title: Hypoxia inducible factor-1alpha mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells.
abstract: OBJECTIVE
  To investigate whether hypoxia inducible factor-1alpha (HIF-1alpha) is linked to the protective effects of ischemic preconditioning (IP) on sinusoidal endothelial cells against ischemia/reperfusion injury.
  
  
  METHODS
  Sinusoidal endothelial cell lines ECV-304 were cultured and divided into four groups: control group, cells were cultured in complete DMEM medium; cold anoxia/warm reoxygenation (A/R) group, cells were preserved in a 4 centigrade UW solution in a mixture of 95% N2 and 5% CO2 for 24 h; anoxia-preconditioning (APC) group, cells were treated with 4 cycles of short anoxia and reoxygenation before prolonged anoxia-preconditioning treatment; and anoxia-preconditioning and hypoxia inducible factor-1alpha (HIF-1alpha) inhibitor (I-HIF-1) group, cells were pretreated with 5 microm of HIF-1alpha inhibitor NS398 in DMEM medium before subjected to the same treatment as group APC. After the anoxia treatment, each group was reoxygenated in a mixture of 95% air and 5% CO2 incubator for 6 h. Cytoprotections were evaluated by cell viabilities from Trypan blue, lactate dehydrogenase (LDH) release rates, and intracellular cell adhesion molecule-1 (ICAM-1) expressions. Expressions of HIF-1alpha mRNA and HIF-1alpha protein from each group were determined by the RT-PCR method and Western blotting, respectively.
  
  
  RESULTS
  Ischemia preconditioning increased cell viability, and reduced LDH release and ICAM-1 expressions. Ischemia preconditioning also upregulated the HIF-1alpha mRNA level and HIF-1alpha protein expression. However, all of these changes were reversed by HIF-1alpha inhibitor NS398.
  
  
  CONCLUSIONS
  Ischemia preconditioning effectively inhibited cold hypoxia/warm reoxygenation injury to endothelial cells, and the authors showed for the first time HIF-1alpha is causally linked to the protective effects of ischemic preconditioning on endothelial cells.
authors.affiliation: Department of Organ Transplantation, Huashan Hospital, Fudan University, No.12, Wulumuqi Road, Shanghai 200040, China. shiliubin@medmail.com.cn
authors.forename: Liu-Bin
authors.initials: LB
authors.lastname: Shi
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Huang
authors.forename: Bao-San
authors.initials: BS
authors.lastname: Han
mesh_terms: D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003080:Cold Temperature; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000502:physiology; D019194:Ischemic Preconditioning; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000502:physiology; D008954:Models, Biological; D009578:Nitrobenzenes / Q000494:pharmacology; D010100:Oxygen / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000502:physiology; D015427:Reperfusion Injury / Q000503:physiopathology / Q000517:prevention & control*; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009578:Nitrobenzenes; D012333:RNA, Messenger; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D018799:Intercellular Adhesion Molecule-1; D007770:L-Lactate Dehydrogenase; D010100:Oxygen