Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation

Abstract

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.

Fig. 1

Schematic drawing of proposed junctional architecture of cerebromicrovessel endothelial cells. Tight junctions (TJ) consisting of the integral membrane proteins occludin and claudin are located towards the apical side of endothelial cells with their corresponding cytoplasmic accessory proteins [i.e., zonula occluden (ZO)-1, ZO-2, ZO-3, and cingulin] connecting TJs to the cytoskeleton (i.e., actin; not shown). Adherens junctions (AJ), located toward the basolateral side of endothelial cells, are composed of the integral membrane bound cadherins and cytoplasmic accessory proteins (i.e., α-and β-catenin).

Fig. 2

Hypoxia-reoxygenation effects on protein expression of bovine brain microvessel endothelial cells (BBMECs). Confluent monolayers were exposed to normoxia (C), 24-h hypoxia (H) or hypoxia with 2-h reoxygenation (HR). Representative Western blots for actin (42 kDa), claudin-1 (44 kDa dimer), occludin (65 kDa), ZO-1 (220 kDa), and ZO-2 (160 kDa) are shown (n = 4).

Fig. 3

Immunofluorescence showing protein localization in BBMECs. Confluent monolayers were exposed to nor-moxic (Control), 24-h hypoxia (Hypoxic), or hypoxia with 2-h reoxygenation (Re-oxygenated) conditions. After treatment, the monolayers were incubated with primary antibodies directed against actin, claudin-1, occludin, ZO-1, and ZO-2, followed by Alexafluor 488-conjugated secondary antibodies. Representative pictures for each treatment group and protein are shown (n = 3). Arrowheads, actin stress tangles; arrows, disruptions in the expression of occludin and ZO-2. *Increased cytosolic staining of ZO-1 and ZO-2.