Postconditioning (intermittent hypoxia at the onset of reperfusion), a strategy known to effectively reduce reperfusion injury, is well proven in cardiomyocytes. However, little is known about postconditioning in endothelial cells and whether it plays a role in anti-apoptosis, which is the predominant form of cell death in endothelial cells. Here the protective effect of postconditioning in endothelial cells and the molecular mechanisms involved were studied, focusing on the inhibitors of apoptosis proteins (IAPs) as potential anti-apoptotic candidates induced by hypoxia. Exposure of human umbilical vein endothelial cells to severe hypoxia (Po2 greater than 1 mmHg) for 2 h caused a 2.1 ± 0.3 fold increase in caspase-3 cleavage, 6 h after reoxygenation and a 2.3 ± 0.2 fold increase in apoptosis (annexin V staining) 24 h after reoxygenation. Postconditioning abolished hypoxia/reoxygenation-induced apoptosis in endothelial cells. Quest for possible anti-apoptotic molecules led to the observation that cIAP2 but not its close homologue cIAP1 or XIAP, is upregulated during hypoxia and reduces to basal level with the onset of reoxygenation. Importantly, cIAP2 could be maintained by postconditioning in an ERK1/2 and PI3-Kinase dependant manner. Hypoxia as well as postconditioning induced an interaction between cIAP2 and procaspase-3 (co-immunoprecipitation and co-localization in immunochemistry), suggesting a mechanism by which cIAP2 counteracts hypoxia/reoxygenation-induced apoptosis. Downregulation of cIAP2 with siRNA enhanced hypoxia/reoxygenation-induced apoptosis and abolished the protective effect of postconditioning. Maintenance of cIAP2 by postconditioning in the intact vessel confirms the patho-physiological significance of the finding. The present study shows for the first time that postconditioning can protect endothelial cells against hypoxia/reoxygenation-induced apoptosis. This protective effect is conferred by the cIAP2, which is expressed during hypoxia and could be maintained at an elevated level by postconditioning, interacting with procaspase-3.
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