Abstract 248: Stabilization of cIAP2 during Postconditioning protects Endothelial Cells from Reperfusion Injury
Hypoxia-reperfusion causes a perturbance in the complex equilibrium of pro and anti apoptotic mechanisms which ultimately leads to apoptosis in the reperfused myocardium. Postconditioning (intermittent hypoxia at the onset of reperfusion) is a proven strategy to reduce reperfusion injury, however, the mechanisms are largely unknown in endothelial cells. Here we analyze the effect of postconditioning in endothelial cells and hypothesize that the ‘inhibitors of apoptosis proteins’ (IAPs), known as antiapoptotic mediators, are key elements of this protective mechanism.
Methods and Results: Exposure of human umbilical vein endothelial cells to severe hypoxia (Po2 < 2 mmHg) for 2 hrs causes a 2.1±0.5-fold increase in caspase 3 activation (western blot analysis; P<0.05, n=3, for all further parameters) and a 2.3±04-fold increase in apoptosis (annexin V staining) after 24 hrs of reoxygenation. cIAP2 but not cIAP1 is rapidly increased during hypoxia in a biphasic manner. Transcription inhibitor, Actinomycin D (20μg/ml) reveals that the 2.5-fold increase within 5 min of hypoxia (first phase) was independent of transcription, but the 3.1-fold increase after 60 min (second phase) was induced by gene transcription. cIAP2 levels dropped down to basal value with the onset of reperfusion. Importantly, cIAP2 levels could be maintained by postconditioning (3 short periods of intermittent hypoxia, 5 minutes each separated by a 5 minute reoxygenation) which abolished hypoxia-reoxygenation-induced apoptosis. Down regulation of IAP2 by siRNA strategy enhanced hypoxia-reoxygenation-induced apoptosis and diminished the protective effect of postconditioning.
Conclusions: 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 stabilized during hypoxia and could be maintained at an elevated level by postconditioning.