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T. J. McFarland, B. Appukuttan, T. J. Stout; Attenuation of VEGF by the RTEF-1 651 Isoform in Primate Primary Mueller Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):497.
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Our group has previously identified novel human retinal specific isoforms of the related transcription enhancer factor (RTEF-1) gene. We have demonstrated that these isoforms are capable of up or down regulating gene expression from the VEGF promoter using an in vitro reporter assay. Mueller cells have been implicated as being a major source of VEGF production in the retina. The current study investigates whether Mueller cell VEGF expression can be attenuated via a specific RTEF-1 isoform in vitro.
Primate Mueller cells were isolated by comparing three different techniques. Cells were examined for GFAP and Vimentin staining by immunocytochemistry. Lentivirus containing GFP (LV-GFP) was used to test efficiency of transduction. Cells were cultured for two weeks, after which cells were plated into 6 well plates at equal densities. One group was transduced with a lentiviral vector containing the RTEF-1 651 isoform, the other group served as the control. After 30 hours of incubation, cells were assayed by semi-quantitative RT-PCR and ELISA of media for VEGF. VEGF levels were normalized to beta-actin mRNA or total media protein
Mueller cells were positive for Vimentin and GFAP, and LV-GFP transduced cells were fluorescent after examination by confocal microscopy. Semi-quantitative RT-PCR for VEGF demonstrated a 37% reduction in VEGF within transduced cells as compared to controls by densitometry. Expression of the RTEF-1 651 isoform was confirmed by RT-PCR from transduced cells. VEGF ELISA demonstrated a 20% decrease in VEGF levels compared to controls.
Up regulation of VEGF gene expression is believed to be a major contributor to the pathogenesis of various retinal diseases. These data suggest that the RTEF-1 651 isoform is capable of VEGF modulation in primary Mueller cells.The possibility of VEGF promoter control using native transcription regulators has attractive therapeutic implications.
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