75 - VEGFR2 Signaling Driven by Cellular Redox Status

Abstract

Angiogenesis (a process of new vessel formation) is triggered by tissue ischemia. Paradoxically, angiogenesis is diminished in cardiovascular diseases (CVDs) due to a decrease in cellular glutathione (GSH) and increase in reactive oxygen species (ROS). Interestingly, our laboratory has demonstrated that a small decrease in cellular GSH promotes vascular endothelial growth factor (VEGF)-driven ischemic angiogenesis while a larger decrease in cellular GSH does not. Thus, we hypothesize VEGF receptor2 (VEGFR2) activation is regulated by intracellular GSH redox state. To specifically study the role of altered GSH redox state on angiogenesis, we used diamide to oxidize GSH to GSSG in human aortic endothelial cells. GSSG can react with protein cysteine thiols to form S-glutathionylated proteins that in turn affect protein function and stability. Our preliminary experiments show that drastic changes in GSH/GSSG activate VEGFR2 independent of VEGF while a moderate change does not activate the receptor. Interestingly, the addition of H2O2 alone did not activate VEGFR2 suggesting a role for GSH/GSSG in regulating VEGFR2. To understand the mechanism of S-glutathionylation in VEGFR2 activation, we used dimedone, a sulfenic acid trap. Pretreatment with dimedone abolished VEGFR2 activation after diamide treatment suggesting that cysteine sulfenic acid formation precedes S-glutathionylation. Local ROS production in the vicinity of proteins can effectively mediate cellular signaling. On that note, O2●– derived from endothelial nitric oxide synthase (eNOS) could activate VEGFR2 in altered GSH/GSSG. S-glutathionylation of eNOS leads to its uncoupling and drives O2●– generation. To test the role of O2●– in VEGFR2 activation in altered GSH redox state, we inhibited eNOS using L-NAME or siRNA and then treated with diamide. VEGFR2 activation decreased after eNOS inhibition suggesting a potential role for eNOS in VEGFR2 activation under altered GSH redox state. We believe a small decrease in GSH/GSSG would result in priming of VEGFR2 signaling pathway while a larger decrease in GSH/GSSG will result in dysfunctional signaling. In conclusion, the GSH/GSSH ratio is important in triggering and promoting VEGFR2 signaling.