplex than this because there are two VLDL subtypes that are differentially408 P. Vollenweider et al.regulated by insulin, the larger and more buoyant VLDL1 particles and the smallerand denser VLDL2 particles. VLDL2 particles are intermediates in the synthesis ofVLDL1 in the liver, but they can also be secreted directly by this organ. Addition-ally, VLDL2 can be generated in the circulation from VLDL1 through lipoproteinlipase activity (Taskinen 2003). Insulin does not appear to affect the hepaticproduction of VLDL2 (at least not in humans, see below) but seems to only inhibithepatic VLDL1 production by inhibiting the conversion of VLDL2 into VLDL1(Taskinen 2003). VLDL1 particles are seen by some authors as “liver-derived”chylomicron-like particles (Taskinen 2003). It makes sense therefore that insulininhibits their production following meals in a situation where chylomicrons areproduced by the intestine. In contrast, VLDL1 particle production could be favoredin the fasting state. While insulin inhibits VLDL1 production, it favors the hepaticproduction of VLDL2 particles because insulin can increase the activity of thesterol regulatory element-binding protein (SREBP) 1c transcription factor thatinduces the expression of enzymes required for fatty acid and TG synthesis, suchas fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) (Hortonet al. 2002). However, insulin was not found to modulate VLDL2 blood levels inhumans (Malmstrom et al. 1997a, b). The physiological importance of the regula-tion of fatty acid and TG synthesis by insulin on VLDL production is thereforeunclear. In type 2 diabetic individuals (i.e., in the presence of an insulin-resistantstate), insulin is no longer able to inhibit VLDL1 production (Malmstromet al. 1997a), and this can thus favor overall VLDL hepatic secretion. This presum-ably contributes to the hypertriglyceridemia seen in type 2 diabetic and metabolicsyndrome patients (Brown and Goldstein 2008). In addition, hyperinsulinemiaresulting from insulin resistance can enhance SREBP1c transcription and therebymicroRNA33b (miR33b) that is encoded by an intron of the SREBP1c gene. BothSREBP1c and miR33b can drive VLDL production and favor hypertriglyceridemiaby stimulating fatty acid and triglyceride synthesis (Rayner et al. 2011).A high production of VLDLs can decrease the stability of HDL particles byincreasing the transfer of TG from TG-rich particles to HDLs via the activity of thecholesteryl ester transfer protein (CETP) enzyme (Rashid et al. 2003). This is onepossible mechanism linking high TG blood levels with decreased HDL cholesterollevels. Insulin participates in the clearance of TG from TG-rich lipoproteins bystimulating the secretion of lipoprotein lipase (LPL) in certain adipose tissues (e.g.,subcutaneous depot) (Fried et al. 1993). Consequently, insulin resistance wouldlead to increased circulating TG-rich particles and augmented TG loading of HDLs.Additionally, reduced TG hydrolysis in VLDLs and chylomicrons will decrease therelease of surface remnants which contribute to the maturation of HDL (Eisenberg1984; Rashid et al. 2003). These two effects combined—decreased surface remnantproduction and enhanced CETP activity—favor the reduction in HDL cholesterollevels via the mechanism mentioned above (i.e., decreased HDL stability due TGenrichment of the particle).Insulin has the potential to directly affect HDL production by the liver, either ina positive or in a negative manner. In the Hep2G liver cell line, insulin can stimulatethe expression of apolipoprotein A-I (ApoA-I) (Murao et al. 1998), the mostHDLs, Diabetes, and Metabolic Syndrome 409abundant apolipoprotein in HDLs (Eisenberg 1984; Rye and Barter 2012), raising thepossibility that hepatic insulin resistance contributes to decreased production ofHDLs. The relevance of this effect has however not been validated in vivo. Incontrast, insulin may also negatively impact on hepatic HDL production by enhanc-ing miR33 transcription. Indeed, miR33 suppresses the production of the adenosinetriphosphate-binding cassette transporter A1 (ABCA1) at the posttranscriptional leveland thereby compromises phospholipid and cholesterol efflux needed for HDLformation (Besler et al. 2012; Rader 2006; Rayner et al. 2011; Tang and Oram 2009).2.2 FFAs and Decreased HDL LevelsIn vitro, FFAs decrease the expression of SREBP1c by inhibiting the transcriptionalactivity of liver X receptor (LXR) (Ou et al. 2001; Yoshikawa et al. 2002). Thisrepresents a negative feedback loop ensuring that the production of VLDLs occurswhen there is a need to deliver TGs to peripheral tissues (muscles, adipose tissue,etc.). Reduction in LXR activity, and consequently VLDL production, would beexpected to lead to increased HDL cholesterol blood levels as a result of increasedHDL stability. However, in the liver, unsaturated FFAs inhibit the expression ofABCA1 (Uehara et al. 2002). Moreover, free fatty acids stimulate the proteasomaldegradation of ABCA1 (Wang and Oram 2005, 2007). So even though FFAs canlower the production of hepatic triglycerides, which will favor HDL stability, FFAs,at the same time, directly inhibit the generation of HDL particles.2.3 HDL Metabolism, Subclasses, and CatabolismNascent HDLs are the precursors of the mature HDL3 and HDL2 particles. They aregenerated from lipid-free ApoA-I that acquires phospholipids and cholesterolextracellularly. ABCA1-mediated lipid efflux from cells is important for this initiallipidation. Further acquisition of lipids and subsequent LCAT-driven cholesterolesterification leads to the formation of mature HDL3 particles. These particles canabsorb more phospholipids and free cholesterol by a mechanism involving ABCG1.Altogether, this transforms the HDL3 particles into larger and less dense HDL2particles. Like ABCA1, this transporter is regulated by LXR and hence suppressedby unsaturated fatty acids at the transcriptional level (Uehara et al. 2007) so that theproduction of larger HDL2 is compromised. In addition, the conversion of HDL2into HDL3 is enhanced in diabetic dyslipidemia by increased activities of CETP andhepatic lipase: First, the transfer rate of cholesteryl esters from HDL to VLDL innormal individuals depends on the concentration of CE acceptors, that is, VLDLparticles, but not on CETP activity (Mann et al. 1991). However, in individuals withhypertriglyceridemia, cholesteryl ester transfer from HDL to VLDL can be aug-mented by increasing CETP plasma activity (Mann et al. 1991). Second, HL andendothelial lipase can convert HDL2 particles back to HDL3 particles that begin anew cycle of maturation or are further degraded by the liver or cleared through the410 P. Vollenweider et al.kidneys (Borggreve et al. 2003; Ginsberg 1998; Rader 2006). HL activity is increasedin T2DM (Tan et al. 1999) and contributes to the production of smaller and denserHDL particles (i.e., HDL2 to HDL3) that are then catabolized as indicated above.3 Does HDL Play a Causal Role in the Pathogenesisof Diabetes?Several epidemiological studies have shown the association between low plasmaconcentrations of HDL-C and increased risks not only of CHD but also T2DM(Di et al. 2009; von Eckardstein et al. 2000).We have seen that some of the metabolic perturbations (hyperglycemia,hypertriglyceridemia, etc.) associated with diabetes (and the metabolic syndrome)are mechanistically linked to decreased HDL cholesterol levels in blood. Tradition-ally, a reduced plasmatic level of HDL cholesterol has been interpreted to be theresult of such metabolic perturbations, which are already present in the prediabeticstate, and hence an innocent bystander of T2DM. However, several more recentstudies suggest that HDLs help to maintain euglycemia via both insulin-dependentand insulin-independent pathways (Drew et al. 2012; von Eckardstein and Sibler2011). The infusion of artificially reconstituted HDL (rHDL) was found to improveglycemia in patients with T2DM (Drew et al. 2009). In a post hoc analysis of datafrom diabetic participants in the ILLUMINATE trial, the addition of the CETPinhibitor torcetrapib to atorvastatin was found to increase HDL-C and to improveglycemic control (Barter et al. 2011). Several in vitro and animal experimentsprovided evidence that HDL improves the function and survival of beta cells aswell as glucose-lowering metabolism in the muscle, liver, and adipose tissue (Drewet al. 2012; von Eckardstein and Sibler 2011; von Eckardstein and Widmann 2014).In mice, the absence of ApoA-I leads to hyperinsulinemia and hyperglycemia aswell as altered responses to insulin tolerance tests. Vice versa, ApoA-I transgenicmice exhibit lower fasting glucose levels and improved glucose tolerance testcompared with wild-type mice (Han et al. 2007; Lehti et al. 2013). These datapoint to a modulating role of HDLs in maintaining normal glucose homeostasis.Moreover, genetically increased HDL cholesterol and ApoA-I levels in miceprevented high-fat diet-induced glucose homeostasis impairment (Lehti et al. 2013).Although it remains to be determined whether this is related to differences inskeletal muscle insulin resistance, adipose tissue mass, or differences in beta cellfunction, these data obtained in mice indicate that ApoA-I plays a modulatory rolein the regulation of glucose homeostasis. This notion is also supported by studies inhumans displaying altered HDL metabolism due to polymorphisms in the ApoA-Iand ABCA1 genes that have been associated with increased risk of diabetes andimpaired glucose uptake (Morcillo et al. 2005; Villarreal-Molina et al. 2008).Heterozygotes for rare mutations in the ABCA1 gene that profoundly decreaseHDL-C levels were found to be less glucose tolerant than their unaffected relatives(Vergeer et al. 2010). However, it should also be noted that larger populationHDLs, Diabetes, and Metabolic Syndrome 411studies did not find any association between heterozygosity for ABCA1 mutationsand risk of diabetes (Schou et al. 2012).3.1 Effects of HDL on ObesityAugmented adipose tissue content is the hallmark of obesity. There is increasingevidence that HDL or ApoA-I regulate adipose tissue content. In humans,polymorphisms within the ApoA-I gene have been associated with increased riskof developing obesity and hypertension in a Brazilian cohort (Chen et al. 2009).More evidence for a potential role of HDL cholesterol on adipose tissue contentregulation and obesity stems from genetic mouse models either lacking oroverexpressing ApoA-I or treated with ApoA-I mimetic peptides. For example,ApoA-I-deficient mice have an increased fat content (Han et al. 2007). On the otherhand, mice overexpressing ApoA-I or treated with the D-4F ApoA-I mimeticpeptide3 (Navab et al. 2005; Van Lenten et al. 2009) showed decreased whiteadipose tissue mass gain when fed a high-fat diet for 3 months (Ruan et al. 2011).ApoA-I transgenic and D-4F-treated mice had slightly improved insulin sensitivitywhen fed a high-fat diet compared to wild-type mice. This was associated withincreased energy expenditure as measured in metabolic cages and related toincreased expression of uncoupling protein 1 (UCP-1) in brown adipose tissueand AMPK phosphorylation (Ruan et al. 2011). Consistent with these results isthe observation that administration of D-4F and L-4F in high-fat diet-fed micereduced weight gain and improved insulin sensitivity when compared withage-matched vehicle-treated mice (Peterson et al. 2009). Taken together, theseobservations implicate a potential antiobesity effect of ApoA-I. In another study,ApoA-I gene transfer in the liver of mice increased adiponectin plasma levels andpotently attenuated the rise of triglycerides and free fatty acids induced by lipopoly-saccharide administration (Van Linthout et al. 2010). In vitro, incubation of 3 T3-L1 adipocytes with HDLs induced adiponectin expression in a phosphatidy-linositol-3-kinase-dependent manner (Van Linthout et al. 2010). This set ofexperiments showed that ApoA-I and HDLs affect adipose tissue metabolism andadiponectin expression. ApoA-I and HDL have been shown to have anti-inflammatory properties in adipocytes similarly to what has been observed inmacrophages and endothelial cells. It was found that both HDL and ApoA-I inhibitpalmitate-mediated gene induction of pro-inflammatory chemotactic factor (e.g.,monocyte chemotactic protein-1, serum amyloid A3) in cultured adipocytes(Umemoto et al. 2013). Furthermore, there was a reduction in chemotactic factor3 This peptide, albeit lacking any sequence homology to ApoA-I, forms a class A amphipatic helixsimilar to those found in ApoA-I. D-4F favors HDL formation and has anti-inflammatory and anti-atherogenic properties. The “D” in D-4F indicates that the peptide was synthesized with nonnaturalD-amino acids (if synthesized with the natural L-amino acids, the peptide is called L-4F). D-aminopeptides are very stable in biological fluids as they are not efficiently recognized by proteases(Chorev and Goodman 1995; Fischer 2003; Michod et al. 2009).412 P. Vollenweider et al.expression and macrophage accumulation in ApoA-I transgenic mice fed a high-fatdiet compared to similarly treated control mice (Umemoto et al. 2013). Becausemacrophage accumulation and adipose tissue inflammation are strongly correlatedwith insulin resistance, these observations provide potential implications for theprevention and management of insulin resistance.The molecular mechanisms involved in the antiobesity effects of ApoA-I arepoorly understood. A recent suggestion is that ApoA-I and HDL might regulateautophagy in adipocytes. Decreased autophagy in pre-adipocytes has beenassociated with a preferential differentiation into “brown-like” adipocytes andimproved energy expenditure, with reduced generation of white adipose tissuethat serves mostly as an energy storage tissue (Wang and Peng 2012). Anotherpotential mechanism is the direct capture of cholesterol from adipocytes by HDLsor ApoA-I (von Eckardstein and Sibler 2011; Zhang et al. 2010).3.2 Effects of HDL on Insulin Sensitivity and Glucose Utilizationby Skeletal MuscleThere is increasing evidence that HDLs regulate glucose uptake in insulin-sensitivetissues. In a set of elegant experiments in patients with T2 DM, Drew et al. showedthat reconstituted HDL (rHDL) infusion over 4 h induced larger decreases ofplasma glucose levels than placebo. This effect was associated with increasedinsulin secretion (Drew et al. 2009). In the C2C12 myocyte cell line, ApoA-Iincreased glucose uptake in an insulin-independent manner and also potentiatedinsulin- and adiponectin-induced glucose uptake (Han et al. 2007). ApoA-I effectson glucose uptake were dependent on the endocytosis of the protein and subsequentphosphorylation of AMPK and acetyl-coenzyme A carboxylase (ACC). In agree-ment with these observations, ApoA-I also increased glucose uptake in isolatedmouse soleus muscle. Furthermore, in ApoA-I-deficient mice, AMPK phosphory-lation status was decreased in skeletal muscle as well as in liver (Han et al. 2007).These data suggest that in vivo, ApoA-I levels are able to regulate AMPK phos-phorylation. In ApoA-I-/- mice, expression of liver enzymes regulating gluconeo-genesis (e.g., phosphoenolpyruvate carboxykinase, glucose-6-phosphatase) wasalso increased (Han et al. 2007), suggesting that hepatic glucose output is increasedin animals lacking the main HDL apolipoprotein.HDLs were also found to directly enhance glucose oxidation by increasingglycolysis and mitochondrial respiration rate in C2C12 muscle cells (Lehtiet al. 2013). ATP synthesis was blunted in mitochondria isolated from the gastroc-nemius muscle of ApoA-I knockout mice. Endurance capacity of ApoA-I knockoutmice was reduced upon exercise exhaustion testing. Conversely, micetransgenically overexpressing human ApoA-I exhibited increased lactate levels,reduced fat mass, and protection against age-induced decline of endurance capacity.Circulating levels of fibroblast growth factor 21, a factor potentially involved inmitochondrial respiratory chain activity and inhibition of white adipose lipolysis,were significantly reduced in ApoA-I transgenic mice (Lehti et al. 2013).HDLs, Diabetes, and Metabolic Syndrome 4133.3 The Beneficial Effects of HDL in Pancreatic Beta CellsHDLs also exert potentially antidiabetogenic functions on pancreatic beta cells bypotently inhibiting stress-induced cell death and possibly enhancing glucose-stimulated insulin secretion.3.3.1 Beneficial Effects of HDL on Insulin SecretionIn vivo and in vitro evidence suggests that HDLs support the insulin secretorycapacity of beta cells (Drew et al. 2012). Infusion of reconstituted HDLs in type2 diabetes patients increased their HOMA-B index, an indirect measurement ofpancreatic beta cell function (Drew et al. 2009). Also, the increase of HDLcholesterol levels in healthy volunteers treated for 2 weeks with a CETP inhibitorwas found to increase postprandial insulin and C-peptide plasma levels (Siebelet al. 2013). The plasma of the CETP inhibited volunteers but not the CETPinhibitor itself also showed an increased capacity to enhance glucose-stimulatedinsulin secretion from MIN6 cells.In vitro data on the effects of HDL or ApoA-I on insulin secretion are howevercontroversial. Fryirs and collaborators showed that stimulation of the Min6insulinoma cell line for 1 h with lipid-free recombinant ApoA-I, ApoA-II, ordiscoidal reconstituted HDLs dose-dependently increased both basal and glucose-stimulated insulin secretion (Fryirs et al. 2010). By RNA interference, the authorsshowed that the stimulatory effect of lipid-free ApoA-I and reconstituted HDLs oninsulin secretion depended on ABCA1 and ABCG1, respectively (Fryirset al. 2010). By contrast, other experiments in Min6 cells as well as islets of miceand humans did not find conclusive evidence that HDL enhances insulin productionor basal and glucose-stimulated insulin secretion (Abderrahmani et al. 2007;Roehrich et al. 2003; Rutti et al. 2009). Therefore and considering the supra-physiological concentrations of lipid-free ApoA-I used by Fryirs et al., it appearsuncertain of whether HDLs have a direct effect on the insulin secretory capacity ofbeta cells.However, there is good evidence that ABCA1 and ABCG1 modulate insulinsecretion from pancreatic beta cells. Mice with a targeted knockout of ABCA1 inpancreatic beta cells and crossbred with hypercholesterolemic LDL-receptorknockout mice were found to be less glucose tolerant than LDL-receptor knockoutonly mice (Brunham et al. 2007). The beta cell-specific ABCA1 knockout mice alsoshowed reduced insulin secretion in response to glucose administration. Isletsisolated from these mice showed altered cholesterol homeostasis and impairedinsulin secretion in vitro (Brunham et al. 2007). In vitro, beta cells lackingABCA1 showed impaired depolarization-induced insulin granule release,disturbances in membrane micro-domain organization, and alteration in Golgiand insulin granule morphology. Acute cholesterol depletion rescued the exocytoticdefect in beta cells lacking ABCA1, suggesting that elevated islet cholesterolaccumulation directly impairs granule fusion and insulin secretion (Kruitet al. 2011). Posttranscriptional suppression of ABCA1 in beta cells by adenoviraloverexpression of miR33a and miR145 also led to increased cholesterol levels and414 P. Vollenweider et al.to decreased glucose-stimulated insulin secretion (Kang et al. 2013; Wijesekaraet al. 2012). This compromised insulin secretion was again rescued by cholesteroldepletion. Inhibition of miR33a expression in apolipoprotein E knockout islets andABCA1 overexpression in beta cell-specific ABCA1 knockout islets rescued nor-mal insulin secretion and reduced islet cholesterol (Wijesekara et al. 2012). ABCG1knockout mice also show glucose intolerance due to reduced insulin secretion(Sturek et al. 2010). In contrast to ABCA1 knockout, this defect was rescued bythe addition of cholesterol rather than by depletion of cholesterol (Stureket al. 2010). These studies show that a delicate balance of cholesterolconcentrations between different subcellular compartments must be achieved toallow optimal beta cell functionality. As the consequence of the nonredundant rolesof ABCA1 and ABCG1 in beta cell activity, the combined inactivation of ABCA1and ABCG1 increased intracellular cholesterol accumulation and induced inflam-mation in beta cells and aggravated the diabetic phenotype found in the singleknockout animals (Kruit et al. 2012).Taken together, these findings indicate that cholesterol homeostasis and itsregulation by ABCA1 and ABCG1 are critical for the secretory function of betacells. However, the human genetic data mentioned before do not unequivocallysupport the findings made in the genetic mouse models. On the one hand, decreasedglucose-induced insulin secretion has been reported in ABCA1-deficient patientswith Tangier disease or heterozygous carriers of ABCA1 mutations (Kosekiet al. 2009; Vergeer et al. 2010). On the other hand, mutations in ABCA1 havenot been associated with increased risk of diabetes (Schou et al. 2012).3.3.2 Effects of HDL on Pancreatic Beta Cell Survival and ER StressSeveral studies have shown that HDLs are very efficient in inhibiting apoptosis ofbeta cells induced by a variety of stimuli including inflammatory cytokines, freefatty acids (e.g., palmitate), thapsigargin, tunicamycin, or protein overexpression(Abderrahmani et al. 2007; Petremand et al. 2012; Roehrich et al. 2003; Ruttiet al. 2009). Many of these stimuli induce endoplasmic reticulum (ER) stress. AsER stress has been proposed to be a driving parameter in beta cell dysfunction anddeath in the course of diabetes development (Eizirik et al. 2008; Oyadomariet al. 2002; Volchuk and Ron 2010), the capacity of HDLs to protect beta cellsfrom ER stressors could be one mechanism underlying their potential ability toprevent T2DM.Pancreatic beta cells are professional secretory cells. Half of the proteins pro-duced by these cells are insulin. These cells have therefore developed an extensiveER network to fulfill their physiological function (Like and Chick 1970; Marshet al. 2001). In response to the development of insulin resistance, triggered byobesity, for example, more insulin needs to be secreted by beta cells to compensatefor insulin resistance and to achieve proper glycemic control. Such a sustaineddemand in insulin production can eventually cause ER dysfunction leading tochronic ER stress. This may promote beta cell dysfunction and ultimately betacell death (Eizirik et al. 2008; Eizirik and Cnop 2010; Volchuk and Ron 2010).There is a wealth of evidence demonstrating that HDLs activate antiapoptoticHDLs, Diabetes, and Metabolic Syndrome 415responses in beta cells (Abderrahmani et al. 2007; Cnop et al. 2002; Petremandet al. 2009; Rutti et al. 2009). HDLs are particularly potent in inhibiting beta celldeath induced by ER stressors (Petremand et al. 2009; Rutti et al. 2009). Recently, itwas shown that HDLs maintain, despite the presence of ER stressors, a proper ERmorphology and functionality in terms of the capacity of the ER to fold proteins andto allow protein trafficking out of the ER. Maintenance of the functionality of theER was shown to be required for the beta cell protective activity of HDLs(Petremand et al. 2012). The ER stressors used included palmitate that is thoughtto mimic pro-diabetogenic conditions found in individuals with impaired glucosetolerance that are at risk of developing diabetes (Biden et al. 2004; Maedleret al. 2001; Shimabukuro et al. 1998). While this work identified a cellular mecha-nism mediating the beneficial effect of HDLs on beta cells against pro-diabetogenicfactors, the underlying molecular mechanisms remain largely unresolved.The agonist for the potentially antidiabetic activity of HDL on beta cell apopto-sis has not yet been identified. The available information is mostly of negativenature. In beta cells, in contrast to endothelial cells, the SR-BI receptor is notrequired for HDL-mediated antiapoptotic responses (Petremand et al. 2012; Ruttiet al. 2009). There is also no evidence that Akt, which plays a pivotal role inmediating the antiapoptotic activity of HDL in endothelial cells, participates in theprotective function of HDLs in beta cells (Puyal et al. 2013). It appears thereforethat the molecular mechanisms underlying the antiapoptotic property of HDLs inbeta cells differ from those employed in endothelial cells. These mechanismsremain unknown (von Eckardstein and Widmann 2014).Conceivably, individual components of HDLs may mediate their protectivefunctions in beta cells. Actually, there is evidence that this could be the case. Inprimary islets, IL1β-induced beta cell apoptosis can be inhibited by either thedelipidated HDL protein or the deproteinated lipid moieties of HDL as well as byApoA-I and by S1P (Rutti et al. 2009). This is a good indication that proteins andlipids found in HDL particles carry pro-survival activities. However, pancreaticislets do not only contain beta cells (70–80 %) but also alpha cells (15–20 %), deltacells, PP cells, and immigrating blood-borne cells such as macrophages. Therefore,it is not clear whether ApoA-I and S1P exert their antiapoptotic effects on beta cellsdirectly or indirectly, for example, by interfering with the release of proapoptoticsignals from non-beta cells. To distinguish direct from indirect effects and tounravel the molecular basis for the antiapoptotic effects, it will be essential tosubstitute the complex model of pancreatic islets with purified beta cells or surro-gate beta cell lines.ConclusionHDL possibly enhances glucose-stimulated insulin secretion (Drew et al. 2012;Fryirs et al. 2010; von Eckardstein and Sibler 2011) and reverses theproapoptotic effects of native and oxidized LDLs, IL1β, or thapsigargin(Abderrahmani et al. 2007; Petremand et al. 2012; Roehrich et al. 2003; Ruttiet al. 2009). In addition, HDL and ApoA-I promote glucose uptake and activateAMP-kinase in primary human skeletal muscle cells and differentiated416 P. Vollenweider et al.adipocytes by an insulin-independent way (Drew et al. 2009; Han et al. 2007;Zhang et al. 2011) and enhance oxidative metabolism in skeletal muscle throughphosphorylation of acetyl-CoA carboxylase (Drew et al. 2009). Unfortunately,the identity of the agonists and their cognate receptors as well as the elicitedsignaling pathways and downstream targets of the antidiabetogenic HDLactivities are yet poorly resolved. Understanding these pathways is a prerequisitefor the development of drugs that stimulate or mimic the antidiabetic effects ofHDLs and thereby help to lower the risk, for example, in overweight patients, tomanifest T2DM.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAbderrahmani A, Niederhauser G, Favre D, Abdelli S, Ferdaoussi M, Yang JY, Regazzi R,Widmann C, Waeber G (2007) Human high-density lipoprotein particles prevent activationof the JNK pathway induced by human oxidised low-density lipoprotein particles in pancreaticb cells. Diabetologia 50:1304–1314Banting FG, Best CH, Collip JB, Campbell WR, Fletcher AA (1922) Pancreatic extracts in thetreatment of diabetes mellitus. Can Med Assoc J 12:141–146Barter PJ, Rye KA, Tardif JC, Waters DD, Boekholdt SM, Breazna A, Kastelein JJ (2011) Effectof torcetrapib on glucose, insulin, and hemoglobin A1c in subjects in the Investigation of LipidLevel Management to Understand its Impact in Atherosclerotic Events (ILLUMINATE) trial.Circulation 124:555–562Besler C, Luscher TF, Landmesser U (2012) Molecular mechanisms of vascular effects of High-density lipoprotein: alterations in cardiovascular disease. EMBO Mol Med 4:251–268Biden TJ, Robinson D, Cordery D, Hughes WE, Busch AK (2004) Chronic effects of fatty acids onpancreatic b-cell function: new insights from functional genomics. Diabetes 53(Suppl 1):S159–S165Borggreve SE, De VR, Dullaart RP (2003) Alterations in high-density lipoprotein metabolism andreverse cholesterol transport in insulin resistance and type 2 diabetes mellitus: role of lipolyticenzymes, lecithin:cholesterol acyltransferase and lipid transfer proteins. Eur J Clin Invest33:1051–1069Bray GA (1996) Methods and obesity research: the radioimmunoassay of insulin. Obes Res4:579–582Brown MS, Goldstein JL (2008) Selective versus total insulin resistance: a pathogenic paradox.Cell Metab 7:95–96Brunham LR, Kruit JK, Pape TD, Timmins JM, Reuwer AQ, Vasanji Z, Marsh BJ, Rodrigues B,Johnson JD, Parks JS, Verchere CB, Hayden MR (2007) b-cell ABCA1 influences insulinsecretion, glucose homeostasis and response to thiazolidinedione treatment. Nat Med13:340–347Chen ES, Mazzotti DR, Furuya TK, Cendoroglo MS, Ramos LR, Araujo LQ, Burbano RR, deArruda Cardoso SM (2009) Apolipoprotein A1 gene polymorphisms as risk factors forhypertension and obesity. Clin Exp Med 9:319–325Chirieac DV, Chirieac LR, Corsetti JP, Cianci J, Sparks CE, Sparks JD (2000) Glucose-stimulatedinsulin secretion suppresses hepatic triglyceride-rich lipoprotein and apoB production. Am JPhysiol Endocrinol Metab 279:E1003–E1011HDLs, Diabetes, and Metabolic Syndrome 417Chorev M, Goodman M (1995) Recent developments in retro peptides and proteins – an ongoingtopochemical exploration. Trends Biotechnol 13:438–445Cnop M, Hannaert JC, Grupping AY, Pipeleers DG (2002) Low density lipoprotein can causedeath of islet beta-cells by its cellular uptake and oxidative modification. Endocrinology143:3449–3453Di AE, Sarwar N, Perry P, Kaptoge S, Ray KK, Thompson A, Wood AM, Lewington S, Sattar N,Packard CJ, Collins R, Thompson SG, Danesh J (2009) Major lipids, apolipoproteins, and riskof vascular disease. JAMA 302:1993–2000Drew BG, Duffy SJ, Formosa MF, Natoli AK, Henstridge DC, Penfold SA, Thomas WG,Mukhamedova N, de Court FJM, Yap FY, Kaye DM, van Hall G, Febbraio MA, Kemp BE,Sviridov D, Steinberg GR, Kingwell BA (2009) High-density lipoprotein modulates glucosemetabolism in patients with type 2 diabetes mellitus. Circulation 119:2103–2111Drew BG, Rye KA, Duffy SJ, Barter P, Kingwell BA (2012) The emerging role of HDL in glucosemetabolism. Nat Rev Endocrinol 8:237–245Eisenberg S (1984) High density lipoprotein metabolism. J Lipid Res 25:1017–1058Eizirik DL, Cnop M (2010) ER stress in pancreatic beta cells: the thin red line between adaptationand failure. Sci Signal 3:e7Eizirik DL, Cardozo AK, Cnop M (2008) The role for endoplasmic reticulum stress in diabetesmellitus. Endocr Rev 29:42–61Fischer PM (2003) The design, synthesis and application of stereochemical and directional peptideisomers: a critical review. Curr Protein Pept Sci 4:339–356Fried SK, Russell CD, Grauso NL, Brolin RE (1993) Lipoprotein lipase regulation by insulin andglucocorticoid in subcutaneous and omental adipose tissues of obese women and men. J ClinInvest 92:2191–2198Fryirs MA, Barter PJ, Appavoo M, Tuch BE, Tabet F, Heather AK, Rye KA (2010) Effects ofhigh-density lipoproteins on pancreatic beta-cell insulin secretion. Arterioscler Thromb VascBiol 30:1642–1648Ginsberg HN (1998) Lipoprotein physiology. Endocrinol Metab Clin North Am 27:503–519Grundy SM (2012) Pre-diabetes, metabolic syndrome, and cardiovascular risk. J Am Coll Cardiol59:635–643Han R, Lai R, Ding Q, Wang Z, Luo X, Zhang Y, Cui G, He J, Liu W, Chen Y (2007)Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabo-lism. Diabetologia 50:1960–1968Horton JD, Goldstein JL, Brown MS (2002) SREBPs: activators of the complete program ofcholesterol and fatty acid synthesis in the liver. J Clin Invest 109:1125–1131Inzucchi SE (2012) Clinical practice. Diagnosis of diabetes. N Engl J Med 367:542–550Kang MH, Zhang LH, Wijesekara N, de Haan W, Butland S, Bhattacharjee A, Hayden MR (2013)Regulation of ABCA1 protein expression and function in hepatic and pancreatic islet cells bymiR-145. Arterioscler Thromb Vasc Biol 33:2724–2732Koseki M, Matsuyama A, Nakatani K, Inagaki M, Nakaoka H, Kawase R, Yuasa-Kawase M,Tsubakio-Yamamoto K, Masuda D, Sandoval JC, Ohama T, Nakagawa-Toyama Y,Matsuura F, Nishida M, Ishigami M, Hirano K, Sakane N, Kumon Y, Suehiro T,Nakamura T, Shimomura I, Yamashita S (2009) Impaired insulin secretion in four Tangierdisease patients with ABCA1 mutations. J Atheroscler Thromb 16:292–296Kruit JK, Wijesekara N, Fox JE, Dai XQ, Brunham LR, Searle GJ, Morgan GP, Costin AJ, Tang R,Bhattacharjee A, Johnson JD, Light PE, Marsh BJ, Macdonald PE, Verchere CB, Hayden MR(2011) Islet cholesterol accumulation due to loss of ABCA1 leads to impaired exocytosis ofinsulin granules. Diabetes 60:3186–3196Kruit JK, Wijesekara N, Westwell-Roper C, Vanmierlo T, de Haan W, Bhattacharjee A, Tang R,Wellington CL, LutJohann D, Johnson JD, Brunham LR, Verchere CB, Hayden MR (2012)Loss of both ABCA1 and ABCG1 results in increased disturbances in islet sterol homeostasis,inflammation, and impaired beta-cell function. Diabetes 61:659–664418 P. Vollenweider et al.Laios K, Karamanou M, Saridaki Z, Androutsos G (2012) Aretaeus of Cappadocia and the firstdescription of diabetes. Hormones (Athens) 11:109–113Lehti M, Donelan E, Abplanalp W, Al-Massadi O, Habegger KM, Weber J, Ress C, Mansfeld J,Somvanshi S, Trivedi C, Keuper M, Ograjsek T, Striese C, Cucuruz S, Pfluger PT, Krishna R,Gordon SM, Silva RA, Luquet S, Castel J, Martinez S, D’Alessio D, Davidson WS, HofmannSM (2013) High-density lipoprotein maintains skeletal muscle function by modulating cellularrespiration in mice. Circulation 128:2364–2371Li N, Fu J, Koonen DP, Kuivenhoven JA, Snieder H, Hofker MH (2014) Are hypertriglyceridemiaand low HDL causal factors in the development of insulin resistance? Atherosclerosis233:130–138Like AA, Chick WL (1970) Studies in the diabetic mutant mouse. II. Electron microscopy ofpancreatic islets. Diabetologia 6:216–242Lin MC, Gordon D, Wetterau JR (1995) Microsomal triglyceride transfer protein (MTP) regula-tion in HepG2 cells: insulin negatively regulates MTP gene expression. J Lipid Res36:1073–1081Maedler K, Spinas GA, Dyntar D, Moritz W, Kaiser N, Donath MY (2001) Distinct effects ofsaturated and monounsaturated fatty acids on beta-cell turnover and function. Diabetes50:69–76Malmstrom R, Packard CJ, Caslake M, Bedford D, Stewart P, Yki-Jarvinen H, Shepherd J,Taskinen MR (1997a) Defective regulation of triglyceride metabolism by insulin in the liverin NIDDM. Diabetologia 40:454–462Malmstrom R, Packard CJ, Watson TD, Rannikko S, Caslake M, Bedford D, Stewart P,Yki-Jarvinen H, Shepherd J, Taskinen MR (1997b) Metabolic basis of hypotriglyceridemiceffects of insulin in normal men. Arterioscler Thromb Vasc Biol 17:1454–1464Mann CJ, Yen FT, Grant AM, Bihain BE (1991) Mechanism of plasma cholesteryl ester transfer inhypertriglyceridemia. J Clin Invest 88:2059–2066Marsh BJ, Mastronarde DN, Buttle KF, Howell KE, McIntosh JR (2001) Organellar relationshipsin the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolutionelectron tomography. Proc Natl Acad Sci USA 98:2399–2406Michod D, Annibaldi A, Schaefer S, Dapples C, Rochat B, Widmann C (2009) Effect of RasGAPN2 fragment-derived peptide on tumor growth in mice. J Natl Cancer Inst 101:828–832Morcillo S, Cardona F, Rojo-Martinez G, Esteva I, Ruiz-de-Adana MS, Tinahones F, Gomez-Zumaquero JM, Soriguer F (2005) Association between MspI polymorphism of the APO AIgene and type 2 diabetes mellitus. Diabet Med 22:782–788Murao K, Wada Y, Nakamura T, Taylor AH, Mooradian AD, Wong NC (1998) Effects of glucoseand insulin on rat apolipoprotein A-I gene expression. J Biol Chem 273:18959–18965Navab M, Anantharamaiah GM, Reddy ST, Hama S, Hough G, Grijalva VR, Yu N, Ansell BJ,Datta G, Garber DW, Fogelman AM (2005) Apolipoprotein A-I mimetic peptides. ArteriosclerThromb Vasc Biol 25:1325–1331Ou J, Tu H, Shan B, Luk A, DeBose-Boyd RA, Bashmakov Y, Goldstein JL, Brown MS (2001)Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c(SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR. Proc Natl Acad SciUSA 98:6027–6032Oyadomari S, Araki E, Mori M (2002) Endoplasmic reticulum stress-mediated apoptosis inpancreatic beta-cells. Apoptosis 7:335–345Peterson SJ, Kim DH, Li M, Positano V, Vanella L, Rodella LF, Piccolomini F, Puri N,Gastaldelli A, Kusmic C, L’Abbate A, Abraham NG (2009) The L-4F mimetic peptideprevents insulin resistance through increased levels of HO-1, pAMPK, and pAKT in obesemice. J Lipid Res 50:1293–1304Petremand J, Bulat N, Butty AC, Poussin C, Rutti S, Au K, Ghosh S, Mooser V, Thorens B, YangJ-Y, Widmann C, Waeber G (2009) Involvement of 4E-BP1 in the protection induced by HDLson pancreatic beta cells. Mol Endocrinol 23:1572–1586Petremand J, Puyal J, Chatton JY, Duprez J, Allagnat F, Frias M, James RW, Waeber G, Jonas JC,Widmann C (2012) HDLs protect pancreatic beta-cells against ER stress by restoring proteinfolding and trafficking. Diabetes 61:1100–1111HDLs, Diabetes, and Metabolic Syndrome 419Puyal J, Petremand J, Dubuis G, Rummel C, Widmann C (2013) HDLs protect the MIN6insulinoma cell line against tunicamycin-induced apoptosis without inhibiting ER stress andwithout restoring ER functionality. Mol Cell Endocrinol 381:291–301Rader DJ (2006) Molecular regulation of HDL metabolism and function: implications for noveltherapies. J Clin Invest 116:3090–3100Rashid S, Watanabe T, Sakaue T, Lewis GF (2003) Mechanisms of HDL lowering in insulinresistant, hypertriglyceridemic states: the combined effect of HDL triglyceride enrichment andelevated hepatic lipase activity. Clin Biochem 36:421–429Rayner KJ, Esau CC, Hussain FN, McDaniel AL, Marshall SM, van Gils JM, Ray TD, Sheedy FJ,Goedeke L, Liu X, Khatsenko OG, Kaimal V, Lees CJ, Fernandez-Hernando C, Fisher EA,Temel RE, Moore KJ (2011) Inhibition of miR-33a/b in non-human primates raises plasmaHDL and lowers VLDL triglycerides. Nature 478:404–407Reaven GM (1988) Banting lecture 1988. Role of insulin resistance in human disease. Diabetes37:1595–1607Reaven GM (2005) Why Syndrome X? From Harold Himsworth to the insulin resistance syn-drome. Cell Metab 1:9–14Roehrich ME, Mooser V, Lenain V, Herz J, Nimpf J, Azhar S, Bideau M, Capponi A, Nicod P,Haefliger JA, Waeber G (2003) Insulin-secreting b-cell dysfunction induced by humanlipoproteins. J Biol Chem 278:18368–18375Rohrer L, Hersberger M, von Eckardstein A (2004) High density lipoproteins in the intersection ofdiabetes mellitus, inflammation and cardiovascular disease. Curr Opin Lipidol 15:269–278Ruan X, Li Z, Zhang Y, Yang L, Pan Y, Wang Z, Feng GS, Chen Y (2011) Apolipoprotein A-Ipossesses an anti-obesity effect associated with increase of energy expenditure andup-regulation of UCP1 in brown fat. J Cell Mol Med 15:763–772Rutti S, Ehses JA, Sibler RA, Prazak R, Rohrer L, Georgopoulos S, Meier DT, Niclauss N,Berney T, Donath MY, von Eckardstein A (2009) Low and high-density lipoproteins modulatefunction, apoptosis and proliferation of primary human and murine pancreatic beta cells.Endocrinology 150:4521–4530Rye KA, Barter PJ (2012) Predictive value of different HDL particles for the protection against orrisk of coronary heart disease. Biochim Biophys Acta 1821:473–480Schou J, Tybjaerg-Hansen A, Moller HJ, Nordestgaard BG, Frikke-Schmidt R (2012) ABCtransporter genes and risk of type 2 diabetes: a study of 40,000 individuals from the generalpopulation. Diabetes Care 35:2600–2606Shimabukuro M, Zhou YT, Levi M, Unger RH (1998) Fatty acid-induced b cell apoptosis: a linkbetween obesity and diabetes. Proc Natl Acad Sci USA 95:2498–2502Siebel AL, Natoli AK, Yap FY, Carey AL, Reddy-Luthmoodoo M, Sviridov D, Weber CI,Meneses-Lorente G, Maugeais C, Forbes JM, Kingwell BA (2013) Effects of high-densitylipoprotein elevation with cholesteryl ester transfer protein inhibition on insulin secretion. CircRes 113:167–175Sturek JM, Castle JD, Trace AP, Page LC, Castle AM, Evans-Molina C, Parks JS, Mirmira RG,Hedrick CC (2010) An intracellular role for ABCG1-mediated cholesterol transport in theregulated secretory pathway of mouse pancreatic beta cells. J Clin Invest 120:2575–2589Tabak AG, Jokela M, Akbaraly TN, Brunner EJ, Kivimaki M, Witte DR (2009) Trajectories ofglycaemia, insulin sensitivity, and insulin secretion before diagnosis of type 2 diabetes: ananalysis from the Whitehall II study. Lancet 373:2215–2221Tan KC, Shiu SW, Chu BY (1999) Roles of hepatic lipase and cholesteryl ester transfer protein indetermining low density lipoprotein subfraction distribution in Chinese patients with non-insulin-dependent diabetes mellitus. Atherosclerosis 145:273–278Tang C, Oram JF (2009) The cell cholesterol exporter ABCA1 as a protector from cardiovasculardisease and diabetes. Biochim Biophys Acta 1791:563–572TaskinenMR (2003) Diabetic dyslipidaemia: from basic research to clinical practice. Diabetologia46:733–749420 P. Vollenweider et al.Uehara Y, Engel T, Li Z, Goepfert C, Rust S, Zhou X, Langer C, Schachtrup C, Wiekowski J,Lorkowski S, Assmann G, von Eckardstein A (2002) Polyunsaturated fatty acids andacetoacetate downregulate the expression of the ATP-binding cassette transporter A1. Diabetes51:2922–2928Uehara Y, Miura S, von Eckardstein A, Abe S, Fujii A, Matsuo Y, Rust S, Lorkowski S,Assmann G, Yamada T, Saku K (2007) Unsaturated fatty acids suppress the expression ofthe ATP-binding cassette transporter G1 (ABCG1) and ABCA1 genes via an LXR/RXRresponsive element. Atherosclerosis 191:11–21Umemoto T, Han CY, Mitra P, Averill MM, Tang C, Goodspeed L, Omer M, Subramanian S,Wang S, Den Hartigh LJ, Wei H, Kim EJ, Kim J, O’Brien KD, Chait A (2013) ApolipoproteinAI and high-density lipoprotein have anti-inflammatory effects on adipocytes via cholesteroltransporters: ATP-binding cassette A-1, ATP-binding cassette G-1, and scavenger receptorB-1. Circ Res 112:1345–1354Van Lenten BJ, Wagner AC, Anantharamaiah GM, Navab M, Reddy ST, Buga GM, FogelmanAM (2009) Apolipoprotein A-I mimetic peptides. Curr Atheroscler Rep 11:52–57Van Linthout S, Foryst-Ludwig A, Spillmann F, Peng J, Feng Y, Meloni M, Van Craeyveld E,Kintscher U, Schultheiss HP, De GB, Tschope C (2010) Impact of HDL on adipose tissuemetabolism and adiponectin expression. Atherosclerosis 210:438–444Vergeer M, Brunham LR, Koetsveld J, Kruit JK, Verchere CB, Kastelein JJ, Hayden MR, StroesES (2010) Carriers of loss-of-function mutations in ABCA1 display pancreatic beta-celldysfunction. Diabetes Care 33:869–874Villarreal-Molina MT, Flores-Dorantes MT, Arellano-Campos O, Villalobos-Comparan M,Rodriguez-Cruz M, Miliar-Garcia A, Huertas-Vazquez A, Menjivar M, Romero-Hidalgo S,Wacher NH, Tusie-Luna MT, Cruz M, Aguilar-Salinas CA, Canizales-Quinteros S (2008)Association of the ATP-binding cassette transporter A1 R230C variant with early-onset type2 diabetes in a Mexican population. Diabetes 57:509–513Volchuk A, Ron D (2010) The endoplasmic reticulum stress response in the pancreatic beta-cell.Diabetes Obes Metab 12(Suppl 2):48–57von Eckardstein A, Sibler RA (2011) Possible contributions of lipoproteins and cholesterol to thepathogenesis of diabetes mellitus type 2. Curr Opin Lipidol 22:26–32von Eckardstein A, Widmann C (2014) HDL, beta cells and diabetes. Cardiovasc Res 103(3):384–394von Eckardstein A, Schulte H, Assmann G (2000) Risk for diabetes mellitus in middle-agedCaucasian male participants of the PROCAM study: implications for the definition of impairedfasting glucose by the American Diabetes Association. J Clin Endocrinol Metab 85:3101–3108Wang Y, Oram JF (2005) Unsaturated fatty acids phosphorylate and destabilize ABCA1 through aphospholipase D2 pathway. J Biol Chem 280:35896–35903Wang Y, Oram JF (2007) Unsaturated fatty acids phosphorylate and destabilize ABCA1 through aprotein kinase Cd pathway. J Lipid Res 48:1062–1068Wang S, Peng D (2012) Regulation of adipocyte autophagy – the potential anti-obesity mechanismof high density lipoprotein and ApolipoproteinA-I. Lipids Health Dis 11:131Wijesekara N, Zhang LH, Kang MH, Abraham T, Bhattacharjee A, Warnock GL, Verchere CB,Hayden MR (2012) miR-33a modulates ABCA1 expression, cholesterol accumulation, andinsulin secretion in pancreatic islets. Diabetes 61:653–658Yoshikawa T, Shimano H, Yahagi N, Ide T, Amemiya-Kudo M, Matsuzaka T, Nakakuki M,Tomita S, Okazaki H, Tamura Y, Iizuka Y, Ohashi K, Takahashi A, Sone H, Osuga JJ,Gotoda T, Ishibashi S, Yamada N (2002) Polyunsaturated fatty acids suppress sterol regulatoryelement-binding protein 1c promoter activity by inhibition of liver X receptor (LXR) bindingto LXR response elements. J Biol Chem 277:1705–1711Zhang Y, McGillicuddy FC, Hinkle CC, O’Neill S, Glick JM, Rothblat GH, Reilly MP (2010)Adipocyte modulation of high-density lipoprotein cholesterol. Circulation 121:1347–1355Zhang Q, Zhang Y, Feng H, Guo R, Jin L, Wan R, Wang L, Chen C, Li S (2011) High densitylipoprotein (HDL) promotes glucose uptake in adipocytes and glycogen synthesis in musclecells. PLoS ONE 6:e23556HDLs, Diabetes, and Metabolic Syndrome 421High-Density Lipoprotein: Structuraland Functional Changes Under UremicConditions and the TherapeuticConsequencesMirjam Schuchardt, Markus Tölle, and Markus van der GietContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4252 Chronic Kidney Disease: Epidemiology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . 4263 Dyslipidemia, Lipid-Modulating Therapy, and Cardiovascular Risk in CKD Patients . . 4283.1 Dyslipidemia in CKD Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4283.2 Lipid-Modulating Therapy in CKD Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4293.3 Protective Effects of HDL on the Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4344 Structural and Functional Modifications of HDL in CKD Patients . . . . . . . . . . . . . . . . . . . . . . . 4364.1 Dysregulation of Proteins in HDL Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4364.2 Changes of HDL Apolipoproteins (Modifications and Levels) . . . . . . . . . . . . . . . . . . . . . 4384.3 Loss of Protective Proteins or Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4404.4 Increase of Molecules within HDL with a Fatal Function in the Vascular Wall . . . 4415 Possibility of Functional Restoration of HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4426 Laboratory Tests to Measure HDL Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443Conclusion and Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444AbstractHigh-density lipoprotein (HDL) has attracted interest as a therapeutic target incardiovascular diseases in recent years. Although many functional mechanismsof the vascular protective effects of HDL have been identified, increasing theHDL plasma level has not been successful in all patient cohorts with increasedcardiovascular risk. The composition of the HDL particle is very complex andM. Schuchardt • M. TölleDepartment of Nephrology, Campus Benjamin Franklin, Charité – Universitaetsmedizin Berlin,Charité Centrum 13, Hindenburgdamm 30, 12203 Berlin, GermanyM. van der Giet (*)Department of Nephrology, Charité – Campus Benjamin Franklin, Hindenburgdamm 30, 12203Berlin, Germanye-mail: Markus.vanderGiet@charite.de# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_13423includes diverse lipids and proteins that can be modified in disease conditions. Inpatients with chronic kidney disease (CKD), the accumulation of uremic toxins,high oxidative stress, and chronic micro-inflammatory conditions contribute tochanges in the HDL composition and may also account for protein/lipidmodifications. These conditions are associated with a decreased protectivefunction of HDL. Therefore, the HDL quantity and the functional quality ofthe particle must be considered.This review summarizes the current knowledge of dyslipidemia in CKDpatients, the effects of lipid-modulating therapy, and the structural modificationsof HDL that are associated with dysfunction.KeywordsChronic kidney disease • Dyslipidemia • High-density lipoprotein • Uremia •Uremic toxinAbbreviationsABCA1 ATP-binding cassette subfamily AADMA Asymmetric dimethylarginineAGE Advanced glycated end productAIM-HIGH Atherothrombosis intervention in metabolic syndrome with lowHDL/high triglycerides: impact on global health outcomesALERT Assessment of LEscol in Renal Transplantationapo ApolipoproteinAURORA A study to evaluate the use of rosuvastatin in subjects on regularhemodialysis: an assessment of survival and cardiovascular eventsCETP Cholesterol ester transfer proteinCKD Chronic kidney diseasecLDL Carbamylated low-density lipoprotein4D Deutsche Diabetes Dialyse StudieDEFINE Determining the Efficacy and Tolerability of CETP inhibitionwith AnacetrapibDMA DimethylarginineeGFR Estimated glomerular filtration rateEPIC European Prospective Investigation into Cancer and NutritionESRD end-stage renal diseaseGFR Glomerular filtration rateHD HemodialysisHDL High-density lipoproteinHDL-C High-density lipoprotein cholesterolHMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme AHPS2-THRIVE Treatment of HDL to Reduce the Incidence of Vascular EventsIDL Intermediate-density lipoprotein424 M. Schuchardt et al.ILLUMINATE Investigation of Lipid Level Management to Understand ItsImpact in Atherosclerotic EventsLCAT Lecithin-cholesterol acyltransferaseLDL Low-density lipoproteinLDL-C Low-density lipoprotein cholesterolLp(a) Lipoprotein aLp-PLA Lipoprotein-associated phospholipaseLPL Lipoprotein lipaseMESA Multi-Ethnic Study of AtherosclerosisMI Myocardial infarctionMPO MyeloperoxidaseNO Nitric oxiden3-PUFA n3-polyunsaturated fatty acidsox-apo Oxidized apolipoproteinPAF-AH Platelet-activating factor acetylhydrolasePD Peritoneal dialysisPLA PhospholipasePON ParaoxonasePPAR Peroxisome proliferator-activated receptorPREVEND-IT Prevention of Renal and Vascular End-Stage Disease Interven-tion TrialRBP4 Retinol-binding protein 4RCT Reverse cholesterol transportRECORD Rosiglitazone evaluated for cardiovascular outcomes in oralagent combination therapy for type 2 diabetesREVEAL Randomized evaluation of the effects of anacetrapib throughlipid-modificationROS Reactive oxygen speciesSAA Serum amyloid ASDMA Symmetric dimethylarginineSHARP Study of Heart and Renal ProtectionS1P Sphingosine-1-phosphatesPLA2 Secretory phospholipase type 2SR-BI Scavenger receptor BIVA-HIT Veterans’ Affairs High-Density Lipoprotein Intervention TrialVLDL Very low-density lipoprotein1 IntroductionPlasma lipoproteins are composed of non-covalent aggregates of different lipidsand proteins. They transport water-insoluble substances in the blood by buildingmicelle-like structures. These structures are classified by their density into theHigh-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 425following groups: very low-density lipoprotein (VLDL), intermediate-density lipo-protein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL).In previous years, several clinical studies have found that HDL cholesterol(HDL-C) plasma levels were inversely correlated with cardiovascular risk, whereashigh LDL-C levels were related to increased cardiovascular mortality(Di Angelantonio et al. 2009; Gordon et al. 1977; Mori et al. 1999). Initially, dueto the crucial role of HDL in reverse cholesterol transport (RCT), HDL wasassociated with a decreased cardiovascular risk. Therefore, the theory of the“good HDL” and “bad LDL” emerged. Further studies, primarily in vitro or animalstudies, identified the pleiotropic cardiovascular protective effects of HDL inaddition to its RCT function: HDL has anti-atherosclerotic, anti-inflammatory,antioxidative, antithrombotic, and endothelial-protective properties (Navabet al. 2011). Accordingly, there is growing interest in the study of HDL metabolismand the cellular and the molecular signaling pathways involved in its vascularprotective effects.Therefore, enhancing HDL plasma levels has been a principal approach for thereduction of cardiovascular risks in different patient cohorts. Pharmacologicallyactive substances, such as cholesterol ester transfer protein (CETP) inhibitors,fibrates, and niacin, have been tested in large clinical trials to evaluate the occur-rence of cardiovascular endpoints (Longenecker et al. 2005; van Capelleveenet al. 2014). However, raising HDL-C plasma levels did not protect against cardio-vascular events in all patient cohorts tested. In addition, despite a significantHDL-C increase in patients, large clinical trials were terminated because of alack of positive effects or, in some cases, an increase in the rate of cardiovascularevents.These findings indicate that a better understanding of lipoprotein modificationsin disease conditions is necessary to establish possible indications and targetmechanisms in the therapy of cardiovascular diseases. Patients with chronic kidneydisease (CKD) suffer from a dramatic increase in cardiovascular morbidity andmortality. The disease condition is associated with dyslipidemia and/ormodifications in lipoprotein composition and function (Keane et al. 2013; Vaziri2006).This review summarizes current knowledge regarding the structural and func-tional changes of HDL in the context of renal dysfunction. A brief overview ofCKD epidemiology and pathophysiology is presented, followed by aspects ofdyslipidemia and its therapy in CKD patients. Subsequently, CKD-dependentstructural and functional changes of HDL are summarized.2 Chronic Kidney Disease: Epidemiologyand PathophysiologyKidney disease is defined as an abnormality of kidney structure or function. Thecriteria for abnormalities include: albuminuria, urine sediment abnormalities, elec-trolyte and other abnormalities due to tubular disorders, abnormalities detected by426 M. Schuchardt et al.histology or imaging, and history of kidney transplantation (Levey et al. 2005).Kidney disease can occur abruptly, and it can either resolve or become chronic.CKD is defined as kidney damage that persists for more than 3 months (Leveyet al. 2005). CKD is one of the major medical concerns associated with prematuremorbidity and mortality, especially due to cardiovascular complications. The epi-demic of CKD is globally driven by demographic aging, as well as an increase inother risk factors leading to CKD (e.g., diabetes mellitus, hypertension, andobesity).CKD is divided into different stages, which are summarized in Fig. 1, based onthe glomerular filtration rate (GFR) (Levey et al. 2005). CKD patients have a highoccurrence of cardiovascular disease. In recent years, several studies haveimplicated an inverse correlation of kidney function and cardiovascular mortality(Tonelli et al. 2006). Thereby, the cardiovascular phenotype of the CKD populationis heterogeneous, and the cardiovascular risk depends on the CKD stage. In additionto traditional risk factors, such as hypertension, diabetes mellitus, and smoking,nontraditional risk factors also contribute to cardiovascular diseases. In CKDpatients, the nontraditional risk factors of anemia and enhanced oxidative stressor uremic toxins play a major role in cardiovascular disease progression(Mizobuchi et al. 2009). Under normal conditions, uremic toxins are cleared bythe kidneys, and many of these toxins have been recently identified (Durantonet al. 2012). Uremic toxins are classified as water-soluble molecules with lowmolecular weight (e.g., uric acid), middle molecules (e.g., parathyroid hormone),and protein-bound toxins (e.g., hippuric acid) (Duranton et al. 2012). A number ofdifferent uremic toxins are elusive, and biological functions have not beenidentified for all toxins. Clinical studies have shown that cardiovascular morbidityand mortality in CKD patients are associated with uremic toxin accumulation,which leads to a progression of vascular alterations. Recently, Moradiet al. summarized the influence of uremic toxins on vascular cells and pathologicalpathways in the vascular wall (Moradi et al. 2013). The activation of leukocytes;Fig. 1 Categories of chronic renal failure. CKD categories were divided into subgroups accordingto KDIGO 2013High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 427endothelial damage, for example, by the disruption of glycocalyx and production ofreactive oxygen species (ROS); the proliferating effects on smooth muscle cells;and platelet activation are only some of the described effects (Moradi et al. 2013).In addition, uremic toxins influence lipoprotein modifications. Urea induces theformation of carbamylated LDL (cLDL) (Apostolov et al. 2010). Some uremictoxins are lipid-bound and may occur in a different subfraction of lipoproteins.Additionally, HDL function may be affected by the accumulation of uremic toxins.Nonetheless, further studies are necessary to characterize the impact of differentcompounds on cardiovascular outcome and the precise signaling pathwaysinvolved. The therapeutic goal should be to remove the solutes associated withthe highest cardiovascular risk to minimize fatal cardiovascular outcomes inpatients with CKD. To date, cardiovascular morbidity in patients with CKDremains high despite the utility of dialysis and renal transplantation (Foleyet al. 1998).3 Dyslipidemia, Lipid-Modulating Therapy,and Cardiovascular Risk in CKD PatientsIn the general population, plasma lipid levels correlate with the level of cardiovas-cular risk (Di Angelantonio et al. 2009). Increased LDL-C levels have beenassociated with cardiovascular mortality. LDL-C is a primary risk biomarker forcardiovascular disease in the general population; however, it loses its prognosticassociation in CKD patients (Kilpatrick et al. 2007), especially for patients withend-stage renal disease (ESRD) (Baigent et al. 2011; Kovesdy et al. 2007). Inaddition, traditional risk factors, such as hypercholesterolemia, hypertension, andobesity, no longer appear to be related to the level of cardiovascular risk in ESRDpatients. One explanation might be the accumulation of nontraditional risk factorsin CKD patients (Appel 2004; Coresh et al. 1998; Muntner et al. 2004). Presumably,the complexity of traditional and nontraditional risk factors determines patientoutcome. An alternative explanation may be that patient outcome is determinedby the numerous quantitative and qualitative lipid abnormalities in triglycerides,phospholipids, and lipoproteins that are observed in CKD patient cohorts (Keaneet al. 2013; Vaziri 2006).3.1 Dyslipidemia in CKD PatientsSeveral studies have demonstrated a characteristic switch in the lipid phenotype atdifferent stages of CKD. Data from the “Multi-Ethnic Study of Atherosclerosis”(MESA) have indicated an elevation of triglyceride-rich lipoproteins (Lamprea-Montealegre et al. 2013). Triglyceride-rich VLDL or other apoB-rich lipoproteinsaccumulate in patients with ESRD as a consequence of increased triglyceride levels(Vaziri and Norris 2011). The VLDL particle increase depends on the CKD stageand is aggravated by a decline in kidney function (Lamprea-Montealegre428 M. Schuchardt et al.et al. 2013). Hypertriglyceridemia is caused by reduced lipoprotein lipase (LPL)levels (Vaziri 2006; Vaziri et al. 2012) and results in the limited delivery oftriglyceride-rich fuel lipoproteins to adipocytes and myocytes (Vaziri et al. 2012).The enrichment of triglyceride-containing lipoprotein particles results in a highersusceptibility to oxidative modifications. These particles are highlypro-inflammatory (Vaziri 2013). In addition, other apoB-rich lipoproteins, such assmall dense LDL or IDL-C, are commonly increased in patients with CKD (Salandand Ginsberg 2007; Vaziri 2006). The reduced clearance rate of LDL particlescontributes to the increase in LDL levels (Ikewaki 2013).Lipoprotein a (Lp(a)), which consists of apoA and apoB and is similar to LDL-C,is increased in CKD patients (Longenecker et al. 2005; Muntner et al. 2004). Lp(a) serves as an independent biomarker that predicts cardiovascular complications(Jacobson 2013; Thompson and Seed 2013).Dyslipidemia is further enhanced by hyperparathyroidism, which frequentlyoccurs in CKD patients (Moe and Sprague 2012). Normal lipid metabolism dependson proper parathyroid function, and hyperparathyroidism tends to result in hypertri-glyceridemia (Liang et al. 1998) as well as in a deficiency in lipid metabolismenzymes, such as hepatic lipase and LPL (Klin et al. 1996). The enrichment ofapoB-containing lipoproteins is correlated with decreased HDL-C levels (Salandand Ginsberg 2007; Vaziri 2006).The lipid profile is altered in CKD patients compared with healthy subjects anddiffers depending on the stage of CKD progression (e.g., stage I to V, pre/post-dialysis, and time on dialysis) and the dialysis procedure (e.g., hemodialysis(HD) or peritoneal dialysis (PD)). As a consequence, the lipid profiles of theCKD population are heterogeneous. Extrinsic factors, including drug administra-tion for kidney disease and associated disorders (hypertension, diabetes mellitus,and malnutrition), contribute to dyslipidemia in this patient cohort.To overcome the complex dyslipidemia in CKD patients, diverse clinical trialshave addressed the influence of lipid-modulating therapies on cardiovascularoutcomes. The results of these therapies in the general population suggest that theincidence of cardiovascular events in CKD patients is expected to decrease (vanCapelleveen et al. 2014).3.2 Lipid-Modulating Therapy in CKD PatientsCurrently, drugs that treat dyslipidemia are in use or under investigation. Statins,which primarily lower the LDL-C level; peroxisome proliferator-activated receptor(PPAR) agonists; inhibitors of lipid metabolism (CETP, niacin); and apoAImimetics have been evaluated in preclinical and/or clinical studies. The currentlyestablished lipidemic drugs have been reviewed elsewhere; therefore, this articlesummarizes aspects of therapy related to CKD. Although lipid-modulatingtherapies have had a positive impact on the cardiovascular outcome in patientswith normal kidney function, the effect in CKD patients has been disappointing.These observations have originated predominantly from statin trials.High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 429Statin treatment leads to a reduction in LDL-C through the inhibition of3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the initialenzyme in endogenous cholesterol biosynthesis. In several clinical trials, it hasbeen clearly demonstrated that for patients without renal disease, a reduction ofLDL-C upon statin therapy is associated with a decreased cardiovascular mortalityrate (The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID)Study Group 1998; Scandinavian Simvastatin Survival Study Group 1994; Baigentet al. 2005; Collins et al. 2003; LaRosa et al. 2005; Sever et al. 2003). Due to thesubstantially high cardiovascular risk in CKD patients, the effect of statin therapywas also evaluated in three large clinical trials (>1,000 patients included): the“Deutsche Diabetes Dialyse Studie” (4D) (Wanner et al. 2005), “A Study toEvaluate the Use of Rosuvastatin in Subjects on Regular Hemodialysis: An Assess-ment of Survival and Cardiovascular Events” (AURORA) (Fellstrom et al. 2009a),and “Study of Heart and Renal Protection” (SHARP) (Baigent et al. 2011). The“Prevention of Renal and Vascular End-Stage Disease Intervention Trial”(PREVEND-IT) (Asselbergs et al. 2004; Brouwers et al. 2011) has also addressedthe effect of statin therapy in a smaller cohort (<1,000 patients) but included alonger follow-up period. The “Assessment of LEscol in Renal Transplantation”(ALERT) (Holdaas et al. 2003) trial investigated cardiovascular outcomes in renaltransplant recipients. The 4D study consisted of a randomized control trial thatexamined Western HD patients with diabetes (Wanner et al. 2005). No benefits inthe primary endpoints were observed in a follow-up period of approximately4 years (Wanner et al. 2005) (Table 1). A subsequent subgroup analysis of the 4Dstudy cohort showed a reduction in cardiovascular disease after statin treatment inpatients with LDL-C >145 mg/dL (Marz et al. 2011). The 4D trial with 1,225patients was followed by the larger AURORA trial, which enrolled 2,776 HDpatients with and without diabetes mellitus. Nonetheless, the reduction in LDL-Cthat was observed did not have an effect on cardiovascular outcomes (Fellstromet al. 2009a). Another large randomized trial, the SHARP trial, enrolled HD patientsand CKD patients in stages III to V: (Baigent et al. 2011). The combination therapyof simvastatin and ezetimibe lowered the LDL-C level but did not reduce theoverall vascular mortality (Baigent et al. 2011). However, the risk for atheroscle-rotic events was reduced in a wide range of CKD patients (Baigent et al. 2011). Thelongest follow-up period, up to 9.5 years, was in the PREVEND-IT trial(Asselbergs et al. 2004; Brouwers et al. 2011). Here, patients withmicroalbuminuria were enrolled and treated with pravastatin and fosinopril. Inthe follow-up period of 46 months, pravastatin treatment had no effect on cardio-vascular endpoints (Asselbergs et al. 2004). The extended follow-up period of9.5 years showed that elevated urinary albumin excretion was associated withincreased cardiovascular morbidity and mortality (Brouwers et al. 2011).Thus, the large clinical trials examining statin therapy in CKD patients failed toshow a significant reduction in overall cardiovascular mortality for patientsundergoing dialysis treatment (Table 1). Based on the data from the US RenalData System, the majority of cardiovascular deaths in CKD patients are due tochronic heart failure and are not influenced by statin treatment (Collins et al. 2013).430 M. Schuchardt et al.Table1CKDpatientsinclinicaltrialsonlipid-modulatingtherapyStudyFollow-up(years)DrugPatientsOutcomeReferencesStatins4D3.9Atorvastatin(20mgdaily)1,255patients,DMtypeIIonHDDecreasedrateofallcardiacevents,increasedrateoffatalstrokeWanneretal.(2005)AURORA3.2Rosuvastatin(10mgdaily)2,776patients,HDwithandwithoutDMNoreductionofcardiovascularmortality,nonfatalMI,nonfatalstrokeFellstrometal.(2009b),Holdaasetal.(2011)PREVEND-IT9.5Pravastatin(40mgdaily),fosinopril(20mgdaily)864patientswithmicroalbuminuriaNoeffectoncardiovasculareventsAsselbergsetal.(2004),Brouwersetal.(2011)SHARP4.9Ezetimibe(10mgdaily)incombinationwithsimvastatin(20mgdaily)9,270CKDpatients(stageIIItoV,includingdialysis)17%reductioninmajoratheroscleroticevents,15%reductioninmajorvasculareventsBaigentetal.(2011)ALERT5–6Fluvastatin2,102renaltransplantrecipientswithstablegraftfunctionFewercardiacdeathsandnonfatalMIs,noreductioninmortalityHoldaasetal.(2003)FibratesandfattyacidsVA-HIT5–7Gemfibrozil(1,200mgdaily)1,046CKDpatientswithcreatinineclearance<75ml/minNoreductionofoverallmortality,decreasedcardiovasculareventsandnonfatalMIsRubinsetal.(1993),Tonellietal.(2004)OPACH2n3-PUFA717HDpatientsNoreductionofcardiovasculareventsordeathSvenssonetal.(2006)Forfullstudynames,pleaseseetextCKDchronickidneydisease,HDhemodialysis,DMdiabetesmellitus,n3-PUFAn3-polyunsaturatedfattyacids,MImyocardialinfarctionHigh-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 431Therefore, other uremia-related pathways and/or the previously existing vasculardamage may contribute to the increase in cardiovascular risk. A trial thatinvestigated the effect of statin therapy in renal transplant recipients observedsimilar effects. In the ALERT trial (Holdaas et al. 2003), statin therapy reducedthe LDL-C plasma concentration in treated patients (Holdaas et al. 2003). Althoughfewer cardiac deaths and nonfatal MIs were observed, no significant reduction inthe primary endpoint was achieved (Holdaas et al. 2003).Based on the findings of the current clinical trials with CKD patients, thetherapies tested had either no real impact on cardiovascular outcome or resultedin fatal effects (Kilpatrick et al. 2007). Interestingly, despite a robust reduction inthe LDL-C level, only negative or neutral effects on acute cardiovascular eventswere measured (Baigent et al. 2011; Fellstrom et al. 2009b; Wanner et al. 2005).Effective prevention of cardiovascular events was only documented in patients withmild to moderate CKD (Baigent et al. 2011).In addition to statins, which primarily lower LDL-C plasma levels, other lipid-modulating drugs are currently used in non-renal-insufficient patients. Here, differ-ent targets in HDL metabolism have been identified to overcome dyslipidemia.Initial studies addressed the effect of enzyme inhibition on lipid metabolism, suchas CETP inhibition. PPAR agonists, niacin, and apoAI mimetics are other thera-peutic options that have been tested in clinical and/or preclinical studies. However,these studies often excluded patients with CKD from recruitment. Therefore,further studies are necessary to determine the effects of these therapies on patientswith renal insufficiency.CETP is responsible for the shuttling of cholesteryl esters between HDL andapoB-containing lipoprotein particles. Inhibition of this enzyme results in a dra-matic increase in HDL levels. Torcetrapib was the first CETP inhibitor examined ina large clinical trial, “Investigation of Lipid Level Management to Understand ItsImpact in Atherosclerotic Events” (ILLUMINATE) which included approximately15,000 patients (Barter et al. 2007). The treatment increased the HDL concentrationby more than 50 % (Barter et al. 2007). However, the study was prematurelyterminated because an increased risk of cardiovascular events was observed (e.g.,increase in arterial blood pressure) (Barter et al. 2007). In the ILLUMINATE trial,patients with severe CKDwere not enrolled. The mean eGFR in patients included inthe study was 79.5 ml/min/1.73 m2 with creatinine concentration of 1 mg/dL(Barter et al. 2007). In subsequent trials with other CETP inhibitors, whichattempted to overcome the off-target effects of torcetrapib, CKD patients wereexcluded. The “Determining the Efficacy and Tolerability of CETP Inhibition withAnacetrapib” (DEFINE) study (Cannon et al. 2010) investigated anacetrapib, and akey exclusion criteria in this study was an eGFR <30 ml/min/1.73 m2 or severerenal impairment (Cannon et al. 2010). In the dal-OUTCOMES trial withdalcetrapib (Schwartz et al. 2009, 2012), the exclusion criteria was a creatininelevel of>2.2 mg/dL (Schwartz et al. 2009). For the “Randomized Evaluation of theEffects of Anacetrapib Through Lipid-Modification” (REVEAL) study, patientrecruitment is ongoing, and the study is expected to be completed in 2017 (Gutsteinet al. 2012). On the basis of the current CETP inhibitor studies, the effects of CETP432 M. Schuchardt et al.inhibition on cardiovascular mortality cannot be determined, and patients withsevere CKD were excluded from all studies.Niacin treatment most effectively raises HDL-C levels and decreases triglycer-ide, LDL-C, and Lp(a) levels. The mechanism underlying the influence of niacin onHDL metabolism is unknown (Linsel-Nitschke and Tall 2005). The beneficialeffects on lipoprotein levels and the corresponding cardiovascular outcome havebeen described in several small-scale clinical trials. Unfortunately, the large-scaletrials AIM-HIGH (AIM-HIGH Investigators 2011; Boden et al. 2011) and HPS2-THRIVE (2013), in which patients were treated with niacin in combination with astatin, were prematurely terminated because of a high prevalence of side effects. Inboth studies, no patients with severe CKD were included. In AIM-HIGH, the meaneGFR was 82.8 ml/min/1.72 m2 (2011), and in HPS2-THRIVE, severe renalinsufficiency was an exclusion criteria for patient recruitment (2013).The family of PPAR transcription factors is involved in the regulation of fattyacid metabolism and influences lipid levels. The family consists of three members:PPAR-α, PPAR-γ, and PPAR-δ. PPAR-γ agonists, such as rosiglitazone andpioglitazone, are high-affinity agonists at the receptor site (Linsel-Nitschke andTall 2005), and their effects on cardiovascular outcome have been tested. However,only a minimal benefit or an increase in heart failure rates was observed in a studypopulation with diabetes in the “Rosiglitazone Evaluated for CardiovascularOutcomes in Oral Agent Combination Therapy for Type 2 Diabetes” (RECORD)trial (Home et al. 2009). The mean serum creatinine level in the study cohort wasapproximately 62 μmol/L (Home et al. 2009). The activation of PPAR-α viafibrates, which are weak agonists of this receptor (Linsel-Nitschke and Tall2005), results in reductions in LDL-C and triglycerides, whereas HDL-C plasmalevels are moderately increased (Linsel-Nitschke and Tall 2005; Sahebkaret al. 2014). The “Veterans’ Affairs High-Density Lipoprotein Intervention Trial”(VA-HIT) (Tonelli et al. 2004) investigated the effects of the fibrate gemfibrozil in acohort of individuals with CKD. Gemfibrozil treatment did not reduce overallmortality, but the rate of major cardiovascular events and nonfatal myocardialinfarctions (MIs) was significantly reduced in patients with mild to moderateCKD (Tonelli et al. 2004). Currently, there are no synthetic ligands for PPAR-δin clinical use (Sahebkar et al. 2014).In the secondary prevention of cardiovascular disease, there is evidence thatn3-polyunsaturated fatty acids (n3-PUFAs) are effective (Studer et al. 2005). Astudy with HD patients failed to show a reduction in cardiovascular events or deathafter treatment with an n3-PUFA (Svensson et al. 2006). However, a significantreduction of MIs was observed in this patient cohort (Svensson et al. 2006).Additional pharmacotherapeutic strategies are currently under development.apoAI-based drugs and reconstituted/engineered HDL particles have been testedin animal and human studies. Recently, van Capelleveen et al. summarized currentknowledge of these novel therapeutics (van Capelleveen et al. 2014). The humantrials were completed with a limited patient number (<200 patients) and a shortfollow-up period (several weeks). However, the clinical impact of these novel drugsis highly interesting and must be evaluated in larger clinical trials. Several trials areHigh-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 433currently under way (van Capelleveen et al. 2014); however, the high treatmentcosts may limit their use in routine clinical practice. After the promising results inanimal studies, treatment with apoAI mimetics did not improve biomarkers thatwere selected to describe HDL function (Watson et al. 2011).To date, the benefit of apoAI-based therapy for CKD patients remains elusive.The studies described indicate that with a decline in renal function, lipid-modulating therapies lose their capacity to prevent the occurrence of cardiovascularnonfatal and fatal events. Consequently, the lipid compositional and correspondingfunctional changes that occur with declining renal function are not fully understood.In Table 1, the different therapeutic approaches in patients with variable renalfunction and the effects on cardiovascular outcome are summarized.Recent guidelines for the management of lipid disorders in patients with chronicrenal failure recommend lipid-lowering therapy only in patients with CKD who arenot undergoing renal replacement therapy (National Kidney Foundation 2003;Tonelli and Wanner 2013). The guidelines clearly indicate our inadequate under-standing of how to address the complex lipid changes in CKD patients.3.3 Protective Effects of HDL on the KidneyBased on the current evidence, the causal association between the HDL level, HDLfunction, and cardiovascular outcome in CKD patients is not fully understood.Dysregulated HDL metabolism might be caused by reduced kidney function.Abnormalities in lipid metabolism enzymes and transport processes have beendescribed (Hirano 2013; Pahl et al. 2009; Vaziri and Norris 2011). The changesinvolve enrichment of free cholesterol, triglycerides, and fatty acids as well as thedepletion of cholesterol esters within HDL. However, there is evidence that HDLmight be an independent protective factor for kidney function. Epidemiologic datafrom a multi-study cohort demonstrated an association between a low HDL-C leveland reduced kidney function (Odden et al. 2013). Additional trials addressed thequestion of whether HDL-C is associated with the progression of CKD. A recentstudy, which included 3,303 patients with CKD stages III to V and a median follow-up period of 2.8 years, supports the hypothesis that dyslipidemia is independentlyassociated with rapid renal progression (Chen et al. 2013). Two meta-analysessuggest that statin therapy has a benefit on GFR and inhibits GFR decline (Friedet al. 2001; Sandhu et al. 2006). However, other studies found no impact on renaloutcome with statin treatment (Atthobari et al. 2006; Rahman et al. 2008). Focusingon HDL-C, Baragetti and coworkers observed an association between low HDL-Cand earlier entry in dialysis or doubling of the plasma creatinine in a patient cohortof 176 subjects with mild to moderate kidney dysfunction (Baragetti et al. 2013).Moreover, a cross-sectional analysis of 4,925 patients with normal kidney functionstrengthened the association between HDL-C and eGFR (Wang et al. 2013).Confounders (e.g., age, blood pressure, and lipid parameters) influenced the rela-tionship (Wang et al. 2013). Furthermore, malnutrition and hypoalbuminemia,434 M. Schuchardt et al.which are frequently present in patients with CKD (Kaysen 2009; Peev et al. 2014),affected the HDL level and functionality (Khovidhunkit et al. 2004).Further studies are necessary to determine whether HDL-targeted therapies arebeneficial for renal outcome. The difference in the HDL quantity and functionalquality in CKD patients must be considered. The results of clinical trials, such asILLUMINATE (Barter et al. 2007) and HPS2-THRIVE (2013), suggest thatincreasing HDL-C plasma levels is not an optimal therapeutic target. Furthermore,as shown in the “European Prospective Investigation into Cancer and Nutrition”(EPIC) study and the MESA study, intima/media thickness and cardiovascularevents had a stronger association with HDL particle number compared with HDLcholesterol levels (Arsenault et al. 2009; El Harchaoui et al. 2009; Mackeyet al. 2012). Figure 2 summarizes the potential protective effects of HDL on kidneyfunction.Additional studies that address the structure-dependent functions of the hetero-geneous HDL particles are necessary to assess the benefit of HDL-increasingtherapy for different disease conditions.Fig. 2 Interaction of HDL level/function with the kidney. HDL has a pleiotropic protectivefunction. A decrease in HDL level/function has an impact on the vascular alterations associatedwith kidney damage. The decline in kidney function induces/strengthens the decrease in HDLlevel/function. For further information and references, refer to the textHigh-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 4354 Structural and Functional Modifications of HDL in CKDPatientsIn the last several years, HDL and its structural composition in disease conditions,especially cardiovascular, metabolic, and renal diseases, have been a focus ofexperimental research. Compositional changes of the proteome, alterations of thelipid moiety, and posttranslational modifications of HDL isolated from these patientcohorts contribute to a lower protective function of the lipoprotein particle(Annema and von Eckardstein 2013). The following section summarizes the currentknowledge of the HDL changes identified in patients with reduced renal function. Itis important to keep in mind that the investigations were performed in a heteroge-neous group of patients with different stages of renal failure. Therefore, it is oftendifficult to define the role of uremia on the functionality of HDL.The HDL particle contains hundreds of different lipids and a multitude ofproteins that form a lipoprotein with different shapes, sizes, and densities through-out its metabolism (Camont et al. 2011; Shah et al. 2013). Because of its amphi-philic character and the presence of specific binding proteins, HDL serves as cargofor vitamins, hormones, toxins, and microRNA (Vickers and Remaley 2014). Thereis evidence of biological activity for many of the lipid and protein components.These components can bind to specific receptors and activate signaling pathways invascular cells, thereby influencing atherogenesis, thrombosis, apoptosis, oxidativereactions, endothelial properties, and inflammatory reactions (Calabresi et al. 2003;Navab et al. 1991; Nofer et al. 2004; Watson et al. 1995). The functions of theassociated proteins and lipids in the vascular wall have been characterized in vitroand in animal studies, as well as in clinical trials. It has been shown that composi-tional changes are related to functionality and are influenced by uremic toxins. Forexample, (1) the dysregulation of proteins in the HDL metabolism affects HDLplasma level and cholesterol clearance. Furthermore, (2) changes in HDL apolipo-protein composition occur, and finally (3) the loss of proteins with a protectivefunction or (4) the accumulation of substances with a fatal function within HDLinfluences its vascular protective properties. The compositional changes may notonly result in a decrease in function but may also lead to increased dysfunction(Fig. 3), and both are associated with an increased cardiovascular risk profile.4.1 Dysregulation of Proteins in HDL MetabolismAs discussed in the preceding paragraph, CKD is associated with a decreasedHDL-C plasma concentration and dyslipidemia. The uremic condition is responsi-ble for the disrupted protein metabolism (Vaziri 2006) due to uremic-induced liverdamage (Yeung et al. 2013). Many proteins important in HDL metabolism arepredominantly produced within the liver, and their production is dysregulated inuremia. For proper HDL maturation and metabolism, enzymes such as LCAT andCETP are responsible. Furthermore, diverse receptors on intestinal and peripheralcells, as well as hepatic cells, are necessary for cholesterol transport via HDL to the436 M. Schuchardt et al.liver for biliary excretion (Fig. 4). There is evidence that the expression of enzymesand receptors in HDL metabolism is altered in patients who suffer from kidneydisease. To date, most studies have been completed with CKD stage V patientsdependent on dialysis (ESRD), and less information on enzyme expression has beenobtained from patients prior to dialysis. LCAT is important for HDL maturation. InESRD patients, the LCAT enzyme concentration and activity were reduced (Miidaet al. 2003; Moradi et al. 2009; Pahl et al. 2009; Shoji et al. 1992; Tolle et al. 2012).Fig. 3 Functional vs. dysfunctional HDL particles. Depending on the substances that accumulatewithin the HDL, the particle may exert functional or dysfunctional propertiesFig. 