The Endothelial Glycocalyx In Vitro: Its Structure and The Role of Heparan Sulfate and Glypican‐1 in eNOS Activation by Flow

Abstract

The endothelial glycocalyx (GCX) mediates flow‐induced nitric oxide release via heparan sulfate (HS), but the GCX structure is unclear and the specific HS core protein(s) involved in this mechanotransduction is unknown. Our study tests the hypotheses that flow regulates GCX thickness and organization and that the HS glypican‐1 core protein mediates flow‐induced activation of endothelial nitric oxide synthase (eNOS). Monolayers of bovine aortic and rat fat pad endothelial cells with intact GCX, enzymatically degraded HS, or RNA‐silenced glypican‐1 were exposed to 15 dyne/cm2 uniform shear stress for 3 hr. Confocal immunocytochemistry and cryo‐transmission electron microscopy revealed a GCX that was 2.5 to 4.0 μm thick. Sheared GCX was well organized and aligned perpendicular to the cell surface, while unsheared GCX was disorganized. Western blot and confocal microscopy demonstrate that when the GCX is intact shear stress increases eNOS activation (ser1177 phosphorylation) and membrane localization, which are blocked by HS degradation. Baseline and flow‐induced levels of activated eNOS are attenuated when glypican‐1 is silenced. This work further elucidates GCX structure and role in endothelial cell mechanotransduction. Supported by NIH: 5T32HL007675, HL73732, HL57093.