VEGFR2 activation during the early onset of flow is ligand‐dependent and mediated by the release of autocrine VEGF

Abstract

Previous studies have shown that VEGFR2 is part of a mechanosensory complex in endothelial cells (ECs) and is activated in response to fluid shear stress in a ligand‐independent manner. Here, we tested the hypothesis that shear stress induces a rapid release of heparan sulfate (HS)‐sequestered VEGF, which subsequently activates VEGFR2 in a ligand‐dependent manner. Our results showed that VEGFR2 is phosphorylated at Y1175 and Y1214 within 15 sec of flow in human coronary artery ECs. Removal of HS by heparinase III digestion led to a decrease in VEGFR2 phosphorylation. Transfection of siRNA against VEGF and blockade of VEGFR2 with a neutralizing antibody were also performed to demonstrate that early activation of VEGFR2 is ligand‐dependent. To determine whether VEGFR2 activation is regulated via the phospholipase C (PLC) pathway, we sheared ECs after pre‐incubation with two structurally different PLC inhibitors, U73122 and Edelfosine. Phosphorylation of VEGFR2 in response to flow was diminished by both drugs, suggesting that VEGFR2 activation may be downstream of PLC activation. Together, these studies aim to identify the role(s) and mechanism(s) by which HSPGs participate in flow‐induced VEGFR2 activation.