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Robert S. Cargill, Trevor J. McFarland, Michael H. Davies, M. J. Phillips, Deborah C. Otteson, Michael R. Powers, J. T. Stout, Binoy Appukuttan; Regulation of VEGF Expression in Hypoxic Mouse Müller Glia. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1856.
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© ARVO (1962-2015); The Authors (2016-present)
Alternate splice variants of RTEF-1, a TEAD-family transcription factor capable of modulating gene expression from the VEGF promoter, have been previously found in human ocular-derived vascular endothelial cells cultured in vitro. This study identifies novel isoforms of RTEF-1 in a mouse model of oxygen-induced retinopathy (OIR) and characterizes their function in regulating VEGF expression in a newly derived mouse Müller glial cell line (C57M10).
RNA was extracted from retinas of P8-P21 control and OIR mice, reverse transcribed, and used to PCR amplify alternatively spliced variants of RTEF-1. Amplicons were gel purified, sequenced, and novel isoforms were cloned into the mammalian expression vector pcDNA3.0. Human and mouse VEGF 5’ flanking promoters (1.1kb) were cloned into pSEAP reporter plasmids upstream of secreted alkaline phosphatase (SEAP). C57M10 cells were co-transfected with pcDNA.RTEF and pSEAP.VEGF plasmids by Amaxa nucleofection. After 48 hours, media was assayed for SEAP. Separately, varying amounts of CoCl2 were tested to determine the best concentration to simulate hypoxia. Following 20h of incubation, total RNA was isolated, reverse transcribed, and analyzed for VEGF transcript levels by quantitative real-time PCR. Protein isolated from triplicate samples was analyzed for RTEF and HIF1a by Western blot and VEGF by ELISA.
Two novel alternately spliced transcripts with premature stop codons resulting in predicted protein lengths of 160 and 203aa were cloned from normal and OIR retinas. When co-transfected in C57M10 Müller glia, both were found to enhance SEAP expression from the human and murine VEGF promoter up to 1.65-fold (p<0.05). By real-time PCR, it was found that 50uM CoCl2 could induce VEGF transcript levels 2.6-fold compared to untreated controls (p<0.01) without inducing significant toxicity.
Two novel RTEF-1 isoforms exist within the OIR and developing murine retina. RTEF-160 and RTEF-203 activation of the VEGF promoter in vitro identifies them as potential targets for therapeutic control of hypoxia-induced VEGF expression. The ability to up-regulate VEGF expression in cultured Müller glia using CoCl2-induced hypoxia provides a novel in vitro model system for studying hypoxic response and developing treatments for hypoxia-induced retinopathies.
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