Molecular mechanisms of high glucose-induced vascular endothelial growth factor expression in retinal endothelial cells

Abstract

Studies in diabetic patients, experimental animal models and tissue culture cells treated with high glucose have shown a close association between pathologic vascular growth, over-expression of the angiogenic factor vascular endothelial growth factor (VEGF) and oxidative stress. Studies of diabetic patients and high glucose treated cells have also shown increased levels of tyrosine nitration, a marker for the formation of the reactive nitrogen species peroxynitrite. Excess formation of reactive oxygen/nitrogen species has been shown to activate two transcription factors that regulate the expression of VEGF, hypoxia-inducible factor-I (HIF-1) and signal transducer and activator of transcription 3 (STAT3). These observations suggest that diabetes causes increases in VEGF expression due to the effects of high glucose in stimulating the formation of peroxynitrite, which l~ads to the activation of the transcription factors HIF-1 and/or ST AT3 and increases in VEGF expression. This hypothesis was tested by experiments using primary cultures of retinal endothelial cells treated with peroxynitrite or high glucose. Both treatments .increased VEGF mRNA and protein levels. Further, pretreatment with the 'specific peroxynitrite decomposition catalyst FeTPPs blocked the increase in VEGF expression. To determine if HIF-1 and/or STAT3 play a role in the peroxynitrite-induced VEGF expression, studies were done to analyze the activation patterns of both transcription factors. These studies showed that peroxynitrite had no effect on the activation or nuclear translocation of HIF-1 a, but did induce a rapid activation and nuclear translocation of STAT3. To further explore the role of STAT3 in the VEGF expression, cells were treated with peroxynitrite or high glucose in the presence or absence of an adenoviral vector expressing dominant-negative STAT3. Overexpression of the dominant-negative STAT3 blocked the effects of either peroxynitrite or high glucose in increasing VEGF mRNA. Further, treatment with FeTPPS blocked the effects of high glucose in stimulating activation of STAT3. A non-receptor tyrosine kinase, cSrc, has been shown to play a role in the activation of ST AT3 as well as the induction of VEGF expression during tumor angiogenesis. To determine if cSrc plays a role in ST AT3 regulated VEGF transcriptional activation, retinal endothelial cells were transduced with an adenovirus over-expressing a constitutively active Src (vSrc). The vSrc transduction induced activation of STAT3 and increased VEGF expression. Further, FeTPPs blocked the effects of peroxynitrite and high glucose in stimulating activation of cSrc. Additionally, the Src inhibitor PPl blocked the effects of peroxynitrite anμ high glucose in· increasing VEGF mRNA and protein expression. This work is the first to show that 1) high glucose-induced peroxynitrite formation increases· VE_GF expression, 2) STAT3 activation by high glucose-induced peroxynitrite formation regulates VEGF expression and 3) cSrc activation by high glucose-induced peroxynitrite formation activates STAT3 and increases VEGF expression.