VEGF Autocrine Gene Expression in Retinal Microvascular Endothelial Cells is Mediated by STAT3

Abstract

Abstract: : Purpose: Retinal microvascular endothelial cells are known to be more sensitive to VEGF's angiogenic actions than macrovascular endothelial cells, but the molecular basis for this difference is not understood. Recently, it has been shown that the transcription factor signal transducer and activator of transcription 3 (STAT3) is a key modulator of VEGF expression in cardiac tissue and in human tumors. We have previously shown that VEGF stimulation induces STAT3 activation in bovine retinal endothelial cells (BREC) uniquely as compared to bovine aortic endothelial cells (BAEC). We hypothesized that STAT3 plays a role in VEGF's autocrine gene expression in BREC. To test this we compared VEGF autocrine gene expression in BREC and BAEC. We also measured VEGF autocrine production in BREC in which we inhibited STAT3 expression Methods: Cultured BREC were stimulated with 25 ng/ml VEGF₁₆₅ for different times. Quantitative real time PCR was used to measure VEGF mRNA formation. STAT3 expression was inhibited by hypertonic loading of phosphorothioate antisense oligonucletides directed against the starting region of the human STAT3 gene. Western blotting analysis was performed to determine STAT3 protein in the treated cells. Results: Quantitative real time PCR analysis of VEGF autocrine gene expression in BREC showed a significant increase in mRNA formation that peaked at 1 hour, declined at 2 hours and returned to the basal level at 3 hours. BAEC treated in the same conditions did not respond to autocrine stimulation of VEGF expression. Antisense-mediated STAT3 depletion was induced in BREC resulting in 75% protein inhibition. VEGF autocrine expression was significantly inhibited in the antisense-transfected but not in the sense-transfected and the control BREC. Conclusions: Our results indicate that STAT3 plays a key role in VEGF autocrine expression in retinal microvascular endothelial cells. The differences between STAT3 activation and VEGF autocrine expression in BREC and BAEC may contribute to the unique sensitivity of retinal endothelial cells to diabetes-induced vascular pathology.

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