Hypoxia inducible factor-1alpha mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells.

ABSTRACT

AIM: 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. CONCLUSION: 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.