4 Dysregulation in HDL metabolism under uremic conditions. # decrease, " increase, $ noeffect. For references, refer to the textHigh-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 437The association between the plasma levels of cholesterol transport proteins, such asCETP and CKD, remains unclear. Some studies have failed to show any significantchanges in the CETP protein level or activity (Pahl et al. 2009), whereas otherstudies have identified an increased (Dullaart et al. 1993) or decreased (Miidaet al. 2003) CETP level. These controversial data may result from the heterogeneityof CKD patients and the analysis of different patient subgroups: increased levelswere identified in proteinuric patients (Dullaart et al. 1993), and unchanged ordecreased levels were identified in HD patients with different lengths of minimumdialysis treatment (minimum of 3 months vs. 1 year) (Miida et al. 2003; Pahlet al. 2009). The expression of receptors important for RCT appears to be affectedby kidney dysfunction. In nephrectomy animal models, SR-BI (Liang and Vaziri1999) and ABCA1 (Zuo et al. 2009) receptor expression is decreased. A reductionof these receptors in humans would subsequently be associated with altered HDLmetabolism in CKD patients.4.2 Changes of HDL Apolipoproteins (Modifications and Levels)HDL contains different apolipoproteins. Under uremic conditions, the compositionof the proteins within HDL changes: some protein levels decrease, whereas otherprotein levels increase. The main apolipoprotein of HDL under normal physiologi-cal conditions is apoAI. The presence of altered apoAI levels in CKD patientsremains controversial. Some studies have identified decreased apoAI levels indialysis patients (Holzer et al. 2011a; Moradi et al. 2009; Vaziri et al. 1999,2009), whereas other studies did not identify a significant change in apoAI concen-tration (Shoji et al. 1992; Tolle et al. 2012). While apoAII and apoCI levelsdecreased in CKD patients (Holzer et al. 2011a), apoCII (Holzer et al. 2011a;Weichhart et al. 2012) and apoCIII (Holzer et al. 2011a) increased. ApoAIV wasnot identified in the HDL obtained from healthy subjects, but was detected indialysis patients via mass spectrometry analysis (Holzer et al. 2011a). No signifi-cant difference was found in apoD and apoE levels, whereas apoM levels decreased(Holzer et al. 2011a). Table 2 summarizes the main apolipoprotein changes in CKDpatients. Currently, most identified changes are based on studies with HD patients.The discrepancy in apolipoprotein levels may be a result of the heterogeneouspatient population (e.g., CKD stage, time on dialysis, medication, or secondarydisease), different HDL isolation protocols (e.g., one-step vs. multistep ultracentri-fugation, chromatography, or differentiation between HDL2/3), and different apo-lipoprotein detection methods (e.g., enzyme-linked immunosorbent assay vs. massspectrometry).In addition to changes in the apolipoprotein levels, modifications of the proteinswithin HDL occurred, which were closely connected to altered functionality. Todate, oxidative modifications, carbamylation, and glycosylation have been detected.These effects may be a consequence of the high level of ROS in patients with renalfailure.438 M. Schuchardt et al.Patients with CKD suffer from increased reactive oxygen stress (Sunget al. 2013; Tucker et al. 2013). The pro-oxidant state is multifactorial but is relatedto high amounts of uremic toxins. Uremic toxins are directly involved in theoxidative response, e.g., phenyl acetic acid is a strong inducer of ROS (Schmidtet al. 2008), and indoxyl sulfate has a dual role that consists of pro-oxidantproperties in the uremic condition and antioxidative properties under normalphysiological conditions (Miyamoto et al. 2011). The increase in highly reactiveradicals contributes to the oxidation of proteins and lipids. For example, theoxidation of LDL is known to occur in CKD patients (Ribeiro et al. 2012;Samouilidou et al. 2012). Furthermore, oxidative modifications of apoAI havebeen identified (Nicholls et al. 2005; Undurti et al. 2009; Zheng et al. 2004).ApoAI oxidation is dependent on the binding activity between apoAI and itsreceptors ABCA1 (Zheng et al. 2004) and SR-BI (Undurti et al. 2009). Therefore,ox-apoAI impairs HDL metabolism. In addition, an association between oxidativemodifications of HDL and a higher risk for cardiovascular events in dialysispatients has been identified (Honda et al. 2012).Another reactive compound that leads to protein modification is reactive cya-nate, which induces the carbamylation of proteins. Cyanates emerge from thedegradation of urea or via myeloperoxidase (MPO) at the sites of inflammation(Holzer et al. 2012; Sirpal 2009). CKD patients are prone to carbamylated proteinsbecause urea levels are increased in the uremic condition, and these patientsexperience increased inflammation. MPO associates with HDL (Nichollset al. 2005); thus, HDL proteins in addition to LDL proteins (Apostolovet al. 2005) become targets for carbamylation. Carbamylation impairs HDL func-tion by decreasing the cholesterol efflux capacity of HDL from macrophages(Hadfield et al. 2013; Holzer et al. 2011b).CKD patients are also prone to glycated protein modifications caused by insulinresistance, which is a condition that is frequently observed with reduced renalfunction (DeFronzo et al. 1981; Kobayashi et al. 2005). Insulin resistance appearsTable 2 Differences in apolipoprotein composition within the HDL in CKD patients comparedwith healthy control subjects (" increase, #decrease, $ no change vs. healthy control)ApolipoproteinChange in the HDLin CKD patients ReferencesapoAI #$ Holzer et al. (2011a), Moradi et al. (2009), Shojiet al. (1992), Tolle et al. (2012), Vaziri et al. (1999),Vaziri et al. (2009)apoAII # Holzer et al. (2011a)apoAIV " Holzer et al. (2011a)apoCI # Holzer et al. (2011a)apoCII " Holzer et al. (2011a), Weichhart et al. (2012)apoCIII " Holzer et al. (2011a)apoD $ Holzer et al. (2011a)apoE $ Holzer et al. (2011a)apoM # Holzer et al. (2011a)High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 439to be associated with a high burden of oxidative stress. The reduction of oxidativestress by treatment with a superoxide dismutase/catalase mimetic reduces not onlyROS production but also insulin resistance in a CKD mouse model (D’Apolitoet al. 2010). The modified proteins are advanced glycated end products (AGEs),which are usually found at high levels in CKD patients. Glycated apoAI is likely anAGE (Lapolla et al. 2008). It has been speculated that these modified apoAIs havereduced RCT activity.Additional proteins are also associated with HDL under physiological andpathophysiological conditions. Many proteins within HDL have been identifiedusing a proteomic approach. Some proteins are increased, whereby other proteinsare decreased in HD patients (Holzer et al. 2011a; Weichhart et al. 2012). However,an association between all of the proteins identified by proteome analysis and HDLfunctionality/dysfunctionality in the vessel wall could not be identified. Retinol-binding protein 4 (RBP4) was detected in the HDL from CKD patients, whereas itwas not found within the HDL of healthy controls (Holzer et al. 2011a). Plasmalevels of RBP4 were increased in dialysis patients (Frey et al. 2008), and theincrease was dependent upon the CKD stage (Henze et al. 2010). Furthermore,surfactant protein B and α-1-microglobulin/bikunin precursor proteins were signif-icantly increased in ESRD patients (Weichhart et al. 2012). Transthyretin was notidentified within the HDL from healthy controls, but was detectable in the HDLfrom dialysis patients (Holzer et al. 2011a). An overview of the identified proteinswas recently presented (Holzer et al. 2011a; Weichhart et al. 2012).4.3 Loss of Protective Proteins or LipidsOxidative stress participates in the pathogenesis and progression of CKD and wasincreased in CKD patients (Mimic-Oka et al. 1999; Puchades et al. 2013). Thisresults from an imbalance of pro-oxidative and antioxidative signals that is favoredby uremic toxins (Mimic-Oka et al. 1999). Navab et al. postulated that HDL itselfcan become pro-inflammatory (Navab et al. 2006). In this condition, serumlipoproteins are prone to oxidative modification. The enzymes linked to HDL thathave an antioxidative capacity are thought to facilitate oxidative protection. Ahigher pro-inflammatory index of HDL in CKD patients resulted in a higheradjusted death hazard ratio (Kalantar-Zadeh et al. 2007). Furthermore, it wasshown that the in vitro antioxidative function of HDL was impaired in CKDpatients (Moradi et al. 2009). The primary antioxidative HDL-linked enzymes arePON and glutathione peroxidase, and of these two enzymes, PON is the moststudied one. The PON family consists of three members: PON1, PON2, andPON3. In contrast to PON2, PON1 and PON3 are secreted in the blood and areassociated within HDL (Macharia et al. 2012). PON1 remains the best-studiedenzyme of this family in cardiovascular disease and kidney dysfunction. In CKDpatients, proteomic analysis did not indicate a significant difference in PON1compared with healthy individuals (Holzer et al. 2011a), whereas its enzymeactivity was reduced in CKD patients (Dantoine et al. 1998; Kennedy et al. 2013;440 M. Schuchardt et al.Moradi et al. 2009). The reduction negatively correlated with the CKD stage(Dantoine et al. 1998). For glutathione peroxidase, decreased concentration andactivity were identified in dialysis patients (Moradi et al. 2009).The loss of this antioxidative capacity combined with an increased level ofpro-oxidative molecules (see below) may, at least in part, account for the increasedoxidative stress in CKD patients.A component that contributes to several of the protective functions of HDL issphingosine-1-phosphate (S1P) (Nofer et al. 2004, 2007; Schuchardt et al. 2011;Theilmeier et al. 2006). HDL-associated apoM binds S1P (Christoffersenet al. 2011). In CKD patients, apoM levels are decreased (Holzer et al. 2011a),which may lead to higher levels of S1P free from HDL. There are severalindications that non-HDL-bound S1P signaling differs compared withHDL-bound S1P (Schuchardt et al. 2011).4.4 Increase of Molecules within HDL with a Fatal Functionin the Vascular WallIn vitro and in vivo studies and/or clinical trials have identified proteins thataccumulate within HDL. Interestingly, the albumin content in HDL from uremicpatients was elevated, and apolipoprotein displacement may occur (Holzeret al. 2011a).The acute phase protein serum amyloid A (SAA), which is secreted by the liverduring inflammation, is primarily transported by HDL within the blood (Uhlar andWhitehead 1999). In uremic patients, elevated SAA (Holzer et al. 2011a; Tolleet al. 2012; Weichhart et al. 2012; Zimmermann et al. 1999) is a sign of a chronicinflammatory status. This is closely related to an increased cardiovascular risk inhumans (Zimmermann et al. 1999). In vitro studies support these findings, as theaccumulation of SAA in HDL was correlated with a reduced anti-inflammatorycapacity of HDL and a pro-inflammatory potential (Tolle et al. 2012; Weichhartet al. 2012). Furthermore, the cholesterol efflux capacity was decreased when theSAA level dramatically increased during acute sepsis (Annema et al. 2010). Inaddition, SAA accumulation results in the replacement of apoAI and influencesHDL remodeling and metabolism. Nonetheless, the replacement was not identifiedin all patient cohorts (Tolle et al. 2012), which may be because of the differentexperimental settings. A displacement of other protective proteins during the acutephase response by SAA was also observed for PON1 and PAF-AH (Van Lentenet al. 1995). Thus, SAA enrichment within HDL affects the anti-inflammatoryresponse. Recently, a pro-inflammatory response of SAA-rich HDL in CKDpatients was observed (Tolle et al. 2012; Weichhart et al. 2012). The effects ofSAA accumulation on the cholesterol efflux function of HDL remain controversial.In some experiments, a reduction of the cholesterol efflux capacity of HDL wasobserved if it was enriched with SAA (Artl et al. 2000; Marsche et al. 2007),whereas other studies found normal efflux capacity even with SAA-rich HDL(Banka et al. 1995; van der Westhuyzen et al. 2005).High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 441Furthermore, phospholipases (PLA) associated with cardiovascular mortality areelevated in uremic patients. For example, lipoprotein-associated PLA (Lp-PLA)2 was increased in uremic patients (Holzer et al. 2011a). The secreted PLA2 (sPLA2) concentration and activity were also higher in ESRD patients. Thiselevation contributes to excessive oxidative stress in these patients (van der Gietet al. 2010a).Dimethylarginines (DMAs), such as asymmetric dimethylarginine (ADMA) andits structural isomer symmetric DMA (SDMA), have been correlated with cardio-vascular risk factors. As uremic toxins, they have been associated with cardiovas-cular outcomes and renal dysfunction (Duranton et al. 2012; Kielstein et al. 2006).DMAs originate from protein proteolysis and are mainly excreted in the urine.DMAs influence nitric oxide (NO) synthesis and negatively influence its vascularprotective effects (Bode-Boger et al. 2006). The accumulation of SDMA in HDLfrom CKD patients and its association with decreased NO have recently beendemonstrated (Speer et al. 2013).MPO is another protein associated with the pathogenesis of cardiovasculardisease because of its pro-oxidative and carbamylating potential (Nichollset al. 2005; Undurti et al. 2009; Zheng et al. 2004). As discussed earlier, MPOassociates with HDL (Nicholls et al. 2005) and contributes to reduced RCT ininflammatory diseases (Annema et al. 2010; Zheng et al. 2004). Furthermore,MPO-derived oxidative products modify apoAI (Hadfield et al. 2013). Themodifications induced by MPO result in a pro-inflammatory HDL particle (Undurtiet al. 2009). In patients with diabetes mellitus type 2, MPO activity was increased inHDL (Sorrentino et al. 2010). Interestingly, in a CKD cohort, the MPO plasma leveldecreased with advancing renal failure (Madhusudhana Rao et al. 2011). Furtherstudies are necessary to determine the role of MPO in HDL dysfunction in a CKDcohort.The HDL from ESRD patients on HD was less effective at accepting cholesterolfrom macrophages compared with the HDL from healthy subjects (Holzeret al. 2011a; Yamamoto et al. 2012). The reverse cholesterol transport may beaffected in uremic patients.5 Possibility of Functional Restoration of HDLHDL function has been a recent focus of experimental research. With respect toweak effects on cardiovascular outcome after an increase of HDL quantity in CKDpatients, HDL function capacity appears important for the design of new therapeu-tic approaches. Therefore, the question arose whether structural modifications andassociated dysfunctions under uremic conditions are reversible. The best approachto test this hypothesis in the case of renal function is to study patients aftersuccessful renal transplantation. A limitation of this model is that these patientsoften require immunosuppressive drugs, which may affect lipid metabolism andfunction (Badiou et al. 2009). Some investigations have attempted to observechanges in HDL function after renal transplantation. Dantoine and coworkers442 M. Schuchardt et al.reported that the PON enzyme activity in patients after kidney transplantation wascomparable to that in control subjects, whereas in dialysis patients, it was decreased(Dantoine et al. 1998). Furthermore, the anti-inflammatory capacity of HDL fromESRD patients compared with healthy controls increased after successful renaltransplantation (van der Giet et al. 2010b). The observed effect appears to be relatedto a decreased SAA level in transplant recipients. Other indicators of the reversibil-ity of HDL dysfunction are based on studies with chronic heart failure patients. Inthis cohort, exercise training, which is an accepted intervention strategy to decreasecardiovascular risk in heart failure patients, resulted in increased HDL. The authorsdemonstrated that changes in HDL function induced by exercise training correlatedwith improved endothelial function (Adams et al. 2013).According to these data it has been suggested that a restoration of HDL ispossible. The lipid metabolism and the composition of different lipoproteinparticles are primarily influenced by the metabolic condition. To date, little infor-mation is available regarding whether the CKD stage before transplantation isdependent on the HDL functionality after kidney transplantation or whether apoint-of-no-return exists for functional restoration, vessel damage, andcardiovascular risk.6 Laboratory Tests to Measure HDL FunctionAccording to previous clinical and experimental trials, it has become clear thatHDL particle modifications in disease conditions are associated with reduced HDLfunction; specific factors that determine HDL functionality remain unclear. It isknown that various structural features are associated with HDL function, which canbe measured by in vitro studies. A limitation arises in the difficulty of comparingexperimental results. The standardization of the experimental design for HDLisolation, separation of proteins/lipids, sample preparation for proteomicapproaches, and detection methods for components within HDL would help toovercome certain limitations that may result in discrepancies between findings. Inaddition, in vitro functionality assays are very complex and further hamper compa-rability. The complexity and cost preclude these tests from routine clinical labora-tory analyses, thus limiting the validation of experimental findings of functionalityin large clinical trials. Standardization, validation, and optimization for highthroughput must first be established.Recently, an overview of the laboratory tests that measure HDL subclasses(shape, density) and several HDL functions was described (Eren et al. 2012).There is a growing need to identify an optimal biomarker that describes HDLfunctionality. The development of reproducible, standardized, and validatedmethods to assess HDL function for routine use is of substantial interest. Theknowledge regarding individual HDL function can help identify patients whomay benefit from HDL-C increasing therapy or patients with a normal HDL-Clevel but at particularly high risk for cardiovascular events.High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 443Conclusion and PerspectiveHDL is a plasma lipoprotein with many pleiotropic protective functions in thevascular wall, including anti-atherosclerotic properties. Emerging evidence fromclinical and laboratory studies indicates that HDL-C plasma levels in humans donot adequately represent HDL function. The pleiotropic protective effects ofHDL depend on its composition, which is influenced by pathophysiologicalconditions. With the decline of renal function, HDL modifications occur. Evi-dence suggests that HDL composition, rather than plasma level, may be animportant determinant for its pleiotropic protective function in the vascularwall. The goal is to identify robust biomarkers that describe HDL functionalityand are measurable in validated, standardized assays that can be used routinely.Improvement of HDL functionality may serve as an interesting therapeutictarget in the future for populations beyond CKD patients.Acknowledgements The authors thank Jaqueline Herrmann for proofreading the manuscript.The research projects of the authors are supported by the EFSD/Boehringer Ingelheim award(M.vdG), the Hans und Gertie Fischer-Stiftung (M.T), and the Peter und Traudl EngelhornStiftung scholarship (M.S). M.vdG is a member of the European Cooperation in Science andTechnology (COST) Action “HDLnet” (BM904).Conflict of Interest Statement None declared.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAdams V, Besler C, Fischer T, Riwanto M, Noack F, Hollriegel R, Oberbach A, Jehmlich N,Volker U, Winzer EB, Lenk K, Hambrecht R, Schuler G, Linke A, Landmesser U, Erbs S(2013) Exercise training in patients with chronic heart failure promotes restoration of high-density lipoprotein functional properties. Circ Res 113(12):1345–1355AIM-HIGH Investigators (2011) The role of niacin in raising high-density lipoprotein cholesterolto reduce cardiovascular events in patients with atherosclerotic cardiovascular disease andoptimally treated low-density lipoprotein cholesterol: baseline characteristics of studyparticipants. The atherothrombosis intervention in metabolic syndrome with low HDL/hightriglycerides: impact on global health outcomes (AIM-HIGH) trial. Am Heart J 161(3):538–543Annema W, von Eckardstein A (2013) High-density lipoproteins. Multifunctional but vulnerableprotections from atherosclerosis. Circ J 77(10):2432–2448Annema W, Nijstad N, Tolle M, de Boer JF, Buijs RV, Heeringa P, van der Giet M, Tietge UJ(2010) Myeloperoxidase and serum amyloid A contribute to impaired in vivo reverse choles-terol transport during the acute phase response but not group IIA secretory phospholipase A(2).J Lipid Res 51(4):743–754Apostolov EO, Shah SV, Ok E, Basnakian AG (2005) Quantification of carbamylated LDL inhuman sera by a new sandwich ELISA. Clin Chem 51(4):719–728444 M. Schuchardt et al.Apostolov EO, Ray D, Savenka AV, Shah SV, Basnakian AG (2010) Chronic uremia stimulatesLDL carbamylation and atherosclerosis. J Am Soc Nephrol 21(11):1852–1857Appel LJ (2004) Beyond (or back to) traditional risk factors: preventing cardiovascular disease inpatients with chronic kidney disease. Ann Intern Med 140(1):60–61Arsenault BJ, Lemieux I, Despres JP, Gagnon P, Wareham NJ, Stroes ES, Kastelein JJ, Khaw KT,Boekholdt SM (2009) HDL particle size and the risk of coronary heart disease in apparentlyhealthy men and women: the EPIC-Norfolk prospective population study. Atherosclerosis 206(1):276–281Artl A, Marsche G, Lestavel S, Sattler W, Malle E (2000) Role of serum amyloid A duringmetabolism of acute-phase HDL by macrophages. Arterioscler Thromb Vasc Biol 20(3):763–772Asselbergs FW, Diercks GF, Hillege HL, van Boven AJ, Janssen WM, Voors AA, de Zeeuw D, deJong PE, van Veldhuisen DJ, van Gilst WH (2004) Effects of fosinopril and pravastatin oncardiovascular events in subjects with microalbuminuria. Circulation 110(18):2809–2816Atthobari J, Brantsma AH, Gansevoort RT, Visser ST, Asselbergs FW, van Gilst WH, de Jong PE,de Jong-van den Berg LT (2006) The effect of statins on urinary albumin excretion andglomerular filtration rate: results from both a randomized clinical trial and an observationalcohort study. Nephrol Dial Transplant 21(11):3106–3114Badiou S, Cristol JP, Mourad G (2009) Dyslipidemia following kidney transplantation: diagnosisand treatment. Curr Diab Rep 9(4):305–311Baigent C, Keech A, Kearney PM, Blackwell L, Buck G, Pollicino C, Kirby A, Sourjina T, Peto R,Collins R, Simes R (2005) Efficacy and safety of cholesterol-lowering treatment: prospectivemeta-analysis of data from 90,056 participants in 14 randomised trials of statins. Lancet 366(9493):1267–1278Baigent C, Landray MJ, Reith C, Emberson J, Wheeler DC, Tomson C, Wanner C, Krane V,Cass A, Craig J, Neal B, Jiang L, Hooi LS, Levin A, Agodoa L, Gaziano M, Kasiske B,Walker R, Massy ZA, Feldt-Rasmussen B, Krairittichai U, Ophascharoensuk V, Fellstrom B,Holdaas H, Tesar V, Wiecek A, Grobbee D, de Zeeuw D, Gronhagen-Riska C, Dasgupta T,Lewis D, Herrington W, Mafham M, Majoni W, Wallendszus K, Grimm R, Pedersen T,Tobert J, Armitage J, Baxter A, Bray C, Chen Y, Chen Z, Hill M, Knott C, Parish S,Simpson D, Sleight P, Young A, Collins R (2011) The effects of lowering LDL cholesterolwith simvastatin plus ezetimibe in patients with chronic kidney disease (Study of Heart andRenal Protection): a randomised placebo-controlled trial. Lancet 377(9784):2181–2192Banka CL, Yuan T, de Beer MC, Kindy M, Curtiss LK, de Beer FC (1995) Serum amyloid A(SAA): influence on HDL-mediated cellular cholesterol efflux. J Lipid Res 36(5):1058–1065Baragetti A, Norata GD, Sarcina C, Rastelli F, Grigore L, Garlaschelli K, Uboldi P, Baragetti I,Pozzi C, Catapano AL (2013) High density lipoprotein cholesterol levels are an independentpredictor of the progression of chronic kidney disease. J Intern Med 274(3):252–262Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJ, Komajda M, Lopez-Sendon J,Mosca L, Tardif JC, Waters DD, Shear CL, Revkin JH, Buhr KA, Fisher MR, Tall AR, BrewerB (2007) Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med 357(21):2109–2122Bode-Boger SM, Scalera F, Kielstein JT, Martens-Lobenhoffer J, Breithardt G, Fobker M,Reinecke H (2006) Symmetrical dimethylarginine: a new combined parameter for renalfunction and extent of coronary artery disease. J Am Soc Nephrol 17(4):1128–1134Boden WE, Probstfield JL, Anderson T, Chaitman BR, Desvignes-Nickens P, Koprowicz K,McBride R, Teo K, Weintraub W (2011) Niacin in patients with low HDL cholesterol levelsreceiving intensive statin therapy. N Engl J Med 365(24):2255–2267Brouwers FP, Asselbergs FW, Hillege HL, de Boer RA, Gansevoort RT, van Veldhuisen DJ, vanGilst WH (2011) Long-term effects of fosinopril and pravastatin on cardiovascular events insubjects with microalbuminuria: ten years of follow-up of Prevention of Renal and VascularEnd-stage Disease Intervention Trial (PREVEND IT). Am Heart J 161(6):1171–1178High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 445Calabresi L, Gomaraschi M, Franceschini G (2003) Endothelial protection by high-densitylipoproteins: from bench to bedside. Arterioscler Thromb Vasc Biol 23(10):1724–1731Camont L, Chapman MJ, Kontush A (2011) Biological activities of HDL subpopulations and theirrelevance to cardiovascular disease. Trends Mol Med 17(10):594–603Cannon CP, Shah S, Dansky HM, Davidson M, Brinton EA, Gotto AM, Stepanavage M, Liu SX,Gibbons P, Ashraf TB, Zafarino J, Mitchel Y, Barter P (2010) Safety of anacetrapib in patientswith or at high risk for coronary heart disease. N Engl J Med 363(25):2406–2415Chen SC, Hung CC, Kuo MC, Lee JJ, Chiu YW, Chang JM, Hwang SJ, Chen HC (2013)Association of dyslipidemia with renal outcomes in chronic kidney disease. PLoS ONE 8(2):e55643Christoffersen C, Obinata H, Kumaraswamy SB, Galvani S, Ahnstrom J, Sevvana M, Egerer-Sieber C, Muller YA, Hla T, Nielsen LB, Dahlback B (2011) Endothelium-protective sphin-gosine-1-phosphate provided by HDL-associated apolipoprotein M. Proc Natl Acad Sci USA108(23):9613–9618Collins R, Armitage J, Parish S, Sleigh P, Peto R (2003) MRC/BHF Heart Protection Study ofcholesterol-lowering with simvastatin in 5963 people with diabetes: a randomised placebo-controlled trial. Lancet 361(9374):2005–2016Collins AJ, Foley RN, Herzog C, Chavers B, Gilbertson D, Ishani A, Johansen K, Kasiske B,Kutner N, Liu J, St Peter W, Ding S, Guo H, Kats A, Lamb K, Li S, Roberts T, Skeans M,Snyder J, Solid C, Thompson B, Weinhandl E, Xiong H, Yusuf A, Zaun D, Arko C, Chen SC,Daniels F, Ebben J, Frazier E, Hanzlik C, Johnson R, Sheets D, Wang X, Forrest B,Constantini E, Everson S, Eggers P, Agodoa L (2013) US renal data system 2012 annualdata report. Am J Kidney Dis 61(1 Suppl 1):A7, e1–e476Coresh J, Longenecker JC, Miller ER 3rd, Young HJ, Klag MJ (1998) Epidemiology of cardio-vascular risk factors in chronic renal disease. J Am Soc Nephrol 9(12 Suppl):S24–S30Dantoine TF, Debord J, Charmes JP, Merle L, Marquet P, Lachatre G, Leroux-Robert C (1998)Decrease of serum paraoxonase activity in chronic renal failure. J Am Soc Nephrol 9(11):2082–2088D’Apolito M, Du X, Zong H, Catucci A, Maiuri L, Trivisano T, Pettoello-Mantovani M,Campanozzi A, Raia V, Pessin JE, Brownlee M, Giardino I (2010) Urea-induced ROSgeneration causes insulin resistance in mice with chronic renal failure. J Clin Invest 120(1):203–213DeFronzo RA, Alvestrand A, Smith D, Hendler R, Hendler E, Wahren J (1981) Insulin resistancein uremia. J Clin Investig 67(2):563–568Di Angelantonio E, Sarwar N, Perry P, Kaptoge S, Ray KK, Thompson A, Wood AM,Lewington S, Sattar N, Packard CJ, Collins R, Thompson SG, Danesh J (2009) Major lipids,apolipoproteins, and risk of vascular disease. JAMA 302(18):1993–2000Dullaart RP, Gansevoort RT, Dikkeschei BD, de Zeeuw D, de Jong PE, Van Tol A (1993) Role ofelevated lecithin: cholesterol acyltransferase and cholesteryl ester transfer protein activities inabnormal lipoproteins from proteinuric patients. Kidney Int 44(1):91–97Duranton F, Cohen G, De Smet R, Rodriguez M, Jankowski J, Vanholder R, Argiles A (2012)Normal and pathologic concentrations of uremic toxins. J Am Soc Nephrol 23(7):1258–1270El Harchaoui K, Arsenault BJ, Franssen R, Despres JP, Hovingh GK, Stroes ES, Otvos JD,Wareham NJ, Kastelein JJ, Khaw KT, Boekholdt SM (2009) High-density lipoprotein particlesize and concentration and coronary risk. Ann Intern Med 150(2):84–93Eren E, Yilmaz N, Aydin O (2012) High density lipoprotein and it’s dysfunction. Open Biochem J6:78–93Fellstrom B, Holdaas H, Jardine AG, Svensson MK, Gottlow M, Schmieder RE, Zannad F (2009a)Cardiovascular disease in patients with renal disease: the role of statins. Curr Med Res Opin 25(1):271–285Fellstrom BC, Jardine AG, Schmieder RE, Holdaas H, Bannister K, Beutler J, Chae DW,Chevaile A, Cobbe SM, Gronhagen-Riska C, De Lima JJ, Lins R, Mayer G, McMahon AW,Parving HH, Remuzzi G, Samuelsson O, Sonkodi S, Sci D, Suleymanlar G, Tsakiris D,446 M. Schuchardt et al.Tesar V, Todorov V, Wiecek A, Wuthrich RP, Gottlow M, Johnsson E, Zannad F (2009b)Rosuvastatin and cardiovascular events in patients undergoing hemodialysis. N Engl J Med360(14):1395–1407Foley RN, Parfrey PS, Sarnak MJ (1998) Epidemiology of cardiovascular disease in chronic renaldisease. J Am Soc Nephrol 9(12 Suppl):S16–S23Frey SK, Nagl B, Henze A, Raila J, Schlosser B, Berg T, Tepel M, Zidek W, Weickert MO,Pfeiffer AF, Schweigert FJ (2008) Isoforms of retinol binding protein 4 (RBP4) are increasedin chronic diseases of the kidney but not of the liver. Lipids Health Dis 7:29Fried LF, Orchard TJ, Kasiske BL (2001) Effect of lipid reduction on the progression of renaldisease: a meta-analysis. Kidney Int 59(1):260–269Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR (1977) High density lipoproteinas a protective factor against coronary heart disease. The Framingham study. Am J Med 62(5):707–714Gutstein DE, Krishna R, Johns D, Surks HK, Dansky HM, Shah S, Mitchel YB, Arena J, WagnerJA (2012) Anacetrapib, a novel CETP inhibitor: pursuing a new approach to cardiovascularrisk reduction. Clin Pharmacol Ther 91(1):109–122Hadfield KA, Pattison DI, Brown BE, Hou L, Rye KA, Davies MJ, Hawkins CL (2013)Myeloperoxidase-derived oxidants modify apolipoprotein A-I and generate dysfunctionalhigh-density lipoproteins: comparison of hypothiocyanous acid (HOSCN) with hypochlorousacid (HOCl). Biochem J 449(2):531–542Henze A, Frey SK, Raila J, Scholze A, Spranger J, Weickert MO, Tepel M, Zidek W, SchweigertFJ (2010) Alterations of retinol-binding protein 4 species in patients with different stages ofchronic kidney disease and their relation to lipid parameters. Biochem Biophys Res Commun393(1):79–83Hirano T (2013) Abnormal lipoprotein metabolism in diabetic nephropathy. Clin Exp Nephrol 1–4Holdaas H, Fellstrom B, Jardine AG, Holme I, Nyberg G, Fauchald P, Gronhagen-Riska C,Madsen S, Neumayer HH, Cole E, Maes B, Ambuhl P, Olsson AG, Hartmann A, Solbu DO,Pedersen TR (2003) Effect of fluvastatin on cardiac outcomes in renal transplant recipients: amulticentre, randomised, placebo-controlled trial. Lancet 361(9374):2024–2031Holdaas H, Holme I, Schmieder RE, Jardine AG, Zannad F, Norby GE, Fellstrom BC (2011)Rosuvastatin in diabetic hemodialysis patients. J Am Soc Nephrol 22(7):1335–1341Holzer M, Birner-Gruenberger R, Stojakovic T, El-Gamal D, Binder V, Wadsack C,Heinemann A, Marsche G (2011a) Uremia alters HDL composition and function. J Am SocNephrol 22(9):1631–1641Holzer M, Gauster M, Pfeifer T, Wadsack C, Fauler G, Stiegler P, Koefeler H, Beubler E,Schuligoi R, Heinemann A, Marsche G (2011b) Protein carbamylation renders high-densitylipoprotein dysfunctional. Antioxid Redox Signal 14(12):2337–2346Holzer M, Zangger K, El-Gamal D, Binder V, Curcic S, Konya V, Schuligoi R, Heinemann A,Marsche G (2012) Myeloperoxidase-derived chlorinating species induce proteincarbamylation through decomposition of thiocyanate and urea: novel pathways generatingdysfunctional high-density lipoprotein. Antioxid Redox Signal 17(8):1043–1052Home PD, Pocock SJ, Beck-Nielsen H, Curtis PS, Gomis R, Hanefeld M, Jones NP, Komajda M,McMurray JJ (2009) Rosiglitazone evaluated for cardiovascular outcomes in oral agentcombination therapy for type 2 diabetes (RECORD): a multicentre, randomised, open-labeltrial. Lancet 373(9681):2125–2135Honda H, Ueda M, Kojima S, Mashiba S, Michihata T, Takahashi K, Shishido K, Akizawa T(2012) Oxidized high-density lipoprotein as a risk factor for cardiovascular events in prevalenthemodialysis patients. Atherosclerosis 220(2):493–501HPS2-THRIVE Collaborative Group (2013) HPS2-THRIVE randomized placebo-controlled trialin 25 673 high-risk patients of ER niacin/laropiprant: trial design, pre-specified muscle andliver outcomes, and reasons for stopping study treatment. Eur Heart J 34(17):1279–1291Ikewaki K (2013) In vivo kinetic studies to further understand pathogenesis of abnormal lipopro-tein metabolism in chronic kidney disease. Clin Exp Nephrol 1–4High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 447Jacobson TA (2013) Lipoprotein(a), cardiovascular disease, and contemporary management.Mayo Clin Proc 88(11):1294–1311Kalantar-Zadeh K, Kopple JD, Kamranpour N, Fogelman AM, Navab M (2007)HDL-inflammatory index correlates with poor outcome in hemodialysis patients. Kidney Int72(9):1149–1156Kaysen GA (2009) Biochemistry and biomarkers of inflamed patients: why look, what to assess.Clin J Am Soc Nephrol 4(Suppl 1):S56–S63Keane WF, Tomassini JE, Neff DR (2013) Lipid abnormalities in patients with chronic kidneydisease: implications for the pathophysiology of atherosclerosis. J Atheroscler Thromb 20(2):123–133Kennedy DJ, Wilson Tang WH, Fan Y, Wu Y, Mann S, Pepoy M, Hazen SL (2013) Diminishedantioxidant activity of high-density lipoprotein-associated proteins in chronic kidney disease. JAm Heart Assoc 2(2):e000104Khovidhunkit W, Kim MS, Memon RA, Shigenaga JK, Moser AH, Feingold KR, Grunfeld C(2004) Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanismsand consequences to the host. J Lipid Res 45(7):1169–1196Kielstein JT, Salpeter SR, Bode-Boeger SM, Cooke JP, Fliser D (2006) Symmetricdimethylarginine (SDMA) as endogenous marker of renal function–a meta-analysis. NephrolDial Transplant 21(9):2446–2451Kilpatrick RD, McAllister CJ, Kovesdy CP, Derose SF, Kopple JD, Kalantar-Zadeh K (2007)Association between serum lipids and survival in hemodialysis patients and impact of race. JAm Soc Nephrol 18(1):293–303Klin M, Smogorzewski M, Ni Z, Zhang G, Massry SG (1996) Abnormalities in hepatic lipase inchronic renal failure: role of excess parathyroid hormone. J Clin Invest 97(10):2167–2173Kobayashi S, Maesato K, Moriya H, Ohtake T, Ikeda T (2005) Insulin resistance in patients withchronic kidney disease. Am J Kidney Dis 45(2):275–280Kovesdy CP, Anderson JE, Kalantar-Zadeh K (2007) Inverse association between lipid levels andmortality in men with chronic kidney disease who are not yet on dialysis: effects of case mixand the malnutrition-inflammation-cachexia syndrome. J Am Soc Nephrol 18(1):304–311Lamprea-Montealegre JA, McClelland RL, Astor BC, Matsushita K, Shlipak M, de Boer IH, SzkloM (2013) Chronic kidney disease, plasma lipoproteins, and coronary artery calcium incidence:the Multi-Ethnic Study of Atherosclerosis. Arterioscler Thromb Vasc Biol 33(3):652–658Lapolla A, Brioschi M, Banfi C, Tremoli E, Cosma C, Bonfante L, Cristoni S, Seraglia R, Traldi P(2008) Nonenzymatically glycated lipoprotein ApoA-I in plasma of diabetic and nephropathicpatients. Ann N Y Acad Sci 1126:295–299LaRosa JC, Grundy SM, Waters DD, Shear C, Barter P, Fruchart JC, Gotto AM, Greten H,Kastelein JJ, Shepherd J, Wenger NK (2005) Intensive lipid lowering with atorvastatin inpatients with stable coronary disease. N Engl J Med 352(14):1425–1435Levey AS, Eckardt KU, Tsukamoto Y, Levin A, Coresh J, Rossert J, De Zeeuw D, Hostetter TH,Lameire N, Eknoyan G (2005) Definition and classification of chronic kidney disease: aposition statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int67(6):2089–2100Liang K, Vaziri ND (1999) Down-regulation of hepatic high-density lipoprotein receptor, SR-B1,in nephrotic syndrome. Kidney Int 56(2):621–626Liang K, Oveisi F, Vaziri ND (1998) Role of secondary hyperparathyroidism in the genesis ofhypertriglyceridemia and VLDL receptor deficiency in chronic renal failure. Kidney Int 53(3):626–630Linsel-Nitschke P, Tall AR (2005) HDL as a target in the treatment of atherosclerotic cardiovas-cular disease. Nat Rev Drug Discov 4(3):193–205Longenecker JC, Klag MJ, Marcovina SM, Liu YM, Jaar BG, Powe NR, Fink NE, Levey AS,Coresh J (2005) High lipoprotein(a) levels and small apolipoprotein(a) size prospectivelypredict cardiovascular events in dialysis patients. J Am Soc Nephrol 16(6):1794–1802448 M. Schuchardt et al.Macharia M, Hassan MS, Blackhurst D, Erasmus RT, Matsha TE (2012) The growing importanceof PON1 in cardiovascular health: a review. J Cardiovasc Med 13(7):443–453Mackey RH, Greenland P, Goff DC Jr, Lloyd-Jones D, Sibley CT, Mora S (2012) High-densitylipoprotein cholesterol and particle concentrations, carotid atherosclerosis, and coronaryevents: MESA (multi-ethnic study of atherosclerosis). J Am Coll Cardiol 60(6):508–516Madhusudhana Rao A, Anand U, Anand CV (2011) Myeloperoxidase in chronic kidney disease.Indian J Clin Biochem 26(1):28–31Marsche G, Frank S, Raynes JG, Kozarsky KF, Sattler W, Malle E (2007) The lipidation status ofacute-phase protein serum amyloid A determines cholesterol mobilization via scavengerreceptor class B, type I. Biochem J 402(1):117–124Marz W, Genser B, Drechsler C, Krane V, Grammer TB, Ritz E, Stojakovic T, Scharnagl H,Winkler K, Holme I, Holdaas H, Wanner C (2011) Atorvastatin and low-density lipoproteincholesterol in type 2 diabetes mellitus patients on hemodialysis. Clin J Am Soc Nephrol 6(6):1316–1325Miida T, Miyazaki O, Hanyu O, Nakamura Y, Hirayama S, Narita I, Gejyo F, Ei I, Tasaki K,Kohda Y, Ohta T, Yata S, Fukamachi I, Okada M (2003) LCAT-dependent conversion ofprebeta1-HDL into alpha-migrating HDL is severely delayed in hemodialysis patients. J AmSoc Nephrol 14(3):732–738Mimic-Oka J, Simic T, Djukanovic L, Reljic Z, Davicevic Z (1999) Alteration in plasma antioxi-dant capacity in various degrees of chronic renal failure. Clin Nephrol 51(4):233–241Miyamoto Y, Watanabe H, Otagiri M, Maruyama T (2011) New insight into the redox propertiesof uremic solute indoxyl sulfate as a pro- and anti-oxidant. Ther Apher Dial 15(2):129–131Mizobuchi M, Towler D, Slatopolsky E (2009) Vascular calcification: the killer of patients withchronic kidney disease. J Am Soc Nephrol 20(7):1453–1464Moe SM, Sprague SM (2012) Chronic kidney disease—mineral bone disorder. In: Taal MW(ed) Brenner and Rector’s the kidney, 9th edn. Elsevier, Philadelphia, pp 2021–2058Moradi H, Pahl MV, Elahimehr R, Vaziri ND (2009) Impaired antioxidant activity of high-densitylipoprotein in chronic kidney disease. Transl Res 153(2):77–85Moradi H, Sica DA, Kalantar-Zadeh K (2013) Cardiovascular burden associated with uremictoxins in patients with chronic kidney disease. Am J Nephrol 38(2):136–148Mori K, Shioi A, Jono S, Nishizawa Y, Morii H (1999) Dexamethasone enhances In vitro vascularcalcification by promoting osteoblastic differentiation of vascular smooth muscle cells.Arterioscler Thromb Vasc Biol 19(9):2112–2118Muntner P, Hamm LL, Kusek JW, Chen J, Whelton PK, He J (2004) The prevalence of nontradi-tional risk factors for coronary heart disease in patients with chronic kidney disease. Ann InternMed 140(1):9–17National Kidney Foundation (2003) I. Introduction. Am J Kidney Dis 41:S11–S21Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, Valente AJ, Berliner JA,Drinkwater DC, Laks H et al (1991) Monocyte transmigration induced by modification oflow density lipoprotein in cocultures of human aortic wall cells is due to induction of monocytechemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest 88(6):2039–2046Navab M, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Fogelman AM (2006)Mechanisms of disease: proatherogenic HDL–an evolving field. Nat Clin Pract EndocrinolMetab 2(9):504–511Navab M, Reddy ST, Van Lenten BJ, Fogelman AM (2011) HDL and cardiovascular disease:atherogenic and atheroprotective mechanisms. Nat Rev Cardiol 8(4):222–232Nicholls SJ, Zheng L, Hazen SL (2005) Formation of dysfunctional high-density lipoprotein bymyeloperoxidase. Trends Cardiovasc Med 15(6):212–219Nofer JR, van der Giet M, Tolle M, Wolinska I, von Wnuck LK, Baba HA, Tietge UJ, Godecke A,Ishii I, Kleuser B, Schafers M, Fobker M, Zidek W, Assmann G, Chun J, Levkau B (2004)HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J ClinInvest 113(4):569–581High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 449Nofer JR, Bot M, Brodde M, Taylor PJ, Salm P, Brinkmann V, van Berkel T, Assmann G, BiessenEA (2007) FTY720, a synthetic sphingosine 1 phosphate analogue, inhibits development ofatherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation 115(4):501–508Odden MC, Tager IB, Gansevoort RT, Bakker SJ, Fried LF, Newman AB, Katz R, Satterfield S,Harris TB, Sarnak MJ, Siscovick D, Shlipak MG (2013) Hypertension and low HDL choles-terol were associated with reduced kidney function across the age spectrum: a collaborativestudy. Ann Epidemiol 23(3):106–111Pahl MV, Ni Z, Sepassi L, Moradi H, Vaziri ND (2009) Plasma phospholipid transfer protein,cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase in end-stage renaldisease (ESRD). Nephrol Dial Transplant 24(8):2541–2546Peev V, Nayer A, Contreras G (2014) Dyslipidemia, malnutrition, inflammation, cardiovasculardisease and mortality in chronic kidney disease. Curr Opin Lipidol 25(1):54–60Puchades MJ, Saez G, Munoz MC, Gonzalez M, Torregrosa I, Juan I, Miguel A (2013) Study ofoxidative stress in patients with advanced renal disease and undergoing either hemodialysis orperitoneal dialysis. Clin Nephrol 80(3):177–186Rahman M, Baimbridge C, Davis BR, Barzilay J, Basile JN, Henriquez MA, Huml A, Kopyt N,Louis GT, Pressel SL, Rosendorff C, Sastrasinh S, Stanford C (2008) Progression of kidneydisease in moderately hypercholesterolemic, hypertensive patients randomized to pravastatinversus usual care: a report from the antihypertensive and lipid-lowering treatment to preventheart attack trial (ALLHAT). Am J Kidney Dis 52(3):412–424Ribeiro S, Faria Mdo S, Silva G, Nascimento H, Rocha-Pereira P, Miranda V, Vieira E, Santos R,Mendonca D, Quintanilha A, Costa E, Belo L, Santos-Silva A (2012) Oxidized low-densitylipoprotein and lipoprotein(a) levels in chronic kidney disease patients under hemodialysis:influence of adiponectin and of a polymorphism in the apolipoprotein(a) gene. Hemodial Int 16(4):481–490Rubins HB, Robins SJ, Iwane MK, Boden WE, Elam MB, Fye CL, Gordon DJ, Schaefer EJ,Schectman G, Wittes JT (1993) Rationale and design of the Department of Veterans AffairsHigh-Density Lipoprotein Cholesterol Intervention Trial (HIT) for secondary prevention ofcoronary artery disease in men with low high-density lipoprotein cholesterol and desirablelow-density lipoprotein cholesterol. Am J Cardiol 71(1):45–52Sahebkar A, Chew GT, Watts GF (2014) New peroxisome proliferator-activated receptor agonists:potential treatments for atherogenic dyslipidemia and non-alcoholic fatty liver disease. ExpertOpin Pharmacother 15(4):493–503Saland JM, Ginsberg HN (2007) Lipoprotein metabolism in chronic renal insufficiency. PediatrNephrol 22(8):1095–1112Samouilidou EC, Karpouza AP, Kostopoulos V, Bakirtzi T, Pantelias K, Petras D, Tzanatou-Exarchou H, Grapsa EJ (2012) Lipid abnormalities and oxidized LDL in chronic kidneydisease patients on hemodialysis and peritoneal dialysis. Ren Fail 34(2):160–164Sandhu S, Wiebe N, Fried LF, Tonelli M (2006) Statins for improving renal outcomes: a meta-analysis. J Am Soc Nephrol 17(7):2006–2016Scandinavian Simvastatin Survival Study Group (1994) Randomised trial of cholesterol loweringin 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study(4S). Lancet 344(8934):1383–1389Schmidt S, Westhoff TH, Krauser P, Zidek W, van der Giet M (2008) The uraemic toxinphenylacetic acid increases the formation of reactive oxygen species in vascular smoothmuscle cells. Nephrol Dial Transplant 23(1):65–71Schuchardt M, Tolle M, Prufer J, van der Giet M (2011) Pharmacological relevance and potentialof sphingosine 1-phosphate in the vascular system. Br J Pharmacol 163(6):1140–1162Schwartz GG, Olsson AG, Ballantyne CM, Barter PJ, Holme IM, Kallend D, Leiter LA,Leitersdorf E, McMurray JJ, Shah PK, Tardif JC, Chaitman BR, Duttlinger-Maddux R,Mathieson J (2009) Rationale and design of the dal-OUTCOMES trial: efficacy and safety ofdalcetrapib in patients with recent acute coronary syndrome. Am Heart J 158(6):896–901 e893450 M. Schuchardt et al.Schwartz GG, Olsson AG, Abt M, Ballantyne CM, Barter PJ, Brumm J, Chaitman BR, Holme IM,Kallend D, Leiter LA, Leitersdorf E, McMurray JJ, Mundl H, Nicholls SJ, Shah PK, Tardif JC,Wright RS (2012) Effects of dalcetrapib in patients with a recent acute coronary syndrome. NEngl J Med 367(22):2089–2099Sever PS, Dahlof B, Poulter NR, Wedel H, Beevers G, Caulfield M, Collins R, Kjeldsen SE,Kristinsson A, McInnes GT, Mehlsen J, Nieminen M, O’Brien E, Ostergren J (2003) Preven-tion of coronary and stroke events with atorvastatin in hypertensive patients who have averageor lower-than-average cholesterol concentrations, in the Anglo-Scandinavian CardiacOutcomes Trial--Lipid Lowering Arm (ASCOT-LLA): a multicentre randomised controlledtrial. Lancet 361(9364):1149–1158Shah AS, Tan L, Long JL, Davidson WS (2013) Proteomic diversity of high density lipoproteins:our emerging understanding of its importance in lipid transport and beyond. J Lipid Res 54(10):2575–2585Shoji T, Nishizawa Y, Nishitani H, Yamakawa M, Morii H (1992) Impaired metabolism of highdensity lipoprotein in uremic patients. Kidney Int 41(6):1653–1661Sirpal S (2009) Myeloperoxidase-mediated lipoprotein carbamylation as a mechanistic pathwayfor atherosclerotic vascular disease. Clin Sci (Lond) 116(9):681–695Sorrentino SA, Besler C, Rohrer L, Meyer M, Heinrich K, Bahlmann FH, Mueller M, Horvath T,Doerries C, Heinemann M, Flemmer S, Markowski A, Manes C, Bahr MJ, Haller H, vonEckardstein A, Drexler H, Landmesser U (2010) Endothelial-vasoprotective effects of high-density lipoprotein are impaired in patients with type 2 diabetes mellitus but are improved afterextended-release niacin therapy. Circulation 121(1):110–122Speer T, Rohrer L, Blyszczuk P, Shroff R, Kuschnerus K, Krankel N, Kania G, Zewinger S,Akhmedov A, Shi Y, Martin T, Perisa D, Winnik S, Muller MF, Sester U, Wernicke G, Jung A,Gutteck U, Eriksson U, Geisel J, Deanfield J, von Eckardstein A, Luscher TF, Fliser D,Bahlmann FH, Landmesser U (2013) Abnormal high-density lipoprotein induces endothelialdysfunction via activation of Toll-like receptor-2. Immunity 38(4):754–768Studer M, Briel M, Leimenstoll B, Glass TR, Bucher HC (2005) Effect of different antilipidemicagents and diets on mortality: a systematic review. Arch Intern Med 165(7):725–730Sung CC, Hsu YC, Chen CC, Lin YF,Wu CC (2013) Oxidative stress and nucleic acid oxidation inpatients with chronic kidney disease. Oxid Med Cell Longev 2013:301982Svensson M, Schmidt EB, Jorgensen KA, Christensen JH (2006) N-3 fatty acids as secondaryprevention against cardiovascular events in patients who undergo chronic hemodialysis: arandomized, placebo-controlled intervention trial. Clin J Am Soc Nephrol 1(4):780–786The long-term intervention with pravastatin in ischaemic disease (LIPID) study group (1998)Prevention of cardiovascular events and death with pravastatin in patients with coronary heartdisease and a broad range of initial cholesterol levels. N Engl J Med 339(19):1349–1357Theilmeier G, Schmidt C, Herrmann J, Keul P, Schafers M, Herrgott I, Mersmann J, Larmann J,Hermann S, Stypmann J, Schober O, Hildebrand R, Schulz R, Heusch G, Haude M, vonWnuckLK, Herzog C, Schmitz M, Erbel R, Chun J, Levkau B (2006) High-density lipoproteins andtheir constituent, sphingosine-1-phosphate, directly protect the heart against ischemia/reperfu-sion injury in vivo via the S1P3 lysophospholipid receptor. Circulation 114(13):1403–1409Thompson GR, Seed M (2013) Lipoprotein(a): the underestimated cardiovascular risk factor.Heart 100(7):534–535Tolle M, Huang T, Schuchardt M, Jankowski V, Prufer N, Jankowski J, Tietge UJ, Zidek W, vander Giet M (2012) High-density lipoprotein loses its anti-inflammatory capacity by accumula-tion of pro-inflammatory-serum amyloid A. Cardiovasc Res 94(1):154–162Tonelli M, Wanner C (2013) Lipid management in chronic kidney disease: synopsis of the kidneydisease: improving global outcomes 2013 clinical practice guideline. Ann Intern Med 160(3):182–189Tonelli M, Collins D, Robins S, Bloomfield H, Curhan GC (2004) Gemfibrozil for secondaryprevention of cardiovascular events in mild to moderate chronic renal insufficiency. Kidney Int66(3):1123–1130High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 451Tonelli M, Wiebe N, Culleton B, House A, Rabbat C, Fok M, McAlister F, Garg AX (2006)Chronic kidney disease and mortality risk: a systematic review. J Am Soc Nephrol 17(7):2034–2047Tucker PS, Dalbo VJ, Han T, Kingsley MI (2013) Clinical and research markers of oxidative stressin chronic kidney disease. Biomarkers 18(2):103–115Uhlar CM,Whitehead AS (1999) Serum amyloid A, the major vertebrate acute-phase reactant. EurJ Biochem 265(2):501–523Undurti A, Huang Y, Lupica JA, Smith JD, DiDonato JA, Hazen SL (2009) Modification of highdensity lipoprotein by myeloperoxidase generates a pro-inflammatory particle. J Biol Chem284(45):30825–30835van Capelleveen JC, Brewer HB, Kastelein JJ, Hovingh GK (2014) Novel therapies focused on thehigh-density lipoprotein particle. Circ Res 114(1):193–204van der Giet M, Tölle M, Pratico D, Lufft V, Schuchardt M, Hörl M, Zidek W, Tietge UF (2010a)Increased type IIA secretory phospholipase A2 expression contributes to oxidative stress inend-stage renal disease. J Mol Med 88(1):75–83van der Giet M, Schuchardt M, Huang T, Prüfer N, Prüfer J, Tölle M (2010b) Dysfunctional HDLfrom patients with end-stage renal disease recover after successful renal transplantation. Am JTransplant 10(Abstract #50):53van der Westhuyzen DR, Cai L, de Beer MC, de Beer FC (2005) Serum amyloid A promotescholesterol efflux mediated by scavenger receptor B-I. J Biol Chem 280(43):35890–35895Van Lenten BJ, Hama SY, de Beer FC, Stafforini DM, McIntyre TM, Prescott SM, La Du BN,Fogelman AM, Navab M (1995) Anti-inflammatory HDL becomes pro-inflammatory duringthe acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wallcell cocultures. J Clin Invest 96(6):2758–2767Vaziri ND (2006) Dyslipidemia of chronic renal failure: the nature, mechanisms, and potentialconsequences. Am J Physiol 290(2):F262–F272Vaziri ND (2013) Role of dyslipidemia in impairment of energy metabolism, oxidative stress,inflammation and cardiovascular disease in chronic kidney disease. Clin Exp Nephrol 1–4Vaziri ND, Norris K (2011) Lipid disorders and their relevance to outcomes in chronic kidneydisease. Blood Purif 31(1–3):189–196Vaziri ND, Deng G, Liang K (1999) Hepatic HDL receptor, SR-B1 and Apo A-I expression inchronic renal failure. Nephrol Dial Transplant 14(6):1462–1466Vaziri ND, Moradi H, Pahl MV, Fogelman AM, Navab M (2009) In vitro stimulation of HDL anti-inflammatory activity and inhibition of LDL pro-inflammatory activity in the plasma ofpatients with end-stage renal disease by an apoA-1 mimetic peptide. Kidney Int 76(4):437–444Vaziri ND, Yuan J, Ni Z, Nicholas SB, Norris KC (2012) Lipoprotein lipase deficiency in chronickidney disease is accompanied by down-regulation of endothelial GPIHBP1 expression. ClinExp Nephrol 16(2):238–243Vickers KC, Remaley AT (2014) HDL and cholesterol: life after the divorce? J Lipid Res 55(1):4–12Wang F, Zheng J, Ye P, Luo L, Bai Y, Xu R, Sheng L, Xiao T, Wu H (2013) Association of high-density lipoprotein cholesterol with the estimated glomerular filtration rate in a community-based population. PLoS ONE 8(11):e79738Wanner C, Krane V, Marz W, Olschewski M, Mann JF, Ruf G, Ritz E (2005) Atorvastatin inpatients with type 2 diabetes mellitus undergoing hemodialysis. N Engl J Med 353(3):238–248Watson AD, Berliner JA, Hama SY, La Du BN, Faull KF, Fogelman AM, Navab M (1995)Protective effect of high density lipoprotein associated paraoxonase. Inhibition of thebiological activity of minimally oxidized low density lipoprotein. J Clin Invest 96(6):2882–2891Watson CE, Weissbach N, Kjems L, Ayalasomayajula S, Zhang Y, Chang I, Navab M, Hama S,Hough G, Reddy ST, Soffer D, Rader DJ, Fogelman AM, Schecter A (2011) Treatment ofpatients with cardiovascular disease with L-4 F, an apo-A1 mimetic, did not improve selectbiomarkers of HDL function. J Lipid Res 52(2):361–373452 M. Schuchardt et al.Weichhart T, Kopecky C, Kubicek M, Haidinger M, Doller D, Katholnig K, Suarna C, Eller P,Tolle M, Gerner C, Zlabinger GJ, van der Giet M, Horl WH, Stocker R, Saemann MD (2012)Serum amyloid A in uremic HDL promotes inflammation. J Am Soc Nephrol 23(5):934–947Yamamoto S, Yancey PG, Ikizler TA, Jerome WG, Kaseda R, Cox B, Bian A, Shintani A, FogoAB, Linton MF, Fazio S, Kon V (2012) Dysfunctional high-density lipoprotein in patients onchronic hemodialysis. J Am Coll Cardiol 60(23):2372–2379Yeung CK, Shen DD, Thummel KE, Himmelfarb J (2013) Effects of chronic kidney disease anduremia on hepatic drug metabolism and transport. Kidney Int 85:522–528Zheng L, Nukuna B, Brennan ML, Sun M, Goormastic M, Settle M, Schmitt D, Fu X, Thomson L,Fox PL, Ischiropoulos H, Smith JD, Kinter M, Hazen SL (2004) Apolipoprotein A-I is aselective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjectswith cardiovascular disease. J Clin Invest 114(4):529–541Zimmermann J, Herrlinger S, Pruy A, Metzger T, Wanner C (1999) Inflammation enhancescardiovascular risk and mortality in hemodialysis patients. Kidney Int 55(2):648–658Zuo Y, Yancey P, Castro I, Khan WN, Motojima M, Ichikawa I, Fogo AB, Linton MF, Fazio S,Kon V (2009) Renal dysfunction potentiates foam cell formation by repressing ABCA1.Arterioscler Thromb Vasc Biol 29(9):1277–1282High-Density Lipoprotein: Structural and Functional Changes Under Uremic. . . 453Impact of Systemic Inflammationand Autoimmune Diseases on apoA-Iand HDL Plasma Levels and FunctionsFabrizio Montecucco, Elda Favari, Giuseppe Danilo Norata,Nicoletta Ronda, Jerzy-Roch Nofer, and Nicolas VuilleumierContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4572 HDL/apoA-I Structure and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4583 HDL and apoA-I as Members of the Innate Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4594 Relationship Between Lipid Raft Modulation and Lymphocyte Function . . . . . . . . . . . . . . . 4605 Modulation of Spinghosine-1-Phosphate (S1P)/S1P-Receptor Axis and LymphocyteFunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4626 Inflammatory Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464F. MontecuccoDivision of Laboratory Medicine, Department of Genetics and Laboratory Medicine,Geneva University Hospitals, 4 rue Gabrielle Perret-Gentil, 1211 Geneva, SwitzerlandDepartment of Human Protein Science, Geneva, SwitzerlandDivision of Cardiology, Geneva University Hospital, Geneva, SwitzerlandE. Favari • N. RondaDepartment of Pharmacy, University of Parma, Parma, ItalyG.D. NorataDepartment of Pharmacological and Biomolecular Sciences, Università degli Studidi Milano, Milan, ItalyCenter for the Study of Atherosclerosis, Società Italiana Studio Aterosclerosi,Ospedale Bassini, Cinisello Balsamo, ItalyThe Blizard Institute, Centre for Diabetes, Barts and The London School ofMedicine & Dentistry, Queen Mary University, London, UKJ.-R. NoferCenter for Laboratory Medicine, University Hospital Münster, Münster, GermanyN. Vuilleumier (*)Division of Laboratory Medicine, Department of Genetics and Laboratory Medicine,Geneva University Hospitals, 4 rue Gabrielle Perret-Gentil, 1211 Geneva, SwitzerlandDepartment of Human Protein Science, Geneva, Switzerlande-mail: nicolas.vuilleumier@hcuge.ch# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_144556.1 Inflammatory Bowel Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4646.2 Vasculitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4646.3 Psoriasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4667 Autoimmune Diseases: The Role of Autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4677.1 Systemic Lupus Erythematosus (SLE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4677.2 Rheumatoid Arthritis (RA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4688 Impact of Anti-inflammatory Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472AbstractThe cholesterol of high-density lipoproteins (HDLs) and its major proteiccomponent, apoA-I, have been widely investigated as potential predictors ofacute cardiovascular (CV) events. In particular, HDL cholesterol levels wereshown to be inversely and independently associated with the risk of acute CVdiseases in different patient populations, including autoimmune and chronicinflammatory disorders. Some relevant and direct anti-inflammatory activitiesof HDL have been also recently identified targeting both immune and vascularcell subsets. These studies recently highlighted the improvement of HDL func-tion (instead of circulating levels) as a promising treatment strategy to reduceinflammation and associated CV risk in several diseases, such as systemic lupuserythematosus and rheumatoid arthritis. In these diseases, anti-inflammatorytreatments targeting HDL function might improve both disease activity andCV risk. In this narrative review, we will focus on the pathophysiologicalrelevance of HDL and apoA-I levels/functions in different acute and chronicinflammatory pathophysiological conditions.KeywordsSystemic inflammation • Autoimmune disease • apoA-I • HDL • InnateimmunityAbbreviationsABCA ATP-binding cassette transportersABCG1 ATP-binding cassette G1APC Antigen-presenting cellsapoA-I Apolipoprotein A-1APR Acute-phase responseBD Behçet’s diseaseCAD Coronary artery diseaseCD Crohn’s diseaseCVD Cardiovascular diseasesEBV Epstein-Barr virusHDL-C High-density lipoprotein cholesterolhs-CRP High-sensitivity C-reactive protein456 F. Montecucco et al.IBD Inflammatory bowel diseasesKD Kawasaki diseaseLPL Lipoprotein lipaseLp-PLA2 Lipoprotein-specific phospholipase A2LPS LipopolysaccharideLTA Lipoteichoic acidMPO MyeloperoxidasepiHDL Proinflammatory HDLPON1 Paraoxonase 1RA Rheumatoid arthritisSAA Serum amyloid ASLE Systemic lupus erythematosusSMAD Small mothers against decapentaplegicSR-BI Scavenger receptor BITh1 T helper type 1TGF-β Transforming growth factor betaUC Ulcerative colitis1 IntroductionSince the seminal publication by Miller and coworkers in 1975, HDL-C hasconsistently been shown to be inversely and independently associated with therisk of acute CVD (such as myocardial infarction) (Miller and Miller 1975; Castelliet al. 1986; Di Angelantonio et al. 2009) and became one of the most commonlymeasured biomarkers (Expert Panel 2001). Nevertheless, the recent failures ofHDL-C-raising therapies to reduce CV complications and atherosclerosis(Davidson 2012) as well as Mendelian randomization studies to demonstrateassociations of genetically determined HDL-C levels with altered CV risk (Voightet al. 2012; Shah et al. 2013; Haase et al. 2012) raised the question whether HDL isan innocent bystander or a mediator of atherogenesis and CVD.Among the important progresses in our understanding about HDL physiopathol-ogy, many studies stress the importance of HDL functions rather than HDL-C bloodlevels. This in turn could be of key importance to understand the negative outcomeof both interventional trials and Mendelian randomization studies which did nottake HDL functionality into account. Indeed, there is a growing body of evidenceindicating that both acute and chronic inflammatory conditions induce posttransla-tional modifications of HDL, impairing both its lipid homoeostasis-regulating andanti-inflammatory properties, and even turn HDL into a proinflammatory molecule(Navab et al. 2011).Those observations are shifting the attention from HDL-C levels to HDLfunction, emphasizing the importance of taking into account the clinical situationsin which HDL-C levels are measured. Therefore, the precise knowledge of theImpact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 457different chronic or acute conditions susceptible to affect both the quantity andquality of HDL is likely to be paramount in order to understand the actual contro-versy about the causal role of HDL in CVD. Furthermore, because many of thefunctional properties of HDL can be recapitulated by apolipoprotein A-1 (apoA-I)(Phillips 2013), we will review the different acute and chronic inflammatorypathophysiological conditions reported to affect both HDL and apoA-I levels andfunctions, as potential modulators of the innate immune system.2 HDL/apoA-I Structure and FunctionSince HDL structure is predominantly affected by the presence of apoA-I (Phillips2013), both molecules will be presented in a joint manner. Depending on the lipidstate of apolipoproteins, HDL molecules are heterogeneous in shape, size, anddensity. The predominant HDL species are spherical HDL, consisting of a core ofcholesteryl ester (CE) and triacylglycerol, encapsulated by a monolayer ofphospholipids, unesterified cholesterol, and different lipoproteins. The latter ismostly represented by apoA-I which constitutes the major protein fraction ofHDL and represents up to 80 % of HDL mass (Heinecke 2009). The second moreabundant lipoprotein of HDL is apoA-II, followed by less abundant proteinsincluding apoCs, D, E, J, and M; several enzymes such as lecithin-cholesterolacyltransferase (LCAT), serum paraoxonase 1 (PON1), and platelet-activatingfactor acetylhydrolase (PAF-AH); and sphingosine-1-phosphate (S1P) (see chapter“Structure of HDL: Particle Subclasses and Molecular Components” for moredetails).The atheroprotective role of HDL on the cardiovascular system has beenattributed to the pleiotropic effects of HDL, including reverse cholesterol transport,vasodilatation, antithrombotic, anticoagulant, and anti-inflammatory effects(Gordon and Davidson 2012). Reflecting those versatile properties, mass spectrom-etry analyses revealed that HDL encompasses up to 80 different proteins.Two-thirds of them are either acute-phase proteins, proteases, antioxidants,antithrombotic enzymes, or proteins involved in complement regulation, and onlyone-third are dedicated to lipid transport (Heinecke 2009).On top of being the major protein fraction of HDL and a limiting factor for HDLformation, apoA-I per se executes many of the HDL-related properties, rangingfrom reverse cholesterol efflux and LPS and LTA scavenging to the inhibition ofdifferent proinflammatory, pro-oxidant, and prothrombotic pathways(Gu et al. 2000; Thuahnai et al. 2003; Yuhanna et al. 2001; Vuilleumieret al. 2013). Thus, HDL and apoA-I appear to contribute to host defense againstmany biological and chemical hazards. Nevertheless, chronic or acute inflammationinduces major changes in HDL-C levels and functions, compromising andperverting the protective activities of HDL to harm.Infection and inflammation generate an acute-phase response (APR), leading toimportant changes affecting the metabolism of different lipids and lipoproteins andthereby resulting in increased plasma levels of triglycerides and very-low-density458 F. Montecucco et al.lipoprotein (VLDL) cholesterol (reviewed in Khovidhunkit et al. 2004). Concomi-tantly, the APR also induces major changes in HDL functions since in this contextapoA-I is replaced by other acute-phase proteins, such as SAA protein, ceruloplas-min, and haptoglobin (Navab et al. 2011), with SAA representing up to 87 % ofHDL proteins and transforming HDL into a proinflammatory molecule (Van Lentenet al. 1995). Furthermore, recent evidence suggests that APR can also inducespecific posttranslational modifications, such as chlorination, nitration, andcarbamylation of amino acids by myeloperoxidase (MPO), oxidation by reactivecarbonyls, as well as glycation, which compromise HDL and apoA-I functions,perverting them to harm (Vuilleumier et al. 2013).3 HDL and apoA-I as Members of the Innate Immune SystemThe innate immune system represents the first line of defense against infectiousagents and modified self-antigens (modified/oxidized lipids and apoptotic cells)consisting of a cellular and a humoral part. Cellular components are representedmostly by antigen-presenting cells (APCs), such as macrophages and dendriticcells, whereas the humoral counterpart is mostly represented by the pentraxinfamily and the complement system. Being extensively conserved throughout theevolution, and existing in early evolutionary species not affected by CVD (Babinet al. 1997), HDLs are believed to be part of the humoral innate immune system,helping mammals to fight against invading pathogens. Indeed, thanks to the pres-ence of different proteins on HDL molecules, such as apoA-I, apoL-1, andhaptoglobin-related proteins, HDLs are known to behave as antimicrobial agentsprotecting mammals against different parasites and bacteria, such as Trypanosomabrucei, Escherichia coli, and Klebsiella pneumoniae (Shiflett et al. 2005; Becket al. 2013) (see chapter “HDL in Infectious Diseases and Sepsis”).Furthermore, on top of interfering directly with invading pathogens, HDL andapoA-I also modulate the APR-induced activation of the innate immune system byboth neutralizing major bacterial membrane components, such as LPS of Gram-negative bacteria and LTA of Gram-positive bacteria, and modulating theproinflammatory signaling at the level of innate receptors, such as Toll-likereceptors (TLRs), scavenger receptors (SRs), and Nod-like receptors (NLRs).Mathison and colleagues demonstrated for the first time in 1979 that HDL couldreduce LPS toxicity in vivo (Mathison and Ulevitch 1979). This observation wassubsequently inferred to the ability of HDL to sequester LPS, preventing the latterto elicit a proinflammatory response through TLR4/CD14 complex interaction(Levine et al. 1993). Later on, this scavenging effect of HDL was attributed to aspecific apoA-I region on the N-terminal segment of apoA-I (aa: 52–74) (Wanget al. 2008). More recently, apoA-I was shown to have a similar ability to bind toLTA and to neutralize its proinflammatory effect (Jiao and Wu 2008). Interestingly,this effect was much weaker with HDL, suggesting that apoA-I is the key effector ofImpact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 459LTA scavenging (Grunfeld et al. 1999). The integrity of innate immune receptorsignaling is largely dependent on their localization into lipid rafts, as well as on theintegrity of the latter (Triantafilou et al. 2002). In this respect, apoA-I has beenshown to deplete cholesterol from lipid rafts, leading to a decrease in TLR4functionality, followed by an inhibition of LPS-induced inflammatory responses(Triantafilou et al. 2002). In the same line of thought, on top of altering the lipid raftcomposition, apoA-I has also been shown to impede TLR4 transport into lipid rafts,thereby preventing its ability to promote an efficient proinflammatory response(Smythies et al. 2010; Cheng et al. 2012). These findings indicate that apoA-I andHDL, albeit to a lesser extent, can effectively modulate TLR activity at thepreceptor, receptor, and post-receptor levels (De Nardo et al. 2014).4 Relationship Between Lipid Raft Modulationand Lymphocyte FunctionAdaptive immune response relies on the activation of lymphocytes and the expan-sion of specific subsets in response to antigens. B lymphocytes represent anessential component of the humoral adaptive response through the synthesis ofimmunoglobulins, while T lymphocytes play a central role in cell-mediated immu-nity, and indeed the different T phenotypes orchestrate the immune responsethrough different mechanisms. Two main aspects suggest a link betweenlipoproteins and adaptive immunity: first, the key receptors of B and T cells(BCR and TCR, respectively) are located in the lipid rafts, and their activity ismodulated upon changes in the lipid raft composition and structure (Gupta andDeFranco 2007; Kabouridis and Jury 2008; Norata et al. 2012); second, B- andT-cell trafficking and T-cell subset differentiation are controlled also by lysosphin-golipids, mainly S1P (Mandala et al. 2002; Liu et al. 2010). Lipid rafts, whichconcentrate specific proteins, thus limiting their ability to freely diffuse over theplasma membrane, act as platforms, bringing together molecules essential for theactivation of immune cells (immunological synapse), but also separating suchmolecules when the conditions for activation are not appropriate (Ehrensteinet al. 2005). Lipid rafts compartmentalize key signaling molecules during thedifferent stages of B-cell activation including BCR-initiated signal transduction,endocytosis of BCR-antigen complexes, loading of antigenic peptides onto MHCclass II molecules, MHC-II-associated antigen presentation to T cells, and receiptof helper signals via the CD40 receptor (Gupta and DeFranco 2007). Criticalregulators of BCR signaling lose their association with membrane rafts in diseaseconditions; for instance, the LMP2A gene product of the EBV constitutively residesin membrane raft of EBV-transformed human B cells and blocks the entry ofligand-clustered BCRs and BCR translocation (Longnecker and Miller 1996).Furthermore, an alteration of Lyn, an accessory protein of BCR signaling, hasbeen reported in patients with systemic lupus erythematosus (SLE) (Flores-Borja460 F. Montecucco et al.et al. 2005). It is therefore reasonable to speculate that HDL, by removing choles-terol from lipid rafts, could affect B-cell function. While a direct evidence for HDLeffects on B-cell function is still lacking, several reports indicate that HDL andapoA-I stimulate cholesterol efflux from cells, leading to cholesterol depletion anddisruption of lipid rafts, which induces profound functional changes (Smythieset al. 2010) in macrophages and also affects antigen presentation and TCR signaling(Gruaz et al. 2010; Norata and Catapano 2012).The response of T lymphocytes to antigen is orchestrated by a number ofmolecules that cluster in lipid rafts. TCR complex integrity is vital for the inductionof optimal and efficient immune responses (Baniyash 2004). In immune-mediateddisorders, such as rheumatoid arthritis (RA) and SLE, and in chronic infectiousdiseases, T cells are dysfunctional with a characteristic loss of expression of theTCRζ chain (also called CD249), a key component of the TCR complex thatcouples surface antigen recognition with intracellular signal transduction (Baniyash2004). Also other molecules associated with the TCR signaling are reduced oraltered in autoimmune disorders, such as lymphocyte-specific protein tyrosinekinase (Lck) (Jury et al. 2006). T-cell immunological synapses are altered incirculating T cells from patients with coronary artery disease (CAD), and increasedmemory T-cell subsets were observed in particular in CAD patients with increasedinflammatory markers (Ammirati et al. 2012a, b). Molecular mechanisms of howreduced levels of blood lipids can affect lipid rafts in immune-mediated disordersstill remain to be addressed. However, atorvastatin reversed many of the signalingdefects characteristic of T cells from patients with SLE (Jury et al. 2006). Thepossibility that atorvastatin targets lipid raft-associated signaling abnormalities inautoreactive T cells has been proposed as the rationale for its use in the therapy ofautoimmune disease (Jury et al. 2006). This is further supported by a large obser-vational study that demonstrated an association between persistence with statintherapy and reduced risk of developing RA (Chodick et al. 2010). More recently,Yvan-Charvet et al. reported that two key proteins involved in HDL cholesterolefflux such as ATP-binding cassette transporters ABCA1 and ABCG1 play a keyrole in hematopoietic stem and multipotential progenitor cell proliferation, thusfurther linking cholesterol efflux and lipid raft modulation to immune cell function(Yvan-Charvet et al. 2010a, b). Finally, a key role for apoA-I in controllingcholesterol-associated lymphocyte activation and proliferation in peripherallymph nodes was observed in animal models (Wilhelm et al. 2009). The prevalenceof classical CD14++/CD16- but not of intermediate CD14++/CD16+ monocytes inhypoalphalipoproteinemia should also be taken into account as could impact on thedifferent polarization on APC cells (Sala et al. 2013). More recently, Wanget al. (2012) demonstrated that HDL and apoA-I-induced cholesterol depletionand consequent disruption of plasma membrane lipid rafts in APCs inhibit theircapacity to stimulate T-cell activation. This mechanism is highly dependent on thereduction of MHC class II molecules present on the cell surface following ABCA1activation and on cholesterol efflux supporting a role for HDL in controlling alsolymphocyte-mediated responses.Impact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 4615 Modulation of Spinghosine-1-Phosphate (S1P)/S1P-Receptor Axis and Lymphocyte FunctionAmong the lipids that concentrate in lipid rafts, sphingolipids represent a majorclass that is metabolized to generate ceramide and subsequently sphingosine that inturn could be phosphorylated by sphingosine kinase (SPHK) (expressed mainly inplatelets and in other peripheral blood cells) to generate S1P (Scanu and Edelstein2008). Free or albumin-bound S1P is more susceptible to degradation than S1Pbound to HDL (Yatomi 2008), which suggests that the latter might have a role indetermining the uptake, cellular degradation, and systemic function. S1P carried byHDL positively correlates with HDL cholesterol, apoA-I, and apoA-II levels;furthermore, S1P is enriched in small dense HDL3 (Scanu and Edelstein 2008).S1P signals through five known G protein-coupled receptors (S1P1–S1P5). Overthe last few years, it become apparent that S1P and the key enzymes SPHKs play acentral role in the pathogenesis of several inflammatory disorders, including rheu-matoid arthritis, asthma, and atherosclerosis by modulating macrophage functionthrough the control of apoptosis as well as cell trafficking (Weigert et al. 2009).Many of these effects might depend on the activation of different S1P receptors.The activation of the lysosphingolipid receptor-PI3K/Akt axis by sphingosine-1-phosphate or other S1P mimetics is responsible for the induction by HDL of severalgenes involved in the immune response including the long pentraxin PTX3 or thetransforming growth factor beta 2 (Norata et al. 2005, 2008). The S1P/S1P-receptoraxis also plays a key role in lymphocyte function. The activation of S1P receptorsand the consequent downstream signaling facilitate the egress of T cells fromlymphoid organs (Mandala et al. 2002; Matloubian et al. 2004) and play a role inthe lineage determination of peripheral T cells (Liu et al. 2010). S1P inhibits thedifferentiation of forkhead box P3 (FoxP3)+ regulatory T cells (Tregs) while pro-moting the development of T helper type 1 (Th1) in a reciprocal manner (Liuet al. 2010). S1P receptor antagonizes TGF-β receptor function through an inhibi-tory effect on SMAD-3 activities to control the dichotomy between these two T-celllineages (Liu et al. 2010). In animal models, apoA-I reduces inflammation in LDLreceptor (/), apoA-I(/) mice by augmenting the effectiveness of the lymphnodes’ Treg response, with an increase in Treg and a decrease in the percentage ofeffector/effector memory T cells (Wilhelm et al. 2010). While marked changes ofTreg number/function (two- to threefold difference) have been associated withatheroprotective functions in animal models (Ait-Oufella et al. 2006; Moret al. 2007), in humans the correlation between Treg, immune, and cardiometabolicdisorders is less clear. We found no association between circulating CD3 +CD4+CD25highCD127low Treg levels and the extent or progression of human athero-sclerotic disease at carotid and coronary sites (Ammirati et al. 2010). In a series ofimmune-mediated diseases such as rheumatoid arthritis, increased Treg levels insynovial fluid of inflamed joints were observed (Mottonen et al. 2005; Bacchettaet al. 2007). Furthermore, Liu et al. observed relatively high Treg-cell levels inpatients with type 1 diabetes mellitus (Liu et al. 2006), and pathogenic T cells havebeen shown to have a paradoxical protective effect in murine autoimmune diabetes462 F. Montecucco et al.by boosting Tregs (Grinberg-Bleyer et al. 2010). Of note, excessive IL-6 orTNF-alpha production was associated with increased Treg levels (Fujimotoet al. 2011; Bilate and Lafaille 2010). This evidence suggests the possibility thatTreg numbers could increase during some stages of disease as an attempt to regulateeffector-cell activity. Unexpectedly, in the general population, we observed aninverse relation between HDL-C and Treg count (Ammirati et al. 2010); whetherthis finding suggests that HDL could influence the polarization of lymphocytesubsets or could be a bystander of the T-cell status remains to be addressed. Recentevidence on the role of S1P in immune surveillance and the discovery of regulatorymechanisms in S1P-mediated immune trafficking has prompted extensive investi-gation in the field of S1P-receptor pharmacology. Fingolimod (FTY720), anS1P-receptor modulator, prevents lymphocyte egress from lymph nodes andmodulates lymphocyte differentiation (Chi 2011). Initially, fingolimod was usedas immunosuppressant in solid organ transplantation (Tedesco-Silva et al. 2006),while MS has been the first disease in which fingolimod was tested. In MS themyelin sheaths around the axons of the brain and spinal cord are damaged byinflammatory processes, leading to demyelination. Current therapeutic approachesare focused on the suppression of the immune system and on the blockage of T-cellblood–brain barrier transmigration into the brain parenchyma. Treatment withfingolimod was effective in reducing the disability progression on a large cohortof patients with relapsing MS (Cohen et al. 2010; Kappos et al. 2010). Of note, MSpatients during the phase of clinical remission showed increased levels of HDL andtotal cholesterol levels (Salemi et al. 2010), whereas in immune-mediated disorderswith increased markers of systemic inflammation, HDL levels are often decreased.Fingolimod was assessed also in diseases with an immunological component, suchas atherosclerosis (Nofer et al. 2007; Keul et al. 2007). In LDL receptor-deficientmice, fingolimod inhibits atherosclerosis by modulating lymphocyte and macro-phage function. In this study plasma lipids remained unchanged during the courseof fingolimod treatment, whereas fingolimod lowered blood lymphocyte count(Nofer et al. 2007). As S1P levels are increased in many inflammatory conditions,such as in asthma and autoimmunity, the exact mechanism by which S1PR agonistscould modulate its function is debated (Rivera et al. 2008). Interestingly,fingolimod is highly active in inducing the internalization, ubiquitination, andsubsequent degradation of S1PR1 (Graler and Goetzl 2004) which suggests thatits inhibitory action on immune cell trafficking might be through receptordownregulation. It is therefore crucial to understand the effect of HDL containedS1P on receptor expression and activity in immune disorders.In spite of the presence of a number of experimental and clinical observationssuggesting a relation between HDL and innate immunity, several questions remainto be addressed. Are the altered HDL-C levels a consequence of the atherogenicprocess, a cause of increased atherosclerosis observed in immune disorders, orindependently related to the latter? How is HDL function altered in these diseases?Does raising HDL-C improve the outcome of immune disorders? As HDLs are areservoir for several biologically active substances that may impact the immunesystem (Norata et al. 2012), how does the fine-tuning of lipid and protein exchangeImpact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 463among lipoproteins affect HDL-related immune functions? Is there a specific HDLsubfraction that is relevant? Addressing these aspects will be critical to understandthe connection between HDL and the immune response.6 Inflammatory Diseases6.1 Inflammatory Bowel DiseasesThe inflammatory bowel diseases (IBD) Crohn’s disease (CD) and ulcerative colitis(UC) are chronic inflammatory diseases whose pathogenesis is not completelyunderstood (Williams et al. 2012) but seems to be linked to autoimmune phenomenain a genetically prone background, following an abnormal immune response tocolonic bacteria, and facilitated by a Western-type diet. IBD are associated withmarked atherosclerosis and increased cardiovascular risk. Infection and chronicinflammation impair and alter lipoprotein metabolism and cause a variety of changesin plasma concentrations of lipids and lipoproteins (Borba et al. 2006; de Carvalhoet al. 2008). An increase in inflammatory cytokines may result in a decrease in LPLenzyme activity, leading to a characteristic lipoprotein profile with decreased HDL-Clevels as seen in patients with IBD that have high circulatory levels of inflammatorycytokines (Williams et al. 2012; Sappati Biyyani et al. 2010).CD is a chronic inflammatory bowel disease that can affect any region of thegastrointestinal tract (Grand et al. 1995) with increased chronic inflammatory cellinfiltrates in the mucosal lesions. The excessive local production of solublemediators from activated monocytes and polymorphonuclear leukocytes has beenimplicated in mediating the tissue injury (Weiss 1989). Important among thesemediators are oxygen free radicals. The chronic gut inflammation promotes animbalance between oxidant and antioxidant mechanisms at the tissue level(Buffinton and Doe 1995) and may even compromise circulating antioxidantconcentrations. A chronic inflammatory state is a risk factor for accelerated athero-genesis (van Leuven et al. 2007). A recent exploratory analysis demonstrated thatCD is associated with an acceleration of the atherosclerotic process, as illustratedby an increased carotid intima-media thickness (IMT) in CD patients compared tohealthy controls. In addition, CD patients were characterized during an inflamma-tory exacerbation by profoundly decreased levels of HDL combined with biochem-ical changes of the HDL particles, such as association with serum amyloid A,suggesting that early detection of atherosclerosis and subsequent cardiovascularprevention in patients with CD might be warranted (Romanato et al. 2009).6.2 VasculitisBehçet’s disease (BD) is a systemic vasculitis, most common in the Mediterraneanarea and in Asia, which can involve nearly every organ system and results from theinterplay between infectious agent exposure and genetic factors. High production of464 F. Montecucco et al.cytokines, T and B lymphocyte activation, autoantibody production, and hyperco-agulable/prothrombotic state are all characteristics of BD. Evidence for acceleratedatherosclerosis in BD has been observed, but the relationship between cardiovas-cular risk factors and accelerated atherosclerosis in patients with BD is stillcontroversial (Messedi et al. 2011). A study published by Messedi and colleaguesdemonstrated that in BD patients, HDL concentration and their subfraction levelsare decreased. The same study reported that the percentage of HDL2 subpopulationwas also decreased and HDL3 subfraction was significantly higher. The LDL-C/HDL-C ratio and CRP level were increased, and HDL and its subfractions werecorrelated with CRP and TG levels, suggesting that all these parameters may beconsidered as important predictors of cardiovascular events in BD patients(Messedi et al. 2011). This study confirms what seen in some previous studies inwhich, compared to control subjects, BD patients were characterized by reducedlevels of HDL (Cimen et al. 2012; Musabak et al. 2005; Orem et al. 2002).Kawasaki disease (KD) is an acute vasculitis that predominantly occurs ininfancy and early childhood. It is commonly thought that KD results from theexposure of a genetically predisposed individual to an as-yet unidentified, possiblyinfectious environmental trigger. Coronary artery aneurysms or ectasia develops inapproximately 15–25 % of affected children (Dhillon et al. 1996; Newburgeret al. 1991; Cheung et al. 2004). There is increasing evidence to suggest thatchildren with a history of KD might be predisposed to premature atherosclerosisand a significant association between carotid IMT and systemic arterial stiffness inchildren after KD has been demonstrated (Cheung et al. 2007). This syndrome isassociated with significant abnormalities in lipid profile. In one of the first studieson this subject, it was shown that in the earliest days of illness, mean plasmaconcentrations of total cholesterol and HDL cholesterol are profoundly depressed,whereas mean triglyceride concentration is very high. Total cholesterol valuesrapidly return to normal and remain stable for more than 3 months after the onsetof illness. HDL concentration recovered more slowly after illness onset, and meanHDL was significantly lower than expected more than 3 years after illness onset(Newburger et al. 1991). The persistence of low HDL for many years suggests amore lasting effect of KD on endothelial function, perhaps attributable to dimin-ished activity of lipoprotein lipase. This enzyme resides on the capillary walls ofmost tissues and functions at the luminal surface of the vascular endothelium (Eckel1989). This observation is confirmed, at least in part, in later studies on the lipidderangement in KD (Cheung et al. 2004; Chiang et al. 1997; Cabana et al. 1997).The results showed that during the acute phase, the concentrations of plasmaHDL-C, apoA-I, and apoA-II were significantly reduced and the reduction ofHDL was mainly related to the lowering of esterified and unesterified cholesterolin HDL2 (Chiang et al. 1997). In parallel, another study demonstrated that the lipidchanges involved not only HDL-C concentration but also HDL composition. Theauthors showed that children with KD have extremely low serum HDL-C andapoA-I levels at the time of the acute illness and that serum amyloid A (SAA) ispresent in the acute stage and is associated mainly with HDL particles of HDL3density (Cabana et al. 1997). Moreover, a more recent study demonstrated aImpact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 465significant induction of MCP-1, CCR2, and iNOS expression in THP-1macrophages in vitro by the serum of children with a history of KD, showing thatthis induction correlated positively with serum high-sensitivity C-reactive protein(hs-CRP) and LDL and negatively with HDL-C levels (Cheung et al. 2005).6.3 PsoriasisPsoriasis is a chronic inflammatory skin disease associated with arthritis in up to40 % of cases. Genetic and environmental factors contribute to the activation oflymphocytes, particularly Th1 and Th17, monocytes/macrophages, and dendriticcells and to the high cytokine content typical of psoriatic lesions. Psoriasis isassociated with increased incidence of stroke and CVD (Yu et al. 2012) and withincreased mean carotid IMT (Troitzsch et al. 2012). The mechanism behind suchassociations is still unknown. It is possible that the chronic