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L. Crosson, R.A. Kroes, J.R. Moskal, R.A. Linsenmeier; Time Course of Hypoxia Inducible Factor–1 alpha (HIF–1) mRNA and Protein Levels in Rat Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5172.
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Purpose: HIF–1α is a key regulator of HIF–1 activity in response to hypoxia. Here we investigated the time course of HIF–1α mRNA and protein expression in the rat retina under normoxic and hypoxic conditions. Methods: Using pigmented rats, we investigated the expression of both HIF–1α mRNA and protein in the retina following hypoxia of varying durations. Hypoxia was induced by placing rats in a Plexiglas chamber in which the oxygen percentage was maintained with a Reming Oxycycler (Biospherix, Redfield, NY). Following the hypoxic episode, animals were euthanized by decapitation without anesthetic, and retinas were removed. HIF–1α mRNA and protein were measured during control conditions (normal air breathing) and immediately following exposure to 6–7% oxygen for 0.5, 1.0, 3.0 and 6.0 hours. For analysis of mRNA, total RNA was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen, Inc., Valencia, CA). Reverse transcription of 1µg RNA generated the substrate for quantitation of mRNA by qRT–PCR. For protein analysis, nuclear extract proteins were prepared and analyzed by Western blotting. Results: No differences were observed between control and hypoxic HIF–1α mRNA samples at any time point. HIF–1α protein levels were higher in hypoxic samples at 0.5, 1.0 and 3.0 hours relative to control (p<0.05). After 0.5 hours of hypoxia, HIF–1α protein levels were elevated approximately 7 fold relative to control. HIF–1α protein levels continued to increase during 3 hours of exposure, showing approximately a 14 fold induction of HIF–1α protein after 3 hours of hypoxia. There was a tendency for HIF–1α protein to be elevated after 6 hours of exposure to hypoxia, but the difference was not significant compared to control. Conclusions: HIF–1α protein levels were shown to not only remain elevated, but also to continue to increase for at least 3 hours after the onset of severe hypoxia. As in other tissues, this occurs with no detectable change in mRNA, implying that control of HIF–1 in the retina is largely posttranscriptional, likely at the level of protein turnover.
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