The structural stability of the endothelial glycocalyx after enzymatic removal of glycosaminoglycans

Abstract

Rationale: It is widely believed that glycosaminoglycans (GAGs) and bound plasma proteins form an interconnected gel-like structure on the surface of endothelial cells (the endothelial glycocalyx layer-EGL) that is stabilized by the interaction of its components. However, the structural organization of GAGs and proteins and the contribution of individual components to the stability of the EGL are largely unknown.

Objective: To evaluate the hypothesis that the interconnected gel-like glycocalyx would collapse when individual GAG components were almost completely removed by a specific enzyme.

Methods and results: Using confocal microscopy, we observed that the coverage and thickness of heparan sulfate (HS), chondroitin sulfate (CS), hyaluronic acid (HA), and adsorbed albumin were similar, and that the thicknesses of individual GAGs were spatially nonuniform. The individual GAGs were degraded by specific enzymes in a dose-dependent manner, and decreased much more in coverage than in thickness. Removal of HS or HA did not result in cleavage or collapse of any of the remaining components. Simultaneous removal of CS and HA by chondroitinase did not affect HS, but did reduce adsorbed albumin, although the effect was not large.

Conclusion: All GAGs and adsorbed proteins are well inter-mixed within the structure of the EGL, but the GAG components do not interact with one another. The GAG components do provide binding sites for albumin. Our results provide a new view of the organization of the endothelial glycocalyx layer and provide the first demonstration of the interaction between individual GAG components.

Figure 1. The methods for image quantification analysis.

The 3D reconstruction of the Z-series stack (A), the split green channel (HS) (B), and blue channel (DAPI) (C). (D–E) XZ-slice images along the dashed line in (A) and (B). (F) The max-intensity Z-projection of HS. The red outline in (C) and the circles in (F) show the nuclei and cell body of the same two cells. The red arrow in (F) indicates the junction between these cells. The white arrow heads in (D) and (E) indicate the cell-cell junctions along the slice. (G) The frequency curves of pixel intensity from the max-intensity Z-projection images of both control and negative control (no antibody). The non-zero point of intersection between the curves was defined as the background threshold. All information equal to or higher than background was selected as a region of interest (ROI). (H) The maximum coverage was usually obtained at threshold. (I) top: the split green channel (HS) XZ-slice; middle: the red region shows the ROI in the XZ-slice at threshold; bottom: negative control, the area of the ROI in XZ-slices was usually zero at threshold. (J) The area of a region bounded by a red outline () and the width of the region’s bounding box (ΔL). The total area and length of all regions represent the area () and the length (L), respectively, of the ROI (;). The average thickness () in this slice is 2.23 µm. (K–L) Partial enlargement of the XZ-slice image with a junctional thickness measure line. The half-maximum intensity value was employed as the 2^nd threshold for junctional thickness detection. In this image, the signal-to-noise ratio is 9.11, and the junction thickness of HS is 3.00 µm.

Figure 2. The distribution of HS on RFPECs after 2 hr heparinase III treatment.

(A) top: Z-projection; bottom: cross-sectional images of stack along the dashed line. The arrow head indicates the cell-cell junction. Scale bar: 20 µm. (B) The changes in coverage; (C) the average thickness; (D) the junction thickness. HepSS-1 epitope HS-antibodies were used. The coverage, average thickness, and junction thickness were significantly decreased with increasing concentration of heparinase III. **P<0.01.

Figure 3. The quantification of HA, CS, and albumin on RFPECs after 2 hr heparinase III treatment.

(A)–(C) The changes in the coverage, the average thickness, and the junction thickness of HA; (D)–(F) CS; (G)–(I) adsorbed albumin. *P<0.05; **P<0.01.

Figure 4. The distribution of HA on RFPECs after 2 hr hyaluronidase treatment.

(A) top: Z-projection; bottom: cross-sectional images of stack along the dashed line. The arrow head indicates the cell-cell junction. Scale bar: 20 µm. (B) The changes in coverage; (C) the average thickness; (D) the junction thickness. **P<0.01.

Figure 5. The quantification of HS, CS, and albumin on RFPECs after 2 hr hyaluronidase treatment.

(A)–(C) The changes in the coverage, the average thickness, and the junction thickness of HS; (D)–(F) CS; (G)–(I) adsorbed albumin.

Figure 6. The distribution of CS on RFPECs after 2 hr chondroitinase ABC treatment.

(A) top: Z-projection; bottom: cross-sectional images of stack along the dashed line. The arrow head indicates the cell-cell junction. Scale bar: 20 µm. (B) The changes in coverage; (C) the average thickness; (D) the junction thickness. **P<0.01.

Figure 7. The quantification of HA, HS, and albumin on RFPECs after 2 hr chondroitinase ABC treatment.

(A)–(C) The changes in the coverage, the average thickness, and the junction thickness of HA; (D)–(F) HS; (G)–(I) adsorbed albumin. **P<0.01.

Figure 8. Hypothesis–Experimental results–Conclusion.

Left: Our hypothesis of an interacting mesh of GAGs and albumin. Middle: A summary of the results of our experiments: removal of HS (heparinase III), HA (hyaluronidase), or both CS and HA (chondroitinase ABC) did not reduce the coverage or thickness of any of the remaining components, indicating that no individual component (HS, HA or CS) is required to stabilize the EGL. Right: The overarching conclusion of our study: GAG components do not interact but do provide binding sites for albumin.