KLF4 alleviates cerebral vascular injury by ameliorating vascular endothelial inflammation and regulating tight junction protein expression following ischemic stroke

Figures

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  Fig. 1. The correlations of infarct volume with the serum levels of three cell adhesion molecules and KLF4 at 48 h after ischemic onset in the acute ischemic stroke patients. Serum levels of three cell adhesion molecules (E-selectin, VCAM-1, and ICAM-1) and KLF4 at 48 h after ischemic onset were measured using ELISA. Diffusion-weighted MRI images were used to calculate the infarct volume. After log transformation, the Pearson analyses revealed that the log infarct volume was significantly positively correlated with serum levels of three cell adhesion molecules (E-selectin, VCAM-1, and ICAM-1) and markedly negatively correlated the serum levels of KLF4 in all the subjects (n = 74)

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  Fig. 2. Increased expression of three cell adhesion molecules and KLF4 following focal cerebral ischemia. a Images show IF staining for three cell adhesion molecules (E-selectin, VCAM-1, and ICAM-1) and KLF4 in ischemic hemisphere from sham-operated mice (S, control) or mice at day 1, 2, 4, 7, and 14 post-ischemia. Scale bar = 100 μm. b Quantification of E-selectin, VCAM-1 and ICAM-1, and KLF4 expressions. Results are expressed as the mean ± standard deviation of the number of positive events per field of view (n = 6 per experimental group). Note that cerebral ischemia induced a strong increase in the expression of all the three cell adhesion molecules in both ischemic penumbra and core, reaching a peak at day 2 and then declining at day 4. While in the ischemic penumbra, the number of KLF4-positive events increased slightly during the first 2 days post-ischemia, but then increased significantly by day 4, and reached a maximum between 7 and 14 days post-ischemia. However, in the ischemic core, the number of KLF4-positive events increased markedly during the first 2 days post-ischemia, before declining at day 4. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control

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  Fig. 3. The mRNA expression of three cell adhesion molecules and KLF4 in the ischemic brain following focal cerebral ischemia. The mRNA levels of E-selectin, VCAM-1, ICAM-1, and KLF4 in the ipsilateral ischemic cerebral cortex from sham-operated mice (S, control) or mice at days 1, 2, 4, 7, and 14 post-ischemia was measured by qPCR. Results are expressed as the mean ± standard deviation (n = 4 per experimental group). Note that cerebral ischemia markedly increased the mRNA expression of all the three cell adhesion molecules including E-selectin, VCAM-1, and ICAM-1 in the ischemic hemisphere, reaching a peak at day 1 or 2 and then declining at day 2 or 4, while cerebral ischemia also induced a strong increase in mRNA expression of KLF4 over the entire 14-day time course, with this effect maximal between 7 and 14 days post-ischemia. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control

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  Fig. 4. Cellular localization of three cell adhesion molecules (E-selectin, VCAM-1, and ICAM-1) expression and co-expression of E-selectin, VCAM-1, and ICAM-1 with KLF4 on cerebral blood vessels after focal cerebral ischemia. Images show the dual-IF staining for E-selectin/VCAM-1/ICAM-1 with CD31, Mac-1, GFAP, or KLF4 in ischemic hemisphere at day 2 post-ischemia. Scale bar = 100 μm (inserts = 40 μm). Note that E-selectin/VCAM-1/ICAM-1 always co-localized with CD31-positive vessels, especially on the leaked blood vessels. In addition, E-selectin/VCAM-1/ICAM-1 co-localized with quite a few Mac-1-positive microglia/inflammatory macrophages in the ischemic hemisphere. Furthermore, E-selectin/VCAM-1/ICAM-1 also co-localized with GFAP-positive astrocytes in the penumbra (penumbra = P), but in the ischemic core (core = C), this was markedly reduced. Of interest, a fraction of E-selectin/VCAM-1/ICAM-1 co-localized with KLF4, but the distribution patterns of their expressions were different, where the high levels of KLF4 were expressed, relatively low levels of E-selectin/VCAM-1/ICAM-1 were expressed on the blood vessels in the ischemic hemisphere, and vice versa.

