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A.T. De Silva, R. Kannan, S. He, S.J. Ryan, D.R. Hinton; RPE and Choroidal Endothelial Cells Differ in their Growth Factor Expression in Response to Oxidant Stress . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1631.
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Purpose: To determine the effect of oxidant stress on expression of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in oxidative stress-induced by BSO (a glutathione (GSH) depletor) and tert-butylhydroperoxide (tBH, a stable H2O2 analog) in human fetal RPE (RPE) cells and bovine choroidal endothelial cells (CEC). Methods: Early passage cultured RPE and CEC in 1% FBS containing DMEM and EGM media were exposed to 500 µM BSO for various times up to 48h or 200 µM tBH (6, 24 and 32h after a change in medium at 1 h). Cellular GSH was measured by ELISA (Oxford Biomedical Research, MI). Protein expression of HGF and VEGF in cell lysates and secretion into medium were determined by Western blot analysis using specific polyclonal antibodies. Results: Cellular GSH in RPE showed a rapid turnover (t1/2 ~3h) and a >90% depletion in 24h with 500µM BSO treatment. Exposure to tBH on the other hand, caused a significant, two-fold increase in cellular GSH at 6h and an insignificant decrease (<10%) at 24h. Similar changes were seen in BSO and tBH-treated CEC. Cell viability was not significantly altered by BSO and tBH during the respective exposure times. Expression of HGF in RPE cells treated with BSO showed an increase in the first 6h (>30%) and a decline thereafter at 24h (25%) and 48h (30%) as compared to untreated controls. However, exposure of RPE to tBH for 6, 24, and 32 h resulted in a modest decrease in HGF and VEGF expression. The expression of HGF and VEGF in BCEC was not significantly different from that of controls with either BSO or tBH treatments. Conclusions: The response to oxidative stress in HGF and VEGF expression differs between choroidal endothelial cells and RPE. The differential expression of HGF with BSO and tBH in RPE suggests a regulatory role for cellular thiol status.
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