Abstract
Purpose: :
To determine whether HIF–1α is responsible for hypoxic preconditioning protection of corneal stromal cells from UV–induced apoptosis.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: apoptosis/cell death • cornea: stroma and keratocytes • hypoxia