title: Hypoxia preconditioning protection of corneal stromal cells requires HIF1alpha but not VEGF.
abstract: OBJECTIVE
  Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1alpha (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-1alpha, and HIF-1alpha siRNA to test if HIF-1alpha 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-1alpha and VEGF in transcriptional and translational levels.
  
  
  RESULTS
  During hypoxia (0.5% O2), 5 muM cadmium chloride completely inhibited HIF-1alpha expression and reversed the protection by hypoxia preconditioning. HIF-1alpha siRNA (15 nM) reduced HIF-1alpha expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1alpha 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-1alpha, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1alpha.
  
  
  CONCLUSIONS
  These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1alpha, but that VEGF is not a component of the protection.
authors.affiliation: School of Optometry, Indiana University, Bloomington, IN 47405, USA.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Xing
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Bonanno
mesh_terms: D000818:Animals; D017209:Apoptosis; D019256:Cadmium Chloride / Q000494:pharmacology; D002417:Cattle; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020287:In Situ Nick-End Labeling; D034622:RNA Interference; D034741:RNA, Small Interfering; D013312:Stress, Physiological; D014466:Ultraviolet Rays; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D019256:Cadmium Chloride