Spatial distribution, temporal development and mechanical properties of the endothelial glycocalyx in vitro

Abstract

The endothelial glycocalyx is a thin layer of macromolecular matrix on the luminal surface of vascular endothelial cells. It determines fluid and solute transport across the vessel wall; affects the mechanotransduction of endothelial cells and contributes to vascular patho-physiology. This thesis investigates the spatiotemporal development of the endothelial glycocalyx in vitro using fluorescence confocal microscopy. The Young’s modulus of the glycocalyx is evaluated using AFM indentation. The glycocalyx on cultured HUVECs shows temporal development: up to day 5 after cell seeding, it covers predominantly the edge of cells and appears on the apical membrane of cells as time progresses. After day 14, the entire cell membrane is covered by the glycocalyx. The thickness of this layer is estimated to be between 300nm and 1μm. AFM indentation result reveals the Young’s modulus of the cell membrane decreases with time. The Young’s modulus of the glycocalyx is deduced from Young’s moduli of cell membranes with and without the glycocalyx layer. The results show the glycocalyx on cultured HUVECs has a Young’s modulus of ~0.39kPa. The thesis further investigates the distribution of the glycocalyx on the endothelial cell membrane following shear flow stimulation. Both the percentage area of the cell membrane that is covered with the glycocalyx and the II fluorescence intensity ratio between the apical and edge areas on endothelial cells are used to measure the glycocalyx distribution. It is observed that the glycocalyx appears near the edge of the endothelial cell following shear stimulation and develops towards the apical area with time. The speed of the recovery of the glycocalyx layer is faster in the earlier period, i.e. 0hr - 4hrs after shear stimulation, than that in the later period, i.e. 8hrs - 24hrs. Additionally, the recovery of the glycocalyx following neuraminidase degradation is investigated under either the static or the shear flow conditions.