Abstract 3658: STAT3-dependent VEGF Expression and Activation of HIF-1 alpha in Endothelial Cells Exposed to Hypoxia
The Signal Transducer and Activator of Transcription 3 (STAT3) has been shown to regulate the expression and activity of Vascular Endothelial Growth fator (VEGF) in a number of tissues including cardiac muscle and vascular endothelial cells. Tissue ischemia is a critical regulator of VEGF expression and it has been mainly attributed to the activity of the transcription factor, Hypoxia-inducible Factor-1 (HIF-1). Here we investigated the specific role of STAT3 in hypoxia-induced VEGF expression and in relation to the activity of HIF. Human umbilical vein endothelial cells (HUVEC) and human coronary endothelial cells in culture were subjected to 2% oxygen levels for different times (10min, 30min, 1h, 3h). Chromatin immunoprecipitation (ChIP) was conducted to identify STAT3 binding to VEGF promoter in hypoxic or normoxic (pO2=23%) conditions. Selective inhibition of STAT3 was achieved by siRNA or over-expression of the inactive mutant STAT3D. Western blotting analysis was used to determine phosphoprotein levels. Hypoxia rapidly (30min) stimulated STAT3 tyrosine phosphorylation (PYSTAT3) and nuclear translocation. Selective inhibition of STAT3 activity significantly blunted hypoxia-induced VEGF expression at mRNA and protein level (70% reduction, p<0.05, n=4) and significantly reduced (65%, p<0.03, n=5), the level of HIF-1’s inducible subunit, HIF-1alpha. Analysis of the VEGF promoter region using ChIP assay revealed that STAT3 binds VEGF promoter in hypoxic conditions in both HCEC and HUVEC. In addition, we found that STAT1, a STAT family member which has been reported to have anti-angiogenic activity, binds VEGF promoter in normoxic conditions but not in hypoxia. Our data show that STAT3 plays a critical role in hypoxia-induced VEGF expression and HIF-1 alpha activation in HCEC and HUVEC. In addition, our results suggest that STAT1 may function as a physiological repressor of VEGF gene expression.