Uncovering glycocalyx mechanosignaling and mechanoremodeling mechanisms

The endothelial glycocalyx (GCX) mediates flow-induced nitric oxide signaling and cellular remodeling via its abundant heparan sulfate (HS) component. Until recently, little was known about the ultrastructural details and the activity of the HS core proteins involved in these GCX mediated mechanotransduction events. Here, we study flow regulation of GCX morphology and HS glypican-1 (GPC1) and syndecan-1 (SDC1) core protein participation in flow-induced activation of endothelial nitric oxide synthase (eNOS), cytoskeleton reorganization, and remodeling of cell shape. Monolayers of endothelial cells with intact GCX, enzymatically degraded HS, RNA-silenced GPC1, or RNA-silenced SDC1 were exposed to physiological shear stress. Confocal immunocytochemistry and rapid freezing/freeze substitution transmission electron microscopy (RF/FS TEM) revealed an abundant GCX and showed that sheared GCX is well organized and aligned perpendicular to the cell surface while unsheared GCX is disorganized. Western blot and confocal microscopy demonstrated that when the GCX is intact, shear stress increases eNOS activation (ser1177 phosphorylation) and membrane localization. Activation of eNOS is blocked by HS degradation and GPC1 reduction but not by SDC1 silencing. By phase contrast and fluorescence microscopy, in the presence of intact GCX, we observed flow-induced EC cytoskeleton reorganization as well as EC elongation and alignment in the direction of flow. Endothelial cell remodeling in response to flow was attenuated by HS degradation and in the absence of SDC1, but preserved with GPC1 knockdown. This work elucidates flow-defined details about GCX ultrastructure and uncovers intricate mechanisms by which specific GCX core proteins mediate endothelial cell mechanotransduction.