Hypertonic Mannitol Loading of NF-kB 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)-kB plays a key role in endothelial cell activation and the inflammatory response. Targeted genetic disruption of NF-kB activation in cerebral endothelial cells may be protective in stroke. We determined whether a NF-kB 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)-a and conditions of hypoxia-reoxygenation (H/R). Mannitol was used to load phosphothiorated oligonucleotides containing 3 copies of the kB binding sequences (TFDs) into cultured HBMEC. An NF-kB TFD, a mutated NF-kB 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-kB TFD but not the mutated or scrambled TFD competed with a kB sequence for binding to nuclear extracts from HBMEC exposed to TNF-a. The NF-kB TFD blocked the TNF-a–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-kB decoy blocks both TNF-a–induced and H/R-induced ICAM-1 upregulation in HBMEC. Targeted genetic disruption of endothelial NF-kB activation may be of benefit in acute ischemic stroke. (Stroke. 2000;31:1179-1186.)