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D. Xing, J.A. Bonanno; HIF–1 Is Responsible for Protection of Corneal Fibroblasts by Hypoxia Preconditioning . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1607.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether HIF–1α is responsible for hypoxic preconditioning protection of corneal stromal cells from UV–induced apoptosis.
Primary culture of bovine corneal fibroblasts was established. Second passage cells were used throughout the entire experiment. Serum was reduced to 0.5% for all experiments. A siRNA for bovine HIF–1α o was designed and tested successfully. Cells were treated with HIF–1α –siRNA or mammalian scrambled sequence control siRNA for 24 hrs and then incubated in a hypoxia chamber (0.5% oxygen) for 4hrs. All cells were then placed in normoxia for 30 minutes and were then UV–irradiated by exposure to a germicidal lamp for 2 minutes. Four hours later, cells were stained for TUNEL assay. Western blot for HIF–1α under each condition was also performed.
After 4 hrs of 0.5% oxygen preconditioning, HIF–1α levels were increased more than 10–fold relative to normoxic incubation. Pretreatment with 75nM HIF –1α siRNA for 24 hours however, reduced levels of HIF–1α after 4 hours of hypoxia by 85%. The hypoxia induced increase in HIF–1α was unaffected by control siRNA. The percent TUNEL positive cells, four hours after UV irradiation, was 50.3% in normoxia control; 16.4% in hypoxia preconditioned control; 11.1% in hypoxia preconditioned control scrambled siRNA transfected cells; and 55.6% in the hypoxia preconditioned HIF–1α siRNA transfected cells.
HIF–1α is a protective factor in hypoxic preconditioning protection of corneal stromal fibroblasts from UV–induced apoptosis. Future studies will focus on exploring potential HIF–1α regulated protective agents.
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