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D.H. Platt, M. Bartoli, A. El–Remessy, M. Al–Shabrawey, T. Lemtalsi, R.B. Caldwell; High Glucose Increases VEGF in Bovine Retinal Endothelial Cells Via Peroxynitrite–induced Activation of Src Kinase . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4195.
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Purpose: In diabetic retinopathy increased expression of VEGF is associated with formation of the peroxynitrite (ONOO –) biomarker nitrotyrosine. Work in our laboratory has also demonstrated that in microvascular endothelial cells high levels of glucose induce VEGF overexpression via ONOO – –mediated activation of the transcription factor STAT3. The purpose of this study was to understand the molecular mechanisms linking high glucose–induced ONOO – formation and STAT3–dependent VEGF expression. Recent studies have shown that constitutively active cSrc induces activation of STAT3 and VEGF production in cancer. Other studies have shown that ONOO– can induce activation of cSrc. Therefore, we hypothesized that the high glucose/ONOO– –induced activation of STAT3 and increased VEGF expression are dependent on cSrc. Methods: We tested our hypothesis by determining the effects of blocking Src on ONOO– and high glucose–induced STAT3 activation and VEGF expression in cultured bovine retinal endothelial (BRE) cells. BRE were treated with high glucose (25mM for 72 hours) or ONOO – (100 µM for one hour) in the presence and absence of the peroxynitrite decomposition catalyst FeTPPs (50µM), the Src kinase inhibitor PP1 (50 µM) or a dominant negative cSrc adenoviral vector (30 MOI for 12 hours). Real–time quantitative PCR and Western analyses were performed to measure expression of VEGF and activation of STAT3 and cSrc. Results: ONOO – induced activation of cSrc. FeTPPS blocked the action of high glucose in activating cSrc. PP1 and dominant negative cSrc blocked the effects of ONOO – and high glucose in stimulating activation of STAT3 and expression of VEGF. Conclusions: Our data indicate that high glucose stimulates VEGF expression in microvascular retinal endothelial cells via peroxynitrite–mediated activation of cSrc. These results suggest that peroxynitrite may be a therapeutic target for the prevention of diabetic retinopathy.
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