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  Fig. 5. Cellular localization of KLF4 expression and co-expression of KLF4 with Claudin-5 on cerebral blood vessels after focal cerebral ischemia. Images show the dual-IF staining for KLF4 with CD31, GFAP, Mac-1, or Claudin-5 in the ischemic hemisphere from sham-operated mice or mice at days 2 and 7 post-ischemia. Scale bar = 100 μm (inserts = 40 μm). Note that KLF4 immunoreactivity was observed in CD31-positive vessels in the ischemic hemisphere at days 2 and 7 post-ischemia. At day 2 post-ischemia, KLF4 co-localized extensively with Mac-1-positive microglia/inflammatory macrophages in the ischemic hemisphere, especially in the core (core = C); at day 7, the co-expression of KLF4 with activated microglia/inflammatory macrophages can still be seen in the ischemic hemisphere, including the penumbra (penumbra = P) and the core (core = C). Furthermore, KLF4 expression always strongly co-localized with cells expressing high levels of GFAP in the ischemic penumbra (penumbra = P) at days 2 and 7 post-ischemia, especially at day 7, but this co-localization was significantly reduced in the ischemic core (core = C) (a). Meanwhile, KLF4 expression co-localized with Claudin-5 on the blood vessels…

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  Fig. 6. The influence of knockdown of KLF4 on the expression of three cell adhesion molecules, phosphorylated NF-κB, tight junction proteins, and TNF-α in bEnd3 cells under OGD/R conditions. a Representative images of western blot for the expression of KLF4 in the bEnd3 cells in the negative control siRNA (siRNA-Ctl) or three different KLF4-specific siRNA (siRNA-KLF4)-treated group. Note that three different specific siRNA against the murine KLF4 all reduced the expression of KLF4 in bEnd3 cells compared to that of siRNA-Ctl-treated bEnd3 cells, respectively, and siRNA-KLF4-2 can target the KLF4 with the highest efficiency. b Representative images of western blot for the expression of KLF4, E-selectin, VCAM-1, ICAM-1, p-NF-κB, Claudin-5, and ZO-1 in the bEnd3 cells of siRNA-Ctl and siRNA-KLF4-2 group at 12 h restoration of OGD or NO-OGD/R. c–i Bar graphs show the quantitative analyses of western blots as ratios of KLF4/β-actin (c), E-selectin/β-actin (d), VCAM-1/β-actin (e), ICAM-1/β-actin (f), phosphorylated NF-κB/total NF-κB (g), Claudin-5/β-actin (h), and ZO-1/β-actin (i) (n = 5 per experimental group). j The mRNA level of TNF-α was determined by qPCR in the BECs of siRNA-Ctl and…

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  Fig. 7. The effect of overexpression of KLF4 on the expression of three cell adhesion molecules, phosphorylated NF-κB, and tight junction proteins in bEnd3 cells under OGD/R conditions. a Representative images of western blot for the expression of KLF4 in the bEnd3 cells in the control plasmid (mock) or KLF4 overexpression group. b Representative images of western blot for the expression of KLF4, E-selectin, VCAM-1, ICAM-1, p-NF-κB, Claudin-5, and ZO-1 in the bEnd3 cells of mock and KLF4 overexpression group at 12 h restoration of OGD or NO-OGD/R. c–i Densitometric analysis shows the ratios of KLF4/β-actin (c), E-selectin/β-actin (d), VCAM-1/β-actin (e), ICAM-1/β-actin (f), phosphorylated NF-κB/total NF-κB (g), Claudin-5/β-actin (h), and ZO-1/β-actin (i). NO-OGD/R mock-treated cells served as control. Data represent mean ± standard deviation and were analyzed by two-way ANOVA (n = 5 per experimental group). Note that the expressions of three cell adhesion molecules including E-selectin, VCAM-1, and ICAM-1 and the phosphorylation of NF-κB on mock-treated bEnd3 cells were significantly induced in response to OGD/R, but the expression of Claudin-5 and ZO-1 markedly decreased compared to…