Vascular Endothelial Growth Factor (VEGF) Receptor-2 Tyrosine 1175 Signaling Controls VEGF-induced von Willebrand Factor Release from Endothelial Cells via Phospholipase C-γ1- and Protein Kinase A-dependent Pathways

Abstract

There is increasing evidence that vascular endothelial growth factor (VEGF) contributes to inflammation independent of its angiogenic functions. Targeting some of the components in endothelial Weibel-Palade bodies (WPBs) effectively inhibits VEGF-induced inflammation, but little is known about how VEGF regulates WPB exocytosis. In this study, we showed that VEGF receptor-2 (VEGFR2), but not VEGFR1, is responsible for VEGF-induced release of von Willebrand factor (vWF), a major marker of WPBs. This is in good contrast to VEGF-stimulated interleukin-6 release from endothelium, which is selectively mediated through VEGFR1. We further demonstrated that VEGFR2-initiated phospholipase C-␥1 (PLC␥1)/calcium signaling is important but insufficient for full vWF release, suggesting the possible participation of another effector pathway. We found that cAMP/protein kinase A (PKA) signaling is required for full vWF release. Importantly, a single mutation of Tyr 1175 in the C terminus of VEGFR2, a tyrosine residue crucial for embryonic vasculogenesis, abolished vWF release, concomitant with defective activations of both PLC␥1 and PKA. These data suggest that Tyr 1175 mediates both PLC␥1-dependent and PKA-dependent signaling pathways. Taken together, our results not only reveal a novel Tyr 1175 -mediated signaling pathway but also highlight a potentially new therapeutic target for the management of vascular inflammation.

Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways

“…Upon stretch (150%), HUVECs rapidly (within 5 min) secreted a significant amount of vWF while their cell membrane remained intact (Supplementary information, Figure S1A and S1B). Stretch induced vWF secretion in a time-and magnitudedependent manner ( Figure 1B and 1C), similar to the responses to chemical agonists, such as thrombin and VEGF [18,19]. When dissecting the time dependency of stretch-induced vWF release rate, it is interesting to note that vWF release rate decreased quickly after an initial burst of secretion within 5 min (Supplementary information, Figure S1B) and continued to decrease gradually thereafter (Supplementary information, Figure S1C), indicating the presence of a possible negative feedback pathway in the regulation of vWF release.…”

“…VEGFR2 is expressed in both venous and arterial ECs. In addition, recent studies, including ours, have shown that VEGF induces WPB exocytosis in both arterial and venous ECs primarily mediated by VEGFR2 [18,19,29]. Therefore, it is not unexpected that stretch exerts a proexocytosis effect on both arteries and veins.…”

“…A VEGFR2/PLCγ1/Ca 2+ pathway positively regulates stretch-induced endothelial exocytosis Our previous work showed that the activation of the VEGFR2/PLCγ1/Ca 2+ -signaling pathway plays a major role in mediating VEGF-induced vWF exocytosis [19]. Therefore, we further explored whether this pathway is activated by stretch.…”

“…Previous work demonstrated that VEGFR2 mediates vWF/WPB exocytosis induced by VEGF [19]. Therefore, we set out to delineate the roles of VEGFRs in stretchtriggered WPB exocytosis.…”

“…In the signaling experiments, p85 was mostly used as the loading control, as we found that general loading controls such as GAPDH, tubulin and actin were too abundant to accurately reflect protein loading in lysates. The vWF ELISA kit was described previously [19]. …”