Hypertonic mannitol loading of NF-κ̄ transcription factor decoys in human brain microvascular endothelial cells blocks upregulation of ICAM-1

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

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

Background and Purpose - An acute inflammatory response exacerbates tissue injury during acute ischemic stroke. The transcription factor nuclear factor (NF)-κB plays a key role in endothelial cell activation and the inflammatory response. Targeted genetic disruption of NF-κB activation in cerebral endothelial cells may be protective in stroke. We determined whether a NF-κB 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)- α and conditions of hypoxia-reoxygenation (H/R). Mannitol was used to load phosphothiorated oligonucleotides containing 3 copies of the κB binding sequences (TFDs) into cultured HBMEC. An NF-κB TFD, a mutated NF-κB 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-κB TFD but not the mutated or scrambled TFD competed with a κB sequence for binding to nuclear extracts from HBMEC exposed to TNF-α. The NF-κB TFD blocked the TNF-α-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-κB decoy blocks both TNF-α-induced and H/R-induced ICAM-1 upregulation in HBMEC. Targeted genetic disruption of endothelial NF-κB activation may be of benefit in acute ischemic stroke.

Original languageEnglish (US)
Pages (from-to)1179-1186
Number of pages8
JournalStroke
Volume31
Issue number5
DOIs
StatePublished - May 2000
Externally publishedYes

Keywords

  • Endothelium
  • Genetic
  • Intercellular adhesion molecule-1
  • Nuclear factor kappa- B
  • Transcription

ASJC Scopus subject areas

  • Clinical Neurology
  • Cardiology and Cardiovascular Medicine
  • Advanced and Specialized Nursing

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