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A. B. El-Remessy, S. Matragoon, R. B. Caldwell; Peroxynitrite Mediates VEGF-Stimulated Focal Adhesion Assembly via Thiol Oxidation in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4460.
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© ARVO (1962-2015); The Authors (2016-present)
VEGF is one of the most potent angiogenic growth factors. Focal adhesion assembly is a key step in cell migration and the angiogenesis response to VEGF. Our previous studies in bovine retinal endothelial (BRE) cells demonstrated that peroxynitrite mediates VEGF-induced tyrosine phosphorylation and angiogenic related function including cell migration and tube formation. We have shown that peroxynitrite (1µM) mimics the effects of VEGF in causing immediate tyrosine phosphorylation of VEGFR2, c-Src and focal adhesion kinase (FAK).The purpose of this study is to elucidate the molecular mechanisms by which peroxynitrite mediates VEGF/FAK activation in BRE cells.
The effects of the specific peroxynitrite decomposition catalyst (FeTPPs, 2.5 µM) or the thiol donor N-acetyl cysteine (NAC, 1mM) were studied on VEGF-mediated phosphorylation of FAK in BRE cells. 5-Iodo Acetamide Fluoresciene (IAF) and Fluoresciene antibody were used to label free thiols of the low molecular weight PTP (LWT-PTP), the specific phosphatase of FAK. Total free thiols were determined by measuring GSH levels using colorimetric assay. VEGF-induced cell migration was determined by wound healing assay.
VEGF-induced activation of FAK peaked at 15 minutes and was blocked by FeTTPs and inhibited partially by Src kinase inhibitor (PP2). FAK activation was also blocked by NAC, suggesting an oxidation-mediated action of peroxynitrite. Treatment of BRE cells with VEGF or 1 µM peroxynitrite resulted in time-dependant depletion of total free thiols that peaked at 15 minutes. IAF labeling of the free thiols of the FAK phosphatase (LMW-PTP) showed cysteine oxidation at 1 and 15 minutes which coincides with the peak of VEGFR2 and FAK phosphorylation. Cell migration assay revealed that FeTTPs and NAC blocked VEGF-stimulated cell migration
Taken together, these results indicate that peroxynitrite mediates VEGF-induced FAK activation partially via Src activation but mainly via oxidative inhibition of LMW-PTP. Thiol oxidation appears to be a key process in mediating VEGF’s angiogenic signal, implying a role for peroxynitrite-mediated oxidation in this process. Treatments targeting peroxynitrite may be effective in anti-angiogenic therapy.
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