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(Circulation. 1999;100:II-361.)
© 1999 American Heart Association, Inc.

Myocardial Protection and Vascular Biology

Oxidative Stress Induces NF-B Nuclear Translocation Without Degradation of IB

Timothy G. Canty, Jr, MD; Edward M. Boyle, Jr, MD; Angie Farr, BS; Elizabeth N. Morgan, MD; Edward D. Verrier, MD; Timothy H. Pohlman, MD

From the Department of Surgery, University of Washington, Seattle.

Correspondence to Timothy H. Pohlman, Department of Surgery, University of Washington, Harborview Medical Center, Box 359796, Seattle, WA 98104-2924. E-mail tpohlman{at}u.washington.edu

Background—Rel/NF-B, an oxidative stress–responsive transcription factor, participates transiently in the control of gene expression. The cellular mechanisms that mediate NF-B activation during ischemia (and during reperfusion in the course of treating ischemia) are not known.

Methods and Results—To investigate the NF-B activation induced during oxidative stress, we examined human cardiac tissue obtained during surgical procedures requiring cardiopulmonary bypass. In vitro, we examined human umbilical vein endothelial cells (HUVECs) exposed to hypoxia, reoxygenation after hypoxia, or a reactive oxygen intermediate (H₂O₂). Electrophoretic mobility shift assays performed on right atrial tissue revealed prominent NF-B activation after hearts had been exposed to ischemia and reperfusion. The assays also showed that NF-B activation was observed in hypoxic HUVECs after reoxygenation and in cultures treated with H₂O₂ (500 µmol/L). Pervanadate (200 µmol/L) also induced marked NF-B activation in HUVECs, indicating that H₂O₂-induced NF-B activation is potentiated by the inhibition of tyrosine phosphatases. Western blotting of cytoplasmic IB demonstrated that NF-B activation induced by oxidative stress was not associated with IB degradation. In contrast, tumor necrosis factor-–induced NF-B activation occurred in concert with degradation of IB. Inhibition of IB degradation with a proteasome inhibitor, MG-115, blocked NF-B activation induced by tumor necrosis factor-; however, MG-115 had no effect on NF-B activation during oxidative stress.

Conclusions—This study demonstrated a stimulus-specific mechanism of NF-B activation in endothelial cells that acts independently of IB degradation and may require tyrosine phosphorylation.

Key Words: cardiopulmonary bypass • ischemia • reperfusion • endothelium