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Jaime Chew, Sung Rhan Weon, Veluchamy A. Barathi, Roger W. Beuerman; Triamcinolone Acetonide (TA) Averts The Effects Of Hypoxic Stress On Mouse Retinal Pigment Epithelial Cells (RPE). Invest. Ophthalmol. Vis. Sci. 2012;53(14):4770.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the effect of TA on the expression of pigment epithelium derived factor (PEDF), thrombospondin-1 (TSP-1) and vascular endothelial growth factor A (VEGF-A) in cultured mouse RPE cells under hypoxic stress.
Primary cultures of mouse RPE cells were grown in culture to passage 2-3 and were then subjected to hypoxic stress. TA (50µg/ml) was used to determine the steroid effect under normoxic (95% air/5 % carbon dioxide) and hypoxic (95% nitrogen/5% carbon dioxide) conditions. Hypoxia was continued for 48 hrs with and without TA. Cells were harvested at 0, 24 and 48 hours and both RNA and protein were extracted. Real Time PCR and western blotting was used to analyze expression levels of PEDF, TSP-1 and VEGF-A.
After 24 hours of hypoxia, mRNA expression levels of both PEDF and TSP-1 were down-regulated by 2-fold. Hypoxic stress for 48 hours, resulted in stronger down-regulation of PEDF and TSP-1; down-regulation of 4.4-fold and 3.3-fold respectively. VEGF-A transcripts levels, on the other hand, showed a slower response to hypoxic stress, an up-regulation of 1.1-fold at 24 hours and 1.5-fold increase at 48 hours. Hypoxic RPE cells, when exposed to TA, led to the rapid recovery of PEDF and TSP-1 transcripts at 24 hours after the initiation of hypoxia. VEGF-A mRNA expression remained at control levels in TA treated hypoxic cells. Western blots showed that both PEDF and TSP-1 protein levels correlated with the PCR results. TA significantly induced the expression of PEDF and TSP-1 protein in hypoxic cells, and the strongest expression was at 48 hours.
Exposure of mouse RPE cells to oxidative stress resulted in significant loss in the expression pattern of PEDF and TSP-1. TA administration rapidly rescued the loss of expression. Hence our results suggest that TA has a protective effect on PEDF and TSP-1 levels, and that a balance of PEDF, TSP-1 and VEGF-A levels, may in turn prevent the progression of neovascularization in the retina.
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