Figure 4: Cytoskeletal reorganization and JP redistribution in endothelial cells causes early paracellular hyperpermeability after oxygen–glucose deprivation in vitro.

HBMECs were infected with Lenti, Lenti-ADF, Lenti-ADFm, Lenti-MLCsc, Lenti-MLCt, Lenti-ROCKsc or Lenti-ROCKt. In separate cultures, HBMECs were treated with vehicle or Y27632. Cells were then subjected to 1 h of OGD. (a,b) HBMECs were stained at 1 or 3 h after OGD for occludin (red) or VE-cadherin (green), and counterstained with DAPI (blue) for nuclear labelling. In ADFm-transfected cells, triple staining was performed for VE-cadherin (green), the HA tag (red) and DAPI nuclear labelling (blue), to show the cytosolic distribution of HA-ADFm. Scale bar, 30 μm. JPs are characteristically located at cell–cell contact sites under physiological, uninjured conditions. OGD resulted in a loss of occludin and VE-cadherin from the points of cell–cell contact, and this effect was inhibited by lentiviral ADFm overexpression or MLC knockdown. (c,d) The immunofluorescent staining intensity of plasma membrane occludin and VE-cadherin was quantified and expressed relative to non-OGD controls. Data represent four independent experiments. *P≤0.05, **P≤0.01 versus Lenti (c) or MLCsc (d). (e,f) Whole-cell extracts, the membrane fraction or the actin cytoskeleton fraction (ACF) were prepared at 1 h after OGD and immunoblotted for occludin (Occl), VE-cadherin (VE-C) and subfraction markers β-actin, CD31 or α-tubulin. Representative images and quantification of blots from the membrane fraction and ACF are presented. Data represent four independent experiments. *P≤0.05, **P≤0.01 versus Lenti (e) or MLCsc (f). (g–j) Lentivirus-infected or drug-treated HBMECs were cultured in the in vitro BBB model, and subjected to 1 h of OGD. The diffusion coefficient of the 4.4 kDa-dextran was measured at 0–3 h after OGD. Data represent four independent experiments. *P≤0.05, **P≤0.01 versus Lenti (g), MLCsc (h), ROCKsc (i) or vehicle (j).