Abstract 5432: Inhibition of Hif Prolyl 4-hydroxylases Protect Against Reoxygenation Injury in Endothelial Cells
Background: Hypoxia inducible factor-1 (Hif-1α) modulates expression of genes essential for cell survival under hypoxia. The oxygen-sensing enzyme prolyl 4-hydroxylase2 (PHD2) targets Hif-1α for destruction under normoxia. Hypoxia/Reoxygenation is associated with p53 activation, a master regulator of apoptosis in many type of cells. We hypothesized that preservation of Hif-1α beyond the period of hypoxia protects endothelial cells from reperfusion injury. To analyze the protective effect of Hif-1α, human umbilical veins endothelial cells were exposed to 1 h of hypoxia (Po2<5mmHg) followed by 24 hrs of reoxygenation (Reox.; Po2=140mmHg). Exposure to hypoxia causes a increase in Hif-1α, p53, and Mdm2 (E3 ubiqitin ligase) by 3.1±0.2, 2±0.3, and 1.9±0.2-fold compared to control, respectively. During Reox. Hif-1α and Mdm2 declined towards basal level, while p53 remained unaltered. Under the same conditions endothelial apoptosis was increased to 47±3% (annexin V staining). Silencing of PHD2, (53% reduction of PHD2 content) lead to an increase of the Hif-1α content during hypoxia and maintained it at that level during Reox. Hif-1α stabilization was accompany by an increase in the Mdm2 content and phosphorylation, whereas expression of p53 was reduced. PHD2 silencing reduced apoptosis to half. Additon of DMOG (1mM dimethyloxalylglycine), a PHD inhibitor, at the onset of Reox had the same effect. Reduction of p53 content was restored when the proteosome inhibitor MG-132 was added. Interaction of Mdm2 and P53 (co-immunoprecipitation) was increased compare to Reox. Downregulation of Hif-1α by siRNA (64% reduction of Hif-1α content) increased apoptosis to 66±5% and abrogate Mdm2-p53 complex formation. Downregulation of Mdm2 by siRNA (38% reduction of content) had no effect on Hif-1α but increased p53 level.
Conclusions: siRNA downregulation as well as pharmacological inhibition of PDH2, manoeuvre which inhibit Hif-1α degradation and maintained it at that level during Reox. protect against reperfusion injury. This effect of Hif-1α involves stimulation of Mdm2 induced degradation of the proapoptotic p53.