inflammatory environ-ment and high cytokine production typical of psoriasis induce abnormal HDLparticle composition (El Harchaoui et al. 2009; McGillicuddy et al. 2009). A similarchronic inflammatory environment is observed in psoriasis (Davidovici et al. 2010)and contributes to the increased incidence of aortic inflammation, stroke, andmyocardial infarction seen in this patient population (Mehta et al. 2011; Gelfandet al. 2006).Lipoprotein profiling by NMR spectroscopy showed a reduction of HDL inpsoriatic patients compared to controls, with an atherogenic profile with respectto HDL particles. In particular, high HDL-C concentration and large HDL size wereassociated with less aortic inflammation, while small HDL particles were moreprevalent in cases with strong inflammation. Such association persisted followingadjustment for CV risk factors, suggesting that HDL particle characteristics mayplay an important role in psoriatic vascular inflammation and CVD (Yu et al. 2012).HDL reduction in psoriatic patients has also been described to be associated to areduction of serum cholesterol efflux capacity compared to control subjects (Mehtaet al. 2012). In this study, psoriasis activity was inversely associated with HDLefflux capacity in a way depending on HDL particle size. Holzer and colleaguesdemonstrated that the protein composition of HDL is markedly altered in patientswith psoriasis. ApoA-I and apoM levels are decreased, whereas the levels of severalacute-phase proteins such as SAA, prothrombin, α-2-HS-glycoprotein, and α-1-acidglycoprotein 1 are increased (Holzer et al. 2012). Additionally, also the lipidcomposition of HDL from patients with psoriasis was altered, with a decrease intotal cholesterol, cholesterol ester, free cholesterol, phosphatidylcholine, andsphingomyelin. These investigators confirmed that HDL from patients with psoria-sis was less efficient in promoting cholesterol efflux from macrophages and that thisdefect in HDL function correlated with the severity of psoriasis. Surprisingly, theantioxidant properties of HDL were similar in control and psoriatic HDL and PONactivity was not altered. However, Lp-PLA2 activity was increased and correlatedwith disease activity (Holzer et al. 2012). This observation does not completelyagree with a report showing that the PON1 55 M allele is a risk factor for psoriasis.466 F. Montecucco et al.Carriers of this allele have high levels of apoB and Lp(a) and a high apoB/apoA-Iratio, indicating that oxidative stress, impairment of the antioxidant system, andabnormal lipid metabolism may play a role in the pathogenesis and progression ofpsoriasis and its related complications (Asefi et al. 2012). Moreover, the paper byHolzer and coworkers demonstrates that a relatively mild chronic inflammatorystate can similarly result in dysfunctional HDL, leading to decreased cholesterolefflux from macrophages (Holzer et al. 2012).7 Autoimmune Diseases: The Role of Autoantibodies7.1 Systemic Lupus Erythematosus (SLE)SLE is a systemic autoimmune disease of multifactorial origin, in which geneticand environmental factors induce innate and acquired immunity derangement, withtype I interferon (INF) production, T- and B-cell dysregulation, autoantibodyproduction, and finally multiple organ damage. SLE is associated with acceleratedatherosclerosis and markedly increased cardiovascular risk (CVR) (Shoenfeldet al. 2005). As in other autoimmune disorders, CVR cannot be fully explainedby traditional risk factors, and various specific immune and inflammatorymechanisms have been demonstrated or proposed. The modifications of HDLlevel and function occurring in SLE have been indicated as possible factorscontributing to cardiovascular damage.Decreased circulating HDL-C levels have been often reported, although notunanimously, in SLE patients with active disease (de Carvalho et al. 2008; Hahnet al. 2008; Kiss et al. 2007), but the actual relevance of this finding with respect toCVR has not been clarified so far. The most important pro-atherogenicmodifications seem to be those relative to composition and function of HDL.HDLs in SLE have impaired anti-inflammatory and antioxidant properties. InSLE, the so-called proinflammatory HDL (piHDL) can be detected in as many as45 % of patients, correlating with increased oxidized LDL formation, carotid plaque,and carotid intima-media thickness (cIMT) (McMahon et al. 2011). piHDL arecharacterized by decreased content in the protective proteins paraoxonase (PON1)and apoA-I and by markedly increased levels of the pro-oxidant SAA (Hahnet al. 2008). These HDLs can be defined as proinflammatory because they actuallyenhance the oxidation of LDL and therefore monocyte attraction/activation, anti-oxLDL antibody production, and immune complex formation (Hahn et al. 2008;Teixeira et al. 2012; Carbone et al. 2013; Vuilleumier et al. 2014). It has been shownthat the reduced activity of PON1 in some cases may be due also to the action of anti-apoA-I and anti-HDL antibodies (O’Neill et al. 2010; Batuca et al. 2007). Anti-apoA-I antibodies, first described in SLE patients but detected also in patients with acutecoronary syndrome, have shown independent association and predictive value withrespect to cardiovascular events in various patient populations (Carbone et al. 2013).Proposed mechanisms for such association include the ability of anti-apoA-Iantibodies to induce HDL dysfunction, scavenger receptor B1 (SR-BI) functionImpact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 467impairment in endothelial cells, neutrophil infiltration and matrix-metalloproteinase9 production in plaques, activation of NFkB via TLR2/CD14 complex interaction,and cytokine release (Carbone et al. 2013). Interestingly, anti-apoA-I and anti-HDLantibodies have been shown to correlate also with disease activity in SLE patients(O’Neill et al. 2010).In addition, HDL dysfunction in SLE involves their capacity to promote cellcholesterol efflux (Ronda et al. 2014). In particular, in SLE patients HDL choles-terol efflux capacity (CEC) is reduced with respect to the ABCG1 and ABCA1transporter pathways, while the SR-BI-mediated CEC is unchanged. In addition thecorrelation between SR-BI-mediated CEC and HDL-C levels was stronger in SLEplasmas as compared to control plasma. This pattern is consistent with a possiblereduction/dysfunction of the small HDL populations (Favari et al. 2009) and a shiftto larger HDL, typical acceptors of cholesterol effluxed by SR-BI. Indeed, HDLs ofSLE patients were found increased in size (Hua et al. 2009; Juárez-Rojaset al. 2008). The impaired ABCA1- and ABCG1-mediated CEC in SLE patientsmay have a great impact because cholesterol efflux not only opposes lipid deposi-tion in vessels but is also crucial for the modulation of macrophage, endothelial, andT-cell inflammatory functions (Prosser et al. 2012; Yvan-Charvet et al. 2010a, b).7.2 Rheumatoid Arthritis (RA)RA is a systemic autoimmune disease characterized by lymphocyte activation,autoantibody production, high serum and tissue cytokine levels, and strong inflam-mation of synovia and vessels; similarly to SLE, it is associated with high CV risk(Shoenfeld et al. 2005). Circulating levels of HDL-C are often reduced, especiallyin active disease, and generally return to normal values during drug-inducedremission; however, as such modifications are mirrored by those relative to LDLcholesterol and total cholesterol, the actual clinical significance of HDL-C variationis not clear. Complex and specific mechanisms underlie lipid metabolism derange-ment in RA, in which cardiovascular risk inversely correlates with circulatingLDL-C levels (the so-called RA lipid paradox) (Myasoedova et al. 2011). As inSLE, the clinical relevance of HDL-C lowering (when present) in RA patients hasyet to be clarified.Composition and functional characteristics of HDL are altered in RA. piHDLlevels are increased in RA patients as compared to healthy controls (Hahnet al. 2008) and correlate positively with disease activity (Charles-Schoemanet al. 2009). Profound modifications of HDL composition in patients with activeRA have been described, particularly with respect to protein content. These changesinclude increased amount of serum amyloid A (SAA), apoJ, fibrinogen, and hapto-globin and reduced PON1 (Charles-Schoeman et al. 2009; Watanabe et al. 2012).As in SLE, anti-apoA-I autoantibodies are detectable in the serum of RA patients.In particular, in this population anti-apoA-I IgG levels have been shown to predictacute cardiovascular events and improve the prognostic power of the FraminghamRisk Score (Carbone et al. 2013).468 F. Montecucco et al.Finally, functional impairment of HDL as cholesterol acceptors for cell choles-terol efflux has been reported in RA (Ronda et al. 2014; Charles-Schoemanet al. 2012), independent of serum HDL-C levels and with a pattern ofmodifications differing from that found in SLE patients with respect to singlecholesterol transporters (Ronda et al. 2014). In particular, ABCG1-mediated CECis impaired in RA patients independently of HDL-C levels but inversely correlatingwith disease activity (Ronda et al. 2014). On the one hand, such correlation mayreflect the impact of inflammation and autoimmunity on HDL function, but on theother hand, it may indicate the adverse effect of reduced ABCG1-mediated CEC onvessel inflammation and immune reaction promotion. In fact, by promoting choles-terol and 7-ketocholesterol release, ABCG1 promotes anti-inflammatoryphenotypes of macrophages and endothelial cells (Terasaka et al. 2007; Hassanet al. 2006; O’Connell et al. 2004).Modifications of HDL occurring in SLE and RA compromise cholesterol efflux-promoting, anti-inflammatory, and antioxidant properties of HDL and thereby mayhave an important role in the progression of both atherosclerosis and autoimmuneas well as inflammatory phenomena typical for these diseases.8 Impact of Anti-inflammatory TreatmentsHDL exerts potent and multifactorial anti-inflammatory effects, which makes it anattractive target for pharmacological intervention. As extensively discussed in thischapter, several molecular principles exploited by HDL particles to interfere withinnate or acquired immunity might be well utilized for designing new drugseffectively combating atherosclerosis and/or autoimmune diseases. By contrast,little efforts have been devoted to assess the influence of medication traditionallyused in acute or chronic inflammatory diseases on HDL-C levels and HDL function.Most information regarding the interrelationship between HDL and anti-inflammatory therapy stems from interventional studies designed to examine theimpact of drugs on the primary disease rather than on lipid metabolism. Despite thislimited approach, currently available data strongly suggest that anti-inflammatorymedication may exert positive adjuvant effects both on HDL quantity and itsfunctionality.It is well known that HDL is a negative acute-phase reactant and that HDL-Clevels decline—sometimes dramatically—at the onset of acute inflammation andinfection. For instance, sepsis, extensive surgery, or viral infections are commonlyassociated with low HDL-C levels that are significantly and rapidly increasedfollowing spontaneous or treatment-related reductions in disease activity (Marik2006; Akgun et al. 1998; Marchesi et al. 2005). In addition, several chronicautoimmune disorders including SLE, RA, Kawasaki disease, and Behçet’s diseaseas well as periodontal disease are accompanied by decreased HDL levels in plasma(Haas and Mooradian 2011). Hence, the administration of nonsteroidal anti-inflammatory drugs (NSAIDs), which represent the most often prescribed class ofanti-inflammatory drugs, might be expected to produce HDL-C elevations by theImpact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 469simple reversal of the acute-phase reaction. However, the effect of NSAIDs on thequantity and quality of HDL remains controversial. In animal studies rising effectsof aspirin on ABCA1 expression and cholesterol efflux as well as on lecithin-cholesterol acyltransferase (LCAT) activity and plasma HDL-C levels were occa-sionally observed (Sethi et al. 2011; Jafarnejad et al. 2008; Viñals et al. 2005).However, these effects apparently did not translate into increased HDL generation,as similar plasma HDL-C levels were noted in a small prospective study in aspirin-and placebo-treated groups (Eritsland et al. 1989). In one study, the administrationof ibuprofen was found to slightly elevate HDL-C levels both in smokers andnonsmokers, whereas in another study treatment with naproxen failed to showany effect (Zapolska-Downar et al. 2000; Young et al. 1995). The influence ofselective cyclooxygenase-2 inhibitors such as celecoxib or rofecoxib on HDL-Cplasma levels has not been reported to date. In a more recent investigation,atreleuton (VIA-2291)—the first clinically tested 5-lipooxygenase inhibitor—wasfound to decrease leukotriene B4 production and CRP levels in blood and toconcomitantly reduce coronary plaque burden, but these favorable effects wereobserved in the absence of any changes in plasma lipid profile including HDL-C(Tardif et al. 2010).In a major contrast to nonsteroidal antiphlogistics, corticosteroids were repeat-edly reported to increase plasma HDL-C levels in patients with chronic autoim-mune diseases including RA, psoriatic arthritis, and SLE as well as in anon-autoimmune chronic inflammation (sarcoidosis) (Ettinger et al. 1987; Salazaret al. 2002; Boers et al. 2003; Sarkissian et al. 2007; Peters et al. 2007; Georgiadiset al. 2008; Garcı́a-Gómez et al. 2008). Furthermore, the addition of corticosteroidsto disease-modifying antirheumatic drugs (DMARDs, such as methotrexate orsulfasalazine), which on their own increase HDL-C in plasma, significantlypotentiated their effects on HDL-C levels (Boers et al. 2003). In one study,corticosteroids were also found to favorably affect HDL subfractions by preferen-tially increasing the amount of HDL2-C over HDL3-C (Garcı́a-Gómez et al. 2008).The beneficial effects exerted by corticosteroids on HDL quantity may appear at thefirst glimpse counterintuitive, since protracted therapy with these compounds isknown to enhance insulin resistance and to produce a prediabetic state, which isalmost obligatorily accompanied by low HDL-C. Actually, endogenous hypercor-tisolism (Cushing disease) has been related both to decreased HDL-C levels and toincreased cardiovascular risk in several studies (Faggiano et al. 2003). However,the relationship between steroid use and cardiovascular risk is complicated by thefact that these drugs tend to be used more often in patients with severe or intractablechronic disease; in such patients, the anti-inflammatory HDL-C elevating effect ofcorticosteroids may outweigh the HDL-C decreasing effect related to aggravationof insulin resistance. The mechanisms underlying modulatory effects ofcorticosteroids on HDL metabolism remain obscure. These compounds werefound to increase the activity of lipoprotein lipase (LPL) and to decrease the activityof hepatic triglyceride lipase, which are both critically involved in the generation ofHDL precursors and in HDL particle remodeling (Ewart et al. 1997; Dolinskyet al. 2004). In addition, corticosteroids may stimulate the production of nascent470 F. Montecucco et al.HDL as they enhance apoA-I gene expression in hepatocytes (Hargroveet al. 1999). Finally, corticosteroids were found to inhibit the activity of cholesterylester transfer protein (CETP), which may well explain their modulatory effect onthe HDL subfraction composition (Georgiadis et al. 2006).Studies involving biological pharmaceuticals, which selectively interfere withproinflammatory signaling pathways, add further evidence underscoring the bene-ficial effects of anti-inflammatory therapies on HDL quantity in plasma. Thegreatest amount of research into the effects of biologicals on lipids has beenperformed with TNF-alpha antagonists, in particular, with infliximab. In general,these studies demonstrated a consistent action of this drug in patients with RA,ankylosing spondylitis, and inflammatory bowel disease characterized by increasesin total cholesterol mostly due to the elevation of HDL-C (Choy and Sattar 2009;Pollono et al. 2010; Mathieu et al. 2010; Koutroubakis et al. 2009; Parmentier-Decrucq et al. 2009). Similar effects on HDL-C were observed in studies utilizingtwo other TNF-alpha antagonists, adalimumab and, most recently, golimumab,whereas the application of etanercept—a TNF-alpha receptor antagonist—pro-duced less consistent results (Pollono et al. 2010; Stagakis et al. 2012; Navarro-Millán et al. 2013; Lestre et al. 2011; Kirkham et al. 2014). Positive effects onplasma lipid profile encompassing the elevation of HDL-C levels were alsoobserved in patients with RA undergoing IL-6 receptor blockade with tocilizumabor CD20 signaling blockade with rituximab (Pollono et al. 2010; Kawashiriet al. 2011; Kerekes et al. 2009). In a major contrast, canakinumab—a compoundneutralizing IL-1beta—failed to change plasma lipid profile in a large randomizedstudy involving 556 men and women at high cardiovascular risk, albeit the treat-ment led to significant reductions in acute-phase proteins such as CRP and fibrino-gen (Ridker et al. 2012). The molecular mechanisms underlying the beneficialeffects of biological therapies on HDL-C levels are poorly understood. While itcannot be entirely excluded that HDL-C increases are at least partly related to theretardation of acute-phase reaction brought about by the inhibition of selectedproinflammatory signaling pathways, it seems more likely that treatment withTNF-alpha or IL-6 antagonists leads to derepression of the APOA1 gene, theactivity of which is known to be downregulated in hepatocytes exposed toproinflammatory cytokines (Haas and Mooradian 2011).In addition to the reduction of HDL quantity, acute-phase reaction wasdemonstrated to profoundly affect HDL composition. HDL particles isolatedfrom subjects suffering from acute or chronic inflammatory diseases were foundto lose proteins and enzymes with established or presumed antiatherogenic functionsuch as apoA-I, LCAT, or PON1 and to concomitantly acquire proinflammatory orpro-oxidative factors such as SAA, ceruloplasmin, Lp-PLA2, or MPO. Suchinflammatory HDL particles are severely impeded in their ability to exert severalantiatherogenic functions including initiation of reversed cholesterol transport,inhibition of pro-oxidative processes, inhibition of leukocyte migration and recruit-ment into arterial wall, or inhibition of thrombocyte activation. The pictureemerging from few recent studies suggests that anti-inflammatory medicationsmay not only elevate HDL quantity in plasma but also help to restore its properImpact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 471composition and thereby the antiatherogenic functionality. For instance, treatmentof RA patients with adalimumab, etanercept, tocilizumab, or rituximab decreasedand increased the content of SAA and apoA-I in HDL particles, respectively(Raterman et al. 2013; McInnes et al. 2014; Jamnitski et al. 2013). In addition,prolonged therapy with methotrexate was found to improve the capacity of HDL toinhibit both LDL oxidation and leukocyte migration in patients with RA (Charles-Schoeman et al. 2009). Increased levels of HDL-associated PON1 and improvedanti-oxidative capacity were seen in patients with psoriasis treated with etanercept(Bacchetti et al. 2013).ConclusionsHDL particles have been shown to play a protective and an anti-inflammatoryrole in autoimmune and inflammatory disorders. Their pathophysiological rele-vance directly implies the regulation of both immune and vascular cell functionsthat influence the common inflammatory processes underlying disease progres-sion and the associated CV risk. Numerous studies have demonstrated thatinflammatory disorders increase the risk of CVD and that this increase cannotbe totally accounted for by traditional risk factors. Alterations in the quantity,composition, and function of HDL may contribute to the promotion of athero-sclerosis process. In this prospective the assessment of HDL function, evaluatedas the capacity to promote cell cholesterol efflux, may offer a better prediction ofCVD than classical HDL-C levels. In addition, HDL function impairment, whichinvolves their anti-inflammatory and antioxidant properties as well as theirability to interact with cellular cholesterol transporters, may have an importantrole in accelerating atherosclerosis but also autoimmune and inflammatorymechanisms typical for these diseases. Therefore, the improvement of HDLfunction (instead of HDL-C levels) represents an interesting therapeutic strategyto reduce inflammation and associated CV risk in several immune diseases, suchas systemic lupus erythematosus and rheumatoid arthritis. We believe thatselective treatments improving HDL function or reducing the adversemodifications of HDL structure might be of pathophysiological relevance.Both basic and clinical studies are needed to validate this promising therapeuticissue in a near future.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAit-Oufella H, Salomon BL, Potteaux S, Robertson AK, Gourdy P, Zoll J, Merval R, Esposito B,Cohen JL, Fisson S, Flavell RA, Hansson GK, Klatzmann D, Tedgui A, Mallat Z (2006)Natural regulatory T cells control the development of atherosclerosis in mice. Nat Med12:178–180472 F. Montecucco et al.Akgun S, Ertel NH, Mosenthal A, Oser W (1998) Postsurgical reduction of serum lipoproteins:interleukin-6 and the acute-phase response. J Lab Clin Med 131:103–108Ammirati E, Cianflone D, Banfi M, Vecchio V, Palini A, De Metrio M, Marenzi G, Panciroli C,Tumminello G, Anzuini A, Palloshi A, Grigore L, Garlaschelli K, Tramontana S, Tavano D,Airoldi F, Manfredi AA, Catapano AL, Norata GD (2010) Circulating CD4+CD25hiCD127loregulatory t-cell levels do not reflect the extent or severity of carotid and coronary atheroscle-rosis. Arterioscler Thromb Vasc Biol 30:1832–1841Ammirati E, Cianflone D, Vecchio V, Banfi M, Vermi AC, De Metrio M, Grigore L, Pellegatta F,Pirillo A, Garlaschelli K, Manfredi AA, Catapano AL, Maseri A, Palini AG, Norata GD(2012a) Effector memory T cells are associated with atherosclerosis in humans and animalmodels. J Am Heart Assoc 1:27–41Ammirati E, Monaco C, Norata GD (2012b) Antigen-dependent and antigen-independentpathways modulate CD4+CD28null T-cells during atherosclerosis. Circ Res 111:e48–e49Asefi M, Vaisi-Raygani A, Bahrehmand F, Kiani A, Rahimi Z, Nomani H, Ebrahimi A,Tavilani H, Pourmotabbed T (2012) Paraoxonase 1 (PON1) 55 polymorphism, lipid profilesand psoriasis. Br J Dermatol 167:1279–1286Babin PJ, Thisse C, Durliat M, Andre M, Akimenko MA, Thisse B (1997) Both apolipoprotein Eand A-I genes are present in a nonmammalian vertebrate and are highly expressed duringembryonic development. Proc Natl Acad Sci USA 94:8622–8627Bacchetta R, Gambineri E, Roncarolo MG (2007) Role of regulatory T cells and FOXP3 in humandiseases. J Allergy Clin Immunol 120:227–235Bacchetti T, Campanati A, Ferretti G, Simonetti O, Liberati G, Offidani AM (2013) Oxidativestress and psoriasis: the effect of antitumour necrosis factor-α inhibitor treatment. Br JDermatol 168:984–989Baniyash M (2004) TCR zeta-chain downregulation: curtailing an excessive inflammatoryimmune response. Nat Rev Immunol 4:675–687Batuca JR, Ames PR, Isenberg DA, Alves JD (2007) Antibodies toward high-density lipoproteincomponents inhibit paraoxonase activity in patients with systemic lupus erythematosus. Ann NY Acad Sci 1108:137–146Beck WH, Adams CP, Biglang-Awa IM, Patel AB, Vincent H, Haas-Stapleton EJ, Weers PM(2013) Apolipoprotein A-I binding to anionic vesicles and lipopolysaccharides: role for lysineresidues in antimicrobial properties. Biochim Biophys Acta 1828:1503–1510Bilate AM, Lafaille JJ (2010) Can TNF-alpha boost regulatory T cells? J Clin Invest120:4190–4192Boers M, Nurmohamed MT, Doelman CJ, Lard LR, Verhoeven AC, Voskuyl AE, Huizinga TW,van de Stadt RJ, Dijkmans BA, van der Linden S (2003) Influence of glucocorticoids anddisease activity on total and high density lipoprotein cholesterol in patients with rheumatoidarthritis. Ann Rheum Dis 62:842–845Borba EF, Carvalho JF, Bonfa E (2006) Mechanisms of dyslipoproteinemias in systemic lupuserythematosus. Clin Dev Immunol 13:203–208Buffinton GD, Doe WF (1995) Depleted mucosal antioxidant defences in inflammatory boweldisease. Free Radic Biol Med 19:911–918Cabana VG, Gidding SS, Getz GS, Chapman J, Shulman ST (1997) Serum amyloid A and highdensity lipoprotein participate in the acute phase response of Kawasaki disease. Pediatr Res42:651–655Carbone F, Nencioni A, Mach F, Vuilleumier N, Montecucco F (2013) Evidence on the pathogenicrole of auto-antibodies in acute cardiovascular diseases. Thromb Haemost 109:854–868Castelli WP, Garrison RJ, Wilson PW, Abbott RD, Kalousdian S, Kannel WB (1986) Incidence ofcoronary heart disease and lipoprotein cholesterol levels. The Framingham study. JAMA256:2835–2838Charles-Schoeman C, Watanabe J, Lee YY, Furst DE, Amjadi S, Elashoff D, Park G,McMahon M, Paulus HE, Fogelman AM, Reddy ST (2009) Abnormal function of high-Impact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 473density lipoprotein is associated with poor disease control and an altered protein cargo inrheumatoid arthritis. Arthritis Rheum 60:2870–2879Charles-Schoeman C, Lee YY, Grijalva V, Amjadi S, FitzGerald J, Ranganath VK, Taylor M,McMahon M, Paulus HE, Reddy ST (2012) Cholesterol efflux by high density lipoproteins isimpaired in patients with active rheumatoid arthritis. Ann Rheum Dis 71:1157–1162Cheng AM, Handa P, Tateya S, Schwartz J, Tang C, Mitra P, Oram JF, Chait A, Kim F (2012)Apolipoprotein A-I attenuates palmitate-mediated NF-κB activation by reducing Toll-likereceptor-4 recruitment into lipid rafts. PLoS ONE 7:e33917Cheung YF, Yung TC, Tam SC, Ho MH, Chau AK (2004) Novel and traditional cardiovascularrisk factors in children after Kawasaki disease: implications for premature atherosclerosis. JAm Coll Cardiol 43:120–124Cheung YF, O K, Tam SC, Siow YL (2005) Induction of MCP1, CCR2, and iNOS expression inTHP-1 macrophages by serum of children late after Kawasaki disease. Pediatr Res58:1306–1310Cheung YF, Wong SJ, Ho MH (2007) Relationship between carotid intima-media thickness andarterial stiffness in children after Kawasaki disease. Arch Dis Child 92:43–47Chi H (2011) Sphingosine-1-phosphate and immune regulation: trafficking and beyond. TrendsPharmacol Sci 32:16–24Chiang AN, Hwang B, Shaw GC, Lee BC, Lu JH, Meng CC, Chou P (1997) Changes in plasmalevels of lipids and lipoprotein composition in patients with Kawasaki disease. Clin Chim Acta260:15–26Chodick G, Amital H, Shalem Y, Kokia E, Heymann AD, Porath A, Shalev V (2010) Persistencewith statins and onset of rheumatoid arthritis: a population-based cohort study. PLoS Med 7:e1000336Choy E, Sattar N (2009) Interpreting lipid levels in the context of high-grade inflammatory stateswith a focus on rheumatoid arthritis: a challenge to conventional cardiovascular risk actions.Ann Rheum Dis 68:460–469Cimen F, Yildirmak ST, Ergen A, Cakmak M, Dogan S, Yenice N, Sezgin F (2012) Serum lipid,lipoprotein and oxidatively modified low density lipoprotein levels in active or inactivepatients with Behcet’s disease. Indian J Dermatol 57:97–101Cohen JA, Barkhof F, Comi G, Hartung HP, Khatri BO, Montalban X, Pelletier J, Capra R,Gallo P, Izquierdo G, Tiel-Wilck K, de Vera A, Jin J, Stites T, Wu S, Aradhye S, Kappos L,TRANSFORMS Study Group (2010) Oral fingolimod or intramuscular interferon for relapsingmultiple sclerosis. N Engl J Med 362:402–415Davidovici BB, Sattar N, Prinz J, Puig L, Emery P, Barker JN, van de Kerkhof P, Stahle M, NestleFO, Girolomoni G, Krueger JG (2010) Psoriasis and systemic inflammatory diseases: potentialmechanistic links between skin disease and co-morbid conditions. J Invest Dermatol130:1785–1796Davidson MH (2012) HDL and CETP inhibition: will this DEFINE the future? Curr Treat OptionsCardiovasc Med 14:384–390de Carvalho JF, Bonfá E, Borba EF (2008) Systemic lupus erythematosus and “lupus dyslipopro-teinemia”. Autoimmun Rev 7:246–250De Nardo D, Labzin LI, Kono H, Seki R, Schmidt SV, Beyer M, Xu D, Zimmer S, Lahrmann C,Schildberg FA, Vogelhuber J, Kraut M, Ulas T, Kerksiek A, Krebs W, Bode N, Grebe A,Fitzgerald ML, Hernandez NJ, Williams BR, Knolle P, Kneilling M, Röcken M, Lütjohann D,Wright SD, Schultze JL, Latz E (2014) High-density lipoprotein mediates anti-inflammatoryreprogramming of macrophages via the transcriptional regulator ATF3. Nat Immunol15:152–160Dhillon R, Clarkson P, Donald AE, Powe AJ, Nash M, Novelli V, Dillon MJ, Deanfield JE (1996)Endothelial dysfunction late after Kawasaki disease. Circulation 94:2103–2106Di Angelantonio E, Sarwar N, Perry P, Kaptoge S, Ray KK, Thompson A, Wood AM,Lewington S, Sattar N, Packard CJ, Collins R, Thompson SG, Danesh J, Emerging Risk474 F. Montecucco et al.Factors Collaboration (2009) Major lipids, apolipoproteins, and risk of vascular disease. JAMA302:1993–2000Dolinsky VW, Douglas DN, Lehner R, Vance DE (2004) Regulation of the enzymes of hepaticmicrosomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexametha-sone. Biochem J 378:967–974Eckel RH (1989) Lipoprotein lipase. A multifunctional enzyme relevant to common metabolicdiseases. N Engl J Med 320:1060–1068Ehrenstein MR, Jury EC, Mauri C (2005) Statins for atherosclerosis–as good as it gets? N Engl JMed 352:73–75El Harchaoui K, Arsenault BJ, Franssen R, Despres JP, Hovingh GK, Stroes ES, Otvos JD,Wareham NJ, Kastelein JJ, Khaw KT, Boekholdt SM (2009) High-density lipoprotein particlesize and concentration and coronary risk. Ann Intern Med 150:84–93Eritsland J, Arnesen H, Smith P, Seljeflot I, Dahl K (1989) Effects of highly concentrated omega-3polyunsaturated fatty acids and acetylsalicylic acid, alone and combined, on bleeding time andserum lipid profile. J Oslo City Hosp 39:97–101Ettinger WH, Klinefelter HF, Kwiterovitch PO (1987) Effect of short-term, low-dosecorticosteroids on plasma lipoprotein lipids. Atherosclerosis 63:167–172Ewart HS, Carroll R, Severson DL (1997) Lipoprotein lipase activity in rat cardiomyocytes isstimulated by insulin and dexamethasone. Biochem J 327:439–442Expert Panel on Detection Evaluation, and Treatment of High Blood Cholesterol in Adults (2001)Executive summary of the third report of the national cholesterol education program (NCEP)expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (AdultTreatment Panel III). JAMA 285:2486–2497Faggiano A, Pivonello R, Spiezia S, De Martino MC, Filippella M, Di Somma C, Lombardi G,Colao A (2003) Cardiovascular risk factors and common carotid artery calibre and stiffness inpatients with Cushing’s disease during active disease and 1 year after disease remission. J ClinEndocrinol Metab 88:2527–2533Favari E, Calabresi L, Adorni MP, Jessup W, Simonelli S, Franceschini G, Bernini F (2009) Smalldiscoidal pre-beta1 HDL particles are efficient acceptors of cell cholesterol via ABCA1 andABCG1. Biochemistry 48:11067–11074Flores-Borja F, Kabouridis PS, Jury EC, Isenberg DA, Mageed RA (2005) Decreased Lynexpression and translocation to lipid raft signaling domains in B lymphocytes from patientswith systemic lupus erythematosus. Arthritis Rheum 52:3955–3965Fujimoto M, Nakano M, Terabe F, Kawahata H, Ohkawara T, Han Y, Ripley B, Serada S,Nishikawa T, Kimura A, Nomura S, Kishimoto T, Naka T (2011) The influence of excessiveIL-6 production in vivo on the development and function of Foxp3+ regulatory T cells. JImmunol 186:32–40Garcı́a-Gómez C, Nolla JM, Valverde J, Narváez J, Corbella E, Pintó X (2008) HighHDL-cholesterol in women with rheumatoid arthritis on low-dose glucocorticoid therapy.Eur J Clin Invest 38:686–692Gelfand JM, Neimann AL, Shin DB, Wang X, Margolis DJ, Troxel AB (2006) Risk of myocardialinfarction in patients with psoriasis. JAMA 296:1735–1741Georgiadis AN, Papavasiliou EC, Lourida ES, Alamanos Y, Kostara C, Tselepis AD (2006)Atherogenic lipid profile is a feature characteristic of patients with early rheumatoid arthritis:effect of early treatment-a prospective, controlled study. Arthritis Res Ther 8:R82Georgiadis AN, Voulgari PV, Argyropoulou MI, Alamanos Y, Elisaf M, Tselepis AD, Drosos AA(2008) Early treatment reduces the cardiovascular risk factors in newly diagnosed rheumatoidarthritis patients. Semin Arthritis Rheum 38:13–19Gordon SM, Davidson WS (2012) Apolipoprotein A-I mimetics and high-density lipoproteinfunction. Curr Opin Endocrinol Diabetes Obes 19:109–114Graler MH, Goetzl EJ (2004) The immunosuppressant FTY720 down-regulates sphingosine1-phosphate G-protein-coupled receptors. FASEB J 18:551–553Impact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 475Grand RJ, Ramakrishna J, Calenda KA (1995) Inflammatory bowel disease in the pediatric patient.Gastroenterol Clin North Am 24:613–632Grinberg-Bleyer Y, Saadoun D, Baeyens A, Billiard F, Goldstein JD, Grégoire S, Martin GH,Elhage R, Derian N, Carpentier W, Marodon G, Klatzmann D, Piaggio E, Salomon BL (2010)Pathogenic T cells have a paradoxical protective effect in murine autoimmune diabetes byboosting Tregs. J Clin Invest 120:4558–4568Gruaz L, Delucinge-Vivier C, Descombes P, Dayer JM, Burger D (2010) Blockade of T cellcontact-activation of human monocytes by high-density lipoproteins reveals a new pattern ofcytokine and inflammatory genes. PLoS ONE 5:e9418Grunfeld C, Marshall M, Shigenaga JK, Moser AH, Tobias P, Feingold KR (1999) Lipoproteinsinhibit macrophage activation by lipoteichoic acid. J Lipid Res 40:245–252Gu X, Lawrence R, Krieger M (2000) Dissociation of the high density lipoprotein and low densitylipoprotein binding activities of murine scavenger receptor class B type I (mSR-BI) usingretrovirus library-based activity dissection. J Biol Chem 275:9120–9130Gupta N, DeFranco AL (2007) Lipid rafts and B cell signaling. Semin Cell Dev Biol 18:616–626Haas MJ, Mooradian AD (2011) Inflammation, high-density lipoprotein and cardiovasculardysfunction. Curr Opin Infect Dis 24:265–272Haase CL, Tybjærg-Hansen A, Qayyum AA, Schou J, Nordestgaard BG, Frikke-Schmidt RLCAT(2012) HDL cholesterol and ischemic cardiovascular disease: a Mendelian randomizationstudy of HDL cholesterol in 54,500 individuals. J Clin Endocrinol Metab 97:E248–E256Hahn BH, Grossman J, Ansell BJ, Skaggs BJ, McMahon M (2008) Altered lipoprotein metabolismin chronic inflammatory states: proinflammatory high-density lipoprotein and acceleratedatherosclerosis in systemic lupus erythematosus and rheumatoid arthritis. Arthritis Res Ther10:213Hargrove GM, Junco A, Wong NC (1999) Hormonal regulation of apolipoprotein AI. J MolEndocrinol 22:103–111Hassan HH, Denis M, Krimbou L, Marcil M, Genest J (2006) Cellular cholesterol homeostasis invascular endothelial cells. Can J Cardiol 22:35B–40BHeinecke JW (2009) The HDL, proteome: a marker–and perhaps mediator–of coronary arterydisease. J Lipid Res 50(Suppl):S167–S171Holzer M, Wolf P, Curcic S, Birner-Gruenberger R, Weger W, Inzinger M, El-Gamal D,Wadsack C, Heinemann A, Marsche G (2012) Psoriasis alters HDL composition and choles-terol efflux capacity. J Lipid Res 53:1618–1624Hua X, Su J, Svenungsson E, Hurt-Camejo E, Jensen-Urstad K, Angelin B, Båvenholm P,Frostegård J (2009) Dyslipidaemia and lipoprotein pattern in systemic lupus erythematosus(SLE) and SLE-related cardiovascular disease. Scand J Rheumatol 38:184–189Jafarnejad A, Bathaie SZ, Nakhjavani M, Hassan MZ (2008) Investigation of the mechanismsinvolved in the high-dose and long-term acetyl salicylic acid therapy of type I diabetic rats. JPharmacol Exp Ther 324:850–857Jamnitski A, Levels JH, van den Oever IA, Nurmohamed MT (2013) High-density lipoproteinprofiling changes in patients with rheumatoid arthritis treated with tumor necrosis factorinhibitors: a cohort study. J Rheumatol 40:825–830Jiao YL, Wu MP (2008) Apolipoprotein A-I diminishes acute lung injury and sepsis in miceinduced by lipoteichoic acid. Cytokine 43:83–87Juárez-Rojas J, Medina-Urrutia A, Posadas-Sánchez R, Jorge-Galarza E, Mendoza-Pérez E,Caracas-Portilla N, Cardoso-Saldaña G, Muñoz-Gallegos G, Posadas-Romero C (2008)High-density lipoproteins are abnormal in young women with uncomplicated systemic lupuserythematosus. Lupus 17:981–987Jury EC, Isenberg DA, Mauri C, Ehrenstein MR (2006) Atorvastatin restores Lck expression andlipid raft-associated signaling in T cells from patients with systemic lupus erythematosus. JImmunol 177:7416–7422Kabouridis PS, Jury EC (2008) Lipid rafts and T-lymphocyte function: implications for autoim-munity. FEBS Lett 582:3711–3718476 F. Montecucco et al.Kappos L, Radue EW, O’Connor P, Polman C, Hohlfeld R, Calabresi P, Selmaj K,Agoropoulou C, Leyk M, Zhang-Auberson L, Burtin P, FREEDOMS Study Group (2010) Aplacebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N Engl J Med362:387–401Kawashiri SY, Kawakami A, Yamasaki S, Imazato T, Iwamoto N, Fujikawa K, Aramaki T,Tamai M, Nakamura H, Ida H, Origuchi T, Ueki Y, Eguchi K (2011) Effects of the anti-interleukin-6 receptor antibody, tocilizumab, on serum lipid levels in patients with rheumatoidarthritis. Rheumatol Int 31:451–456Kerekes G, Soltész P, Dér H, Veres K, Szabó Z, Végvári A, Szegedi G, Shoenfeld Y, Szekanecz Z(2009) Effects of rituximab treatment on endothelial dysfunction, carotid atherosclerosis, andlipid profile in rheumatoid arthritis. Clin Rheumatol 28:705–710Keul P, Tölle M, Lucke S, von Wnuck LK, Heusch G, Schuchardt M, van der Giet M, Levkau B(2007) The sphingosine-1-phosphate analogue FTY720 reduces atherosclerosis in apolipopro-tein E-deficient mice. Arterioscler Thromb Vasc Biol 27:607–613Khovidhunkit W, Kim MS, Memon RA, Shigenaga JK, Moser AH, Feingold KR, Grunfeld C(2004) Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanismsand consequences to the host. J Lipid Res 45:1169–1196Kirkham BW, Wasko MC, Hsia EC, Fleischmann RM, Genovese MC, Matteson EL, Liu H,Rahman MU (2014) Effects of golimumab, an anti-tumour necrosis factor-α human monoclo-nal antibody, on lipids and markers of inflammation. Ann Rheum Dis 73:161–169Kiss E, Seres I, Tarr T, Kocsis Z, Szegedi G, Paragh G (2007) Reduced paraoxonase1 activity is arisk for atherosclerosis in patients with systemic lupus erythematosus. Ann N Y Acad Sci1108:83–91Koutroubakis IE, Oustamanolakis P, Malliaraki N, Karmiris K, Chalkiadakis I, Ganotakis E,Karkavitsas N, Kouroumalis EA (2009) Effects of tumor necrosis factor alpha inhibitionwith infliximab on lipid levels and insulin resistance in patients with inflammatory boweldisease. Eur J Gastroenterol Hepatol 21:283–288Lestre S, Diamantino F, Veloso L, Fidalgo A, Ferreira A (2011) Effects of etanercept treatment onlipid profile in patients with moderate-to-severe chronic plaque psoriasis: a retrospectivecohort study. Eur J Dermatol 21:916–920Levine DM, Parker TS, Donnelly TM, Walsh A, Rubin AL (1993) In vivo protection againstendotoxin by plasma high density lipoprotein. Proc Natl Acad Sci USA 90:12040–12044Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gingeras TR,Fazekas de St Groth B, Clayberger C, Soper DM, Ziegler SF, Bluestone JA (2006) CD127expression inversely correlates with FoxP3 and suppressive function of human CD4+ T regcells. J Exp Med 203:1701–1711Liu G, Yang K, Burns S, Shrestha S, Chi H (2010) The S1P(1)-mTOR axis directs the reciprocaldifferentiation of T(H)1 and T(reg) cells. Nat Immunol 11:1047–1056Longnecker R, Miller CL (1996) Regulation of Epstein-Barr virus latency by latent membraneprotein 2. Trends Microbiol 4:38–42Mandala S, Hajdu R, Bergstrom J, Quackenbush E, Xie J, Milligan J, Thornton R, Shei GJ, Card D,Keohane C, Rosenbach M, Hale J, Lynch CL, Rupprecht K, Parsons W, Rosen H (2002)Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science296:346–349Marchesi S, Lupattelli G, Lombardini R, Sensini A, Siepi D, Mannarino M, Vaudo G, MannarinoE (2005) Acute inflammatory state during influenza infection and endothelial function. Ath-erosclerosis 178:345–350Marik PE (2006) Dyslipidemia in the critically ill. Crit Care Clin 22:151–159Mathieu S, Dubost JJ, Tournadre A, Malochet-Guinamand S, Ristori JM, Soubrier M (2010)Effects of 14 weeks of TNF alpha blockade treatment on lipid profile in ankylosing spondylitis.Joint Bone Spine 77:50–52Mathison JC, Ulevitch RJ (1979) The clearance, tissue distribution, and cellular localization ofintravenously injected lipopolysaccharide in rabbits. J Immunol 123:2133–2143Impact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 477Matloubian M, Lo CG, Cinamon G, Lesneski MJ, Xu Y, Brinkmann V, Allende ML, Proia RL,Cyster JG (2004) Lymphocyte egress from thymus and peripheral lymphoid organs is depen-dent on S1P receptor 1. Nature 427:355–360McGillicuddy FC, de la Llera MM, Hinkle CC, Joshi MR, Chiquoine EH, Billheimer JT, RothblatGH, Reilly MP (2009) Inflammation impairs reverse cholesterol transport in vivo. Circulation119:1135–1145McInnes IB, Thompson L, Giles JT, Bathon JM, Salmon JE, Beaulieu AD, Codding CE, CarlsonTH, Delles C, Lee JS, Sattar N (2014) Effect of interleukin-6 receptor blockade on surrogatesof vascular risk in rheumatoid arthritis: MEASURE, a randomised, placebo-controlled study.Ann Rheum Dis. doi:10.1136/annrheumdis-2013-204345 (in press)McMahon M, Skaggs BJ, Sahakian L, Grossman J, Fitzgerald J, Ragavendra N, Charles-Schoeman C, Chernishof M, Gorn A, Witztum JL, Wong WK, Weisman M, Wallace DJ, LaCava A, Hahn BH (2011) High plasma leptin levels confer increased risk of atherosclerosis inwomen with systemic lupus erythematosus, and are associated with inflammatory oxidisedlipids. Ann Rheum Dis 70:1619–1624Mehta NN, Yu Y, Saboury B, Foroughi N, Krishnamoorthy P, Raper A, Baer A, Antigua J, VanVoorhees AS, Torigian DA, Alavi A, Gelfand JM (2011) Systemic and vascular inflammationin patients with moderate to severe psoriasis as measured by [18F]-fluorodeoxyglucose posi-tron emission tomography-computed tomography (FDG-PET/CT): a pilot study. ArchDermatol 147:1031–1039Mehta NN, Li R, Krishnamoorthy P, Yu Y, Farver W, Rodrigues A, Raper A, Wilcox M, Baer A,DerOhannesian S, Wolfe M, Reilly MP, Rader DJ, Van Voorhees A, Gelfand JM (2012)Abnormal lipoprotein particles and cholesterol efflux capacity in patients with psoriasis.Atherosclerosis 224:218–221Messedi M, Jamoussi K, Frigui M, Laporte F, Turki M, Chaabouni K, Mnif E, Jaloulli M,Kaddour N, Bahloul Z, Ayedi F (2011) Atherogenic lipid profile in Behcet’s disease: evidenceof alteration of HDL subclasses. Arch Med Res 42:211–218Miller GJ, Miller NE (1975) Plasma-high-density-lipoprotein concentration and development ofischaemic heart-disease. Lancet 1:16–19Mor A, Planer D, Luboshits G, Afek A, Metzger S, Chajek-Shaul T, Keren G, George J (2007)Role of naturally occurring CD4+ CD25+ regulatory T cells in experimental atherosclerosis.Arterioscler Thromb Vasc Biol 27:893–900Mottonen M, Heikkinen J, Mustonen L, Isomaki P, Luukkainen R, Lassila O (2005) CD4+ CD25+T cells with the phenotypic and functional characteristics of regulatory T cells are enriched inthe synovial fluid of patients with rheumatoid arthritis. Clin Exp Immunol 140:360–367Musabak U, Baylan O, Cetin T, Yesilova Z, Sengul A, Saglam K, Inal A, Kocar IH (2005) Lipidprofile and anticardiolipin antibodies in Behcet’s disease. Arch Med Res 36:387–392Myasoedova E, Crowson CS, Kremers HM, Roger VL, Fitz-Gibbon PD, Therneau TM, Gabriel SE(2011) Lipid paradox in rheumatoid arthritis: the impact of serum lipid measures and systemicinflammation on the risk of cardiovascular disease. Ann Rheum Dis 70:482–487Navab M, Reddy ST, Van Lenten BJ, Fogelman AM (2011) HDL and cardiovascular disease:atherogenic and atheroprotective mechanisms. Nat Rev Cardiol 8:222–232Navarro-Millán I, Charles-Schoeman C, Yang S, Bathon JM, Bridges SL Jr, Chen L, Cofield SS,Dell’Italia LJ, Moreland LW, O’Dell JR, Paulus HE, Curtis JR (2013) Changes in lipoproteinsassociated with methotrexate or combination therapy in early rheumatoid arthritis: results fromthe treatment of early rheumatoid arthritis trial. Arthritis Rheum 65:1430–1438Newburger JW, Burns JC, Beiser AS, Loscalzo J (1991) Altered lipid profile after Kawasakisyndrome. Circulation 84:625–631Nofer JR, Bot M, Brodde M, Taylor PJ, Salm P, Brinkmann V, van Berkel T, Assmann G, BiessenEA (2007) FTY720, a synthetic sphingosine 1 phosphate analogue, inhibits development ofatherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation 115:501–508Norata GD, Catapano AL (2012) HDL and adaptive immunity: a tale of lipid rafts. Atherosclerosis225:34–35478 F. Montecucco et al.Norata GD, Callegari E, Marchesi M, Chiesa G, Eriksson P, Catapano AL (2005) High-densitylipoproteins induce transforming growth factor-beta2 expression in endothelial cells. Circula-tion 111:2805–2811Norata GD, Marchesi P, Pirillo A, Uboldi P, Chiesa G, Maina V, Garlanda C, Mantovani A,Catapano AL (2008) Long pentraxin 3, a key component of innate immunity, is modulated byhigh-density lipoproteins in endothelial cells. Arterioscler Thromb Vasc Biol 28:925–931Norata GD, Pirillo A, Ammirati E, Catapano AL (2012) Emerging role of high density lipoproteinsas a player in the immune system. Atherosclerosis 220:11–21O’Connell BJ, Denis M, Genest J (2004) Cellular physiology of cholesterol efflux in vascularendothelial cells. Circulation 110:2881–2888O’Neill SG, Giles I, Lambrianides A, Manson J, D’Cruz D, Schrieber L, March LM, LatchmanDS, Isenberg DA, Rahman A (2010) Antibodies to apolipoprotein A-I, high-density lipopro-tein, and C-reactive protein are associated with disease activity in patients with systemic lupuserythematosus. Arthritis Rheum 62:845–854Orem A, Yandi YE, Vanizor B, Cimsit G, Uydu HA, Malkoc M (2002) The evaluation ofautoantibodies against oxidatively modified low-density lipoprotein (LDL), susceptibility ofLDL to oxidation, serum lipids and lipid hydroperoxide levels, total antioxidant status,antioxidant enzyme activities, and endothelial dysfunction in patients with Behcet’s disease.Clin Biochem 35:217–224Parmentier-Decrucq E, Duhamel A, Ernst O, Fermont C, Louvet A, Vernier-Massouille G,Cortot A, Colombel JF, Desreumaux P, Peyrin-Biroulet L (2009) Effects of infliximab therapyon abdominal fat and metabolic profile in patients with Crohn’s disease. Inflamm Bowel Dis15:1476–1484Peters MJ, Vis M, van Halm VP, Wolbink GJ, Voskuyl AE, Lems WF, Dijkmans BA, Twisk JW,de Koning MH, van de Stadt RJ, Nurmohamed MT (2007) Changes in lipid profile duringinfliximab and corticosteroid treatment in rheumatoid arthritis. Ann Rheum Dis 66:958–961Phillips MC (2013) New insights into the determination of HDL structure by apolipoproteins:thematic review series: high density lipoprotein structure, function, and metabolism. J LipidRes 54:2034–2048Pollono EN, Lopez-Olivo MA, Lopez JA, Suarez-Almazor ME (2010) A systematic review of theeffect of TNF-alpha antagonists on lipid profiles in patients with rheumatoid arthritis. ClinRheumatol 29:947–955Prosser HC, Ng MK, Bursill CA (2012) The role of cholesterol efflux in mechanisms of endothe-lial protection by HDL. Curr Opin Lipidol 23:182–189Raterman HG, Levels H, Voskuyl AE, Lems WF, Dijkmans BA, Nurmohamed MT (2013) HDLprotein composition alters from proatherogenic into less atherogenic and proinflammatory inrheumatoid arthritis patients responding to rituximab. Ann Rheum Dis 72:560–565Ridker PM, Howard CP, Walter V, Everett B, Libby P, Hensen J, Thuren T, CANTOS PilotInvestigative Group (2012) Effects of interleukin-1β inhibition with canakinumab on hemo-globin A1c, lipids, C-reactive protein, interleukin-6, and fibrinogen: a phase IIb randomized,placebo-controlled trial. Circulation 126:2739–2748Rivera J, Proia RL, Olivera A (2008) The alliance of sphingosine-1-phosphate and its receptors inimmunity. Nat Rev Immunol 8:753–763Romanato G, Scarpa M, Angriman I, Faggian D, Ruffolo C, Marin R, Zambon S, Basato S,Zanoni S, Filosa T, Pilon F, Manzato E (2009) Plasma lipids and inflammation in activeinflammatory bowel diseases. Aliment Pharmacol Ther 29:298–307Ronda N, Favari E, Borghi MO, Ingegnoli F, Gerosa M, Chighizola C, Zimetti F, Adorni MP,Bernini F, Meroni PL (2014) Impaired serum cholesterol efflux capacity in rheumatoid arthritisand systemic lupus erythematosus. Ann Rheum Dis 73(3):609–615. doi:10.1136/annrheumdis-2012-202914Sala F, Cutuli L, Grigore L, Pirillo A, Chiesa G, Catapano AL, Norata GD (2013) Prevalence ofclassical CD14++/CD16 but not of intermediate CD14++/CD16+ monocytes in hypoalphali-poproteinemia. Int J Cardiol 168:2886–2889Impact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 479Salazar A, Mana J, Pinto X, Argimon JM, Hurtado I, Pujol R (2002) Corticosteroid therapyincreases HDL-cholesterol concentrations in patients with active sarcoidosis and hypoalpha-lipoproteinemia. Clin Chim Acta 320:59–64Salemi G, Gueli MC, Vitale F, Battaglieri F, Guglielmini E, Ragonese P, Trentacosti A, MassentiMF, Savettieri G, Bono A (2010) Blood lipids, homocysteine, stress factors, and vitamins inclinically stable multiple sclerosis patients. Lipids Health Dis 9:19Sappati Biyyani RS, Putka BS, Mullen KD (2010) Dyslipidemia and lipoprotein profiles inpatients with inflammatory bowel disease. J Clin Lipidol 4:478–482Sarkissian T, Beyene J, Feldman B, McCrindle B, Silverman ED (2007) Longitudinal examinationof lipid profiles in pediatric systemic lupus erythematosus. Arthritis Rheum 56:631–638Scanu AM, Edelstein C (2008) HDL: bridging past and present with a look at the future. FASEB J22:4044–4054Sethi A, Parmar HS, Kumar A (2011) The effect of aspirin on atherogenic diet-induced diabetesmellitus. Basic Clin Pharmacol Toxicol 108:371–377Shah S, Casas JP, Drenos F, Whittaker J, Deanfield J, Swerdlow DI, Holmes MV, Kivimaki M,Langenberg C, Wareham N, Gertow K, Sennblad B, Strawbridge RJ, Baldassarre D, Veglia F,Tremoli E, Gigante B, de Faire U, Kumari M, Talmud PJ, Hamsten A, Humphries SE,Hingorani AD (2013) Causal relevance of blood lipid fractions in the development of carotidatherosclerosis: Mendelian randomization analysis. Circ Cardiovasc Genet 6:63–72Shiflett AM, Bishop JR, Pahwa A, Hajduk SL (2005) Human high density lipoproteins areplatforms for the assembly of multi-component innate immune complexes. J Biol Chem280:32578–32585Shoenfeld Y, Gerli R, Doria A, Matsuura E, Cerinic MM, Ronda N, Jara LJ, Abu-Shakra M,Meroni PL, Sherer Y (2005) Accelerated atherosclerosis in autoimmune rheumatic diseases.Circulation 112:3337–3347Smythies LE, White CR, Maheshwari A, Palgunachari MN, Anantharamaiah GM, Chaddha M,Kurundkar AR, Datta G (2010) Apolipoprotein A-I mimetic 4F alters the function of humanmonocyte-derived macrophages. Am J Physiol Cell Physiol 298:C1538–C1548Stagakis I, Bertsias G, Karvounaris S, Kavousanaki M, Virla D, Raptopoulou A, Kardassis D,Boumpas DT, Sidiropoulos PI (2012) Anti-tumor necrosis factor therapy improves insulinresistance, beta cell function and insulin signaling in active rheumatoid arthritis patients withhigh insulin resistance. Arthritis Res Ther 14:R141Tardif JC, L’allier PL, Ibrahim R, Grégoire JC, Nozza A, Cossette M, Kouz S, Lavoie MA,Paquin J, Brotz TM, Taub R, Pressacco J (2010) Treatment with 5-lipoxygenase inhibitorVIA-2291 (Atreleuton) in patients with recent acute coronary syndrome. Circ CardiovascImaging 3:298–307Tedesco-Silva H, Pescovitz MD, Cibrik D, Rees MA, Mulgaonkar S, Kahan BD, Gugliuzza KK,Rajagopalan PR, Esmeraldo Rde M, Lord H, Salvadori M, Slade JM, FTY720 Study Group(2006) Randomized controlled trial of FTY720 versus MMF in de novo renal transplantation.Transplantation 82:1689–1697Teixeira PC, Cutler P, Vuilleumier N (2012) Autoantibodies to apolipoprotein A-1 in cardiovas-cular diseases: current perspectives. Clin Dev Immunol 2012:868251. doi:10.1155/2012/868251Terasaka N, Wang N, Yvan-Charvet L, Tall AR (2007) High-density lipoprotein protectsmacrophages from oxidized low-density lipoprotein-induced apoptosis by promoting effluxof 7-ketocholesterol via ABCG1. Proc Natl Acad Sci USA 104:15093–15098Thuahnai ST, Lund-Katz S, Anantharamaiah GM, Williams DL, Phillips MC (2003) A quantita-tive analysis of apolipoprotein binding to SR-BI: multiple binding sites for lipid-free and lipid-associated apolipoproteins. J Lipid Res 44:1132–1142Triantafilou M, Miyake K, Golenbock DT, Triantafilou K (2002) Mediators of innate immunerecognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cellactivation. J Cell Sci 115:2603–2611480 F. Montecucco et al.Troitzsch P, Paulista Markus MR, Dorr M, Felix SB, Junger M, Schminke U, Schmidt CO,Volzke H, Baumeister SE, Arnold A (2012) Psoriasis is associated with increased intima-media thickness–the Study of Health in Pomerania (SHIP). Atherosclerosis 225:486–490Van Lenten BJ, Hama SY, de Beer FC, Stafforini DM, McIntyre TM, Prescott SM, La Du BN,Fogelman AM, Navab M (1995) Anti-inflammatory HDL becomes pro-inflammatory duringthe acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wallcell cocultures. J Clin Invest 96:2758–2767van Leuven SI, Hezemans R, Levels JH, Snoek S, Stokkers PC, Hovingh GK, Kastelein JJ, StroesES, de Groot E, Hommes DW (2007) Enhanced atherogenesis and altered high densitylipoprotein in patients with Crohn’s disease. J Lipid Res 48:2640–2646Viñals M, Bermúdez I, Llaverias G, Alegret M, Sanchez RM, Vázquez-Carrera M, Laguna JC(2005) Aspirin increases CD36, SR-BI, and ABCA1 expression in human THP-1macrophages. Cardiovasc Res 66:141–149Voight BF, Peloso GM, Orho-Melander M, Frikke-Schmidt R, Barbalic M, Jensen MK, Hindy G,Hólm H, Ding EL, Johnson T, Schunkert H, Samani NJ, Clarke R, Hopewell JC, Thompson JF,Li M, Thorleifsson G, Newton-Cheh C, Musunuru K, Pirruccello JP, Saleheen D, Chen L,Stewart A, Schillert A, Thorsteinsdottir U, Thorgeirsson G, Anand S, Engert JC, Morgan T,Spertus J, Stoll M, Berger K, Martinelli N, Girelli D, McKeown PP, Patterson CC, Epstein SE,Devaney J, Burnett MS, Mooser V, Ripatti S, Surakka I, Nieminen MS, Sinisalo J, Lokki ML,Perola M, Havulinna A, de Faire U, Gigante B, Ingelsson E, Zeller T, Wild P, de Bakker PI,Klungel OH, Maitland-van der Zee Zee AH, Peters BJ, de Boer A, Grobbee DE, KamphuisenPW, Deneer VH, Elbers CC, Onland-Moret NC, Hofker MH, Wijmenga C, Verschuren WM,Boer JM, van der Schouw YT, Rasheed A, Frossard P, Demissie S, Willer C, Do R, OrdovasJM, Abecasis GR, Boehnke M, Mohlke KL, Daly MJ, Guiducci C, Burtt NP, Surti A,Gonzalez E, Purcell S, Gabriel S, Marrugat J, Peden J, Erdmann J, Diemert P, Willenborg C,König IR, Fischer M, Hengstenberg C, Ziegler A, Buysschaert I, Lambrechts D, Van deWerf F, Fox KA, El Mokhtari NE, Rubin D, Schrezenmeir J, Schreiber S, Schäfer A,Danesh J, Blankenberg S, Roberts R, McPherson R, Watkins H, Hall AS, Overvad K,Rimm E, Boerwinkle E, Tybjaerg-Hansen A, Cupples LA, Reilly MP, Melander O, MannucciPM, Ardissino D, Siscovick D, Elosua R, Stefansson K, O’Donnell CJ, Salomaa V, Rader DJ,Peltonen L, Schwartz SM, Altshuler D, Kathiresan S (2012) Plasma HDL cholesterol and riskof myocardial infarction: a mendelian randomisation study. Lancet 380:572–580Vuilleumier N, Dayer JM, von Eckardstein A, Roux-Lombard P (2013) Pro- or anti-inflammatoryrole of apolipoprotein A-1 in high-density lipoproteins? Swiss Med Wkly 143:w13781Vuilleumier N, Montecucco F, Hartley O (2014) Autoantibodies to apolipoprotein A-1 as abiomarker of cardiovascular autoimmunity. World J Cardiol 6(5):314–326. doi:10.4330/wjc.v6.i5.314Wang Y, Zhu X, Wu G, Shen L, Chen B (2008) Effect of lipid-bound apoA-I cysteine mutants onlipopolysaccharide-induced endotoxemia in mice. J Lipid Res 49:1640–1645Wang SH, Yuan SG, Peng DQ, Zhao SP (2012) HDL and ApoA-I inhibit antigen presentation-mediated T cell activation by disrupting lipid rafts in antigen presenting cells. Atherosclerosis225:105–114Watanabe J, Charles-Schoeman C, Miao Y, Elashoff D, Lee YY, Katselis G, Lee TD, Reddy ST(2012) Proteomic profiling following immunoaffinity capture of high-density lipoprotein:association of acute-phase proteins and complement factors with proinflammatory high-densitylipoprotein in rheumatoid arthritis. Arthritis Rheum 64:1828–1837Weigert A, Weis N, Brune B (2009) Regulation of macrophage function by sphingosine-1-phosphate. Immunobiology 214:748–760Weiss SJ (1989) Tissue destruction by neutrophils. N Engl J Med 320:365–376Wilhelm AJ, Zabalawi M, Grayson JM, Weant AE, Major AS, Owen J, Bharadwaj M, Walzem R,Chan L, Oka K, Thomas MJ, Sorci-Thomas MG (2009) Apolipoprotein A-I and its role inlymphocyte cholesterol homeostasis and autoimmunity. Arterioscler Thromb Vasc Biol29:843–849Impact of Systemic Inflammation and Autoimmune Diseases on apoA-I and HDL. . . 481Wilhelm AJ, Zabalawi M, Owen JS, Shah D, Grayson JM,Major AS, Bhat S, Gibbs DP Jr, ThomasMJ, Sorci-Thomas MG (2010) Apolipoprotein A-I modulates regulatory T cells in autoimmuneLDLr-/-, ApoA-I-/- mice. J Biol Chem 285:36158–36169Williams HR, Willsmore JD, Cox IJ, Walker DG, Cobbold JF, Taylor-Robinson SD, Orchard TR(2012) Serum metabolic profiling in inflammatory bowel disease. Dig Dis Sci 57:2157–2165Yatomi Y (2008) Plasma sphingosine 1-phosphate metabolism and analysis. Biochim BiophysActa 1780:606–611Young D, Peterson C, Basch C, Halladay SC (1995) Effects of naproxen and nabumetone on serumcholesterol levels in patients with osteoarthritis. Clin Ther 17:231–240Yu Y, Sheth N, Krishnamoorthy P, Saboury B, Raper A, Baer A, Ochotony R, Doveikis J,Derohannessian S, Voorhees AS, Torigian DA, Alavi A, Gelfand JM, Mehta NN (2012) Aorticvascular inflammation in psoriasis is associated with HDL particle size and concentration: apilot study. Am J Cardiovasc Dis 2:285–292Yuhanna IS, Zhu Y, Cox BE, Hahner LD, Osborne-Lawrence S, Lu P, Marcel YL, Anderson RG,Mendelsohn ME, Hobbs HH, Shaul PW (2001) High-density lipoprotein binding to scavengerreceptor-BI activates endothelial nitric oxide synthase. Nat Med 7:853–857Yvan-Charvet L, Pagler T, Gautier EL, Avagyan S, Siry RL, Han S, Welch CL, Wang N,Randolph GJ, Snoeck HW, Tall AR (2010a) ATP-binding cassette transporters and HDLsuppress hematopoietic stem cell proliferation. Science 328:1689–1693Yvan-Charvet L, Wang N, Tall AR (2010b) Role of HDL, ABCA1, and ABCG1 transporters incholesterol efflux and immune responses. Arterioscler Thromb Vasc Biol 30:139–143Zapolska-Downar D, Naruszewicz M, Zapolski-Downar A, Markiewski M, Bukowska H, Millo B(2000) Ibuprofen inhibits adhesiveness of monocytes to endothelium and reduces cellularoxidative stress in smokers and non-smokers. Eur J Clin Invest 30:1002–1010482 F. Montecucco et al.HDL in Infectious Diseases and SepsisAngela Pirillo, Alberico Luigi Catapano, and Giuseppe DaniloNorataContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4852 HDL and Bacterial Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4862.1 Interaction of HDL with LPS and Gram-Negative Bacteria . . . . . . . . . . . . . . . . . . . . . . . . 4882.2 Interaction of HDL with LTA and Gram-Positive Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . 4902.3 HDL and Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4912.4 General Innate Host Defense Mechanisms Exerted by HDL After BacterialInfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4913 HDL and Parasitic Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4944 HDL and Viral Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4954.1 HIV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4954.2 HCV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500AbstractDuring infection significant alterations in lipid metabolism and lipoproteincomposition occur. Triglyceride and VLDL cholesterol levels increase, whilereduced HDL cholesterol (HDL-C) and LDL cholesterol (LDL-C) levels areobserved. More importantly, endotoxemia modulates HDL composition andsize: phospholipids are reduced as well as apolipoprotein (apo) A-I, whileA. Pirillo • A.L. CatapanoSISA Center for the Study of Atherosclerosis, Bassini Hospital, Cinisello Balsamo, ItalyIRCCS, Multimedica, Milan, ItalyG.D. Norata (*)SISA Center for the Study of Atherosclerosis, Bassini Hospital, Cinisello Balsamo, ItalyDepartment of Pharmacological Sciences, Università degli Studi di Milano, Milan, ItalyDepartment of Pharmacological and Biomolecular Sciences, University of Milan,Via Balzaretti 9, 20133 Milan, Italye-mail: Danilo.Norata@unimi.it# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_15483serum amyloid A (SAA) and secretory phospholipase A2 (sPLA2) dramaticallyincrease, and, although the total HDL particle number does not change, asignificant decrease in the number of small- and medium-size particles isobserved. Low HDL-C levels inversely correlate with the severity of septicdisease and associate with an exaggerated systemic inflammatory response.HDL, as well as other plasma lipoproteins, can bind and neutralize Gram-negative bacterial lipopolysaccharide (LPS) and Gram-positive bacteriallipoteichoic acid (LTA), thus favoring the clearance of these products. HDLsare emerging also as a relevant player during parasitic infections, and a specificcomponent of HDL, namely, apoL-1, confers innate immunity against trypano-some by favoring lysosomal swelling which kills the parasite. During virusinfections, proteins associated with the modulation of cholesterol bioavailabilityin the lipid rafts such as ABCA1 and SR-BI have been shown to favor virus entryinto the cells. Pharmacological studies support the benefit of recombinant HDLor apoA-I mimetics during bacterial infection, while apoL-1–nanobodycomplexes were tested for trypanosome infection. Finally, SR-BI antagonismrepresents a novel and forefront approach interfering with hepatitis C virus entrywhich is currently tested in clinical studies. From the coming years, we have toexpect new and compelling observations further linking HDL to innate immu-nity and infections.KeywordsHDL • Infections • Bacteria • Parasites • VirusAbbreviationsABCA-1 ATP-binding cassette transporter 1ApoA-I Apolipoprotein A-IApoE Apolipoprotein EApoL-1 Apolipoprotein L-1ApoM Apolipoprotein MIL-1β Interleukin-1βIL-6 Interleukin-6HCV Hepatitis C virusHIV Human immunodeficiency virusHDL-C HDL cholesterolLBP LPS-binding proteinLCAT Lecithin-cholesterol acyltransferaseLDL-C LDL cholesterolLPS Gram-negative bacterial lipopolysaccharideLTA Gram-positive bacterial lipoteichoic acidPAF-AH Platelet-activating factor acetylhydrolase484 A. Pirillo et al.PON1 Serum paraoxonasePTX3 Pentraxin 3rHDL Reconstituted HDLSAA Serum amyloid AS1P Sphingosine-1-phosphatesPLA2 Secretory phospholipase A2SR-BI Scavenger receptor class B member 1TG TriglyceridesTLF-1 Trypanosome lytic factor-1TLRs Toll-like receptorsTNF-α Tumor necrosis factor-α1 IntroductionHDLs are heterogeneous particles generated by the continuous remodeling bylipolytic enzymes and lipid transporters and by lipid and apolipoprotein exchangewith other circulating lipoproteins and tissues (Kontush and Chapman 2006).Mature HDL particles have a hydrophobic core containing cholesteryl esters andtriglycerides, while proteins are embedded in a lipid monolayer composed mainlyof phospholipids and free cholesterol. HDLs contain two main proteins, apolipo-protein A-I (apoA-I) and apoA-II, but several other minor apoproteins as well asenzymes such as lecithin-cholesterol acyltransferase (LCAT), serum paraoxonase(PON1), and platelet-activating factor acetylhydrolase (PAF-AH) are associatedwith HDL particles (Navab et al. 2004). HDLs possess several biological functions(Pirillo et al. 2013), but the role of HDL in innate immunity has emerged in the1970s with the first observation associating HDL cholesterol (HDL-C) plasmalevels to protection against sepsis. In the coming years, it has emerged that theability of HDL to modulate cholesterol bioavailability in the lipid rafts, membranemicrodomains enriched in glycosphingolipids and cholesterol, is evolutionaryconserved and affects the properties of cells involved in the innate and adaptiveimmune response, tuning inflammatory response and antigen presentation functionsin macrophages as well as activation of B and T cells. In the context of infections,HDL and their components have been linked with protection toward Gram-negativeand Gram-positive bacteria and parasites, while the role during virus infection isdebated (Fig. 1). Furthermore, HDLs influence humoral innate immunity by tuningthe activation of the complement system and the expression of pentraxin 3 (PTX3).HDLs are critical not only during sepsis but also in other bacterial, parasitic, andviral infection. The aim of this chapter is to discuss the relevant findings on the linkbetween HDL and immune response, shedding a new light on the role of theselipoproteins during sepsis and infectious disease.HDL in Infectious Diseases and Sepsis 4852 HDL and Bacterial InfectionsIncreasing observations suggest that persistent low-grade inflammation isassociated with the pathogenesis of severe chronic diseases such as atherosclerosis,diabetes, and other aging-related neurological diseases. Low levels of circulatingGram-negative bacterial endotoxin lipopolysaccharide (LPS) appear to sustain anon-resolving low-grade inflammation. As a consequence, low-gradeendotoxinemia may skew host immune environment into a mild non-resolvingpro-inflammatory state, which eventually leads to the pathogenesis and progressionof inflammatory diseases.During infection, significant changes in the lipid metabolism are observed. Atfirst, plasma levels of lipid and lipoproteins may change: triglyceride (TG) andVLDL cholesterol levels increase due to several mechanisms, including reductionof TG hydrolysis, LPS- and pro-inflammatory cytokines-induced de novo free fattyacid production, and TG synthesis in the liver and reduction of lipoprotein lipaseactivity thus resulting in reduced VLDL clearance and increased TG levels (Wendelet al. 2007). In addition, the increase in free fatty acids induces insulin resistance,thus contributing to increased glucose levels during systemic inflammation. On theother hand, HDL-C and LDL-C levels decrease during sepsis, and a low plasmaBacteriaParasitesVirusHDLTYPE OF INFECTION HDL-MEDIATED EFFECT•Favor LPS/LTA binding and neutralizaon.•Favor LPS/LTA clearance• Inhibit LPS (LTA)-induced cytokine release• Inhibit of LPS (LTA)-induced cell acvaon• Induce an early inflammatory response•Support ApoL1, Apo-AI and HRP interacon to form the trypanosoma lyc factor-1 (TLF-1). complex. ApoL1 then traffics to the trypanosomal lysosome, where causes swelling which kills the trypanosome.•Dampen (ApoA-1 mimec pepdes) the ABCA-1 impairment induced by the HIV-1 Nef protein.• Inhibit cell fusion, both in HIV-1-infected T cells and in recombinant vaccinia-virus-infected CD4+ HeLa cells.•Compete with Hepas C virus on SRBI interacon to dampen virus entry?Fig. 1 HDL and infections486 A. Pirillo et al.HDL-C level (associated with a low plasma apoA-I level) is a poor prognosticfactor for severe sepsis, as it is associated with increased mortality and adverseclinical outcomes (Chien et al. 2005); more importantly, significant alterations inlipoprotein composition are observed, and increased levels of acute-phase proteins,including serum amyloid A (SAA) and secretory phospholipase A2 (sPLA2), maycontribute to decreased HDL-C levels, by replacing some structural and functionalHDL components (Fig. 2).Endotoxinemia also modulates HDL composition and size: phospholipids arereduced while SAA dramatically increases and apoA-I decreases, and, although thetotal HDL particle number does not change, a significant decrease in the numbers ofsmall- and medium-size particles is observed (de la Llera et al. 2012). The apoA-Icontent is reduced due to the rapid association of SAA, which displaces apoA-I andbecomes the main protein of acute-phase HDL (Coetzee et al. 1986; Khovidhunkitet al. 2004); the content of other proteins associated with HDL (PON1, PAF-AH) isaltered, resulting in reduced antioxidant properties of HDL (Feingold et al. 1998)and increased content of pro-atherogenic lipids (Cao et al. 1998; Memonet al. 1999). Also the lipid composition of HDL is altered during the acute-phaseresponse (Khovidhunkit et al. 2004). Endotoxemia induces the increase of someenzymes involved in HDL remodeling, including endothelial lipase (Badellinoet al. 2008) and secretory phospholipase A2 (de la Llera et al. 2012), and thedecrease of other, such as CETP and LCAT (de la Llera et al. 2012; Wendelet al. 2007). Altogether these changes result in the loss of functional properties ofHDL (Banka et al. 1995; de la Llera et al. 2012; McGillicuddy et al. 2009).Infecon/SepsisAcute phase-HDLAn-inflammatoryacvityMature HDLHDLPAF-AH AP-HDLFig. 2 Acute-phase HDLHDL in Infectious Diseases and Sepsis 4872.1 Interaction of HDL with LPS and Gram-Negative BacteriaSepsis is a major cause of death in hospitalized patients. Mortality is mainly due tothe cytotoxic actions of lipid components of the bacterial outer membraneLipopolysaccharide (LPS) is the toxic component of endotoxin in the outer mem-brane of Gram-negative bacteria. Lipoteichoic acid (LTA) is a heat-stable compo-nent of the cell membrane and wall of most Gram-positive bacteria that sharesstructural similarities with LPS and induces cytokine cascades alike LPS(Grunfeld et al. 1999).LPS, the major pathogenic factor in Gram-negative sepsis, is an essentialcomponent of the bacterial cell wall, and it is not toxic when incorporated intothe membrane; after release in the blood following bacterial cell reproduction, lysis,or death, lipid A, the most essential part of LPS, induces an inflammatory response(Van Amersfoort et al. 2003). This is mediated by pro-inflammatory cytokinesreleased primarily from monocytes/macrophages and neutrophils, such as tumornecrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) (Levineet al. 1993). LPS is recognized mainly by toll-like receptor 4 (TLR4) in cooperationwith other proteins including MD-2, CD14, and LPS-binding protein (LBP) (Jerala2007). LBP catalyzes the transfer of LPS to CD14, thus enhancing LPS-inducedcell activation (Van Amersfoort et al. 2003). To prevent exaggerated responses toLPS, the host has developed several control mechanisms that include inhibitoryLPS-binding proteins and plasma lipoproteins (Van Amersfoort et al. 2003).In patients with severe sepsis, HDL-C decreases rapidly, and SAA is the majorprotein present in HDL (45 %) at the start of sepsis and is slowly replaced by apoA-Iduring recovery (van Leeuwen et al. 2003). Low HDL-C levels inversely correlatewith the severity of septic disease and associate with an exaggerated systemicinflammatory response (Wendel et al. 2007), although it is difficult to establishwhether changes in plasma lipoproteins simply reflect the severity of disease or theycan directly modify the host response to inflammation. Also in healthy subjects lowHDL levels are associated with increased inflammatory response on endotoxinchallenge compared to subjects with normal or high HDL levels (Birjmohunet al. 2007), without differences in the HDL proteome (Levels et al. 2011). Theseobservations indicate a positive role of HDL in the protection against sepsis.Several mechanisms are involved in HDL-mediated protection. HDL, as well asother plasma lipoproteins (LDL, TG-rich lipoproteins), can bind and neutralizeGram-negative bacterial LPS as well as Gram-positive bacterial lipoteichoic acid(LTA) (Grunfeld et al. 1999; Murch et al. 2007). ApoA-I knockout mice, whichlack HDL, exhibit decreased LPS neutralization in the serum compared with serumfrom control mice (Guo et al. 2013); overexpression of apoA-I moderatelyimproves survival compared to controls, suggesting that HDL elevation mayprotect against septic death. In endotoxemic rats, in which LPS has been infusedafter HDL administration, HDLs attenuate LPS-induced organ damageaccompanied by lower TNF-α and nitric oxide production (Lee et al. 2007). Inaddition to its role in LPS neutralization, HDLs exert its protection againstsepsis also by promoting LPS clearance; in fact, almost all LPS exists in the488 A. Pirillo et al.complex LPS–HDL in the blood (Ulevitch et al. 1979, 1981), the HDL receptorSR-BI binds and mediates LPS uptake, and HDLs promote SR-BI-mediated LPSuptake (Vishnyakova et al. 2003).Administration of reconstituted HDL (rHDL) efficiently inhibits theLPS-induced cytokine release from the whole-blood system in vitro (Parkeret al. 1995); however, the first study that demonstrated the LPS-neutralizing abilityof rHDL in humans was performed by intravenous infusion of rHDL beforeinduction of endotoxemia in healthy volunteers (Pajkrt et al. 1996). rHDL signifi-cantly reduced endotoxemia-induced inflammatory response, as it reduced clinicalsymptoms, reduced inflammatory cytokine (TNF-α, IL-6, IL-8) production, andattenuated LPS-induced leukocyte activation, in part due to the downregulation ofthe main LPS receptor monocyte-bound CD14 (Pajkrt et al. 1996). InLPS-challenged macrophages, HDLs selectively inhibit the activation of type IIFN response genes (Suzuki et al. 2010), which play a critical role in the antiviralresponse of cells, although emerging evidence also implicates this response in hostdefense during bacterial infection. This inhibitory effect of HDL does not requireLPS binding to lipoproteins (Suzuki et al. 2010). HDLs (and apoA-I) attenuate alsoLPS-induced neutrophil activation (Murphy et al. 2011). More recently Di Nardoet al. (2014) have shown that HDLs promote the expression of ATF3 inmacrophages, a transcriptional regulator which inhibits TLR2 expression. Of notethe protective effects of HDL against TLR-induced inflammation were shown to befully dependent on ATF3 in vitro and in vivo.Another mechanism by which HDLs exert a protection against sepsis is byinducing an early inflammatory response to Gram-negative bacteria, thus helpingto maintain a sensitive host response to LPS; HDLs exert this effect by suppressingthe inhibitory activity of LBP (Thompson and Kitchens 2006). In contrast to nativeHDL, recombinant HDL did not increase cell response at early time points and arestrongly inhibitory of cell response; this different effect is due to the composition ofrHDL, which contain only apoA-I and PC and are optimized for LPS binding andneutralization (Thompson and Kitchens 2006).Indeed apoA-I is a major HDL component that plays a central role in the anti-inflammatory functions of this lipoprotein class and exhibits protective effectsagainst sepsis. ApoA-I in fact can directly inactivate bacterial endotoxin byprotein–protein interaction (Emancipator et al. 1992), being the C-terminal halfof apoA-I the main domain responsible for LPS neutralization (Henninget al. 2011), but also inhibits LPS-induced cytokine release from human monocytes(Flegel et al. 1993); in addition, apoA-I reduces TNF-α levels during LPS challengein rats and increases the survival rates (Humphries et al. 2006), suggesting thatapoA-I might inhibit LPS binding to macrophages thus inhibiting the production ofinflammatory cytokines that are related to sepsis. In mice, the overexpression ofapoA-I (that results in an increased serum level of both apoA-I and HDL) attenuatesLPS-induced acute injury in lung and kidney (Li et al. 2008); LPS-induced pro-inflammatory cytokines decrease, as well as CD14 expression in liver and lung,resulting in a protective effect against systemic inflammation and multiple organdamage (Li et al. 2008). Similar to what reported in mice, subjects with low plasmaHDL in Infectious Diseases and Sepsis 489HDL levels (hypoalphalipoproteinemia) present an increased prevalence of classi-cal CD14++/CD16 but not of intermediate CD14++/CD16+ monocytes, furtherlinking HDL- to LPS-mediated responses (Sala et al. 2013). Septic HDLs arealmost depleted of apoC-I (Barlage et al. 2001). ApoC-I contains a consensusLPS-binding motif and is able to enhance the biological response to LPS thusreducing mortality in mice with Gram-negative-induced sepsis (Berbeeet al. 2006). In fact, apoC-I binds to LPS and prevents its clearance by the liverand spleen, resulting in the stimulation of the LPS-induced pro-inflammatoryresponse and protection against septic death (Berbee et al. 2006). These findingssuggest that when LPS is released into the plasma following bacteria proliferationin the blood, apoC-I binds to LPS and presents it to responsive cells such asmacrophages, leading to a rapid and enhanced production of pro-inflammatorycytokines, which are essential for effective eradication of the bacterial infection.2.2 Interaction of HDL with LTA and Gram-Positive BacteriaThe effects of HDL on components of the bacterial outer membrane are notrestricted to Gram-negative bacteria but also involve cell wall components ofGram-positive bacteria such as LTA which is able to induce an inflammatoryresponse similar to that induced by LPS. Indeed it induces a massive productionof mediators of inflammation that may result in systemic inflammatory responsesyndrome, septic shock, and multiorgan damage (Bhakdi et al. 1991; De Kimpeet al. 1995). LTA may also trigger disturbances of lipid metabolism, interferingwith both lipoprotein production and lipoprotein clearance (Grunfeld et al. 1999).All lipoproteins can bind LTA, but the majority of LTA is found in the HDLfraction (Levels et al. 2003), suggesting that HDLs have the highest affinity. Incontrast to LPS, HDLs (as well as other lipoproteins) alone do not inhibit thecytokine production induced by LTA, but require the presence of lipoprotein-depleted plasma (Grunfeld et al. 1999), suggesting that lipoproteins containcofactors in sufficient amounts to facilitate LPS binding to lipoproteins but notLTA binding. LBP is a plasma component (normally bound to HDL, but it can befound in lipoprotein-depleted plasma following ultracentrifugation) whichenhances the activation of macrophages by LPS in the absence of lipoproteinsand facilitates LPS binding to lipoproteins; LBP can also bind LTA (Tobiaset al. 1989) and allows HDL to efficiently inactivate LTA (Grunfeld et al. 1999).The inhibition of LBP with neutralizing antibodies significantly decreases (53 %)the ability of lipoprotein-depleted plasma to facilitate LTA inactivation, but alsosuggests the presence of other plasma factors playing a role in HDL inactivation ofLTA (Grunfeld et al. 1999).ApoA-I has been shown to bind directly LTA in vitro and to attenuateLTA-induced NF-kB activation (Jiao and Wu 2008); apoA-I dose-dependentlyinhibits L-929 cell death induced by LTA-activated macrophages, andlipoprotein-depleted plasma strengthened this effect of apoA-I (Jiao and Wu2008). In mice, apoA-I attenuates LTA-induced acute lung injury and significantlyinhibits LTA-induced pro-inflammatory cytokine production (Jiao and Wu 2008).490 A. Pirillo et al.These findings suggest that apoA-I can inhibit LTA activation by multiplemechanisms, by direct binding to LTA, and by interfering with LTA-mediatedinflammatory response.2.3 HDL and MycobacteriaHDLs were reported to be protective also toward intracellular bacteria such asmycobacteria (Cruz et al. 2008). Intracellular pathogens survive by evading the hostimmune system and accessing host metabolic pathways to obtain nutrients for theirgrowth. Mycobacterium leprae, the causative agent of leprosy, is thought to be themycobacterium most dependent on host metabolic pathways, including host-derived lipids. Although fatty acids and phospholipids accumulate in the lesionsof individuals with the lepromatous (also known as disseminated) form of humanleprosy (L-lep), the origin and significance of these lipids remains unclear. Host-derived oxidized phospholipids were detected in macrophages within L-lep lesions,and one specific oxidized phospholipid, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3phosphorylcholine (PEIPC), accumulates in macrophages infectedwith live mycobacteria (Cruz et al. 2008). Mycobacterial infection and host-derivedoxidized phospholipids both inhibited innate immune responses, and this inhibitionwas reversed by the addition of normal HDL, a scavenger of oxidizedphospholipids, but not by HDL from patients with L-lep (Cruz et al. 2008). Theaccumulation of host-derived oxidized phospholipids in L-lep lesions is strikinglysimilar to observations in atherosclerosis, which suggests that the link between hostlipid metabolism and innate immunity could contribute to the pathogenesis of bothmicrobial infection and metabolic disease.2.4 General Innate Host Defense Mechanisms Exerted by HDLAfter Bacterial InfectionIn addition to limiting LPS or LTA responses during infection, HDLs exert addi-tional functions during the innate immune response. In normal plasma, about 5 % ofHDL particles contain apolipoprotein M (apoM) which binds sphingosine-1-phos-phate (S1P), an important bioactive lipid mediator known to be associated withHDL (Christoffersen et al. 2011). The inhibition of apoM production results in thedecrease of HDL-C levels and changes in HDL size, subclass profile, and functions(Wolfrum et al. 2005); apoM is a negative acute-phase protein that decreases duringinfection and inflammation (Feingold et al. 2008); in patients with severe sepsis andsystemic inflammatory response syndrome (SIRS), a leading cause of mortality innon-coronary intensive care units, apoM plasma levels decrease dramaticallysuggesting a reduction of the vasculoprotective effects of apoM and its ligandS1P, with a strong correlation between apoM decrease and the severity of disease(Kumaraswamy et al. 2012). It is still unclear whether apoM and S1P levels mayhave prognostic value and whether changes in apoM levels contribute to thepathogenesis of SIRS and septic shock.HDL in Infectious Diseases and Sepsis 491Another key player during the immune response regulated by HDL is the longpentraxin 3 (PTX3). This protein belongs, together with the C-reactive protein(CRP) and other acute-phase proteins, to the pentraxin superfamily: soluble, multi-functional, pattern recognition proteins. Pentraxins share a common C-terminalpentraxin domain, which in the case of PTX3 is coupled to an unrelated longN-terminal domain (Bonacina et al. 2013). PTX3, which is the prototypic longpentraxin, was identified in the early 1990s, as a molecule rapidly induced by IL-1in endothelial cells (ECs) or by TNF-α in both ECs and fibroblasts (Breviarioet al. 1992; Lee et al. 1993). The protein presents a high degree of conservationfrom mouse to human (82 % identical and 92 % conserved amino acids) and isinduced in a variety of somatic and innate immunity cells by primary inflammatorystimuli (Garlanda et al. 2005). PTX3 is a key player of the humoral arm of theinnate immunity, and its physiological functions are associated with the recognitionand binding to different ligands, including microbial moieties, complementcomponents, and P-selectin. This protein plays also a key role in cardiovasculardiseases including atherosclerosis (Norata et al. 2009, 2010). Similarly to shortpentraxins, PTX3 recognizes the highly conserved pathogen-associated molecularpatterns (PAMPs) expressed by microorganisms (Iwasaki and Medzhitov 2010) andbinds a number of bacteria, fungi, and viruses. A specific binding has been observedto conidia of Aspergillus fumigatus (Garlanda et al. 2002), Paracoccidioidesbrasiliensis, and zymosan (Diniz et al. 2004), to selected Gram-positive andGram-negative bacteria (Bozza et al. 2006; Garlanda et al. 2002; Jeanninet al. 2005), and finally to some viral strains, including human and murine cyto-megalovirus and influenza virus type A (IVA) (Bozza et al. 2006; Readinget al. 2008). Both short pentraxin and PTX3 bind apoptotic cells and facilitatetheir clearance (Doni et al. 2012; Jaillon et al. 2009). Surface bound CRP activatesthe classical pathway of complement through interaction with C1q, thus leading tocell elimination (Nauta et al. 2003). Cell-bound PTX3 might favor the clearance ofapoptotic cells (Jaillon et al. 2009; Poon et al. 2010) by enhancing the deposition ofboth C1q and C3 on cell surfaces (Nauta et al. 2003). On the contrary, when in thefluid phase, PTX3 interacts with C1q and dampens the deposition on apoptotic cellsand the resulting phagocytosis by dendritic cells and phagocytes (Baruahet al. 2006; Gershov et al. 2000; Rovere et al. 2000; van Rossum et al. 2004). Inaddition to PTX3, C1q recognizes and binds to ficolin-2 and mannose-bindinglectin (MBL), thus modulating the classical and the lectin pathways of complementactivation (Bottazzi et al. 1997). The best described and characterized ligand ofPTX3 is the first component of the classical complement system C1q (Bottazziet al. 1997; Nauta et al. 2003); PTX3 interacts with the globular head of the protein(Roumenina et al. 2006) thus resulting in the activation of the classical complementcascade only when C1q is plastic immobilized, a situation that mimics C1q boundto a microbial surface. Anti-inflammatory molecules were shown to modulatePTX3 expression. Under inflammatory conditions, glucocorticoid hormones(GCs) induce and enhance the protein expression in fibroblasts but not in myeloid492 A. Pirillo et al.cells (Doni et al. 2008). Also HDLs, which possess a series of vascular protectiveactivities, induce PTX3 expression in endothelial cells (Norata et al. 2008). Thelatter mechanism requires the activation of the PI3K/Akt pathway throughG-coupled lysosphingolipid receptors and is mimicked by sphingosine-1-phosphateand others S1P mimetics (Norata et al. 2008), physiologically present in HDL andresponsible for some of the activities linking HDL to the immunoinflammatoryresponse (Norata et al. 2005, 2012). In vivo, an increased expression of PTX3mRNA was detected in the aorta of transgenic mice overexpressing human apoA-I,compared to apoA-I knockout mice, and plasma levels of PTX3 are significantlyincreased in C57BL/6 mice injected with HDL (Norata et al. 2008). These datasuggest that some of the beneficial effects in immunity of HDLmay result also fromthe modulation of molecules that act as sensors of the immunoinflammatorybalance.In summary, all the observations showing that the increase of HDL is associatedwith an attenuation of LPS-induced inflammatory response (Levine et al. 1993; vanLeeuwen et al. 2003) strongly favor the hypothesis that raising plasma HDL mayrepresent a therapeutic approach in the treatment of sepsis and its complications.The picture however is more complicated, and not only HDL quantity but also HDLquality/composition is critical. For instance, the increase in sepsis-related mortalityof the ILLUMINATE trial observed in the torcetrapib (a cholesteryl ester transferprotein inhibitor, increasing HDL-C levels) arm (Barter et al. 2007) was unlikelydue to a direct effect of torcetrapib on LBP or bactericidal/permeability increasingprotein function nor to inhibition of an interaction of CETP with LPS (Clarket al. 2010). It is rather possible that changes in plasma lipoprotein compositiondespite increased HDL levels, or the known off-target effects of torcetrapib, such asaldosterone elevation, could have aggravated the effects of sepsis (Clarket al. 2010). Apolipoprotein mimetic peptides represent an emerging area of HDLtherapy; the most effective apoA-I mimetic peptide is 4F, which has been shown toimprove HDL quality/function (Sherman et al. 2010; White et al. 2009). 