Hypoxia-inducible factor and nuclear factor kappa-B activation in blood–brain barrier endothelium under hypoxic/reoxygenation stress

Abstract

This investigation focuses on transcription factor involvement in blood–brain barrier (BBB) endothelial cell-induced alterations under conditions of hypoxia and post-hypoxia/reoxygenation (H/R), using established in vivo/ex vivo and in vitro BBB models. Protein/DNA array analyses revealed a correlation in key transcription factor activation during hypoxia and H/R, including NFjB and hypoxia-inducible factor (HIF)1. Electrophoretic mobility shift assays confirmed NFjB and HIF1 binding activity ex vivo and in vitro, under conditions of hypoxia and H/R. Hypoxia- and H/R-treated BBB endothelium showed increased HIF1a protein expression in both cytoplasmic and nuclear fractions, in ex vivo and in vitro models. Co-immunoprecipitation of HIF1a and HIF1b was shown in the nuclear fraction under conditions of hypoxia and H/R in both models. Hypoxia- and H/R-treated BBB endothelium showed increased expression of NFjB-p65 protein in both cytoplasmic and nuclear fractions. Co-immunoprecipitation of NFjB-p65. with NFjB-p50 was shown in the nuclear fraction under conditions of hypoxia and H/R in the ex vivo model, and after H/R in the in vitro model. These data offer novel avenues in which to alter and/or investigate BBB activity across model systems and to further our understanding of upstream regulators during hypoxia and H/R.