title: Hypertonic mannitol loading of NF-kappaB transcription factor decoys in human brain microvascular endothelial cells blocks upregulation of ICAM-1.
abstract: OBJECTIVE
  An acute inflammatory response exacerbates tissue injury during acute ischemic stroke. The transcription factor nuclear factor (NF)-kappaB plays a key role in endothelial cell activation and the inflammatory response. Targeted genetic disruption of NF-kappaB activation in cerebral endothelial cells may be protective in stroke. We determined whether a NF-kappaB 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)-alpha and conditions of hypoxia-reoxygenation (H/R). Mannitol was used to load phosphothiorated oligonucleotides containing 3 copies of the kappaB binding sequences (TFDs) into cultured HBMEC. An NF-kappaB TFD, a mutated NF-kappaB 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-kappaB TFD but not the mutated or scrambled TFD competed with a kappaB sequence for binding to nuclear extracts from HBMEC exposed to TNF-alpha. The NF-kappaB TFD blocked the TNF-alpha-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-kappaB decoy blocks both TNF-alpha-induced and H/R-induced ICAM-1 upregulation in HBMEC. Targeted genetic disruption of endothelial NF-kappaB activation may be of benefit in acute ischemic stroke.
authors.affiliation: Neuroscience Service Line, VA Medical Center, Augusta, GA 30904, USA. dhess@neuro.mcg.edu
authors.forename: D C
authors.initials: DC
authors.lastname: Hess
authors.forename: E
authors.initials: E
authors.lastname: Howard
authors.forename: C
authors.initials: C
authors.lastname: Cheng
authors.forename: J
authors.initials: J
authors.lastname: Carroll
authors.forename: W D
authors.initials: WD
authors.lastname: Hill
authors.forename: C Y
authors.initials: CY
authors.lastname: Hsu
mesh_terms: D015687:Cell Hypoxia; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000503:physiopathology; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity; D015854:Up-Regulation
chemical_list: D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1