4F mimicsalso anti-inflammatory properties of HDL: in vitro, 4F inhibits the expression ofpro-inflammatory mediators in LPS-treated cells by directly binding to LPS, thusresulting in the inhibition of LPS binding to LBP (Gupta et al. 2005). Inendotoxemic rats, the administration of 4F after LPS injection results in theattenuation of acute lung injury and increased survival, probably due to the preser-vation of circulating HDL-C and the downregulation of inflammatory pathways(Kwon et al. 2012); 4F also prevents defects in vascular functions and is associatedwith a decrease in plasma endotoxin activity in rats (Dai et al. 2010) and improvedcardiac performance in LPS-treated rats (Datta et al. 2011). These observationsindicate that, by scavenging LPS, 4F may prevent LPS-induced release ofpro-inflammatory cytokines and changes in HDL composition resulting in aneffective reduction of clinical complications associated with sepsis. Althoughpromising, future studies are warranted to translate these findings into the clinicalsetting.HDL in Infectious Diseases and Sepsis 4933 HDL and Parasitic InfectionsThe connection between HDL and parasitic infections mainly relates on the abilityof specific apolipoproteins, which circulate as part of the HDL3 complex, to limitTrypanosoma brucei or Leishmania infection. The most relevant apolipoprotein inthis context is apoL-1 which was discovered in 1997 (Duchateau et al. 1997) andwas shown to be a part of a primate-specific complex named trypanosome lyticfactor-1 (TLF-1) that also contains apoA-I and haptoglobin-related protein as themain protein components. TLF-1 has lytic activity toward African Trypanosomabrucei brucei and renders humans and most other primates resistant to infectionwith this parasite causing endemic infections of African cattle. The Trypanosomaspecies brucei rhodesiense and brucei gambiense, however, are resistant to TLF-1and cause sleeping sickness in humans. Sleeping sickness is fatal when untreatedand thus is an important health problem in many African countries. The trypano-some lytic activity has been associated with apoL-1 (Vanhamme et al. 2003); theapoL-1-containing complex is taken up by T. b. brucei via a receptor that bindshemoglobin and haptoglobin-related protein (Hb-Hpr). ApoL-1 traffics to thetrypanosomal lysosome, where the acidic pH causes a conformational change,leading to activation of anion channel function in the apoL-1 N terminus. Lyso-somal swelling kills the trypanosome (Perez-Morga et al. 2005). Thus, apoL-1confers innate immunity against this parasite (Wheeler 2010). Over time, T. b.brucei developed a virulence factor called SRA that can inactivate apoL-1 protein,although the cellular location of this interaction is unknown. These SRA-expressingtrypanosomes evolved into T. b. rhodesiense, the etiologic agent that causes acuteAfrican sleeping sickness (Wheeler 2010). This discovery has now been used toengineer a potential fusion protein drug for treating sleeping sickness caused bybrucei rhodesiense in human (Baral et al. 2006). The active component in the novelprotein drug candidate is a recombinant apoL-1 where the C-terminal SRA-bindingregion of wild-type apoL-1 has been deleted. This truncated apoL-1 alone probablyhas low biological activity when injected into plasma due to competition withendogenous apoL-1 (~6 mg/l) for uptake by trypanosomes. To circumvent thisproblem and effectively target recombinant apoL-1 to trypanosomes, theSRA-resistant apoL-1 without the C-terminal sequences was fused with a fragmentof a high-affinity camel antibody (designated nanobody) specifically recognizingconserved epitopes of variant surface glycoprotein on trypanosomes (Baralet al. 2006). In vitro, the recombinant apoL-1–nanobody specifically bound totrypanosomes and was capable of lysing them in vivo. When mice infected withTrypanosoma brucei brucei were treated with the apoL-1–nanobody fusion protein,the parasites were promptly cleared from the circulation.Another trypanosomatida, Leishmania, is also targeted by apoL-1 (Samanovicet al. 2009). It is certainly possible that apoL-1 has broad innate immunityproperties, shaping the relative frequencies of the APOL1 alleles. Of note thechronic kidney disease (CKD)-associated G1 and G2 variants (Parsa et al. 2013)encode forms of apoL-1 that evade SRA and remain active against T. b.rhodesiense. This, and/or other biological effects, may have conferred a selective494 A. Pirillo et al.advantage to G1 and G2 heterozygotes, causing a selective sweep. The contributionof apoL-1 to CKD could also be related to the HDL functionality (Baragettiet al. 2013) under specific immunopathological conditions such as those observedduring CKD. Hence, HDL could represent the bridge between apoL-1 and CKD andshould be taken into consideration when exploring the contribution of apoL-1 to thedisease. The delineation of how primates in late evolution have exploited HDL tofight parasite infections highlights the need of investigations on other potential rolesof plasma HDL beyond those in lipid metabolism and reverse cholesterol transport.4 HDL and Viral InfectionsChanges in plasma HDL-C levels have been reported to occur also during infectionwith viruses including human immunodeficiency virus (HIV) and hepatitis C virus(HCV). Furthermore, proteins related to HDL life cycle, such as SR-BI, were shownto play a key role in HCV infection. So far HDLs were proposed to increase virusinfection and inhibit virus neutralizing antibodies; however, recent findings arechallenging previous data and proposing a more complicated picture based on theability of virus to take advantage of HDL–lipid transfer activity in host cells. Mostof the available evidences linking HDL to viral infection are available for HIVand HCV.4.1 HIV InfectionHIV infects and depletes CD4 lymphocytes, resulting in immunodeficiency and aslowly progressive disease. HIV is associated with dyslipidemia, namely,hypocholesterolemia, low levels of LDL, and hypertriglyceridemia (Riddleret al. 2003; Shor-Posner et al. 1993). HIV infection is commonly associated alsowith hypoalphalipoproteinemia; however, it is unclear whether virion replicationplays a causative role in these changes. Some data suggest that hypoalphalipopro-teinemia in patients with HIV is likely to be secondary to HIV infection itself (Roseet al. 2006). Systemic inflammation has been shown to lower the antioxidant andanti-inflammatory activity by transforming HDL to a pro-oxidant,pro-inflammatory acute-phase HDL (Kelesidis et al. 2013; Norata et al. 2006). Asmall pilot study of HIV-1-infected individuals with suppressed viremia on combi-nation antiretroviral therapy showed that oxidative stress and inflammation inHIV-1 are associated with a marked reduction of HDL antioxidant–anti-inflamma-tory activities. In vitro, these abnormalities were significantly improved by treat-ment with the apoA-1 mimetic peptide 4F (Kelesidis et al. 2011). HIV infection isassociated with modified HDL metabolism redirecting cholesterol to the apoB-containing lipoproteins and likely reducing the functionality of reverse cholesteroltransport (Rose et al. 2008). Of note, the HIV-1 Nef protein can impair ABCA1cholesterol efflux from macrophages, thus supporting atherosclerosis. This viralinhibition of efflux was correlated with a direct interaction between ABCA1 andNef (Fitzgerald et al. 2010; Mujawar et al. 2006). More recently it was shown thatHDL in Infectious Diseases and Sepsis 495Nef downregulates ABCA1 function by a posttranslational mechanism thatstimulates ABCA1 degradation but does not require the ability of Nef to bindABCA1 (Mujawar et al. 2010).Not all data are however concordant on this, and although HDL cholesterol andpreβ1-HDL were significantly lower in all HIV-infected groups ( p< 0.05), meanlevels of apoA-I and the ability of plasma to promote cholesterol efflux were similarin treatment-naı̈ve HIV-infected patients or in HIV-infected patients on longtherapy break. Of note a positive correlation between apoA-I and levels of CD4+cells was also observed (r2¼ 0.3, p< 0.001) (Rose et al. 2008). Furthermore apoA-I, the major protein component of high-density lipoprotein, and its amphipathicpeptide analogue were found to inhibit cell fusion, both in HIV-1-infected T cellsand in recombinant vaccinia-virus-infected CD4+ HeLa cells expressing HIVenvelope protein on their surfaces (Srinivas et al. 1990). The amphipathic peptidesinhibited the infectivity of HIV-1. The inhibitory effects were manifest when thevirus, but not cells, was pretreated with the peptides. Also, a reduction inHIV-induced cell killing was observed when virus-infected cell cultures weremaintained in the presence of amphipathic peptides. These results have potentialimplications for HIV biology and therapy (Srinivas et al. 1990).An aspect debated is how much the HIV infection and/or treatment contribute tothe changes in HDL-C levels. With highly active antiretroviral therapy (HAART)intervention, mortality due to HIV was greatly reduced (Madamanchi et al. 2002).However, there have been several reports of increases in cardiovascularcomplications in patients with HIV. It is now established that some HAARTregimens cause severe dyslipidemia, characterized by high levels of TC andLDL-C, hypertriglyceridemia, and hypoalphalipoproteinemia (Riddleret al. 2003). This clearly pro-atherogenic lipoprotein profile is associated with arise in the incidence of CAD (Depairon et al. 2001). The rate of inflammationpredicts changes in HDL-C and apoA-I following the initiation of antiretroviraltherapy and indeed in a subgroup of participants not taking ART at study entry whowere randomized in the Strategies for Management of Antiretroviral Therapy(SMART) to immediately initiate ART (“VS group”) or to defer it (“DC group”);HDL-C and ApoA-I levels increased among VS participants (n¼ 128) after startingART compared to DC. The effect of starting ART on changes in HDL-C and apoA-Iwas greater for those with higher versus lower baseline levels of IL-6 or hsCRPindicating that the activation of inflammatory pathways could contribute toHIV-associated changes in HDL (Baker et al. 2011). Also non-nucleoside reversetranscriptase inhibitors (NNRTI), such as nevirapine (NVP), were shown toincrease apoA-I production, which contributes to the HDL-C increase after intro-duction of NVP-containing regimens. In view of the potent anti-atherogenic effectsof apoA-I, the observed increase was suggested to contribute to the favorablecardiovascular profile of NVP (Franssen et al. 2009). Also efavirenz, anotherNNRTI antiretroviral treatment, was associated with HDL particles with a betterantioxidant function, i.e., with a higher PON-1 activity. The PON-1 activity ofblack patients is higher than that found in whites regardless of treatment suggestingthat ethnicity should be taken into consideration when studying drug effects onPON-1 activity (Pereira et al. 2009).496 A. Pirillo et al.Overall the available evidences suggest that HIV infection could be associatedwith modified HDL metabolism redirecting cholesterol to the apoB-containinglipoproteins and likely reducing the functionality of reverse cholesterol transportand promote atherosclerosis. Additional pro-atherogenic mechanisms could beassociated with a decrease in the anti-inflammatory properties of HDL.4.2 HCV InfectionHCV is a major cause of liver cirrhosis and hepatocellular carcinoma. Viral entry isrequired for initiation, spread, and maintenance of infection and thus is a promisingtarget for antiviral therapy. HCV exists in heterogenous forms in human serum andmay be associated with VLDL, LDL, and HDL which can shield the virus fromneutralizing antibodies targeting the HCV envelope glycoproteins (Agnelloet al. 1999; Hijikata et al. 1993; Nielsen et al. 2006; Thomssen et al. 1992). HCVbinding and entry into hepatocytes is a complex process involving the viral enve-lope glycoproteins E1 and E2, as well as several host factors, including highlysulfated heparan sulfate, CD81, the low-density lipoprotein receptor, claudin-1,occludin, and receptor tyrosine kinases (Lupberger et al. 2011; Zeisel et al. 2011).Also SR-BI which binds a variety of lipoproteins and mediates the selectiveuptake of HDL cholesterol ester (CE) as well as bidirectional free cholesteroltransport at the cell membrane emerged as a critical receptor affecting HCVentry. SR-BI directly binds HCV E2 (Bartosch et al. 2003; Evans et al. 2007;Scarselli et al. 2002), but virus-associated lipoproteins, including apoB containing,also contribute to host cell binding and uptake (Dao Thi et al. 2012; Maillardet al. 2006). Moreover, physiological SR-BI ligands modulate HCV infection(Bartosch et al. 2005; Voisset et al. 2005; von Hahn et al. 2006), suggesting theexistence of a complex interplay between lipoproteins (not only HDL), SR-BI andHCV envelope glycoproteins for HCV entry. Earlier studies using small moleculeinhibitors indicated a role for SR-BI lipid transfer function in HCV infection andHDL-mediated entry enhancement (Bartosch et al. 2003; Dreux et al. 2009; Syderet al. 2011; Voisset et al. 2005). A human anti-SR-BI mAb has been reported toinhibit HDL binding, to interfere with cholesterol efflux, and to decrease cellculture-derived HCV (HCVcc) entry during attachment steps without having arelevant impact on SR-BI-mediated post-binding steps (Catanese et al. 2007,2010). However, SR-BI mediates the uptake of HDL-C in a two-step processincluding HDL binding and subsequent transfer of CE into the cell without inter-nalization of HDL; a novel emerging hypothesis suggests that the interference withSR-BI lipid transfer function may be relevant for both initiation of HCV infectionand viral dissemination independently of HDL function (Zahid et al. 2013).Indeed SR-BI has also been demonstrated to mediate post-binding events duringHCV entry (Haberstroh et al. 2008; Syder et al. 2011; Zeisel et al. 2007). HCV–SR-BI interaction during post-binding steps occurs at similar time points as the HCVutilization of CD81 and claudin-1 suggesting that HCV entry may be mediatedthrough the formation of co-receptor complexes (Harris et al. 2008; KriegerHDL in Infectious Diseases and Sepsis 497et al. 2010; Zeisel et al. 2007). Also the SR-BI partner PDZK1 was shown tofacilitate hepatitis C virus entry (Eyre et al. 2010).These data suggest that SR-BI plays a multifunctional role during HCV entry atboth binding and post-binding steps (Catanese et al. 2010). Furthermore the HCVpost-binding function of human SR-BI can be dissociated from its E2-bindingfunction. Murine SR-BI does not bind E2 although it is capable of promotingHCV entry (Catanese et al. 2010; Ploss et al. 2009), and SR-BI, although able todirectly bind E2 and virus-associated lipoproteins, could play additional functionsduring HCV infection (Bartosch et al. 2003; Dreux et al. 2009; Zeisel et al. 2007).Although the addition of HDL enhances the efficiency of HCVcc infection, anti-SR-BI antibodies and SR-BI-specific siRNA efficiently inhibited HCV infectionindependently of lipoproteins (Zeisel et al. 2007). In this context, the post-bindingactivity of SR-BI is of key relevance for cell-free HCV infection as well as cell-to-cell transmission and by using antibodies which do not inhibit HDL binding toSR-BI; it was observed that post-binding function of SR-BI appears to be unrelatedto HDL interaction but to be directly linked to its lipid transfer function (Zahidet al. 2013). So far small molecules and mAbs targeting SR-BI and interfering withHCV infection have been described (Bartosch et al. 2003; Catanese et al. 2007;Syder et al. 2011). A codon-optimized version of this mAb has been demonstratedto prevent HCV spread in vivo (Meuleman et al. 2012), and ITX5061, a SR-BIinhibitor, is in clinical development as HCV entry inhibitor (phase I, http://clinicaltrials.gov/ct2/show/NCT01560468?term¼ITX+5061&rank¼3).Despite this promising approach, some open questions remain. First, it has beenshown in vitro that apoA-I is required for HCV production (Mancone et al. 2011)and that serum amyloid A has antiviral activities against HCV which are reducedwhen HDL are co-incubated with SAA (Lavie et al. 2006). Given the changesbetween apoA-I and SAA occurring in HDL during the acute phase (Norataet al. 2012), it is still unknown whether this mechanism could be the consequenceof viral infection or may represent part of the immune response which HCV learnedto escape. Second, as the inhibition of SR-BI represents one of the most promisingtargets for HCV infection, the potential side effects on the impairment of HDLfunction should be carefully evaluated. The new data showing that HCV infectiondoes not require receptor-E2–HDL interactions, coupled with the observation thatHCV entry and dissemination can be inhibited without blocking HDL–SR-BIbinding (Zahid et al. 2013), open a novel perspective for the design of entryinhibitors interfering specifically with the proviral function of SR-BI.ConclusionHDLs are emerging as a relevant player in both innate and adaptive immunity(Norata et al. 2011, 2012; Sala et al. 2012). HDL activities rely not only on theability to modulate cholesterol availability in immune cells but also on the roleof specific molecules shuttled by HDL. During infections and acute conditions,HDL-C levels decrease very rapidly, and HDL particles undergo changes thatdramatically alter their composition and function. Whether this is the conse-quence of a humoral innate response aimed at scavenging lipid bacterial498 A. Pirillo et al.products such as LPS from the circulation and driving them into the liver forcatabolism and elimination is still debated. Experimental evidence in geneticallymanipulated animal models suggests, however, that alterations in HDL struc-ture/composition are associated with poor prognosis following endotoxemia orsepsis, further supporting a protective role for HDL. The same is true for someparasitic infections, where the key player appears to be a specific and minorapolipoprotein of HDL–apoL-1. For viral infections, the landscape is morecomplicated; SR-BI was clearly indicated as a player favoring virus entry;however, it is not clear whether viruses, such as HCV, evolved to take advantageof the HDL–SR-BI interaction to entry liver cells as in vitro studies suggest orwhether in vivo HDL can compete for the interaction between HCV and SR-BI.Further studies are warranted in this context. Despite this, proteins related toHDL physiology represent already a target in clinical development forinfections, and SR-BI antagonism represents a novel and forefront approach tointerfere with hepatitis C virus entry.All the observations showing that the increase of HDL-C is associated with anattenuation of LPS-induced inflammatory responses strongly favor the hypothe-sis that raising plasma HDL may represent a therapeutic approach in the treat-ment of sepsis and its complications (Table 1). HDL-related therapies are ofgreat interest also in the context of parasites and virus infections (Table 1). As ofbeginning of 2014, the research in the field of HDL and immunity is in itsinfancy compared to the body of data available for HDL and atherosclerosis.From the coming years, we have to expect new and compelling observationsfurther linking HDL to innate immunity.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.Table 1 HDL-related therapies and infectionsCompound ReferencesEndotoxemia/LPS scavengingRecombinantHDLParker et al. (1995),Pajkrt et al. (1996)ApoA-ImimeticsGupta et al. (2005)ParasitesinfectionsTrypanosomaLeishmaniaApoL-1–nanobodyBaral et al. (2006),Samanovic et al. (2009)Hepatitis C virusentrySR-BImonoclonalantibodiesBartosch et al. (2003), Catanese et al. (2007),Meuleman et al. (2012), Syder et al. (2011)HDL in Infectious Diseases and Sepsis 499ReferencesAgnello V, Abel G, Elfahal M, Knight GB, Zhang QX (1999) Hepatitis C virus and otherflaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci U SA 96:12766–12771Badellino KO, Wolfe ML, Reilly MP, Rader DJ (2008) Endothelial lipase is increased in vivo byinflammation in humans. Circulation 117:678–685. doi:10.1161/CIRCULATIONAHA.107.707349Baker JV, Neuhaus J, Duprez D, Cooper DA, Hoy J, Kuller L, Lampe FC, Liappis A, Friis-Moller N, Otvos J, Paton NI, Tracy R, Neaton JD, Group ISS (2011) Inflammation predictschanges in high-density lipoprotein particles and apolipoprotein A1 following initiation ofantiretroviral therapy. AIDS 25:2133–2142. doi:10.1097/QAD.0b013e32834be088Banka CL, Yuan T, de Beer MC, Kindy M, Curtiss LK, de Beer FC (1995) Serum amyloid A(SAA): influence on HDL-mediated cellular cholesterol efflux. J Lipid Res 36:1058–1065Baragetti A, Norata GD, Sarcina C, Rastelli F, Grigore L, Garlaschelli K, Uboldi P, Baragetti I,Pozzi C, Catapano AL (2013) High density lipoprotein cholesterol levels are an independentpredictor of the progression of chronic kidney disease. J Intern Med 274:252–262. doi:10.1111/joim.12081Baral TN, Magez S, Stijlemans B, Conrath K, Vanhollebeke B, Pays E, Muyldermans S, DeBaetselier P (2006) Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor. Nat Med 12:580–584. doi:10.1038/nm1395Barlage S, Frohlich D, Bottcher A, Jauhiainen M, Muller HP, Noetzel F, Rothe G, Schutt C, LinkeRP, Lackner KJ, Ehnholm C, Schmitz G (2001) ApoE-containing high density lipoproteins andphospholipid transfer protein activity increase in patients with a systemic inflammatoryresponse. J Lipid Res 42:281–290Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJ, Komajda M, Lopez-Sendon J,Mosca L, Tardif JC, Waters DD, Shear CL, Revkin JH, Buhr KA, Fisher MR, Tall AR, BrewerB (2007) Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med357:2109–2122. doi:10.1056/NEJMoa0706628Bartosch B, Vitelli A, Granier C, Goujon C, Dubuisson J, Pascale S, Scarselli E, Cortese R,Nicosia A, Cosset FL (2003) Cell entry of hepatitis C virus requires a set of co-receptors thatinclude the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem 278:41624–41630. doi:10.1074/jbc.M305289200Bartosch B, Verney G, Dreux M, Donot P, Morice Y, Penin F, Pawlotsky JM, Lavillette D, CossetFL (2005) An interplay between hypervariable region 1 of the hepatitis C virus E2 glycopro-tein, the scavenger receptor BI, and high-density lipoprotein promotes both enhancement ofinfection and protection against neutralizing antibodies. J Virol 79:8217–8229. doi:10.1128/JVI.79.13.8217-8229.2005Baruah P, Dumitriu IE, Peri G, Russo V, Mantovani A, Manfredi AA, Rovere-Querini P (2006)The tissue pentraxin PTX3 limits C1q-mediated complement activation and phagocytosis ofapoptotic cells by dendritic cells. J Leukoc Biol 80:87–95. doi:10.1189/jlb.0805445Berbee JF, van der Hoogt CC, Kleemann R, Schippers EF, Kitchens RL, van Dissel JT, Bakker-Woudenberg IA, Havekes LM, Rensen PC (2006) Apolipoprotein CI stimulates the response tolipopolysaccharide and reduces mortality in gram-negative sepsis. Faseb J 20:2162–2164Bhakdi S, Klonisch T, Nuber P, Fischer W (1991) Stimulation of monokine production bylipoteichoic acids. Infect Immun 59:4614–4620Birjmohun RS, van Leuven SI, Levels JH, van’t Veer C, Kuivenhoven JA, Meijers JC, Levi M,Kastelein JJ, van der Poll T, Stroes ES (2007) High-density lipoprotein attenuates inflamma-tion and coagulation response on endotoxin challenge in humans. Arterioscler Thromb VascBiol 27:1153–1158. doi:10.1161/ATVBAHA.106.136325Bonacina F, Baragetti A, Catapano AL, Norata GD (2013) Long pentraxin 3: experimental andclinical relevance in cardiovascular diseases. Mediators Inflamm 2013:725102. doi:10.1155/2013/725102500 A. Pirillo et al.Bottazzi B, Vouret-Craviari V, Bastone A, De Gioia L, Matteucci C, Peri G, Spreafico F, Pausa M,D’Ettorre C, Gianazza E, Tagliabue A, Salmona M, Tedesco F, Introna M, Mantovani A (1997)Multimer formation and ligand recognition by the long pentraxin PTX3. Similarities anddifferences with the short pentraxins C-reactive protein and serum amyloid P component. JBiol Chem 272:32817–32823Bozza S, Bistoni F, Gaziano R, Pitzurra L, Zelante T, Bonifazi P, Perruccio K, Bellocchio S,Neri M, Iorio AM, Salvatori G, De Santis R, Calvitti M, Doni A, Garlanda C, Mantovani A,Romani L (2006) Pentraxin 3 protects from MCMV infection and reactivation through TLRsensing pathways leading to IRF3 activation. Blood 108:3387–3396. doi:10.1182/blood-2006-03-009266Breviario F, d’Aniello EM, Golay J, Peri G, Bottazzi B, Bairoch A, Saccone S, Marzella R,Predazzi V, Rocchi M et al (1992) Interleukin-1-inducible genes in endothelial cells. Cloningof a new gene related to C-reactive protein and serum amyloid P component. J Biol Chem267:22190–22197Cao Y, Stafforini DM, Zimmerman GA, McIntyre TM, Prescott SM (1998) Expression of plasmaplatelet-activating factor acetylhydrolase is transcriptionally regulated by mediators of inflam-mation. J Biol Chem 273:4012–4020Catanese MT, Graziani R, von Hahn T, Moreau M, Huby T, Paonessa G, Santini C, Luzzago A,Rice CM, Cortese R, Vitelli A, Nicosia A (2007) High-avidity monoclonal antibodies againstthe human scavenger class B type I receptor efficiently block hepatitis C virus infection in thepresence of high-density lipoprotein. J Virol 81:8063–8071. doi:10.1128/JVI.00193-07Catanese MT, Ansuini H, Graziani R, Huby T, Moreau M, Ball JK, Paonessa G, Rice CM,Cortese R, Vitelli A, Nicosia A (2010) Role of scavenger receptor class B type I in hepatitisC virus entry: kinetics and molecular determinants. J Virol 84:34–43. doi:10.1128/JVI.02199-08Chien JY, Jerng JS, Yu CJ, Yang PC (2005) Low serum level of high-density lipoproteincholesterol is a poor prognostic factor for severe sepsis. Crit Care Med 33:1688–1693Christoffersen C, Obinata H, Kumaraswamy SB, Galvani S, Ahnstrom J, Sevvana M, Egerer-Sieber C, Muller YA, Hla T, Nielsen LB, Dahlback B (2011) Endothelium-protective sphin-gosine-1-phosphate provided by HDL-associated apolipoprotein M. Proc Natl Acad Sci USA108:9613–9618. doi:10.1073/pnas.1103187108Clark RW, Cunningham D, Cong Y, Subashi TA, Tkalcevic GT, Lloyd DB, Boyd JG, ChrunykBA, Karam GA, Qiu X, Wang IK, Francone OL (2010) Assessment of cholesteryl ester transferprotein inhibitors for interaction with proteins involved in the immune response to infection. JLipid Res 51:967–974. doi:10.1194/jlr.M002295Coetzee GA, Strachan AF, van der Westhuyzen DR, Hoppe HC, Jeenah MS, de Beer FC (1986)Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipopro-tein composition. J Biol Chem 261:9644–9651Cruz D, Watson AD, Miller CS, Montoya D, Ochoa MT, Sieling PA, Gutierrez MA, Navab M,Reddy ST, Witztum JL, Fogelman AM, Rea TH, Eisenberg D, Berliner J, Modlin RL (2008)Host-derived oxidized phospholipids and HDL regulate innate immunity in human leprosy. JClin Invest 118:2917–2928. doi:10.1172/JCI34189Dai L, Datta G, Zhang Z, Gupta H, Patel R, Honavar J, Modi S, Wyss JM, Palgunachari M,Anantharamaiah GM, White CR (2010) The apolipoprotein A-I mimetic peptide 4F preventsdefects in vascular function in endotoxemic rats. J Lipid Res 51:2695–2705. doi:10.1194/jlr.M008086Dao Thi VL, Granier C, Zeisel MB, Guerin M, Mancip J, Granio O, Penin F, Lavillette D,Bartenschlager R, Baumert TF, Cosset FL, Dreux M (2012) Characterization of hepatitis Cvirus particle subpopulations reveals multiple usage of the scavenger receptor BI for entrysteps. J Biol Chem 287:31242–31257. doi:10.1074/jbc.M112.365924Datta G, Gupta H, Zhang Z, Mayakonda P, Anantharamaiah GM, White CR (2011) HDL mimeticpeptide administration improves left ventricular filling and cardiac output inlipopolysaccharide-treated rats. J Clin Exp Cardiolog 2:pii: 1000172. doi:10.4172/2155-9880.1000172HDL in Infectious Diseases and Sepsis 501De Kimpe SJ, Kengatharan M, Thiemermann C, Vane JR (1995) The cell wall componentspeptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to causeshock and multiple organ failure. Proc Natl Acad Sci USA 92:10359–10363de la Llera MM, McGillicuddy FC, Hinkle CC, Byrne M, Joshi MR, Nguyen V, Tabita-Martinez J,Wolfe ML, Badellino K, Pruscino L, Mehta NN, Asztalos BF, Reilly MP (2012) Inflammationmodulates human HDL composition and function in vivo. Atherosclerosis 222:390–394.doi:10.1016/j.atherosclerosis.2012.02.032/S0021-9150(12)00146-3Depairon M, Chessex S, Sudre P, Rodondi N, Doser N, Chave JP, Riesen W, Nicod P, Darioli R,Telenti A, Mooser V, Swiss HIVCS (2001) Premature atherosclerosis in HIV-infectedindividuals – focus on protease inhibitor therapy. AIDS 15:329–334Diniz SN, Nomizo R, Cisalpino PS, Teixeira MM, Brown GD, Mantovani A, Gordon S, Reis LF,Dias AA (2004) PTX3 function as an opsonin for the dectin-1-dependent internalization ofzymosan by macrophages. J Leukoc Biol 75:649–656. doi:10.1189/jlb.0803371Doni A, Mantovani G, Porta C, Tuckermann J, Reichardt HM, Kleiman A, Sironi M, Rubino L,Pasqualini F, Nebuloni M, Signorini S, Peri G, Sica A, Beck-Peccoz P, Bottazzi B, MantovaniA (2008) Cell-specific regulation of PTX3 by glucocorticoid hormones in hematopoietic andnonhematopoietic cells. J Biol Chem 283:29983–29992. doi:10.1074/jbc.M805631200Doni A, Garlanda C, Bottazzi B, Meri S, Garred P, Mantovani A (2012) Interactions of thehumoral pattern recognition molecule PTX3 with the complement system. Immunobiology217:1122–1128. doi:10.1016/j.imbio.2012.07.004/S0171-2985(12)00168-4Dreux M, Dao Thi VL, Fresquet J, Guerin M, Julia Z, Verney G, Durantel D, Zoulim F,Lavillette D, Cosset FL, Bartosch B (2009) Receptor complementation and mutagenesis revealSR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellulardomains. PLoS Pathog 5:e1000310. doi:10.1371/journal.ppat.1000310Duchateau PN, Pullinger CR, Orellana RE, Kunitake ST, Naya-Vigne J, O’Connor PM, MalloyMJ, Kane JP (1997) Apolipoprotein L, a new human high density lipoprotein apolipoproteinexpressed by the pancreas. Identification, cloning, characterization, and plasma distribution ofapolipoprotein L. J Biol Chem 272:25576–25582Emancipator K, Csako G, Elin RJ (1992) In vitro inactivation of bacterial endotoxin by humanlipoproteins and apolipoproteins. Infect Immun 60:596–601Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wolk B, Hatziioannou T, McKeatingJA, Bieniasz PD, Rice CM (2007) Claudin-1 is a hepatitis C virus co-receptor required for alate step in entry. Nature 446:801–805. doi:10.1038/nature05654Eyre NS, Drummer HE, Beard MR (2010) The SR-BI partner PDZK1 facilitates hepatitis C virusentry. PLoS Pathog 6:e1001130. doi:10.1371/journal.ppat.1001130Feingold KR, Memon RA, Moser AH, Grunfeld C (1998) Paraoxonase activity in the serum andhepatic mRNA levels decrease during the acute phase response. Atherosclerosis 139:307–315Feingold KR, Shigenaga JK, Chui LG, Moser A, Khovidhunkit W, Grunfeld C (2008) Infectionand inflammation decrease apolipoprotein M expression. Atherosclerosis 199:19–26. doi:10.1016/j.atherosclerosis.2007.10.007/S0021-9150(07)00660-0Fitzgerald ML, Mujawar Z, Tamehiro N (2010) ABC transporters, atherosclerosis and inflamma-tion. Atherosclerosis 211:361–370. doi:10.1016/j.atherosclerosis.2010.01.011Flegel WA, Baumstark MW, Weinstock C, Berg A, Northoff H (1993) Prevention of endotoxin-induced monokine release by human low- and high-density lipoproteins and by apolipoproteinA-I. Infect Immun 61:5140–5146Franssen R, Sankatsing RR, Hassink E, Hutten B, Ackermans MT, Brinkman K, Oesterholt R,Arenas-Pinto A, Storfer SP, Kastelein JJ, Sauerwein HP, Reiss P, Stroes ES (2009) Nevirapineincreases high-density lipoprotein cholesterol concentration by stimulation of apolipoproteinA-I production. Arterioscler Thromb Vasc Biol 29:1336–1341. doi:10.1161/ATVBAHA.109.192088Garlanda C, Hirsch E, Bozza S, Salustri A, De Acetis M, Nota R, Maccagno A, Riva F, Bottazzi B,Peri G, Doni A, Vago L, Botto M, De Santis R, Carminati P, Siracusa G, Altruda F, Vecchi A,Romani L, Mantovani A (2002) Non-redundant role of the long pentraxin PTX3 in anti-fungalinnate immune response. Nature 420:182–186502 A. Pirillo et al.Garlanda C, Bottazzi B, Bastone A, Mantovani A (2005) Pentraxins at the crossroads betweeninnate immunity, inflammation, matrix deposition, and female fertility. Annu Rev Immunol23:337–366Gershov D, Kim S, Brot N, Elkon KB (2000) C-Reactive protein binds to apoptotic cells, protectsthe cells from assembly of the terminal complement components, and sustains anantiinflammatory innate immune response: implications for systemic autoimmunity. J ExpMed 192:1353–1364Grunfeld C, Marshall M, Shigenaga JK, Moser AH, Tobias P, Feingold KR (1999) Lipoproteinsinhibit macrophage activation by lipoteichoic acid. J Lipid Res 40:245–252Guo L, Ai J, Zheng Z, Howatt DA, Daugherty A, Huang B, Li XA (2013) High density lipoproteinprotects against polymicrobe-induced sepsis in mice. J Biol Chem 288:17947–17953. doi:10.1074/jbc.M112.442699Gupta H, Dai L, Datta G, Garber DW, Grenett H, Li Y, Mishra V, Palgunachari MN, Handattu S,Gianturco SH, Bradley WA, Anantharamaiah GM, White CR (2005) Inhibition oflipopolysaccharide-induced inflammatory responses by an apolipoprotein AI mimetic peptide.Circ Res 97:236–243Haberstroh A, Schnober EK, Zeisel MB, Carolla P, Barth H, Blum HE, Cosset FL,Koutsoudakis G, Bartenschlager R, Union A, Depla E, Owsianka A, Patel AH, Schuster C,Stoll-Keller F, Doffoel M, Dreux M, Baumert TF (2008) Neutralizing host responses inhepatitis C virus infection target viral entry at postbinding steps and membrane fusion.Gastroenterology 135(1719–1728):e1. doi:10.1053/j.gastro.2008.07.018Harris HJ, Farquhar MJ, Mee CJ, Davis C, Reynolds GM, Jennings A, Hu K, Yuan F, Deng H,Hubscher SG, Han JH, Balfe P, McKeating JA (2008) CD81 and claudin 1 coreceptor associa-tion: role in hepatitis C virus entry. J Virol 82:5007–5020. doi:10.1128/JVI.02286-07Henning MF, Herlax V, Bakas L (2011) Contribution of the C-terminal end of apolipoprotein AI toneutralization of lipopolysaccharide endotoxic effect. Innate Immun 17:327–337Hijikata M, Shimizu YK, Kato H, Iwamoto A, Shih JW, Alter HJ, Purcell RH, Yoshikura H (1993)Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immunecomplexes. J Virol 67:1953–1958Humphries SE, Whittall RA, Hubbart CS, Maplebeck S, Cooper JA, Soutar AK, Naoumova R,Thompson GR, Seed M, Durrington PN, Miller JP, Betteridge DJ, Neil HA (2006) Geneticcauses of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levelsand coronary heart disease risk. J Med Genet 43:943–949Iwasaki A, Medzhitov R (2010) Regulation of adaptive immunity by the innate immune system.Science 327:291–295. doi:10.1126/science.1183021/327/5963/291Jaillon S, Jeannin P, Hamon Y, Fremaux I, Doni A, Bottazzi B, Blanchard S, Subra JF,Chevailler A, Mantovani A, Delneste Y (2009) Endogenous PTX3 translocates at the mem-brane of late apoptotic human neutrophils and is involved in their engulfment by macrophages.Cell Death Differ 16:465–474. doi:10.1038/cdd.2008.173Jeannin P, Bottazzi B, Sironi M, Doni A, Rusnati M, Presta M, Maina V, Magistrelli G, Haeuw JF,Hoeffel G, Thieblemont N, Corvaia N, Garlanda C, Delneste Y, Mantovani A (2005) Com-plexity and complementarity of outer membrane protein A recognition by cellular and humoralinnate immunity receptors. Immunity 22:551–560. doi:10.1016/j.immuni.2005.03.008/S1074-7613(05)00103-2Jerala R (2007) Structural biology of the LPS recognition. Int J Med Microbiol 297:353–363.doi:10.1016/j.ijmm.2007.04.001/S1438-4221(07)00068-9Jiao YL, Wu MP (2008) Apolipoprotein A-I diminishes acute lung injury and sepsis in miceinduced by lipoteichoic acid. Cytokine 43:83–87. doi:10.1016/j.cyto.2008.04.002/S1043-4666(08)00099-9Kelesidis T, Yang OO, Currier JS, Navab K, Fogelman AM, Navab M (2011) HIV-1 infectedpatients with suppressed plasma viremia on treatment have pro-inflammatory HDL. LipidsHealth Dis 10:35. doi:10.1186/1476-511X-10-35HDL in Infectious Diseases and Sepsis 503Kelesidis T, Yang OO, Kendall MA, Hodis HN, Currier JS (2013) Dysfunctional HDL andprogression of atherosclerosis in HIV-1-infected and -uninfected adults. Lipids Health Dis12:23. doi:10.1186/1476-511X-12-23Khovidhunkit W, Kim MS, Memon RA, Shigenaga JK, Moser AH, Feingold KR, Grunfeld C(2004) Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanismsand consequences to the host. J Lipid Res 45:1169–1196Kontush A, Chapman MJ (2006) Functionally defective high-density lipoprotein: a new therapeu-tic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis. Pharmacol Rev58:342–374Krieger SE, Zeisel MB, Davis C, Thumann C, Harris HJ, Schnober EK, Mee C, Soulier E,Royer C, Lambotin M, Grunert F, Dao Thi VL, Dreux M, Cosset FL, McKeating JA,Schuster C, Baumert TF (2010) Inhibition of hepatitis C virus infection by anti-claudin-1antibodies is mediated by neutralization of E2-CD81-claudin-1 associations. Hepatology51:1144–1157. doi:10.1002/hep.23445Kumaraswamy SB, Linder A, Akesson P, Dahlback B (2012) Decreased plasma concentrations ofapolipoprotein M in sepsis and systemic inflammatory response syndromes. Crit Care 16:R60.doi:10.1186/cc11305/cc11305Kwon WY, Suh GJ, Kim KS, Kwak YH, Kim K (2012) 4F, apolipoprotein AI mimetic peptide,attenuates acute lung injury and improves survival in endotoxemic rats. J Trauma Acute CareSurg 72:1576–1583. doi:10.1097/TA.0b013e3182493ab4/01586154-201206000-00023Lavie M, Voisset C, Vu-Dac N, Zurawski V, Duverlie G, Wychowski C, Dubuisson J (2006)Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in acell culture system. Hepatology 44:1626–1634. doi:10.1002/hep.21406Lee GW, Lee TH, Vilcek J (1993) TSG-14, a tumor necrosis factor- and IL-1-inducible protein, isa novel member of the pentaxin family of acute phase proteins. J Immunol 150:1804–1812Lee RP, Lin NT, Chao YF, Lin CC, Harn HJ, Chen HI (2007) High-density lipoprotein preventsorgan damage in endotoxemia. Res Nurs Health 30:250–260. doi:10.1002/nur.20187Levels JH, Abraham PR, van Barreveld EP, Meijers JC, van Deventer SJ (2003) Distribution andkinetics of lipoprotein-bound lipoteichoic acid. Infect Immun 71:3280–3284Levels JH, Geurts P, Karlsson H, Maree R, Ljunggren S, Fornander L, Wehenkel L, Lindahl M,Stroes ES, Kuivenhoven JA, Meijers JC (2011) High-density lipoprotein proteome dynamics inhuman endotoxemia. Proteome Sci 9:34. doi:10.1186/1477-5956-9-34Levine DM, Parker TS, Donnelly TM, Walsh A, Rubin AL (1993) In vivo protection againstendotoxin by plasma high density lipoprotein. Proc Natl Acad Sci USA 90:12040–12044Li Y, Dong JB, Wu MP (2008) Human ApoA-I overexpression diminishes LPS-induced systemicinflammation and multiple organ damage in mice. Eur J Pharmacol 590:417–422. doi:10.1016/j.ejphar.2008.06.047/S0014-2999(08)00656-0Lupberger J, Zeisel MB, Xiao F, Thumann C, Fofana I, Zona L, Davis C, Mee CJ, Turek M,Gorke S, Royer C, Fischer B, Zahid MN, Lavillette D, Fresquet J, Cosset FL, Rothenberg SM,Pietschmann T, Patel AH, Pessaux P, Doffoel M, Raffelsberger W, Poch O, McKeating JA,Brino L, Baumert TF (2011) EGFR and EphA2 are host factors for hepatitis C virus entry andpossible targets for antiviral therapy. Nat Med 17:589–595. doi:10.1038/nm.2341Madamanchi NR, Patterson C, Runge MS (2002) HIV therapies and atherosclerosis: answers orquestions? Arterioscler Thromb Vasc Biol 22:1758–1760Maillard P, Huby T, Andreo U, Moreau M, Chapman J, Budkowska A (2006) The interaction ofnatural hepatitis C virus with human scavenger receptor SR-BI/Cla1 is mediated by ApoB-containing lipoproteins. FASEB J 20:735–737. doi:10.1096/fj.05-4728fjeMancone C, Steindler C, Santangelo L, Simonte G, Vlassi C, Longo MA, D’Offizi G, DiGiacomo C, Pucillo LP, Amicone L, Tripodi M, Alonzi T (2011) Hepatitis C virus productionrequires apolipoprotein A-I and affects its association with nascent low-density lipoproteins.Gut 60:378–386. doi:10.1136/gut.2010.211292504 A. Pirillo et al.McGillicuddy FC, de la Llera MM, Hinkle CC, Joshi MR, Chiquoine EH, Billheimer JT, RothblatGH, Reilly MP (2009) Inflammation impairs reverse cholesterol transport in vivo. Circulation119:1135–1145Memon RA, Fuller J, Moser AH, Feingold KR, Grunfeld C (1999) In vivo regulation of plasmaplatelet-activating factor acetylhydrolase during the acute phase response. Am J Physiol 277:R94–R103Meuleman P, Catanese MT, Verhoye L, Desombere I, Farhoudi A, Jones CT, Sheahan T, Grzyb K,Cortese R, Rice CM, Leroux-Roels G, Nicosia A (2012) A human monoclonal antibodytargeting scavenger receptor class B type I precludes hepatitis C virus infection and viralspread in vitro and in vivo. Hepatology 55:364–372. doi:10.1002/hep.24692Mujawar Z, Rose H, Morrow MP, Pushkarsky T, Dubrovsky L, Mukhamedova N, Fu Y, Dart A,Orenstein JM, Bobryshev YV, Bukrinsky M, Sviridov D (2006) Human immunodeficiencyvirus impairs reverse cholesterol transport from macrophages. PLoS Biol 4:e365. doi:10.1371/journal.pbio.0040365Mujawar Z, Tamehiro N, Grant A, Sviridov D, Bukrinsky M, Fitzgerald ML (2010) Mutation ofthe ATP cassette binding transporter A1 (ABCA1) C-terminus disrupts HIV-1 Nef binding butdoes not block the Nef enhancement of ABCA1 protein degradation. Biochemistry 49:8338–8349. doi:10.1021/bi100466qMurch O, Collin M, Hinds CJ, Thiemermann C (2007) Lipoproteins in inflammation and sepsis.I. Basic science. Intensive Care Med 33:13–24Murphy AJ, Woollard KJ, Suhartoyo A, Stirzaker RA, Shaw J, Sviridov D, Chin-Dusting JP(2011) Neutrophil activation is attenuated by high-density lipoprotein and apolipoprotein a-I inin vitro and in vivo models of inflammation. Arterioscler Thromb Vasc Biol 31:1333–1341Nauta AJ, Bottazzi B, Mantovani A, Salvatori G, Kishore U, Schwaeble WJ, Gingras AR,Tzima S, Vivanco F, Egido J, Tijsma O, Hack EC, Daha MR, Roos A (2003) Biochemicaland functional characterization of the interaction between pentraxin 3 and C1q. Eur J Immunol33:465–473. doi:10.1002/immu.200310022Navab M, Ananthramaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Fonarow GC,Vahabzadeh K, Hama S, Hough G, Kamranpour N, Berliner JA, Lusis AJ, Fogelman AM(2004) The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL.J Lipid Res 45:993–1007Nielsen SU, Bassendine MF, Burt AD, Martin C, Pumeechockchai W, Toms GL (2006) Associa-tion between hepatitis C virus and very-low-density lipoprotein (VLDL)/LDL analyzed iniodixanol density gradients. J Virol 80:2418–2428. doi:10.1128/JVI.80.5.2418-2428.2006Norata GD, Callegari E, Marchesi M, Chiesa G, Eriksson P, Catapano AL (2005) High-densitylipoproteins induce transforming growth factor-beta2 expression in endothelial cells. Circula-tion 111:2805–2811Norata GD, Pirillo A, Catapano AL (2006) Modified HDL: biological and physiopathologicalconsequences. Nutr Metab Cardiovasc Dis 16(5):371–386Norata GD, Marchesi P, Pirillo A, Uboldi P, Chiesa G, Maina V, Garlanda C, Mantovani A,Catapano AL (2008) Long pentraxin 3, a key component of innate immunity, is modulated byhigh-density lipoproteins in endothelial cells. Arterioscler Thromb Vasc Biol 28:925–931.doi:10.1161/ATVBAHA.107.160606Norata GD, Marchesi P, Pulakazhi Venu VK, Pasqualini F, Anselmo A, Moalli F, Pizzitola I,Garlanda C, Mantovani A, Catapano AL (2009) Deficiency of the long pentraxin PTX3promotes vascular inflammation and atherosclerosis. Circulation 120:699–708. doi:10.1161/CIRCULATIONAHA.108.806547Norata GD, Garlanda C, Catapano AL (2010) The long pentraxin PTX3: a modulator of theimmunoinflammatory response in atherosclerosis and cardiovascular diseases. TrendsCardiovasc Med 20:35–40. doi:10.1016/j.tcm.2010.03.005/S1050-1738(10)00036-8Norata GD, Pirillo A, Catapano AL (2011) HDLs, immunity, and atherosclerosis. Curr OpinLipidol 22:410–416. doi:10.1097/MOL.0b013e32834adac3HDL in Infectious Diseases and Sepsis 505Norata GD, Pirillo A, Ammirati E, Catapano AL (2012) Emerging role of high density lipoproteinsas a player in the immune system. Atherosclerosis 220:11–21. doi:10.1016/j.atherosclerosis.2011.06.045Pajkrt D, Doran JE, Koster F, Lerch PG, Arnet B, van der Poll T, ten Cate JW, van Deventer SJ(1996) Antiinflammatory effects of reconstituted high-density lipoprotein during humanendotoxemia. J Exp Med 184:1601–1608Parker TS, Levine DM, Chang JC, Laxer J, Coffin CC, Rubin AL (1995) Reconstituted high-density lipoprotein neutralizes gram-negative bacterial lipopolysaccharides in human wholeblood. Infect Immun 63:253–258Parsa A, Kao WH, Xie D, Astor BC, Li M, Hsu CY, Feldman HI, Parekh RS, Kusek JW, GreeneTH, Fink JC, Anderson AH, Choi MJ, Wright JT Jr, Lash JP, Freedman BI, Ojo A, WinklerCA, Raj DS, Kopp JB, He J, Jensvold NG, Tao K, Lipkowitz MS, Appel LJ, AASK StudyInvestigators, CRIC Study Investigators (2013) APOL1 risk variants, race, and progression ofchronic kidney disease. N Engl J Med 369(23):2183–2196. doi:10.1056/NEJMoa1310345Pereira SA, Batuca JR, Caixas U, Branco T, Delgado-Alves J, Germano I, Lampreia F, MonteiroEC (2009) Effect of efavirenz on high-density lipoprotein antioxidant properties inHIV-infected patients. Br J Clin Pharmacol 68:891–897. doi:10.1111/j.1365-2125.2009.03535.xPerez-Morga D, Vanhollebeke B, Paturiaux-Hanocq F, Nolan DP, Lins L, Homble F,Vanhamme L, Tebabi P, Pays A, Poelvoorde P, Jacquet A, Brasseur R, Pays E (2005)Apolipoprotein L-I promotes trypanosome lysis by forming pores in lysosomal membranes.Science 309:469–472. doi:10.1126/science.1114566Pirillo A, Norata GD, Catapano AL (2013) Treating high density lipoprotein cholesterol (HDL-C):quantity versus quality. Curr Pharm Des 19:3841–3857Ploss A, Evans MJ, Gaysinskaya VA, Panis M, You H, de Jong YP, Rice CM (2009) Humanoccludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature457:882–886. doi:10.1038/nature07684Poon IK, Hulett MD, Parish CR (2010) Molecular mechanisms of late apoptotic/necrotic cellclearance. Cell Death Differ 17:381–397. doi:10.1038/cdd.2009.195Reading PC, Bozza S, Gilbertson B, Tate M, Moretti S, Job ER, Crouch EC, Brooks AG, BrownLE, Bottazzi B, Romani L, Mantovani A (2008) Antiviral activity of the long chain pentraxinPTX3 against influenza viruses. J Immunol 180:3391–3398Riddler SA, Smit E, Cole SR, Li R, Chmiel JS, Dobs A, Palella F, Visscher B, Evans R, KingsleyLA (2003) Impact of HIV infection and HAART on serum lipids in men. JAMA 289:2978–2982. doi:10.1001/jama.289.22.2978Rose H, Woolley I, Hoy J, Dart A, Bryant B, Mijch A, Sviridov D (2006) HIV infection and high-density lipoprotein: the effect of the disease vs the effect of treatment. Metabolism 55:90–95.doi:10.1016/j.metabol.2005.07.012Rose H, Hoy J, Woolley I, Tchoua U, Bukrinsky M, Dart A, Sviridov D (2008) HIV infection andhigh density lipoprotein metabolism. Atherosclerosis 199:79–86. doi:10.1016/j.atherosclerosis.2007.10.018Roumenina LT, Ruseva MM, Zlatarova A, Ghai R, Kolev M, Olova N, Gadjeva M, Agrawal A,Bottazzi B, Mantovani A, Reid KB, Kishore U, Kojouharova MS (2006) Interaction of C1qwith IgG1, C-reactive protein and pentraxin 3: mutational studies using recombinant globularhead modules of human C1q A, B, and C chains. Biochemistry 45:4093–4104. doi:10.1021/bi052646fRovere P, Peri G, Fazzini F, Bottazzi B, Doni A, Bondanza A, Zimmermann VS, Garlanda C,Fascio U, Sabbadini MG, Rugarli C, Mantovani A, Manfredi AA (2000) The long pentraxinPTX3 binds to apoptotic cells and regulates their clearance by antigen-presenting dendriticcells. Blood 96:4300–4306Sala F, Catapano AL, Norata GD (2012) High density lipoproteins and atherosclerosis: emergingaspects. J Geriatr Cardiol 9:401–407. doi:10.3724/SP.J.1263.2011.12282506 A. Pirillo et al.Sala F, Cutuli L, Grigore L, Pirillo A, Chiesa G, Catapano AL, Norata GD (2013) Prevalence ofclassical CD14++/CD16 but not of intermediate CD14++/CD16+ monocytes in hypoalphali-poproteinemia. Int J Cardiol 168:2886–2889. doi:10.1016/j.ijcard.2013.03.103Samanovic M, Molina-Portela MP, Chessler AD, Burleigh BA, Raper J (2009) Trypanosome lyticfactor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection. PLoSPathog 5:e1000276. doi:10.1371/journal.ppat.1000276Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, Traboni C, Nicosia A,Cortese R, Vitelli A (2002) The human scavenger receptor class B type I is a novel candidatereceptor for the hepatitis C virus. EMBO J 21:5017–5025Sherman CB, Peterson SJ, Frishman WH (2010) Apolipoprotein A-I mimetic peptides: a potentialnew therapy for the prevention of atherosclerosis. Cardiol Rev 18:141–147. doi:10.1097/CRD.0b013e3181c4b508Shor-Posner G, Basit A, Lu Y, Cabrejos C, Chang J, Fletcher M, Mantero-Atienza E, Baum MK(1993) Hypocholesterolemia is associated with immune dysfunction in early human immuno-deficiency virus-1 infection. Am J Med 94:515–519Srinivas RV, Birkedal B, Owens RJ, Anantharamaiah GM, Segrest JP, Compans RW (1990)Antiviral effects of apolipoprotein A-I and its synthetic amphipathic peptide analogs. Virology176:48–57Suzuki M, Pritchard DK, Becker L, Hoofnagle AN, Tanimura N, Bammler TK, Beyer RP,Bumgarner R, Vaisar T, de Beer MC, de Beer FC, Miyake K, Oram JF, Heinecke JW (2010)High-density lipoprotein suppresses the type I interferon response, a family of potent antiviralimmunoregulators, in macrophages challenged with lipopolysaccharide. Circulation122:1919–1927Syder AJ, Lee H, Zeisel MB, Grove J, Soulier E, Macdonald J, Chow S, Chang J, Baumert TF,McKeating JA, McKelvy J, Wong-Staal F (2011) Small molecule scavenger receptor BIantagonists are potent HCV entry inhibitors. J Hepatol 54:48–55. doi:10.1016/j.jhep.2010.06.024Thompson PA, Kitchens RL (2006) Native high-density lipoprotein augments monocyte responsesto lipopolysaccharide (LPS) by suppressing the inhibitory activity of LPS-binding protein. JImmunol 177:4880–4887. doi:10.4049/jimmunol.177.7.4880Thomssen R, Bonk S, Propfe C, Heermann KH, Kochel HG, Uy A (1992) Association of hepatitisC virus in human sera with beta-lipoprotein. Med Microbiol Immunol 181:293–300Tobias PS, Soldau K, Ulevitch RJ (1989) Identification of a lipid A binding site in the acute phasereactant lipopolysaccharide binding protein. J Biol Chem 264:10867–10871Ulevitch RJ, Johnston AR, Weinstein DB (1979) New function for high density lipoproteins. Theirparticipation in intravascular reactions of bacterial lipopolysaccharides. J Clin Invest 64:1516–1524. doi:10.1172/JCI109610Ulevitch RJ, Johnston AR, Weinstein DB (1981) New function for high density lipoproteins.Isolation and characterization of a bacterial lipopolysaccharide-high density lipoprotein com-plex formed in rabbit plasma. J Clin Invest 67:827–837. doi:10.1172/JCI110100Van Amersfoort ES, Van Berkel TJ, Kuiper J (2003) Receptors, mediators, and mechanismsinvolved in bacterial sepsis and septic shock. Clin Microbiol Rev 16:379–414van Leeuwen HJ, Heezius EC, Dallinga GM, van Strijp JA, Verhoef J, van Kessel KP (2003)Lipoprotein metabolism in patients with severe sepsis. Crit Care Med 31:1359–1366van Rossum AP, Fazzini F, Limburg PC, Manfredi AA, Rovere-Querini P, Mantovani A,Kallenberg CG (2004) The prototypic tissue pentraxin PTX3, in contrast to the short pentraxinserum amyloid P, inhibits phagocytosis of late apoptotic neutrophils by macrophages. ArthritisRheum 50:2667–2674. doi:10.1002/art.20370Vanhamme L, Paturiaux-Hanocq F, Poelvoorde P, Nolan DP, Lins L, Van Den Abbeele J, Pays A,Tebabi P, Van Xong H, Jacquet A, Moguilevsky N, Dieu M, Kane JP, De Baetselier P,Brasseur R, Pays E (2003) Apolipoprotein L-I is the trypanosome lytic factor of humanserum. Nature 422:83–87. doi:10.1038/nature01461HDL in Infectious Diseases and Sepsis 507Vishnyakova TG, Bocharov AV, Baranova IN, Chen Z, Remaley AT, Csako G, Eggerman TL,Patterson AP (2003) Binding and internalization of lipopolysaccharide by Cla-1, a humanorthologue of rodent scavenger receptor B1. J Biol Chem 278:22771–22780. doi:10.1074/jbc.M211032200Voisset C, Callens N, Blanchard E, Op De Beeck A, Dubuisson J, Vu-Dac N (2005) High densitylipoproteins facilitate hepatitis C virus entry through the scavenger receptor class B type I. JBiol Chem 280:7793–7799. doi:10.1074/jbc.M411600200von Hahn T, Lindenbach BD, Boullier A, Quehenberger O, Paulson M, Rice CM, McKeating JA(2006) Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepa-toma cells. Hepatology 43:932–942. doi:10.1002/hep.21139Wendel M, Paul R, Heller AR (2007) Lipoproteins in inflammation and sepsis. II. Clinical aspects.Intensive Care Med 33:25–35. doi:10.1007/s00134-006-0433-xWheeler RJ (2010) The trypanolytic factor-mechanism, impacts and applications. Trends Parasitol26:457–464. doi:10.1016/j.pt.2010.05.005White CR, Datta G, Mochon P, Zhang Z, Kelly O, Curcio C, Parks D, Palgunachari M, Handattu S,Gupta H, Garber DW, Anantharamaiah GM (2009) Vasculoprotective effects of apolipoproteinmimetic peptides: an evolving paradigm in HDL therapy. Vasc Dis Prev 6:122–130. doi:10.2174/1567270000906010122Wolfrum C, Poy MN, Stoffel M (2005) Apolipoprotein M is required for prebeta-HDL formationand cholesterol efflux to HDL and protects against atherosclerosis. Nat Med 11:418–422.doi:10.1038/nm1211Zahid MN, Turek M, Xiao F, Thi VL, Guerin M, Fofana I, Bachellier P, Thompson J, Delang L,Neyts J, Bankwitz D, Pietschmann T, Dreux M, Cosset FL, Grunert F, Baumert TF, Zeisel MB(2013) The postbinding activity of scavenger receptor class B type I mediates initiation ofhepatitis C virus infection and viral dissemination. Hepatology 57:492–504. doi:10.1002/hep.26097Zeisel MB, Koutsoudakis G, Schnober EK, Haberstroh A, Blum HE, Cosset FL, Wakita T,Jaeck D, Doffoel M, Royer C, Soulier E, Schvoerer E, Schuster C, Stoll-Keller F,Bartenschlager R, Pietschmann T, Barth H, Baumert TF (2007) Scavenger receptor class Btype I is a key host factor for hepatitis C virus infection required for an entry step closely linkedto CD81. Hepatology 46:1722–1731. doi:10.1002/hep.21994Zeisel MB, Fofana I, Fafi-Kremer S, Baumert TF (2011) Hepatitis C virus entry into hepatocytes:molecular mechanisms and targets for antiviral therapies. J Hepatol 54:566–576. doi:10.1016/j.jhep.2010.10.014508 A. Pirillo et al.High-Density Lipoproteins in StrokeOlivier MeilhacContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5102 Pleiotropic Effects of HDLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5113 HDL Potential Effects on the Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5113.1 Reconstituted HDLs and HDL Mimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5124 Part 1: Pleiotropic Effects of HDLs—In Vitro and In Vivo Data . . . . . . . . . . . . . . . . . . . . . . . . 5124.1 HDLs and Nitric Oxide (NO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5124.2 HDLs and Sphingosine 1-Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5134.3 Antioxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5144.4 Anti-inflammatory and Antiprotease Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5144.5 Endothelial Cell Integrity, EPCs and Antiapoptotic Action . . . . . . . . . . . . . . . . . . . . . . . . 5154.6 Antithrombotic Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5165 HDLs in the Cerebral Compartment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5175.1 Lipoproteins in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5175.2 Plasma HDLs in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5186 Part 2: HDLs and Acute Stroke (In Vivo Data) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5186.1 HDLs and Acute Stroke: Experimental Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520AbstractBesides their well-documented function of reverse transport of cholesterol, high-density lipoproteins (HDLs) display pleiotropic effects due to their antioxidant,antithrombotic, anti-inflammatory and antiapoptotic properties that may play aO. Meilhac (*)Inserm U1148, Paris, FranceUniversité de La Réunion, Saint-Denis, Ile de la Réunion, FranceCHU de La Réunion, Saint-Denis, Ile de la Réunion, FranceLaboratory for Vascular Translational Research, Inserm U1148,46 Rue Henri Huchard, 75018 Paris, Francee-mail: olivier.meilhac@inserm.fr# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_16509major protective role in acute stroke, in particular by limiting the deleteriouseffects of ischaemia on the blood–brain barrier (BBB) and on the parenchymalcerebral compartment. HDLs may also modulate leukocyte and platelet activa-tion, which may also represent an important target that would justify the use ofHDL-based therapy in acute stroke. In this review, we will present an update ofall the recent findings in HDL biology that could support a potential clinical useof HDL therapy in ischaemic stroke.KeywordsHDL • Ischaemic stroke • Endothelium • Animal models1 IntroductionStroke is usually divided according to the type of cerebrovascular lesion intoischaemic and haemorrhagic stroke. Ischaemia is the leading cause of stroke, dueto obstruction within a blood vessel supplying blood to the brain. It accounts forabout 80–90 % of all stroke cases. Haemorrhagic stroke occurs when a weakenedvessel ruptures due to an aneurysm or arteriovenous malformations. Uncontrolledhypertension is often associated with haemorrhagic stroke.Numerous epidemiological studies demonstrate that HDLs are a strong indepen-dent negative predictor of vascular events (Miller and Miller 1975; Noferet al. 2002), supporting HDL-raising therapies. The SPARCL study (Stroke Pre-vention by Aggressive Reduction in Cholesterol Levels) was designed to assess theeffects of atorvastatin 80 mg/day in patients who previously experienced a stroke ortransient ischaemic attack, but without known coronary heart disease (Amarencoet al. 2003). In this study, a negative correlation between HDL-C levels and strokerecurrence was found among these 4,731 patients, but only when ischaemic strokewas considered. Only baseline HDL-C and LDL/HDL ratio were associated with agood outcome in the ischaemic stroke subgroup (Amarenco et al. 2009). Each13.7 mg/dL (0.35 mmol/L) increment in HDL-C was associated with a 13 %reduction in the risk of ischaemic stroke (Amarenco et al. 2008). Interestingly,decreased LDL levels and high HDL levels did not have any effect on haemorrhagicstroke. In a different study, low HDL-C levels have been also associated with aworse outcome after ischaemic stroke. Of 489 patients treated with rtPA for IS, thelow concentration of HDL-C was also associated with a poor prognosis at 3 months(Makihara et al. 2012). More recently, another study in patients with atheroscleroticischaemic stroke reported that low levels of HDL-C (35 mg/dL) at admissionwere associated with higher stroke severity and poor clinical outcome duringfollow-up (Yeh et al. 2013).Data from studies investigating the effect of HDL subfractions on vascularprognosis are conflicting, mainly due to the variety of techniques used to assessHDL particle size (Camont et al. 2011; Krauss 2010). Only the inverse relationshipbetween large HDLs (alpha1) and vascular risk was found in several studies510 O. Meilhac(Asztalos et al. 2004; Schaefer and Asztalos 2007; Asztalos and Schaefer 2003).Two large cohorts comprising a total of more than 160,000 people used theMendelian randomisation single nucleotide polymorphism associated withHDL-C to assess a causative role of HDLs in preventing myocardial infarction(Voight et al. 2012). The authors found no significant relationship and thereforeconcluded that HDL-C was not causal in vascular disease but merely a risk marker.Another interpretation could be that HDL-C, i.e. cholesterol associated with HDL,is not an appropriate prognostic marker or therapeutic target. Given the pleiotropicfunctions of HDLs (see below), in particular their protective action on the endothe-lium (Tran-Dinh et al. 2013), their functionality should be evaluated in addition tothe concentration of HDL cholesterol.The long-term beneficial effect of HDLs has been largely attributed to their rolein reverse cholesterol transport and atherosclerotic burden regression. However,short-term HDL elevation has also been shown to be beneficial: in patients withacute coronary syndromes, each 1 mg/dL increment of HDL-C during the course ofa 16-week treatment with atorvastatin resulted in a 1.4 % risk reduction forrecurrent adverse events (Olsson et al. 2005).2 Pleiotropic Effects of HDLsHDLs represent a heterogeneous class of lipoproteins as far as size, shape andcomposition are concerned. Whereas their principal function is to transport choles-terol from peripheral tissues to the liver, additional functions have been attributed toHDLs, which may depend on their composition in lipids, proteins, vitamins orantioxidant molecules. In this review, we will focus on pleiotropic effects of HDLsother than reverse transport of cholesterol that can impact on stroke at differentlevels and particularly on ischaemic stroke.3 HDL Potential Effects on the Blood–Brain BarrierThe blood–brain barrier (BBB) is composed of endothelial cells interconnected bymeans of tight junctions lining the microvasculature of the central nervous system(CNS), pericytes, astrocytes, end feet and neuron processes. The first barrierseparating the blood and cerebral compartments is represented by endothelialcells, which play a pivotal role in regulating the traffic of molecules and bloodcells. Many protective properties of HDLs have been documented in endothelialcells (Tran-Dinh et al. 2013) and are developed below, including vasodilatation,antioxidant, anti-inflammatory and antiapoptotic effects. However, most of thein vitro results have been obtained using HUVECs (human umbilical vein endothe-lial cells) or BAEC (bovine aortic endothelial cells), which do not present the samecharacteristics as endothelial cells composing the BBB, in terms of permeabilityand establishment of tight junctions. In this review, we will summarise the differenteffects attributed to HDLs that could support their use as therapeutic tools in acuteHigh-Density Lipoproteins in Stroke 511stroke or in secondary prevention. Effects of reconstituted HDLs, HDL mimeticsand HDLs isolated from plasma will be presented.3.1 Reconstituted HDLs and HDL MimeticsApo A-1 is the major protein of HDLs, able to recruit lipids and organise HDLparticles. Reconstituted HDLs consist of an in vitro combination of apo A-I andphospholipids, producing disc-shaped particles resembling nascent HDL. Apo A-Imay be either purified from human plasma or produced by recombinant technology.Carriers of the apo A-I Milano mutation, characterised by the replacement of anarginine by a cysteine at position 173, have reduced plasma levels of apo A-I andHDL-C without increased cardiovascular disease (Sirtori et al. 2001). This obser-vation has led to a strong interest in using apo A-I Milano peptides or proteins aspotential therapeutic agent to treat cardiovascular disease (Nissen et al. 2003).Intravenous injection of apo A-I Milano in humans was shown to reduce theatheromatous plaque volume in both murine models and in humans.However, controversial data have been published as to whether apo A-I Milanois more effective than apo A-I in reverse transporting cholesterol. In mice, genetransfer of wild-type human apo A-I and human apo A-I Milano inhibited theprogression of native atherosclerosis and allograft vasculopathy to a similar extent.An equivalent increase in endothelial progenitor cell (EPC) number/function andendothelial regeneration in allografts was also observed (Feng et al. 2009). The useof either native or mutated apo A-I may however be beneficial for endothelialprotection and repair in pathological situations such as ischaemic stroke.The lipid-binding activity of apo A-1 was attempted to be mimicked by syntheticpeptides containing the class A amphipathic helix modified by phenylalanineresidues on the hydrophobic face. Improvement of the apo A-1 mimetic peptidestability was reached by using D-amino acids instead of L-amino acids which aremore readily degraded in plasma. For example, D4F was shown to reduce athero-sclerotic lesions in apo E-null mice (Navab et al. 2005) and to improve the anti-inflammatory properties of HDL in humans (Bloedon et al. 2008). Very few data arecurrently available on the potential beneficial effects, in stroke, of reconstitutedHDLs containing wild-type or mutated apo A-I.4 Part 1: Pleiotropic Effects of HDLs—In Vitroand In Vivo Data4.1 HDLs and Nitric Oxide (NO)Hypertension represents a major risk factor for stroke, and polymorphismsgoverning endothelial NO synthase (NOS3) gene expression/function have beenreported to increase the risk of stroke (Berger et al. 2007; Howard et al. 2005). A512 O. Meilhacrecent meta-analysis indicates that NOS3 gene 4b/a, T-786C and G894T polymor-phism could be associated with IS (Wang et al. 2013). NO may participate inneuroprotection in cerebral ischaemia by its vasodilatory effects but also byincreasing erythrocyte membrane fluidity which could limit the microviscosity(Tsuda et al. 2000). In experimental stroke, administration of NO appears to reducethe infarct volume in both permanent and transient models (Willmot et al. 2005).The results of ongoing clinical studies using NO donors are needed to validateexperimental data (Sare et al. 2009). Several lines of evidence suggest that HDLsincrease NO bioavailability, in particular via the induction of NOS3 [increasedexpression and activity (Mineo et al. 2006)]. NO exerts many beneficial effects onthe microvasculature during reperfusion including vasodilatation, reflow anddecreased permeability (Schulz et al. 2004). The results of the trial “Efficacy ofNitric Oxide in Stroke” are still not available. This study should give more insightinto the effects of the glyceryl trinitrate patch (transdermal diffusion of NO) on theoutcome post-stroke (ENOS Trial Investigators, 2006).4.2 HDLs and Sphingosine 1-PhosphateSphingosine 1-Phosphate (S1P) is an important lipid component identified in HDLs(Kimura et al. 2001) that may account for NO-mediated vasodilatory effects ofHDLs (Nofer et al. 2004). S1P is secreted by activated platelets (Yatomi et al. 1995)but mainly transported in plasma by HDL particles (54 %) (Argraves et al. 2008).HDLs, and in particular S1P, were demonstrated to directly protect the heartagainst ischaemia/reperfusion in a mouse model (Theilmeier et al. 2006). Theauthors report that this protection could be attributed to inhibition of neutrophilrecruitment and cardiomyocyte apoptosis in the infarcted area by a mechanisminvolving NO and S1P signalling.S1P has been shown to promote endothelial barrier function in cultured pulmo-nary endothelial cells (Garcia et al. 2001) by enhancing tight junction formation[in HUVECs (Lee et al. 2006)] and cortical actin assembly (Garcia et al. 2001),resulting in a decreased permeability. HDL-associated S1P was also reported topromote endothelial motility, a process of potential importance in case of vascularinjury, via Gi-coupled S1P receptors and the Akt signalling pathway (in HUVECs(Argraves et al. 2008)).In a model of transient middle cerebral artery occlusion in rats, an agonist or theS1P receptor 1 was shown to be neuroprotective, after intraperitoneal injection atthe time of reperfusion (Hasegawa et al. 2010). Most studies using this agonist(fingolimod or FTY720) report reduced infarct volumes and improved functionaloutcomes, as summarised in a recent meta-analysis (Liu et al. 2013). This protectiveeffect could be due to a reduced cerebral lymphocyte infiltration (Rollandet al. 2013). In a thromboembolic model, FTY720 was reported to reduce rtPA-associated haemorrhagic transformations (Campos et al. 2013). Since HDLparticles are the major transporter of S1P, it could be expected that at least part oftheir beneficial effect on the BBB could be attributed to this sphingolipid.High-Density Lipoproteins in Stroke 5134.3 Antioxidative StressIn stroke, a plethora of studies have shown that oxidative stress is associated withischaemia and reperfusion and still more so after rtPA treatment. Many experimen-tal studies report that antioxidant treatment displays neuroprotective effects(Margaill et al. 2005). However, hitherto, no concluding clinical study couldprovide evidence of a potential benefit of antioxidant therapy in stroke (Amaroand Chamorro 2011).Antiatherogenic functions of HDLs have been related, at least in part, to theirantioxidant properties. HDLs contain lipid-soluble vitamins, antioxidants andenzymes such as paraoxonase 1 (PON1), platelet-activating factor acetylhydrolase(PAF-AH) and glutathione phospholipid peroxidase (Florentin et al. 2008). Apo A-Iand apo A-II also display antioxidant properties. This antioxidant arsenal confers toHDLs the capacity to limit LDL oxidation and to scavenge lipid hydroperoxides(Navab et al. 2000; Negre-Salvayre et al. 2006). In a rat model of renal ischaemia/reperfusion, rHDLs were shown to reduce the severity of acute ischaemic renalfailure associated with decreased malondialdehyde, suggesting attenuation of lipidperoxidation subsequent to oxidative stress (Thiemermann et al. 2003).4.4 Anti-inflammatory and Antiprotease PropertiesAcute stroke is characterised by an activated endothelium favouring the recruitmentof leukocytes. In particular, neutrophil proteases such as elastase and matrixmetalloprotease-9 may induce BBB breakdown and produce deleterious effectson the parenchymal compartment (Stowe et al. 2009). Proinflammatory cytokinessuch as tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-6 areincreased in plasma of patients with acute stroke (Tuttolomondo et al. 2008).HDLs may exert anti-inflammatory effects on the endothelium but also onleukocytes. In endothelial cells, HDLs (both native and rHDLs) inhibited TNFαand IL-1 induction of leukocyte adhesion molecules VCAM-1, ICAM-1 andE-selectin but had no effect on the expression of platelet endothelial cell adhesionmolecule (PECAM) (Cockerill et al. 1995). More recently, McGrath and colleaguesreported that the modulatory effects of rHDL on endothelial cells stimulatedTNF-alpha and were mediated by DHCR24, an antioxidant enzyme involved incholesterol biosynthesis (McGrath et al. 2009). Reconstituted HDLs were alsoreported to limit PMN adhesion to endothelial cells stimulated by TNFα and LPS(Moudry et al. 1997). These effects were later confirmed in vitro using both nativeand rHDL on HUVECs but also in vivo, in a rat model of haemorrhagic shock(Cockerill et al. 2001). In monocytes, HDL reduced activation of CD11b inducedby PMA leading to decreased adhesion to an endothelial cell monolayer, monocyticspreading under shear flow, and transmigration. This process was mimicked by apoA-1 and reported to be ABCA1-dependent (Murphy et al. 2008). More recently, thesame group reported that plasma HDLs were potent inhibitors of neutrophil activa-tion both in vitro and in vivo, using mice models of inflammation (Murphy514 O. Meilhacet al. 2011). In patients with peripheral vascular disease treated by rHDL, theyshowed a decreased expression of CD11b by neutrophils 5–7 days post-injectionversus saline-injected patients. HDLs can also neutralise circulating inflammatorymolecules such as C-reactive protein (Wadham et al. 2004) and LPS (Wurfelet al. 1994).In addition, proteomic studies have shown that HDLs may transport differentantiproteases (Karlsson et al. 2005; Vaisar et al. 2007) and in particular alpha-1antitrypsin (AAT), the natural inhibitor of elastase (Ortiz-Munoz et al. 2009). HDLsdisplayed antielastase activity and protected vascular cells against elastase-inducedapoptosis. Using an in vitro model of BBB (Weksler et al. 2005), we have recentlyreported that HDLs could limit the deleterious, elastase-mediated role of activatedneutrophils under oxygen-glucose deprivation conditions leading to BBB disrup-tion (Bao Dang et al. 2013). In stroke, inhibition of neutrophil elastase may be atherapeutic target, as shown by using specific inhibitors (Ikegame et al. 2010).4.5 Endothelial Cell Integrity, EPCs and Antiapoptotic ActionApoptosis is an important mechanism involved in BBB breakdown and associatedcerebral damage. In vivo imaging of apoptotic cells using annexin V was reportedto be correlated with BBB permeability in patients with acute stroke (Lorberboymet al. 2006). Prevention of apoptosis of all cells composing the neurovascular unit istherefore of major importance to reduce deleterious effects of ischaemia. PlasmaHDL-C and apo A-I were reported to prevent apoptosis of endothelial cells inducedby mildly oxidised LDL independently of paraoxonase activity (Suc et al. 1997). Inthis in vitro model (bovine and human endothelial cell lines), HDL binding wasspecific (receptor-mediated) and HDLs blocked the intracellular calcium increasepreceding apoptosis (Escargueil-Blanc et al. 1997). It was suggested that the Apo A-Imoiety mediates this cytoprotective effect rather than SP1 (de Souza et al. 2010).Hypoxia has been reported to induce an autophagic process in endothelial cells(Zhang et al. 2011). Autophagy can be regarded as an adaptive response of the cellto deleterious environmental conditions that could delay apoptosis but could alsorepresent an early step of the apoptotic process. HDLs were shown to inhibitendoplasmic reticulum stress and autophagic response induced by oxidised LDLsin endothelial cells (Muller et al. 2011), suggesting that they impact on very earlyevents of the apoptotic cascade. In the brain, autophagy was significantly increasedin the cortex immediately following experimental subarachnoid haemorrhage(SAH) (Lee et al. 2009). During SAH, the BBB is disrupted subsequent to endothe-lial cell death by apoptosis. In this model, inhibition of apoptosis was shown tosignificantly reduce the formation of cerebral oedema and associated mortality(Yan et al. 2011). HDLs could thus modulate apoptosis of BBB endothelial cellsby preventing autophagy.Endothelial cell progenitor (EPC) therapy has been envisaged as a potentialtherapy in stroke [for review, see (Rouhl et al. 2008)]. EPCs can repair damagedvessels and form new ones that could promote recovery after ischaemic injury. In aHigh-Density Lipoproteins in Stroke 515mouse model of transient middle cerebral artery occlusion (tMCAO), systemicdelivery of EPCs limited brain damage associated with ischaemic injury(i.e. improved neurovascular repair and long-term neurobehavioral outcomes)(Fan et al. 2010). In rats, EPCs injected 24 h after tMCAO were shown to reachthe injured area and improved functional recovery, potentially attributable toantiapoptotic factors secreted by EPCs (Moubarik et al. 2011). In humans, erythro-poietin therapy significantly increased the number of circulating EPCs andimproved a 90-day major adverse neurological effect (Yip et al. 2011). Differentstudies report that reconstituted HDLs may increase circulating EPC number (Tsoet al. 2006) and their differentiation from mononuclear cells as well as theirangiogenic capacity (Sumi et al. 2007). This was shown in mouse models ofendothelial injury in response to LPS and hind limb ischaemia but also in humansafter injection of rHDLs in type 2 diabetic patients (van Oostrom et al. 2007). Thisincrease in circulating EPCs was significant at day 7 post-injection. In hypercho-lesterolemic subjects, HDL concentration was linked to EPC number and function(Rossi et al. 2010). Low EPC number was also reported to be an independent riskfactor for endothelial dysfunction in these patients. Petoumenos et al. reported acorrelation between circulating EPCs and HDL concentrations in patients withcoronary artery disease (Petoumenos et al. 2009). In addition, they have shownthat recombinant HDLs improved the function of circulating EPCs in a model ofendothelial denudation, which could be one explanation for the vasculoprotectiveactions of HDLs.4.6 Antithrombotic ActionsIn stroke, haemostasis disorders are associated with dysfunction of vascular endo-thelium, or with abnormalities of or interference with anticoagulant proteins(i.e. protein C, protein S and antithrombin III) (Coull and Clark 1993). However,all factors favouring clot formation may increase the risk of ischaemic stroke,involving all cellular components (endothelium, erythrocytes, platelets andleukocytes).HDLs may modulate haemostasis by impacting on platelet, red blood cell (RBC)and endothelial functions (Mineo et al. 2006). For example, thrombin-inducedendothelial tissue factor expression in vitro was shown to be downregulated byrHDLs (Viswambharan et al. 2004). Since tissue factor induction on endothelialcells is regulated by NO production, HDLs may inhibit its production in response toendotoxins or cytokines, by increasing the synthesis of NO (Yang and Loscalzo2000).Apo A-I, its amphipathic peptide analogues and HDLs were also shown toprotect erythrocytes against the generation of procoagulant activity (Epandet al. 1994). HDL2 subfraction was reported to be inversely correlated with eryth-rocyte aggregation in hypercholesterolemic patients (Razavian et al. 1994). Amultiple regression analysis demonstrated that this association was independentof fibrinogen, the major determinant of erythrocyte aggregation. The mechanisms516 O. Meilhacby which HDL2 may prevent RBC aggregability have not been elucidated, but thiseffect is relevant in small-sized arteries where increased blood viscosity may triggerclot formation. Apo A-I, its amphipathic peptide analogues and HDLs were alsoshown to protect erythrocytes against the generation of procoagulant activity(Epand et al. 1994). HDLs may inhibit phospholipid flip-flop and the associatedprocoagulant activity. In healthy volunteers receiving low doses of LPS, injectionof rHDLs reduced collagen-induced platelet aggregation and modified theprocoagulant state associated with endotoxemia (Pajkrt et al. 1997). More recently,reconstituted HDL infusion in patients with type 2 diabetes induced a 50–75 %attenuation of platelet aggregation in response to ADP and collagen (Calkinet al. 2009). In vitro, this effect was shown to be dose-dependent and remainedafter removal of rHDLs. Ex vivo, under blood flow conditions, rHDL limitedthrombus formation on a matrix of collagen. This effect was attributed to thephospholipid moiety of rHDLs, since it was not reproduced by apo A-I, whereasphosphatidylcholine reached the same level of reduction of aggregation as intactrHDLs. However, in this study, plasma HDLs did not display similar in vitro effectson platelet aggregation. The authors suggest that this may be due to limited efflux ofcholesterol from platelet membranes induced by native HDLs.5 HDLs in the Cerebral Compartment5.1 Lipoproteins in the BrainVarious studies have described the capacity of the brain to synthesise lipoproteinsthat are different from plasma lipoproteins (for review, see Wang and Eckel 2014).Lipoproteins found in the cerebrospinal fluid (CSF) mainly originate fromastrocytes and are characterised by a small size (resembling the size and densityof HDLs) and contain mainly apo E (Ladu et al. 2000; Koch et al. 2001). Apo E, J,D and A-I are the main apolipoproteins synthesised in the brain, in an age-dependentmanner for apo J and E (Elliott et al. 2010). Apo E and more particularly apo J arethought to be involved in the clearance of amyloid β-peptide by astrocytes andmicroglial cells (Mulder et al. 2014; Calero et al. 2000). Most of lipoproteinsisolated from the CSF are associated with amyloid β-peptide and may participatein its polymerisation, transport and clearance (Ladu et al. 2000). The BBB requiresa local homeostasis of cholesterol. Whereas astrocytes are thought to be the majorlipoprotein factory, neurons may also participate in the regulation of their synthesisand redistribution in the brain (Pfrieger and Ungerer 2011). After cortical spreadingdepression (a stimulus that provides long-lasting ischaemic tolerance), apo Jexpression is increased, suggesting that this apolipoprotein may participate inneuroprotection subsequently to an ischaemic episode (Wiggins et al. 2003). In amodel of middle cerebral artery occlusion, apo J-deficient mice displayed a worsestructural restoration in the vicinity of the infarct scar as compared to WT mice(Imhof et al. 2006).High-Density Lipoproteins in Stroke 5175.2 Plasma HDLs in the BrainUnder physiological conditions, it is not clear whether HDLs reach the cerebralparenchymal compartment. Based on in vitro results using bovine cerebral endo-thelial cells, HDLs have been suggested to cross the BBB via paracellular transport(de Vries et al. 1995). More recent data describe an active process of transcytosisinvolving receptors for HDLs at the surface of endothelial cells. In particular,SRB1seems to be involved in HDL uptake whereas ABCA1 (ATP-binding cassettetransporter A1) is responsible for internalisation of apo A-1 alone (for review, seevon Eckardstein and Rohrer 2009). However, most studies on transendothelialtransport of HDLs were performed on aortic endothelial cells that may differfrom endothelial cells of the BBB. Few studies are available on HDL actions onneurons and astrocytes. Ferretti et al. reported that HDLs reduced oxidative stressand cell death induced by copper ions in rat astrocytes (Ferretti et al. 2003). In rattype I astrocytes and glioma cells, HDLs stimulated DNA synthesis and expressionof fibroblast growth factor-2, a potent neurotrophic factor, which was associatedwith the activation of proliferative intracellular signalling (Malchinkhuuet al. 2003). It is likely that in pathological conditions (haemorrhage or transientincrease of BBB permeability), HDLs have an improved access to the cerebralparenchymal compartment and then act on both neurons and astrocytes (Lapergueet al. 2010, 2013).6 Part 2: HDLs and Acute Stroke (In Vivo Data)Under acute stroke conditions, the lipid profile has been reported to be modified(Santos-Silva et al. 2002). These authors have shown that patients with diagnosedischaemic stroke (CT imaging, n¼ 21) had lower plasma concentrations of bothHDL and apo A-I than healthy subjects with no history of cardiovascular events andnormal haematologic values (n¼ 29). Blood was sampled after a 12-h fastingperiod. In these patients, leukocyte count was increased relative to controls, butthe concentration of granulocytes accounted for most of this increase (monocyteswere moderately augmented whereas lymphocyte count was decreased in strokepatients). More importantly, markers of neutrophil activation, including lactoferrinand elastase, were strongly increased in plasma of stroke patients when comparedto controls.6.1 HDLs and Acute Stroke: Experimental ModelsFew studies have attempted to use HDL therapy in acute stroke. Based on theobservation that low HDL cholesterol levels were associated with cerebrovascularevents (Sacco et al. 2001; Wannamethee et al. 2000), Paterno et al. showed thatpretreatment with reconstituted HDL reduced neuronal damage in two experimen-tal models of stroke in rats (Paterno et al. 2004). They showed that high doses of518 O. MeilhacrHDLs (120 mg/kg) infused 2 h before the onset of stroke reduced the brain necroticarea by 61 and 76 %, respectively, in an excitotoxic (NMDA) and a transientMCAO model of stroke in rats. More recently, we have shown that plasma HDLs(10 mg/kg), injected immediately or at 3 or 5 h after stroke, also reduced cerebralinfarct volume by 74, 68 and 70 %, respectively, in a rat model of thromboembolicstroke (Lapergue et al. 2010). This was associated