Abstract P315: S-Glutathiolation of Low-Molecular-Weight PTP Regulates VEGF-Angiogenic Signal

Abstract

While reparative angiogenesis improves outcome of stroke and myocardial infarction; pathological angiogenesis aggravates tumors, psoriasis and diabetic retinopathy. We have previously shown that low levels of peroxynitrite (PN, 1µM) are required to mediate VEGF’s angiogenic signal. Prior studies showed that low molecular weight protein tyrosine phosphatase (LMW-PTP) can regulate activation of VEGFR2 and focal adhesion kinase (FAK), key proteins involved in VEGF angiogenic signal. Here, we test the hypothesis that VEGF-induced peroxynitrite regulates LMW-PTP activity via reversible S-glutathiolation. Our results using human microvascular endothelial (HME) cells showed that VEGF (20 ng/ml) caused immediate (1,5 min) and transient negative shift in redox-state assessed by reduced-glutathione (GSH) level that was restored back to baseline by 15–30 minutes. In parallel, total free thiols of LMW-PTP; assessed by 5-iodoacetamide; were immediately oxidized (1, 5 min) and recovered after 15–30 minutes. PN (1μM) mimicked VEGF’ actions by inducing oxidative inhibition of LMW-PTP and FAK phosphorylation that peaked at 15 min. Studies using anti-GSH antibody demonstrated strong s-glutathiolation of LMW-PTP thiols that peaked at (5–10min) under non-reducing conditions and were completely abrogated in DTT-treated samples. These effects were associated with impaired tyrosine phosphorylation of LMW-PTP. PN caused concentration-dependent significant inhibition of LMW-PTP phosphatase activity reaching maximum at 0.5mM. Under pathological conditions recapitulated by combining with peroxynitrite (0.5mM), VEGF caused immediate yet permanent negative shift in cellular redox-state, LMW-PTP inactivation and sustained FAK activation in HME cells. Modulating cellular redox-state to reductive stress using N-acetyl cysteine (NAC, 1mM) or FeTPPS (2.5μM) prevented VEGF-induced angiogenesis in vitro and in vivo using hypoxia-induced neovascularization mouse model. Taken together, shifting redox-state to oxidative stress or reductive stress can impair VEGF’s signal and achieving a balanced redox-state is critical to facilitate VEGF’ angiogenic signal.