Hypoxia preconditioning protection of corneal stromal cells requires HIF1α but not VEGF

PURPOSE: Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1α (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor). METHODS: Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1α, and HIF-1α siRNA to test if HIF-1α activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1α and VEGF in transcriptional and translational levels. RESULTS: During hypoxia (0.5% O2), 5 μM cadmium chloride completely inhibited HIF-1α expression and reversed the protection by hypoxia preconditioning. HIF-1α siRNA (15 nM) reduced HIF-1α expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1α dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning. VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning. However, the transcription and translation of VEGF were paradoxically increased by siHIF-1α, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1α. CONCLUSIONS: These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1α, but that VEGF is not a component of the protection.