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 Edwin Carroll, David W. Hill

Research output: Contribution to journalArticle

39 Citations (Scopus)

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 - Jan 1 2000

Fingerprint

Mannitol
Intercellular Adhesion Molecule-1
Transcription Factors
Up-Regulation
Endothelial Cells
Brain
Tumor Necrosis Factor-alpha
Stroke
Oligonucleotides
Messenger RNA
Cell Nucleus
Reperfusion
Transfection
Ischemia
Enzyme-Linked Immunosorbent Assay
3'-(1-butylphosphoryl)adenosine
Wounds and Injuries

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

Cite this

Hypertonic mannitol loading of NF-κ̄ transcription factor decoys in human brain microvascular endothelial cells blocks upregulation of ICAM-1. / Hess, David C; Howard, Eugene; Cheng, Charles; Carroll, James Edwin; Hill, David W.

In: Stroke, Vol. 31, No. 5, 01.01.2000, p. 1179-1186.

Research output: Contribution to journalArticle

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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.",
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AU - Cheng, Charles

AU - Carroll, James Edwin

AU - Hill, David W.

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N2 - 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.

AB - 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.

KW - Endothelium

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KW - Nuclear factor kappa- B

KW - Transcription

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