Hyaluronan Immobilized via Peptide Self-Assembled Monolayers to Study the Behaviour of Endothelial Cells and Reveal Endovascular Therapies.

PhD Theses.The glycocalyx is the immediate pericellular matrix that surrounds many cell types, including endothelial cells (ECs), and is typically composed of glycans (glycosaminoglycans, proteoglycans, and glycoproteins). The endothelial glycocalyx is rich in hyaluronic acid (HA), the only non-sulfated glycosaminoglycan. HA plays an important role in the maintenance of vascular integrity and influences various behaviours of ECs. The role of HA molecular weight (MW) on cell cultures has been investigated in many studies, but HA is usually probed in the soluble form, and the impact of peptide-bound HA has not yet been exploited. Here, the contribution of HA to the regulation of endothelial function is investigated using model surfaces. In Chapter I, the fundamental background of this project is introduced. It starts with a general description of the formation and function of vascular endothelium, followed by an overview of the endothelial glycocalyx and self-assembled monolayers (SAMs) to develop model surfaces. The next chapter (Chapter II) presents the materials and methods used in this research. In Chapter III, a 2D platform formed by SAMs of a HA-binding peptide is used as an anchor for HA immobilization. The peptide sequence GAHWQFNALTVR, previously identified by phage display with strong binding affinity for HA and named Pep-1, is thiolated at the N-terminus to form SAMs on gold (Au) substrates. Acetylated Pep-1 and a scrambled sequence of Pep-1 are used as controls. Mixed SAMs, consisting of thiolated Pep-1 (HS-Pep-1) and 1-octanethiol, are prepared and characterized using ellipsometry and contact angle measurement. Atomic force microscopy (AFM) characterization shows that HA modified surfaces have a higher roughness. Ellipsometry shows that HA immobilized on Pep-1 SAMs has a better swelling and higher thicknesses in wet condition. Contact angle measurements indicate that functionalization of gold surfaces with Pep-1 and HA leads to more hydrophilic surfaces, and full density Pep-1 SAMs are more hydrophilic and bind more HA than mixed SAMs. The SAMs and HA-coated surfaces are characterized by X-ray photoelectron spectroscopy (XPS) and quartz crystal microbalance with dissipation (QCM-D) monitoring, which confirm the binding and presence of thiolated peptides on the Au surfaces and the deposition of HA. Micro-contact printing (μCP) is used to develop patterned surfaces for the controlled spatial presentation of HA and fluorescence microscopy shows the localization of fluorescently labelled HA only on areas printed with Pep-1 SAMs. Chapter IV aims to comprehensively study the roles of immobilized HA on the regulation of human umbilical vein endothelial cells (HUVECs) behaviour compared to the more conventional use of soluble HA. Cell spreading and migration are enhanced by immobilized low MW (LMW) HA, which also facilitates cell alignment and elongation under laminar flow conditions, and potentially drives directional migration. However, immobilized high MW (HMW) HA reduces cell attachment and migration. Such biomimetic surfaces provide new insights into the role of HA in regulating the spreading and phenotype of ECs. Cells seeded on bare gold substrates or Pep-1 SAMs are also investigated in the presence of soluble HA, while the effect of immobilized HA on cell function is more significant than soluble HA. This effect is not mediated by the expression of CD44 and immobilized LMW HA is found to accelerate assembly of focal adhesions. Chapter V concludes the main findings of this thesis and outlines new research directions and potential applications. This research provides insight into the use of SAMs for the controlled presentation of HA with defined size in cultures of HUVECs to study their functions. These studies will facilitate progress on developing potential HA-based therapeutic solutions for vascular diseases