Inhibition of HIF prolyl 4-hydroxylases protect endothelial cells from apoptosis
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In the present study a new approach against the ongoing apoptotic cell death duringhypoxia/reoxygenation (H/R) in endothelial cells was established. Particularly, theactivation of hypoxia-inducible factor (HIF-1alpha), plays an essential role in triggeringcellular protection during hypoxia, but it s rapidly degradation during reoxygenation, maylimit its effect on cell survival to the hypoxic period alone. Regulation of HIF-1alphaexpression is controlled by repression of oxygen-dependent prolyl 4-hydroxylases (PHD)during hypoxia. The present study was conducted to elucidate the molecular mechanismby which inhibition of PHD pathway leads to protection of endothelial cells againstongoing apoptotic cell death during H/R. The study was performed using an establishedmodel of cultured monolayers of human umbilical vein endothelial cells to test thehypothesis that stabilization of HIF-1alpha beyond hypoxia exerts anti-apoptotic effectsduring H/R by preventing p53-mediated apoptosis.Cells were serum starved for 12 h, then exposed to 1 h of hypoxia (Po2 < 5mmHg)followed by 24 h of reoxygenation (Po2=140mmHg). Exposure to hypoxia caused anincrease in HIF-1alpha and p53 content. During reoxygenation HIF-1alpha declined towardsbasal level, while p53 remained unaltered. Under the same conditions, endothelialapoptosis was increased to 58% (annexin V staining). Silencing of PHD2, led to anincrease of the HIF-1alpha content during hypoxia and maintained it at that level duringreoxygenation. HIF-1alpha stabilization was associated by an increase in the Mdm2 content,whereas expression of p53 was reduced. PHD2 silencing reduced apoptosis to half.Additon of DMOG (1mM, dimethyloxalyl glycine), a PHD inhibitor, at the onset ofreoxygenation had the same effect. Reduction of p53 content was restored when theproteosome inhibitor MG-132 was added. Interaction of Mdm2 and p53 (coimmunoprecipitation)was increased compared to reoxygenation. Downregulation of HIF-1alpha by siRNA increased apoptosis to 60% and abrogated Mdm2-p53 complex formation.Downregulation of Mdm2 by siRNA had no effect on HIF-1alpha but increased p53 level.Stabilization of HIF-1alpha due to PHD inhibition beyond the period of hypoxia defines anovel mechanism that exerts anti-apoptotic effects during H/R injury by preventing p53-mediated apoptosis and identifies PHD2 as a new molecular target for therapeuticintervention.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler