The Short Term Response of PECAM-1 to Mechanical Loading: Cyclooxygenase-2 and Nitric Oxide Production with AFM Pulling and Shear Stress

Abstract

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a transmembrane protein located at the intercellular junction of endothelial cells (EC). PECAM-1 in adjacent EC bind to each other through homophilic interactions and regulate endothelial function. Recent studies have identified the role of PECAM-1 in mechanotransduction, sensing luminal shear stress and coordinating the production of both prostacyclin/prostaglandin I2 (PGI2) and nitric oxide. While most of these studies use inhibition methods, such as PECAM-1 knockdown cells or knockout animals, our group has implemented novel methods to selectively activate PECAM-1 on the EC surface for 10-30 minutes. Using AFM pulling with PECAM-1 antibody coated cantilevers on rat fat pad endothelial cell (RFPEC) monolayers, we have found significant activation of cyclooxygenase-2 (COX-2), the mediating enzyme of prostacyclin (PGI2) production. Conversely, AFM pulling with PECAM-1 coated probes did not induce any significant change in NO production. These data suggest that fluid shear stress acts directly on PECAM-1 to induce PGI2 production but indirectly to induce NO production. We have shown that pulling with probes targeting the heparan sulfate proteoglycan glypican-1 initiates NO production, suggesting that glypican-1 is upstream of PECAM-1 in the mechanotransduction cascade that produces NO. We are currently investigating the potential interaction between glypican-1 and PECAM-1 in shear-induced NO production. Heparin and surfen were used to inhibit extracellular heparan sulfate-to-PECAM-1 binding and shear-induced NO production was studied at both 10 and 30 minutes. The inhibition of PECAM-1 expression and intermediate intracellular signaling pathways may further illuminate the role of PECAM-1 in glycocalyx-dependent shear-induced NO production. This work aims to identify critical mechanosensing structures on the surface of endothelial cells that mediate the production of vasoregulatory and anti-atherogenic substances for the treatment of vascular disease.