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(Stroke. 2000;31:1179.)
© 2000 American Heart Association, Inc.

Original Contributions

Hypertonic Mannitol Loading of NF-B Transcription Factor Decoys in Human Brain Microvascular Endothelial Cells Blocks Upregulation of ICAM-1

Presented in part at the 24th International Joint Conference on Stroke and Cerebral Circulation (American Heart Association), Nashville, Tenn, February 46, 1999.

David C. Hess, MD; Eugene Howard, PhD; Charles Cheng, PhD; James Carroll, MD W. David Hill, PhD

From Neuroscience Service Line, VA Medical Center (D.C.H.), and the Departments of Neurology (D.C.H., J.C.), Biochemistry and Molecular Biology (E.H., C.C.), and Anatomy and Cell Biology (W.D.H.), Medical College of Georgia, Augusta.

Correspondence to David C. Hess, MD, Neuroscience (27), VA Medical Center, Augusta, GA 30904. E-mail dhess{at}neuro.mcg.edu

Background and Purpose—An acute inflammatory response exacerbates tissue injury during acute ischemic stroke. The transcription factor nuclear factor (NF)-B plays a key role in endothelial cell activation and the inflammatory response. Targeted genetic disruption of NF-B activation in cerebral endothelial cells may be protective in stroke. We determined whether a NF-B transcription factor decoy (TFD) could block intercellular adhesion molecule (ICAM)-1 upregulation, an indicator of endothelial cell activation.

Methods—We modeled ischemia-reperfusion in vitro by exposing cultured human brain microvascular endothelial cells (HBMEC) to tumor necrosis factor (TNF)- and conditions of hypoxia-reoxygenation (H/R). Mannitol was used to load phosphothiorated oligonucleotides containing 3 copies of the B binding sequences (TFDs) into cultured HBMEC. An NF-B TFD, a mutated NF-B TFD, and a scrambled TFD were studied for their effect on ICAM-1 mRNA levels and surface ICAM-1 by ELISA.

Results—Hyperosmolar loading with mannitol permitted rapid transfection of TFD into endothelial cell nuclei. The NF-B TFD but not the mutated or scrambled TFD competed with a B sequence for binding to nuclear extracts from HBMEC exposed to TNF-. The NF-B TFD blocked the TNF-–induced and H/R-induced increase in ICAM-1 mRNA levels and the upregulation of surface ICAM-1.

Conclusions—Mannitol delivers phosphothiorated oligonucleotides into cultured HBMEC. An NF-B decoy blocks both TNF-–induced and H/R-induced ICAM-1 upregulation in HBMEC. Targeted genetic disruption of endothelial NF-B activation may be of benefit in acute ischemic stroke.

Editorial Comment

Presented in part at the 24th International Joint Conference on Stroke and Cerebral Circulation (American Heart Association), Nashville, Tenn, February 46, 1999.

Chung Y. Hsu, MD, PhD, Guest Editor

Department of Neurology Washington University School of Medicine St Louis, Missouri

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