Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (
claudin-1 and
occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2)
proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with
ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of
hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined
hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ
proteins using primary bovine brain microvessel endothelial cells (BBMEC).
Hypoxia induced a 2.6-fold increase in [(14)C]
sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After
hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ
protein expression immediately after
hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in
occludin, ZO-1, and ZO-2
protein localization during
hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show
hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.