with a reduced BBB breakdownand decreased neutrophil recruitment in the infarct area. In this model, injection offluorescent-labelled HDLs showed a staining of endothelial cells (but also glialcells), suggesting that their primary protective effect was on the BBB. Thus, HDLsmay be a powerful neuroprotective tool for the treatment of cerebrovasculardiseases by preventing BBB breakdown. We have confirmed these in vivo resultsby a study using a model of BBB in vitro (Bao Dang et al. 2013).In humans, recombinant tissue plasminogen activator (rtPA) is the only effectivefibrinolytic treatment in ischaemic stroke. The disruption of the BBB is involved inoedema and haemorrhagic transformation following tPA treatment. We assessedwhether HDL-rtPA combined treatment could improve the safety and efficacy oftPA in experimental stroke. We showed that HDL injection decreased tPA-inducedhaemorrhagic transformation in two models of focal middle cerebral artery occlu-sion (MCAO) (embolic and 4-h monofilament MCAO). Both the blood–brainbarrier in vitro model and in vivo results support the vasculoprotective action ofHDLs on BBB under ischaemic conditions. Finally, HDLs do not interfere with thefibrinolytic activity of rtPA (Lapergue et al. 2013).ConclusionIn addition to reverse transport of cholesterol, HDL particles display differentprotective effects that could support their use in the acute phase of stroke.Different neuroprotective drugs proven to be effective in animal models havefailed to translate into clinical settings (Xu and Pan 2013). Early treatments thatcould be used without interfering with fibrinolytic treatments should be consid-ered as a good option to limit the deleterious effects of ischaemic stroke. HDLsmay represent a good candidate, particularly with respect to their protectiveeffects on the endothelium in ischaemia/reperfusion conditions. As discussed byNavab et al., HDL mimetic peptides should be more appropriate for use inchronic settings since they can be administrated orally or by subcutaneousroute, whereas reconstituted HDLs may be more suitable for acute treatmentby intravenous injection (Navab et al. 2010). Whether apo A-I combined withphospholipids are sufficient to prevent the effects of ischaemia is questionable. Itis likely that enrichment of reconstituted HDLs with protective molecules suchas antioxidants or antiproteases should provide an optimal beneficial effect asobserved when using plasma HDLs isolated from healthy subjects in differentanimal models of stroke.Acknowledgements OM’s group at Inserm is supported by the Fondation de France, theFondation Coeur et Artères and the Agence Nationale de la Recherche (ANR JCJC 2010).High-Density Lipoproteins in Stroke 519Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAmarenco P, Bogousslavsky J, Callahan AS, Goldstein L, Hennerici M, Sillsen H, Welch MA,Zivin J (2003) Design and baseline characteristics of the stroke prevention by aggressivereduction in cholesterol levels (SPARCL) study. Cerebrovasc Dis 16(4):389–395Amarenco P, Labreuche J, Touboul PJ (2008) High-density lipoprotein-cholesterol and risk ofstroke and carotid atherosclerosis: a systematic review. Atherosclerosis 196(2):489–496.doi:10.1016/j.atherosclerosis.2007.07.033Amarenco P, Goldstein LB, Messig M, O’Neill BJ, Callahan A 3rd, Sillesen H, Hennerici MG,Zivin JA, Welch KM, Investigators S (2009) Relative and cumulative effects of lipid and bloodpressure control in the stroke prevention by aggressive reduction in cholesterol levels trial.Stroke 40(7):2486–2492. doi:10.1161/STROKEAHA.108.546135Amaro S, Chamorro A (2011) Translational stroke research of the combination of thrombolysisand antioxidant therapy. Stroke 42(5):1495–1499. doi:10.1161/STROKEAHA.111.615039Argraves KM, Gazzolo PJ, Groh EM, Wilkerson BA, Matsuura BS, Twal WO, Hammad SM,Argraves WS (2008) High density lipoprotein-associated sphingosine 1-phosphate promotesendothelial barrier function. J Biol Chem 283(36):25074–25081Asztalos BF, Schaefer EJ (2003) High-density lipoprotein subpopulations in pathologicconditions. Am J Cardiol 91(7A):12E–17EAsztalos BF, Cupples LA, Demissie S, Horvath KV, Cox CE, Batista MC, Schaefer EJ (2004)High-density lipoprotein subpopulation profile and coronary heart disease prevalence in maleparticipants of the Framingham Offspring Study. Arterioscler Thromb Vasc Biol 24(11):2181–2187. doi:10.1161/01.ATV.0000146325.93749.a8Bao Dang Q, Lapergue B, Tran-Dinh A, Diallo D, Moreno JA, Mazighi M, Romero IA,Weksler B,Michel JB, Amarenco P, Meilhac O (2013) High-density lipoproteins limit neutrophil-induceddamage to the blood-brain barrier in vitro. J Cereb Blood Flow Metab 33(4):575–582. doi:10.1038/jcbfm.2012.206Berger K, Stogbauer F, Stoll M, Wellmann J, Huge A, Cheng S, Kessler C, John U, Assmann G,Ringelstein EB, Funke H (2007) The glu298asp polymorphism in the nitric oxide synthase3 gene is associated with the risk of ischemic stroke in two large independent case-controlstudies. Hum Genet 121(2):169–178. doi:10.1007/s00439-006-0302-2Bloedon LT, Dunbar R, Duffy D, Pinell-Salles P, Norris R, DeGroot BJ, Movva R, Navab M,Fogelman AM, Rader DJ (2008) Safety, pharmacokinetics, and pharmacodynamics of oralapoA-I mimetic peptide D-4F in high-risk cardiovascular patients. J Lipid Res 49(6):1344–1352Calero M, Rostagno A, Matsubara E, Zlokovic B, Frangione B, Ghiso J (2000) Apolipoprotein J(clusterin) and Alzheimer’s disease. Microsc Res Tech 50(4):305–315. doi:10.1002/1097-0029(20000815)50:4<305::AID-JEMT10>3.0.CO;2-LCalkin AC, Drew BG, Ono A, Duffy SJ, Gordon MV, Schoenwaelder SM, Sviridov D, CooperME, Kingwell BA, Jackson SP (2009) Reconstituted high-density lipoprotein attenuatesplatelet function in individuals with type 2 diabetes mellitus by promoting cholesterol efflux.Circulation 120(21):2095–2104. doi:10.1161/CIRCULATIONAHA.109.870709Camont L, Chapman MJ, Kontush A (2011) Biological activities of HDL subpopulations and theirrelevance to cardiovascular disease. Trends Mol Med 17(10):594–603. doi:10.1016/j.molmed.2011.05.013Campos F, Qin T, Castillo J, Seo JH, Arai K, Lo EH, Waeber C (2013) Fingolimod reduceshemorrhagic transformation associated with delayed tissue plasminogen activator treatment in520 O. Meilhaca mouse thromboembolic model. Stroke 44(2):505–511. doi:10.1161/STROKEAHA.112.679043Cockerill GW, Rye KA, Gamble JR, Vadas MA, Barter PJ (1995) High-density lipoproteins inhibitcytokine-induced expression of endothelial cell adhesion molecules. Arterioscler Thromb VascBiol 15(11):1987–1994Cockerill GW, McDonald MC, Mota-Filipe H, Cuzzocrea S, Miller NE, Thiemermann C (2001)High density lipoproteins reduce organ injury and organ dysfunction in a rat model ofhemorrhagic shock. Faseb J 15(11):1941–1952Coull BM, Clark WM (1993) Abnormalities of hemostasis in ischemic stroke. Med Clin North Am77(1):77–94de Souza JA, Vindis C, Negre-Salvayre A, Rye KA, Couturier M, Therond P, Chantepie S,Salvayre R, Chapman MJ, Kontush A (2010) Small, dense HDL 3 particles attenuate apoptosisin endothelial cells: pivotal role of apolipoprotein A-I. J Cell Mol Med 14(3):608–620. doi:10.1111/j.1582-4934.2009.00713.x/JCMM713de Vries HE, Breedveld B, Kuiper J, de Boer AG, Van Berkel TJ, Breimer DD (1995) High-density lipoprotein and cerebral endothelial cells in vitro: interactions and transport. BiochemPharmacol 50(2):271–273. doi:10.1016/0006-2952(95)00127-LElliott DA, Weickert CS, Garner B (2010) Apolipoproteins in the brain: implications for neuro-logical and psychiatric disorders. Clin Lipidol 51(4):555–573. doi:10.2217/CLP.10.37ENOS Trial Investigators ET (2006) Glyceryl trinitrate vs. control, and continuing vs. stoppingtemporarily prior antihypertensive therapy, in acute stroke: rationale and design of the efficacyof nitric oxide in stroke (ENOS) trial (ISRCTN99414122). Int J Stroke 1(4):245–249. doi:10.1111/j.1747-4949.2006.00059.xEpand RM, Stafford A, Leon B, Lock PE, Tytler EM, Segrest JP, Anantharamaiah GM (1994)HDL and apolipoprotein A-I protect erythrocytes against the generation of procoagulantactivity. Arterioscler Thromb 14(11):1775–1783Escargueil-Blanc I, Meilhac O, Pieraggi MT, Arnal JF, Salvayre R, Negre-Salvayre A (1997)Oxidized LDLs induce massive apoptosis of cultured human endothelial cells through acalcium-dependent pathway. Prevention by aurintricarboxylic acid. Arterioscler ThrombVasc Biol 17(2):331–339Fan Y, Shen F, Frenzel T, Zhu W, Ye J, Liu J, Chen Y, Su H, Young WL, Yang GY (2010)Endothelial progenitor cell transplantation improves long-term stroke outcome in mice. AnnNeurol 67(4):488–497. doi:10.1002/ana.21919Feng Y, Van Craeyveld E, Jacobs F, Lievens J, Snoeys J, De Geest B (2009) Wild-type apo A-Iand apo A-I(Milano) gene transfer reduce native and transplant arteriosclerosis to a similarextent. J Mol Med 87(3):287–297Ferretti G, Bacchetti T, Moroni C, Vignini A, Curatola G (2003) Protective effect of human HDLagainst Cu(2+)-induced oxidation of astrocytes. J Trace Elem Med Biol 17(Suppl 1):25–30Florentin M, Liberopoulos EN, Wierzbicki AS, Mikhailidis DP (2008) Multiple actions of high-density lipoprotein. Curr Opin Cardiol 23(4):370–378. doi:10.1097/HCO.0b013e3283043806/00001573-200807000-0001Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, English D(2001) Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependentcytoskeletal rearrangement. J Clin Invest 108(5):689–701Hasegawa Y, Suzuki H, Sozen T, Rolland W, Zhang JH (2010) Activation of sphingosine1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats. Stroke 41(2):368–374. doi:10.1161/STROKEAHA.109.568899Howard TD, Giles WH, Xu J, Wozniak MA, Malarcher AM, Lange LA, Macko RF, Basehore MJ,Meyers DA, Cole JW, Kittner SJ (2005) Promoter polymorphisms in the nitric oxide synthase3 gene are associated with ischemic stroke susceptibility in young black women. Stroke 36(9):1848–1851. doi:10.1161/01.STR.0000177978.97428.53Ikegame Y, Yamashita K, Hayashi S, Yoshimura S, Nakashima S, Iwama T (2010) Neutrophilelastase inhibitor prevents ischemic brain damage via reduction of vasogenic edema.Hypertens Res 33(7):703–707. doi:10.1038/hr.2010.58High-Density Lipoproteins in Stroke 521Imhof A, Charnay Y, Vallet PG, Aronow B, Kovari E, French LE, Bouras C, Giannakopoulos P(2006) Sustained astrocytic clusterin expression improves remodeling after brain ischemia.Neurobiol Dis 22(2):274–283. doi:10.1016/j.nbd.2005.11.009Karlsson H, Leanderson P, Tagesson C, Lindahl M (2005) Lipoproteomics II: mapping of proteinsin high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry.Proteomics 5(5):1431–1445. doi:10.1002/pmic.200401010Kimura T, Sato K, Kuwabara A, Tomura H, Ishiwara M, Kobayashi I, Ui M, Okajima F (2001)Sphingosine 1-phosphate may be a major component of plasma lipoproteins responsible for thecytoprotective actions in human umbilical vein endothelial cells. J Biol Chem 276(34):31780–31785. doi:10.1074/jbc.M104353200Koch S, Donarski N, Goetze K, Kreckel M, Stuerenburg HJ, Buhmann C, Beisiegel U (2001)Characterization of four lipoprotein classes in human cerebrospinal fluid. J Lipid Res 42(7):1143–1151Krauss RM (2010) Lipoprotein subfractions and cardiovascular disease risk. Curr Opin Lipidol 21(4):305–311. doi:10.1097/MOL.0b013e32833b7756LaduMJ, Reardon C, Van Eldik L, Fagan AM, Bu G, Holtzman D, Getz GS (2000) Lipoproteins inthe central nervous system. Ann N Y Acad Sci 903:167–175Lapergue B, Moreno JA, Dang BQ, Coutard M, Delbosc S, Raphaeli G, Auge N, Klein I,Mazighi M, Michel JB, Amarenco P, Meilhac O (2010) Protective effect of high-densitylipoprotein-based therapy in a model of embolic stroke. Stroke 41(7):1536–1542. doi:10.1161/STROKEAHA.110.581512Lapergue B, Dang BQ, Desilles JP, Ortiz-Munoz G, Delbosc S, Loyau S, Louedec L, Couraud PO,Mazighi M, Michel JB, Meilhac O, Amarenco P (2013) High-density lipoprotein-based therapyreduces the hemorrhagic complications associated with tissue plasminogen activator treatmentin experimental stroke. Stroke 44(3):699–707. doi:10.1161/STROKEAHA.112.667832Lee JF, Zeng Q, Ozaki H, Wang L, Hand AR, Hla T, Wang E, Lee MJ (2006) Dual roles of tightjunction-associated protein, zonula occludens-1, in sphingosine 1-phosphate-mediated endo-thelial chemotaxis and barrier integrity. J Biol Chem 281(39):29190–29200Lee JY, He Y, Sagher O, Keep R, Hua Y, Xi G (2009) Activated autophagy pathway inexperimental subarachnoid hemorrhage. Brain Res 1287:126–135. doi:10.1016/j.brainres.2009.06.028/S0006-8993(09)01185-8Liu J, Zhang C, Tao W, Liu M (2013) Systematic review and meta-analysis of the efficacy ofsphingosine-1-phosphate (S1P) receptor agonist FTY720 (fingolimod) in animal models ofstroke. Int J Neurosci 123(3):163–169. doi:10.3109/00207454.2012.749255Lorberboym M, Blankenberg FG, Sadeh M, Lampl Y (2006) In vivo imaging of apoptosis inpatients with acute stroke: correlation with blood-brain barrier permeability. Brain Res 1103(1):13–19. doi:10.1016/j.brainres.2006.05.073/S0006-8993(06)01491-0Makihara N, Okada Y, Koga M, Shiokawa Y, Nakagawara J, Furui E, Kimura K, Yamagami H,Hasegawa Y, Kario K, Okuda S, Naganuma M, Toyoda K (2012) Effect of serum lipid levelson stroke outcome after rt-PA therapy: SAMURAI rt-PA registry. Cerebrovasc Dis 33(3):240–247. doi:10.1159/000334664Malchinkhuu E, Sato K, Muraki T, Ishikawa K, Kuwabara A, Okajima F (2003) Assessment of therole of sphingosine 1-phosphate and its receptors in high-density lipoprotein-induced stimula-tion of astroglial cell function. Biochem J 370(Pt 3):817–827. doi:10.1042/BJ20020867Margaill I, Plotkine M, Lerouet D (2005) Antioxidant strategies in the treatment of stroke. FreeRadic Biol Med 39(4):429–443. doi:10.1016/j.freeradbiomed.2005.05.003/S0891-5849(05)00266-2McGrath KC, Li XH, Puranik R, Liong EC, Tan JT, Dy VM, DiBartolo BA, Barter PJ, Rye KA,Heather AK (2009) Role of 3beta-hydroxysteroid-delta 24 reductase in mediatingantiinflammatory effects of high-density lipoproteins in endothelial cells. Arterioscler ThrombVasc Biol 29(6):877–882. doi:10.1161/ATVBAHA.109.184663Miller GJ, Miller NE (1975) Plasma-high-density-lipoprotein concentration and development ofischaemic heart-disease. Lancet 1(7897):16–19522 O. MeilhacMineo C, Deguchi H, Griffin JH, Shaul PW (2006) Endothelial and antithrombotic actions ofHDL. Circ Res 98(11):1352–1364Moubarik C, Guillet B, Youssef B, Codaccioni JL, Piercecchi MD, Sabatier F, Lionel P, Dou L,Foucault-Bertaud A, Velly L, Dignat-George F, Pisano P (2011) Transplanted late outgrowthendothelial progenitor cells as cell therapy product for stroke. Stem Cell Rev 7(1):208–220.doi:10.1007/s12015-010-9157-yMoudry R, Spycher MO, Doran JE (1997) Reconstituted high density lipoprotein modulatesadherence of polymorphonuclear leukocytes to human endothelial cells. Shock 7(3):175–181Mulder SD, Nielsen HM, Blankenstein MA, Eikelenboom P, Veerhuis R (2014) Apolipoproteins Eand J interfere with amyloid-beta uptake by primary human astrocytes and microglia in vitro.Glia. doi:10.1002/glia.22619Muller C, Salvayre R, Negre-Salvayre A, Vindis C (2011) HDLs inhibit endoplasmic reticulumstress and autophagic response induced by oxidized LDLs. Cell Death Differ 18(5):817–828.doi:10.1038/cdd2010149Murphy AJ, Woollard KJ, Hoang A, Mukhamedova N, Stirzaker RA, McCormick SP, RemaleyAT, Sviridov D, Chin-Dusting J (2008) High-density lipoprotein reduces the human monocyteinflammatory response. Arterioscler Thromb Vasc Biol 28(11):2071–2077. doi:10.1161/ATVBAHA.108.168690Murphy AJ, Woollard KJ, Suhartoyo A, Stirzaker RA, Shaw J, Sviridov D, Chin-Dusting JP(2011) Neutrophil activation is attenuated by high-density lipoprotein and apolipoprotein A-Iin in vitro and in vivo models of inflammation. Arterioscler Thromb Vasc Biol 31(6):1333–1341. doi:10.1161/ATVBAHA.111.226258Navab M, Hama SY, Cooke CJ, Anantharamaiah GM, Chaddha M, Jin L, Subbanagounder G,Faull KF, Reddy ST, Miller NE, Fogelman AM (2000) Normal high density lipoprotein inhibitsthree steps in the formation of mildly oxidized low density lipoprotein: step 1. J Lipid Res 41(9):1481–1494Navab M, Anantharamaiah GM, Hama S, Hough G, Reddy ST, Frank JS, Garber DW, Handattu S,Fogelman AM (2005) D-4F and statins synergize to render HDL antiinflammatory in mice andmonkeys and cause lesion regression in old apolipoprotein E-null mice. Arterioscler ThrombVasc Biol 25(7):1426–1432Navab M, Shechter I, Anantharamaiah GM, Reddy ST, Van Lenten BJ, Fogelman AM (2010)Structure and function of HDL mimetics. Arterioscler Thromb Vasc Biol 30(2):164–168Negre-Salvayre A, Dousset N, Ferretti G, Bacchetti T, Curatola G, Salvayre R (2006) Antioxidantand cytoprotective properties of high-density lipoproteins in vascular cells. Free Radic BiolMed 41(7):1031–1040. doi:10.1016/j.freeradbiomed.2006.07.006/S0891-5849(06)00430-8Nissen SE, Tsunoda T, Tuzcu EM, Schoenhagen P, Cooper CJ, Yasin M, Eaton GM, Lauer MA,Sheldon WS, Grines CL, Halpern S, Crowe T, Blankenship JC, Kerensky R (2003) Effect ofrecombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronarysyndromes: a randomized controlled trial. JAMA 290(17):2292–2300Nofer JR, Kehrel B, Fobker M, Levkau B, Assmann G, von Eckardstein A (2002) HDL andarteriosclerosis: beyond reverse cholesterol transport. Atherosclerosis 161(1):1–16Nofer JR, van der Giet M, Tolle M, Wolinska I, von Wnuck LK, Baba HA, Tietge UJ, Godecke A,Ishii I, Kleuser B, Schafers M, Fobker M, Zidek W, Assmann G, Chun J, Levkau B (2004)HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J ClinInvest 113(4):569–581Olsson AG, Schwartz GG, Szarek M, Sasiela WJ, Ezekowitz MD, Ganz P, Oliver MF, Waters D,Zeiher A (2005) High-density lipoprotein, but not low-density lipoprotein cholesterol levelsinfluence short-term prognosis after acute coronary syndrome: results from the MIRACL trial.Eur Heart J 26(9):890–896. doi:10.1093/eurheartj/ehi186Ortiz-Munoz G, Houard X, Martin-Ventura JL, Ishida BY, Loyau S, Rossignol P, Moreno JA,Kane JP, Chalkley RJ, Burlingame AL, Michel JB, Meilhac O (2009) HDL antielastase activityprevents smooth muscle cell anoikis, a potential new antiatherogenic property. Faseb J 23(9):3129–3139. doi:10.1096/fj.08-127928High-Density Lipoproteins in Stroke 523Pajkrt D, Lerch PG, van der Poll T, Levi M, Illi M, Doran JE, Arnet B, van den Ende A, ten CateJW, van Deventer SJ (1997) Differential effects of reconstituted high-density lipoprotein oncoagulation, fibrinolysis and platelet activation during human endotoxemia. Thromb Haemost77(2):303–307Paterno R, Ruocco A, Postiglione A, Hubsch A, Andresen I, Lang MG (2004) Reconstituted high-density lipoprotein exhibits neuroprotection in two rat models of stroke. Cerebrovasc Dis 17(2–3):204–211Petoumenos V, Nickenig G, Werner N (2009) High-density lipoprotein exerts vasculoprotectionvia endothelial progenitor cells. J Cell Mol Med 13(11–12):4623–4635Pfrieger FW, Ungerer N (2011) Cholesterol metabolism in neurons and astrocytes. Prog Lipid Res50(4):357–371. doi:10.1016/j.plipres.2011.06.002Razavian SM, Atger V, Giral P, Cambillau M, Del-Pino M, Simon AC, Moatti N, Levenson J(1994) Influence of HDL subfractions on erythrocyte aggregation in hypercholesterolemicme. PCVMETRA group. Arterioscler Thromb 14(3):361–366Rolland WB, Lekic T, Krafft PR, Hasegawa Y, Altay O, Hartman R, Ostrowski R, Manaenko A,Tang J, Zhang JH (2013) Fingolimod reduces cerebral lymphocyte infiltration in experimentalmodels of rodent intracerebral hemorrhage. Exp Neurol 241:45–55. doi:10.1016/j.expneurol.2012.12.009Rossi F, Bertone C, Montanile F, Miglietta F, Lubrano C, Gandini L, Santiemma V (2010) HDLcholesterol is a strong determinant of endothelial progenitor cells in hypercholesterolemicsubjects. Microvasc Res 80(2):274–279. doi:10.1016/j.mvr.2010.05.003/S0026-2862(10)00091-9Rouhl RP, van Oostenbrugge RJ, Damoiseaux J, Tervaert JW, Lodder J (2008) Endothelialprogenitor cell research in stroke: a potential shift in pathophysiological and therapeuticalconcepts. Stroke 39(7):2158–2165. doi:10.1161/STROKEAHA.107.507251Sacco RL, Benson RT, Kargman DE, Boden-Albala B, Tuck C, Lin IF, Cheng JF, Paik MC,Shea S, Berglund L (2001) High-density lipoprotein cholesterol and ischemic stroke in theelderly: the Northern Manhattan Stroke Study. JAMA 285(21):2729–2735Santos-Silva A, Rebelo I, Castro E, Belo L, Catarino C, Monteiro I, Almeida MD, Quintanilha A(2002) Erythrocyte damage and leukocyte activation in ischemic stroke. Clin Chim Acta 320(1–2):29–35Sare GM, Gray LJ, Wardlaw J, Chen C, Bath PM (2009) Is lowering blood pressure hazardous inpatients with significant ipsilateral carotid stenosis and acute ischaemic stroke? Interimassessment in the ‘Efficacy of Nitric Oxide in Stroke’ trial. Blood Press Monit 14(1):20–25.doi:10.1097/MBP.0b013e32831e30bdSchaefer EJ, Asztalos BF (2007) Increasing high-density lipoprotein cholesterol, inhibition ofcholesteryl ester transfer protein, and heart disease risk reduction. Am J Cardiol 100(11 A):n25–n31. doi:10.1016/j.amjcard.2007.08.010Schulz R, Kelm M, Heusch G (2004) Nitric oxide in myocardial ischemia/reperfusion injury.Cardiovasc Res 61(3):402–413Sirtori CR, Calabresi L, Franceschini G, Baldassarre D, Amato M, Johansson J, Salvetti M,Monteduro C, Zulli R, Muiesan ML, Agabiti-Rosei E (2001) Cardiovascular status of carriersof the apolipoprotein A-I(Milano) mutant: the Limone sul Garda study. Circulation 103(15):1949–1954Stowe AM, Adair-Kirk TL, Gonzales ER, Perez RS, Shah AR, Park TS, Gidday JM (2009)Neutrophil elastase and neurovascular injury following focal stroke and reperfusion. NeurobiolDis 35(1):82–90. doi:10.1016/j.nbd.2009.04.006/S0969-9961(09)00083-7Suc I, Escargueil-Blanc I, Troly M, Salvayre R, Negre-Salvayre A (1997) HDL and ApoA preventcell death of endothelial cells induced by oxidized LDL. Arterioscler Thromb Vasc Biol 17(10):2158–2166Sumi M, Sata M, Miura S, Rye KA, Toya N, Kanaoka Y, Yanaga K, Ohki T, Saku K, Nagai R(2007) Reconstituted high-density lipoprotein stimulates differentiation of endothelial progen-itor cells and enhances ischemia-induced angiogenesis. Arterioscler Thromb Vasc Biol 27(4):813–818. doi:10.1161/01.ATV.0000259299.38843.64524 O. MeilhacTheilmeier G, Schmidt C, Herrmann J, Keul P, Schafers M, Herrgott I, Mersmann J, Larmann J,Hermann S, Stypmann J, Schober O, Hildebrand R, Schulz R, Heusch G, Haude M, vonWnuckLK, Herzog C, Schmitz M, Erbel R, Chun J, Levkau B (2006) High-density lipoproteins andtheir constituent, sphingosine-1-phosphate, directly protect the heart against ischemia/reperfu-sion injury in vivo via the S1P3 lysophospholipid receptor. Circulation 114(13):1403–1409Thiemermann C, Patel NS, Kvale EO, Cockerill GW, Brown PA, Stewart KN, Cuzzocrea S,Britti D, Mota-Filipe H, Chatterjee PK (2003) High density lipoprotein (HDL) reduces renalischemia/reperfusion injury. J Am Soc Nephrol 14(7):1833–1843Tran-Dinh A, Diallo D, Delbosc S, Varela-Perez LM, Dang QB, Lapergue B, Burillo E, Michel JB,Levoye A, Martin-Ventura JL, Meilhac O (2013) HDL and endothelial protection. Br JPharmacol 169(3):493–511. doi:10.1111/bph.12174Tso C, Martinic G, Fan WH, Rogers C, Rye KA, Barter PJ (2006) High-density lipoproteinsenhance progenitor-mediated endothelium repair in mice. Arterioscler Thromb Vasc Biol 26(5):1144–1149. doi:10.1161/01.ATV.0000216600.37436.cfTsuda K, Kimura K, Nishio I, Masuyama Y (2000) Nitric oxide improves membrane fluidity oferythrocytes in essential hypertension: an electron paramagnetic resonance investigation.Biochem Biophys Res Commun 275(3):946–954. doi:10.1006/bbrc.2000.3408(S0006-291X(00)93408-9)Tuttolomondo A, Di Raimondo D, di Sciacca R, Pinto A, Licata G (2008) Inflammatory cytokinesin acute ischemic stroke. Curr Pharm Des 14(33):3574–3589Vaisar T, Pennathur S, Green PS, Gharib SA, Hoofnagle AN, Cheung MC, Byun J, Vuletic S,Kassim S, Singh P, Chea H, Knopp RH, Brunzell J, Geary R, Chait A, Zhao XQ, Elkon K,Marcovina S, Ridker P, Oram JF, Heinecke JW (2007) Shotgun proteomics implicates proteaseinhibition and complement activation in the antiinflammatory properties of HDL. J Clin Invest117(3):746–756. doi:10.1172/JCI26206van Oostrom O, Nieuwdorp M, Westerweel PE, Hoefer IE, Basser R, Stroes ES, Verhaar MC(2007) Reconstituted HDL increases circulating endothelial progenitor cells in patients withtype 2 diabetes. Arterioscler Thromb Vasc Biol 27(8):1864–1865. doi:10.1161/ATVBAHA.107.143875Viswambharan H, Ming XF, Zhu S, Hubsch A, Lerch P, Vergeres G, Rusconi S, Yang Z (2004)Reconstituted high-density lipoprotein inhibits thrombin-induced endothelial tissue factorexpression through inhibition of RhoA and stimulation of phosphatidylinositol 3-kinase butnot Akt/endothelial nitric oxide synthase. Circ Res 94(7):918–925. doi:10.1161/01.RES.0000124302.20396.B7Voight BF, Peloso GM, Orho-Melander M, Frikke-Schmidt R, Barbalic M, Jensen MK, Hindy G,Holm H, Ding EL, Johnson T, Schunkert H, Samani NJ, Clarke R, Hopewell JC, Thompson JF,Li M, Thorleifsson G, Newton-Cheh C, Musunuru K, Pirruccello JP, Saleheen D, Chen L,Stewart A, Schillert A, Thorsteinsdottir U, Thorgeirsson G, Anand S, Engert JC, Morgan T,Spertus J, Stoll M, Berger K, Martinelli N, Girelli D, McKeown PP, Patterson CC, Epstein SE,Devaney J, Burnett MS, Mooser V, Ripatti S, Surakka I, Nieminen MS, Sinisalo J, Lokki ML,Perola M, Havulinna A, de Faire U, Gigante B, Ingelsson E, Zeller T, Wild P, de Bakker PI,Klungel OH, Maitland-van der Zee AH, Peters BJ, de Boer A, Grobbee DE, Kamphuisen PW,Deneer VH, Elbers CC, Onland-Moret NC, Hofker MH, Wijmenga C, Verschuren WM, BoerJM, van der Schouw YT, Rasheed A, Frossard P, Demissie S, Willer C, Do R, Ordovas JM,Abecasis GR, Boehnke M, Mohlke KL, Daly MJ, Guiducci C, Burtt NP, Surti A, Gonzalez E,Purcell S, Gabriel S, Marrugat J, Peden J, Erdmann J, Diemert P, Willenborg C, Konig IR,Fischer M, Hengstenberg C, Ziegler A, Buysschaert I, Lambrechts D, Van de Werf F, Fox KA,El Mokhtari NE, Rubin D, Schrezenmeir J, Schreiber S, Schafer A, Danesh J, Blankenberg S,Roberts R, McPherson R, Watkins H, Hall AS, Overvad K, Rimm E, Boerwinkle E, Tybjaerg-Hansen A, Cupples LA, Reilly MP, Melander O, Mannucci PM, Ardissino D, Siscovick D,Elosua R, Stefansson K, O’Donnell CJ, Salomaa V, Rader DJ, Peltonen L, Schwartz SM,Altshuler D, Kathiresan S (2012) Plasma HDL cholesterol and risk of myocardial infarction: amendelian randomisation study. Lancet 380(9841):572–580. doi:10.1016/S0140-6736(12)60312-2High-Density Lipoproteins in Stroke 525von Eckardstein A, Rohrer L (2009) Transendothelial lipoprotein transport and regulation ofendothelial permeability and integrity by lipoproteins. Curr Opin Lipidol 20(3):197–205.doi:10.1097/MOL.0b013e32832afd63Wadham C, Albanese N, Roberts J, Wang L, Bagley CJ, Gamble JR, Rye KA, Barter PJ, VadasMA, Xia P (2004) High-density lipoproteins neutralize C-reactive protein proinflammatoryactivity. Circulation 109(17):2116–2122. doi:10.1161/01.CIR.0000127419.45975.26Wang H, Eckel RH (2014) What are lipoproteins doing in the brain? Trends Endocrinol Metab 25(1):8–14. doi:10.1016/j.tem.2013.10.003Wang M, Jiang X, WuW, Zhang D (2013) Endothelial NO synthase gene polymorphisms and riskof ischemic stroke in Asian population: a meta-analysis. PLoS ONE 8(3):e60472. doi:10.1371/journal.pone.0060472Wannamethee SG, Shaper AG, Ebrahim S (2000) HDL-cholesterol, total cholesterol, and the riskof stroke in middle-aged British men. Stroke 31(8):1882–1888Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, Leveque M, Tricoire-Leignel H,Nicotra A, Bourdoulous S, Turowski P, Male DK, Roux F, Greenwood J, Romero IA, CouraudPO (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line.Faseb J 19(13):1872–1874. doi:10.1096/fj.04-3458fjeWiggins AK, Shen PJ, Gundlach AL (2003) Delayed, but prolonged increases in astrocyticclusterin (ApoJ) mRNA expression following acute cortical spreading depression in the rat:evidence for a role of clusterin in ischemic tolerance. Brain Res Mol Brain Res 114(1):20–30Willmot M, Gray L, Gibson C, Murphy S, Bath PM (2005) A systematic review of nitric oxidedonors and L-arginine in experimental stroke; effects on infarct size and cerebral blood flow.Nitric Oxide 12(3):141–149. doi:10.1016/j.niox.2005.01.003/S1089-8603(05)00004-2Wurfel MM, Kunitake ST, Lichenstein H, Kane JP, Wright SD (1994) Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS. JExp Med 180(3):1025–1035Xu SY, Pan SY (2013) The failure of animal models of neuroprotection in acute ischemic stroke totranslate to clinical efficacy. Med Sci Monit Basic Res 19:37–45Yan J, Li L, Khatibi NH, Yang L, Wang K, Zhang W, Martin RD, Han J, Zhang J, Zhou C (2011)Blood-brain barrier disruption following subarachnoid hemorrhage may be facilitated throughPUMA induction of endothelial cell apoptosis from the endoplasmic reticulum. Exp Neurol230(2):240–247. doi:10.1016/j.expneurol.2011.04.022/S0014-4886(11)00153-1Yang Y, Loscalzo J (2000) Regulation of tissue factor expression in human microvascularendothelial cells by nitric oxide. Circulation 101(18):2144–2148Yatomi Y, Ruan F, Hakomori S, Igarashi Y (1995) Sphingosine-1-phosphate: a platelet-activatingsphingolipid released from agonist-stimulated human platelets. Blood 86(1):193–202Yeh PS, Yang CM, Lin SH, Wang WM, Chen PS, Chao TH, Lin HJ, Lin KC, Chang CY, ChengTJ, Li YH (2013) Low levels of high-density lipoprotein cholesterol in patients with athero-sclerotic stroke: a prospective cohort study. Atherosclerosis 228(2):472–477. doi:10.1016/j.atherosclerosis.2013.03.015Yip HK, Tsai TH, Lin HS, Chen SF, Sun CK, Leu S, Yuen CM, Tan TY, Lan MY, Liou CW, LuCH, Chang WN (2011) Effect of erythropoietin on level of circulating endothelial progenitorcells and outcome in patients after acute ischemic stroke. Crit Care 15(1):R40. doi:10.1186/cc10002Zhang R, Zhu F, Ren J, Huang L, Liu P, Wu G (2011) Beclin1/PI3K-mediated autophagy preventshypoxia-induced apoptosis in EAhy926 cell line. Cancer Biother Radiopharm 26(3):335–343.doi:10.1089/cbr.2010.0814526 O. MeilhacTherapeutic Potential of HDLin Cardioprotection and Tissue RepairSophie Van Linthout, Miguel Frias, Neha Singh, and Bart De GeestContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5292 In Vitro Effects of HDL on Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5302.1 Effects of HDL in Rat Neonatal Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5312.2 Adult Mouse Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5312.3 Apo A-I and Sphingosine-1-Phosphate Mediate the Cytoprotective Effectsof HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5323 HDL Confer Protection Against Ischaemia Reperfusion Injury . . . . . . . . . . . . . . . . . . . . . . . . 5333.1 IRI in the Isolated Heart Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5343.2 IRI in In Vivo Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5343.3 Apo A-I and S1P Confer Cardioprotective Effects on HDL . . . . . . . . . . . . . . . . . . . . . . 5354 Human Apo A-I Gene Transfer Attenuates Diabetic Cardiomyopathy . . . . . . . . . . . . . . . . . 5364.1 Human Apo A-I Gene Transfer Influences Metabolic Parametersin Streptozotocin-Induced Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5384.2 Human Apo A-I Gene Transfer Attenuates Diabetes-Associated OxidativeStress, Cardiac Fibrosis, and Endothelial Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . 5384.3 Human Apo A-I Gene Transfer Reduces Diabetes-InducedCardiac Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5404.4 Human Apo A-I Gene Transfer Reduces Diabetes-Associated Cardiac Apoptosisand Improves the Cardiac Endothelial Integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5425 HDL and Tissue Repair: Modulation of EPC Biology via SR-BI . . . . . . . . . . . . . . . . . . . . . . 5436 Development of Topical HDL Therapy for Cutaneous Wound Healing . . . . . . . . . . . . . . . 547S. Van LinthoutCharité-University-Medicine Berlin, Campus Virchow, Berlin-Brandenburg Center forRegenerative Therapy (BCRT), Berlin, GermanyM. FriasLaboratoire des Lipides, Hôpitaux Universitaires de Genève, Geneva, SwitzerlandN. Singh • B. De Geest (*)Molecular and Vascular Biology, Department of Cardiovascular Sciences, Center forMolecular and Vascular Biology, Katholieke Universiteit Leuven, Campus Gasthuisberg,Herestraat 49 bus 911, 3000 Leuven, Belgiume-mail: bart.degeest@med.kuleuven.be# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_175277 Beneficial Effects of Selective HDL-Raising Gene Transfer on Cardiac Remodellingand Cardiac Function After Myocardial Infarction in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554AbstractEpidemiological studies support a strong association between high-density lipo-protein (HDL) cholesterol levels and heart failure incidence. Experimental evi-dence from different angles supports the view that low HDL is unlikely aninnocent bystander in the development of heart failure. HDL exerts directcardioprotective effects, which are mediated via its interactions with the myocar-dium and more specifically with cardiomyocytes. HDL may improve cardiacfunction in several ways. Firstly, HDL may protect the heart against ischaemia/reperfusion injury resulting in a reduction of infarct size and thus in myocardialsalvage. Secondly, HDL can improve cardiac function in the absence ofischaemic heart disease as illustrated by beneficial effects conferred by theselipoproteins in diabetic cardiomyopathy. Thirdly, HDL may improve cardiacfunction by reducing infarct expansion and by attenuating ventricularremodelling post-myocardial infarction. These different mechanisms aresubstantiated by in vitro, ex vivo, and in vivo intervention studies that appliedtreatment with native HDL, treatment with reconstituted HDL, or human apo A-Igene transfer. The effect of human apo A-I gene transfer on infarct expansion andventricular remodelling post-myocardial infarction illustrates the beneficialeffects of HDL on tissue repair. The role of HDL in tissue repair is furtherunderpinned by the potent effects of these lipoproteins on endothelial progenitorcell number, function, and incorporation, which may in particular be relevantunder conditions of high endothelial cell turnover. Furthermore, topical HDLtherapy enhances cutaneous wound healing in different models. In conclusion,the development of HDL-targeted interventions in these strategically chosentherapeutic areas is supported by a strong clinical rationale and significantpreclinical data.KeywordsHigh-density lipoproteins • Apolipoprotein A-I • Heart failure • Ventricularremodelling • Drug development • Ischaemia/reperfusion injury • Tissue repair •Wound healing • Diabetic ulcerAbbreviationsApo ApolipoproteinAT1R Angiotensin II receptor, type 1BOEC Blood outgrowth endothelial cells528 S. Van Linthout et al.CAV Cardiac allograft vasculopathyECFCs Endothelial colony-forming cellseNOS Endothelial nitric oxide synthaseEPC Endothelial progenitor cellERK Extracellular signal-regulated kinaseHDL High-density lipoproteinICAM Intercellular adhesion moleculeIDL Intermediate-density lipoproteinHFpEF Heart failure with preserved ejection fractionHFrEF Heart failure with reduced ejection fractionIL InterleukinIRI Ischaemia/reperfusion injuryJNK c-Jun N-terminal kinaseLDL Low-density lipoproteinL-NAME L-NG-nitroarginine methyl esterNADPH Nicotinamide adenine dinucleotide phosphateNO Nitric oxidep38 MAPK p38 mitogen-activated protein kinasePI3K Phosphoinositide 3-kinaseS1P Sphingosine-1-phosphateSTAT3 Signal transducer and activator of transcription 3SR-BI Scavenger receptor class B, type ITBARS Thiobarbituric acid reactive substancesTLR4 Toll-like receptor 4TNF-α Tumour necrosis factor-αVCAM Vascular cell adhesion moleculeVEGF Vascular endothelial growth factorVLDL Very-low-density lipoprotein1 IntroductionHeart failure is the pathophysiological state in which cardiac dysfunction is respon-sible for failure of the heart to pump blood at a rate commensurate with therequirements of metabolising tissues. This clinical syndrome is characterised bysymptoms and signs of increased tissue and organ water and of decreased tissue andorgan perfusion. Heart failure is a growing public health problem, the leading causeof hospitalisation and a major cause of mortality (Rathi and Deedwania 2012). Theprevalence of heart failure can be estimated at 2 % in the Western world, and theincidence approaches 5–10 per 1,000 persons per year (Mosterd and Hoes 2007).However, the prevalence and incidence progressively increase with age. Theprevalence of heart failure is 7 % in the age group 75–84 years and over 10 % inthose older than 85 years (Mosterd et al. 1999). Projections show that by 2030, theTherapeutic Potential of HDL in Cardioprotection and Tissue Repair 529prevalence of heart failure will increase 25 % from 2013 estimates (Go et al. 2013).The 5-year age-adjusted mortality rates after onset of heart failure are 50 % in menand 46 % in women (Roger et al. 2004).Epidemiological studies support a strong association between high-density lipo-protein (HDL) cholesterol levels and heart failure incidence. In Framingham HeartStudy participants free of coronary heart disease at baseline, low HDL cholesterollevels were independently associated with heart failure incidence after adjustmentfor interim myocardial infarction and clinical covariates (Velagaleti et al. 2009).These seminal prospective data are in agreement with earlier cross-sectional studiesshowing that post-infarct ejection fraction is lower in patients with low HDLcholesterol levels (Kempen et al. 1987; Wang et al. 1998). Low HDL cholesterollevels and low levels of apolipoprotein (apo) A-I, the main apo of HDL, indicate anunfavourable prognosis in patients with heart failure independent of the aetiology(Iwaoka et al. 2007; Mehra et al. 2009). An intriguing observation is that apo A-I isexpressed in the human heart and in the heart of human apo A-I transgenic mice(Baroukh et al. 2004; Lowes et al. 2006).The observed relationship between HDL and heart failure in epidemiologicalstudies might be due to residual confounding. Low HDL may be an integratedbiomarker of adverse metabolic processes including abnormal metabolism oftriglyceride-rich lipoproteins, insulin resistance, and ongoing tissue inflammation.Crosstalk between inflammatory processes and metabolic dysregulation may be acritical player in the pathogenesis of heart failure (Palomer et al. 2013). Here, wewill present experimental evidence from different angles, which support the viewthat low HDL is unlikely an innocent bystander in the development of heart failure.As will be discussed in the next sections, HDL exerts direct cardioprotectiveeffects, which are mediated via their interactions with the myocardium and morespecifically with cardiomyocytes (Sect. 2). HDL may improve cardiac function inseveral ways. Firstly, HDL may protect the heart against ischaemia/reperfusioninjury resulting in a reduction of infarct size and thus in myocardial salvage(Sect. 3). Secondly, HDL can improve cardiac function in the absence of ischaemicheart disease as illustrated by its beneficial effects in diabetic cardiomyopathy(Sect. 4). Thirdly, HDL may improve cardiac function by reducing infarct expan-sion and by attenuating ventricular remodelling (Sect. 7). Since this latter effect isan example of the role of HDL in tissue repair, the reparative functions of HDL willbe further dealt with in Sect. 5 on HDL and endothelial progenitor cells (EPCs) andin Sect. 6 on topical HDL therapy for cutaneous wound healing.2 In Vitro Effects of HDL on CardiomyocytesHDL may exert an indirect protective influence on the heart derived from theirability to limit atherosclerotic plaque formation, thus preventing myocardial ischae-mia and loss of myocardial tissue in acute coronary syndromes. Data are nowaccumulating to indicate that HDL have a more immediate and direct effect oncardiomyocytes. Discussion of these direct effects in this section will provide a530 S. Van Linthout et al.more complete picture of the cardioprotective role of these lipoproteins and willalso raise questions about the use of the HDL complex itself as a therapeutic agent.Although HDL is found in plasma, they are also present in the interstitial spacein amounts that correspond to approximately 25 % of their plasma concentration(Parini et al. 2006). It is therefore physiologically relevant to evaluate their directimpact on cells. The direct impact of HDL on cardiomyocytes has mainly beenstudied in two in vitro models: (1) rat neonatal cardiomyocytes and (2) adult mousecardiomyocytes. These models allow the investigation of the specific actions ofHDL on cardiomyocytes independent of the systemic parameters and of endothelialcells whose activity is regulated by HDL. Although studies investigating the directrole of HDL on cardiomyocytes are relatively limited, they all show a beneficialimpact of HDL. These protective effects have been attributed to the activation ofintracellular signalling cascades involved in pro-survival cell fate.2.1 Effects of HDL in Rat Neonatal CardiomyocytesIn cultured rat neonatal cardiomyocytes, HDL treatment inhibits the cytotoxiceffects induced by oxidative stress from glucose and growth factor depletion ordoxorubicin incubation. This protective effect has mainly been evaluated by itscapacity to counteract the proapoptotic signals such as caspase 3 activation andDNA fragmentation (Frias et al. 2010; Theilmeier et al. 2006). HDL incubationinduces the phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt, extracellularsignal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and the transcription factor signal transducer and activator of transcription3 (STAT3) (Frias et al. 2009). Although the precise mechanism of action has notbeen completely elucidated, experiments using specific inhibitors have defined theirrole in counteracting apoptotic signals induced by doxorubicin. These studiessuggest that the cardioprotective effect involves the activation of the pro-survivalproteins ERK1/2 and STAT3, but not p38 MAPK. HDL also induces the phosphor-ylation of connexin 43 (Morel et al. 2012). This phosphorylation leads to areduction in gap junction permeability, which may limit the spread of mediatorsimplicated in death pathways. Connexin 43 phosphorylation requires the activationof protein kinase C (Morel et al. 2012). Although there is not yet direct proof of theimplication of connexin 43 in the protective effects of HDL, connexin 43 has beenshown to be implicated in ischaemic conditioning (Schulz et al. 2007; Schulz andHeusch 2004).2.2 Adult Mouse CardiomyocytesIn mouse adult cardiomyocytes, the protective actions of HDL have beendemonstrated using a model of hypoxia and reperfusion. Incubation ofcardiomyocytes with HDL before the hypoxia period (preconditioning) improvedcell survival (Frias et al. 2013). This protection involves the preservation ofTherapeutic Potential of HDL in Cardioprotection and Tissue Repair 531mitochondrial integrity, as HDL treatment inhibits mitochondrial permeabilitytransition pore opening. Similarly to the results obtained in neonatalcardiomyocytes, HDL induces that activation of intracellular signalling pathwayssuch as PI3K/Akt, ERK1/2, and STAT3, which play a role in this protection. Thisprotective action of HDL is significantly inhibited in cardiomyocytes fromcardiomyocyte-restricted STAT3 knockout mice (Frias et al. 2013). Similarly,cardiomyocytes treated with HDL after hypoxia (at reperfusion) show improvedcell survival compared to non-treated cells. In this context, the effects of HDL wereattributed to PI3K/Akt and ERK1/2 (Tao et al. 2010). The possible role of STAT3has not been investigated in this reperfusion protocol.Taken together, HDL-induced activation of several target proteins incardiomyocytes appears to be cardioprotective. These beneficial effects cannot beattributed to its role in cholesterol transport.2.3 Apo A-I and Sphingosine-1-Phosphate Mediatethe Cytoprotective Effects of HDLAs mentioned above, HDL can modulate the activation of signalling pathwaysin cardiac cells. HDL is a complex particle, which is composed of numerousproteins and bioactive lipids. Among these constituents, apo A-I and sphingosine-1-phosphate (S1P) are the two major effectors that have been involved in theactivation of signalling pathways. Activation occurs via the binding of the HDLparticle to scavenger receptor class B, type I (SR-BI), and of S1P to S1P receptors.S1P binds to five (S1P1–S1P5) high-affinity G protein-coupled receptors generatingmultiple downstream signals. Of the different S1P receptor subtypes, only S1P1,S1P2, and S1P3 receptors are expressed in the heart (Means and Brown 2009). S1Phas been shown to be cytoprotective and to confer cardioprotection. For a moredetailed discussion of the cardioprotective role of S1P, the interested reader isreferred to recent reviews (Karliner 2013; Sattler and Levkau 2009).In vitro, most of the beneficial effects of HDL mentioned above have beenattributed to S1P. Data from experiments using pharmacological antagonists spe-cific for S1P1, S1P2, and S1P3 receptors demonstrate an inhibition of the HDLactions. For example, several experiments suggest that S1P3 is involved in PI3K/Akt signalling and that S1P2 and/or S1P1 might be involved in ERK1/2 and STAT3signalling (Frias et al. 2009; Tao et al. 2010). S1P2 was shown to play a key role inthe HDL-induced protection against the apoptotic effects of doxorubicin (Friaset al. 2010). In the protection against hypoxia reperfusion injury, a predominant,mediatory role was indicated for the S1P1 and S1P3 receptors, via ERK1/2 andPI3K/Akt activation. No role was attributed to S1P2, but neither was it investigated(Tao et al. 2010). The evidence that reinforces the cytoprotective role of S1P comesfrom experiments, where addition of S1P to reconstituted HDL (rHDL) containingapo A-I improved the protection against the apoptotic action of doxorubicin (Friaset al. 2010).532 S. Van Linthout et al.It should be acknowledged that S1P cannot freely circulate in plasma and thatthe major plasma lipoprotein source of this lipid is HDL. Therefore, it is notsurprising that the effects of HDL can be mediated by S1P. However, conflictingdata suggest S1P-independent effects of apo A-I. One explanation proposedinitially by Nofer and colleagues (2004) is that SR-BI can anchor the HDL particleat the cell membrane and allow S1P to interact with its specific receptors. In thiscontext, Means and colleagues (Means et al. 2008) have shown that S1P1 is presentin lipid raft structures from adult mouse cardiomyocytes. Lipid rafts are known toharbour SR-BI (Babitt et al. 1997). Although increasing evidence suggests a majorrole of S1P, the mechanism of signal transduction from HDL particle to cell is stillunder intensive investigation, and a possible role of SR-BI cannot be eliminated.Crosstalk between intracellular signalling pathways increases the complexity ofthe regulation of HDL-induced cascades and impedes identification of the specificrole of HDL constituents. Nevertheless, it is important to keep in mind that suchcomplexity can result in differing and conflicting data. Further investigations willbe necessary to elucidate the precise mechanism of action of HDL incardiomyocytes in vitro.3 HDL Confer Protection Against Ischaemia ReperfusionInjuryWhereas the previous section was dedicated to HDL-induced signalling incardiomyocytes and to cytoprotective effects of HDL in vitro, the role of HDL inthis section is extended to cardiac ischaemia/reperfusion injury (IRI).Ischaemic heart disease is the leading cause of death in the world. Aftermyocardial infarction, early reperfusion is the most effective strategy to limit celldeath and subsequent complications that can lead to heart failure. However,re-establishing perfusion is paradoxically detrimental to the heart, giving rise toIRI. This phenomenon is termed lethal reperfusion injury and is defined as amyocardial injury caused by the restoration of coronary flow after an ischaemicepisode. This injury culminates in the death of cardiomyocytes that were viableimmediately before the myocardial reperfusion. Combating IRI is a notable focus ofattempts to improve treatment strategies.Experimental animal models of IRI are adequate to investigate the precisemechanisms of action and the potential targets of therapeutic drugs. Mostinvestigations used the isolated rodent heart (ex vivo model) or transient ligatureof the left anterior descending coronary artery (in vivo model). All studies thatevaluated the effects of HDL on the heart submitted to the IRI protocol areunanimous and demonstrate a beneficial effect on cardiac function. Among thesestudies, a few demonstrated similar effects using rHDL, composed of apo A-I andphospholipids (palmitoyl-oleoyl-phosphatidylcholine). Some studies used rHDLcontaining the apo A-I Milano mutant.Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 5333.1 IRI in the Isolated Heart ModelAn early study using HDL perfusion in isolated hearts suggested that HDL signifi-cantly reduced post-ischaemic arrhythmias (Mochizuki et al. 1991). In contrast,perfusion with low-density lipoproteins (LDL) had no effects indicating HDLspecificity. The mechanism was not defined, but it was hypothesised that thelipoprotein stabilised bioactive arachidonic acid metabolites (Mochizukiet al. 1991). These antiarrhythmogenic effects were later confirmed in vivo whentreatment with rHDL decreased the incidence of post-ischaemic arrhythmias(Imaizumi et al. 2008). Surprisingly, treatment with apo A-I or phospholipids didnot significantly reduce these arrhythmogenic responses. The effect of rHDL wasmediated via the production of nitric oxide (NO), through an Akt/ERK/NO pathwayin endothelial cells (Imaizumi et al. 2008). Unfortunately, the direct impact oncardiomyocytes was not investigated.The direct cardioprotective impact of HDL on IRI has been studied by Calabresiand colleagues (Calabresi et al. 2003). They reported that treatment with rHDLimproved post-ischaemic functional recovery and decreased creatine kinase releasein the coronary effluent. Lipid-free apo A-I or phosphatidylcholine liposomes werenot effective in protecting the heart from IRI. rHDL caused a dose-dependentreduction of ischaemia-induced cardiac tumour necrosis factor-α (TNF-α) expres-sion and content, which correlated with the improved functional recovery(Calabresi et al. 2003). In agreement with this prior study, rHDL improved cardiacfunction (Rossoni et al. 2004) and reduced infarct size compared to non-treatedanimals (Gu et al. 2007). Recent studies showed that treatment with HDL signifi-cantly reduced the infarct size in rodents submitted to IRI compared to non-treatedIRI animals (Frias et al. 2013; Morel et al. 2012). The protective impact of HDL orrHDL did not depend on the timing of injection. Indeed, the treatment at themoment of reperfusion was also effective, but to a lesser extent than the treatmentbefore the ischaemic period.The precise mechanism of HDL-induced cardioprotection is under intensiveinvestigation, and the activation of intracellular signalling pathways plays animportant role in such cardioprotection. In this context, the protective role ofSTAT3, previously shown in cardiomyocytes, has been confirmed in the isolatedheart model. The reduction of the infarct size induced by HDL was significantlyinhibited in STAT3 knockout mice (Frias et al. 2013). Mitochondria play animportant role in cell survival and may be the key player in cardioprotection.Interestingly, HDL and rHDL containing apo A-I Milano have been shown topreserve mitochondrial integrity (Frias et al. 2013; Marchesi et al. 2008), consistentwith a direct impact of HDL and rHDL on cardiac cells.3.2 IRI in In Vivo ModelsThe data obtained ex vivo were extended to in vivo models. The first in vivo data oncardiac function after HDL injection demonstrated that HDL injection stimulates534 S. Van Linthout et al.myocardial perfusion. This effect was mediated via the activation of endothelialnitric oxide synthase (eNOS) (Nofer et al. 2004). Although this study did notinvestigate IRI, it underlines the potential response of the heart to HDL in vivo.The effect of HDL was extended to an in vivo transient ligature of the left anteriordescending coronary artery model (Theilmeier et al. 2006). One single injection ofnative HDL before ischaemia reduced infarct size, apoptotic cell death, and neutro-phil infiltration. These protective effects were also significantly inhibited in thepresence of the eNOS inhibitor L-NG-nitroarginine methyl ester (L-NAME) andwere partially inhibited in S1P receptor subtype 3 KOmice (Theilmeier et al. 2006).Taken together, these results suggest that the HDL action is mediated via NOproduction and is dependent on the S1P content of HDL.Similarly to the results obtained in the isolated heart, the protective role of rHDLcontaining apo A-I Milano during ischaemia or at reperfusion significantly reducedthe infarct size induced by IRI in rabbits (Marchesi et al. 2004). Protective effects ofapo A-I were also demonstrated (Gu et al. 2007). Injection of apo A-I 10 min beforereperfusion significantly improved cardiac function, which was associated with areduction of myocardial TNF-α and interleukin (IL)-6 levels. Histological analysisof cardiac tissue from rats treated with apo A-I showed a decrease in intercellularadhesion molecule (ICAM) expression and neutrophil infiltration compared tonon-treated animals (Gu et al. 2007). It should be acknowledged that apo A-I andrHDL can absorb phospholipids, including S1P, in plasma. This could modulatetheir impact on IRI.3.3 Apo A-I and S1P Confer Cardioprotective Effects on HDLBased on all published reports, it is not easy to delineate the precise role of S1P orapo A-I in the cardioprotective effects of HDL. Both constituents have beeninvestigated individually, and both have been shown to protect against IRI individ-ually. However, their precise impact on the actions of HDL has not always beeninvestigated in the same study or in the same model. Interestingly, levels ofHDL-associated S1P were diminished in patients with coronary artery disease(Sattler et al. 2010). Very recent data demonstrate a key role of apolipoprotein M(apoM) in S1P binding to HDL particles (Christoffersen et al. 2011). Indeed, theamount of S1P contained in HDL was very low in apoM KO and higher in apoMoverexpressing mice. Unfortunately, IRI in these mice has not yet been investigated.With respect to S1P, given that its plasma concentration is many times higherthan its affinity for S1P receptors, it has been suggested that HDL might play therole of neutralising and limiting the actions of circulating S1P (Murata et al. 2000).The impact of S1P association with HDL on the function of HDL still remains to beelucidated. It is probable that in the HDL particle, both are necessary to obtain themaximal positive response.The mechanisms of HDL-induced cardioprotection against IRI are complex andcannot be attributed to the role of HDL in reverse cholesterol transport. Moreprecise knowledge on the role of different HDL constituents in intracellularTherapeutic Potential of HDL in Cardioprotection and Tissue Repair 535signalling and cardioprotection would help to design a therapeutic compound,which replicates all the beneficial aspects of HDL in the heart. Figure 1 illustratesconceptually the direct actions of HDL on cardiomyocytes.4 Human Apo A-I Gene Transfer Attenuates DiabeticCardiomyopathyDiabetic cardiomyopathy is a cardiac disorder, which takes place in the absence ofcoronary artery disease and hypertension, and is characterised by impaired leftventricular function due to cardiac inflammation, oxidative stress, cardiomyocyteapoptosis, cardiac perivascular and interstitial fibrosis, intramyocardialmicroangiopathy, endothelial dysfunction, disturbed cardiac substrate metabolism,cardiomyocyte hypertrophy, abnormal intracellular Ca2+ handling, and impairedfunctionality of cardiac stem cells. This cardiac entity has first been recognised byRubler et al. (1972), and its existence has been confirmed during the last threedecades via epidemiological, clinical, and experimental studies. Hyperglycaemia,hyperinsulinaemia, and dyslipidaemia each contribute to the pathogenesis of dia-betic cardiomyopathy via triggering cellular signalling and subsequent specificalterations in cardiac structure. However, hyperglycaemia-induced oxidative stresshas been proposed to be the key determinant in the development of diabeticcardiomyopathy. Left ventricular diastolic dysfunction represents the earliest pre-clinical manifestation of diabetic cardiomyopathy, preceding systolic dysfunction,which can progress to symptomatic heart failure (Cosson and Kevorkian 2003;Freire et al. 2007; Raev 1994).Fig. 1 Proposed protective intracellular signalling pathways induced by HDL in cardiomyocytes.HDL induces the activation of intracellular signalling pathways in cardiomyocytes. This activationleads to cardioprotection. Most of the activated signalling pathways induced by HDL involve itsS1P constituent (HDL-S1P) and the participation of S1P receptor subtypes. Further investigationsare necessary to elucidate the precise role of SR-BI in cardioprotection induced by HDL. Thearrows indicate intracellular signalling as described in cardiomyocytes. Cx 43; connexin 43; PKCprotein kinase C; MEK1/2: ERK (MAPK) kinase 1/2536 S. Van Linthout et al.HDLs have antidiabetic properties. They reduce hyperglycaemia via their capac-ity to decrease pancreatic β-cell apoptosis (Abderrahmani et al. 2007; Fryirset al. 2010; Rutti et al. 2009) and to stimulate glucose disposal in skeletal muscle(Han et al. 2007). They decrease dyslipidaemia via lowering adipocyte lipolysis(Van Linthout et al. 2010a) and induce insulin sensitivity (Berg et al. 2001) viaupregulating the expression of adiponectin (Van Linthout et al. 2010a), whichabrogates the development of hyperinsulinaemia. Consequently, increased HDLmay, from a theoretical point of view, antagonise the main triggers of diabeticcardiomyopathy.The main hallmarks of diabetic cardiomyopathy, including cardiac oxidativestress, interstitial inflammation, apoptosis, fibrosis, and endothelial dysfunction, onthe one hand and the pleiotropic effects of HDL on the other hand further supportthe hypothesis that an increase in HDL may reduce the development of diabeticcardiomyopathy (Fig. 2). Via a human apo A-I gene transfer strategy, thisFig. 2 HDL reduces the development of diabetic cardiomyopathy. Type 2 diabetes mellitus isassociated with hyperglycaemia, dyslipidaemia, and hyperinsulinaemia. Hyperglycaemia is themain trigger that contributes to the development of diabetic cardiomyopathy via the induction ofoxidative stress. Hallmarks of diabetic cardiomyopathy are, besides cardiac oxidative stress,cardiac fibrosis, inflammation, apoptosis, and endothelial dysfunction, which reciprocally affectone another (interconnected grey lines). HDLs have antidiabetic features (dotted lines): theyreduce hyperglycaemia and dyslipidaemia via—among other mechanisms—their capacity todecrease pancreatic β-cell apoptosis and adipocyte lipolysis, respectively, and they increaseinsulin sensitivity via the induction of adiponectin, abrogating the development of hyperinsu-linaemia. Besides interference with the triggers that lead to diabetic cardiomyopathy, HDLs reducethe features of diabetic cardiomyopathy via their pleiotropic effects, including their anti-oxidative,antifibrotic, anti-inflammatory, antiapoptotic, and endothelial-protective effects ( full lines)Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 537hypothesis has been evaluated in an experimental rat model of streptozotocin-induced diabetes mellitus associated with severe hyperglycaemia and an LDL toHDL ratio of 1:1 (Van Linthout et al. 2007; Young et al. 1988).4.1 Human Apo A-I Gene Transfer Influences MetabolicParameters in Streptozotocin-Induced Diabetes MellitusApo A-I transfer resulted in a 60 % increase in HDL cholesterol, which wasparalleled with a significant decline in very-low-density lipoprotein (VLDL) cho-lesterol, intermediate-density lipoprotein (IDL) cholesterol, and triglycerides,whereas LDL cholesterol was unaffected (Van Linthout et al. 2008). Despite thewell-described antidiabetic effects of HDL involving their ability to reduce pancre-atic β-cell apoptosis (Abderrahmani et al. 2007; Fryirs et al. 2010; Rutti et al. 2009),apo A-I transfer did not reduce blood glucose levels (Van Linthout et al. 2008),potentially due to the severity of the streptozotocin model, which is associated witha remaining insulin production below 1 % (Hughes et al. 2001). The decline intriglycerides and in the triglyceride-rich lipoproteins VLDL and IDL (Sztalryd andKraemer 1995) after apo A-I transfer suggests an HDL-mediated decrease inlipolysis in adipose tissue, leading to less free fatty acids in the circulation, lesstriglyceride synthesis in the liver, and subsequent less VLDL and IDL synthesis(Tunaru et al. 2003). This hypothesis is corroborated by the ability of HDL toreduce the expression of hormone-sensitive lipase (Van Linthout et al. 2010a), therate-limiting enzyme of adipocyte lipolysis in abdominal fat (Sztalryd and Kraemer1995), and to increase the phosphorylation of the PI3K downstream target Akt inabdominal fat (Van Linthout et al. 2010a). Akt is involved in the anti-lipolytic andlipogenic effects of insulin in adipose tissue (Whiteman et al. 2002) and in theregulation of the expression of the adipokine adiponectin (Cong et al. 2007; Pereiraand Draznin 2005), which is known to improve insulin sensitivity under diabetes(Peterson et al. 2008). However, an involvement of HDL in the hepatic expressionof genes involved in triglyceride metabolism may not be excluded. The decrease incardiac glycogen content (Van Linthout et al. 2008) suggests that apo A-I transfer instreptozotocin rats partly restored glucose metabolism as an energy source in theheart instead of nearly exclusive reliance on fatty acid metabolism for production ofATP (Kota et al. 2011).4.2 Human Apo A-I Gene Transfer Attenuates Diabetes-Associated Oxidative Stress, Cardiac Fibrosis,and Endothelial DysfunctionHyperglycaemia induces oxidative stress via creating a disbalance between thegeneration of reactive oxygen species and their resolution by antioxidant enzymes,like superoxide dismutases, which convert O2 anions into molecular oxygen andhydrogen peroxide (Nishikawa et al. 2000). Reactive oxygen species initiate diverse538 S. Van Linthout et al.stress-signalling pathways including ERK, c-Jun N-terminal kinase (JNK),and p38 MAPK, alter cellular proteins, and induce lipid peroxidation. Indiabetic cardiomyocytes, reactive oxygen species are predominantly generatedby mitochondria, due to mitochondrial oxidation of fatty acids (Kota et al. 2011),and by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases(Li et al. 2010b). The pathological importance of p38 MAPK in the diabetic heartfollows from the observation that p38 MAPK inhibition reduces cardiac inflamma-tion and improves left ventricular dysfunction in streptozotocin-induced diabeticmice (Westermann et al. 2006). Besides decreasing the activity of p38 MAPK, apoA-I transfer reduced cardiac oxidative stress via upregulating the expression of thediabetes-downregulated mitochondrial superoxide dismutase-2 and extracellular-superoxide dismutase (Van Linthout et al. 2008). Both forms of superoxidedismutase are important for the heart as outlined in studies whereby cardiacoverexpression of superoxide dismutase-2 protected the mitochondrial respiratoryfunction and blocked apoptosis induction (Shen et al. 2006; Suzuki et al. 2002) andoverexpression of extracellular-superoxide dismutase decreased macrophage infil-tration and fibrosis and improved left ventricular dysfunction (Dewald et al. 2003).These studies postulate that the decrease in cardiac fibrosis after apo A-I transfer instreptozotocin-diabetic rats can be partly explained by the reduction in oxidativestress and inflammation, including downregulated expression of pro-fibroticcytokines (inflammatory fibrosis) (Tschope et al. 2005), as well as by the decreasein cardiac apoptosis (cfr. supra) and subsequent replacement fibrosis, resulting inimproved left ventricular function. The anti-inflammatory and antifibrotic effects ofHDL are supported by findings in apo A-I knockout mice, which are associated withincreased inflammatory cells and collagen deposition in the lung (Wang et al. 2010),and from observations with the mimetic apo A-I peptide 4F demonstrating anL-4F-mediated decreased endothelial cell matrix production (Ou et al. 2005).Since cardiac NADPH oxidases play a predominant role in the development ofdiabetic cardiomyopathy (Guo et al. 2007; Wold et al. 2006) and in diabetic cardiacremodelling (Li et al. 2010b), decreased cardiac NADPH oxidase activity followingapo A-I transfer (Van Linthout et al. 2009) may be a critical mediator of reducedcardiac oxidative stress and subsequent fibrosis in the myocardium.Systemically, apo A-I transfer decreased the oxidative stress in rats withstreptozotocin-induced diabetes as indicated by the decline in serum levels ofthiobarbituric acid reactive substances (TBARS) (Van Linthout et al. 2008), amarker of lipid peroxidation (Tschope et al. 2005). This finding is corroboratedby Mackness et al. (1991), who reported that HDLs decrease the formation ofTBARS on oxidised LDL. This is likely mediated by an increase in the activity ofparaoxonase or platelet-activating factor-acetyl hydrolase, 2 enzymes with anti-oxidative features, which are associated with HDL and which are known to beinduced after apo A-I gene transfer (De Geest et al. 2000).In the vasculature of diabetic rats, apo A-I transfer decreased oxidative stress asindicated by the reduction in diabetes-enhanced NADPH oxidase activity andeNOS uncoupling (Van Linthout et al. 2009), a phenomenon occurring wheneNOS produces O2 rather than NO. Uncoupling of eNOS is, besides NADPHTherapeutic Potential of HDL in Cardioprotection and Tissue Repair 539oxidases (Cai 2005), an important source of reactive oxygen species in diseased,including diabetic, blood vessels (Hink et al. 2001). Consistent with thedemonstrated role of the angiotensin II receptor, type 1 (AT1R) in mediatingincreased NADPD oxidase activity, and eNOS uncoupling in diabetes (Oak andCai 2007), the downregulation in diabetes-induced AT1R (Hodroj et al. 2007; Nybyet al. 2007) after apo A-I transfer (Van Linthout et al. 2009) was postulated to be thepredominant mediator of reduced NADPH oxidase activity and eNOS uncoupling.This hypothesis is further supported by in vitro findings showing that theHDL-mediated reduction in AT1R expression in human aortic endothelial cellswas associated with a decline in hyperglycaemia-induced oxidative stress and areduced responsiveness to angiotensin II (Van Linthout et al. 2009). Theseobservations underline the finding of Tolle et al. (2008), who showed that HDLreduce NADPH oxidase-dependent reactive oxygen species generation via inhibi-tion of the activation of Rac1, which is a downstream AT1R-dependent mediator ofangiotensin II (Ohtsu et al. 2006). The exact mechanism by which HDL affectAT1R regulation under diabetes mellitus requires further fundamental studies.However, since oxidised LDL (Li et al. 2000) and reactive oxygen species(Gragasin et al. 2003) play a role in the induction of the AT1R in human aorticendothelial cells, one may speculate that HDL via intrinsic anti-oxidative features(cfr. supra, via paraoxonase and platelet-activating factor-acetyl hydrolase) maycontribute to the downregulation of the AT1R under diabetes mellitus, whichresults in less NADPH oxidase activity and reactive oxygen formation and in turnmay decrease AT1R expression. Concomitant with the reduced vascular oxidativestress, including decreased eNOS uncoupling, apo A-I transfer in streptozotocin ratsresulted in an enhanced NO bioavailability (Chalupsky and Cai 2005) and conse-quently in a decrease in endothelial dysfunction (Van Linthout et al. 2009), which isanother hallmark of diabetic cardiomyopathy.4.3 Human Apo A-I Gene Transfer Reduces Diabetes-InducedCardiac InflammationIn agreement with the direct anti-inflammatory properties of HDL (Cockerillet al. 2001; Hyka et al. 2001), apo A-I transfer decreased the diabetes-inducedintramyocardial inflammation (Westermann et al. 2007b, c) as indicated by thereduction in ICAM-1, vascular cell adhesion molecule (VCAM)-1, and TNF-αmRNA expression (Van Linthout et al. 2008) and VCAM-1 protein expression(Van Linthout et al. 2010b). Downregulation of VCAM-1/ICAM-1 expressionsuppresses monocyte-endothelial cell adhesion and subsequent transendothelialmigration of inflammatory cells. This in turn reduced local expression of cytokineslike TNF-α and consequently may attenuate the potentiation of the intramyocardialinflammatory reaction. Hyperglycaemia (Li et al. 2010a; Piga et al. 2007), oxidisedLDL (Lee et al. 2010) (Hodgkinson et al. 2008; Peterson et al. 2007), and increasedangiotensin II (Alvarez et al. 2004; Rius et al. 2010) under diabetes mellitusupregulate the expression of VCAM-1/ICAM-1. Since HDL did not affect blood540 S. Van Linthout et al.glucose levels, the reduction in adhesion molecule expression was not an indirectconsequence of an HDL-mediated antidiabetic effect. However, the reduced levelsof TBARS, which are also retrieved on oxidised LDL, in diabetic rats treated withapo A-I transfer compared to diabetic rats treated with control vector suggest thatapo A-I transfer decreases oxidised LDL and subsequent cardiac inflammation.Oxidised LDLs are agonists of Toll-like receptor 4 (TLR4), which is expressed onthe cell surface of cardiac cells, including cardiomyocytes, smooth muscle cells,and endothelial cells. A role for TLR4 in the development of diabetic cardiomyop-athy has recently been suggested (Zhang et al. 2010). The documentedHDL-mediated reduction in endothelial TLR4 expression and subsequent decreasein NF-κB activity (Van Linthout et al. 2011) suggest that the reduction in cardiacinflammation after apo A-I transfer is further partly mediated via a decrease inendothelial TLR4 expression, limiting oxidised LDL-endothelial TLR4 interactionsand subsequent activation of NF-κB. Furthermore, the activation of the AT1R,whose expression is upregulated under diabetes mellitus (Cohen 1993; Nybyet al. 2007), contributes to the development of diabetic cardiomyopathy. After all,AT1R antagonism under diabetes mellitus improves endothelial function(Cheetham et al. 2000) and reduces cardiac inflammation and fibrosis, resulting inan improvement in cardiac function (Westermann et al. 2007a). The observationsthat apo A-I transfer reduces aortic AT1R expression in streptozotocin-induceddiabetic rats and that HDLs decrease the hyperglycaemia-induced AT1R expressionin endothelial cells (Van Linthout et al. 2009) suggest that apo A-I transfer reducescardiac endothelial AT1R and subsequent angiotensin II responsiveness includingthe induction of VCAM-1/ICAM-1 expression and subsequent monocyte-endothelial cell adhesion (Alvarez et al. 2004; Rius et al. 2010). Downregulationof the expression of the anti-inflammatory adipokine adiponectin (Ouchiet al. 2000, 2001; Ouedraogo et al. 2007) as observed in diabetic patients and instreptozotocin rats (Thule et al. 2006) has been implicated in chronic inflammatoryphenotype in these subjects/rats. The positive correlation between apo A-I/HDLplasma levels and adiponectin concentrations (Verges et al. 2006) and the findingthat apo A-I transfer increases the expression of adiponectin in an experimentalmodel of extreme inflammation (Van Linthout et al. 2010a) further suggest that theeffects of HDL on adiponectin expression may contribute to its anti-inflammatoryeffects and to the attenuation of cardiac inflammation in rats with streptozotocin-induced diabetes. Finally, since apo A-I induces regulatory T cells (Wilhelmet al. 2010), which protect against the pro-inflammatory status of endothelial cells(He et al. 2010), an induction in regulatory T cells following apo A-I transfer mayalso partly account for the reduced cardiac inflammation in diabetic rats.Since inflammation triggers fibrosis (Ismahil et al. 2013; Savvatis et al. 2012;Westermann et al. 2011), the less pronounced diabetes-induced cardiac fibrosis (cfr.infra) following apo A-I transfer might partly be explained by the decrease incardiac inflammation. In this context, the influence of a systemic HDL-mediatedimmunomodulation on cardiac inflammation and fibrosis needs furtherinvestigation.Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 5414.4 Human Apo A-I Gene Transfer Reduces Diabetes-AssociatedCardiac Apoptosis and Improves the Cardiac EndothelialIntegrityCardiac apoptosis is another hallmark of diabetic cardiomyopathy. The incidence ofcardiac apoptosis is higher in diabetic patients (Frustaci et al. 2000) and in diabeticanimals (Cai et al. 2002) compared to non-diabetic controls and is directly linked tohyperglycaemia-induced oxidative stress (Cai et al. 2002). Mitochondria play animportant role in oxidative stress-induced apoptosis. Caspase 3 and caspase 7 areessential mediators in the mitochondrial processes of apoptosis (Lakhaniet al. 2006). In accordance with the reduction in oxidative stress, including theupregulation of cardiac mitochondrial superoxide dismutase-2 expression, apo A-Itransfer decreased the induced caspase 3/7 activity in streptozotocin rats (VanLinthout et al. 2008). Concomitantly, apo A-I transfer raised the ratio of theantiapoptotic Bcl-2, a ‘guardian’ against mitochondrial initiation of caspase activa-tion (Susin et al. 1996), towards the proapoptotic Bax. This ratio is a marker ofincreased cardiomyocyte survival probability (Condorelli et al. 1999). Furthermore,apo A-I transfer normalised the diabetes-reduced phosphorylation of thepro-survival protein kinase B Akt (Montanari et al. 2005; Uchiyama et al. 2004)and of its effector eNOS to levels found in non-diabetic hearts (Van Linthoutet al. 2008). This finding was further corroborated by experiments incardiomyocytes. HDL supplementation on cardiomyocytes in the presence ofin vitro hyperglycaemia reduced apoptosis in a PI3K- and NO-dependent manner(Van Linthout et al. 2008). The antiapoptotic effects of apo A-I transfer werereflected at ultrastructural level by a reduced number of cardiomyocytes withswollen mitochondria and apoptotic bodies and a more intact endothelium andbasement membrane (Van Linthout et al. 2008). These findings and the presence ofactivated Akt in cardiomyocytes, as well as in cardiac endothelial cells documentedvia immunofluorescence staining, underscore the importance of Akt activation afterapo A-I transfer in the reduction of cardiomyocyte apoptosis as well as in theimprovement in endothelial integrity. The ameliorated integrity of the endotheliumsuggests a potential restoration of the microvascular homeostasis, which has beendemonstrated to reduce cardiomyocyte apoptosis and to result in the recovery ofcardiac function in diabetic cardiomyopathy (Yoon et al. 2005). The significance ofthe cardiac endothelium on the contractile state and Ca2+ handling of subjacentcardiomyocytes (Brutsaert 2003; Nishida et al. 1993; Ramaciotti et al. 1992)suggests that part of the HDL-mediated improvement in left ventricular functionin rats with streptozotocin-induced diabetes was indirectly due to their protectiveeffect on the cardiac endothelium. This hypothesis is supported by the improvementin endothelial function found after apo A-I transfer in streptozotocin-induceddiabetic rats (Van Linthout et al. 2009). In addition, HDL supplementation onisolated cardiomyocytes directly improved their contractility underhyperglycaemia-induced stress in a PI3K- and NO-dependent manner (VanLinthout et al. 2008).542 S. Van Linthout et al.Besides the direct HDL-mediated cardiomyocyte-protective effects and theendothelial-protective features, the decrease in cardiac apoptosis after apo A-Itransfer in diabetic rats might be attributed to several other in parallelHDL-mediated triggered processes: (1) HDL may act as biological buffers capableof rapidly removing active proapoptotic TNF-α from the heart; (2) they have thepotential to increase (Rossoni et al. 2004), stabilise, and activate prostaglandins(Aoyama et al. 1990). This enhanced prostanoid availability/activity may contrib-ute to the HDL-mediated cardioprotection, by acting directly on cardiomyocytes(Zacharowski et al. 1999) and/or by inhibiting cardiac TNF-α production(Shinomiya et al. 2001); (3) HDL may reduce the expression of TLR 4 (VanLinthout et al. 2011), which has recently been shown to play an important role incardiac apoptosis in diabetic cardiomyopathy.In conclusion, apo A-I transfer attenuates the development of experimentaldiabetic cardiomyopathy via its anti-oxidative, anti-inflammatory, antifibrotic,and antiapoptotic actions (Fig. 2). Beyond beneficial vascular/cardioprotectivelong-term effects, direct myocardial effects of HDL may contribute to the improve-ment of cardiac function under severe streptozotocin-induced stress. Further studiesare required to investigate the potential of apo A-I transfer to ameliorate establisheddiabetic cardiomyopathy, especially in the context of type 2 diabetes mellitus, andto improve the function of HDL, which is impaired under diabetic conditions(Hedrick et al. 2000; Persegol et al. 2006; Sorrentino et al. 2009; Zhenget al. 2004). Finally, also the impact of the apo A-I/HDL-induced immunomodula-tory effects on diabetes-associated endothelial dysfunction and cardiac fibrosisneeds further clarification.5 HDL and Tissue Repair: Modulation of EPC Biology viaSR-BIThe term EPC is most often used in a very broad sense. There is no consensusdefinition of EPCs, which is a major source of confusion in the literature. EPCsencompass different categories of cells with different phenotypic and functionalproperties that affect neovascularisation and reendothelialisation. In a narrow,literally correct, and unambiguous sense, EPCs are immature precursor cells capa-ble of differentiating into mature endothelial cells in vivo (Hagensen et al. 2010).Thus, an EPC sensu stricto corresponds to an in vivo category and is defined from afunctional point of view. However, there are very significant methodologicalchallenges to detect incorporation of circulating progenitor cells in the endothelium(Hagensen et al. 2010). Incorporation of EPCs is in general a relatively rare event(Feng et al. 2008, 2009a, b; Hagensen et al. 2010). High-resolution multichannelsequential confocal scanning microscopy provides a platform for colocalisationanalysis with high specificity, which is required in lege artis cell tracking studies(De Geest 2009). Alternatively, EPCs may act in a paracrine way by releasingangiogenic factors and proteases to stimulate sprouting of local vessels or promotereendothelialisation indirectly (Rehman et al. 2003; Takakura et al. 2000; UrbichTherapeutic Potential of HDL in Cardioprotection and Tissue Repair 543et al. 2005). To make a distinction with EPCs sensu stricto, these cells may bedesignated as proangiogenic haematopoietic progenitor cells (Fig. 3).Different protocols for short-term culture of blood mononuclear cells on fibro-nectin (with or without gelatin)-coated plates have been established by Vasaet al. (2001) and Hill et al. (2003). These cells are stimulated by in vitro cultureconditions to mimic many features of endothelial cells (Hirschi et al. 2008). How-ever, the putative EPCs identified by these assays do not give rise to a lineage ofendothelial progeny, but cultured cells in these assays consist of monocytes or apopulation of haematopoietic cells with monocyte-macrophage or T-cell lineagecommitment. Nevertheless, these so-called early EPCs or early outgrowth EPCsmay regulate the angiogenic response in a paracrine way (Hirschi et al. 2008). InFig. 