Overexpression of Ref-1 Inhibits Hypoxia and Tumor Necrosis Factor–Induced Endothelial Cell Apoptosis Through Nuclear Factor-kB–Independent and –Dependent Pathways

We hypothesized that a redox-sensitive transcription factor, redox factor-1 (Ref-1) (HAP1, APE, and APEX), was critical in the regulation of endothelial cell survival in response to hypoxia and cytokines, including tumor necrosis factor (TNF)-a. Hypoxia resulted in a significant decrease in Ref-1 protein expression in both human umbilical vein endothelial cells and calf pulmonary artery endothelial cells. The hypoxia-induced decrease in Ref-1 expression was followed by a significant induction of apoptosis as measured by caspase 3 activity and nuclear morphology. Transient upregulation of Ref-1 significantly inhibited hypoxia-induced apoptosis. However, deletion of the redox-sensitive domain of Ref-1 abolished the antiapoptotic effect. We postulated that the antiapoptotic effects of Ref-1 were mediated through nuclear factor-kB (NF-kB). However, blockade of NF-kB with a dominant-negative IkB (S32A/S36A) expression vector had no effect on Ref-1–mediated survival under hypoxic conditions. The second aim of this study was to test the cytoprotective ability of Ref-1 upregulation in response to TNF-induced apoptosis. Ref-1 inhibition of TNF-induced death was associated with a significant potentiation of NF-kB activity. Deletion of the redox-sensitive domain of Ref-1 significantly inhibited TNF-induced NF-kB activation. Moreover, loss of the redox-sensitive domain also abolished the antiapoptotic effect of Ref-1 in response to TNF. To test whether Ref-1 induced activation of NF-kB was necessary to promote survival, we blocked NF-kB activity with a dominant-negative IkB (S32A/S36A). Indeed, blockade of NF-kB activity abolished the ability of Ref-1 to rescue TNF-induced apoptosis. In conclusion, upregulation of Ref-1 promotes endothelial cell survival in response to hypoxia and TNF through NF-kB–independent and NF-kB–dependent signaling cascades, respectively. Moreover, it seems that Ref-1 may act as a critical cofactor, mediating the TNF-induced NF-kB response in the vascular endothelium. (Circ Res. 2001;88:1247-1253.)