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

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