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(Circulation Research. 2001;88:1247.)
© 2001 American Heart Association, Inc.

Molecular Medicine

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

Jennifer L. Hall, Xiaohong Wang, Van Adamson, Ying Zhao, Gary H. Gibbons

From the Division of Cardiovascular Medicine (J.L.H., G.H.G.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass, and Cardiovascular Research Institute (J.L.H., X.W., V.A., Y.Z., G.H.G.), Morehouse School of Medicine, Atlanta, Ga.

Correspondence to Jennifer L. Hall, PhD, Division of Molecular Cardiology, Department of Medicine, University of Minnesota, MMC, Mayo, 420 Delaware, Minneapolis, MN 55455. E-mail hallx{at}tc.umn.edu

Abstract

Abstract— 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)-. 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-B (NF-B). However, blockade of NF-B with a dominant-negative IB (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-B activity. Deletion of the redox-sensitive domain of Ref-1 significantly inhibited TNF-induced NF-B 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-B was necessary to promote survival, we blocked NF-B activity with a dominant-negative IB (S32A/S36A). Indeed, blockade of NF-B 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-B–independent and NF-B–dependent signaling cascades, respectively. Moreover, it seems that Ref-1 may act as a critical cofactor, mediating the TNF-induced NF-B response in the vascular endothelium.

Key Words: endothelium • apoptosis • hypoxia • redox • nuclear factor-B

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