Mechanisms of flow-dependent endothelial COX-2 and PGI2 expression

The luminal surfaces of blood vessels are covered with several membrane-bound glycosaminoglycans (GAGs) that constitute the endothelial glycocalyx layer (EGL) of endothelial cells(ECs). ECs, like all anchorage-dependent cells, adhere to the extracellular matrix at focal adhesions. By virtue of their cellular location and signaling abilities, it has been suggested that focal adhesions and the EGL may function as mechanoreceptors, by sensing and facilitating the transfer of “outside in”, mechanical signals across the plasma membrane. However, the mechanism by which ECs detect fluid shear stress (FSS) and transduce signals across the membrane to activate signaling pathways such as the induction of COX-2 and release of PGI₂ is still poorly understood. To directly test the role that focal adhesions (apical and basal) and the EGL play in mediating the “outside in”, COX-2 expression and PGI₂ mechanotransduction, we first limited the formation of focal adhesions by culturing ECs on fibronectin in the presence of soluble fibronectin fragments H-Gly-Arg-Gly-Asp-Ser-Pro-OH (GRGDSP) peptide, which competitively disrupts the fibronectin-integrin interactions, and selectively degraded the glycocalyx component heparan sulfate (HS) using the EGL degrading enzyme Heparanase III (HepIII). ECs were then subjected to fluid shear stress and COX-2 expression and PGI₂ release measured. Surprisingly, the expression and release of both COX-2 and PGI₂ in response to FSS were not significantly decreased in ECs treated with either GRGDSP or EGL degrading enzyme HepIII. These results suggest that the “outside in” pathway is not the major mechanotransduction route by which shear stress activates COX-2 and release of PGI₂ in ECs.