A central role of p38 MAPK and JNK in bone morphogenic protein-4 induced endothelial cell apoptosis.

Yung, Lai Hang.Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.Includes bibliographical references (leaves 93-115).Abstract also in Chinese.Declaration --- p.iAcknowledgements --- p.iiAbbreviations --- p.iiiAbstract in English --- p.vAbstract in Chinese --- p.ixContents --- p.xiChapter Chapter I - --- IntroductionChapter 1.1) --- Endothelial cells function --- p.1Chapter 1.2) --- Oxidative stress in the vascular wall --- p.2Chapter 1.2.1) --- Sources of ROS --- p.3Chapter 1.2.2) --- Actions of ROS --- p.3Chapter 1.2.2.1) --- Impaired endothelium-dependent vasodilatation --- p.3Chapter 1.2.2.2) --- VSMC migration --- p.4Chapter 1.2.2.3) --- Programmed cell death (cell apoptosis) --- p.4Chapter 1.3) --- Endothelial cell apoptosis --- p.7Chapter 1.3.1) --- Apoptosis and cardiovascular diseases --- p.7Chapter 1.3.2) --- Mechanisms of endothelial cells apoptosis --- p.7Chapter 1.3.2.1) --- What are caspases? --- p.8Chapter 1.3.2.2) --- Death receptor-mediated apoptosis --- p.9Chapter 1.3.2.3) --- Mitochondria-dependent pathway --- p.9Chapter 1.3.3) --- Regulations of endothelial cells apoptosis --- p.10Chapter 1.3.3.1) --- Oxidative stress --- p.10Chapter 1.3.3.2) --- Shear Stress --- p.11Chapter 1.3.3.3) --- Growth factors --- p.12Chapter 1.3.3.4) --- NO --- p.12Chapter 1.3.3.5) --- Inflammatory mediators --- p.13Chapter 1.4) --- Mitogen activated kinases signaling in apoptosis --- p.15Chapter 1.5) --- Bone morphogenic proteins (BMPs) --- p.17Chapter 1.5.1) --- BMPs functions and cardiovascular system --- p.17Chapter 1.5.2) --- BMPs signaling pathways --- p.18Chapter 1.5.2.1) --- Smad-dependent pathway --- p.18Chapter 1.5.2.2) --- MAPKs and SAPKs pathways --- p.19Chapter 1.5.2.3) --- Antagonists of BMPs signaling --- p.20Chapter 1.5.3) --- BMP4 and cardiovascular diseases --- p.20Chapter 1.6) --- "Justification, long-term significance and objectives of the present project" --- p.23Chapter Chapter II - --- Methods and MaterialsChapter 2.1) --- Animal handling --- p.24Chapter 2.2) --- Endothelial cell isolation and culture --- p.24Chapter 2.2.1) --- Primary culture of rat endothelial cells --- p.24Chapter 2.2.2) --- Culture of human umbilical cord vein endothelial cells… --- p.25Chapter 2.3) --- Apoptosis assessment --- p.25Chapter 2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.25Chapter 2.3.2) --- Cell death detection ELISA kit --- p.26Chapter 2.3.3) --- Flow cytometry --- p.27Chapter 2.4) --- Western blot analysis --- p.28Chapter 2.4.1) --- Sample preparation --- p.28Chapter 2.4.2) --- SDS-PAGE and transfer --- p.28Chapter 2.5) --- DHE fluorescence --- p.29Chapter 2.6) --- "Drugs, chemicals and other reagents" --- p.30Chapter 2.6.1) --- Drugs and chemicals used in the present experiments --- p.30Chapter 2.6.2) --- Reagents for Western blot analysis --- p.30Chapter 2.6.3) --- Primary antibodies --- p.33Chapter 2.7) --- Small interfering RNA experiment --- p.34Chapter 2.8) --- Statistical analysis --- p.34Chapter Chapter III - --- BMP4 induces endothelial cell apoptosis in ROS related p38 MAPK and JNK mediated caspase-3 dependent pathwayChapter 3.1) --- Introduction --- p.35Chapter 3.2) --- Methods and materials --- p.39Chapter 3.2.1) --- Isolation and culture of endothelial cells --- p.39Chapter 3.2.2) --- Drugs treatment --- p.39Chapter 3.2.3) --- Assay for cell apoptosis --- p.40Chapter 3.2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.40Chapter 3.2.3.2) --- Cell death detection ELISA kit --- p.41Chapter 3.2.3.3) --- Flow cytometric analysis --- p.41Chapter 3.2.4) --- Western blot analysis --- p.41Chapter 3.2.5) --- Dihydroethidium (DHE) staining --- p.42Chapter 3.2.6) --- Statistical analysis --- p.42Chapter 3.3) --- Results --- p.43Chapter 3.3.1) --- Dose- and time-dependent effect of BMP4 --- p.43Chapter 3.3.2) --- Role of caspases in apoptosis of RAECs and HUVECs --- p.43Chapter 3.3.3) --- Roles of BMP4 and ROS in endothelial cell apoptosis --- p.44Chapter 3.3.3.1) --- Noggin antagonism of BMP4-induced effect --- p.44Chapter 3.3.3.2) --- NAD(P)H oxidase-mediated ROS production --- p.44Chapter 3.3.3.3) --- Inhibition of endothelial cell apoptosis by ROS scavengers --- p.45Chapter 3.3.4) --- Roles of MAPKs/SAPKs in BMP4-induced endothelial cell apoptosis --- p.45Chapter 3.3.5) --- Relationship between ROS and MAPKs/SAPKs --- p.46Chapter 3.3.6) --- Relationship between p38 MAPK and JNK --- p.46Chapter 3.4) --- Discussion --- p.82Chapter 3.4.1) --- Caspase-dependent pathways --- p.82Chapter 3.4.2) --- Oxidative stress --- p.85Chapter 3.4.3) --- Role of MAPKs activation in BMP4-induced endothelial cell apoptosis --- p.87Chapter 3.4.4) --- ROS mediates BMP4-induced activation of MAPKs --- p.88Chapter 3.4.5) --- Role of p38 MAPK in the activation of JNK 1 --- p.89Chapter 3.5) --- Concluding remarks --- p.91References --- p.93Publications and Awards --- p.11