Activation of STAT3 in the Ischemic Retina and in Retinal Endothelial Cells Subjected to Hypoxia Is Dependent on NAD(P)H Oxidase Activity

Abstract

Abstract: : Purpose: The signal transducer and activator of transcription 3 (STAT3) is an important modulator of VEGF expression and activity. We have previously shown that STAT3 is activated in the ischemic retina and in bovine retinal endothelial cells (BREC) subjected to hypoxia. Recent evidence has suggested that production of reactive oxygen species (ROS) by NAD(P)H oxidase plays a critical role in the induction of hypoxia–induced VEGF expression and retinal neovascularization (RNV). Therefore, we hypothesized that hypoxia/ischemia induction of STAT3 activation involves NAD(P)H oxidase activity Methods:Retinal ischemia was induced in neonatal mice by exposure to 75% oxygen for 5 days (vaso–obliteration period) followed by 5 days in normoxia (relative ischemia), a time in which neovascularization occurs. During the time of relative ischemia mice were treated with the NAD(P)H oxidase specific inhibitor apocynin (10mg/kg/day). BREC were subjected to 6h of hypoxia (pO2<2%) in the presence or absence of 30µM apocynin, 50U/ml cell permeable superoxide dismutase (PEG–SOD), and 1mg/ml gp91phox–ds–tat peptide inhibitor which specifically targets endothelial NAD(P)H oxidase. Western blotting was used to measure phospho–(Tyr 705)–STAT3 (PYSTAT3), the activated form of STAT3, in the different experimental conditions. Results: PY STAT3 formation was increased in the ischemic retinas by 3 fold (p<0.01, n=7 versus normal age–matched control mice). The apocynin treatment reduced PYSTAT3 levels to near control levels (p<0.05, n=4 versus untreated mice in relative ischemia). Furthermore, exposure of BREC to hypoxia rapidly induced PYSTAT3. This effect was completely blocked by pre–treatment of the cells with gp91phox–ds–tat and significantly reduced by the apocynin treatment. The PYSTAT3 was only slightly reduced by PEG–SOD, suggesting that hydrogen peroxide can also activate STAT3. Conclusions: These results support our working hypothesis that NAD(P)H oxidase activity is required for STAT3 activation and provide new insights about the molecular mechanisms linking ROS formation and VEGF expression in the ischemic retina.

Keywords: growth factors/growth factor receptors • hypoxia • retinal neovascularization