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W. Wu, L. Xu, J. Penn, F. Recchia; Ascorbate Decreases Vascular Endothelial Growth Factor Production in Rat Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):53.
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Oxidative stress plays a critical role in expression of vascular endothelial growth factor (VEGF) through the hypoxia-inducible factor 1-alpha (hif1) pathway and may thus contribute to retinal neovascularization. The stability of hif1 determines the level of VEGF. Prolyl hydroxylase is a catalytic enzyme that alters the stability of hif 1 in response to changes in oxygen tension. Ascorbate, an antioxidant, is a cofactor for prolyl hydroxylase. The purpose of this study was to evaluate effect of ascorbate on the hif1 pathway in retinal cells following exposure to hypoxia.
Primary cultures of rat retinal Muller cells (RMCs) and retinal microvascular endothelial cells (RMECs) were used for all experiments. Confluent cells were grown at 37ºC for 24 hours in either serum-free medium or 1% fetal bovine serum (FBS) medium containing varying concentrations (0 µM, 5 µM, 25 µM, 125 µM, 250 µM, and 500 µM) of ascorbate under both normoxic and hypoxic (O2 < 5%) conditions. VEGF mRNA expression in RMCs was measured by quantitative rt-PCR. Protein expression of hif1 and vascular endothelial growth factor receptor type-2 (VEGFR2) in RMECs were measured by Western blotting.
In RMCs, expression of VEGF mRNA was reduced by 40% following treatment as little as 25µM ascorbate. In RMECs, expression of VEGFR2 protein was reduced by 50% following treatment with 125 µM ascorbate. In both RMCs and RMECs, treatment with ascorbate led to a dose-dependent reduction in expression of hif1 protein (40% following 125µM ascorbate and 70% following 500 µM ascorbate).
These in vitro results indicate that ascorbate may play a strong modulatory role in expression of VEGF and its expression in retinal tissue via a hypoxia-inducible pathway.
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