3 Role of bone marrow-derived proangiogenic haematopoietic progenitor cells and EPCssensu stricto in endothelial repair and neovascularisation. Proangiogenic haematopoietic progeni-tor cells, the in vivo equivalent of ‘early EPCs’, contribute to vascularisation and endothelial repairindirectly by secreting paracrine factors such as VEGF, hepatocyte growth factor (HGF), granulo-cyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), placental growth factor (PLGF), and IL-8. These factors stimulate local endothelialcell proliferation. On the other hand, EPCs sensu stricto enhance neovascularisation and endothe-lial repair directly by incorporation into the endothelium. EPCs sensu stricto can be considered toconstitute the in vivo equivalent of ECFCs or BOECs (‘late outgrowth EPCs’). ECs endothelialcells; SMCs smooth muscle cells544 S. Van Linthout et al.contrast, endothelial colony-forming cells (ECFCs) (also called ‘late outgrowthEPCs’ or ‘blood outgrowth endothelial cells’ (BOECs)) are immature precursorcells capable of differentiating into mature endothelial cells in vivo. They arederived from long-term culture of adherent peripheral blood mononuclear cells inendothelial conditions (‘late outgrowth EPCs’) and consist of colonies that displaya cobblestone morphology (Ingram et al. 2004). ECFCs express cell surfaceantigens like primary endothelium, clonally propagate, can be replated into second-ary and tertiary ECFCs, form capillary-like structures in vitro, and become endo-thelial cells in vivo (Ingram et al. 2004). ECFCs display postnatal vasculogenicactivity upon transplantation in a matrix scaffold (Richardson and Yoder 2011).ECFCs could be considered to be similar to EPCs sensu stricto: they have apotential for postnatal vasculogenesis and endothelial repair at sites of endothelialdamage. However, one should always consider that cell categories that are definedbased on in vitro cell culture conditions have always a rather artificial character.HDLs enhance EPC-mediated repair (Feng et al. 2008, 2009a, b; Tsoet al. 2006). The effect of HDL on EPC number, function, and incorporation occursvia SR-BI (Feng et al. 2009b). Since HDLs have been demonstrated to increasevascular endothelial growth factor (VEGF) production by early EPCs in vitro,HDLs may improve reendothelialisation by its effects on these circulatingproangiogenic cells (Feng et al. 2011). On the other hand, HDLs also improveECFC function in vitro, and these effects are dependent on signalling via SR-BI,ERKs, and NO and on increased β1 integrin expression (Feng et al. 2011). Takentogether, HDL exerts potent effects on different functional categories of EPCs viaSR-BI.The endothelial-protective and endothelial-reparative properties of HDL may bepathophysiologically and clinically relevant under conditions of high endothelialturnover. In healthy individuals, the endothelial layer is renewed at a low replica-tion rate of 0–1 % per day (Erdbruegger et al. 2006). In pathological conditions ofarteriosclerosis, endothelial turnover may be higher. Arteriosclerosis is a broadterm that encompasses all diseases that lead to arterial hardening, including nativeatherosclerosis, post-injury neointima formation, cardiac allograft vasculopathy(CAV), and vein graft atherosclerosis. Areas of low shear stress in human arterieshave an increased rate of endothelial turnover (Tricot et al. 2000). Augmentedendothelial turnover is also observed in atherosclerosis-prone areas in apo E/mice (Foteinos et al. 2008). However, the highest degree of endothelial turnover islikely observed after arterial injury, in allografts (Rahmani et al. 2006), and in veingrafts. Although there is significant evidence that HDLs exert beneficial effects inmodels of angioplasty- or injury-induced arteriosclerosis (Ameli et al. 1994; DeGeest et al. 1997; Shah and Amin 1992), we will focus here on allograftvasculopathy and vein graft atherosclerosis.Orthotopic heart transplantation is a well-established therapy for selectedpatients with end-stage congestive heart failure. The long-term success of hearttransplantation is limited by CAV. EPC-mediated repair may inhibit the progres-sion of CAV. Although CAV is primarily the result of chronic rejection,non-immunological factors are modifiers of CAV progression. In the ‘response toTherapeutic Potential of HDL in Cardioprotection and Tissue Repair 545injury’ concept of CAV, vascular lesions are considered to be the result of cumula-tive endothelial injury both by alloimmune responses and by non-alloimmuneinsults (Vassalli et al. 2003). T-cell alloimmunity, antibody-mediated immuneattack, and non-immune factors induce endothelial cell death or endothelial dys-function. Since the endothelium regulates vascular tone, inflammation, smoothmuscle cell proliferation, and thrombosis (Behrendt and Ganz 2002), restorationof endothelial integrity and function is pivotal to attenuate the development of CAV(Pinney and Mancini 2004). Markers of endothelial injury have been shown todiscriminate between CAV-positive and CAV-negative heart transplant recipients(Singh et al. 2012), which is consistent with a postulated pivotal role of endothelialinjury in the pathogenesis of CAV.Current strategies to prevent or treat CAV are clearly insufficient. Disruption ofthe endothelial lining due to endothelial cell apoptosis can be restored by prolifera-tion of adjacent endothelial cells but also by incorporation of circulating bonemarrow-derived EPCs (Friedrich et al. 2006; Walter et al. 2002; Werneret al. 2002, 2003). Hu et al. (2003) have demonstrated that EPCs contribute toendothelial regeneration in murine allografts. Moreover, increased HDL induced byhepatocyte-directed human apo A-I gene transfer in C57BL/6 apo E/ miceenhanced the incorporation of bone marrow-derived EPCs into the injured endo-thelium of allografts from donor BALB/c mice and reduced neointima formation inthese allografts (Feng et al. 2008). In subsequent experiments using BALB/callografts in wild-type C57BL/6 mice, it was demonstrated that increased EPCincorporation and attenuation of allograft vasculopathy induced by hepatocyte-directed human apo A-I gene transfer are strictly dependent on SR-BI expressionin bone marrow and bone marrow-derived cells. This conclusion was reached basedon experiments in mice transplanted with wild-type bone marrow or alternativelywith SR-BI/ bone marrow. These data suggest that the effect of HDL on EPCincorporation and/or paracrine effects of EPCs are critical for the attenuation ofallograft vasculopathy in this model. Nevertheless, the implications of these datafor clinical CAV are unknown since murine experiments are performed in theabsence of immunosuppressive therapies.Surgical revascularisation using autologous vein grafts is limited by vein graftfailure, which is frequently due to an aggressive form of atherosclerosis (Campeauet al. 1984). Vein graft failure is observed in 30–50 % of coronary bypass graftswithin 10 years (Fitzgibbon et al. 1996). Hypercholesterolaemia is a risk factor forvein graft failure (Goldman et al. 2004), which may be related to adverse effects onendothelial integrity and function (Raja et al. 2004). Previously, it has beendemonstrated that endothelial regeneration is retarded, and neointima formation isaccelerated in vein grafts in hypercholesterolaemic apo E/ mice compared tonormocholesterolaemic controls (Dietrich et al. 2000; Xu et al. 2003). Topical HDLtherapy reduced vein graft atherosclerosis in apo E/ mice (Feng et al. 2011).Topical HDL application involves formulation of HDL in 20 % pluronic F-127 gel(pH 7.2) (Feng et al. 2011). Pluronic F-127 is a biocompatible and non-toxicsubstance and is characterised by thermoreversible gel formation at temperaturesabove 21 C (Hu et al. 1999). One of the main advantages of topical HDL therapy is546 S. Van Linthout et al.that the ‘distribution volume’ of the therapeutic agent is small compared to sys-temic administration. In addition, the extracellular protein concentration of HDL is300–400 μg/ml (Parini et al. 2006; Sloop et al. 1987). This is approximately 20 % ofthe plasma protein concentration. Therefore, therapeutic effects of topical HDL canbe expected at HDL concentrations that are substantially lower than the plasmaconcentration. Topical HDL administration on the adventitial side of vein graftsimproved vein graft patency and function (Feng et al. 2011). Caval veins of C57BL/6 apo E/ mice were grafted to the right carotid arteries of recipient C57BL/6TIE2-LacZ/apo E/ mice. HDL in 20 % pluronic F-127 gel was applied on theadventitial side of vein grafts. Topical HDL application reduced intimal area by55 % ( p< 0.001) at day 28 compared to control mice. Blood flow quantified bymicro-magnetic resonance imaging at day 28 was 2.8-fold ( p< 0.0001) higher ingrafts of topical HDL-treated mice than in control mice. Topical HDL potentlyreduced intimal inflammation and resulted in enhanced endothelial regeneration asevidenced by an increase in the number of CD31-positive endothelial cells. Asstated supra, HDL potently enhanced migration and adhesion of ECFCs in vitro,and these effects were dependent on signalling via SR-BI, ERK, and NO and onincreased β1-integrin expression. Correspondingly, the number of CD31β-galactosidase double-positive cells, reflecting incorporated circulating progenitorcells, was 3.9-fold ( p< 0.01) higher in grafts of HDL-treated mice than in controlgrafts. Importantly, the effects of topical HDL therapy on vein graft atherosclerosiswere similar compared to the effects of systemically increased HDL cholesterolafter human apo A-I gene transfer. Taken together, topical HDL application is anew paradigm of HDL therapy.Whereas this section was focused on HDL- and EPC-mediated endothelialrepair, the next two sections will deal with HDL and tissue repair. In thesetherapeutic areas, the effect of HDL on EPCs may be an important mediator ofthe beneficial effects of HDL-targeted therapies.6 Development of Topical HDL Therapy for CutaneousWound HealingWound healing results from complex interactions between extracellular matrixmolecules, soluble mediators, resident skin cells, and infiltrating leukocytes aswell as infiltrating EPCs. Rather artificially, wound healing can be divided in aninflammation phase, a phase of tissue formation with accumulation of granulationtissue and reepithelialisation, and finally a phase of tissue remodelling (Diegelmannand Evans 2004).Granulation tissue contains tissue macrophages, fibroblasts, numerous newvessels, and extracellular matrix molecules. Macrophages stimulate fibroplasiaand angiogenesis by secretion of various growth factors. Fibroblasts deposit andremodel the extracellular matrix of wounds. Blood vessels are needed to provideoxygen and nutrients, whereas extracellular matrix provides a conduit for cellmigration and cell ingrowth. Formation of granulation tissue starts a few daysTherapeutic Potential of HDL in Cardioprotection and Tissue Repair 547after injury and is mainly triggered by growth factors such as platelet-derivedgrowth factor and transforming growth factor-β, which stimulate fibroblast prolif-eration and migration (Singer and Clark 1999). Fibroblasts start to deposit andremodel extracellular matrix, which initially consists of fibronectin and hyaluronanbut is later on replaced by proteoglycans and type I and III collagens (Toriseva andKahari 2009). The formation of new blood vessels is a critical component of togranulation tissue formation. Angiogenesis is initially induced by tissue destructionand hypoxia and is subsequently stimulated by various molecules such as basicfibroblast growth factor, VEGF, and transforming growth factor-β, secreted bymacrophages, keratinocytes, and endothelial cells. A role of EPCs in granulationtissue formation is directly suggested by experiments demonstrating that celltherapy with EPCs enhances wound healing in mice (Suh et al. 2005). The releaseof various growth factors such as VEGF and platelet-derived growth factor by EPCsappears to enhance wound healing (Suh et al. 2005). Reepithelialisation is the termused to describe the appearance of a new epithelial layer on top of a healing skinwound. It is dependent on the formation of a provisional wound bed matrix andinvolves the migration and proliferation of keratinocytes, the differentiation andstratification of new epithelium, and finally the reformation of the basementmembrane.Typical chronic cutaneous wounds are diabetic ulcers, ischaemic ulcers, andpressure ulcers. Diabetic ulcers occur in patients with type 1 and type 2 diabetes,whereas ischaemic ulcers are observed in patients with peripheral arterial diseases.Pressure ulcers occur in patients with paralysis or in other conditions that inhibitmovement of body parts. Pressure ulcers or decubitus wounds typically occur onsacrum, shoulder blades, and heels. Several factors contribute to deficient woundhealing in patients with diabetes: deficient growth factor production (Galkowskaet al. 2006), impaired neovascularisation (Galiano et al. 2004b), attenuatedkeratinocyte and fibroblast proliferation and migration (Gibran et al. 2002), andaltered balance between extracellular matrix accumulation and remodelling of theextracellular matrix by matrix metalloproteinases (Lobmann et al. 2002). Footulcers occur in a diabetic population with a prevalence of 5 % and lifetimeincidence of 15 % (Abbott et al. 2002; Muller et al. 2002). Even with lege artistreatment, amputation is required in 14–24 % of cases. Every 30 s, a lower limb islost due to diabetes. Approximately 85 % of these amputations are preceded by anulcer. Mortality is up to 50 % in the first 3 years after amputation. Various studiesshow that costs to treat diabetic foot ulcers are extremely high, especially ifhospitalisation is required (Matricali et al. 2007).HDL may beneficially affect wound healing by accelerating resolution ofinflammation, by enhancing granulation tissue formation involving increasedEPC incorporation and increased paracrine effects of EPCs, and by acceleratingreepithelialisation. Keratinocytes express SR-BI, and SR-BI expression isupregulated in dividing keratinocytes (Tsuruoka et al. 2002). Whereas in humanshealing is primarily the result of reepithelialisation and granulation tissue forma-tion, wound healing in mice occurs predominantly by wound contraction(Greenhalgh 2003). In the excisional wound healing model (Galiano548 S. Van Linthout et al.et al. 2004a), a circular full-thickness wound is applied on the back of each mouse.Subsequently, a silicone splint is fixed around the wound with nylon sutures tocounteract wound contraction. Consequently, wound healing in this model occursby granulation tissue formation and reepithelialisation from the border. Granulationtissue formation and reepithelialisation were significantly delayed in C57BL/6 apoE/ mice compared to C57BL/6 mice (Gordts et al. 2014). Topical administrationof HDL (protein concentration 800 μg/ml; volume 80 μl) formulated in 20 %pluronic F-127 gel (pH 7.2) every 2 days on wounds in C57BL/6 apo E/ micesignificantly improved granulation tissue formation and reepithelialisation. Woundhealing in mice treated with topical HDL therapy was very similar compared toC57BL/6 mice. Topical gel without HDL did not enhance wound healing.Improved wound healing induced by topical HDL therapy was also observed inC57BL/6 mice with streptozotocin-induced diabetes mellitus and in male type2 diabetic leptin receptor-deficient mice (unpublished data).Further preclinical studies are required to evaluate the robustness of this strategyin other models of delayed wound healing. However, murine models of diabeticwound healing differ in many respects to clinical diabetic wound healing. They donot take into account that there is a significant degree of heterogeneity of ulcers inpatients with diabetes. Three categories can be discerned: ‘purely neurotrophic’diabetic ulcers, ‘purely ischaemic’ ulcers in patients with diabetes, and poorlyhealing ulcers in diabetic patients with microangiopathy. The ‘purely neurotrophic’diabetic ulcers heal well after off-loading. The ‘purely ischaemic’ ulcers should betreated with revascularisation strategies. Typically, diabetic ulcers in patients withmore pronounced microangiopathy and macroangiopathy are characterised bydefects in granulation tissue formation and constitute a potential therapeutic areafor HDL-targeted therapies. However, no murine model can adequately mimicthese chronic diabetic ulcers. Decubitus wounds constitute another target for topicalHDL therapy but lack of adequate animal models hinders preclinical development.7 Beneficial Effects of Selective HDL-Raising Gene Transferon Cardiac Remodelling and Cardiac Function AfterMyocardial Infarction in MiceLoss of myocardial tissue following acute myocardial infarction results in adecreased systolic ejection and increased left ventricular end-diastolic volumeand pressure. The Frank-Starling mechanism, implying that an increasedend-diastolic volume results in an increased pressure developed during systole,may help to restore cardiac output. However, the concomitant increased wall stressmay induce regional hypertrophy in the non-infarcted segment, whereas in theinfarcted area expansion and thinning may occur. Experimental animal studiesshow that the infarcted ventricle hypertrophies and that the degree of hypertrophyis dependent on the infarct size (Anversa and Sonnenblick 1990). Taken together,architectural remodelling is characterised by the formation of a discrete collagenscar, ventricular dilatation, and ventricular hypertrophy. This process may continueTherapeutic Potential of HDL in Cardioprotection and Tissue Repair 549for weeks or months until the distending forces are counterbalanced by the tensilestrength of the collagen scar. Remodelling post-myocardial infarction is complexsince it involves the infarct area, the infarct border zone, and the non-infarctedmyocardium (Sutton and Sharpe 2000). Following myocardial infarction, dyski-netic bulging may occur in infarct area. The myocardial fibres contiguous to thissegment in the infarct border zone become exposed to a more pronounced increasein wall stress because of the more prominent change in the radius of curvatureinduced by the regional dilatation. Cardiomyocytes in the border zone of the infarctbecome larger than cardiomyocytes in the remote area of the ventricle. This isconsistent with the view that the infarct-induced stress on the chamber walls is animportant determinant of cardiomyocyte hypertrophy (Cohn 1993).Post-infarct remodelling occurs in the setting of volume overload, since thestretched and dilated infarcted tissue increases the left ventricular volume. Anincreased ventricular volume not only implies increased preload (passive ventricu-lar wall stress at the end of diastole) but also increased afterload (total myocardialwall stress during systolic ejection). Afterload is increased since the systolic radiusis increased. Since both systolic and diastolic wall stress are increased, remodellingand hypertrophy post-myocardial infarction are characterised by mixed features ofpressure overload and volume overload.The myocardium consists of 3 integrated components: cardiomyocytes, extra-cellular matrix, and capillary microcirculation. All 3 components are involved inthe remodelling process. The role of the extracellular matrix is distinct in the earlyphase of remodelling (within 72 h) and the late phase (beyond 72 h). Neutrophilinfiltration of matrix metalloproteinases induces degradation of intermyocyte col-lagen struts and cardiomyocyte slippage. This leads to infarct expansioncharacterised by the disproportionate thinning and dilatation of the infarct segment(Erlebacher et al. 1984). Infarct expansion predisposes to myocardial rupture andcongestive heart failure (Eaton et al. 1979; Erlebacher et al. 1982; Jugdutt andMichorowski 1987; Schuster and Bulkley 1979). Increased wall stress as a result ofinfarct expansion leads to mechanical stretch-elicited local angiotensin II releaseand activation of a fetal gene programme (Sutton and Sharpe 2000). Local angio-tensin II release promotes cardiomyocyte hypertrophy. In later stages ofremodelling, interstitial fibrosis is induced. Transforming growth factor-β1transforms fibroblasts into myofibroblasts and induces activation of tissue inhibitorsof metalloproteinases and production of type I and type III collagen. The resultantinterstitial fibrosis negatively affects the diastolic properties of the heart.Post-infarct ejection fraction is lower in patients with low HDL cholesterollevels (Kempen et al. 1987; Wang et al. 1998). Although this could be due todifferences in atherosclerosis or in the microvasculature or could be related to adecrease in infarct size due to beneficial effects of HDL in IRI, the possibility thatHDL beneficially affects infarct expansion and ventricular remodelling should beconsidered. This can be investigated in models of permanent ligation of, e.g. the leftanterior descending coronary artery. An attenuation of post-infarct left ventricularremodelling and improved infract healing was induced by infusion of rHDL once aweek for 4 weeks in rats following ligation of the proximal left coronary artery550 S. Van Linthout et al.(Kiya et al. 2009). Interestingly, rHDL-treated rats also showed an increase ofphosphorylation of ERK1/2 in the left ventricular tissue, but not of p38 MAPK orJNK (Kiya et al. 2009). Gordts et al. (2013) have recently shown that human apoA-I gene transfer in C57BL/6 LDL receptor-deficient mice increases survival,decreases infarct expansion, attenuates left ventricular dilatation, and improvescardiac function following permanent ligation of the left anterior descendingcoronary artery. Gene transfer in C57BL/6 LDL receptor-deficient mice wasperformed with the E1E3E4-deleted adenoviral vector AdA-I, inducinghepatocyte-specific expression of human apo A-I, or with the control vector Adnull.A ligation of the left anterior descending coronary artery was performed 2 weeksafter transfer or saline injection. Permanent ligation of the left anterior descendingcoronary artery excludes salutary coronary effects of HDL on myocardial salvageas observed in models of IRI (Theilmeier et al. 2006). Consequently, the infarct areaand the area at risk were nearly identical at 24 h after ligation implying that theinitial increase in loading conditions was not different between human apo A-I genetransfer myocardial infarction mice and control myocardial infarction mice. HDLcholesterol levels were persistently 1.5 times ( p< 0.0001) higher in AdA-I micecompared to controls. Survival was increased ( p< 0.01) in AdA-I myocardialinfarction mice compared to control myocardial infarction mice during the28-day follow-up period (hazard ratio for mortality 0.42; 95 % CI 0.24–0.76).Longitudinal morphometric analysis demonstrated attenuated infarct expansion(reduced infract length and increased infarct thickness of the infarct in AdA-Imyocardial infarction mice) and inhibition of left ventricular dilatation in AdA-Imyocardial infarction mice compared to controls. AdA-I transfer exerted immuno-modulatory effects and increased neovascularisation in the infarct zone. IncreasedHDL after AdA-I transfer significantly improved systolic and diastolic cardiacfunction post-MI and led to a preservation of peripheral blood pressure.The effects of AdA-I transfer on remodelling have significant consequences forthe progressive development of heart failure. Ventricular dilatation increases theloading conditions of the heart. After all, ‘preload’ at the organ level corresponds topassive ventricular wall stress at the end of diastole (preloadLV¼ (EDPLV)(EDRLV)/2WTLV where EDP is end-diastolic pressure, EDR is end-diastolic radius,and WT is wall thickness). ‘Afterload’ at the organ level reflects myocardial wallstress during systolic ejection (afterloadLV¼ (SPLV) (SRLV)/2WTLV where SP issystolic pressure, SR is systolic radius, and WT is wall thickness). Thus, theattenuation of infarct expansion and ventricular dilatation induced by increasedHDL is critical for long-term preservation of left ventricular function. Although anincrease of end-diastolic volume may be physiologically beneficial in terms of theFrank–Starling mechanism, the increase in afterload in a larger ventricle is clearlynot beneficial. The increase in afterload corresponds at the cellular level to anincrease in tension that each muscle fibre must develop, and correspondingly,oxygen consumption increases. In addition, ventricular hypertrophy may beassociated with an imbalance between the vascular and cardiomyocyte compart-ment in the myocardium (Shimizu et al. 2010; Tirziu et al. 2007). Increased oxygenconsumption together with a decrease in relative vascularity may lead to tissueTherapeutic Potential of HDL in Cardioprotection and Tissue Repair 551hypoxia, cardiomyocyte dysfunction, and late cardiomyocyte death. There was atrend for a higher capillary density and a higher relative vascularity at day 28 inAdA-I myocardial infarction mice compared to control myocardial infarction mice.Taken together, ventricular dilatation initially produced by an external stimulus(ligation of the left anterior descending coronary artery) induces a vicious cyclewhere an increase of loading conditions leads to an intrinsic pathological heartmuscle (cardiomyopathy) characterised by cardiomyocyte death and dysfunction(reduced intrinsic myocardial contractility and relaxation), collagen deposition, andprogressive dilatation of the heart. Although no follow-up was performed in thecurrent study after 28 days, we speculate that differences in ventricular functionbetween control MI mice and human apo A-I gene transfer MI mice will be moremarked after longer follow-up.Neovascularisation may play a role in scar formation and scar tissue remodelling(Wang et al. 2005). Increased neovascularisation was observed in the infarcts ofAdA-I myocardial infarction mice at day 28 after gene transfer. HDL exerts potenteffects on the endothelium. These effects occur via enhanced endothelial survival(Nofer et al. 2001), endothelial cell migration (Seetharam et al. 2006), andEPC-mediated repair (Feng et al. 2009a, b; Tso et al. 2006). Beneficial effects onEPCs may have contributed to improved infarct healing.Taken together, attenuation of remodelling following human apo A-I genetransfer may be considered to constitute the cardiac equivalent of enhancedwound healing and tissue repair induced by HDL.ConclusionsOne of the pitfalls in the field of HDL-targeted interventions is the lack ofselective HDL-raising drugs. The development of gene transfer technologieswith a sufficiently high therapeutic index may pave the road for a selective andeffective HDL-targeted therapeutic intervention (Jacobs et al. 2012; VanCraeyveld et al. 2010). Nathwani et al. (2011) performed a landmark studythat demonstrated for the first time long-term expression of a transgene productat therapeutic levels following systemic hepatocyte-directed gene transfer inhumans. In this study (Nathwani et al. 2011), haemophilia B was successfullytreated with an adeno-associated viral serotype 8 human FIX expressing vector.These seminal data highlight that hepatocyte-directed adeno-associated viralserotype 8 gene transfer may become a clinical reality in the next decades.The recent positive assessment by the European Medicines Agency’s Committeefor Medicinal Products for Human Use of the marketing authorisation for thefirst gene transfer product in Europe (alipogene tiparvovec (Glybera®))highlights an important paradigm shift by regulatory agencies, as well as thebiotechnological entrepreneurs and investors. Nevertheless, there is no short-term perspective for widespread clinical use of gene transfer technologies.Three modalities discussed in this review are selective HDL-targetedtherapies: infusion of rHDL, human apo A-I gene transfer, and topical HDLtherapy. The main strategic questions with regard to HDL-targeted therapies arerelated to the choice of therapeutic areas in which a real clinical difference could552 S. Van Linthout et al.be made. These questions are inextricably linked to the current state of the art ofevidence-based medicine. Since a significant part of this review is focused onheart failure, we will illustrate this point on the basis of a very significantdichotomous classification of these patients.Among patients with hospitalised heart failure, approximately 50 % haveheart failure with reduced ejection fraction (HFrEF), and 50 % have heart failurewith preserved ejection fraction (HFpEF) with the proportion of patients withHFpEF increasing with time (Liu et al. 2013; Vaduganathan and Fonarow 2013).The cutoff value for preserved ejection fraction is 0.5. Most clinical heart failuretrials have been focused on patients with HFrEF. Inhibition of the renin-angiotensin-aldosterone and sympathetic nervous systems improves survivaland decreases hospitalisations in patients with HFrEF (Reed et al. 2014). Incontrast to these significant advances in treatment and reduction in mortalityin patients with HFrEF, drug strategies with strong evidence in HFrEF haveproved unsuccessful in HFpEF, and the mortality in patients with HFpEF hasremained unchanged (Liu et al. 2013). HFpEF is a complex clinical syndromethat is characterised by classical heart failure symptoms with increased leftventricular filling pressures and preserved left ventricular ejection fraction.Increased end-diastolic ventricular stiffness is observed in HFpEF, but theexact mechanisms that induce HFpEF are unknown. This heart failure subtypedisproportionately affects women and the elderly and is commonly associatedwith other cardiovascular comorbidities, such as hypertension and diabetes.Most therapeutic gains can likely be made in the field HFpEF. Interestingly,human apo A-I gene transfer has been shown to improve diastolic function inC57BL/6 LDL receptor-deficient mice (Gordts et al. 2012). The same strategicquestions should also be raised in relation to the role of HDL in prevention andtreatment of coronary artery disease. One might speculate that perspectives foran HDL-targeted therapy in patients with stable coronary artery disease arelimited, whereas beneficial coronary and myocardial effects in the setting ofacute coronary syndromes may still constitute a window of opportunity.Conflict of Interest None.Acknowledgments Dr. Sophie Van Linthout received grant support from the European Founda-tion for the Study of Diabetes (EFSD). Research in the labs of Dr. Frias was supported by grantsfrom the Fondation Gustave et Simone Prévot, Wolfermann Nageli Stiftung, Novartis ConsumableFoundation, Jubiläumsstiftung, and Fondation pour la luttre contre le cancer et investigationsmédico-biologiques. Dr. Bart De Geest received grant support of the FWO-Vlaanderen (grantsG.0529.10N and G0A3114N) and of the KU Leuven (grant OT/13/090). Dr. Frias and Dr. DeGeest participated in and received financial support from COST Action BM0904 ‘HDL—frombiological understanding to clinical exploitation’.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 553ReferencesAbbott CA, Carrington AL, Ashe H, Bath S, Every LC, Griffiths J, Hann AW, Hussein A,Jackson N, Johnson KE, Ryder CH, Torkington R, Van Ross ER, Whalley AM, Widdows P,Williamson S, Boulton AJ (2002) The North-West Diabetes Foot Care Study: incidence of, andrisk factors for, new diabetic foot ulceration in a community-based patient cohort. Diabet Med19:377–384Abderrahmani A, Niederhauser G, Favre D, Abdelli S, Ferdaoussi M, Yang JY, Regazzi R,Widmann C, Waeber G (2007) Human high-density lipoprotein particles prevent activationof the JNK pathway induced by human oxidised low-density lipoprotein particles in pancreaticbeta cells. Diabetologia 50:1304–1314Alvarez A, Cerda-Nicolas M, Naim Abu Nabah Y, Mata M, Issekutz AC, Panes J, Lobb RR, SanzMJ (2004) Direct evidence of leukocyte adhesion in arterioles by angiotensin II. Blood104:402–408Ameli S, Hultgardh-Nilsson A, Cercek B, Shah PK, Forrester JS, Ageland H, Nilsson J (1994)Recombinant apolipoprotein A-I Milano reduces intimal thickening after balloon injury inhypercholesterolemic rabbits. Circulation 90:1935–1941Anversa P, Sonnenblick EH (1990) Ischemic cardiomyopathy: pathophysiologic mechanisms.Prog Cardiovasc Dis 33:49–70Aoyama T, Yui Y, Morishita H, Kawai C (1990) Prostaglandin I2 half-life regulated by highdensity lipoprotein is decreased in acute myocardial infarction and unstable angina pectoris.Circulation 81:1784–1791Babitt J, Trigatti B, Rigotti A, Smart EJ, Anderson RG, Xu S, Krieger M (1997) Murine SR-BI, ahigh density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated andfatty acylated and colocalizes with plasma membrane caveolae. J Biol Chem 272:13242–13249Baroukh N, Lopez CE, Saleh MC, Recalde D, Vergnes L, Ostos MA, Fiette L, Fruchart JC,Castro G, Zakin MM, Ochoa A (2004) Expression and secretion of human apolipoprotein A-Iin the heart. FEBS Lett 557:39–44Behrendt D, Ganz P (2002) Endothelial function. From vascular biology to clinical applications.Am J Cardiol 90:40L–48LBerg AH, Combs TP, Du X, Brownlee M, Scherer PE (2001) The adipocyte-secreted proteinAcrp30 enhances hepatic insulin action. Nat Med 7:947–953Brutsaert DL (2003) Cardiac endothelial-myocardial signaling: its role in cardiac growth, contrac-tile performance, and rhythmicity. Physiol Rev 83:59–115Cai H (2005) NAD(P)H oxidase-dependent self-propagation of hydrogen peroxide and vasculardisease. Circ Res 96:818–822Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ (2002) Hyperglycemia-induced apoptosis inmouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway.Diabetes 51:1938–1948Calabresi L, Rossoni G, Gomaraschi M, Sisto F, Berti F, Franceschini G (2003) High-densitylipoproteins protect isolated rat hearts from ischemia-reperfusion injury by reducing cardiactumor necrosis factor-alpha content and enhancing prostaglandin release. Circ Res 92:330–337Campeau L, Enjalbert M, Lesperance J, Bourassa MG, Kwiterovich P Jr, Wacholder S, SnidermanA (1984) The relation of risk factors to the development of atherosclerosis in saphenous-veinbypass grafts and the progression of disease in the native circulation. A study 10 years afteraortocoronary bypass surgery. N Engl J Med 311:1329–1332Chalupsky K, Cai H (2005) Endothelial dihydrofolate reductase: critical for nitric oxide bioavail-ability and role in angiotensin II uncoupling of endothelial nitric oxide synthase. Proc NatlAcad Sci USA 102:9056–9061Cheetham C, Collis J, O’Driscoll G, Stanton K, Taylor R, Green D (2000) Losartan, an angiotensintype 1 receptor antagonist, improves endothelial function in non-insulin-dependent diabetes. JAm Coll Cardiol 36:1461–1466554 S. Van Linthout et al.Christoffersen C, Obinata H, Kumaraswamy SB, Galvani S, Ahnstrom J, Sevvana M, Egerer-Sieber C, Muller YA, Hla T, Nielsen LB, Dahlback B (2011) Endothelium-protective sphin-gosine-1-phosphate provided by HDL-associated apolipoprotein M. Proc Natl Acad Sci USA108:9613–9618Cockerill GW, Huehns TY, Weerasinghe A, Stocker C, Lerch PG, Miller NE, Haskard DO (2001)Elevation of plasma high-density lipoprotein concentration reduces interleukin-1-inducedexpression of E-selectin in an in vivo model of acute inflammation. Circulation 103:108–112Cohen RA (1993) Dysfunction of vascular endothelium in diabetes mellitus. Circulation 87:V67–V76Cohn JN (1993) Post-MI remodeling. Clin Cardiol 16:II21–II24Condorelli G, Morisco C, Stassi G, Notte A, Farina F, Sgaramella G, de Rienzo A, Roncarati R,Trimarco B, Lembo G (1999) Increased cardiomyocyte apoptosis and changes in proapoptoticand antiapoptotic genes bax and bcl-2 during left ventricular adaptations to chronic pressureoverload in the rat. Circulation 99:3071–3078Cong L, Chen K, Li J, Gao P, Li Q, Mi S, Wu X, Zhao AZ (2007) Regulation of adiponectin andleptin secretion and expression by insulin through a PI3K-PDE3B dependent mechanism in ratprimary adipocytes. Biochem J 403:519–525Cosson S, Kevorkian JP (2003) Left ventricular diastolic dysfunction: an early sign of diabeticcardiomyopathy? Diabetes Metab 29:455–466De Geest B (2009) The origin of intimal smooth muscle cells: are we on a steady road back to thepast? Cardiovasc Res 81:7–8De Geest B, Zhao Z, Collen D, Holvoet P (1997) Effects of adenovirus-mediated human apo A-Igene transfer on neointima formation after endothelial denudation in apo E-deficient mice.Circulation 96:4349–4356De Geest B, Stengel D, Landeloos M, Lox M, Le Gat L, Collen D, Holvoet P, Ninio E (2000)Effect of overexpression of human apo A-I in C57BL/6 and C57BL/6 apo E-deficient mice on2 lipoprotein-associated enzymes, platelet-activating factor acetylhydrolase and paraoxonase.Comparison of adenovirus-mediated human apo A-I gene transfer and human apo A-Itransgenesis. Arterioscler Thromb Vasc Biol 20:E68–E75Dewald O, Frangogiannis NG, Zoerlein M, Duerr GD, Klemm C, Knuefermann P, Taffet G,Michael LH, Crapo JD, Welz A, Entman ML (2003) Development of murine ischemiccardiomyopathy is associated with a transient inflammatory reaction and depends on reactiveoxygen species. Proc Natl Acad Sci USA 100:2700–2705Diegelmann RF, Evans MC (2004) Wound healing: an overview of acute, fibrotic and delayedhealing. Front Biosci 9:283–289Dietrich H, Hu Y, Zou Y, Huemer U, Metzler B, Li C, Mayr M, Xu Q (2000) Rapid developmentof vein graft atheroma in ApoE-deficient mice. Am J Pathol 157:659–669Eaton LW, Weiss JL, Bulkley BH, Garrison JB, Weisfeldt ML (1979) Regional cardiac dilatationafter acute myocardial infarction: recognition by two-dimensional echocardiography. N Engl JMed 300:57–62Erdbruegger U, Haubitz M, Woywodt A (2006) Circulating endothelial cells: a novel marker ofendothelial damage. Clin Chim Acta 373:17–26Erlebacher JA,Weiss JL, Eaton LW, Kallman C,Weisfeldt ML, Bulkley BH (1982) Late effects ofacute infarct dilation on heart size: a two dimensional echocardiographic study. Am J Cardiol49:1120–1126Erlebacher JA, Weiss JL, Weisfeldt ML, Bulkley BH (1984) Early dilation of the infarctedsegment in acute transmural myocardial infarction: role of infarct expansion in acute leftventricular enlargement. J Am Coll Cardiol 4:201–208Feng Y, Jacobs F, Van Craeyveld E, Brunaud C, Snoeys J, Tjwa M, Van Linthout S, De Geest B(2008) Human ApoA-I transfer attenuates transplant arteriosclerosis via enhancedincorporation of bone marrow-derived endothelial progenitor cells. Arterioscler ThrombVasc Biol 28:278–283Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 555Feng Y, Van Craeyveld E, Jacobs F, Lievens J, Snoeys J, De Geest B (2009a) Wild-type apo A-Iand apo A-I(Milano) gene transfer reduce native and transplant arteriosclerosis to a similarextent. J Mol Med 87:287–297Feng Y, van Eck M, Van Craeyveld E, Jacobs F, Carlier V, Van Linthout S, Erdel M, Tjwa M, DeGeest B (2009b) Critical role of scavenger receptor-BI-expressing bone marrow-derivedendothelial progenitor cells in the attenuation of allograft vasculopathy after human apo A-Itransfer. Blood 113:755–764Feng Y, Gordts SC, Chen F, Hu Y, Van Craeyveld E, Jacobs F, Carlier V, Zhang Z, Xu Q, Ni Y, DeGeest B (2011) Topical HDL administration reduces vein graft atherosclerosis in apo Edeficient mice. Atherosclerosis 214:271–278Fitzgibbon GM, Kafka HP, Leach AJ, Keon WJ, Hooper GD, Burton JR (1996) Coronary bypassgraft fate and patient outcome: angiographic follow-up of 5,065 grafts related to survival andreoperation in 1,388 patients during 25 years. J Am Coll Cardiol 28:616–626Foteinos G, Hu Y, Xiao Q, Metzler B, Xu Q (2008) Rapid endothelial turnover in atherosclerosis-prone areas coincides with stem cell repair in apolipoprotein E-deficient mice. Circulation117:1856–1863Freire CM, Moura AL, Barbosa Mde M, Machado LJ, Nogueira AI, Ribeiro-Oliveira A Jr (2007)Left ventricle diastolic dysfunction in diabetes: an update. Arq Bras Endocrinol Metabol51:168–175Frias MA, James RW, Gerber-Wicht C, Lang U (2009) Native and reconstituted HDL activateStat3 in ventricular cardiomyocytes via ERK1/2: role of sphingosine-1-phosphate. CardiovascRes 82:313–323Frias MA, Lang U, Gerber-Wicht C, James RW (2010) Native and reconstituted HDL protectcardiomyocytes from doxorubicin-induced apoptosis. Cardiovasc Res 85:118–126Frias MA, Pedretti S, Hacking D, Somers S, Lacerda L, Opie LH, James RW, Lecour S (2013)HDL protects against ischemia reperfusion injury by preserving mitochondrial integrity.Atherosclerosis 228:110–116Friedrich EB, Walenta K, Scharlau J, Nickenig G, Werner N (2006) CD34-/CD133+/VEGFR-2+endothelial progenitor cell subpopulation with potent vasoregenerative capacities. Circ Res 98:e20–e25Frustaci A, Kajstura J, Chimenti C, Jakoniuk I, Leri A, Maseri A, Nadal-Ginard B, Anversa P(2000) Myocardial cell death in human diabetes. Circ Res 87:1123–1132Fryirs MA, Barter PJ, Appavoo M, Tuch BE, Tabet F, Heather AK, Rye KA (2010) Effects ofhigh-density lipoproteins on pancreatic beta-cell insulin secretion. Arterioscler Thromb VascBiol 30:1642–1648Galiano RD, Jt M, Dobryansky M, Levine JP, Gurtner GC (2004a) Quantitative and reproduciblemurine model of excisional wound healing. Wound Repair Regen 12:485–492Galiano RD, Tepper OM, Pelo CR, Bhatt KA, Callaghan M, Bastidas N, Bunting S, Steinmetz HG,Gurtner GC (2004b) Topical vascular endothelial growth factor accelerates diabetic woundhealing through increased angiogenesis and by mobilizing and recruiting bone marrow-derivedcells. Am J Pathol 164:1935–1947Galkowska H, Wojewodzka U, Olszewski WL (2006) Chemokines, cytokines, and growth factorsin keratinocytes and dermal endothelial cells in the margin of chronic diabetic foot ulcers.Wound Repair Regen 14:558–565Gibran NS, Jang YC, Isik FF, Greenhalgh DG, Muffley LA, Underwood RA, Usui ML, Larsen J,Smith DG, Bunnett N, Ansel JC, Olerud JE (2002) Diminished neuropeptide levels contributeto the impaired cutaneous healing response associated with diabetes mellitus. J Surg Res108:122–128Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Borden WB, Bravata DM, Dai S, FordES, Fox CS, Franco S, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, HuffmanMD, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Magid D, Marcus GM,Marelli A, Matchar DB, McGuire DK, Mohler ER, Moy CS, Mussolino ME, Nichol G, PaynterNP, Schreiner PJ, Sorlie PD, Stein J, Turan TN, Virani SS, Wong ND, Woo D, Turner MB556 S. Van Linthout et al.(2013) Heart disease and stroke statistics–2013 update: a report from the American HeartAssociation. Circulation 127:e6–e245Goldman S, Zadina K, Moritz T, Ovitt T, Sethi G, Copeland JG, Thottapurathu L, Krasnicka B,Ellis N, Anderson RJ, Henderson W (2004) Long-term patency of saphenous vein and leftinternal mammary artery grafts after coronary artery bypass surgery: results from a Departmentof Veterans Affairs Cooperative Study. J Am Coll Cardiol 44:2149–2156Gordts SC, Van Craeyveld E, Muthuramu I, Singh N, Jacobs F, De Geest B (2012) Lipid loweringand HDL raising gene transfer increase endothelial progenitor cells, enhance myocardialvascularity, and improve diastolic function. PLoS ONE 7:e46849Gordts SC, Muthuramu I, Nefyodova E, Jacobs F, Van Craeyveld E, De Geest B (2013) Beneficialeffects of selective HDL raising gene transfer on survival, cardiac remodelling, and cardiacfunction after myocardial infarction in mice. Gene Ther 20:1053–1061Gordts SC, Muthuramu I, Amin R, Jacobs F, De Geest B (2014) The impact of lipoproteins onwound healing: topical HDL therapy corrects delayed wound healing in apolipoprotein Edeficient mice. Pharmaceuticals 7(4):419–432Gragasin FS, Xu Y, Arenas IA, Kainth N, Davidge ST (2003) Estrogen reduces angiotensinII-induced nitric oxide synthase and NAD(P)H oxidase expression in endothelial cells.Arterioscler Thromb Vasc Biol 23:38–44Greenhalgh DG (2003) Wound healing and diabetes mellitus. Clin Plast Surg 30:37–45Gu SS, Shi N, WuMP (2007) The protective effect of ApolipoproteinA-I on myocardial ischemia-reperfusion injury in rats. Life Sci 81:702–709Guo Z, Xia Z, Jiang J, McNeill JH (2007) Downregulation of NADPH oxidase, antioxidantenzymes, and inflammatory markers in the heart of streptozotocin-induced diabetic rats byN-acetyl-L-cysteine. Am J Physiol Heart Circ Physiol 292:H1728–H1736Hagensen MK, Shim J, Thim T, Falk E, Bentzon JF (2010) Circulating endothelial progenitor cellsdo not contribute to plaque endothelium in murine atherosclerosis. Circulation 121:898–905Han R, Lai R, Ding Q, Wang Z, Luo X, Zhang Y, Cui G, He J, Liu W, Chen Y (2007)Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabo-lism. Diabetologia 50:1960–1968He S, Li M, Ma X, Lin J, Li D (2010) CD4+CD25+Foxp3+ regulatory T cells protect theproinflammatory activation of human umbilical vein endothelial cells. Arterioscler ThrombVasc Biol 30:2621–2630Hedrick CC, Thorpe SR, Fu MX, Harper CM, Yoo J, Kim SM, Wong H, Peters AL (2000)Glycation impairs high-density lipoprotein function. Diabetologia 43:312–320Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T (2003)Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl JMed 348:593–600Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RA,Warnholtz A, Meinertz T, Griendling K, Harrison DG, Forstermann U, Munzel T (2001)Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res 88:E14–E22Hirschi KK, Ingram DA, Yoder MC (2008) Assessing identity, phenotype, and fate of endothelialprogenitor cells. Arterioscler Thromb Vasc Biol 28:1584–1595Hodgkinson CP, Laxton RC, Patel K, Ye S (2008) Advanced glycation end-product of low densitylipoprotein activates the toll-like 4 receptor pathway implications for diabetic atherosclerosis.Arterioscler Thromb Vasc Biol 28:2275–2281Hodroj W, Legedz L, Foudi N, Cerutti C, Bourdillon MC, Feugier P, Beylot M, Randon J, BriccaG (2007) Increased insulin-stimulated expression of arterial angiotensinogen and angiotensintype 1 receptor in patients with type 2 diabetes mellitus and atheroma. Arterioscler ThrombVasc Biol 27:525–531Hu Y, Zou Y, Dietrich H, Wick G, Xu Q (1999) Inhibition of neointima hyperplasia of mouse veingrafts by locally applied suramin. Circulation 100:861–868Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 557Hu Y, Davison F, Zhang Z, Xu Q (2003) Endothelial replacement and angiogenesis in arterioscle-rotic lesions of allografts are contributed by circulating progenitor cells. Circulation108:3122–3127Hughes SJ, Powis SH, Press M (2001) Surviving native beta-cells determine outcome of syngeneicintraportal islet transplantation. Cell Transplant 10:145–151Hyka N, Dayer JM, Modoux C, Kohno T, Edwards CK 3rd, Roux-Lombard P, Burger D (2001)Apolipoprotein A-I inhibits the production of interleukin-1beta and tumor necrosis factor-alpha by blocking contact-mediated activation of monocytes by T lymphocytes. Blood97:2381–2389Imaizumi S, Miura S, Nakamura K, Kiya Y, Uehara Y, Zhang B, Matsuo Y, Urata H, Ideishi M,Rye KA, Sata M, Saku K (2008) Antiarrhythmogenic effect of reconstituted high-densitylipoprotein against ischemia/reperfusion in rats. J Am Coll Cardiol 51:1604–1612Ingram DA, Mead LE, Tanaka H, Meade V, Fenoglio A, Mortell K, Pollok K, Ferkowicz MJ,Gilley D, Yoder MC (2004) Identification of a novel hierarchy of endothelial progenitor cellsusing human peripheral and umbilical cord blood. Blood 104:2752–2760Ismahil MA, Hamid T, Bansal SS, Patel B, Kingery JR, Prabhu SD (2013) Remodeling of themononuclear phagocyte network underlies chronic inflammation and disease progression inheart failure: critical importance of the cardiosplenic axis. Circ Res 114(2):266–282Iwaoka M, Obata JE, Abe M, Nakamura T, Kitta Y, Kodama Y, Kawabata K, Takano H,Fujioka D, Saito Y, Kobayashi T, Hasebe H, Kugiyama K (2007) Association of low serumlevels of apolipoprotein A-I with adverse outcomes in patients with nonischemic heart failure. JCard Fail 13:247–253Jacobs F, Gordts S, Muthuramu I, De Geest B (2012) The liver as a target organ for gene therapy:state of the art, challenges, and future perspectives. Pharmaceuticals 5:1372–1392Jugdutt BI, Michorowski BL (1987) Role of infarct expansion in rupture of the ventricular septumafter acute myocardial infarction: a two-dimensional echocardiographic study. Clin Cardiol10:641–652Karliner JS (2013) Sphingosine kinase and sphingosine 1-phosphate in the heart: a decade ofprogress. Biochim Biophys Acta 1831:203–212Kempen HJ, van Gent CM, Buytenhek R, Buis B (1987) Association of cholesterol concentrationsin low-density lipoprotein, high-density lipoprotein, and high-density lipoprotein subfractions,and of apolipoproteins AI and AII, with coronary stenosis and left ventricular function. J LabClin Med 109:19–26Kiya Y, Miura S, Imaizumi S, Uehara Y, Matsuo Y, Abe S, Jimi S, Urata H, Rye KA, Saku K(2009) Reconstituted high-density lipoprotein attenuates postinfarction left ventricularremodeling in rats. Atherosclerosis 203:137–144Kota SK, Kota SK, Jammula S, Panda S, Modi KD (2011) Effect of diabetes on alteration ofmetabolism in cardiac myocytes: therapeutic implications. Diabetes Technol Ther13:1155–1160Lakhani SA, Masud A, Kuida K, Porter GA Jr, Booth CJ, Mehal WZ, Inayat I, Flavell RA (2006)Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311:847–851Lee WJ, Ou HC, Hsu WC, Chou MM, Tseng JJ, Hsu SL, Tsai KL, Sheu WH (2010) Ellagic acidinhibits oxidized LDL-mediated LOX-1 expression, ROS generation, and inflammation inhuman endothelial cells. J Vasc Surg 52:1290–1300Li D, Saldeen T, Romeo F, Mehta JL (2000) Oxidized LDL upregulates angiotensin II type1 receptor expression in cultured human coronary artery endothelial cells: the potential roleof transcription factor NF-kappaB. Circulation 102:1970–1976Li J, Jin HB, Sun YM, Su Y, Wang LF (2010a) KB-R7943 inhibits high glucose-inducedendothelial ICAM-1 expression and monocyte-endothelial adhesion. Biochem Biophys ResCommun 392:516–519Li J, Zhu H, Shen E, Wan L, Arnold JM, Peng T (2010b) Deficiency of rac1 blocks NADPHoxidase activation, inhibits endoplasmic reticulum stress, and reduces myocardial remodelingin a mouse model of type 1 diabetes. Diabetes 59:2033–2042558 S. Van Linthout et al.Liu Y, Haddad T, Dwivedi G (2013) Heart failure with preserved ejection fraction: currentunderstanding and emerging concepts. Curr Opin Cardiol 28:187–196Lobmann R, Ambrosch A, Schultz G, Waldmann K, Schiweck S, Lehnert H (2002) Expression ofmatrix-metalloproteinases and their inhibitors in the wounds of diabetic and non-diabeticpatients. Diabetologia 45:1011–1016Lowes BD, Zolty R, Minobe WA, Robertson AD, Leach S, Hunter L, Bristow MR (2006) Serialgene expression profiling in the intact human heart. J Heart Lung Transplant 25:579–588Mackness MI, Arrol S, Durrington PN (1991) Paraoxonase prevents accumulation oflipoperoxides in low-density lipoprotein. FEBS Lett 286:152–154Marchesi M, Booth EA, Davis T, Bisgaier CL, Lucchesi BR (2004) Apolipoprotein A-IMilano and1-palmitoyl-2-oleoyl phosphatidylcholine complex (ETC-216) protects the in vivo rabbit heartfrom regional ischemia-reperfusion injury. J Pharmacol Exp Ther 311:1023–1031Marchesi M, Booth EA, Rossoni G, Garcia RA, Hill KR, Sirtori CR, Bisgaier CL, Lucchesi BR(2008) Apolipoprotein A-IMilano/POPC complex attenuates post-ischemic ventricular dys-function in the isolated rabbit heart. Atherosclerosis 197:572–578Matricali GA, Dereymaeker G, Muls E, Flour M, Mathieu C (2007) Economic aspects of diabeticfoot care in a multidisciplinary setting: a review. Diabetes Metab Res Rev 23:339–347Means CK, Brown JH (2009) Sphingosine-1-phosphate receptor signalling in the heart.Cardiovasc Res 82:193–200Means CK, Miyamoto S, Chun J, Brown JH (2008) S1P1 receptor localization confers selectivityfor Gi-mediated cAMP and contractile responses. J Biol Chem 283:11954–11963Mehra MR, Uber PA, Lavie CJ, Milani RV, Park MH, Ventura HO (2009) High-density lipopro-tein cholesterol levels and prognosis in advanced heart failure. J Heart Lung Transplant28:876–880Mochizuki S, Okumura M, Tanaka F, Sato T, Kagami A, Tada N, Nagano M (1991) Ischemia-reperfusion arrhythmias and lipids: effect of human high- and low-density lipoproteins onreperfusion arrhythmias. Cardiovasc Drugs Ther 5(Suppl 2):269–276Montanari D, Yin H, Dobrzynski E, Agata J, Yoshida H, Chao J, Chao L (2005) Kallikrein genedelivery improves serum glucose and lipid profiles and cardiac function in streptozotocin-induced diabetic rats. Diabetes 54:1573–1580Morel S, Frias MA, Rosker C, James RW, Rohr S, Kwak BR (2012) The natural cardioprotectiveparticle HDL modulates connexin43 gap junction channels. Cardiovasc Res 93:41–49Mosterd A, Hoes AW (2007) Clinical epidemiology of heart failure. Heart 93:1137–1146Mosterd A, Hoes AW, de Bruyne MC, Deckers JW, Linker DT, Hofman A, Grobbee DE (1999)Prevalence of heart failure and left ventricular dysfunction in the general population. TheRotterdam study. Eur Heart J 20:447–455Muller IS, de Grauw WJ, van Gerwen WH, Bartelink ML, van Den Hoogen HJ, Rutten GE (2002)Foot ulceration and lower limb amputation in type 2 diabetic patients in dutch primary healthcare. Diabetes Care 25:570–574Murata N, Sato K, Kon J, Tomura H, Yanagita M, Kuwabara A, Ui M, Okajima F (2000)Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins,regulates the lipid receptor-mediated actions. Biochem J 352(Pt 3):809–815Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, Chowdary P,Riddell A, Pie AJ, Harrington C, O’Beirne J, Smith K, Pasi J, Glader B, Rustagi P, Ng CY, KayMA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM,Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss UM, NienhuisAW, Davidoff AM (2011) Adenovirus-associated virus vector-mediated gene transfer inhemophilia B. N Engl J Med 365:2357–2365Nishida M, Springhorn JP, Kelly RA, Smith TW (1993) Cell-cell signaling between adult ratventricular myocytes and cardiac microvascular endothelial cells in heterotypic primaryculture. J Clin Invest 91:1934–1941Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 559Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D,Oates PJ, Hammes HP, Giardino I, Brownlee M (2000) Normalizing mitochondrial superoxideproduction blocks three pathways of hyperglycaemic damage. Nature 404:787–790Nofer JR, Levkau B, Wolinska I, Junker R, Fobker M, von Eckardstein A, Seedorf U, Assmann G(2001) Suppression of endothelial cell apoptosis by high density lipoproteins (HDL) andHDL-associated lysosphingolipids. J Biol Chem 276:34480–34485Nofer JR, van der Giet M, Tolle M, Wolinska I, von Wnuck LK, Baba HA, Tietge UJ, Godecke A,Ishii I, Kleuser B, Schafers M, Fobker M, Zidek W, Assmann G, Chun J, Levkau B (2004)HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J ClinInvest 113:569–581Nyby MD, Abedi K, Smutko V, Eslami P, Tuck ML (2007) Vascular Angiotensin type 1 receptorexpression is associated with vascular dysfunction, oxidative stress and inflammation infructose-fed rats. Hypertens Res 30:451–457Oak JH, Cai H (2007) Attenuation of angiotensin II signaling recouples eNOS and inhibitsnonendothelial NOX activity in diabetic mice. Diabetes 56:118–126Ohtsu H, Suzuki H, Nakashima H, Dhobale S, Frank G, Motley E, Eguchi S (2006) Angiotensin IIsignal transduction through small GTP-binding proteins: mechanism and significance invascular smooth muscle cells. Hypertension 48:534–540Ou J, Wang J, Xu H, Ou Z, Sorci-Thomas MG, Jones DW, Signorino P, Densmore JC, Kaul S,Oldham KT, Pritchard KA Jr (2005) Effects of D-4F on vasodilation and vessel wall thicknessin hypercholesterolemic LDL receptor-null and LDL receptor/apolipoprotein A-I double-knockout mice on Western diet. Circ Res 97:1190–1197Ouchi N, Kihara S, Arita Y, Okamoto Y, Maeda K, Kuriyama H, Hotta K, Nishida M,Takahashi M, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Funahashi T, MatsuzawaY (2000) Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-kappaBsignaling through a cAMP-dependent pathway. Circulation 102:1296–1301Ouchi N, Kihara S, Arita Y, Nishida M, Matsuyama A, Okamoto Y, Ishigami M, Kuriyama H,Kishida K, Nishizawa H, Hotta K, Muraguchi M, Ohmoto Y, Yamashita S, Funahashi T,Matsuzawa Y (2001) Adipocyte-derived plasma protein, adiponectin, suppresses lipid accu-mulation and class A scavenger receptor expression in human monocyte-derived macrophages.Circulation 103:1057–1063Ouedraogo R, Gong Y, Berzins B, Wu X, Mahadev K, Hough K, Chan L, Goldstein BJ, Scalia R(2007) Adiponectin deficiency increases leukocyte-endothelium interactions via upregulationof endothelial cell adhesion molecules in vivo. J Clin Invest 117:1718–1726Palomer X, Salvado L, Barroso E, Vazquez-Carrera M (2013) An overview of the crosstalkbetween inflammatory processes and metabolic dysregulation during diabetic cardiomyopathy.Int J Cardiol 168:3160–3172Parini P, Johansson L, Broijersen A, Angelin B, Rudling M (2006) Lipoprotein profiles in plasmaand interstitial fluid analyzed with an automated gel-filtration system. Eur J Clin Invest36:98–104Pereira RI, Draznin B (2005) Inhibition of the phosphatidylinositol 30-kinase signaling pathwayleads to decreased insulin-stimulated adiponectin secretion from 3T3-L1 adipocytes. Metabo-lism 54:1636–1643Persegol L, Verges B, Foissac M, Gambert P, Duvillard L (2006) Inability of HDL from type2 diabetic patients to counteract the inhibitory effect of oxidised LDL on endothelium-dependent vasorelaxation. Diabetologia 49:1380–1386Peterson SJ, Husney D, Kruger AL, Olszanecki R, Ricci F, Rodella LF, Stacchiotti A, Rezzani R,McClung JA, Aronow WS, Ikehara S, Abraham NG (2007) Long-term treatment with theapolipoprotein A1 mimetic peptide increases antioxidants and vascular repair in type I diabeticrats. J Pharmacol Exp Ther 322:514–520Peterson SJ, Drummond G, Kim DH, Li M, Kruger AL, Ikehara S, Abraham NG (2008) L-4Ftreatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity inobese mice. J Lipid Res 49:1658–1669560 S. Van Linthout et al.Piga R, Naito Y, Kokura S, Handa O, Yoshikawa T (2007) Short-term high glucose exposureinduces monocyte-endothelial cells adhesion and transmigration by increasing VCAM-1 andMCP-1 expression in human aortic endothelial cells. Atherosclerosis 193:328–334Pinney SP, Mancini D (2004) Cardiac allograft vasculopathy: advances in understanding itspathophysiology, prevention, and treatment. Curr Opin Cardiol 19:170–176Raev DC (1994) Which left ventricular function is impaired earlier in the evolution of diabeticcardiomyopathy? An echocardiographic study of young type I diabetic patients. Diabetes Care17:633–639Rahmani M, Cruz RP, Granville DJ, McManus BM (2006) Allograft vasculopathy versus athero-sclerosis. Circ Res 99:801–815Raja SG, Haider Z, Ahmad M, Zaman H (2004) Saphenous vein grafts: to use or not to use? HeartLung Circ 13:403–409Ramaciotti C, Sharkey A, McClellan G, Winegrad S (1992) Endothelial cells regulate cardiaccontractility. Proc Natl Acad Sci USA 89:4033–4036Rathi S, Deedwania PC (2012) The epidemiology and pathophysiology of heart failure. Med ClinNorth Am 96:881–890Reed BN, Rodgers JE, Sueta CA (2014) Polypharmacy in heart failure: drugs to use and avoid.Heart Fail Clin 10(4):577–590Rehman J, Li J, Orschell CM,March KL (2003) Peripheral blood “endothelial progenitor cells” arederived from monocyte/macrophages and secrete angiogenic growth factors. Circulation107:1164–1169Richardson MR, Yoder MC (2011) Endothelial progenitor cells: quo vadis? J Mol Cell Cardiol 50(2):266–272Rius C, Abu-Taha M, Hermenegildo C, Piqueras L, Cerda-Nicolas JM, Issekutz AC, Estan L,Cortijo J, Morcillo EJ, Orallo F, Sanz MJ (2010) Trans- but not cis-resveratrol impairsangiotensin-II-mediated vascular inflammation through inhibition of NF-kappaB activationand peroxisome proliferator-activated receptor-gamma upregulation. J Immunol185:3718–3727Roger VL, Weston SA, Redfield MM, Hellermann-Homan JP, Killian J, Yawn BP, Jacobsen SJ(2004) Trends in heart failure incidence and survival in a community-based population. JAMA292:344–350Rossoni G, Gomaraschi M, Berti F, Sirtori CR, Franceschini G, Calabresi L (2004) Synthetic high-density lipoproteins exert cardioprotective effects in myocardial ischemia/reperfusion injury. JPharmacol Exp Ther 308:79–84Rubler S, Dlugash J, Yuceoglu YZ, Kumral T, Branwood AW, Grishman A (1972) New type ofcardiomyopathy associated with diabetic glomerulosclerosis. Am J Cardiol 30:595–602Rutti S, Ehses JA, Sibler RA, Prazak R, Rohrer L, Georgopoulos S, Meier DT, Niclauss N,Berney T, Donath MY, von Eckardstein A (2009) Low- and high-density lipoproteins modu-late function, apoptosis, and proliferation of primary human and murine pancreatic beta-cells.Endocrinology 150:4521–4530Sattler K, Levkau B (2009) Sphingosine-1-phosphate as a mediator of high-density lipoproteineffects in cardiovascular protection. Cardiovasc Res 82:201–211Sattler KJ, Elbasan S, Keul P, Elter-Schulz M, Bode C, Graler MH, Brocker-Preuss M, Budde T,Erbel R, Heusch G, Levkau B (2010) Sphingosine 1-phosphate levels in plasma and HDL arealtered in coronary artery disease. Basic Res Cardiol 105:821–832Savvatis K, van Linthout S, Miteva K, Pappritz K, Westermann D, Schefold JC, Fusch G,Weithauser A, Rauch U, Becher PM, Klingel K, Ringe J, Kurtz A, Schultheiss HP, TschopeC (2012) Mesenchymal stromal cells but not cardiac fibroblasts exert beneficial systemicimmunomodulatory effects in experimental myocarditis. PLoS ONE 7:e41047Schulz R, Heusch G (2004) Connexin 43 and ischemic preconditioning. Cardiovasc Res62:335–344Schulz R, Boengler K, Totzeck A, Luo Y, Garcia-Dorado D, Heusch G (2007) Connexin 43 inischemic pre- and postconditioning. Heart Fail Rev 12:261–266Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 561Schuster EH, Bulkley BH (1979) Expansion of transmural myocardial infarction: a pathophysio-logic factor in cardiac rupture. Circulation 60:1532–1538Seetharam D, Mineo C, Gormley AK, Gibson LL, Vongpatanasin W, Chambliss KL, Hahner LD,Cummings ML, Kitchens RL, Marcel YL, Rader DJ, Shaul PW (2006) High-density lipopro-tein promotes endothelial cell migration and reendothelialization via scavenger receptor-Btype I. Circ Res 98:63–72Shah PK, Amin J (1992) Low high density lipoprotein level is associated with increased restenosisrate after coronary angioplasty. Circulation 85:1279–1285Shen X, Zheng S, Metreveli NS, Epstein PN (2006) Protection of cardiac mitochondria byoverexpression of MnSOD reduces diabetic cardiomyopathy. Diabetes 55:798–805Shimizu I, Minamino T, Toko H, Okada S, Ikeda H, Yasuda N, Tateno K, Moriya J, YokoyamaM,Nojima A, Koh GY, Akazawa H, Shiojima I, Kahn CR, Abel ED, Komuro I (2010) Excessivecardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload inrodents. J Clin Invest 120:1506–1514Shinomiya S, Naraba H, Ueno A, Utsunomiya I, Maruyama T, Ohuchida S, Ushikubi F, Yuki K,Narumiya S, Sugimoto Y, Ichikawa A, Oh-ishi S (2001) Regulation of TNFalpha andinterleukin-10 production by prostaglandins I(2) and E(2): studies with prostaglandinreceptor-deficient mice and prostaglandin E-receptor subtype-selective synthetic agonists.Biochem Pharmacol 61:1153–1160Singer AJ, Clark RA (1999) Cutaneous wound healing. N Engl J Med 341:738–746Singh N, Van Craeyveld E, Tjwa M, Ciarka A, Emmerechts J, Droogne W, Gordts SC, Carlier V,Jacobs F, Fieuws S, Vanhaecke J, Van Cleemput J, De Geest B (2012) Circulating apoptoticendothelial cells and apoptotic endothelial microparticles independently predict the presenceof cardiac allograft vasculopathy. J Am Coll Cardiol 60:324–331Sloop CH, Dory L, Roheim PS (1987) Interstitial fluid lipoproteins. J Lipid Res 28:225–237Sorrentino SA, Besler C, Rohrer L, Meyer M, Heinrich K, Bahlmann FH, Mueller M, Horvath T,Doerries C, Heinemann M, Flemmer S, Markowski A, Manes C, Bahr MJ, Haller H, vonEckardstein A, Drexler H, Landmesser U (2009) Endothelial-vasoprotective effects of high-density lipoprotein are impaired in patients with type 2 diabetes mellitus but are improved afterextended-release niacin therapy. Circulation 121(1):110–122Suh W, Kim KL, Kim JM, Shin IS, Lee YS, Lee JY, Jang HS, Lee JS, Byun J, Choi JH, Jeon ES,Kim DK (2005) Transplantation of endothelial progenitor cells accelerates dermal woundhealing with increased recruitment of monocytes/macrophages and neovascularization. StemCells 23:1571–1578Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A, Daugas E, Geuskens M,Kroemer G (1996) Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J ExpMed 184:1331–1341Sutton MG, Sharpe N (2000) Left ventricular remodeling after myocardial infarction: pathophysi-ology and therapy. Circulation 101:2981–2988Suzuki K, Murtuza B, Sammut IA, Latif N, Jayakumar J, Smolenski RT, Kaneda Y, Sawa Y,Matsuda H, Yacoub MH (2002) Heat shock protein 72 enhances manganese superoxidedismutase activity during myocardial ischemia-reperfusion injury, associated with mitochon-drial protection and apoptosis reduction. Circulation 106:I270–I276Sztalryd C, Kraemer FB (1995) Regulation of hormone-sensitive lipase in streptozotocin-induceddiabetic rats. Metabolism 44:1391–1396Takakura N, Watanabe T, Suenobu S, Yamada Y, Noda T, Ito Y, Satake M, Suda T (2000) A rolefor hematopoietic stem cells in promoting angiogenesis. Cell 102:199–209Tao R, Hoover HE, Honbo N, Kalinowski M, Alano CC, Karliner JS, Raffai R (2010) High-densitylipoprotein determines adult mouse cardiomyocyte fate after hypoxia-reoxygenation throughlipoprotein-associated sphingosine 1-phosphate. Am J Physiol Heart Circ Physiol 298:H1022–H1028Theilmeier G, Schmidt C, Herrmann J, Keul P, Schafers M, Herrgott I, Mersmann J, Larmann J,Hermann S, Stypmann J, Schober O, Hildebrand R, Schulz R, Heusch G, Haude M, vonWnuck562 S. Van Linthout et al.LK, Herzog C, Schmitz M, Erbel R, Chun J, Levkau B (2006) High-density lipoproteins andtheir constituent, sphingosine-1-phosphate, directly protect the heart against ischemia/reperfu-sion injury in vivo via the S1P3 lysophospholipid receptor. Circulation 114:1403–1409Thule PM, Campbell AG, Kleinhenz DJ, Olson DE, Boutwell JJ, Sutliff RL, Hart CM (2006)Hepatic insulin gene therapy prevents deterioration of vascular function and improvesadipocytokine profile in STZ-diabetic rats. Am J Physiol Endocrinol Metab 290:E114–E122Tirziu D, Chorianopoulos E, Moodie KL, Palac RT, Zhuang ZW, Tjwa M, Roncal C, Eriksson U,Fu Q, Elfenbein A, Hall AE, Carmeliet P, Moons L, Simons M (2007) Myocardial hypertrophyin the absence of external stimuli is induced by angiogenesis in mice. J Clin Invest117:3188–3197Tolle M, Pawlak A, Schuchardt M, Kawamura A, Tietge UJ, Lorkowski S, Keul P, Assmann G,Chun J, Levkau B, van der Giet M, Nofer JR (2008) HDL-associated lysosphingolipids inhibitNAD(P)H oxidase-dependent monocyte chemoattractant protein-1 production. ArteriosclerThromb Vasc Biol 28:1542–1548Toriseva M, Kahari VM (2009) Proteinases in cutaneous wound healing. Cell Mol Life Sci66:203–224Tricot O, Mallat Z, Heymes C, Belmin J, Leseche G, Tedgui A (2000) Relation betweenendothelial cell apoptosis and blood flow direction in human atherosclerotic plaques. Circula-tion 101:2450–2453Tschope C, Walther T, Escher F, Spillmann F, Du J, Altmann C, Schimke I, Bader M, Sanchez-Ferrer CF, Schultheiss HP, Noutsias M (2005) Transgenic activation of the kallikrein-kininsystem inhibits intramyocardial inflammation, endothelial dysfunction and oxidative stress inexperimental diabetic cardiomyopathy. Faseb J 19:2057–2059Tso C, Martinic G, Fan WH, Rogers C, Rye KA, Barter PJ (2006) High-density lipoproteinsenhance progenitor-mediated endothelium repair in mice. Arterioscler Thromb Vasc Biol26:1144–1149Tsuruoka H, Khovidhunkit W, Brown BE, Fluhr JW, Elias PM, Feingold KR (2002) Scavengerreceptor class B type I is expressed in cultured keratinocytes and epidermis. Regulation inresponse to changes in cholesterol homeostasis and barrier requirements. J Biol Chem277:2916–2922Tunaru S, Kero J, Schaub A, Wufka C, Blaukat A, Pfeffer K, Offermanns S (2003) PUMA-G andHM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect. Nat Med 9:352–355Uchiyama T, Engelman RM, Maulik N, Das DK (2004) Role of Akt signaling in mitochondrialsurvival pathway triggered by hypoxic preconditioning. Circulation 109:3042–3049Urbich C, Aicher A, Heeschen C, Dernbach E, Hofmann WK, Zeiher AM, Dimmeler S (2005)Soluble factors released by endothelial progenitor cells promote migration of endothelial cellsand cardiac resident progenitor cells. J Mol Cell Cardiol 39:733–742Vaduganathan M, Fonarow GC (2013) Epidemiology of hospitalized heart failure: differences andsimilarities between patients with reduced versus preserved ejection fraction. Heart Fail Clin9:271–276, vVan Craeyveld E, Gordts S, Jacobs F, De Geest B (2010) Gene therapy to improve high-densitylipoprotein metabolism and function. Curr Pharm Des 16:1531–1544Van Linthout S, Riad A, Dhayat N, Spillmann F, Du J, Dhayat S, Westermann D, Hilfiker-Kleiner D, Noutsias M, Laufs U, Schultheiss HP, Tschope C (2007) Anti-inflammatory effectsof atorvastatin improve left ventricular function in experimental diabetic cardiomyopathy.Diabetologia 50:1977–1986Van Linthout S, Spillmann F, Riad A, Trimpert C, Lievens J, Meloni M, Escher F, Filenberg E,Demir O, Li J, Shakibaei M, Schimke I, Staudt A, Felix SB, Schultheiss HP, De Geest B,Tschope C (2008) Human apolipoprotein A-I gene transfer reduces the development ofexperimental diabetic cardiomyopathy. Circulation 117:1563–1573Van Linthout S, Spillmann F, Lorenz M, Meloni M, Jacobs F, Egorova M, Stangl V, De Geest B,Schultheiss HP, Tschope C (2009) Vascular-protective effects of high-density lipoproteininclude the downregulation of the angiotensin II type 1 receptor. Hypertension 53:682–687Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 563Van Linthout S, Foryst-Ludwig A, Spillmann F, Peng J, Feng Y, Meloni M, Van Craeyveld E,Kintscher U, Schultheiss HP, De Geest B, Tschope C (2010a) Impact of HDL on adipose tissuemetabolism and adiponectin expression. Atherosclerosis 210:438–444Van Linthout S, Spillmann F, Schultheiss HP, Tschope C (2010b) High-density lipoprotein at theinterface of type 2 diabetes mellitus and cardiovascular disorders. Curr Pharm Des16:1504–1516Van Linthout S, Spillmann F, Graiani G, Miteva K, Peng J, Van Craeyveld E, Meloni M, Tolle M,Escher F, Subasiguller A, Doehner W, Quaini F, De Geest B, Schultheiss HP, Tschope C(2011) Down-regulation of endothelial TLR4 signalling after apo A-I gene transfer contributesto improved survival in an experimental model of lipopolysaccharide-induced inflammation. JMol Med (Berl) 89:151–160Vasa M, Fichtlscherer S, Adler K, Aicher A, Martin H, Zeiher AM, Dimmeler S (2001) Increase incirculating endothelial progenitor cells by statin therapy in patients with stable coronary arterydisease. Circulation 103:2885–2890Vassalli G, Gallino A, Weis M, von Scheidt W, Kappenberger L, von Segesser LK, Goy JJ (2003)Alloimmunity and nonimmunologic risk factors in cardiac allograft vasculopathy. Eur Heart J24:1180–1188Velagaleti RS, Massaro J, Vasan RS, Robins SJ, Kannel WB, Levy D (2009) Relations of lipidconcentrations to heart failure incidence: the Framingham Heart Study. Circulation120:2345–2351Verges B, Petit JM, Duvillard L, Dautin G, Florentin E, Galland F, Gambert P (2006) Adiponectinis an important determinant of apoA-I catabolism. Arterioscler Thromb Vasc Biol26:1364–1369Walter DH, Rittig K, Bahlmann FH, Kirchmair R, Silver M, Murayama T, Nishimura H, LosordoDW, Asahara T, Isner JM (2002) Statin therapy accelerates reendothelialization: a novel effectinvolving mobilization and incorporation of bone marrow-derived endothelial progenitor cells.Circulation 105:3017–3024Wang TD, Wu CC, Chen WJ, Lee CM, Chen MF, Liau CS, Sung FC, Lee YT (1998)Dyslipidemias have a detrimental effect on left ventricular systolic function in patients witha first acute myocardial infarction. Am J Cardiol 81:531–537Wang B, Ansari R, Sun Y, Postlethwaite AE, Weber KT, Kiani MF (2005) The scarneovasculature after myocardial infarction in rats. Am J Physiol Heart Circ Physiol 289:H108–H113WangW, Xu H, Shi Y, Nandedkar S, Zhang H, Gao H, Feroah T, Weihrauch D, Schulte ML, JonesDW, Jarzembowski J, Sorci-Thomas M, Pritchard KA Jr (2010) Genetic deletion of apolipo-protein A-I increases airway hyperresponsiveness, inflammation, and collagen deposition inthe lung. J Lipid Res 51:2560–2570Werner N, Priller J, Laufs U, Endres M, Bohm M, Dirnagl U, Nickenig G (2002) Bone marrow-derived progenitor cells modulate vascular reendothelialization and neointimal formation:effect of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibition. Arterioscler ThrombVasc Biol 22:1567–1572Werner N, Junk S, Laufs U, Link A, Walenta K, Bohm M, Nickenig G (2003) Intravenoustransfusion of endothelial progenitor cells reduces neointima formation after vascular injury.Circ Res 93:e17–e24Westermann D, Rutschow S, Van Linthout S, Linderer A, Bucker-Gartner C, Sobirey M, Riad A,Pauschinger M, Schultheiss HP, Tschope C (2006) Inhibition of p38 mitogen-activated proteinkinase attenuates left ventricular dysfunction by mediating pro-inflammatory cardiac cytokinelevels in a mouse model of diabetes mellitus. Diabetologia 49:2507–2513Westermann D, Rutschow S, Jager S, Linderer A, Anker S, Riad A, Unger T, Schultheiss HP,Pauschinger M, Tschope C (2007a) Contributions of inflammation and cardiac matrixmetalloproteinase activity to cardiac failure in diabetic cardiomyopathy: the role of angiotensintype 1 receptor antagonism. Diabetes 56:641–646564 S. Van Linthout et al.Westermann D, Van Linthout S, Dhayat S, Dhayat N, Escher F, Bucker-Gartner C, Spillmann F,Noutsias M, Riad A, Schultheiss HP, Tschope C (2007b) Cardioprotective and anti-inflammatory effects of interleukin converting enzyme inhibition in experimental diabeticcardiomyopathy. Diabetes 56:1834–1841Westermann D, Van Linthout S, Dhayat S, Dhayat N, Schmidt A, Noutsias M, Song XY,Spillmann F, Riad A, Schultheiss HP, Tschope C (2007c) Tumor necrosis factor-alphaantagonism protects from myocardial inflammation and fibrosis in experimental diabeticcardiomyopathy. Basic Res Cardiol 102:500–507Westermann D, Lindner D, Kasner M, Zietsch C, Savvatis K, Escher F, von Schlippenbach J,Skurk C, Steendijk P, Riad A, Poller W, Schultheiss HP, Tschope C (2011) Cardiac inflamma-tion contributes to changes in the extracellular matrix in patients with heart failure and normalejection fraction. Circ Heart Fail 4:44–52Whiteman EL, Cho H, Birnbaum MJ (2002) Role of Akt/protein kinase B in metabolism. TrendsEndocrinol Metab 13:444–451Wilhelm AJ, Zabalawi M, Owen JS, Shah D, Grayson JM,Major AS, Bhat S, Gibbs DP Jr, ThomasMJ, Sorci-Thomas MG (2010) Apolipoprotein A-I modulates regulatory T cells in autoimmuneLDLr-/-, ApoA-I-/- mice. J Biol Chem 285(46):36158–36169Wold LE, Ceylan-Isik AF, Fang CX, Yang X, Li SY, Sreejayan N, Privratsky JR, Ren J (2006)Metallothionein alleviates cardiac dysfunction in streptozotocin-induced diabetes: role of Ca2+cycling proteins, NADPH oxidase, poly(ADP-Ribose) polymerase and myosin heavy chainisozyme. Free Radic Biol Med 40:1419–1429Xu Q, Zhang Z, Davison F, Hu Y (2003) Circulating progenitor cells regenerate endothelium ofvein graft atherosclerosis, which is diminished in ApoE-deficient mice. Circ Res 93:e76–e86Yoon YS, Uchida S, Masuo O, Cejna M, Park JS, Gwon HC, Kirchmair R, Bahlman F, Walter D,Curry C, Hanley A, Isner JM, Losordo DW (2005) Progressive attenuation of myocardialvascular endothelial growth factor expression is a seminal event in diabetic cardiomyopathy:restoration of microvascular homeostasis and recovery of cardiac function in diabetic cardio-myopathy after replenishment of local vascular endothelial growth factor. Circulation111:2073–2085Young NL, Lopez DR, McNamara DJ (1988) Contributions of absorbed dietary cholesterol andcholesterol synthesized in small intestine to hypercholesterolemia in diabetic rats. Diabetes37:1151–1156Zacharowski K, Olbrich A, Piper J, Hafner G, Kondo K, Thiemermann C (1999) Selectiveactivation of the prostanoid EP(3) receptor reduces myocardial infarct size in rodents.Arterioscler Thromb Vasc Biol 19:2141–2147Zhang Y, Peng T, Zhu H, Zheng X, Zhang X, Jiang N, Cheng X, Lai X, Shunnar A, Singh M,Riordan N, Bogin V, Tong N, Min WP (2010) Prevention of hyperglycemia-inducedmyocardial apoptosis by gene silencing of Toll-like receptor-4. J Transl Med 8:133Zheng L, Nukuna B, Brennan ML, Sun M, Goormastic M, Settle M, Schmitt D, Fu X, Thomson L,Fox PL, Ischiropoulos H, Smith JD, Kinter M, Hazen SL (2004) Apolipoprotein A-I is aselective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjectswith cardiovascular disease. J Clin Invest 114:529–541Therapeutic Potential of HDL in Cardioprotection and Tissue Repair 565Part IVTreatments for Dyslipidemias andDysfunction of HDLHDL and Lifestyle InterventionsJoan Carles Escolà-Gil, Josep Julve, Bruce A. Griffin, Dilys Freeman,and Francisco Blanco-VacaContents1 HDL and Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5711.1 Effects of Substituting Dietary Saturated Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5711.2 Dietary MUFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5721.3 n-3 Polyunsaturated Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5721.4 Carbohydrate and Extrinsic Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5731.5 Effects of Dietary Fatty Acids and Cholesterol on HDL Function . . . . . . . . . . . . . . . . . 5732 Weight Loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5742.1 Impact of Adiposity on HDL Concentration and Function . . . . . . . . . . . . . . . . . . . . . . . . . 5742.2 Effect of Weight-Loss Therapies on HDL Concentration and Function . . . . . . . . . . . 5753 Effects of Regular Aerobic Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5763.1 Effects of Exercise on HDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5763.2 Effects of Exercise on Prevention and Treatment of Cardiometabolic Risk . . . . . . . 5774 Smoking Cessation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5784.1 Smoking and HDL-C Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5794.2 Potential Mechanisms for the Smoking-Related Reduction in HDL-C . . . . . . . . . . . . . 5794.3 Smoking Cessation Intervention to Increase Plasma HDL Concentration . . . . . . . . . 