High Glucose Increases VEGF in Bovine Retinal Endothelial Cells Via Peroxynitrite–induced Activation of Src Kinase

Abstract

Abstract: : 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.

Keywords: growth factors/growth factor receptors • gene/expression • vascular cells