Abstract
RationaleIt 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.ObjectiveTo 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 ResultsUsing 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.ConclusionAll 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.
Highlights
The luminal surfaces of vascular endothelium are covered by an endothelial glycocalyx layer (EGL) which serves as a transport and adhesion barrier and plays important roles in the mechanotransduction of shear stress [1,2,3,4]
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
We previously demonstrated that a 2 hr pretreatment with 15 mU/mL heparinase III, 15 mU/mL chondroitinase ABC, 15 mU/mL neuraminidase, and 1.5 U/mL hyaluronidase, respectively removed 42.6% heparan sulfate (HS), 44.2% chondroitin sulfate (CS), 38.4% sialic acids (SA), and 61.7% hyaluronic acid (HA) in cultured bovine aortic endothelial cells (BAEC), and found that shear-induced nitric oxide (NO) production was blocked in the presence of those enzymes except for chondroitinase ABC [25]
Summary
The luminal surfaces of vascular endothelium are covered by an endothelial glycocalyx layer (EGL) which serves as a transport and adhesion barrier and plays important roles in the mechanotransduction of shear stress [1,2,3,4]. GAGs are linear polydisperse heteropolysaccharides, characterized by distinct disaccharide unit repeats. Specific combinations of these give rise to different GAG families, including heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronic acid (HA, known as hyaluronan). The pixel intensity histograms from the FOV of max-intensity Z-projection images of control and negative control were obtained using the ImageJ histogram tool, and converted to curves (Figure 1G). The point of intersection between the curves was defined as the background noise value (threshold). The area of the ROI in the max-intensity Z-projection image was calculated using ImageJ. The percent coverage was defined as the ratio of the area of the ROI and FOV, COV ~ DAT |100%
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