581J.C. Escolà-Gil • J. Julve • F. Blanco-Vaca (*)Institut d’Investigació Biomèdica (IIB) Sant Pau, Barcelona, SpainCIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM),Barcelona, SpainDepartament de Bioquı́mica i Biologia Molecular, Universitat Autònomade Barcelona, Barcelona, Spaine-mail: fblancova@santpau.catB.A. GriffinDepartment of Nutritional Sciences, University of Surrey, Surrey, UKD. FreemanInstitute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_185695 Effects of Alcohol on HDL and Cardiovascular Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5825.1 Alcohol Intake and HDL-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5825.2 Effects of Alcohol on HDL Atheroprotective Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584AbstractThe main lifestyle interventions to modify serum HDL cholesterol includephysical exercise, weight loss with either caloric restriction or specific dietaryapproaches, and smoking cessation. Moderate alcohol consumption can bepermitted in some cases. However, as these interventions exert multiple effects,it is often difficult to discern which is responsible for improvement in HDLoutcomes. It is particularly noteworthy that recent data questions the use of HDLcholesterol as a risk factor and therapeutic target since randomised interventionsand Mendelian randomisation studies failed to provide evidence for such anapproach. Therefore, these current data should be considered when reading andinterpreting this review. Further studies are needed to document the effect oflifestyle changes on HDL structure–function and health.KeywordsAlcohol • Exercise • HDL • Fat • Smoking • WeightAbbreviationsABC Adenosine triphosphate-binding cassette transporterApo ApolipoproteinCVD Cardiovascular diseaseCETP Cholesteryl ester transfer proteinHL Hepatic lipaseHDL-C High-density lipoprotein cholesterolLCAT Lecithin–cholesterol acyltransferaseLDL-C Low-density lipoprotein cholesterolLPL Lipoprotein lipaseLp-PLA2 Lipoprotein-associated phospholipase A2MUFA Monounsaturated fatty acidsPLTP Phospholipid transfer proteinPON1 Paraoxonase 1PUFA Polyunsaturated fatty acidsRCT Reverse cholesterol transportSFA Saturated fatty acidsSR-BI Scavenger receptor class-BITAG Triacylglycerol570 J.C. Escolà-Gil et al.1 HDL and DietThe landmark cross-cultural and migration studies of Ancel Keys in the 1950s werethe first to identify associations between serum HDL cholesterol (HDL-C) and dietand lifestyle factors in free-living populations (Keys 1980a). These early studieslaid the foundation for the development of the diet–heart hypothesis, by revealinglinks between the incidence of coronary heart disease, raised serum cholesterol andenergy derived from saturated fat. They also helped to establish diet as a determi-nant of low HDL in obesity (Keys 1980b). It has been established that certaindietary saturated fatty acids (SFA), with a chain length of between 12 and16 carbons, and dietary cholesterol can increase both serum low-density lipoproteincholesterol (LDL-C) and, somewhat paradoxically with respect to cardiovasculardisease (CVD) risk, HDL-C (Grundy and Denke 1990). Conversely, the replace-ment of SFA with either polyunsaturated (PUFA) or monounsaturated fatty acids(MUFA) has been shown to reduce serum HDL-C. Moreover, the replacement ofdietary fat with carbohydrate has also been shown to produce significant reductionsin serum HDL-C (Katan et al. 1997; Stanhope et al. 2013). Heightened awareness ofthe risk associated with dietary sugars in the early 1970s (Yudkin 1972) hasresurfaced recently, in part, through escalation of obesity-related cardiometabolicrisk and increased understanding of how extrinsic sugars influence HDL byadversely affecting the metabolism of triacylglycerol (TAG) (Lustig 2010). Thehistory of diet and HDL has evolved in the era of the LDL-lowering statins, whichare relatively ineffective in raising HDL-C, and leave behind what is oftendescribed as untreated ‘residual risk’ (Belsey et al. 2008). This situation has createda need for alternative HDL-targeted drugs and also increased awareness of theimportance of diet as a modifier of HDL structure and function.1.1 Effects of Substituting Dietary Saturated Fatty AcidsSerum HDL-C has been shown to be influenced by both the amount and quality ofdietary fatty acids and carbohydrate. In the past, the relative effects of dietary fattyacids on HDL-C have been described in absolute terms, that is, SFA tends to raiseHDL-C, whilst PUFA, trans fatty acids and carbohydrate, all tend to reduceHDL-C, with MUFA being relatively neutral (Grundy and Denke 1990). However,in reality the absolute effect of fatty acids, or any macronutrient, on HDL-C cannotbe measured, as its addition or replacement may be counter-affected by whateverfatty acid or carbohydrate takes its place to maintain a feasible diet. This phenome-non of substitution limits the ability to interpret the impact of dietarymacronutrients on HDL and CVD risk to ‘relative’, rather than ‘absolute’ effects.Meta-analyses of intervention studies have provided strong evidence to show thatthe iso-energetic replacement of SFA with PUFA, MUFA and carbohydratedecreases HDL-C, with carbohydrate exerting the greatest impact in loweringboth HDL-C and the total cholesterol–HDL-C ratio (Mensink et al. 2003). Incontrast, the iso-energetic replacement of carbohydrate with all dietary fattyHDL and Lifestyle Interventions 571acids, other than trans fatty acids, tends to raise HDL-C (Micha and Mozaffarian2010). Estimates for the relative magnitude of change in the concentration of serumHDL-C in response to these dietary substitutions are directly proportional to theamount of energy being exchanged. An iso-energetic exchange of 5 % energy fromcarbohydrate to SFA, MUFA and PUFA is associated with increases in HDL-C of0.05, 0.04 and 0.03 mmol/L, respectively, with trans fatty acids reducing HDL by0.02 mmol/L. In addition, individual SFA is known to exert differential effects onserum HDL-C, so that replacing 5 % energy as carbohydrate with lauric, myristic,palmitic and stearic, increases HDL-C by 0.13, 0.09, 0.05 and <0.01 mmol/L,respectively. These associations between dietary fatty acids and serum HDL-C arestatistically robust and have been used to formulate dietary guidelines. However,because they rely heavily upon data from measures of dietary intake, as estimatedfrom dietary recall, food frequency questionnaires or diet diaries, this can limit theirvalue in predicting the biological effects of complex foods on HDL (Astrupet al. 2011). In other words, foods are not single nutrients, but complex mixturesof nutrients within a food matrix, all of which interact together to produce abiological effect. Because a food is rich in one particular fatty acid, it does notmean that consumption of this food will result in an effect on HDL that is typical ofthat fatty acid.1.2 Dietary MUFAInterest in the potential health benefits of dietary MUFA originated from the use ofolive oil in the Mediterranean diet. Whilst substitution of SFA with MUFA is lesseffective in lowering LDL cholesterol than n-6 PUFA, there is evidence to suggestthat this substitution may be more effective in preventing the decrease in HDL-Cthat accompanies the removal of SFA (Schwingshackl and Hoffmann 2012). Thisfinding is supported by data to show that MUFA is relatively less effective instimulating cholesteryl ester transfer protein (CETP), and thus the remodelling andincreased clearance of HDL from serum, than either SFA or n-6 PUFA (Groeneret al. 1991; Lagrost et al. 1999). This finding is in accord with data from a recentstudy which concluded that dietary MUFA reduced the catabolic rate of theprincipal apolipoprotein (apo) in HDL, apo A-I (Labonte et al. 2013).1.3 n-3 Polyunsaturated Fatty AcidsThe principal essential fatty acid of the n-3 series, α-linolenic acid (18:2) is the mostabundant fatty acid on earth, but is consumed in significantly less quantity byhumans than linoleic acid (18:2 n-6) (National Diet and Nutrition Survey. Depart-ment of Health 2011). When fed in physiologically relevant amounts, α-linolenicacid has been shown to be equivalent to linoleic acid as a substitute for SFA inlowering LDL cholesterol (Harris 1997). However, even though a recent meta-analysis indicated a benefit of α-linolenic acid intake on CVD risk (Pan et al. 2012),572 J.C. Escolà-Gil et al.human interventions with α-linolenic acid-enriched diets have shown variableeffects on serum HDL-C (Harper et al. 2006; Goyens and Mensink 2006; Kaulet al. 2008; Griffin et al. 2006). The longer-chain derivatives of α-linolenic acid,chiefly eicosapentaenoic and docosahexaenoic acids (EPA, DHA), which inhumans are mainly obtained directly from oily fish, exert only a moderate elevatingeffect on HDL-C (Harris 1989). This is perhaps surprising given the potentTAG-lowering effect of these long-chain fatty acids.1.4 Carbohydrate and Extrinsic SugarsThe replacement of dietary fat with carbohydrate in low-fat, high-carbohydratediets has long been associated with a reduction in serum HDL-C that may be linkedto the carbohydrate-induced increase in TAG (Katan et al. 1997). The latter isknown to promote lipid exchanges between HDL- and TAG-rich lipoproteins thatremodel HDL into smaller and denser particles with an increased catabolic rate andthus reduced residence time in serum. Adverse effects of carbohydrate on HDL-C inthe longer term may also be mediated through increased body weight and theaccumulation of body fat (Stanhope et al. 2013). Whilst the reduction in HDL-Chas been attributed to diets with a high glycaemic index (Frost et al. 1999), there isnow little doubt that the extrinsic sugars, sucrose and fructose make a majorcontribution to this effect (Lustig 2010). The findings of a recent meta-analysiswhich concluded that a very high intake of fructose (>100 g/d) increases serumLDL cholesterol, but has no significant effects on HDL-C, are somewhat surprisingin both respects (Zhang et al. 2013) and in contrast to the outcome of dietaryinterventions with beverages sweetened with fructose. One example of the lattershowed marked increases in cardiometabolic risk factors, including significantreductions in serum HDL-C, relative to glucose-sweetened beverages (Stanhopeet al. 2009). A vital question to be answered is whether populations are consumingamounts of extrinsic sugars which are sufficient to elicit these adverse changes inHDL in the long term. Dietary intakes in the United Kingdom (National Diet andNutrition Survey. Department of Health 2011) indicate that the upper 2.5th percen-tile of the population may be approaching intakes of extrinsic sugars which have thepotential to induce adverse effects on metabolism and increase body weight. Theoverconsumption of sweetened beverages in adolescents is of particular concern inpromoting premature obesity and lowering of HDL-C, as shown in a recentAustralian study (Ambrosini et al. 2013).1.5 Effects of Dietary Fatty Acids and Cholesterol on HDLFunctionHDL-C is a surrogate marker of HDL particle size and number and may conveylittle or no information about the anti-atherosclerotic properties of HDL in theprocess of reverse cholesterol. There is evidence to suggest that paradoxicalHDL and Lifestyle Interventions 573increase in HDL-C induced by dietary SFA and cholesterol in mice (Escola-Gilet al. 2011) and in egg-fed humans (Andersen et al. 2013) results in a beneficialincrease in cholesterol efflux capacity. Beneficial effects of dietary fatty acids oncholesterol efflux capacity have also been described for EPA and DHA supplemen-tation in hamsters (Kasbi Chadli et al. 2013) and MUFA-rich extra-virgin olive oilconsumption in humans (Helal et al. 2013). Conversely, several intervention studiesin humans showed no effect on cholesterol efflux capacity of replacing of SFA witheither PUFA (Kralova Lesna et al. 2008) or carbohydrate (De Vries et al. 2005), ordifferences between diets enriched with trans fatty acids (8.3 % energy), SFA(13.2 % energy) and PUFA (14.6 %) elicited by either total plasma HDL or HDLsubfractions (Buonacorso et al. 2007).In conclusion, evidence from meta-analyses to support the relative effects ofdietary fatty acids on HDL-C may be statistically incontrovertible, but they do notnecessarily translate directly to the effects of complex foods and diets on HDL-Cand CVD risk. Reduction in HDL-C induced by the overconsumption of dietaryextrinsic sugars in sugar-sweetened beverages may have major implications forcardiometabolic health, especially in adolescents. Finally, evidence for the effectsof dietary components on the anti-atherogenic, functional properties of HDL isinconclusive and warrants further study.2 Weight LossObesity, defined as an excess of body fat, is a major health problem in Westernsocieties (Tchernof and Despres 2013; Bays et al. 2013). Excess body fat is also anindependent risk factor for CVD (Tchernof and Despres 2013). Excessive adiposetissue accumulation, but more importantly its distribution in the body, particularlyvisceral obesity, has been identified as a main correlate of cardiometabolicdisorders including not only alterations in glucose tolerance and insulin sensitivitybut also in blood lipids, including HDL-C.2.1 Impact of Adiposity on HDL Concentration and FunctionAbnormally low plasma HDL-C concentrations accompany hypertriacylgly-cerolaemia and are a common trait in many obese subjects that contributes to thedevelopment of atherosclerosis (Bays et al. 2013; Tchernof and Despres 2013). Inobesity, the low plasma HDL-C concentration has been mainly attributed toincreased HDL clearance, in part, due to enhanced uptake of HDL by adipocytes(Wang and Peng 2011; Rashid and Genest 2007). Plasma concentrations of largerHDL particles are decreased in patients with high cardiovascular risk (Pirilloet al. 2013) and, similarly, appear to be low in overweight and obese subjects(Tian et al. 2006, 2011). Moreover, a switch towards a higher proportion of smallerHDL particles in relation to larger HDL has been reported in the plasma of obesesubjects with ectopic fat and dysfunctional adipose tissue (Tchernof and Despres574 J.C. Escolà-Gil et al.2013). Obesity is commonly associated with increased oxidative stress (Wang andPeng 2011), and, in this context, there may be impairment of HDL functions,including reverse cholesterol transport and protection against lipoprotein oxidation.In this regard, the ability of HDL to mediate cholesterol efflux from cells, which isconsidered the first step of reverse cholesterol transport (RCT) (Escola-Gilet al. 2009), is reduced in the obese (Sasahara et al. 1998; Vazquez et al. 2012).In addition, although the protection against LDL oxidation conferred by HDL hasnot been assessed to date, the antioxidant action of apoA-I has been reported to beattenuated in scenarios of increased oxidative stress (Kontush and Chapman 2010),such as that present in obesity (Wang and Peng 2011). Other HDL-associatedproteins such as paraoxonase 1 (PON1) and lipoprotein-associated phospholipaseA2 (Lp-PLA2), which are also main contributors to the antioxidant properties ofHDL (Kontush and Chapman 2010), have been found to be altered in obesity (Sereset al. 2010; da Silva et al. 2013).2.2 Effect of Weight-Loss Therapies on HDL Concentrationand FunctionWeight loss has been widely thought to be an effective means of achieving asubstantial reduction in cardiometabolic risk (Vest et al. 2012). Obesity-associatedmetabolic disorders can, in theory, be treated by lifestyle modifications includingreduced energy intake and/or increased regular, moderate physical activity (Sackset al. 2009; Bays et al. 2013). A further strategy to achieve excess weight loss inseverely obese patients is bariatric surgery (Bays et al. 2013). In particular, theRoux-en-Y gastric bypass (RYGBP) has been reported to induce a sustained weightreduction without relapse in morbidly obese patients. In contrast, the metabolicresults of lifestyle interventions are variable and insufficient to achieve sustainedweight loss (Vest et al. 2012; Bays et al. 2013), whilst surgery has been shown tostably improve the lipid profile in most randomised, prospective trials (Bayset al. 2013; Vest et al. 2012). However, in both approaches, only modest effectson plasma HDL-C concentrations were reported in most recent systematic reviewsand meta-analyses of stabilised weight-loss clinical trials which examined theeffects of different dietary (Bays et al. 2013; Chapman et al. 2011; Dattilo andKris-Etherton 1992; Hu et al. 2012; Sacks et al. 2009; Schwingshackl andHoffmann 2013; Singh et al. 2007; Poobalan et al. 2004) or bariatric surgeryprogrammes (Bays et al. 2013; Vest et al. 2012; Poobalan et al. 2004). There arefew reports directly assessing the effect of weight reduction on HDL remodellingand its relationship, if any, with its anti-atherogenic properties. In this regard, recentstudies showed that weight loss exerts beneficial changes in HDL subpopulations inthe obese (Aron-Wisnewsky et al. 2011; Asztalos et al. 2010), leading to a switchtowards predominantly larger HDL particles and increased circulating plasma HDLmass despite no significant impact on plasma HDL-C concentrations. Interestingly,in one of these studies, an elevation in HDL mass was strongly associated with anincreased capacity of sera from postoperative patients to mediate cholesterol effluxHDL and Lifestyle Interventions 575via adenosine triphosphate-binding cassette transporter (ABC) G1 and scavengerreceptor class-BI (SR-BI) pathways, relative to baseline values (Aron-Wisnewskyet al. 2011), thus suggesting that the removal of excess cholesterol from cells byHDL particles might be enhanced after surgery. Excess weight loss also improvesoxidative stress (Uzun et al. 2004; Gletsu-Miller et al. 2009; Rector et al. 2007).However, whilst the impact of weight loss on HDL’s antioxidant properties inseverely obese patients undergoing bariatric surgery has been reported in observa-tional studies, it has not yet been examined in detail (Gletsu-Miller et al. 2009). Forinstance, increased PON1 activity levels have been found in serum of patientsundergoing gastric surgery (Uzun et al. 2004), whereas the Lp-PLA2 mass concen-tration did not change in another study (Hanusch-Enserer et al. 2009). Given thatthe distribution of Lp-PLA2 activity between serum lipoproteins is still uncertain,and evidence to suggest that Lp-PLA2 may be anti-atherogenic when associatedwith HDL (Kontush and Chapman 2010), it is reasonable to speculate that theincrease in plasma concentration of larger HDL particles following weight lossmight lead to a proportional increase in anti-atherogenic HDL-associated Lp-PLA2.Taken together, HDL changes may, in part, account for the significant improve-ment in the cardiovascular risk observed in obese patients after weight reduction.Thus, in summary, weight loss has long-term beneficial effects on plasma lipids inthe obese. Regarding the plasma concentration of HDL-C, the impact of lifestyleinterventions appears to be variable and modest in response to different weight-losstreatments. However, data on the effect of weight-reducing lifestyle interventionson the functional properties of HDL are scant. Bariatric surgery is becoming afrequent approach for reducing adiposity and improving the lipid profile in theseverely obese. The excess weight loss following intervention may provide aninteresting scenario for assessing the potential favourable changes in HDLremodelling and functionality.3 Effects of Regular Aerobic ExerciseSedentary behaviour is a major health problem that is associated with increasedcardiometabolic risk. Regular aerobic physical activity results in improved exerciseperformance which depends on an increased ability to utilise oxygen to deriveenergy for work. These effects usually require exercise intensities ranging from40 to 85 % of maximal oxygen consumption (VO2 max) or heart rate reserve, withhigher exercise intensities being necessary for higher levels of initial fitness, andvice versa (Chapman et al. 2011).3.1 Effects of Exercise on HDLRegular aerobic physical activity has a positive effect on many of the establishedrisk factors for CVD by causing a long-lasting reduction in TAGs by up to 20 % andincreasing HDL-C by up to 10 %. It also increases the size of LDL and HDLparticles. All these changes are considered potentially protective against576 J.C. Escolà-Gil et al.atherosclerosis (Kraus et al. 2002; Tall 2002). Given the close interrelation betweenplasma TAG-rich particles and HDL, changes in HDL-C after regular aerobicexercise are probably dependent on an increased HDL synthesis induced by raisedTAG lipolysis (Tall 2002; Olchawa et al. 2004) and, perhaps, also on a reducedHDL catabolism secondary to decreased hepatic lipase (HL) activity (Bergeronet al. 2001).The effect of physical activity on TAG levels varies depending on baselineTAGs and exercise characteristics such as intensity, caloric expenditure and dura-tion (Chapman et al. 2011; Miller et al. 2011; Kraus et al. 2002; Kodamaet al. 2007). The reduction in plasma TAGs of 20 % induced by daily exercisewas similar to that obtained by caloric restriction in nonobese subjects (Fontanaet al. 2007). Daily exercise blocked, at least in the short term, the increase in TAGsinduced by a high-carbohydrate diet in healthy postmenopausal women (Koutsariet al. 2001). Even with a variable adherence to diet, exercise did have a significanteffect on plasma TAG and HDL-C levels (Huffman et al. 2012). In healthy,non-smoking men, the increase in HDL-C was dependent on exercise intensity(for instance, miles run per week) (Kokkinos et al. 1995; Tambalis et al. 2009;Duncan et al. 2005), thus suggesting a dose–response relationship. Between 1,200and 2,200 Kcal of energy expenditure per week (15–20 miles/week of brisk walkingor jogging) has been estimated to be the threshold for obtaining a significant changein plasma TAGs and HDL-C (Durstine et al. 2002). The TAG decrease was greaterin individuals with fasting TAGs >1.69 mmol/L than in those with TAGs<1.69 mmol/L (Couillard et al. 2001). HDL-C increased, particularly in menwith TAGs >1.69 mmol/L and low HDL-C, compared with those with lowHDL-C and TAGs <1.69 mmol/L, and was associated with a reduction in abdomi-nal adipose tissue (Couillard et al. 2001). This improvement may have beenassociated with decreased insulin resistance, adiposity and TAG levels, at least inpart, through increased fatty acid oxidation by the muscle under the control of Rev-erb-alpha (Tall 2002; Woldt et al. 2013). Whilst data regarding the changes in HDLfunctionality induced by regular aerobic training are to date scant and inconsistent,there is evidence to implicate beneficial changes in some functions of HDL (Iborraet al. 2008; Kazeminasab et al. 2013; Meissner et al. 2010, 2011).3.2 Effects of Exercise on Prevention and Treatmentof Cardiometabolic RiskObviously, exercise exerts many different effects at a molecular level, rendering itvery difficult to attribute the final effects on disease prevention or treatment to oneeffect, including HDL-C.Several prospective cohort trials have shown a reduction in the risk of type2 diabetes in individuals participating in physical activity of moderate intensitycompared with being sedentary (Wing 2010; Uusitupa et al. 2009). This differencewas also found in people who walked regularly (typically >2.5 h of brisk walking)(reviewed by Jeon et al. 2007).HDL and Lifestyle Interventions 577Regular aerobic exercise is widely considered an important tool for primary andsecondary cardiovascular prevention since it is thought to reduce the risk of fataland nonfatal events in the general population (Chapman et al. 2011; Heranet al. 2011). Although the volume of moderate-intensity physical activity able toprovide a reduction in cardiovascular mortality was initially considered to rangefrom 2.5 to 5 h/week, similar results appear to be obtainable with 1–1.5 h/week ofvigorous-intensity or a combination of vigorous- and moderate-intensity exercises(Wen et al. 2011; Sattelmair et al. 2011). However, a systematic review of exercise-based rehabilitation found a decrease in total and cardiovascular mortality but nodifference in the number of new myocardial infarctions (Heran et al. 2011). Further,the Look AHEAD Trial which had, as a goal, to achieve and maintain a weight lossof at least 7 % by both reducing caloric intake and increasing physical activity intype 2 diabetic patients found no reduction in cardiovascular events in a 10-yearfollow-up (Wing et al. 2013). Similar negative results were obtained by the FinnishDiabetes Prevention Study after a 10-year lifestyle intervention (diet + exercise) inmiddle-aged, overweight people with impaired glucose tolerance (Uusitupaet al. 2009). To interpret these discrepant results, one has to consider differencesin patient disease (type 2 diabetes versus established coronary artery disease) versusthe general population, follow-up time and patient intervention type(s) (Sattelmairet al. 2011; Wen et al. 2011; Heran et al. 2011; Uusitupa et al. 2009).Exercise improved the components of the metabolic syndrome in affectedpatients (Pattyn et al. 2013). In patients with established type 2 diabetes mellitusincluded in the Look AHEAD Trial, increased physical activity had beneficialeffects on glycaemic control and cardiovascular risk factors, including HDL-Cand TAGs (Wing 2010). However, in other studies, aerobic and/or resistancetraining was shown to improve glycaemic control, but did not change HDL-C orTAGs in adults with type 2 diabetes (Sigal et al. 2007). There were majordifferences in the number of patients and follow-up time between studies thatshould be considered when interpreting these different results (Wing 2010; Sigalet al. 2007). Furthermore, one study included a hypo-caloric diet together withexercise (Wing 2010), whereas the other only included exercise (Sigal et al. 2007).In summary, there appears to be a consensus that regular aerobic exercise raisesHDL-C in a way closely related to plasma TAG reduction. Regular aerobic exercisemay prevent type 2 diabetes and CVD, probably with more intensity in primary thanin secondary prevention. In particular, aerobic exercise does not seem to preventCVD in patients with established type 2 diabetes. The role of exercise-inducedchanges in HDL in these outcomes is largely unknown.4 Smoking CessationSmoking is a well-documented risk factor for CVD that is amenable to intervention.A potential mechanism for the atherogenic effect of cigarette smoke is via plasmaHDL. Most research to date has looked at smoking effects on HDL-C concentrationrather than HDL structure or function. One issue that is particularly difficult toaddress in observational smoking research is the presence of confounding lifestyle578 J.C. Escolà-Gil et al.behaviours that may cluster with smoking behaviour (such as low physical activity,poor diet and increased alcohol consumption). Thus, comparison of the propertiesof HDL isolated from smokers and non-smokers does not allow direct causalrelationships to be determined, and there is a need for more direct experimentalapproaches to dissect the biological effects of smoking and to identify specificmediating factors (i.e. the combustible products of tobacco such as nicotine, carbonmonoxide and other gaseous products and free radicals). To further complicate theissue, smoking has effects on TAG metabolism, via increased sympathetic drive,insulin resistance or both, leading to increased plasma TAG concentrations andconsequent remodelling effects of plasma HDL resulting in higher plasma turnover[reviewed in Freeman and Packard (1995)].4.1 Smoking and HDL-C ConcentrationAmeta-analysis carried out almost 25 years ago indicated that smoking has a strongindependent effect on plasma HDL-C levels with smokers having on average 6 %lower HDL-C concentrations compared to non-smokers (Craig et al. 1989). A morerecent meta-analysis, which compared within individual differences before andafter stopping smoking, indicated that the absolute HDL-C concentration differencewas between 0.06 and 0.11 mmol/L (Forey et al. 2013). There also appears to be alarger smoking effect in women than men (Freeman and Packard 1995). HDL-Cconcentrations rise after stopping smoking and fall on restarting, and the magnitudeof the effect on HDL-C is related to the number of cigarettes smoked (Fortmannet al. 1986; Moffatt et al. 1995; Stubbe et al. 1982; Tuomilehto et al. 1986). Thereare no long-lasting effects of smoking on HDL-C concentration after cessation(Forey et al. 2013), and there is no association between number of years stoppedand plasma HDL-C (Wilson et al. 1983). Passive smoking is also associated withlower plasma HDL (Moffatt et al. 1995; Neufeld et al. 1997), and a reduction inHDL-C has been observed acutely only 6 h after exposure to environmental tobaccosmoke (Moffatt et al. 2004). The reduction in plasma HDL-C levels appears to belimited to a 0.15 mmol/L reduction in the larger HDL2 particles, independent ofconfounders, as determined by analytical ultracentrifugation (Freeman et al. 1993).This has been confirmed by others (Moriguchi et al. 1991; Shennan et al. 1985;Moffatt et al. 2004), although one study attributed the acute effects of smoking tothe smaller HDL3 fraction (Gnasso et al. 1984). Plasma apoA-I levels are also 6 %lower in smokers (Craig et al. 1989), and in some studies apoA-II has also beenshown to be reduced in smokers (Berg et al. 1979; Haffner et al. 1985) andex-smokers (Richard et al. 1997).4.2 Potential Mechanisms for the Smoking-RelatedReduction in HDL-CBecause of the intrinsic link between plasma TAG and HDL metabolism,demonstrated by their negative association in populations, and the biological effectsof smoking on plasma TAG metabolism (Freeman and Packard 1995), it is likelyHDL and Lifestyle Interventions 579that changes in plasma HDL-C concentration could be an indirect effect ofsmoking-related increases in plasma TAG concentration. Indeed, much of the effectof smoking on plasma HDL is lost after statistical correction for changes in plasmaTAGs (Freeman et al. 1993; Phillips et al. 1981). Cigarette smoking affects theactivities of plasma enzymes involved in regulating HDL size and turnover:lecithin–cholesterol acyltransferase (LCAT) (Freeman et al. 1998; Haffneret al. 1985) and lipoprotein lipase (LPL) (Freeman et al. 1998; Elkeleset al. 1983) activities are reduced, whilst HL (Moriguchi et al. 1991) and CETP(Dullaart et al. 1994) activities are increased. These changes result in a shift in thesize distribution of HDL into smaller particles which have increased clearance fromthe plasma compartment (Brinton et al. 1994). However, there is a residual effect ofsmoking after correction for changes in plasma TAG, suggesting TAG-independenteffects of smoking on plasma HDL concentration also exist. TheseTAG-independent effects appear to be more important in men than women (Free-man et al. 1993).There is evidence for structural/compositional changes in HDL brought about bysmoking. Ex vivo experiments, in which human plasma was acutely exposed tocigarette smoke, resulted in cross-linking of apoA-I and apoA-II (McCallet al. 1994) that may impair activation of LCAT. Similarly, chemically cross-linked HDL has an increased clearance in rodents (Senault et al. 1990). Smokingis associated with a reduction in the HDL content of Lp-PLA2, an enzyme thoughtto play an anti-atherogenic role in HDL (Tselepis et al. 2009). Early studies showedthat chronic inhalation of cigarette smoke in pigeons inhibits liver HDL uptake(Mulligan et al. 1983). Recent data have shown that a by-product of cigarettesmoking, benzo(a)pyrene, inhibited apoA-I synthesis in HepG2 cells, via activationof the aryl hydrocarbon nuclear steroid receptor, whilst nicotine had no effect(Naem et al. 2012). Modelling of the monocyte transcriptome in smokers comparedto non-smokers identified SLC39A8 to be on a causal pathway between smokingand plaque formation (Verdugo et al. 2013). This gene is known to be associatedwith the cellular uptake of cadmium from tobacco and was negatively associatedwith HDL cholesterol levels in this study.There are very few studies on the effects of smoking on HDL function. HDLisolated from smokers showed reduced ability to induce cholesterol efflux frommacrophages, possibly via apoA-I-mediated effects (Kralova Lesna et al. 2012). Exvivo cigarette smoke treated HDL, which resulted in an increased conjugated dieneand denatured apoA-I content, reduced the efflux capacity of HDL to a level similarto that of copper-oxidised HDL (Ueyama et al. 1998). Co-incubation with superox-ide dismutase prevented approximately half of the impairment and reduced thelevel of conjugated dienes, but not the apoA-I denaturation. There is also someevidence that plasma thiocyanates found in high levels in smokers could cause HDLoxidation and reduced apoA-I cholesterol efflux ability (Hadfield et al. 2013).Smoking is associated with reduced activity and concentration of PON1, anHDL-associated antioxidant enzyme, effects which are reversed after smokingcessation (James et al. 2000). The mechanistic data for the effects of smoking onHDL function are far from comprehensive and merit further investigation.580 J.C. Escolà-Gil et al.4.3 Smoking Cessation Intervention to Increase Plasma HDLConcentrationThe majority of smoking cessation studies are small, and most, but not all, havebeen shown to result in a rise in HDL concentration. A meta-analysis of 27 studiesincorporating over 6,000 subjects indicated that HDL-C increased by 0.10 mmol/Lafter smoking cessation, whilst plasma total cholesterol, LDL-C and TAG did notchange (Maeda et al. 2003).A number of lifestyle changes may also occur when smokers cease smoking, andat least one study has shown that the rise in HDL after smoking cessation is notindependent of the change in diet (Quensel et al. 1989b). However, refuting this, arecent large randomised, double-blind controlled trial was carried out in over 1,500smokers smoking an average of 21 cigarettes per day (Gepner et al. 2011). Patientswere randomised to one of six treatments: nicotine lozenge, nicotine patch,sustained-release bupropion, nicotine patch plus nicotine lozenge, sustained-releasebupropion plus nicotine lozenge, or placebo. Of the 923 participants who returnedafter 1 year intervention, 36 % who had stopped smoking showed a significantlygreater rise in HDL-C than those who did not, despite gaining an average of 4 kgmore weight than those who continued to smoke. The effects of smoking cessationin this study were particularly evident in women.Transdermal nicotine replacement therapy is a commonly used approach tosupport smoking cessation. A small study was carried out to compare the effectsof transdermal nicotine patches, used as part of a smoking cessation intervention, onplasma HDL-C levels (Moffatt et al. 2000). Subjects who used transdermal nicotinepatches over 35 days showed no improvement in HDL-C concentration. However,if the use of patches was then stopped, HDL-C concentrations rose to normal,non-smoking levels over the next 42 days. Others have observed contrasting effectsin a larger study where smoking cessation, accompanied by the use of higher-dosenicotine transdermal patches, resulted in an immediate increase in HDL, whereas incontrast, low-dose nicotine did not (Allen et al. 1994). These latter findings aresomewhat contradictory to data indicating that nicotine administration tonon-smokers, via chewing gum, does not affect HDL-C levels (Quenselet al. 1989a). The potential effects of nicotine patches during smoking cessationon plasma HDL have yet to be clarified.In summary, smoking leads to a reduction in plasma HDL-C, HDL2-C, apo A-Iand probably apoA-II concentrations. The effects of smoking on HDL are dosedependent and reversed upon smoking cessation. Much of the effect of smokingmay be TAG dependent where increased plasma TAG concentration leads toremodelling of HDL to a smaller particle size which is more rapidly cleared fromthe circulation. TAG-independent effects have not yet been thoroughly investigatedbut include modification of apoA-I, reductions in HDL antioxidant enzyme activityand reduced ability of HDL to promote cholesterol efflux. The effect of nicotineaids, used to support smoking cessation interventions, on HDL-C concentration andfunction is unclear.HDL and Lifestyle Interventions 5815 Effects of Alcohol on HDL and Cardiovascular RiskMultiple studies have established a known J-shaped relationship between alcoholintake and cardiovascular risk including coronary heart disease and ischaemicstroke (Krenz and Korthuis 2012). The term ‘drink’ is imprecise; however, theamount of alcohol in one drink is similar for wine, spirits or beer. Thus, a 100 mlglass of table wine at 13 % alcohol, 35 ml of distilled spirits at 40 % alcohol and300 ml of beer at 5 % alcohol all contain around 10–12 g of pure ethyl alcohol. Thegeneral consensus is that men consuming two standard alcoholic drinks per day andwomen consuming half that intake appear to have a lower cardiovascular event ratethan persons abstaining from alcohol (Krenz and Korthuis 2012). Estimates of therisk reduction associated with moderate alcohol intake drinkers compared withthose abstaining from alcohol range from 25 to 30 % (Krenz and Korthuis 2012).5.1 Alcohol Intake and HDL-CSeveral plausible mechanisms have been proposed to explain the positive moderatealcohol intake-mediated effects on CVDs: reduced thrombogenic and coagulationfactors (platelet aggregation and fibrinogen levels), low plasma concentrations ofinflammation markers (C-reactive protein, interleukin-6 and adiponectin), loweredblood pressure, increased insulin sensitivity and lipoprotein profile via a loweringof LDL-C and increase in HDL-C (Klatsky 2010). However, this alcohol–CVDrelationship is nonlinear, and excessive alcohol consumption has adverse effects onhypertension and other pro-inflammatory factors, despite increasing HDL-C(Foerster et al. 2009).Some reports have suggested that wine carries a lower risk of mortality than beeror spirits and that this may be related to the ability of nonalcoholic phenoliccompounds to inhibit LDL oxidation and its pro-inflammatory effects (Frankelet al. 1993; Gronbaek et al. 2000; Klatsky et al. 2003). However, other studieshave failed to identify an additional advantage associated with red wine(Di Castelnuovo et al. 2002; Mukamal et al. 2003), thereby suggesting that ethylalcohol is the main factor for the cardioprotective effect.A recent meta-analysis demonstrated that alcohol exerts favourable effects onseveral cardiovascular biomarkers (higher HDL-C, apoA-I and adiponectin levelsand lower fibrinogen levels) which seems to be independent of the alcoholicbeverage type (Brien et al. 2011). This systematic review found that 1–2 drinksper day increased HDL-C by 0.10 mmol/L (Brien et al. 2011). However, thecontribution of alcohol-induced HDL-C-raising effects to cardiovascular riskremains largely unknown. Early studies indicated that the atheroprotective effectswere mediated, largely, by the increase in HDL-C, since the addition of HDL-C tothe multivariate model attenuated the inverse association between alcohol intakeand myocardial infarction (Gaziano et al. 1993). Nevertheless, one recent reportdescribing a large Norwegian cohort with extensive control for confounding factorsshowed that HDL-C levels were not on the causal pathway connecting alcohol to582 J.C. Escolà-Gil et al.the lower risk of death from coronary heart disease (Magnus et al. 2011). Thesefindings raise the question of which specific HDL subpopulation particles areaffected by alcohol intake and their significance for coronary heart diseaseprotection.5.2 Effects of Alcohol on HDL Atheroprotective FunctionsStudies on the effect of alcohol consumption on HDL particle size are inconsistent.In some studies, moderate alcohol consumption increased small HDL subfractions,mainly HDL3 (Haskell et al. 1984; Gardner et al. 2000; Nishiwaki et al. 1994), andlipid-poor preβ-HDL particles (Beulens et al. 2004). These effects on HDL sizecould be related to the ability of alcohol to stimulate LPL activity (Nishiwakiet al. 1994). However, other studies on moderate drinkers found increasedconcentrations of both HDL2 and HDL3 (Gaziano et al. 1993; Clevidenceet al. 1995; De Oliveira et al. 2000), and human kinetic studies demonstrated thatmoderate alcohol consumption resulted in dose-dependent increases in HDL-C,apoA-I and apoA-II through an increase in the HDL apolipoprotein transport rate,but without altering their catabolic rate and LPL activity (De Oliveira et al. 2000).These changes might explain the ability of HDL from moderate alcohol drinkers toenhance macrophage cholesterol efflux (Beulens et al. 2004). However, cholesterolefflux was also enhanced in heavy drinkers who usually show larger HDL (Makelaet al. 2008); these changes were associated with decreased CETP activity andincreased phospholipid transfer protein (PLTP) activity (Makela et al. 2008). Itshould be noted that these macrophage cholesterol efflux assays only quantified thefirst-step RCT pathway without addressing the efficiency of the rest of the RCTsteps or the other HDL cardioprotective functions. Nevertheless, moderate alcoholintake was not associated with changes in the activities of other HDL remodellingfactors, such as LCAT or CETP (Nishiwaki et al. 1994; Riemens et al. 1997;Sierksma et al. 2004), and, furthermore, the ability of HDL to deliver cholesterolinto the liver cells seemed to be decreased in both moderate and heavy drinkers(Rao et al. 2000; Marmillot et al. 2007).Beyond the RCT pathway, moderate alcohol consumption increased serumPON1 (van der Gaag et al. 1999; Sierksma et al. 2002; Rao et al. 2003), alongwith liver PON1 mRNA levels (Rao et al. 2003), although this effect was not foundin one study with moderate red wine drinkers (Sarandol et al. 2003). In contrast,heavy alcohol drinking produced the opposite effects (Rao et al. 2003). Further-more, alcohol promotes the conversion of phosphatidylcholine into phosphatidy-lethanol, and it was reported that HDL-associated phosphatidylethanol increasedendothelial secretion of the vascular endothelial growth factor (Liisanantti andSavolainen 2009), which could partly explain the positive effect of alcohol onangiogenesis induction.In summary, the inverse relationship between moderate and regular alcoholintake and cardiovascular risk as well as the ability of alcohol to increase HDL-Cis well documented in the literature. Although some studies reported favourableHDL and Lifestyle Interventions 583effects of alcohol on HDL cardioprotective functions, it is unclear how many of thealcohol-mediated cardioprotective effects were mediated by the HDL increase.Finally, the American Heart Association recommendation that alcoholic beverageconsumption should be limited to no more than 2 drinks per day for men and 1 drinkper day for women and should be consumed with meals should be taken intoaccount. Alcohol should not be considered a therapeutic option for cardiovascularrisk reduction (Lichtenstein et al. 2006).Acknowledgements This work was partly funded by European Cooperation in Science andTechnology (COST) action BM0904, Ministerio de Sanidad y Consumo, Instituto de Salud CarlosIII, FIS 10-00277 and CP13-00070 (to J.J.), FIS 11-0176 (to F.V-B.) and FIS 12-00291 (to J.C.E-G). CIBER de Diabetes y Enfermedades Metabólicas Asociadas is an Instituto de Salud Carlos IIIProject.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAllen SS, Hatsukami D, Gorsline J (1994) Cholesterol changes in smoking cessation using thetransdermal nicotine system. Transdermal nicotine study group. Prev Med 23(2):190–196Ambrosini GL, Oddy WH, Huang RC, Mori TA, Beilin LJ, Jebb SA (2013) Prospectiveassociations between sugar-sweetened beverage intakes and cardiometabolic risk factors inadolescents. Am J Clin Nutr 98(2):327–334Andersen CJ, Blesso CN, Lee J, Barona J, Shah D, Thomas MJ, Fernandez ML (2013) Eggconsumption modulates HDL lipid composition and increases the cholesterol-accepting capac-ity of serum in metabolic syndrome. Lipids 48(6):557–567Aron-Wisnewsky J, Julia Z, Poitou C, Bouillot JL, Basdevant A, Chapman MJ, Clement K, GuerinM (2011) Effect of bariatric surgery-induced weight loss on SR-BI-, ABCG1-, and ABCA1-mediated cellular cholesterol efflux in obese women. J Clin Endocrinol Metab 96(4):1151–1159Astrup A, Dyerberg J, Elwood P, Hermansen K, Hu FB, Jakobsen MU, Kok FJ, Krauss RM, LecerfJM, LeGrand P, Nestel P, Riserus U, Sanders T, Sinclair A, Stender S, Tholstrup T, Willett WC(2011) The role of reducing intakes of saturated fat in the prevention of cardiovascular disease:where does the evidence stand in 2010? Am J Clin Nutr 93(4):684–688Asztalos BF, Swarbrick MM, Schaefer EJ, Dallal GE, Horvath KV, Ai M, Stanhope KL,Austrheim-Smith I, Wolfe BM, Ali M, Havel PJ (2010) Effects of weight loss, induced bygastric bypass surgery, on HDL remodeling in obese women. J Lipid Res 51(8):2405–2412Bays HE, Toth PP, Kris-Etherton PM, Abate N, Aronne LJ, Brown WV, Gonzalez-Campoy JM,Jones SR, Kumar R, La Forge R, Samuel VT (2013) Obesity, adiposity, and dyslipidemia: aconsensus statement from the National Lipid Association. J Clin Lipidol 7(4):304–383Belsey J, de Lusignan S, Chan T, van Vlymen J, Hague N (2008) Abnormal lipids in high-riskpatients achieving cholesterol targets: a cross-sectional study of routinely collected UK generalpractice data. Curr Med Res Opin 24(9):2551–2560Berg K, Borresen AL, Dahlen G (1979) Effect of smoking on serum levels of HDL apoproteins.Atherosclerosis 34(3):339–343Bergeron J, Couillard C, Despres JP, Gagnon J, Leon AS, Rao DC, Skinner JS, Wilmore JH,Bouchard C (2001) Race differences in the response of postheparin plasma lipoprotein lipase584 J.C. Escolà-Gil et al.and hepatic lipase activities to endurance exercise training in men: results from the HERI-TAGE Family Study. Atherosclerosis 159(2):399–406Beulens JW, Sierksma A, van Tol A, Fournier N, van Gent T, Paul JL, Hendriks HF (2004)Moderate alcohol consumption increases cholesterol efflux mediated by ABCA1. J Lipid Res45(9):1716–1723Brien SE, Ronksley PE, Turner BJ, Mukamal KJ, Ghali WA (2011) Effect of alcohol consumptionon biological markers associated with risk of coronary heart disease: systematic review andmeta-analysis of interventional studies. BMJ 342:d636Brinton EA, Eisenberg S, Breslow JL (1994) Human HDL cholesterol levels are determined byapoA-I fractional catabolic rate, which correlates inversely with estimates of HDL particlesize. Effects of gender, hepatic and lipoprotein lipases, triglyceride and insulin levels, and bodyfat distribution. Arterioscler Thromb 14(5):707–720Buonacorso V, Nakandakare ER, Nunes VS, Passarelli M, Quintao EC, Lottenberg AM (2007)Macrophage cholesterol efflux elicited by human total plasma and by HDL subfractions is notaffected by different types of dietary fatty acids. Am J Clin Nutr 86(5):1270–1277Chapman MJ, Ginsberg HN, Amarenco P, Andreotti F, Boren J, Catapano AL, Descamps OS,Fisher E, Kovanen PT, Kuivenhoven JA, Lesnik P, Masana L, Nordestgaard BG, Ray KK,Reiner Z, Taskinen MR, Tokgozoglu L, Tybjaerg-Hansen A, Watts GF (2011) Triglyceride-rich lipoproteins and high-density lipoprotein cholesterol in patients at high risk of cardiovas-cular disease: evidence and guidance for management. Eur Heart J 32(11):1345–1361Clevidence BA, Reichman ME, Judd JT, Muesing RA, Schatzkin A, Schaefer EJ, Li Z, Jenner J,Brown CC, Sunkin M et al (1995) Effects of alcohol consumption on lipoproteins ofpremenopausal women. A controlled diet study. Arterioscler Thromb Vasc Biol 15(2):179–184Couillard C, Despres JP, Lamarche B, Bergeron J, Gagnon J, Leon AS, Rao DC, Skinner JS,Wilmore JH, Bouchard C (2001) Effects of endurance exercise training on plasma HDLcholesterol levels depend on levels of triglycerides: evidence from men of the Health, RiskFactors, Exercise Training and Genetics (HERITAGE) Family Study. Arterioscler ThrombVasc Biol 21(7):1226–1232Craig WY, Palomaki GE, Haddow JE (1989) Cigarette smoking and serum lipid and lipoproteinconcentrations: an analysis of published data. BMJ 298(6676):784–788da Silva IT, Timm Ade S, Damasceno NR (2013) Influence of obesity and cardiometabolic makerson lipoprotein-associated phospholipase A2 (Lp-PLA2) activity in adolescents: the healthyyoung cross-sectional study. Lipids Health Dis 12:19Dattilo AM, Kris-Etherton PM (1992) Effects of weight reduction on blood lipids and lipoproteins:a meta-analysis. Am J Clin Nutr 56(2):320–328De Oliveira ESER, Foster D, McGee Harper M, Seidman CE, Smith JD, Breslow JL, Brinton EA(2000) Alcohol consumption raises HDL cholesterol levels by increasing the transport rate ofapolipoproteins A-I and A-II. Circulation 102(19):2347–2352De Vries R, Beusekamp BJ, Kerstens MN, Groen AK, Van Tol A, Dullaart RP (2005) Alow-saturated-fat, low-cholesterol diet decreases plasma CETP activity and pre beta-HDLformation but does not affect cellular cholesterol efflux to plasma from type 1 diabetic patients.Scand J Clin Lab Invest 65(8):729–737Di Castelnuovo A, Rotondo S, Iacoviello L, Donati MB, De Gaetano G (2002) Meta-analysis ofwine and beer consumption in relation to vascular risk. Circulation 105(24):2836–2844Dullaart RP, Hoogenberg K, Dikkeschei BD, van Tol A (1994) Higher plasma lipid transferprotein activities and unfavorable lipoprotein changes in cigarette-smoking men. ArteriosclerThromb 14(10):1581–1585Duncan GE, Anton SD, Sydeman SJ, Newton RL Jr, Corsica JA, Durning PE, Ketterson TU,Martin AD, Limacher MC, Perri MG (2005) Prescribing exercise at varied levels of intensityand frequency: a randomized trial. Arch Intern Med 165(20):2362–2369Durstine JL, Grandjean PW, Cox CA, Thompson PD (2002) Lipids, lipoproteins, and exercise. JCardiopulm Rehabil 22(6):385–398HDL and Lifestyle Interventions 585Elkeles RS, Khan SR, Chowdhury V, Swallow MB (1983) Effects of smoking on oral fat toleranceand high density lipoprotein cholesterol. Clin Sci (Lond) 65(6):669–672Escola-Gil JC, Rotllan N, Julve J, Blanco-Vaca F (2009) In vivo macrophage-specific RCT andantioxidant and antiinflammatory HDL activity measurements: new tools for predicting HDLatheroprotection. Atherosclerosis 206(2):321–327Escola-Gil JC, Llaverias G, Julve J, Jauhiainen M, Mendez-Gonzalez J, Blanco-Vaca F (2011) Thecholesterol content of Western diets plays a major role in the paradoxical increase in high-density lipoprotein cholesterol and upregulates the macrophage reverse cholesterol transportpathway. Arterioscler Thromb Vasc Biol 31(11):2493–2499Foerster M, Marques-Vidal P, Gmel G, Daeppen JB, Cornuz J, Hayoz D, Pecoud A, Mooser V,Waeber G, Vollenweider P, Paccaud F, Rodondi N (2009) Alcohol drinking and cardiovascularrisk in a population with high mean alcohol consumption. Am J Cardiol 103(3):361–368Fontana L, Villareal DT, Weiss EP, Racette SB, Steger-May K, Klein S, Holloszy JO (2007)Calorie restriction or exercise: effects on coronary heart disease risk factors. A randomized,controlled trial. Am J Physiol Endocrinol Metab 293(1):E197–E202Forey BA, Fry JS, Lee PN, Thornton AJ, Coombs KJ (2013) The effect of quitting smoking onHDL-cholesterol – a review based on within-subject changes. Biomark Res 1(1):26Fortmann SP, Haskell WL, Williams PT (1986) Changes in plasma high density lipoproteincholesterol after changes in cigarette use. Am J Epidemiol 124(4):706–710Frankel EN, Kanner J, German JB, Parks E, Kinsella JE (1993) Inhibition of oxidation of humanlow-density lipoprotein by phenolic substances in red wine. Lancet 341(8843):454–457Freeman DJ, Packard CJ (1995) Smoking and plasma lipoprotein metabolism. Clin Sci (Lond) 89(4):333–342Freeman DJ, Griffin BA, Murray E, Lindsay GM, Gaffney D, Packard CJ, Shepherd J (1993)Smoking and plasma lipoproteins in man: effects on low density lipoprotein cholesterol levelsand high density lipoprotein subfraction distribution. Eur J Clin Invest 23(10):630–640Freeman DJ, Caslake MJ, Griffin BA, Hinnie J, Tan CE, Watson TD, Packard CJ, Shepherd J(1998) The effect of smoking on post-heparin lipoprotein and hepatic lipase, cholesteryl estertransfer protein and lecithin:cholesterol acyl transferase activities in human plasma. Eur J ClinInvest 28(7):584–591Frost G, Leeds AA, Dore CJ, Madeiros S, Brading S, Dornhorst A (1999) Glycaemic index as adeterminant of serum HDL-cholesterol concentration. Lancet 353(9158):1045–1048Gardner CD, Tribble DL, Young DR, Ahn D, Fortmann SP (2000) Associations of HDL, HDL(2),and HDL(3) cholesterol and apolipoproteins A-I and B with lifestyle factors in healthy womenand men: the Stanford Five City Project. Prev Med 31(4):346–356Gaziano JM, Buring JE, Breslow JL, Goldhaber SZ, Rosner B, VanDenburgh M, Willett W,Hennekens CH (1993) Moderate alcohol intake, increased levels of high-density lipoproteinand its subfractions, and decreased risk of myocardial infarction. N Engl J Med 329(25):1829–1834Gepner AD, Piper ME, Johnson HM, Fiore MC, Baker TB, Stein JH (2011) Effects of smoking andsmoking cessation on lipids and lipoproteins: outcomes from a randomized clinical trial. AmHeart J 161(1):145–151Gletsu-Miller N, Hansen JM, Jones DP, Go YM, Torres WE, Ziegler TR, Lin E (2009) Loss oftotal and visceral adipose tissue mass predicts decreases in oxidative stress after weight-losssurgery. Obesity (Silver Spring) 17(3):439–446Gnasso A, Haberbosch W, Schettler G, Schmitz G, Augustin J (1984) Acute influence of smokingon plasma lipoproteins. Klin Wochenschr 62(Suppl 2):36–42Goyens PL, Mensink RP (2006) Effects of alpha-linolenic acid versus those of EPA/DHA oncardiovascular risk markers in healthy elderly subjects. Eur J Clin Nutr 60(8):978–984Griffin MD, Sanders TA, Davies IG, Morgan LM, Millward DJ, Lewis F, Slaughter S, Cooper JA,Miller GJ, Griffin BA (2006) Effects of altering the ratio of dietary n-6 to n-3 fatty acids oninsulin sensitivity, lipoprotein size, and postprandial lipemia in men and postmenopausalwomen aged 45–70 y: the OPTILIP Study. Am J Clin Nutr 84(6):1290–1298586 J.C. Escolà-Gil et al.Groener JE, van Ramshorst EM, Katan MB, Mensink RP, van Tol A (1991) Diet-inducedalteration in the activity of plasma lipid transfer protein in normolipidemic human subjects.Atherosclerosis 87(2–3):221–226Gronbaek M, Becker U, Johansen D, Gottschau A, Schnohr P, Hein HO, Jensen G, Sorensen TI(2000) Type of alcohol consumed and mortality from all causes, coronary heart disease, andcancer. Ann Intern Med 133(6):411–419Grundy SM, Denke MA (1990) Dietary influences on serum lipids and lipoproteins. J Lipid Res 31(7):1149–1172Hadfield KA, Pattison DI, Brown BE, Hou L, Rye KA, Davies MJ, Hawkins CL (2013)Myeloperoxidase-derived oxidants modify apolipoprotein A-I and generate dysfunctionalhigh-density lipoproteins: comparison of hypothiocyanous acid (HOSCN) with hypochlorousacid (HOCl). Biochem J 449(2):531–542Haffner SM, Applebaum-Bowden D, Wahl PW, Hoover JJ, Warnick GR, Albers JJ, Hazzard WR(1985) Epidemiological correlates of high density lipoprotein subfractions, apolipoproteinsA-I, A-II, and D, and lecithin cholesterol acyltransferase. Effects of smoking, alcohol, andadiposity. Arteriosclerosis 5(2):169–177Hanusch-Enserer U, Zorn G, Wojta J, Kopp CW, Prager R, Koenig W, Schillinger M, Roden M,Huber K (2009) Non-conventional markers of atherosclerosis before and after gastric bandingsurgery. Eur Heart J 30(12):1516–1524Harper CR, Edwards MC, Jacobson TA (2006) Flaxseed oil supplementation does not affectplasma lipoprotein concentration or particle size in human subjects. J Nutr 136(11):2844–2848Harris WS (1989) Fish oils and plasma lipid and lipoprotein metabolism in humans: a criticalreview. J Lipid Res 30(6):785–807Harris WS (1997) n-3 fatty acids and serum lipoproteins: human studies. Am J Clin Nutr 65(5 Suppl):1645S–1654SHaskell WL, Camargo C Jr, Williams PT, Vranizan KM, Krauss RM, Lindgren FT, Wood PD(1984) The effect of cessation and resumption of moderate alcohol intake on serum high-density-lipoprotein subfractions. A controlled study. N Engl J Med 310(13):805–810Helal O, Berrougui H, Loued S, Khalil A (2013) Extra-virgin olive oil consumption improves thecapacity of HDL to mediate cholesterol efflux and increases ABCA1 and ABCG1 expression inhuman macrophages. Br J Nutr 109(10):1844–1855Heran BS, Chen JM, Ebrahim S, Moxham T, Oldridge N, Rees K, Thompson DR, Taylor RS(2011) Exercise-based cardiac rehabilitation for coronary heart disease. Cochrane DatabaseSyst Rev 7, CD001800Hu T, Mills KT, Yao L, Demanelis K, Eloustaz M, Yancy WS Jr, Kelly TN, He J, Bazzano LA(2012) Effects of low-carbohydrate diets versus low-fat diets on metabolic risk factors: a meta-analysis of randomized controlled clinical trials. Am J Epidemiol 176(Suppl 7):S44–S54Huffman KM, Hawk VH, Henes ST, Ocampo CI, Orenduff MC, Slentz CA, Johnson JL, HoumardJA, Samsa GP, Kraus WE, Bales CW (2012) Exercise effects on lipids in persons with varyingdietary patterns-does diet matter if they exercise? Responses in Studies of a Targeted RiskReduction Intervention through Defined Exercise I. Am Heart J 164(1):117–124Iborra RT, Ribeiro IC, Neves MQ, Charf AM, Lottenberg SA, Negrao CE, Nakandakare ER,Passarelli M (2008) Aerobic exercise training improves the role of high-density lipoproteinantioxidant and reduces plasma lipid peroxidation in type 2 diabetes mellitus. Scand J Med SciSports 18(6):742–750James RW, Leviev I, Righetti A (2000) Smoking is associated with reduced serum paraoxonaseactivity and concentration in patients with coronary artery disease. Circulation 101(19):2252–2257Jeon CY, Lokken RP, Hu FB, van Dam RM (2007) Physical activity of moderate intensity and riskof type 2 diabetes: a systematic review. Diabetes Care 30(3):744–752Kasbi Chadli F, Nazih H, Krempf M, Nguyen P, Ouguerram K (2013) Omega 3 fatty acidspromote macrophage reverse cholesterol transport in hamster fed high fat diet. PLoS ONE 8(4):e61109HDL and Lifestyle Interventions 587Katan MB, Grundy SM, Willett WC (1997) Should a low-fat, high-carbohydrate diet berecommended for everyone? Beyond low-fat diets. N Engl J Med 337(8):563–566, discussion566-567Kaul N, Kreml R, Austria JA, Richard MN, Edel AL, Dibrov E, Hirono S, Zettler ME, Pierce GN(2008) A comparison of fish oil, flaxseed oil and hempseed oil supplementation on selectedparameters of cardiovascular health in healthy volunteers. J Am Coll Nutr 27(1):51–58Kazeminasab F, Marandi M, Ghaedi K, Esfarjani F, Moshtaghian J (2013) Endurance trainingenhances LXRalpha gene expression in Wistar male rats. Eur J Appl Physiol 113(9):2285–2290Keys A (1980a) Alpha lipoprotein (HDL) cholesterol in the serum and the risk of coronary heartdisease and death. Lancet 2(8195 pt 1):603–606Keys A (1980b) W. O. Atwater memorial lecture: overweight, obesity, coronary heart disease andmortality. Nutr Rev 38(9):297–307Klatsky AL (2010) Alcohol and cardiovascular health. Physiol Behav 100(1):76–81Klatsky AL, Friedman GD, Armstrong MA, Kipp H (2003) Wine, liquor, beer, and mortality. Am JEpidemiol 158(6):585–595Kodama S, Tanaka S, Saito K, Shu M, Sone Y, Onitake F, Suzuki E, Shimano H, Yamamoto S,Kondo K, Ohashi Y, Yamada N, Sone H (2007) Effect of aerobic exercise training on serumlevels of high-density lipoprotein cholesterol: a meta-analysis. Arch Intern Med 167(10):999–1008Kokkinos PF, Holland JC, Narayan P, Colleran JA, Dotson CO, Papademetriou V (1995) Miles runper week and high-density lipoprotein cholesterol levels in healthy, middle-aged men. A dose-response relationship. Arch Intern Med 155(4):415–420Kontush A, Chapman MJ (2010) Antiatherogenic function of HDL particle subpopulations: focuson antioxidative activities. Curr Opin Lipidol 21(4):312–318Koutsari C, Karpe F, Humphreys SM, Frayn KN, Hardman AE (2001) Exercise prevents theaccumulation of triglyceride-rich lipoproteins and their remnants seen when changing to ahigh-carbohydrate diet. Arterioscler Thromb Vasc Biol 21(9):1520–1525Kralova Lesna I, Suchanek P, Kovar J, Stavek P, Poledne R (2008) Replacement of dietarysaturated FAs by PUFAs in diet and reverse cholesterol transport. J Lipid Res 49(11):2414–2418Kralova Lesna I, Poledne R, Pagacova L, Stavek P, Pitha J (2012) HDL and apolipoprotein A1concentrations as markers of cholesterol efflux in middle-aged women: interaction withsmoking. Neuro Endocrinol Lett 33(Suppl 2):38–42Kraus WE, Houmard JA, Duscha BD, Knetzger KJ, Wharton MB, McCartney JS, Bales CW,Henes S, Samsa GP, Otvos JD, Kulkarni KR, Slentz CA (2002) Effects of the amount andintensity of exercise on plasma lipoproteins. N Engl J Med 347(19):1483–1492Krenz M, Korthuis RJ (2012) Moderate ethanol ingestion and cardiovascular protection: fromepidemiologic associations to cellular mechanisms. J Mol Cell Cardiol 52(1):93–104Labonte ME, Jenkins DJ, Lewis GF, Chiavaroli L, Wong JM, Kendall CW, Hogue JC, Couture P,Lamarche B (2013) Adding MUFA to a dietary portfolio of cholesterol-lowering foods reducesapoAI fractional catabolic rate in subjects with dyslipidaemia. Br J Nutr 110(3):426–436Lagrost L, Mensink RP, Guyard-Dangremont V, Temme EH, Desrumaux C, Athias A, Hornstra G,Gambert P (1999) Variations in serum cholesteryl ester transfer and phospholipid transferactivities in healthy women and men consuming diets enriched in lauric, palmitic or oleicacids. Atherosclerosis 142(2):395–402Lichtenstein AH, Appel LJ, Brands M, Carnethon M, Daniels S, Franch HA, Franklin B, Kris-Etherton P, Harris WS, Howard B, Karanja N, Lefevre M, Rudel L, Sacks F, Van Horn L,Winston M, Wylie-Rosett J (2006) Diet and lifestyle recommendations revision 2006: ascientific statement from the American Heart Association Nutrition Committee. Circulation114(1):82–96Liisanantti MK, Savolainen MJ (2009) Phosphatidylethanol mediates its effects on the vascularendothelial growth factor via HDL receptor in endothelial cells. Alcohol Clin Exp Res 33(2):283–288588 J.C. Escolà-Gil et al.Lustig RH (2010) Fructose: metabolic, hedonic, and societal parallels with ethanol. J Am DietAssoc 110(9):1307–1321Maeda K, Noguchi Y, Fukui T (2003) The effects of cessation from cigarette smoking on the lipidand lipoprotein profiles: a meta-analysis. Prev Med 37(4):283–290Magnus P, Bakke E, Hoff DA, Hoiseth G, Graff-Iversen S, Knudsen GP, Myhre R, Normann PT,Naess O, Tambs K, Thelle DS, Morland J (2011) Controlling for high-density lipoproteincholesterol does not affect the magnitude of the relationship between alcohol and coronaryheart disease. Circulation 124(21):2296–2302Makela SM, Jauhiainen M, Ala-Korpela M, Metso J, Lehto TM, Savolainen MJ, Hannuksela ML(2008) HDL2 of heavy alcohol drinkers enhances cholesterol efflux from raw macrophages viaphospholipid-rich HDL 2b particles. Alcohol Clin Exp Res 32(6):991–1000Marmillot P, Munoz J, Patel S, Garige M, Rosse RB, Lakshman MR (2007) Long-term ethanolconsumption impairs reverse cholesterol transport function of high-density lipoproteins bydepleting high-density lipoprotein sphingomyelin both in rats and in humans. Metabolism 56(7):947–953McCall MR, van den Berg JJ, Kuypers FA, Tribble DL, Krauss RM, Knoff LJ, Forte TM (1994)Modification of LCAT activity and HDL structure. New links between cigarette smoke andcoronary heart disease risk. Arterioscler Thromb 14(2):248–253Meissner M, Nijstad N, Kuipers F, Tietge UJ (2010) Voluntary exercise increases cholesterolefflux but not macrophage reverse cholesterol transport in vivo in mice. Nutr Metab (Lond)7:54Meissner M, Lombardo E, Havinga R, Tietge UJ, Kuipers F, Groen AK (2011) Voluntary wheelrunning increases bile acid as well as cholesterol excretion and decreases atherosclerosis inhypercholesterolemic mice. Atherosclerosis 218(2):323–329Mensink RP, Zock PL, Kester AD, Katan MB (2003) Effects of dietary fatty acids andcarbohydrates on the ratio of serum total to HDL cholesterol and on serum lipids andapolipoproteins: a meta-analysis of 60 controlled trials. Am J Clin Nutr 77(5):1146–1155Micha R, Mozaffarian D (2010) Saturated fat and cardiometabolic risk factors, coronary heartdisease, stroke, and diabetes: a fresh look at the evidence. Lipids 45(10):893–905Miller M, Stone NJ, Ballantyne C, Bittner V, Criqui MH, Ginsberg HN, Goldberg AC, HowardWJ, Jacobson MS, Kris-Etherton PM, Lennie TA, Levi M, Mazzone T, Pennathur S (2011)Triglycerides and cardiovascular disease: a scientific statement from the American HeartAssociation. Circulation 123(20):2292–2333Moffatt RJ, Stamford BA, Biggerstaff KD (1995) Influence of worksite environmental tobaccosmoke on serum lipoprotein profiles of female nonsmokers. Metabolism 44(12):1536–1539Moffatt RJ, Biggerstaff KD, Stamford BA (2000) Effects of the transdermal nicotine patch onnormalization of HDL-C and its subfractions. Prev Med 31(2 Pt 1):148–152Moffatt RJ, Chelland SA, Pecott DL, Stamford BA (2004) Acute exposure to environmentaltobacco smoke reduces HDL-C and HDL2-C. Prev Med 38(5):637–641Moriguchi EH, Fusegawa Y, Tamachi H, Goto Y (1991) Effects of smoking on HDL subfractionsin myocardial infarction patients: effects on lecithin-cholesterol acyltransferase and hepaticlipase. Clin Chim Acta 195(3):139–143Mukamal KJ, Conigrave KM, Mittleman MA, Camargo CA Jr, Stampfer MJ, Willett WC, RimmEB (2003) Roles of drinking pattern and type of alcohol consumed in coronary heart disease inmen. N Engl J Med 348(2):109–118Mulligan JJ, Cluette JE, Kew RR, Stack DJ, Hojnacki JL (1983) Cigarette smoking impairs hepaticuptake of high density lipoproteins. Biochem Biophys Res Commun 112(3):843–850Naem E, Alcalde R, Gladysz M, Mesliniene S, Jaimungal S, Sheikh-Ali M, Haas MJ, Wong NC,Mooradian AD (2012) Inhibition of apolipoprotein A-I gene by the aryl hydrocarbon receptor:a potential mechanism for smoking-associated hypoalphalipoproteinemia. Life Sci 91(1–2):64–69National Diet and Nutrition Survey (2011) Headline results from Years 1 and 2 (combined) of therolling programme 2008/9–2009/10. http://www.dh.gov.uk/prod_consum_dh/groups/dh_digitalassets/documents/digitalasset/dh_128550.pdf.HDL and Lifestyle Interventions 589Neufeld EJ, Mietus-Snyder M, Beiser AS, Baker AL, Newburger JW (1997) Passive cigarettesmoking and reduced HDL cholesterol levels in children with high-risk lipid profiles. Circula-tion 96(5):1403–1407Nishiwaki M, Ishikawa T, Ito T, Shige H, Tomiyasu K, Nakajima K, Kondo K, Hashimoto H,Saitoh K, Manabe M et al (1994) Effects of alcohol on lipoprotein lipase, hepatic lipase,cholesteryl ester transfer protein, and lecithin:cholesterol acyltransferase in high-density lipo-protein cholesterol elevation. Atherosclerosis 111(1):99–109Olchawa B, Kingwell BA, Hoang A, Schneider L, Miyazaki O, Nestel P, Sviridov D (2004)Physical fitness and reverse cholesterol transport. Arterioscler Thromb Vasc Biol 24(6):1087–1091Pan A, Chen M, Chowdhury R, Wu JH, Sun Q, Campos H, Mozaffarian D, Hu FB (2012) alpha-Linolenic acid and risk of cardiovascular disease: a systematic review and meta-analysis. Am JClin Nutr 96(6):1262–1273Pattyn N, Cornelissen VA, Eshghi SR, Vanhees L (2013) The effect of exercise on the cardiovas-cular risk factors constituting the metabolic syndrome: a meta-analysis of controlled trials.Sports Med 43(2):121–133Phillips NR, Havel RJ, Kane JP (1981) Levels and interrelationships of serum and lipoproteincholesterol and triglycerides. Association with adiposity and the consumption of ethanol,tobacco, and beverages containing caffeine. Arteriosclerosis 1(1):13–24Pirillo A, Norata GD, Catapano AL (2013) High-density lipoprotein subfractions–what theclinicians need to know. Cardiology 124(2):116–125Poobalan A, Aucott L, Smith WC, Avenell A, Jung R, Broom J, Grant AM (2004) Effects ofweight loss in overweight/obese individuals and long-term lipid outcomes–a systematicreview. Obes Rev 5(1):43–50Quensel M, Agardh CD, Nilsson-Ehle P (1989a) Nicotine does not affect plasma lipoproteinconcentrations in healthy men. Scand J Clin Lab Invest 49(2):149–153Quensel M, Soderstrom A, Agardh CD, Nilsson-Ehle P (1989b) High density lipoproteinconcentrations after cessation of smoking: the importance of alterations in diet. Atherosclerosis75(2–3):189–193Rao MN, Liu QH, Marmillot P, Seeff LB, Strader DB, Lakshman MR (2000) High-densitylipoproteins from human alcoholics exhibit impaired reverse cholesterol transport function.Metabolism 49(11):1406–1410Rao MN, Marmillot P, Gong M, Palmer DA, Seeff LB, Strader DB, Lakshman MR (2003) Light,but not heavy alcohol drinking, stimulates paraoxonase by upregulating liver mRNA in rats andhumans. Metabolism 52(10):1287–1294Rashid S, Genest J (2007) Effect of obesity on high-density lipoprotein metabolism. Obesity(Silver Spring) 15(12):2875–2888Rector RS, Warner SO, Liu Y, Hinton PS, Sun GY, Cox RH, Stump CS, Laughlin MH,Dellsperger KC, Thomas TR (2007) Exercise and diet induced weight loss improves measuresof oxidative stress and insulin sensitivity in adults with characteristics of the metabolicsyndrome. Am J Physiol Endocrinol Metab 293(2):E500–E506Richard F, Marecaux N, Dallongeville J, Devienne M, Tiem N, Fruchart JC, Fantino M,Zylberberg G, Amouyel P (1997) Effect of smoking cessation on lipoprotein A-I and lipopro-tein A-I:A-II levels. Metabolism 46(6):711–715Riemens SC, van Tol A, Hoogenberg K, van Gent T, Scheek LM, Sluiter WJ, Dullaart RP (1997)Higher high density lipoprotein cholesterol associated with moderate alcohol consumption isnot related to altered plasma lecithin:cholesterol acyltransferase and lipid transfer proteinactivity levels. Clin Chim Acta 258(1):105–115Sacks FM, Bray GA, Carey VJ, Smith SR, Ryan DH, Anton SD, McManus K, Champagne CM,Bishop LM, Laranjo N, Leboff MS, Rood JC, de Jonge L, Greenway FL, Loria CM,Obarzanek E, Williamson DA (2009) Comparison of weight-loss diets with differentcompositions of fat, protein, and carbohydrates. N Engl J Med 360(9):859–873Sarandol E, Serdar Z, Dirican M, Safak O (2003) Effects of red wine consumption on serumparaoxonase/arylesterase activities and on lipoprotein oxidizability in healthy-men. J NutrBiochem 14(9):507–512590 J.C. Escolà-Gil et al.Sasahara T, Nestel P, Fidge N, Sviridov D (1998) Cholesterol transport between cells and highdensity lipoprotein subfractions from obese and lean subjects. J Lipid Res 39(3):544–554Sattelmair J, Pertman J, Ding EL, Kohl HW 3rd, Haskell W, Lee IM (2011) Dose responsebetween physical activity and risk of coronary heart disease: a meta-analysis. Circulation124(7):789–795Schwingshackl L, Hoffmann G (2012) Monounsaturated fatty acids and risk of cardiovasculardisease: synopsis of the evidence available from systematic reviews and meta-analyses.Nutrients 4(12):1989–2007Schwingshackl L, Hoffmann G (2013) Long-term effects of low-fat diets either low or high inprotein on cardiovascular and metabolic risk factors: a systematic review and meta-analysis.Nutr J 12:48Senault C, Mahlberg FH, Renaud G, Girard-Globa A, Chacko GK (1990) Effect of apoproteincross-linking on the metabolism of human HDL3 in rat. Biochim Biophys Acta 1046(1):81–88Seres I, Bajnok L, Harangi M, Sztanek F, Koncsos P, Paragh G (2010) Alteration of PON1 activityin adult and childhood obesity and its relation to adipokine levels. Adv Exp Med Biol660:129–142Shennan NM, Seed M, Wynn V (1985) Variation in serum lipid and lipoprotein levels associatedwith changes in smoking behaviour in non-obese Caucasian males. Atherosclerosis 58(1–3):17–25Sierksma A, van der Gaag MS, van Tol A, James RW, Hendriks HF (2002) Kinetics of HDLcholesterol and paraoxonase activity in moderate alcohol consumers. Alcohol Clin Exp Res 26(9):1430–1435Sierksma A, Vermunt SH, Lankhuizen IM, van der Gaag MS, Scheek LM, Grobbee DE, vanTol A, Hendriks HF (2004) Effect of moderate alcohol consumption on parameters of reversecholesterol transport in postmenopausal women. Alcohol Clin Exp Res 28(4):662–666Sigal RJ, Kenny GP, Boule NG, Wells GA, Prud’homme D, Fortier M, Reid RD, Tulloch H,Coyle D, Phillips P, Jennings A, Jaffey J (2007) Effects of aerobic training, resistance training,or both on glycemic control in type 2 diabetes: a randomized trial. Ann Intern Med 147(6):357–369Singh IM, Shishehbor MH, Ansell BJ (2007) High-density lipoprotein as a therapeutic target: asystematic review. JAMA 298(7):786–798Stanhope KL, Schwarz JM, Keim NL, Griffen SC, Bremer AA, Graham JL, Hatcher B, Cox CL,Dyachenko A, Zhang W, McGahan JP, Seibert A, Krauss RM, Chiu S, Schaefer EJ, Ai M,Otokozawa S, Nakajima K, Nakano T, Beysen C, Hellerstein MK, Berglund L, Havel PJ (2009)Consuming fructose-sweetened, not glucose-sweetened, beverages increases visceral adiposityand lipids and decreases insulin sensitivity in overweight/obese humans. J Clin Invest 119(5):1322–1334Stanhope KL, Schwarz JM, Havel PJ (2013) Adverse metabolic effects of dietary fructose: resultsfrom the recent epidemiological, clinical, and mechanistic studies. Curr Opin Lipidol 24(3):198–206Stubbe I, Eskilsson J, Nilsson-Ehle P (1982) High-density lipoprotein concentrations increase afterstopping smoking. BMJ 284(6328):1511–1513Tall AR (2002) Exercise to reduce cardiovascular risk – how much is enough? N Engl J Med 347(19):1522–1524Tambalis K, Panagiotakos DB, Kavouras SA, Sidossis LS (2009) Responses of blood lipids toaerobic, resistance, and combined aerobic with resistance exercise training: a systematicreview of current evidence. Angiology 60(5):614–632Tchernof A, Despres JP (2013) Pathophysiology of human visceral obesity: an update. Physiol Rev93(1):359–404Tian L, Jia L, Mingde F, Tian Y, Xu Y, Tian H, Yang Y (2006) Alterations of high densitylipoprotein subclasses in obese subjects. Lipids 41(8):789–796Tian L, Xu Y, Fu M, Peng T, Liu Y, Long S (2011) The impact of plasma triglyceride andapolipoproteins concentrations on high-density lipoprotein subclasses distribution. LipidsHealth Dis 10:17HDL and Lifestyle Interventions 591Tselepis AD, Panagiotakos DB, Pitsavos C, Tellis CC, Chrysohoou C, Stefanadis C (2009)Smoking induces lipoprotein-associated phospholipase A2 in cardiovascular disease freeadults: the ATTICA Study. Atherosclerosis 206(1):303–308Tuomilehto J, Tanskanen A, Salonen JT, Nissinen A, Koskela K (1986) Effects of smoking andstopping smoking on serum high-density lipoprotein cholesterol levels in a representativepopulation sample. Prev Med 15(1):35–45Ueyama K, Yokode M, Arai H, Nagano Y, Li ZX, Cho M, Kita T (1998) Cholesterol efflux effectof high density lipoprotein is impaired by whole cigarette smoke extracts through lipidperoxidation. Free Radic Biol Med 24(1):182–190Uusitupa M, Peltonen M, Lindstrom J, Aunola S, Ilanne-Parikka P, Keinanen-Kiukaanniemi S,Valle TT, Eriksson JG, Tuomilehto J (2009) Ten-year mortality and cardiovascular morbidityin the Finnish Diabetes Prevention Study – secondary analysis of the randomized trial. PLoSONE 4(5):e5656Uzun H, Zengin K, Taskin M, Aydin S, Simsek G, Dariyerli N (2004) Changes in leptin,plasminogen activator factor and oxidative stress in morbidly obese patients following openand laparoscopic Swedish adjustable gastric banding. Obes Surg 14(5):659–665van der GaagMS, van Tol A, Scheek LM, James RW, Urgert R, Schaafsma G, Hendriks HF (1999)Daily moderate alcohol consumption increases serum paraoxonase activity; a diet-controlled,randomised intervention study in middle-aged men. Atherosclerosis 147(2):405–410Vazquez E, Sethi AA, Freeman L, Zalos G, Chaudhry H, Haser E, Aicher BO, Aponte A,Gucek M, Kato GJ, Waclawiw MA, Remaley AT, Cannon RO 3rd (2012) High-densitylipoprotein cholesterol efflux, nitration of apolipoprotein A-I, and endothelial function inobese women. Am J Cardiol 109(4):527–532Verdugo RA, Zeller T, Rotival M, Wild PS, Munzel T, Lackner KJ, Weidmann H, Ninio E,Tregouet DA, Cambien F, Blankenberg S, Tiret L (2013) Graphical modeling of gene expres-sion in monocytes suggests molecular mechanisms explaining increased atherosclerosis insmokers. PLoS ONE 8(1):e50888Vest AR, Heneghan HM, Agarwal S, Schauer PR, Young JB (2012) Bariatric surgery andcardiovascular outcomes: a systematic review. Heart 98(24):1763–1777Wang H, Peng DQ (2011) New insights into the mechanism of low high-density lipoproteincholesterol in obesity. Lipids Health Dis 10:176Wen CP, Wai JP, Tsai MK, Yang YC, Cheng TY, Lee MC, Chan HT, Tsao CK, Tsai SP, Wu X(2011) Minimum amount of physical activity for reduced mortality and extended life expec-tancy: a prospective cohort study. Lancet 378(9798):1244–1253Wilson PW, Garrison RJ, Abbott RD, Castelli WP (1983) Factors associated with lipoproteincholesterol levels. The Framingham study. Arteriosclerosis 3(3):273–281Wing RR (2010) Long-term effects of a lifestyle intervention on weight and cardiovascular riskfactors in individuals with type 2 diabetes mellitus: four-year results of the Look AHEAD trial.Arch Intern Med 170(17):1566–1575Wing RR, Bolin P, Brancati FL, Bray GA, Clark JM, Coday M, Crow RS, Curtis JM, Egan CM,Espeland MA, Evans M, Foreyt JP, Ghazarian S, Gregg EW, Harrison B, Hazuda HP, Hill JO,Horton ES, Hubbard VS, Jakicic JM, Jeffery RW, Johnson KC, Kahn SE, Kitabchi AE,Knowler WC, Lewis CE, Maschak-Carey BJ, Montez MG, Murillo A, Nathan DM,Patricio J, Peters A, Pi-Sunyer X, Pownall H, Reboussin D, Regensteiner JG, Rickman AD,Ryan DH, Safford M, Wadden TA, Wagenknecht LE, West DS, Williamson DF, Yanovski SZ(2013) Cardiovascular effects of intensive lifestyle intervention in type 2 diabetes. N Engl JMed 369(2):145–154Woldt E, Sebti Y, Solt LA, Duhem C, Lancel S, Eeckhoute J, Hesselink MK, Paquet C, Delhaye S,Shin Y, Kamenecka TM, Schaart G, Lefebvre P, Neviere R, Burris TP, Schrauwen P, Staels B,Duez H (2013) Rev-erb-alpha modulates skeletal muscle oxidative capacity by regulatingmitochondrial biogenesis and autophagy. Nat Med 19(8):1039–1046Yudkin J (1972) Sugar and disease. Nature 239(5369):197–199Zhang YH, An T, Zhang RC, Zhou Q, Huang Y, Zhang J (2013) Very high fructose intakeincreases serum LDL-cholesterol and total cholesterol: a meta-analysis of controlled feedingtrials. J Nutr 143(9):1391–1398592 J.C. Escolà-Gil et al.Effects of Established Hypolipidemic Drugson HDL Concentration, SubclassDistribution, and FunctionMonica Gomaraschi, Maria Pia Adorni, Maciej Banach, FrancoBernini, Guido Franceschini, and Laura CalabresiContents1 Effects on Plasma HDL-C Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5951.1 Statins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5951.2 Fibrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5951.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5962 Effects on HDL Subclass Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5962.1 Statins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5962.2 Fibrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5972.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5973 Effects on Cholesterol Efflux Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5973.1 Statins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5983.2 Fibrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5993.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5994 Effect on HDL Ability to Preserve Endothelial Cell Homeostasis . . . . . . . . . . . . . . . . . . . . . . . 6004.1 Statins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6014.2 Fibrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6024.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6025 Effect on HDL Antioxidant Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6035.1 Statins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6045.2 Fibrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6045.3 Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607M. Gomaraschi • G. Franceschini • L. Calabresi (*)Center E. Grossi Paoletti, Department of Pharmacological and Biomolecular Sciences,University of Milano, Via Balzaretti 9, 20133 Milan, Italye-mail: monica.gomaraschi@unimi.it; guido.franceschini@unimi.it; laura.calabresi@unimi.itM.P. Adorni • F. BerniniDepartment of Pharmacy, University of Parma, Parma, Italye-mail: mariapia.adorni@unipr.it; fbernini@unipr.itM. BanachMedical University of Lodz, Łódź, Polande-mail: maciejbanach@aol.co.uk# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_19593AbstractThe knowledge of an inverse relationship between plasma high-density lipopro-tein cholesterol (HDL-C) concentrations and rates of cardiovascular disease hasled to the concept that increasing plasma HDL-C levels would be protectiveagainst cardiovascular events. Therapeutic interventions presently available tocorrect the plasma lipid profile have not been designed to specifically act onHDL, but have modest to moderate effects on plasma HDL-C concentrations.Statins, the first-line lipid-lowering drug therapy in primary and secondarycardiovascular prevention, have quite modest effects on plasma HDL-Cconcentrations (2–10 %). Fibrates, primarily used to reduce plasma triglyceridelevels, also moderately increase HDL-C levels (5–15 %). Niacin is the mostpotent available drug in increasing HDL-C levels (up to 30 %), but its use islimited by side effects, especially flushing.The present chapter reviews the effects of established hypolipidemic drugs(statins, fibrates, and niacin) on plasma HDL-C levels and HDL subclass distri-bution, and on HDL functions, including cholesterol efflux capacity, endothelialprotection, and antioxidant properties.KeywordsHDL • HDL subclasses • Cell cholesterol efflux • Endothelial cells • Statins •Fibrates • NiacinAbbreviationsABCA1 ATP-binding cassette A1CETP Cholesteryl ester transfer proteinCEC Cholesterol efflux capacityCRP Reactive proteineNOS Endothelial nitric oxide synthaseEPC Endothelial progenitor cellsFMD Flow-mediated vasodilationGSPx Glutathione selenoperoxidaseHDL-C HDL cholesterolLDL-C LDL cholesterolLCAT Lecithin:cholesterol acyltransferaseLOOHs Lipoprotein lipid hydroperoxidesLpA-I HDL containing apoA-I onlyLpA-I:A-II HDL containing apoA-I and apoA-IILp-PLA2 Lipoprotein-associated phospholipase A2NO Nitric oxidePGI2 ProstacyclinPON1 Paraoxonase 1594 M. Gomaraschi et al.ROS Reactive oxygen speciesSR-BI Scavenger receptor class B type IS1P Sphingolipid sphingosine-1-phosphateT2DM Type 2 diabetesTNFα Tumor necrosis factor alpha1 Effects on Plasma HDL-C Concentration1.1 StatinsStatins are inhibitors of the hydroxymethylglutaryl coenzyme A (HMG-CoA)reductase enzyme, a key enzyme in cholesterol synthesis. In addition to effectivelyreducing low-density lipoprotein cholesterol (LDL-C), all statins modestly increaseHDL-C levels across the range of doses used, with important differences betweendifferent statins (Jones et al. 2003). The increase in HDL-C is dose dependent withsome statins, e.g., simvastatin and rosuvastatin, but not with others. In the case ofatorvastatin, the highest increase in HDL-C levels is observed at the lowest doseused. Statin-induced changes in HDL-C are normally paralleled by an increase inapoA-I levels (Barter et al. 2010). Interestingly, variations in LDL-C and in HDL-Clevels are independent from each other (Barter et al. 2010). The mechanism bywhich statins increase plasma HDL-C and apoA-I levels remains unknown, and it isapparently unrelated to the inhibition of the HMG-CoA reductase enzyme. Theability of statins to upregulate hepatic ABCA1 gene expression (Tamehiroet al. 2007), as well as the described statin-induced inhibition of cholesteryl estertransfer protein (CETP) (Guerin et al. 2000; Kassai et al. 2007) can contribute toexplain the effect of statin on HDL-C levels.1.2 FibratesFibrates (fibric acid derivates), synthetic ligands for PPAR-α, significantly reduceplasma triglyceride levels (30–50 %) and moderately increase HDL-Cconcentrations (5–15 %), with no major differences among available molecules(Khoury and Goldberg 2011). Activation of PPAR-α leads to β-oxidation of freefatty acids in the liver reducing VLDL synthesis and secretion. Furthermore, theexpression of the gene coding for lipoprotein lipase is increased and apolipoproteinC-III expression in the liver is decreased, resulting in increased hydrolysis oftriglyceride-rich lipoproteins. HDL-C raises results from the increased expressionof apoA-II and, at a much lesser extent, apoA-I (Yetukuri et al. 2011), as well as tothe enhanced cholesterol efflux via the induction of ABCA1 in the liver (Bergeret al. 2005). In addition, animal studies have shown that fenofibrate reduces CETPhepatic expression (van der Hoogt et al. 2007).Effects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 5951.3 NiacinNiacin (nicotinic acid), a GPR109a agonist used clinically for more than 50 years tolower cholesterol and triglycerides, increases HDL-C levels up to 30 % (Vega andGrundy 1994). All the different niacin formulations (regular and extended release,ER) are equally effective in increasing HDL-C levels, whereas acipimox(a nicotinic acid analog) only induces a 7 % increase (Birjmohun et al. 2005).Niacin side-effect profile, including flushing, gastrointestinal upset, and liver func-tion testing abnormalities, has, however, limited its use, especially in warmerMediterranean countries (Birjmohun et al. 2005). Coadministration of the prosta-glandin receptor antagonist laropiprant significantly reduces but does not eliminateniacin-induced skin symptoms (Yadav et al. 2012). It is not quite clear how niacinleads to an increase in HDL-C levels, and various theories have been proposed(Soudijn et al. 2007). These include the ability of niacin to inhibit CETP(Hernandez et al. 2007), to induce ABCA1 expression (Rubic et al. 2004), and toreduce HDL catabolism (Bodor and Offermanns 2008).2 Effects on HDL Subclass DistributionPlasma HDL is highly heterogeneous and can be separated in several subclassesaccording to density, size, shape, and lipid and protein composition (Calabresiet al. 2010). According to density, two HDL subclasses can be identified, the lessdense HDL2 and the more dense HDL3 (De Lalla and Gofman 1954). HDL2 andHDL3 could be further fractionated in five distinct subclasses on the basis ofparticle size (from large to small: HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c)(Nichols et al. 1986). According to surface charge, HDL could be separated intoα-HDL and preβ-HDL. The separation by charge and size, using a two-dimensional(2-D) gel electrophoresis, allow the identification of up to 12 distinct apoA-I-containing HDL subclasses (Asztalos and Schaefer 2003). Finally, according tothe protein component, HDL can be separated into particles containing only apoA-I(LpA-I) or containing also apoA-II (LpA-I:A-II).2.1 StatinsThe reported effects of statins on HDL subclass distribution are not consistent,likely depending on the technique used for the analysis. Some studies showed noeffect of statins on HDL subclasses (Alaupovic et al. 1994; Bard et al. 1989;Franceschini et al. 2007; Tomas et al. 2000), and on HDL particle size distribution(Franceschini et al. 1989; Homma et al. 1995). On the contrary, studies evaluatingHDL subclasses by 2-D gel electrophoresis showed that all statins, although withdifferent potency, increase the levels of the large HDL particles and decrease thelevels of small particles (Asztalos et al. 2002a, b, 2007).596 M. Gomaraschi et al.2.2 FibratesA number of studies have shown that treatment with fibrates causes a shift of HDLfrom large to small particles in low HDL (Franceschini et al. 2007; Otvoset al. 2006), hypertriglyceridemic (Miida et al. 2000; Sasaki et al. 2002), andhypercholesterolemic (Franceschini et al. 1989) patients. These effects could beexplained by the fibrate-induced increase in hepatic lipase activity (Miidaet al. 2000; Patsch et al. 1984). In addition, bezafibrate has been shown to increasesmall discoidal preβ1-HDL particles in hypertriglyceridemic patients (Miidaet al. 2000). The analysis of HDL subclasses by 2-D electrophoresis in the largecohort of the VA-HIT trial confirmed that gemfibrozil treatment is associated with adecrease in large (α-1 and α-2) HDL and with an increase in the small, lipid-poorHDL particles (Asztalos et al. 2008).2.3 NiacinSheperd et al. were the first to demonstrate in the late 1970s that niacin (3 g/day) inhealthy volunteers significantly increases the large HDL2 particles, associated witha parallel reduction of the small HDL3 (Shepherd et al. 1979). Several studies haveconfirmed this observation in patients with different types of dyslipidemia treatedwith 2, 3, or 4 g/day of regular or ER niacin (Johansson and Carlson 1990; Knoppet al. 1998; Morgan et al. 2003; Wahlberg et al. 1990) and in patients with coronaryartery disease (Kuvin et al. 2006). A very recent study showed that also 1 g/day ofER niacin increases large HDL particles in dyslipidemic patients, with a concomi-tant significant increase in mean HDL size (Franceschini et al. 2013). The analysisof HDL subclasses by 2-D gel electrophoresis recently confirmed the observationthat niacin promotes the maturation of HDL into large particles (Lamon-Favaet al. 2008), likely explained by a reduction in CETP activity. In addition, Sakaiet al. reported that niacin selectively increases LpA-I particles in patients with lowHDL-C, due to a prolongation of apoA-I residence time (Sakai et al. 2001).3 Effects on Cholesterol Efflux CapacityThe most relevant antiatherogenic function of HDL is the ability to promote reversecholesterol transport, the physiological process by which excess cholesterol isremoved from peripheral tissues and transported to the liver for excretion (Cucheland Rader 2006). The first and limiting step of this process is the efflux of cellularcholesterol from lipid-laden macrophages of the arterial wall (Lewis and Rader2005). Multiple mechanisms for efflux of cholesterol exist in macrophages (Adorniet al. 2007). The release of free cholesterol via passive diffusion occurs in all celltypes and is accelerated when the scavenger receptor class B type I (SR-BI) ispresent on the cell plasma membrane. Both passive diffusion and SR-BI-mediatedefflux occur to phospholipid-containing acceptors (i.e., HDL and lipidatedEffects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 597apolipoproteins). ATP-binding cassette A1 (ABCA1) belongs to the ATP-bindingcassette transporter family and promotes unidirectional efflux of membrane choles-terol and phospholipids to lipid-poor apolipoproteins. Macrophages may alsorelease cholesterol via the ABCG1 pathway, another ABC transporter that mediatesnet mass efflux of cellular cholesterol to mature HDL (Favari et al. 2009). HDLcholesterol efflux capacity (CEC) from macrophages can be measured in vitro andprovides a reliable measure of HDL functionality (Khera et al. 2011).3.1 StatinsThe impact of statin therapy on HDL ability to promote cholesterol efflux has beeninvestigated in a number of studies. Khera et al. measured HDL CEC in patientstreated with 10 and 80 mg atorvastatin or 40 mg pravastatin for 16 weeks andreported no effects of statin therapy on HDL CEC from macrophages expressing allthe known cholesterol efflux pathways (ABCA1, ABCG1, SR-BI, and passivediffusion) (Khera et al. 2011). As opposed to these results, Guerin et al. haveshown that atorvastatin therapy for 12 weeks (10 and 40 mg daily) could increaseSR-BI-dependent CEC of plasma from patients with type IIB hyperlipidemia, in adose-dependent manner (Guerin et al. 2002). The effect of simvastatin on HDL’sability to promote cholesterol efflux was assessed in a randomized, double-blind,parallel group trial carried out in dyslipidemic patients with low HDL-C levelsreceiving either fenofibrate (160 mg/day) or simvastatin (40 mg/day) for 8 weeks(Franceschini et al. 2007). Simvastatin led to a small, significant increase in thecapacity of plasma to promote SR-BI-mediated cholesterol efflux, likely related tothe slight increase in HDL-C plasma levels that occurred after simvastatin treat-ment. The latter results are in contrast with data obtained by de Vries et al. whodemonstrated that HDL from moderately hypercholesterolemic type 1 diabeticpatients after 10, 20, and 40 mg simvastatin treatment does not increase their abilityto promote cellular cholesterol efflux via SR-BI (De Vries et al. 2005). The effect ofsimvastatin on HDL functionality was further investigated recently in a smallnumber of diabetic patients treated with simvastatin 40 mg/day or bezafibrate400 mg/day, alone or in combination. CEC of apoB-depleted plasma samplesfrom cholesterol-loaded macrophages, a cellular model which expresses simulta-neously all the pathways of known relevance in cholesterol efflux, was increased by14 % in response to simvastatin compared to placebo (Triolo et al. 2013a). Sviridovet al. showed that treatment with 40 mg/day rosuvastatin in overweight subjectswith defined metabolic syndrome did not significantly modify HDL capacity topromote cholesterol efflux from LXR-activated human macrophages (Sviridovet al. 2008). Finally, a recent study showed a moderate ability of pitavastatin toenhance HDL CEC from macrophage foam cells in dyslipidemic subjects(Miyamoto-Sasaki et al. 2013). Overall, the impact of statin therapy on HDLCEC does not appear to be substantial, in agreement with the modest effect ofstatins on HDL-C concentration and on HDL subclass distribution.598 M. Gomaraschi et al.3.2 FibratesThe effect of fibrates on HDL cholesterol efflux capacity was assessed by Guerinet al. showing a significant ciprofibrate-mediated 13 % elevation in the capacity ofplasma from type IIB hyperlipidemic subjects to mediate cholesterol efflux fromSR-BI-expressing Fu5AH hepatoma cells (Guerin et al. 2003). The study byFranceschini et al., comparing fenofibrate and simvastatin in dyslipidemic patients(Franceschini et al. 2007), provided evidence that fenofibrate increases the plasmacapacity to promote ABCA1-mediated efflux with no changes in SR-BI efflux.These observed results might be explained by a shift of HDL from large to smallparticles, the preferential cholesterol acceptors for ABCA1 (Adorni et al. 2007).This result was not confirmed in a more recent paper by the same group(Franceschini et al. 2013), likely because of the different HDL particle profile inthe two investigated populations of patients. In the first study (Guerin et al. 2002),patients displayed a greater content of large HDL particles, whereas in patients withdyslipidemia of the last study (Franceschini et al. 2013), the small, ABCA1-interacting HDL was the predominant fraction, and no further increase in thisHDL population was observed after fenofibrate therapy, consistent with the lackof changes in ABCA1-mediated CEC. Whether fenofibrate therapy modulate theability of plasma or HDL to facilitate cholesterol efflux from macrophages has beeninvestigated also in a subset of the FIELD study showing that cholesterol effluxvalues from macrophage foam cells to HDL and plasma in the fenofibrate group(200 mg/day for 5 years) were comparable to those of the placebo group (Maranghiet al. 2011). Finally, in a small study carried out in diabetic patients, it was shownthat bezafibrate increases CEC of apoB-depleted plasma samples from cholesterol-loaded THP-1 macrophages (Triolo et al. 2013a). Overall, studies on the ability offibrates to modulate HDL cholesterol efflux capacity seem not consistent, likely dueto the different cell models used in the different studies and also to the differenttypes of patients.3.3 NiacinThe impact of niacin on HDL cholesterol efflux capacity was investigated in aplacebo-controlled study carried out by Khera et al. who measured HDL CECbefore and after ER niacin therapy in a small number of patients with carotidatherosclerosis (Khera et al. 2013). The study showed that niacin treatment led tofavorable changes in patients’ lipid profiles without significantly improving CEC ofHDL from macrophages. In the study by Franceschini et al., the effect of ER niacintherapy on plasma CEC was evaluated in a population of dyslipidemic subjects(Franceschini et al. 2013). The tendency of ER niacin in enhancing passive diffu-sion, SR-BI-, and ABCG1-mediated CEC observed in this study is in line with theresults of a previous investigation, which demonstrated that niacin increased bothHDL-C levels and CEC from human macrophages (Yvan-Charvet et al. 2010). Inthis study the increase in CEC achieved statistical significance, possibly because ofa greater increase in plasma HDL-C levels compared with that observed in study byEffects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 599Franceschini et al., because passive diffusion, SR-BI-, and partially ABCG1-mediated CECs are known to be dependent on HDL-C concentrations. Consis-tently, a previous investigation by Morgan et al. reported that ER niacin therapy inpatients with a history of primary dyslipoproteinemia had a beneficial effect on SR-BI-mediated efflux and that it was related to the change in level of HDL-C (Morganet al. 2007). Another study evaluated specific cholesterol efflux pathway-mediatedCECs (ABCA1, SR-BI, and ABCG1) of serum from men with HDL deficiencybefore and after treatment with niacin, but no changes were reported in all theconsidered efflux pathways (Alrasadi et al. 2008). Taken together, available data onthe effect of niacin on the HDL-mediated ability to promote cell cholesterol effluxshow modest effects on the pathways involving large HDL, in agreement with thechanges observed on HDL particles.4 Effect on HDL Ability