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

Huiyan Zeng,Yan Xiong,Changgeng Ruan,Chaoliang Wei,Masabumi Shibuya,Jincai Luo,Xiaofan Lu,Yingqing Huo,Zhenqian Hu,Chao Chen,Xiaoyu Zhang

doi:10.1074/jbc.m109.019679

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-gamma1 (PLCgamma1)/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 PLCgamma1 and PKA. These data suggest that Tyr(1175) mediates both PLCgamma1-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.

Highlights

In addition to its role in promoting angiogenesis, there is increasing evidence that vascular endothelial growth factor (VEGF) contributes to inflammation independent of its angiogenic functions, the molecular basis for this effect is incompletely understood (6 – 8)
We showed that VEGF receptor-2 (VEGFR2), but not VEGFR1, is responsible for VEGF-induced release of von Willebrand factor, a major marker of Weibel-Palade bodies (WPBs)
VEGFR2, but Not VEGFR1, Mediates VEGF-induced von Willebrand factor (vWF) Release—A previous study showed that VEGF stimulates WPB exocytosis from Human aortic endothelial cells (HAECs), as evaluated by vWF release as well as P-selectin translocation [20]

Summary

EXPERIMENTAL PROCEDURES

Additional procedures are described in the supplemental Materials and Methods. Cell Culture—Human umbilical vein endothelial cells (HUVECs) were grown in medium 199 (Invitrogen) containing fibroblast growth factor, heparin, and 20% fetal bovine serum (Hyclone, Ogden, UT). Human aortic endothelial cells (HAECs) were grown in endothelial growth medium 2-MV (Clonetics, San Diego, CA). RNA Interference—To silence phospholipase C-␥1 (PLC␥1) and protein kinase A (PKA) (catalytic ␣ and ␤ subunits), we used a lentiviral system (Sigma) for delivering short hairpin RNAs (shRNAs) into HUVECs. The target sequences (see supplemental Materials and Methods) and control scrambled sequences were selected according to an open program on the official website of Massachusetts Institute of Technology. Measurement of vWF and IL-8 by Standard Enzyme-linked Immunosorbent Assays (ELISAs)—HUVECs or HAECs were grown until confluent on 48-well plates and serum-starved for 5 h before stimulation with agonists.

RESULTS

Turn on vWF Release through

DISCUSSION

This is in contrast to the role that