Figure 7: ADFm overexpression blunts early cytoskeletal alterations in ECs after tFCI.

WT or Tg-ADFm mice received 1-h tFCI or sham (S) operation, and brain microvessel extracts were isolated at 0.5–3 h of reperfusion for western blotting analysis. The F/G-actin ratio (a) and pMLC (b) in ipsilateral cortical microvessels of WT mice were quantified. **P≤0.01, ***P≤0.001 versus sham. (c) Double-label immunostaining for pMLC and the endothelial marker CD31 in ipsilateral cortex. The leakage of the 3 kDa Alexa555-dextran is shown for each time point. Rectangle: the region enlarged in high-power images. Scale bar, 100 μm. (d) The F/G-actin ratio in microvessels at 1 h of reperfusion. **P≤0.01 versus WT. (e) Representative images showing the formation of F-actin⁺ stress fibres in CD31⁺ microvessels (arrow) in the ischaemic cortex. Scale bar, 10 μm. ADFm overexpression inhibited tFCI-induced formation of stress fibres. (f) Whole-cell extracts, the membrane fraction or the ACF were prepared from brain microvessel extracts at 1 h after tFCI and subsequently immunoblotted. Representative images and quantification of blots (normalized to WT contralateral) are presented. Endothelial ADFm overexpression suppressed tFCI-induced redistribution of JPs from the membrane fraction to the ACF. n=6 mice per group. *P≤0.05, **P≤0.01 versus WT. (g) Double-label immunostaining for VE-cadherin and CD31 in ipsilateral cortex at 1 h of reperfusion or after sham operation. Low-power images were shown (left top panel) with white lines delineating the shape of the vessels. Rectangles indicate regions where the high-power images were taken (shown in the left bottom panel). In non-ischaemic controls (both WT and Tg-ADFm brains), VE-cadherin immunofluorescence (arrows) was present predominantly at endothelial cell–cell contacts (cell membrane and extracellular space between cells; surrounding the cytosolic CD31 immunofluorescence). In WT brains, tFCI reduced the level of VE-cadherin immunofluorescence (arrows) in the cell–cell contacts of CD31⁺ endothelial cells but increased intracellular VE-cadherin immunofluorescence (asterisk) compared with non-ischaemic control brains. These changes were less frequently observed in Tg-ADFm brains. The enlarged images of VE-cadherin immunofluorescence (arrows) at endothelial cell–cell contacts (extracted from the rectangle regions at the left bottom panel) are shown on the right panel. Scale bar, 5 μm.