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R.E. Burton, R. Kannan, S. He, S.J. Ryan, D.R. Hinton; The Effect of Glutathione Depletion in Human RPE on Expression of HGF and VEGF . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1630.
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Purpose: To study changes in mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor-beta2 (TGF-ß2) in human fetal retinal pigment epithelial cells (RPE) in response to oxidant stress induced by dl-buthionine-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis. Methods: RPE cells cultured in DMEM containing 1% FBS (passage 3) were treated with 500 µM BSO for 0-48h. After total RNA isolation, cDNA was synthesized for real-time polymerase chain reaction (PCR) analysis. Gene specific primers for HGF, VEGF, and TGF-ß2 were used to quantitate and compare mRNA samples from control and BSO- treated RPE. Cell viability of RPE was evaluated by Trypan Blue exclusion staining. The effect of BSO on expression of the above growth factors was determined using immunocytochemical staining. Results: BSO treatment for 6 and 48 hours caused a 2-fold and a 5-fold increase in VEGF mRNA expression from control levels in RPE, respectively. In the initial 6 hours of treatment, HGF mRNA decreased 60% followed by a return to control levels in 24 hours and a 4-fold increase in 48 hours. TGF-ß2 mRNA levels were not significantly changed by BSO treatment. Viability of RPE (93-95%) was little affected with BSO treatment for up to 48 hours. Immunochemical staining generally confirmed the pattern observed in mRNA studies. Conclusions: HGF and VEGF are differentially regulated in oxidant stress induced by GSH depletion in RPE while TGF-ß2 expression is not affected.
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