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Abstract
Achieving therapeutic angiogenesis remains an unrealized goal in cardiovascular diseases. We have previously shown that while low levels of peroxynitrite are required to transduce vascular endothelial growth factor (VEGF) signal, high levels of peroxynitrite can impair its signal, suggesting that changes in cellular redox-state can dictate the outcome for the VEGF signal. The overall goal of this project is to examine the impact and elucidate the molecular mechanisms by which shifting the cellular redox-state modulates the outcome of the VEGF angiogenic signal. Using an ischemic retinopathy mouse model, we demonstrated that excessive peroxynitrite formation impaired the VEGF survival signal via tyrosine nitration of the p85 regulatory subunit of the PI-3 kinase, which resulted in retinal endothelial cell death. Next, we assessed the impact of acute reductive stress by knocking down the expression of thioredoxin inhibiting protein (TXNIP), the endogenous inhibitor of the major antioxidant, thioredoxin. TXNIP deficient mice (TKO) experienced systemic and retinal reductive stress and expressed similar VEGF levels compared to wild type. However, retinal revascularization and VEGFR2 phosphorylation were significantly impaired in TKO mice. Low molecular weight protein tyrosine phosphatase (LMW-PTP), a redox-regulated phosphatase, regulates activation of VEGFR2 and focal adhesion kinase (FAK), key mediators of VEGF-mediated cell migration. In vitro studies demonstrated that VEGF transiently oxidizes cellular glutathione (GSH) levels to facilitate S-glutathionylation and oxidative inhibition of LMW-PTP resulting in activation of FAK and cell migration. Mild shift of redox-state to oxidative stress augmented VEGFs angiogenic response while it was blunted by acute oxidative stress. Shifting redox-state to reductive stress using antioxidants or silencing TXNIP expression blunted S-glutathionylation of LMW-PTP, activation of VEGFR2 and angiogenic response. In summary, our results showed that while mild oxidative stress augments VEGF signal and angiogenic response, acute oxidative stress and acute reductive stress impair VEGF signal. Our findings identified S-glutathionylation of LMW-PTP as a novel therapeutic target to regulate VEGF angiogenic response.