Peroxynitrite Induces Transcriptional Activation of Vascular Endothelial Growth Factor in Bovine Retinal Endothelial Cells

Abstract

Abstract: : Purpose: Increased vascular endothelial growth factor (VEGF) expression is associated with retinal neovascularization as seen in ischemic retinopathy. Work in our laboratory has demonstrated a positive correlation between VEGF expression and the formation of peroxynitrite (ONOO^-) in models of diabetic retinopathy and retinal neovascularization. Because some reactive oxygen species have been shown to induce transcriptional activation of VEGF in other cell types, we hypothesized that ONOO^- may directly activate transcription in retinal endothelial cells. We tested this by determining the effects of ONOO^- treatment on VEGF expression in retinal microvascular endothelial cells. We also assessed the ONOO^- -induced activation of two transcription factors known to be involved in the regulation of VEGF gene expression, signal transducer and activator of transcription 3 (STAT3) and hypoxia inducible factor-1 alpha (Hif-1α). Methods: Cultured bovine retinal endothelial cells (BRE) were stimulated with ONOO^- at various concentrations and times. Real-time quantitative PCR and immunoprobe techniques were performed to measure VEGF expression in BRE. STAT3 activation and Hif-1α induction were monitored by western analysis and quantitative PCR respectively. Results: ONOO^- stimulation of BRE resulted in significant time and dose-dependent increases in VEGF mRNA which peaked at 60 minutes and 100 µM ONOO^-. This effect was followed by a significant increase in VEGF protein. STAT3 was activated in a time and dose-dependent manner as shown by its tyrosine phosphorylation as early as 5 minutes at 100 µM ONOO^- whereas Hif-1α mRNA expression was increased only after 8 hours. Conclusions: Our results indicate that ONOO^- stimulates VEGF expression in retinal microvascular endothelial cells. This effect positively correlates with increased tyrosine phosphorylation and activation of the transcription factor STAT3. Hif-1α is induced after increased VEGF expression and STAT3 phosphorylation, suggesting that STAT3 is the primary mediator of early ONOO^- -induced VEGF gene transcriptional activation.

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