to Preserve Endothelial CellHomeostasisSeveral in vitro and in vivo evidences indicate that the anti-atherosclerotic activityof HDL is also due to their ability to preserve endothelial cell homeostasis(Calabresi et al. 2003). HDL is able to promote vasorelaxation, to inhibit theproduction of cell adhesion and proinflammatory molecules, and to favor theintegrity and repair of the endothelial layer, thus preventing and correcting themain features of endothelial dysfunction typical of the atherosclerotic process.HDL can modulate vascular tone by promoting the release of vasoactive moleculeslike nitric oxide (NO) and prostacyclin (PGI2) which also exert powerfulantithrombotic effects. HDL is able to increase the protein abundance and to inducethe activation of endothelial nitric oxide synthase (eNOS) (Kuvin et al. 2002;Yuhanna et al. 2001). This latter effect is driven by the interaction of HDL withthe scavenger receptor SR-BI and by the subsequent activation of the PI3K/Aktsignaling pathway leading to eNOS phosphorylation; in addition, the sphingolipidsphingosine-1-phosphate (S1P) carried by HDL can also mediate eNOS activationby binding with its receptor S1P3 (Nofer et al. 2004). PGI2 is a metabolite ofarachidonate and HDL was shown to increase its production by differentmechanisms. HDL can provide endothelial cells with the substrate arachidonateor promote its release from cellular phospholipids by activating calcium-sensitivemembrane-bound phospholipases (Calabresi et al. 2003). Another feature of endo-thelial dysfunction is the increased production of molecules favoring blood celladhesion to the endothelial layer and their consequent extravasation and activation.Studies on endothelial cells have shown that HDL are able to inhibit the cytokine-induced expression of cell adhesion molecules (Calabresi et al. 1997; Cockerillet al. 1995), but the precise mechanism responsible for this effect has not been fullyelucidated to date (Barter et al. 2002). Different studies have shown anHDL-mediated inhibition of sphingosine kinase activity and NF-kB nuclear trans-location and an increased expression of heme-oxygenase 1 mediated by SR-BI(McGrath et al. 2009; Xia et al. 1999). HDL was also shown to inhibit the600 M. Gomaraschi et al.production of proinflammatory cytokines, as interleukin-6 and chemokines(Gomaraschi et al. 2005). Finally, HDL was shown to promote endothelial cellmigration and proliferation, to inhibit cell apoptosis through different mechanisms(Mineo and Shaul 2012), and to promote endothelial progenitor cells (EPC) differ-entiation and survival (Mineo and Shaul 2012; Petoumenos et al. 2009).In certain pathological conditions HDL may lose their ability to protect theendothelium and even become dysfunctional. In particular, conditions associatedwith both acute and chronic inflammatory processes, such as ischemic vascularevents, infections, type 2 diabetes (T2DM), obesity, metabolic syndrome, autoim-mune disorders, and chronic kidney disease, have been associated with HDLdisplaying impaired endothelial protective activities (Riwanto and Landmesser2013). Indeed, HDL isolated from these patients failed to promote NO production,to exert antioxidant and anti-inflammatory effects, and to favor endothelial integrityin vitro (Riwanto and Landmesser 2013).The measurement of vasodilation in response to changes in forearm blood flow(flow-mediated vasodilation, FMD) is currently considered the best method toevaluate endothelial function in vivo (Deanfield et al. 2007) and is commonlyused to assess in vivo vascular condition and to evaluate the effects of pharmaco-logical treatments. In addition, plasma levels of the soluble forms of adhesionmolecules and of proinflammatory cytokines can be used as in vivo markers ofendothelial inflammatory activation. Recently, circulating EPC number and migra-tory capacity emerged as novel biomarkers of endothelial repair (Petoumenoset al. 2009).4.1 StatinsSeveral studies have shown that statins are able to improve FMD, to reduce theplasma levels of inflammatory markers, and to increase EPC number indyslipidemic subjects (Antonopoulos et al. 2012; Liu et al. 2012; Rerianiet al. 2011). These effects are likely not related to statin-induced changes inHDL. In vitro experiments have indeed shown that statin-mediated improvementof endothelial function is due to a direct effect of the drug on endothelial cells.Incubation of endothelial cells with different statins promotes a significant increaseexpression of SR-BI and S1P receptors and of eNOS, with a consequent increasedproduction of NO after incubation with HDL (Igarashi et al. 2007; Kimuraet al. 2008). A recent study performed on subjects with isolated low HDL-C showedthat FMD was significantly improved after 4 weeks treatment with pravastatin, andmultiple regression analysis revealed that changes in HDL-C and EPC number wereindependent predictors of FMD increase (Higashi et al. 2010). Despite thelimitations due to the small number of analyzed subjects, this latter study suggestsa potential role of statins on HDL capacity to maintain endothelial homeostasis.Effects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 6014.2 FibratesDifferent fibrates have been tested for their ability to modulate endothelial functionin dyslipidemic patients. Fenofibrate was shown to improve FMD and to decreaseinflammatory biomarkers, as C-reactive protein (CRP), tumor necrosis factor alpha(TNFα), interleukin-1, and soluble CD40 ligand, in patients with combined hyper-lipidemia (Malik et al. 2001; Wang et al. 2003). FMD improvement was inverselyrelated to baseline HDL-C levels and indeed fenofibrate significantly increasedFMD only in the subgroup with low HDL-C and not in subjects with normalHDL-C, in which a statin was instead effective (Wang et al. 2003). Fenofibrate,alone or in combination with a statin, was also effective in modulating FMD anddecreasing plasma levels of CRP and TNFα in hypertriglyceridemic subjects (Kohet al. 2004, 2005), and a significant decrease of plasma levels of sCAMs wasobserved in subjects with low HDL-C levels (Calabresi et al. 2002); in this latterstudy, the decrease of plasma sCAMs was inversely related to the fenofibrate-induced increase of HDL-C, while no correlation was found with triglyceridereduction (Calabresi et al. 2002).Recently, particular attention has been devoted to the effects of fibrates insubjects with T2DM. Four studies consistently demonstrated that different fibrates,i.e., ciprofibrate, gemfibrozil, fenofibrate, and bezafibrate, were all able to signifi-cantly improve FMD in T2DM, in a way that was not dependent on changes in lipidparameters, including HDL-C (Avogaro et al. 2001; Evans et al. 2000; Ghaniet al. 2013; Triolo et al. 2013b). Interestingly, after 12 weeks of treatment withciprofibrate, T2DM subjects displayed improved FMD measured in fastingconditions and in the postprandial phase (Evans et al. 2000). Ciprofibrate treatmentalso markedly modified lipid metabolism: the fasting triglyceride content of alllipoproteins was reduced and the increase during the postprandial phase blunted;the improvement of fasting FMD was correlated with the decreased triglyceridecontent in VLDL and HDL (but not with changes of HDL-C), while in thepostprandial phase changes of FMD were correlated with the reduced lipoproteinTG content (Evans et al. 2000).4.3 NiacinThe effect of niacin treatment on endothelial function has been assessed in a varietyof clinical conditions, including established coronary artery disease, T2DM, meta-bolic syndrome, and low HDL-C. Overall, when niacin was used as monotherapy,the studies showed an increase in FMD and a reduction in inflammatory biomarkersbut failed in demonstrating a correlation with the niacin-mediated increase ofHDL-C levels. In 2002, Kuvin et al. demonstrated that a 3-month treatment withniacin improved FMD in patients with CAD and low HDL-C and that this improve-ment could be related to the HDL ability to increase eNOS protein abundancein vitro (Kuvin et al. 2002). Some years later, the same authors also showed that instatin treated patients with established CAD, the addition of niacin caused a602 M. Gomaraschi et al.reduction of the plasma levels of inflammatory biomarkers as CRP and lipoprotein-associated phospholipase A2 (Kuvin et al. 2006); unfortunately, likely due to thesmall number of participants, correlation analyses between changes of endothelialfunction parameters and the increase of HDL-C or other lipid variables were notperformed (Kuvin et al. 2006). Later on, the ability of niacin to improve FMD andreduce inflammatory biomarkers as CRP was confirmed in subjects with isolatedlow HDL-C (Benjo et al. 2006), with the metabolic syndrome (Thoenes et al. 2007),and in patients after myocardial infarction (Bregar et al. 2013); the relationshipbetween increase of HDL-C and changes of FMD was analyzed only in the lattercase, and no correlation was found with any lipid parameter. The absence ofcorrelation between niacin-mediated changes of HDL-C and endothelial functioncould be explained by the direct antioxidant, anti-inflammatory, and anti-adhesiveactivities of niacin which are independent from its role on lipid metabolism (Yadavet al. 2012). However, a recent study clearly showed that HDL isolated fromsubjects with T2DM after 3 months of treatment with niacin displayed an improvedability to stimulate NO production, to reduce reactive oxygen species, and topromote EPC-mediated endothelial repair in vitro when compared to HDL isolatedat baseline (Sorrentino et al. 2010), further strengthening the concept that plasmaHDL-C and its change may not be a reliable indicator of HDL function. Recentstudies analyzed the effect of niacin treatment in patients with established CAD ontop of the existing statin therapy; two of them failed to detect any significantimprovement of FMD, while in one case FMD was significantly increased only inthe subgroup of patients with a low baseline HDL-C level (Lee et al. 2009; Philpottet al. 2013; Warnholtz et al. 2009).5 Effect on HDL Antioxidant PropertiesApoA-I plays a major role in HDL-mediated protection from oxidative damage(Rosenson et al. 2013). Many studies have indicated that the antioxidant function ofHDL also depends upon such enzymes as lipoprotein-associated phospholipase A2(Lp-PLA2), paraoxonase 1 (PON1), lecithin-cholesterol acyltransferase (LCAT),and glutathione selenoperoxidase (GSPx) (Florentin et al. 2008; Otocka-Kmieciket al. 2012; Podrez 2010). GSPx can reduce lipoprotein lipid hydroperoxides(LOOHs) to the corresponding hydroxides and thereby detoxify them (Rosensonet al. 2013). Besides apoA-I, other apolipoproteins such as apoE, apoJ, apoA-II, andapoA-IV also display antioxidant properties but to a less degree than apoA-I(Otocka-Kmiecik et al. 2012). An antioxidant mechanism of HDL may also resultfrom their ability to accept phospholipid-containing hydroperoxides (PLOOHs) andother lipid peroxidation products from oxidized LDL (Stremler et al. 1991) andtheir subsequent reduction by redox-active methionine residues of apoA-I, with theformation of redox-inactive phospholipid hydroxides (Kontush and Chapman2010). In addition, HDL functions as an antioxidant enzyme by hydrolyzingoxidized phospholipids, such as F2-isoprostanes, formed during the oxidativemodification of LDL. Antioxidant properties differ for the different HDL subclassesEffects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 603(Kontush and Chapman 2010). Activities of HDL-associated enzymes—LCAT,PON1, and platelet-activating factor acetyl hydrolase (PAF-AH)—are elevated insmall, dense HDL3c (Davidson et al. 2009), which thus seem to have a potent rolein protecting LDL from oxidation (Podrez 2010). It is worth emphasizing thatsmall, dense HDL3 particles are also more resistant to oxidative modificationcompared with large, light HDL2 (Shuhei et al. 2010).5.1 StatinsThe role of statins in improving plasma oxidative status has been reported in anumber of studies, although this effect was not always related to drug-inducedchanges in HDL. Different statins have been shown to significantly enhance theantioxidant activity of PON1. Atorvastatin has a positive impact on plasma totalantioxidant status and PON1 activity by reducing plasma susceptibility to lipidperoxidation induced by free radicals (Fuhrman et al. 2002; Harangi et al. 2004,2009; Nagila et al. 2009). Pitavastatin was also shown to increase the promoteractivity and protein expression of PON1 in vitro via p44/42 mitogen-activatedprotein kinase-mediated phosphorylation of sterol-regulatory-element-bindingprotein-2 and binding of Sp1 to PON1 DNA (Arii et al. 2009; Yamashitaet al. 2010). The improvement of antioxidant properties of HDL, via increasedPON1 activity, was also observed after simvastatin and fluvastatin therapy(Bergheanu et al. 2007; Mirdamadi et al. 2008; Muacevic-Katanec et al. 2007).Atorvastatin also suppresses the enhanced cellular uptake of oxidized LDL bymacrophages and reduces expression of cellular scavenger receptors (Fuhrmanet al. 2002; Otocka-Kmiecik et al. 2012). Similar results were observed in thestudy by Kassai et al. which showed a significant increase in serum PON1 specificactivity, PON/HDL ratio, and LCAT activity, as well as significant reduction ofoxidized LDL and CETP activities after atorvastatin therapy (Kassai et al. 2007).Finally, statin treatment enhances the defective antioxidative activity of HDL3 inpatients with acute coronary syndrome by increasing the activity ofHDL-associated enzymes (Otocka-Kmiecik et al. 2012).5.2 FibratesTsimihodimos et al. reported that fenofibrate induces redistribution of Lp-PLA2from apoB-containing lipoproteins to HDL in dyslipidemic patients (Tsimihodimoset al. 2003). This effect enhanced the anti-inflammatory and antioxidant potential ofthe enzyme. In a recent study, Vazanna et al. examined whether HDL levels arerelated to in vivo oxidative stress and platelet activation in patients with coronaryheart disease treated with fenofibrate (Vazzana et al. 2013). The authors measuredurinary 8-iso-prostaglandin (PG) F2α and 11-dehydrothromboxane (TX) B2—in vivo markers of oxidative stress and platelet activation, respectively. Theyobserved significantly higher urinary 8-iso-PGF2α and 11-dehydro-TXB2 in604 M. Gomaraschi et al.patients with low HDL than in individuals with higher HDL levels, as well as aninverse relation between the markers of oxidation and HDL concentrations.Fenofibrate treatment significantly reduced the 2 eicosanoids in healthy subjects,in parallel with an HDL increase (Vazzana et al. 2013). Tkac et al. showed asignificant reduction of circulating conjugated dienes, a nonsignificant decrease inthe production of malondialdehyde, and an increase in the GPx activity in patientswith combined hyperlipidemia treated with fenofibrate (Tkac et al. 2006). Similarresults were obtained in patients with visceral obesity and dyslipidemia treated withmicronized fenofibrate (Broncel et al. 2006). Few studies have evaluated the effectof fibrates on PON1 activity. Paragh et al. evaluated this effect in a group ofcoronary patients with type IIb hyperlipidemia; after 3 months of treatment,HDL-C and apoA-I levels increased significantly, and PON1 specific activity alsoincreased significantly (Paragh et al. 2003). Very similar results were obtained byPhuntuwate et al., who evaluated the effect of fenofibrate on PON1 levels, as wellas the effects of the PON1 polymorphisms on lipid and PON1 responses to the drug,in dyslipidemic patients with low HDL (Phuntuwate et al. 2008). A significantincreases in PON1 concentration and activity was observed after treatment, and asignificant positive correlation between changes in HDL-C and changes in PON1concentration/activity was detected (Phuntuwate et al. 2008). The response of lipidparameters to fenofibrate was independent of PON1 polymorphisms; however,PON1 Q192R and T-108C polymorphisms significantly affected the increase inPON1 activity and concentration (Phuntuwate et al. 2008). An increase of PON1activity, which was significantly correlated with the changes in HDL-C, was alsoobserved in patients with combined hyperlipidemia (Yesilbursa et al. 2005). On thecontrary, Dullart et al. did not observe such increase after short-term (8 weeks)administration of simvastatin and bezafibrate, even when combined in diabeticpatients (Dullaart et al. 2009). It needs to be also emphasized that besides theabove mentioned positive HDL associated antioxidant properties of fibrates, theyalso elevate plasma total homocysteine, which is known to induce oxidative stressand endothelial dysfunction. Among others, a subgroup analysis of the FIELDstudy showed that fenofibrate treatment was associated with an increase of homo-cysteine levels, with no changes of serum PON-1 mass (Maranghi et al. 2011).5.3 NiacinThere is still very limited data on the potential role of niacin therapy onHDL-related antioxidant properties. Recent studies indicate that niacin increasesvascular endothelial cell redox state, resulting in the inhibition of oxidative stress(Kamanna and Kashyap 2008). These effects were investigated by Ganjiet al. in vitro, who showed that in cultured endothelial cells niacin increasesNADPH levels and reduces GSH (Ganji et al. 2009). In the same study, niacinalso inhibited angiotensin II-induced reactive oxygen species (ROS) production(Ganji et al. 2009). These findings indicate for the first time that niacin inhibitsvascular inflammation by decreasing endothelial ROS production and subsequentEffects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 605LDL oxidation (Ganji et al. 2009). Kaplon et al. have recently tested the hypothesisthat vascular endothelial function and oxidative stress are related to dietary niacinintake among healthy middle-aged and older adults (Kaplon et al. 2014). Theyobserved that plasma ox-LDL was inversely related to niacin intake. Moreover, inendothelial cells sampled from the brachial artery of a subgroup patients, dietaryniacin intake was inversely related to nitrotyrosine, a marker of oxidative damage,leading to the hypothesis that higher dietary niacin intake is associated with greatervascular endothelial function related to lower systemic and vascular oxidative stress(Kaplon et al. 2014). The antioxidant properties of niacin were also observedpatients with hypercholesterolemia and low HDL-C treated with niacin for12 weeks (Hamoud et al. 2013). Subjects with low HDL-C levels exhibited higheroxidative stress compared with subjects with normal HDL-C levels. Niacin treat-ment in hypercholesterolemic patients caused a significant increase in HDL-C andapoA-I levels and a decrease in TG levels. Niacin also significantly reducedoxidative stress, as measured by a significant decrease in the serum content ofthiobarbituric acid reactive substances and lipid peroxides and increase of PONactivity, compared with the levels before treatment (Hamoud et al. 2013).Conclusions and PerspectivesCurrently, optimal statin treatment manages to achieve a risk reduction forcardiovascular mortality of approximately 25–35 %, mainly due to a reductionof LDL-C. This leaves an important residual risk that needs to be considered forthe optimal management of patients at risk of cardiovascular disease. RaisingHDL-C was seen as a viable and promising way to further reduce the risk ofcardiovascular mortality. However, things look to be difficult with HDL (Table 1).Although all fibrates have been shown to increase HDL-C significantly, theirbeneficial effect on cardiovascular and all-cause mortality remains controversial(Saha et al. 2007). The early VA-HIT and BIP secondary prevention trials haveshown that treating CHD patients with low plasma HDL-C levels and especiallyTable 1 Effects of established hypolipidemic drugs on HDL concentration and functionStatins Fibrates NiacinHDL-C " 2–10 % " 5–15 % " 15–30 %HDL subclasses " large HDL particles " small HDL particles " large HDL particlesCell cholesterolefflux" up to 14 % (mainlySR-BI mediated)" up to 21 % (mainlyABCA1 mediated)" up to 25 % (mainlySR-BI mediated)Endothelial cellhomeostasis" FMDa" HDL-induced NOproductionb" FMDa# markers ofinflammation" FMD" EPC-mediatedendothelial repair" HDL-induced NOproductionAntioxidantactivity" PON1 activity " PON1 activity" HDL-bound LpPLA2" PON1 activityaNot necessarily related to HDL changesbIn vitro data606 M. Gomaraschi et al.those with moderate triglyceride elevations and some degree of insulin resis-tance (Rubins et al. 2002; The Bezafibrate Infarction Prevention (BIP) StudyGroup 2000) with a fibrate leads to significant reductions in the risk of arecurrent cardiovascular event, part of the effect being mediated by the fibrate-induced increase in HDL-C (Robins et al. 2001). In the FIELD study in peoplewith type 2 diabetes mellitus, fenofibrate induced a highly significant reductionof cardiovascular events in patients with low HDL-C levels, versus a minimaleffectiveness of the drug in patients with higher HDL-C (The FIELD studyinvestigators 2005). The more recent ACCORD trial shows no benefits of addingfibrate therapy to statin on cardiovascular events except possibly in patients withresidual atherogenic dyslipidemia (Ginsberg et al. 2010).Earlier studies with niacin suggested a favorable effect of the drug oncardiovascular mortality (Canner et al. 1986), but the AIM-HIGH trial, whichcompared the combination niacin/simvastatin with simvastatin alone, wasstopped for lack of efficacy, as niacin on the top of statin, despite increasingHDL-C levels, failed to determine an incremental clinical benefit in patients withLDL-C values at target (Boden et al. 2011). The more recent and much largerHPS2-THRIVE trial, also designed to assess the effect of adding niacin to astatin therapy in patients with established cardiovascular disease, also gavedisappointing results (HPS2-THRIVE Collaborative Group 2013).Novel HDL-raising drugs targeting new players in the HDL system, and morepotent than the available drugs, are urgently needed. The promising CETPinhibitors, despite substantially increasing HDL-C levels, did not give so farpositive results on cardiovascular risk (Barter et al. 2007; Schwartz et al. 2013).Different is the case of the HDL mimetics, which appears to be more promising,at least from results on surrogate end points (Nissen et al. 2003; Tardifet al. 2007), awaiting morbidity and mortality studies.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesAdorni MP, Zimetti F, Billheimer JT, Wang N, Rader DJ, Phillips MC, Rothblat GH (2007) Theroles of different pathways in the release of cholesterol from macrophages. J Lipid Res48:2453–2462Alaupovic P, Hodis HN, Knight Gibson C, Mack WJ, LaBree L, Cashin Hemphill L, Corder CN,Kramsch DM, Blankenhorn DH (1994) Effects of lovastatin on ApoA- and ApoB-containinglipoproteins families in a subpopulation of patients participating in the monitored atheroscle-rosis regression study (MARS). Arterioscler Thromb 14:1906–1913Alrasadi K, Awan Z, Alwaili K, Ruel I, Hafiane A, Krimbou L, Genest J (2008) Comparison oftreatment of severe high-density lipoprotein cholesterol deficiency in men with dailyatorvastatin (20 mg) versus fenofibrate (200 mg) versus extended-release niacin (2 g). Am JCardiol 102:1341–1347Effects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 607Antonopoulos AS, Margaritis M, Lee R, Channon K, Antoniades C (2012) Statins as anti-inflammatory agents in atherogenesis: molecular mechanisms and lessons from the recentclinical trials. Curr Pharm Des 18:1519–1530Arii K, Suehiro T, Ota K, Ikeda Y, Kumon Y, Osaki F, Inoue M, Inada S, Ogami N, Takata H,Hashimoto K, Terada Y (2009) Pitavastatin induces PON1 expression through p44/42mitogen-activated protein kinase signaling cascade in Huh7 cells. Atherosclerosis202:439–445Asztalos BF, Schaefer EJ (2003) High-density lipoprotein subpopulations in pathologicconditions. Am J Cardiol 91:12E–17EAsztalos BF, Horvath K, McNamara J, Roheim P, Rubinstein J, Schaefer E (2002a) Comparing theeffects of five different statins on the HDL subpopulation profiles of coronary heart diseasepatients. Atherosclerosis 164:361–369Asztalos BF, Horvath KV, McNamara JR, Roheim PS, Rubinstein JJ, Schaefer EJ (2002b) Effectsof atorvastatin on the HDL subpopulation profile of coronary heart disease patients. J Lipid Res43:1701–1707Asztalos BF, Le MF, Dallal GE, Stein E, Jones PH, Horvath KV, McTaggart F, Schaefer EJ (2007)Comparison of the effects of high doses of rosuvastatin versus atorvastatin on thesubpopulations of high-density lipoproteins. Am J Cardiol 99:681–685Asztalos BF, Collins D, Horvath KV, Bloomfield HE, Robins SJ, Schaefer EJ (2008) Relation ofgemfibrozil treatment and high-density lipoprotein subpopulation profile with cardiovascularevents in the veterans affairs high-density lipoprotein intervention trial. Metabolism 57:77–83Avogaro A, Miola M, Favaro A, Gottardo L, Pacini G, Manzato E, Zambon S, Sacerdoti D, deKreutzenberg S, Piliego T, Tiengo A, Del Prato S (2001) Gemfibrozil improves insulinsensitivity and flow-mediated vasodilatation in type 2 diabetic patients. Eur J Clin Invest31:603–609Bard JM, Luc G, Douste-Blazy P, Drouin P, Ziegler O, Jacotot B, Dachet C, de Gennes JL,Fruchart JC (1989) Effect of simvastatin on plasma lipids, apolipoproteins and lipoproteinparticles in patients with primary hypercholesterolaemia. Eur J Clin Pharmacol 37:545–550Barter PJ, Baker PW, Rye KA (2002) Effect of high-density lipoproteins on the expression ofadhesion molecules in endothelial cells. Curr Opin Lipidol 13:285–288Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJ, Komajda M, Lopez-Sendon J,Mosca L, Tardif JC, Waters DD, Shear CL, Revkin JH, Buhr KA, Fisher MR, Tall AR, BrewerB (2007) Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med357:2109–2122Barter PJ, Brandrup-Wognsen G, Palmer MK, Nicholls SJ (2010) Effect of statins on HDL-C: acomplex process unrelated to changes in LDL-C: analysis of the VOYAGER Database. J LipidRes 51:1546–1553Benjo AM, Maranhao RC, Coimbra SR, Andrade AC, Favarato D, Molina MS, Brandizzi LI, daLuz PL (2006) Accumulation of chylomicron remnants and impaired vascular reactivity occurin subjects with isolated low HDL cholesterol: effects of niacin treatment. Atherosclerosis187:116–122Berger JP, Akiyama TE, Meinke PT (2005) PPARs: therapeutic targets for metabolic disease.Trends Pharmacol Sci 26:244–251Bergheanu SC, Van Tol A, Dallinga-Thie GM, Liem A, Dunselman PH, Van der Bom JG, JukemaJW (2007) Effect of rosuvastatin versus atorvastatin treatment on paraoxonase-1 activity inmen with established cardiovascular disease and a low HDL-cholesterol. Curr Med Res Opin23:2235–2240Birjmohun RS, Hutten BA, Kastelein JJ, Stroes ES (2005) Efficacy and safety of high-densitylipoprotein cholesterol-increasing compounds. A meta-analysis of randomized controlledtrials. J Am Coll Cardiol 45:185–197Boden WE, Probstfield JL, Anderson T, Chaitman BR, Desvignes-Nickens P, Koprowicz K,McBride R, Teo K, Weintraub W (2011) Niacin in patients with low HDL cholesterol levelsreceiving intensive statin therapy. N Engl J Med 365:2255–2267Bodor ET, Offermanns S (2008) Nicotinic acid: an old drug with a promising future. Br JPharmacol 153(Suppl 1):S68–S75608 M. Gomaraschi et al.Bregar U, Jug B, Keber I, Cevc M, Sebestjen M (2013) Extended-release niacin/laropiprantimproves endothelial function in patients after myocardial infarction. Heart Vessel 29(3):313–319Broncel M, Cieslak D, Koter-Michalak M, Duchnowicz P, Mackiewicz K, Chojnowska-JezierskaJ (2006) The anti-inflammatory and antioxidants effects of micronized fenofibrate in patientswith visceral obesity and dyslipidemia. Pol Merkur Lekarski 20:547–550Calabresi L, Franceschini G, Sirtori CR, de Palma A, Saresella M, Ferrante P, Taramelli D (1997)Inhibition of VCAM-1 expression in endothelial cells by reconstituted high densitylipoproteins. Biochem Biophys Res Commun 238:61–65Calabresi L, Gomaraschi M, Villa B, Omoboni L, Dmitrieff C, Franceschini G (2002) Elevatedsoluble cellular adhesion molecules in subjects with low HDL-cholesterol. ArteriosclerThromb Vasc Biol 22:656–661Calabresi L, Gomaraschi M, Franceschini G (2003) Endothelial protection by high-densitylipoproteins: from bench to bedside. Arterioscler Thromb Vasc Biol 23:1724–1731Calabresi L, Gomaraschi M, Franceschini G (2010) High-density lipoprotein quantity or qualityfor cardiovascular prevention? Curr Pharm Des 16:1494–1503Canner PL, Berge KG, Wenger NK, Stamler J, Friedman L, Prineas RJ, Friedewald WT (1986)Fifteen year mortality in coronary drug project patients: longterm benefit with niacin. J AmColl Cardiol 8:1245–1255Cockerill GW, Rye KA, Gamble JR, Vadas MA, Barter PJ (1995) High-density lipoproteins inhibitcytokine-induced expression of endothelial cell adhesion molecules. Arterioscler Thromb VascBiol 15:1987–1994Cuchel M, Rader DJ (2006) Macrophage reverse cholesterol transport: key to the regression ofatherosclerosis? Circulation 113:2548–2555Davidson WS, Silva RA, Chantepie S, Lagor WR, Chapman MJ, Kontush A (2009) Proteomicanalysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance toantioxidative function. Arterioscler Thromb Vasc Biol 29:870–876De Lalla OF, Gofman JW (1954) Ultracentrifugal analysis of serum lipoproteins. MethodsBiochem Anal 1:459–478De Vries R, Kerstens MN, Sluiter WJ, Groen AK, Van TA, Dullaart RP (2005) Cellular cholesterolefflux to plasma from moderately hypercholesterolaemic type 1 diabetic patients is enhanced,and is unaffected by simvastatin treatment. Diabetologia 48:1105–1113Deanfield JE, Halcox JP, Rabelink TJ (2007) Endothelial function and dysfunction: testing andclinical relevance. Circulation 115:1285–1295Dullaart RP, De VR, Voorbij HA, Sluiter WJ, Van TA (2009) Serum paraoxonase-I activity isunaffected by short-term administration of simvastatin, bezafibrate, and their combination intype 2 diabetes mellitus. Eur J Clin Invest 39:200–203Evans M, Anderson RA, Graham J, Ellis GR, Morris K, Davies S, Jackson SK, Lewis MJ,Frenneaux MP, Rees A (2000) Ciprofibrate therapy improves endothelial function and reducespostprandial lipemia and oxidative stress in type 2 diabetes mellitus. Circulation101:1773–1779Favari E, Calabresi L, Adorni MP, Jessup W, Simonelli S, Franceschini G, Bernini F (2009) Smalldiscoidal pre-beta1 HDL particles are efficient acceptors of cell cholesterol via ABCA1 andABCG1. Biochemistry 48:11067–11074Florentin M, Liberopoulos EN, Wierzbicki AS, Mikhailidis DP (2008) Multiple actions of high-density lipoprotein. Curr Opin Cardiol 23:370–378Franceschini G, Sirtori M, Vaccarino V, Gianfranceschi G, Chiesa G, Sirtori CR (1989) Plasmalipoprotein changes after treatment with pravastatin and gemfibrozil in patients with familialhypercholesterolemia. J Lab Clin Med 114:250–259Franceschini G, Calabresi L, Colombo C, Favari E, Bernini F, Sirtori CR (2007) Effects offenofibrate and simvastatin on HDL-related biomarkers in low-HDL patients. Atherosclerosis195:385–391Franceschini G, Favari E, Calabresi L, Simonelli S, Bondioli A, Adorni MP, Zimetti F,Gomaraschi M, Coutant K, Rossomanno S, Niesor EJ, Bernini F, Benghozi R (2013) Differ-ential effects of fenofibrate and extended-release niacin on high-density lipoprotein particleEffects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 609size distribution and cholesterol efflux capacity in dyslipidemic patients. J Clin Lipidol7:414–422Fuhrman B, Koren L, Volkova N, Keidar S, Hayek T, Aviram M (2002) Atorvastatin therapy inhypercholesterolemic patients suppresses cellular uptake of oxidized-LDL by differentiatingmonocytes. Atherosclerosis 164:179–185Ganji SH, Qin S, Zhang L, Kamanna VS, Kashyap ML (2009) Niacin inhibits vascular oxidativestress, redox-sensitive genes, and monocyte adhesion to human aortic endothelial cells.Atherosclerosis 202:68–75Ghani RA, Bin YI, Wahab NA, Zainudin S, Mustafa N, Sukor N, Wan Mohamud WN, Kadir KA,Kamaruddin NA (2013) The influence of fenofibrate on lipid profile, endothelial dysfunction,and inflammatory markers in type 2 diabetes mellitus patients with typical and mixeddyslipidemia. J Clin Lipidol 7:446–453Ginsberg HN, Elam MB, Lovato LC, Crouse JR III, Leiter LA, Linz P, Friedewald WT, Buse JB,Gerstein HC, Probstfield J, Grimm RH, Ismail-Beigi F, Bigger JT, Goff DC Jr, Cushman WC,Simons-Morton DG, Byington RP (2010) Effects of combination lipid therapy in type 2 diabe-tes mellitus. N Engl J Med 362:1563–1574Gomaraschi M, Basilico N, Sisto F, Taramelli D, Eligini S, Colli S, Sirtori CR, Franceschini G,Calabresi L (2005) High-density lipoproteins attenuate interleukin-6 production in endothelialcells exposed to pro-inflammatory stimuli. Biochim Biophys Acta 1736:136–143Guerin M, Lassel TS, Le Goff W, Farnier M, Chapman MJ (2000) Action of atorvastatin incombined hyperlipidemia : preferential reduction of cholesteryl ester transfer from HDL toVLDL1 particles. Arterioscler Thromb Vasc Biol 20:189–197Guerin M, Egger P, Soudant C, Le Goff W, van Tol A, Dupuis R, Chapman MJ (2002) Dose-dependent action of atorvastatin in type IIB hyperlipidemia: preferential and progressivereduction of atherogenic apoB-containing lipoprotein subclasses (VLDL-2, IDL, small denseLDL) and stimulation of cellular cholesterol efflux. Atherosclerosis 163:287–296Guerin M, Le Goff W, Frisdal E, Schneider S, Milosavljevic D, Bruckert E, Chapman MJ (2003)Action of ciprofibrate in type IIb hyperlipoproteinemia: modulation of the atherogenic lipo-protein phenotype and stimulation of high-density lipoprotein-mediated cellular cholesterolefflux. J Clin Endocrinol Metab 88:3738–3746Hamoud S, Kaplan M, Meilin E, Hassan A, Torgovicky R, Cohen R, Hayek T (2013) Niacinadministration significantly reduces oxidative stress in patients with hypercholesterolemia andlow levels of high-density lipoprotein cholesterol. Am J Med Sci 345:195–199Harangi M, Seres I, Varga Z, Emri G, Szilvassy Z, Paragh G, Remenyik E (2004) Atorvastatineffect on high-density lipoprotein-associated paraoxonase activity and oxidative DNA damage.Eur J Clin Pharmacol 60:685–691Harangi M, Mirdamadi HZ, Seres I, Sztanek F, Molnar M, Kassai A, Derdak Z, Illyes L, Paragh G(2009) Atorvastatin effect on the distribution of high-density lipoprotein subfractions andhuman paraoxonase activity. Transl Res 153:190–198Hernandez M, Wright SD, Cai TQ (2007) Critical role of cholesterol ester transfer protein innicotinic acid-mediated HDL elevation in mice. Biochem Biophys Res Commun355:1075–1080Higashi Y, Matsuoka H, Umei H, Sugano R, Fujii Y, Soga J, Kihara Y, Chayama K, Imaizumi T(2010) Endothelial function in subjects with isolated low HDL cholesterol: role of nitric oxideand circulating progenitor cells. Am J Physiol Endocrinol Metab 298:E202–E209Homma Y, Ozawa H, Kobayashi T, Yamaguchi H, Sakane H, Nakamura H (1995) Effects ofsimvastatin on plasma lipoprotein subfractions, cholesterol esterification rate, and cholesterylester transfer protein in type II hyperlipoproteinemia. Atherosclerosis 114:223–234HPS2-THRIVE Collaborative Group (2013) HPS2-THRIVE randomized placebo-controlled trialin 25 673 high-risk patients of ER niacin/laropiprant: trial design, pre-specified muscle andliver outcomes, and reasons for stopping study treatment. Eur Heart J 34:1279–1291Igarashi J, Miyoshi M, Hashimoto T, Kubota Y, Kosaka H (2007) Statins induce S1P(1) receptorsand enhance endothelial nitric oxide production in response to high-density lipoproteins. Br JPharmacol 150:470–479610 M. Gomaraschi et al.Johansson J, Carlson LA (1990) The effects of nicotinic acid treatment on high density lipoproteinparticle size subclass levels in hyperlipidaemic subjects. Atherosclerosis 83:207–216Jones PH, Davidson MH, Stein EA, Bays HE, McKenney JM, Miller E, Cain VA, Blasetto JW(2003) Comparison of the efficacy and safety of rosuvastatin versus atorvastatin, simvastatin,and pravastatin across doses (STELLAR* Trial). Am J Cardiol 92:152–160Kamanna VS, Kashyap ML (2008) Mechanism of action of niacin. Am J Cardiol 101:20B–26BKaplon RE, Gano LB, Seals DR (2014) Vascular endothelial function and oxidative stress arerelated to dietary niacin intake among healthy middle-aged and older adults. J Appl Physiol(1985) 116:156–163Kassai A, Illyes L, Mirdamadi HZ, Seres I, Kalmar T, Audikovszky M, Paragh G (2007) The effectof atorvastatin therapy on lecithin:cholesterol acyltransferase, cholesteryl ester transfer proteinand the antioxidant paraoxonase. Clin Biochem 40:1–5Khera AV, Cuchel M, De LL-M, Rodrigues A, Burke MF, Jafri K, French BC, Phillips JA,Mucksavage ML, Wilensky RL, Mohler ER, Rothblat GH, Rader DJ (2011) Cholesterol effluxcapacity, high-density lipoprotein function, and atherosclerosis. N Engl J Med 364:127–135Khera AV, Patel PJ, Reilly MP, Rader DJ (2013) The addition of niacin to statin therapy improveshigh-density lipoprotein cholesterol levels but not metrics of functionality. J Am Coll Cardiol62:1909–1910Khoury N, Goldberg AC (2011) The use of fibric acid derivatives in cardiovascular prevention.Curr Treat Option Cardiovasc Med 13:335–342Kimura T, Mogi C, Tomura H, Kuwabara A, Im DS, Sato K, Kurose H, Murakami M, Okajima F(2008) Induction of scavenger receptor class B type I is critical for simvastatin enhancement ofhigh-density lipoprotein-induced anti-inflammatory actions in endothelial cells. J Immunol181:7332–7340Knopp RH, Alagona P, Davidson M, Goldberg AC, Kafonek SD, Kashyap M, Sprecher D,Superko HR, Jenkins S, Marcovina S (1998) Equivalent efficacy of a time-release form ofniacin (Niaspan) given once-a-night versus plain niacin in the management of hyperlipidemia.Metabolism 47:1097–1104Koh KK, Yeal AJ, Hwan HS, Kyu JD, Sik KH, Cheon LK, Kyun SE, Sakuma I (2004) Effects offenofibrate on lipoproteins, vasomotor function, and serological markers of inflammation,plaque stabilization, and hemostasis. Atherosclerosis 174:379–383Koh KK, Quon MJ, Han SH, Chung WJ, Ahn JY, Seo YH, Choi IS, Shin EK (2005) Additivebeneficial effects of fenofibrate combined with atorvastatin in the treatment of combinedhyperlipidemia. J Am Coll Cardiol 45:1649–1653Kontush A, Chapman MJ (2010) Antiatherogenic function of HDL particle subpopulations: focuson antioxidative activities. Curr Opin Lipidol 21:312–318Kuvin JT, Ramet ME, Patel AR, Pandian NG, Mendelsohn ME, Karas RH (2002) A novelmechanism for the beneficial vascular effects of high-density lipoprotein cholesterol: enhancedvasorelaxation and increased endothelial nitric oxide synthase expression. Am Heart J144:165–172Kuvin JT, Dave DM, Sliney KA, Mooney P, Patel AR, Kimmelstiel CD, Karas RH (2006) Effectsof extended-release niacin on lipoprotein particle size, distribution, and inflammatory markersin patients with coronary artery disease. Am J Cardiol 98:743–745Lamon-Fava S, Diffenderfer MR, Barrett PH, Buchsbaum A, NyakuM, Horvath KV, Asztalos BF,Otokozawa S, Ai M, Matthan NR, Lichtenstein AH, Dolnikowski GG, Schaefer EJ (2008)Extended-release niacin alters the metabolism of plasma apolipoprotein (Apo) A-I and ApoB-containing lipoproteins. Arterioscler Thromb Vasc Biol 28:1672–1678Lee JM, Robson MD, Yu LM, Shirodaria CC, Cunnington C, Kylintireas I, Digby JE, Bannister T,Handa A, Wiesmann F, Durrington PN, Channon KM, Neubauer S, Choudhury RP (2009)Effects of high-dose modified-release nicotinic acid on atherosclerosis and vascular function arandomized, placebo-controlled, magnetic resonance imaging study. J Am Coll Cardiol54:1787–1794Effects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 611Lewis GF, Rader DJ (2005) New insights into the regulation of HDL metabolism and reversecholesterol transport. Circ Res 96:1221–1232Liu Y, Wei J, Hu S, Hu L (2012) Beneficial effects of statins on endothelial progenitor cells. Am JMed Sci 344:220–226Malik J, Melenovsky V, Wichterle D, Haas T, Simek J, Ceska R, Hradec J (2001) Both fenofibrateand atorvastatin improve vascular reactivity in combined hyperlipidaemia (fenofibrate versusatorvastatin trial – FAT). Cardiovasc Res 52:290–298Maranghi M, Hiukka A, Badeau R, Sundvall J, Jauhiainen M, Taskinen MR (2011) Macrophagecholesterol efflux to plasma and HDL in subjects with low and high homocysteine levels: aFIELD substudy. Atherosclerosis 219:259–265McGrath KC, Li XH, Puranik R, Liong EC, Tan JT, Dy VM, DiBartolo BA, Barter PJ, Rye KA,Heather AK (2009) Role of 3beta-hydroxysteroid-Delta 24 reductase in mediatingantiinflammatory effects of high-density lipoproteins in endothelial cells. Arterioscler ThrombVasc Biol 29:877–882Miida T, Sakai K, Ozaki K, Nakamura Y, Yamaguchi T, Tsuda T, Kashiwa T, Murakami T,Inano K, Okada M (2000) Bezafibrate increases Prebeta1-HDL at the expense of HDL(2b) inhypertriglyceridemia. Arterioscler Thromb Vasc Biol 20:2428–2433Mineo C, Shaul PW (2012) Novel biological functions of high-density lipoprotein cholesterol.Circ Res 111:1079–1090Mirdamadi HZ, Sztanek F, Derdak Z, Seres I, Harangi M, Paragh G (2008) The humanparaoxonase-1 phenotype modifies the effect of statins on paraoxonase activity and lipidparameters. Br J Clin Pharmacol 66:366–374Miyamoto-Sasaki M, Yasuda T, Monguchi T, Nakajima H, Mori K, Toh R, Ishida T, Hirata K(2013) Pitavastatin increases HDL particles functionally preserved with cholesterol effluxcapacity and antioxidative actions in dyslipidemic patients. J Atheroscler Thromb 20:708–716Morgan JM, Capuzzi DM, Baksh RI, Intenzo C, Carey CM, Reese D, Walker K (2003) Effects ofextended-release niacin on lipoprotein subclass distribution. Am J Cardiol 91:1432–1436Morgan JM, De LL-M, Capuzzi DM (2007) Effects of niacin and Niaspan on HDL lipoproteincellular SR-BI-mediated cholesterol efflux. J Clin Lipidol 1:614–619Muacevic-Katanec D, Bradamante V, Poljicanin T, Reiner Z, Babic Z, Simeon-Rudolf V, KatanecD (2007) Clinical study on the effect of simvastatin on paraoxonase activity. Arzneimittel-forschung 57:647–653Nagila A, Permpongpaiboon T, Tantrarongroj S, Porapakkham P, Chinwattana K, Deakin S,Porntadavity S (2009) Effect of atorvastatin on paraoxonase1 (PON1) and oxidative status.Pharmacol Rep 61:892–898Nichols AV, Krauss RM, Musliner TA (1986) Nondenaturing polyacrylamide gradient gel elec-trophoresis. Methods Enzymol 128:417–431Nissen SE, Tsunoda T, Tuzcu EM, Schoenhagen P, Cooper CJ, Yasin M, Eaton GM, Lauer MA,Sheldon WS, Grines CL, Halpern S, Crowe T, Blankenship JC, Kerensky R (2003) Effect ofrecombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronarysyndromes: a randomized controlled trial. JAMA 290:2292–2300Nofer JR, van der Giet M, Tolle M, Wolinska I, von Wnuck LK, Baba HA, Tietge UJ, Godecke A,Ishii I, Kleuser B, Schafers M, Fobker M, Zidek W, Assmann G, Chun J, Levkau B (2004)HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J ClinInvest 113:569–581Otocka-Kmiecik A, Mikhailidis DP, Nicholls SJ, Davidson M, Rysz J, Banach M (2012) Dys-functional HDL: a novel important diagnostic and therapeutic target in cardiovascular disease?Prog Lipid Res 51:314–324Otvos JD, Collins D, Freedman DS, Shalaurova I, Schaefer EJ, McNamara JR, Bloomfield HE,Robins SJ (2006) Low-density lipoprotein and high-density lipoprotein particle subclassespredict coronary events and are favorably changed by gemfibrozil therapy in the veteransaffairs high-density lipoprotein intervention trial. Circulation 113:1556–1563612 M. Gomaraschi et al.Paragh G, Seres I, Harangi M, Balogh Z, Illyes L, Boda J, Szilvassy Z, Kovacs P (2003) The effectof micronised fenofibrate on paraoxonase activity in patients with coronary heart disease.Diabetes Metab 29:613–618Patsch JR, Prasad S, Gotto AM Jr, Bengtsson-Olivecrona G (1984) Postprandial lipemia. A key forthe conversion of high density lipoprotein2 into high density lipoprotein3 by hepatic lipase. JClin Invest 74:2017–2023Petoumenos V, Nickenig G, Werner N (2009) High-density lipoprotein exerts vasculoprotectionvia endothelial progenitor cells. J Cell Mol Med 13:4623–4635Philpott AC, Hubacek J, Sun YC, Hillard D, Anderson TJ (2013) Niacin improves lipid profile butnot endothelial function in patients with coronary artery disease on high dose statin therapy.Atherosclerosis 226:453–458Phuntuwate W, Suthisisang C, Koanantakul B, Chaloeiphap P, Mackness B, Mackness M (2008)Effect of fenofibrate therapy on paraoxonase1 status in patients with low HDL-C levels.Atherosclerosis 196:122–128Podrez EA (2010) Anti-oxidant properties of high-density lipoprotein and atherosclerosis. ClinExp Pharmacol Physiol 37:719–725Reriani MK, Dunlay SM, Gupta B, West CP, Rihal CS, Lerman LO, Lerman A (2011) Effects ofstatins on coronary and peripheral endothelial function in humans: a systematic review andmeta-analysis of randomized controlled trials. Eur J Cardiovasc Prev Rehabil 18:704–716Riwanto M, Landmesser U (2013) High density lipoproteins and endothelial functions: mechanis-tic insights and alterations in cardiovascular disease. J Lipid Res 54:3227–3243Robins SJ, Collins D, Wittes JT, Papademetriou V, Deedwania PC, Schaefer EJ, McNamara JR,Kashyap ML, Hershman JM, Wexler LF, Rubins HB (2001) Relation of gemfibrozil treatmentand lipid levels with major coronary events: VA-HIT: a randomized controlled trial. JAMA285:1585–1591Rosenson RS, Brewer HB Jr, Ansell B, Barter P, ChapmanMJ, Heinecke JW, Kontush A, Tall AR,Webb NR (2013) Translation of high-density lipoprotein function into clinical practice: currentprospects and future challenges. Circulation 128:1256–1267Rubic T, Trottmann M, Lorenz RL (2004) Stimulation of CD36 and the key effector of reversecholesterol transport ATP-binding cassette A1 in monocytoid cells by niacin. BiochemPharmacol 67:411–419Rubins HB, Robins SJ, Collins D, Nelson DB, Elam MB, Schaefer EJ, Faas FH, Anderson JW(2002) Diabetes, plasma insulin, and cardiovascular disease: subgroup analysis from thedepartment of veterans affairs high-density lipoprotein intervention trial (VA-HIT). ArchIntern Med 162:2597–2604Saha SA, Kizhakepunnur LG, Bahekar A, Arora RR (2007) The role of fibrates in the prevention ofcardiovascular disease–a pooled meta-analysis of long-term randomized placebo-controlledclinical trials. Am Heart J 154:943–953Sakai T, Kamanna VS, Kashyap ML (2001) Niacin, but not gemfibrozil, selectively increasesLP-AI, a cardioprotective subfraction of HDL, in patients with low HDL cholesterol.Arterioscler Thromb Vasc Biol 21:1783–1789Sasaki J, Yamamoto K, Ageta M (2002) Effects of fenofibrate on high-density lipoprotein particlesize in patients with hyperlipidemia: a randomized, double-blind, placebo-controlled, multi-center, crossover study. Clin Ther 24:1614–1626Schwartz GG, Olsson AG, Barter PJ (2013) Dalcetrapib in patients with an acute coronarysyndrome. N Engl J Med 368:869–870Shepherd J, Packard CJ, Patsch JR, Gotto AM Jr, Taunton OD (1979) Effects of nicotinic acidtherapy on plasma high density lipoprotein subfraction distribution and composition onapolipoprotein A metabolism. J Clin Invest 63:858–867Shuhei N, Soderlund S, Jauhiainen M, Taskinen MR (2010) Effect of HDL composition andparticle size on the resistance of HDL to the oxidation. Lipids Health Dis 9:104Sorrentino SA, Besler C, Rohrer L, Meyer M, Heinrich K, Bahlmann FH, Mueller M, Horvath T,Doerries C, Heinemann M, Flemmer S, Markowski A, Manes C, Bahr MJ, Haller H, Von EA,Effects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 613Drexler H, Landmesser U (2010) Endothelial-vasoprotective effects of high-density lipopro-tein are impaired in patients with type 2 diabetes mellitus but are improved after extended-release niacin therapy. Circulation 121:110–122Soudijn W, van Wijngaarden I, Ijzerman AP (2007) Nicotinic acid receptor subtypes and theirligands. Med Res Rev 27:417–433Stremler KE, Stafforini DM, Prescott SM, McIntyre TM (1991) Human plasma platelet-activatingfactor acetylhydrolase. Oxidatively fragmented phospholipids as substrates. J Biol Chem266:11095–11103Sviridov D, Hoang A, Ooi E, Watts G, Barrett PH, Nestel P (2008) Indices of reverse cholesteroltransport in subjects with metabolic syndrome after treatment with rosuvastatin. Atherosclero-sis 197:732–739Tamehiro N, Shigemoto-Mogami Y, Kakeya T, Okuhira K, Suzuki K, Sato R, Nagao T,Nishimaki-Mogami T (2007) Sterol regulatory element-binding protein-2- and liver Xreceptor-driven dual promoter regulation of hepatic ABC transporter A1 gene expression:mechanism underlying the unique response to cellular cholesterol status. J Biol Chem282:21090–21099Tardif JC, Gregoire J, L’Allier PL, Ibrahim R, Lesperance J, Heinonen TM, Kouz S, Berry C,Basser R, Lavoie MA, Guertin MC, Rodes-Cabau J (2007) Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial.JAMA 297:1675–1682The Bezafibrate Infarction Prevention (BIP) Study Group (2000) Secondary prevention by raisingHDL cholesterol and reducing triglycerides in patients with coronary artery disease: theBezafibrate Infarction Prevention (BIP) study. Circulation 102:21–27The FIELD study investigators (2005) Effects of long-term fenofibrate therapy on cardiovascularevents in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlledtrial. Lancet 366:1849–1861Thoenes M, Oguchi A, Nagamia S, Vaccari CS, Hammoud R, Umpierrez GE, Khan BV (2007)The effects of extended-release niacin on carotid intimal media thickness, endothelial functionand inflammatory markers in patients with the metabolic syndrome. Int J Clin Pract61:1942–1948Tkac I, Molcanyiova A, Javorsky M, Kozarova M (2006) Fenofibrate treatment reduces circulatingconjugated diene level and increases glutathione peroxidase activity. Pharmacol Res53:261–264Tomas M, Senti M, Garcia-Faria F, Vila J, Torrents A, Covas M, Marrugat J (2000) Effect ofsimvastatin therapy on paraoxonase activity and related lipoproteins in familial hypercholes-terolemic patients. Arterioscler Thromb Vasc Biol 20:2113–2119Triolo M, Annema W, de Boer JF, Tietge UJ, Dullaart RP (2013a) Simvastatin and bezafibrateincrease cholesterol efflux in men with type 2 diabetes. Eur J Clin Invest 44(3):240–248Triolo M, Kwakernaak AJ, Perton FG, De Vries R, Dallinga-Thie GM, Dullaart RP (2013b) Lownormal thyroid function enhances plasma cholesteryl ester transfer in type 2 diabetes mellitus.Atherosclerosis 228:466–471Tsimihodimos V, Kakafika A, Tambaki AP, Bairaktari E, Chapman MJ, Elisaf M, Tselepis AD(2003) Fenofibrate induces HDL-associated PAF-AH but attenuates enzyme activityassociated with apoB-containing lipoproteins. J Lipid Res 44:927–934van der Hoogt CC, de Haan W, Westerterp M, Hoekstra M, Dallinga-Thie GM, Romijn JA,Princen HM, Jukema JW, Havekes LM, Rensen PC (2007) Fenofibrate increasesHDL-cholesterol by reducing cholesteryl ester transfer protein expression. J Lipid Res48:1763–1771Vazzana N, Ganci A, Cefalu AB, Lattanzio S, Noto D, Santoro N, Saggini R, Puccetti L,Averna M, Davi G (2013) Enhanced lipid peroxidation and platelet activation as potentialcontributors to increased cardiovascular risk in the low-HDL phenotype. J Am Heart Assoc 2:e000063614 M. Gomaraschi et al.Vega GL, Grundy SM (1994) Lipoprotein responses to treatment with lovastatin, gemfibrozil, andnicotinic acid in normolipidemic patients with hypoalphalipoproteinemia. Arch Intern Med154:73–82Wahlberg G, Walldius G, Olsson AG, Kirstein P (1990) Effects of nicotinic acid on serumcholesterol concentrations of high density lipoprotein subfractions HDL2 and HDL3 inhyperlipoproteinaemia. J Intern Med 228:151–157Wang TD, Chen WJ, Lin JW, Cheng CC, Chen MF, Lee YT (2003) Efficacy of fenofibrate andsimvastatin on endothelial function and inflammatory markers in patients with combinedhyperlipidemia: relations with baseline lipid profiles. Atherosclerosis 170:315–323Warnholtz A, Wild P, Ostad MA, Elsner V, Stieber F, Schinzel R, Walter U, Peetz D, Lackner K,Blankenberg S, Munzel T (2009) Effects of oral niacin on endothelial dysfunction in patientswith coronary artery disease: results of the randomized, double-blind, placebo-controlled INEFstudy. Atherosclerosis 204:216–221Xia P, Vadas MA, Rye KA, Barter PJ, Gamble JR (1999) High density lipoproteins (HDL)interrupt the sphingosine kinase signaling pathway. A possible mechanism for protectionagainst atherosclerosis by HDL. J Biol Chem 274:33143–33147Yadav R, France M, Younis N, Hama S, Ammori BJ, Kwok S, Soran H (2012) Extended-releaseniacin with laropiprant : a review on efficacy, clinical effectiveness and safety. Expert OpinPharmacother 13:1345–1362Yamashita S, Tsubakio-Yamamoto K, Ohama T, Nakagawa-Toyama Y, Nishida M (2010)Molecular mechanisms of HDL-cholesterol elevation by statins and its effects on HDLfunctions. J Atheroscler Thromb 17:436–451Yesilbursa D, Serdar A, Saltan Y, Serdar Z, Heper Y, Guclu S, Cordan J (2005) The effect offenofibrate on serum paraoxonase activity and inflammatory markers in patients with com-bined hyperlipidemia. Kardiol Pol 62:526–530Yetukuri L, Huopaniemi I, Koivuniemi A, Maranghi M, Hiukka A, Nygren H, Kaski S, TaskinenMR, Vattulainen I, Jauhiainen M, Oresic M (2011) High density lipoprotein structural changesand drug response in lipidomic profiles following the long-term fenofibrate therapy in theFIELD substudy. PLoS ONE 6:e23589Yuhanna IS, Zhu Y, Cox BE, Hahner LD, Osborne-Lawrence S, Lu P, Marcel YL, Anderson RG,Mendelsohn ME, Hobbs HH, Shaul PW (2001) High-density lipoprotein binding to scavengerreceptor-BI activates endothelial nitric oxide synthase. Nat Med 7:853–857Yvan-Charvet L, Kling J, Pagler T, Li H, Hubbard B, Fisher T, Sparrow CP, Taggart AK, Tall AR(2010) Cholesterol efflux potential and antiinflammatory properties of high-density lipoproteinafter treatment with niacin or anacetrapib. Arterioscler Thromb Vasc Biol 30:1430–1438Effects of Established Hypolipidemic Drugs on HDL Concentration, Subclass. . . 615Emerging Small Molecule DrugsSophie Colin, Giulia Chinetti-Gbaguidi, Jan A. Kuivenhoven,and Bart StaelsContents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6182 Cholesteryl Ester Transfer Protein Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6192.1 Biological Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6192.2 Current State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6192.3 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6203 Novel PPAR Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6213.1 Biological Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6213.2 Current State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6223.3 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6224 Novel LXR Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6234.1 Biological Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6234.2 Current State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6244.3 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6245 RVX-208 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6255.1 Biological Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6255.2 Current State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626S. Colin • G. Chinetti-Gbaguidi • B. Staels (*)Université Lille 2, F-59000 Lille, FranceInserm, U1011, F-59000 Lille, FranceInstitut Pasteur de Lille, F-59019 Lille, FranceEuropean Genomic Institute for Diabetes (EGID), FR 3508, F-59000 Lille, Francee-mail: bart.staels@pasteur-lille.frJ.A. KuivenhovenSection Molecular Genetics, Department of Pediatrics, University Medical CenterGroningen, University of Groningen, 9713 AV Groningen, The Netherlands# The Author(s) 2015A. von Eckardstein, D. Kardassis (eds.), High Density Lipoproteins, Handbook ofExperimental Pharmacology 224, DOI 10.1007/978-3-319-09665-0_20617AbstractDyslipidaemia is a major risk factor for cardiovascular diseases. Pharmacologi-cal lowering of LDL-C levels using statins reduces cardiovascular risk. How-ever, a substantial residual risk persists especially in patients with type 2 diabetesmellitus. Because of the inverse association observed in epidemiological studiesof HDL-C with the risk for cardiovascular diseases, novel therapeutic strategiesto raise HDL-C levels or improve HDL functionality are developed as comple-mentary therapy for cardiovascular diseases. However, until now most therapiestargeting HDL-C levels failed in clinical trials because of side effects or absenceof clinical benefits. This chapter will highlight the emerging small moleculescurrently developed and tested in clinical trials to pharmacologically modulateHDL-C and functionality including new CETP inhibitors (anacetrapib,evacetrapib), novel PPAR agonists (K-877, CER-002, DSP-8658, INT131 andGFT505), LXR agonists (ATI-111, LXR-623, XL-652) and RVX-208.KeywordsHDL therapy • CETP inhibitors • PPAR • LXR • RVX-208AbbreviationsABCA1 ATP-binding cassette transporter A1ABCG1 ATP-binding cassette transporter G1ACC Acetyl-CoA carboxylaseApo apolipoproteinCETP Cholesteryl ester transfer proteinCVD Cardiovascular diseaseFAS Fatty acid synthaseHDL High-density lipoproteinLDL Low-density lipoproteinLXR Liver X receptorMTTP Microsomal triglyceride transfer proteinPPAR Peroxisome proliferator-activated receptorPCSK9 Proprotein convertase subtilisin/kexin type 9SCD-1 Stearoyl-CoA desaturase-1SREBP-1 Sterol regulatory element-binding protein1 IntroductionDyslipidaemia is a major risk factor for cardiovascular diseases, a main cause ofmorbidity and mortality worldwide, with 17.3 million deaths per year (Laslettet al. 2012). LDL-C-lowering therapy, especially with statins, has shown to be an618 S. Colin et al.efficient approach to reduce cardiovascular risk on average by 25–35 %. Althoughlowering LDL-C with statins has beneficial effects and reduces cardiovascularevents, significant numbers of residual cardiovascular events remain in high-riskpatients, prompting the search for alternative complementary approaches. Amongthese strategies, new agents combined with statins, such as proprotein convertasesubtilisin/kexin type 9 (PCSK9) inhibitors, microsomal triglyceride transfer protein(MTTP) inhibitors or ezetimibe can provide additional lowering LDL-C effects.Because HDL-C levels are inversely correlated with cardiovascular risk (Gordonet al. 1977), raising HDL-C levels has spawned high hopes as additional therapy forcardiovascular diseases. However, so far none of the pharmacological interventionsaimed at raising HDL-C levels has yielded convincing results with respect toreduction of cardiovascular risk.In this chapter we will focus on these emerging small molecule drugs indevelopment, including cholesteryl ester transfer protein (CETP) inhibitors, novelperoxisome proliferator-activated receptor (PPAR) agonists, liver X receptor(LXR) agonists and RVX-208. For each drug, the biological mechanisms of thesemolecules, an overview of the current state of the clinical trials and the futureperspectives will be provided.2 Cholesteryl Ester Transfer Protein Inhibitors2.1 Biological MechanismsThe cholesteryl ester transfer protein (CETP) promotes the transfer of triglyceridesfrom apoB-containing lipoproteins (LDL, IDL and VLDL) to HDL-C in exchangefor cholesteryl esters. Interest in CETP inhibitor development came from studies infamilies with CETP deficiency with hyperalphalipoproteinaemia CETP deficiency(Inazu 1990) and epidemiological studies showing that CETP gene variants areassociated with increased HDL-C levels and a lower risk of coronary heart diseaseevents (Curb et al. 2004).2.2 Current StateFour CETP inhibitors have been developed in humans: torcetrapib, anacetrapib,dalcetrapib and evacetrapib. The first CETP inhibitor designed by Pfizer and testedin phase III clinical trials was torcetrapib. The initial results of the ILLUMINATE(Investigation of Lipid Level Management to Understand its Impact in Atheroscle-rotic Events) trial were encouraging, with a 72 % increase in HDL-C and a 25 %decrease in LDL-C in patients with high CVD risk treated with torcetrapib on top ofatorvastatin (Kastelein et al. 2007). However, all studies were interrupted becauseof an increased risk of cardiovascular events and an excess of mortality upontorcetrapib usage, possibly due to an increase in aldosterone level and bloodpressure (Barter et al. 2007). Further analyses have demonstrated that the effectEmerging Small Molecule Drugs 619on blood pressure was independent of CETP inhibition. Indeed, torcetrapibincreases blood pressure in mice that do not express CETP (Forrest et al. 2008).Furthermore, genetic association studies in 58,948 subjects with polymorphism inCETP gene report that CETP genotype was not associated with systolic nordiastolic blood pressure (Sofat et al. 2010). These results suggest that toxicityupon torcetrapib treatment could be CETP independent.Even though the dal-OUTCOMES trial with dalcetrapib (Roche) showed anincrease of HDL-C by 30 %, the results showed futility and trials were halted due tothe absence of obvious benefit (Schwartz et al. 2012). Furthermore, the effect ofdalcetrapib on vessel wall structure and vascular inflammation was investigatedafter a 2-year treatment in the dal-PLAQUE trial (Fayad et al. 2011). No significantbenefit was reported in dalcetrapib-treated patients, but in patients with low HDL-Cat baseline, beneficial effects on endothelial function were observed. However, inthe dal-VESSEL study, designed to validate these results, the beneficial effectswere not confirmed (Lüscher et al. 2012). Nevertheless, despite these failures, novascular toxicity and blood pressure increase were observed on top of dalcetrapibtreatment (Lüscher et al. 2012). Based on these results, two other more potent CETPinhibitors, anacetrapib and evacetrapib, have been developed and are still in phaseIII clinical trials.2.3 Future PerspectivesAnacetrapib is a potent CETP inhibitor developed by Merck. In a first clinical trial,anacetrapib increased HDL-C by 138 % and reduced LDL-C by 40 % in patientswith coronary artery disease or at high risk for coronary heart disease on statintherapy. This clinical trial with the acronym DEFINE (Determining the EFficacyand Tolerability of CETP Inhibition with Anacetrapib), demonstrated the safety ofanacetrapib and absence of significant changes in blood pressure, aldosterone andelectrolyte levels (Cannon et al. 2010). Currently, a phase III clinical trial isongoing, acronym REVEAL (Randomized EValuation of the Effects ofAnacetrapib Through Lipid-modification), which will determine whether lipidmodification on anacetrapib therapy reduces the risk of coronary death, myocardialinfarction (MI) or coronary revascularisation in patients with circulatory problemsand low LDL-C on statin therapy (ClinicalTrials.gov identifier: NCT01252953).The results of this trial are expected at the beginning of 2017.Evacetrapib, designed by Eli Lilly & Company, is the fourth member of theCETP inhibitor class that is tested in clinical trials. A phase II clinical trialevaluated the efficacy of evacetrapib as monotherapy or in combination with themost prescribed statins in patients with either hypercholesterolaemia or low HDL-Clevels. As monotherapy, evacetrapib (30 mg, 100 mg or 500 mg/day) dose-dependently reduced LDL-C from 14 to 36 % and increased HDL-C from 54 to129 %. Although the decrease of LDL-C was higher in combination with statins(49 % vs. 24 %), the increase of HDL-C was not stronger when compared toevacetrapib monotherapy (Nicholls et al. 2011). No adverse effects were observedin this trial, with no changes in blood pressure or aldosterone levels. Thus620 S. Colin et al.evacetrapib appears to be well-tolerated (Nicholls et al. 2011). The benefits ofevacetrapib in combination with statins will be determined in a phase IIIrandomised outcome trial, acronym ACCELERATE (Assessment of ClinicalEffects of Cholesteryl Ester Transfer Protein Inhibition with Evacetrapib in Patientsat a High-Risk for Vascular Outcomes) (Estimated end date: January 2016;ClinicalTrials.gov identifier: NCT01687998).3 Novel PPAR Agonists3.1 Biological MechanismsPeroxisome proliferator-activated receptors (PPARs) are a nuclear receptor sub-family with three members, PPARα, PPARγ and PPARβ/δ, encoded by distinctgenes. The three isoforms display distinct patterns of expression with PPARα beinghighly expressed in liver, kidney, heart, muscle and brown adipose tissue, PPARγ ismost abundant in adipose tissue, whereas PPARβ/δ is ubiquitously expressed(Lefebvre et al. 2006). Upon heterodimerisation with the retinoic X receptor,PPARs bind to PPAR response elements (PPRE) located in the promoters of theirtarget genes and thus exert negative or positive control on their transcription.The role of PPARα was initially studied in the liver where it enhances fatty acidoxidation, regulates gluconeogenesis through an increase of pyruvate dehydroge-nase kinase 4 expression and ketone body production in response to the fastingstate. Through its natural ligands, such as long-chain unsaturated fatty acids,arachidonic acid derivatives and oxidised phospholipids, PPARα regulates alsosome genes involved in lipid and lipoprotein metabolism. The increase of apoAVand lipoprotein lipase expression by PPARα activation associated with the reduc-tion of apoCIII expression contributes to reduced plasma triglyceride levels inhumans. Moreover, plasma HDL cholesterol levels increase as a result of thestimulation of two major HDL-associated apolipoproteins, apoAI and apoAII, byPPARα (Staels et al. 1998). In addition to these hepatic effects, PPARα activationenhances reverse cholesterol transport related to the increase of ATP-bindingcassette transporter A1 (ABCA1) in macrophages (Chinetti et al. 2001) and exertsmany pleiotropic effects on vascular remodelling and inflammatory responses.PPARγ is activated by natural ligands such as polyunsaturated fatty acids,15-deoxy-Δ12,14-prostaglandin J2 and oxidised fatty acids. In addition, pharmaco-logical PPARγ agonists, the thiazolidinediones, are used as insulin sensitisers.Furthermore, PPARγ is the major regulator of adipogenesis (Tontonoz andSpiegelman 2008). PPARγ regulates the expression of adipokines such asadiponectin (Yu et al. 2002). In addition to this adipogenic effect, PPARγ displaysanti-inflammatory actions and promotes the polarisation of monocytes towardsalternative M2 macrophages (Bouhlel et al. 2007).PPARβ/δ is activated by long-chain unsaturated fatty acids, and several syn-thetic ligands have been designed including L-165041, GW501516 and GW0742.However, no PPARβ/δ agonists are in clinical use yet. PPARβ/δ is highly expressedin skeletal muscle where it increases the expression of fatty acid oxidation-relatedEmerging Small Molecule Drugs 621genes. This nuclear receptor also improves lipid metabolism by reducingtriglycerides and LDL-C levels and by increasing HDL-C levels. Moreover,PPARβ/δ activation increases insulin sensitivity (Oliver et al. 2001).Overall, PPARs are involved in the control of lipid lipoprotein and glucosemetabolism as well as in the inflammatory response (Lefebvre et al. 2006).3.2 Current StateAmong the pharmacologically used PPARα ligands are the fibrates, which arecurrently clinically used as hypolipidaemic drugs. Clinical benefits of fibrateswere reported in primary and secondary intervention trials and reviewed in ameta-analysis (Jun et al. 2010). In line with their capacity to increase HDL-C andreduce triglycerides and LDL-C (Jun et al. 2010), their effects on the incidence ofcoronary heart disease appear most pronounced in patients with high triglycerides(>200 mg/dL) and/or low HDL-C (<40 mg/dL), although this has not yet beenformally proven in a dedicated trial in type 2 diabetes patients (Suh et al. 2012;Keech et al. 2005).Each PPAR isoform displays specific roles, with PPARα controlling lipidmetabolism, whereas PPARδ improves also glucose metabolism. An interestingstrategy consisted in the development of dual agonists with synergistic effects ondifferent PPAR isoforms and minimal side effects (Rosenson et al. 2012). In thiscontext, Roche developed a dual PPAR α/γ agonist, aleglitazar. Despite promisingresults in the synchrony study, with an improvement of lipid and glucoseparameters in type 2 diabetes patients (Henry et al. 2009), the AleCardio phaseIII trial failed due to adverse effects on heart failure. Subsequently, all trials withaleglitazar were stopped (press report: http://www.roche.com/media/media_releases/med-cor-2013-07-10.htm).3.3 Future PerspectivesSome limitations of fibrate therapy are their relatively weak activity on PPARα andtheir efficacy that depends on the targeted population (Staels 2010). To addressthese issues, several highly selective and potent PPAR agonists were developed(Fruchart 2013). The KOWA Company is developing K-877, a potent PPARαagonist. In a comparative clinical trial (International Clinical Trials identifier:JPRN-JapicCTI-121764), patients with hypertriglyceridaemia and low HDL-Cwere treated with K-877 or fenofibrate for 12 weeks. The first results showed thatthe increase of HDL-C is stronger by K-877 than by fenofibrate. In addition, none ofthe adverse effects induced by fenofibrate, such as increased levels of serumhomocysteine and creatinine, were observed in K-877-treated patients (http://www.kenes.com/eas2012/abstracts/pdf/525.pdf). This molecule is currently inphase II in the USA and EU (International Clinical Trials identifier:EUCTR2013-001517-32-SE) and in phase III clinical trials for atheroscleroticdyslipidaemia in Japan (International Clinical Trials identifier: JPRN-JapicCTI-132067).622 S. Colin et al.A HDL-inducer developed by Cerenis Therapeutics is a specific PPARδ agonist,CER-002. The phase I clinical trial demonstrated that CER-002 is well-toleratedwithout major adverse effects (press report: http://www.drugs.com/clinical_trials/cerenis-therapeutics-announces-successful-completion-phase-clinical-trial-cer-002-cardiovascular-4272.html). Another selective PPARδ agonist, HPP593, is cur-rently tested in healthy subjects, and its effect on LDL and HDL cholesterol as well astriglycerides will be determined (press report: http://www.ttpharma.com/TherapeuticAreas/MetabolicDisorders/Dyslipidemia/HPP593/tabid/118/Default.aspx).The synthetic, non-thiazolidinedione PPARγ compound, INT131, improvesglucose tolerance in rodent models of diabetes to a similar extent as rosiglitazone.However, no major adverse events, such as weight gain, haemodilution or plasmavolume increase, were observed in INT131-treated rats compared to rosiglitazone-treated rats (Motani et al. 2009). In a randomised, double-blind study, type 2 diabe-tes patients were treated 4 weeks with 1 or 10 mg of INT131. Consistent with thein vitro data, INT131 displayed a glucose-lowering activity and increased HDL-Clevels without changing other lipid parameters (Dunn et al. 2011). This molecule iscurrently tested in a comparative clinical study in type 2 diabetes patients treatedfor 24 weeks with INT131 or pioglitazone (ClinicalTrials.gov Identifier:NCT00631007).In addition, a phase I clinical trial is ongoing to evaluate safety, tolerability andpharmacokinetic behaviour of a new PPAR α/γ modulator, DSP-8658, in type2 diabetes mellitus and healthy subjects (ClinicalTrials.gov Identifier:NCT01042106). DSP-8658 is a non-thiazolidinedione compound which exhibitspotent anti-hyperglycaemic effects, reduces plasma triglycerides and increasesHDL-C levels with less side effects on, e.g., body weight gain (press report:www.ds-pharma.com/ir/library/presentation/pdf).Finally, the new dual PPAR α/δ agonist GFT505, developed by Genfit, reducedplasma triglycerides and increased HDL-C levels in abdominally obese patientswith either dyslipidaemia or prediabetes (Cariou et al. 2011). A phase IIb clinicaltrial is ongoing to evaluate the efficacy of GFT505 in patients with non-alcoholicsteatohepatitis (ClinicalTrials.gov identifier: NCT01694849), an unmet clinicalneed because of the continuous increasing incidence of fatty liver disease due toabdominal obesity (Tailleux et al. 2012).These novel selective PPAR agonists, K-877, CER-002, DSP-8658, INT131 andGFT505 appear promising drugs to treat the cardiovascular risk associated withmetabolic syndrome and type 2 diabetes.4 Novel LXR Agonists4.1 Biological MechanismsLiver X receptors (LXRs) are nuclear receptors. There are two isoforms, LXRα andLXRβ, with LXRα mainly expressed in the liver, intestine, kidney and spleen,whereas LXRβ is ubiquitously expressed (Repa and Mangelsdorf 2000). OxysterolsEmerging Small Molecule Drugs 623and other cholesterol metabolites are natural ligands for LXRs, and several syn-thetic ligands were also developed (T0901317, GW3965). After their binding,LXRs modulate the expression of genes involved in cholesterol metabolism andtransport and glucose metabolism (Schultz et al. 2000; Laffitte et al. 2003). Activa-tion of LXR in macrophages induces ABCA1, ABCG1 and apoE expression whichpromotes cholesterol efflux and reverses cholesterol transport (Sabol et al. 2005;Venkateswaran et al. 2000; Laffitte et al. 2001). Besides their effects on lipidmetabolism, LXRs display anti-inflammatory properties (Joseph et al. 2003) andimprove glucose tolerance. In contrast to their beneficial effects, LXR activationinduces fatty acid synthesis (de novo lipogenesis) related to a modulation of thehepatic expression of sterol regulatory element-binding protein (SREBP-1),stearoyl-CoA desaturase-1 (SCD-1), fatty acid synthase (FAS) and acetyl-CoAcarboxylase (ACC) (Schultz et al. 2000). This upregulation of hepatic SREBP-1cexpression may contribute to the elevation of plasma triglycerides.4.2 Current StateT0901317 and GW3965, the most studied agonists, have been extensivelydescribed to exert beneficial effects in preclinical animal models of cardiovasculardiseases, neurodegenerative diseases and inflammation (Terasaka et al. 2003;Joseph et al. 2002, 2003; Zelcer et al. 2007). However, LXR ligands have not yetbeen tested in clinical trials because of their adverse effects such as an increase ofhepatic lipogenesis, hypertriglyceridaemia and hepatosteatosis (Calkin andTontonoz 2012). These lipogenic effects have been assigned to LXRα which ishighly expressed in the liver (Lehrke et al. 2005; Bradley et al. 2007). Therapeuticstrategies are now focusing on the development of selective LXR modulators,LXRβ-specific agonists and/or ligands which act selectively in specific tissues inorder to maintain positive effects on cholesterol metabolism and minimise the lipidside effects.LXR-623 (WAY-252623) is a novel synthetic ligand with higher potency forLXRβ, which induces plaque regression in combination with statins in a rabbitmodel of atherosclerosis (Giannarelli et al. 2012). LXR-623 entered in a phase Itrial (NCT00366522) to test tolerance and safety in humans (Katz et al. 2009).However, its development was interrupted because of adverse effects in the centralnervous system with potential induction of psychiatric disorders.4.3 Future PerspectivesA novel synthetic, steroidal LXR ligand, ATI-111, has been developed. This mole-cule is most potent on LXRα with modest effects on LXRβ. The higher efficiency onLXRα allows its utilisation at lower concentrations than T0901317, which probablyreduces cytotoxicity and also adverse effects such as hypertriglyceridaemia. Todetermine whether ATI-111 does not provoke hypertriglyceridaemia, mice were624 S. Colin et al.treated with 5 mg.kg-1.day-1 of ATI-111 for 8 weeks. Interestingly, a decrease ofplasma triglyceride levels and VLDL cholesterol was observed in ATI-111-treatedmice (Peng et al. 2011). In addition, ATI-111 exhibits anti-inflammatory propertieswith a decrease of LPS-induced inflammatory gene expression. Furthermore,ATI-111 reduced atherosclerotic lesions in LDL-receptor-deficient mice (Penget al. 2011). Altogether, accumulating proofs from in vitro and in vivo animal studiesshow beneficial effects of ATI-111 on atherosclerosis with anti-inflammatory effects,a reduction of hypertriglyceridaemia and a consequential decrease of atheroscleroticlesions. Further molecular investigations are necessary to assess the full potential ofATI-111 in clinical trials.A phase I clinical trial with XL-652 (XL-014), a novel LXR ligand, is currentlyongoing to evaluate its safety (www.exelixis.com/pipeline/xl652).5 RVX-2085.1 Biological MechanismsAn alternative strategy to raise the serum level of HDL-C is to increase one of themajor HDL proteins, apolipoprotein AI (apoAI). In this context, after a screeningassay in HepG2 cells to identify molecules inducing apoAI, Resverlogix Corpora-tion has selected and developed an oral quinazoline molecule, RVX-208(RVX-000222). This molecule, a derivative of resveratrol, increases hepaticapoAI production (Bailey et al. 2010). Thus RVX-208 is a small molecule forpotential treatment of cardiovascular diseases.5.2 Current StateFirst in vitro experiments have shown that RVX-208 increases apoAI mRNA,protein and the release of apoAI in the medium of HepG2 cells (Baileyet al. 2010). To test its efficiency in vivo, male monkeys were orally treated withRVX-208 for 63 days. This in vivo study in nonhuman primates demonstrated thatRVX-208 increases in a dose-dependent manner serum levels of apoAI and pre-β-HDL-C (Bailey et al. 2010). No adverse effects were observed in monkeys. Inhumans, 7 days of treatment increased preβ-HDL, apoAI and cholesterol efflux(Bailey et al. 2010). The efficacy and safety of RVX-208 was furthermoreinvestigated in a phase II randomised trial including 299 statin-treated patientswith coronary artery diseases (ASSERT study: NTC01058018). Twice daily admin-istration of RVX-208 (50, 100 and 150 mg) was well tolerated; however, a transientelevation in transaminase levels was found in some RVX-208-treated patients.Furthermore, only a modest increase of HDL-C level (3.2–8.3 %) was observed(Nicholls et al. 2011). In a next phase IIb clinical study, the ASSURE (ApoAISynthesis Stimulation and Intravascular Ultrasound for Coronary AtheromaRegression Evaluation) trial, the effect of 26 weeks treatment with RVX-208Emerging Small Molecule Drugs 625(100 mg) was determined on the progression of coronary atherosclerosis in323 patients with symptomatic coronary artery disease and low HDL-C levels.The primary end point, change in atheroma volume determined by intravascularultrasound (IVUS), was not met. No significant change in plaque regression wasobserved between placebo and RVX-208 group. The question is whether this lackof beneficial effect on plaque regression is due to the weak efficacy of RVX-208 orthe impossibility to confer beneficial effects on top of statins, since 84 % of patientswere on statin therapy. Furthermore, the increases of apoAI and HDL-C levels didnot differ from placebo, whereas transaminases were again found to be elevated.Thus, so far, no clinical benefit has been demonstrated with RVX-208 (press report:communication Nicholls SJ www.clinicaltrialresults.org/Nicholls_ASSURE).ConclusionTo reduce residual cardiovascular risk persisting after statin therapy, noveltherapeutic strategies based on raising HDL-C are currently under investigation.However, the utility of increasing HDL-C is not yet established, and pharma-ceutical manipulation of HDL-C appears to be less efficient than loweringLDL-C to reduce cardiovascular risk. Moreover, the failure of several clinicaltrials with the first members of the CETP inhibitor class, torcetrapib anddalcetrapib, and the lack of beneficial effects of an increase of HDL-C and thereduction of cardiovascular events raises doubts about the relevance of the“HDL-C hypothesis”. These disappointing results highlight the complexity ofHDL metabolism in contrast to that of LDL.Many biological activities of HDL, such as antioxidant, anti-inflammatoryand antiapoptotic properties, are mediated by different HDL subclasses, andsolely increasing HDL-C may not enhance these functions. Moreover, plasmaHDL contains heterogeneous particle subpopulations whose composition,metabolism and functionality differ depending on the metabolic status (Besleret al. 2011). Thus far, the larger randomised placebo controlled phase III clinicaltrials have shown that an increase in HDL-C does not benefit the patient, andthus, future strategies should aim at improving HDL function.Open Access This chapter is distributed under the terms of the Creative Commons AttributionNoncommercial License, which permits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source are credited.ReferencesBailey D, Jahagirdar R, Gordon A, Hafiane A, Campbell S, Chatur S, Wagner GS, Hansen HC,Chiacchia FS, Johansson J, Krimbou L, Wong NCW, Genest J (2010) RVX-208: a smallmolecule that increases apolipoprotein AI and high-density lipoprotein cholesterol in vitro andin vivo. J Am Coll Cardiol 55:2580–2589Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJP, Komajda M, Lopez-Sendon J,Mosca L, Tardif J, Waters DD, Shear CL, Revkin JH, Buhr KA, Fisher MR, Tall AR, Brewer B626 S. Colin et al.(2007) Effects of Torcetrapib in patients at high risk for coronary events. N Engl J Med357:2109–2122Besler C, Heinrich K, Rohrer L, Doerries C, Riwanto M, Shih DM, Chroni A, Yonekawa K,Stein S, Schaefer N, Mueller M, Akhmedov A, Daniil G, Manes C, Templin C, Wyss C,Maier W, Tanner FC, Matter CM, Corti R, Furlong C, Lusis AJ, von Eckardstein A, FogelmanAM, Lüscher TF, Landmesser U (2011) Mechanisms underlying adverse effects of HDL onenos-activating pathways in patients with coronary artery disease. J Clin Invest 121:2693–2708Bouhlel MA, Derudas B, Rigamonti E, Dièvart R, Brozek J, Haulon S, Zawadzki C, Jude B,Torpier G, Marx N, Staels B, Chinetti-Gbaguidi G (2007) PPARgamma activation primeshuman monocytes into alternative m2 macrophages with anti-inflammatory properties. CellMetab 6:137–143Bradley MN, Hong C, Chen M, Joseph SB, Wilpitz DC, Wang X, Lusis AJ, Collins A, Hseuh WA,Collins JL, Tangirala RK, Tontonoz P (2007) Ligand activation of LXR beta reverses athero-sclerosis and cellular cholesterol overload in mice lacking LXR alpha and apoE. J Clin Invest117:2337–2346Calkin AC, Tontonoz P (2012) Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR. Nat Rev Mol Cell Biol 13:213–224Cannon CP, Shah S, Dansky HM, Davidson M, Brinton EA, Gotto AM, Stepanavage M, Liu SX,Gibbons P, Ashraf TB, Zafarino J, Mitchel Y, Barter P (2010) Safety of Anacetrapib in patientswith or at high risk for coronary heart disease. N Engl J Med 363:2406–2415Cariou B, Zaı̈r Y, Staels B, Bruckert E (2011) Effects of the new dual PPAR α/δ agonist GFT505on lipid and glucose homeostasis in abdominally obese patients with combined dyslipidemia orimpaired glucose metabolism. Diabetes Care 34:2008–2014Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Torra IP, Teissier E, Minnich A, Jaye M,Duverger N, Brewer HB, Fruchart JC, Clavey V, Staels B (2001) PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells throughstimulation of the ABCA1 pathway. Nat Med 7:53–58Curb JD, Abbott RD, Rodriguez BL, Masaki K, Chen R, Sharp DS, Tall AR (2004) A prospectivestudy of HDL-C and cholesteryl ester transfer protein gene mutations and the risk of coronaryheart disease in the elderly. J Lipid Res 45:948–953Dunn FL, Higgins LS, Fredrickson J, DePaoli AM (2011) Selective modulation of PPARγ activitycan lower plasma glucose without typical thiazolidinedione side-effects in patients with type2 diabetes. J Diabetes Complicat 25:151–158Fayad ZA, Mani V, Woodward M, Kallend D, Abt M, Burgess T, Fuster V, Ballantyne CM, SteinEA, Tardif J, Rudd JHF, Farkouh ME, Tawakol A (2011) Safety and efficacy of Dalcetrapib onatherosclerotic disease using novel non-invasive multimodality imaging (dal-plaque): arandomised clinical trial. Lancet 378:1547–1559Forrest MJ, Bloomfield D, Briscoe RJ, Brown PN, Cumiskey AM, Ehrhart J, Hershey JC, KellerWJ, Ma X, McPherson HE, Messina E, Peterson LB, Sharif-Rodriguez W, Siegl PK, SinclairPJ, Sparrow CP, Stevenson AS, Sun SY, Tsai C, Vargas H, Walker M 3rd, West SH, White V,Woltmann RF (2008) Torcetrapib-induced blood pressure elevation is independent of cetpinhibition and is accompanied by increased circulating levels of aldosterone. Br J Pharmacol154:1465–1473Fruchart J (2013) Selective peroxisome proliferator-activated receptor αmodulators (SPPARMα):the next generation of peroxisome proliferator-activated receptor α-agonists. CardiovascDiabetol 12:82Giannarelli C, Cimmino G, Connolly TM, Ibanez B, Ruiz JMG, Alique M, Zafar MU, Fuster V,Feuerstein G, Badimon JJ (2012) Synergistic effect of Liver X Receptor activation andsimvastatin on plaque regression and stabilization: an magnetic resonance imaging study in amodel of advanced atherosclerosis. Eur Heart J 33:264–273Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR (1977) High density lipoproteinas a protective factor against coronary heart disease the framingham study. Am J Med62:707–714Emerging Small Molecule Drugs 627Henry RR, Lincoff AM, Mudaliar S, Rabbia M, Chognot C, Herz M (2009) Effect of the dualperoxisome proliferator-activated receptor-alpha/gamma agonist aleglitazar on risk of cardio-vascular disease in patients with type 2 diabetes (synchrony): a phase II, randomised, dose-ranging study. Lancet 374:126–135http://www.drugs.com/clinical_trials/cerenis-therapeutics-announces-successful-co

High Density Lipoproteins

Abstract

Extracted data

High Density Lipoproteins

Abstract

Extracted data

Related items

Related items