Spatio-temporal development of the endothelial glycocalyx layer and its mechanical property in vitro

Abstract

The endothelial glycocalyx is a thin layer of polysaccharide matrix on the luminal surface of endothelial cells (ECs), which contains sulphated proteoglycans and glycoproteins. It is a mechanotransducer and functions as an amplifier of the shear stress on ECs. It controls the vessel permeability and mediates the blood-endothelium interaction. This study investigates the spatial distribution and temporal development of the glycocalyx on cultured ECs, and evaluates mechanical properties of the glycocalyx using atomic force microscopy (AFM) nano-indentation. The glycocalyx on human umbilical vein endothelial cells (HUVECs) is observed under a confocal microscope. Manipulation of the glycocalyx is achieved using heparanase or neuraminidase. The Young's modulus of the cell membrane is calculated from the force-distance curve during AFM indentation. Results show that the glycocalyx appears predominantly on the edge of cells in the early days in culture, e.g. up to day 5 after seeding. On day 7, the glycocalyx is also seen in the apical area of the cell membrane. The thickness of the glycocalyx is approximately 300 nm-1 μm. AFM indentation reveals the Young's modulus of the cell membrane decreases from day 3 (2.93 ± 1.16 kPa) to day 14 (0.35 ± 0.15 kPa) and remains unchanged to day 21 (0.33 ± 0.19 kPa). Significant difference in the Young's modulus is also seen between the apical (1.54 ± 0.58 kPa) and the edge (0.69 ± 0.55 kPa) of cells at day 7. By contrast, neuraminidase-treated cells (i.e. without the glycocalyx) have similar values between day 3 (3.18 ± 0.88 kPa), day 14 (2.12 ± 0.78 kPa) and day 21 (2.15 ± 0.48 kPa). The endothelial glycocalyx in vitro shows temporal development in the early days in culture. It covers predominantly the edge of cells initially and appears on the apical membrane of cells as time progresses. The Young's modulus of the glycocalyx is deduced from Young's moduli of cell membranes with and without the glycocalyx layer. Our results show the glycocalyx on cultured HUVECs has a Young's modulus of approximately 0.39 kPa.

Figure 1.

Ex vivo HepSS-I (green) and CD144 (red) staining of the mouse thoracic aortic endothelium. (a) Control, (b) after HepIII treatment, (c) change in the green dye intensity following enzyme perfusion of the aorta. (a,b) Scale bars, 10 µm.

Figure 2.

Three-dimensional confocal images of the glycocalyx layer on freshly harvest and unfixed mouse thoracic aorta. The main panel shows the enface image at a given z-depth. The bottom and side panels show the x–z and y–z cross-sectional images, respectively. Scale bar, 10 μm, in the main panel. (a) Control, (b) after neuraminidase perfusion of the aorta.

Figure 3.

Spatial distribution of the glycocalyx layer on live HUVECs in vitro and its temporal development from day 1 to day 21. The main panel shows the enface image at a given z-depth. The bottom and side panels show the x–z and y–z cross-sectional images, respectively. Scale bar, 10 μm, in the main panel. (a) Control, (b) after neuraminidase treatment.

Figure 4.

(a) Schematics of AFM probing of HUVECs in vitro, (b) rectangular cantilever, pyramidal-shaped tip (enlarged panel), the end of the tip is semi-spherical (further enlarged panel), (c) height image of HUVECs, (d) phase image of HUVECs. (Online version in colour.)

Figure 5.

The Young's modulus of the HUVEC membrane in vitro. HUVECs cultured for 3, 7, 14 and 21 days are tested using AFM indentation. Comparisons are made between different locations (i.e. apical, middle and edge) on the cell membrane, as well as between the control groups (Con) and neuraminidase-treated groups (Nase) at different days. Black bars, apical; grey bars, middle; white bars, edge.