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

Background Although in�ammatory cell adhesion molecules (CAMs) and anti-in�ammation factor-kruppel-like transcription factor (KLF) 4 have all been reported to be induced after cerebral ischemic stroke (CIS), the close temporal and spatial relationship between expressions of CAMs and KLF4 following CIS, and whether and how CAMs and KLF-4 contribute to development of CIS-induced vascular injury are still unclear. Methods Here, we �rst examined the correlation between serum levels of CAMs/KLF4 and infarct volume in acute CIS patients. Then, we determined relationship between CAMs and KLF4 in mice after focal cerebral ischemia. Finally, we investigated the mechanism of KLF4 in protecting against oxygen-glucose deprivation-induced brain endothelial cell injury. Results Our results demonstrated that patients with moderate to severe CIS had higher serum levels of three CAMs including E-selectin, inter-cellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) but lower levels of KLF4 at 48 hours after acute event as compared to patients with minor CIS. The expression levels of three CAMs as well as KLF4 all correlated well with the infarct volume in all the CIS subjects at that time. Although the expressions of three CAMs and KLF4 were all induced in the ischemic hemisphere following focal cerebral ischemia, but the peak timing and distribution patterns of their expression were different: the induction of KLF4 lagged behind that of the CAMs in the ischemic penumbra, furthermore, the dual immuno�uorescent studies displayed that high expression of KLF4 was always associated with relatively less cerebral vascular endothelial in�ammation response in the ischemic hemisphere, and vice versa. Mechanistic analyses revealed that KLF4 alleviated CIS-induced cerebral vascular injury by regulating endothelial expressions of CAMs, nuclear factor-kB and tight junction proteins. Conclusions These data indicate that KLF4 confers vascular protection against cerebral injury, suggesting of CIS. Claudin-5 and ZO-1 in the BECs 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-kB/total NF-kB (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 siRNA-KLF4-2 group at 12 h restoration of OGD or NO-OGD/R (n=4 per experimental group). NO-OGD/R siRNA-Ctl treated cells served as control. Data represent mean ±standard deviation and were analyzed by two-way ANOVA. Note that the expressions of three cell adhesion molecules including E-selectin, VCAM-1 and ICAM-1 and the phosphorylation of NF-kB from the siRNA-Ctl-treated BECs were signi�cantly induced in response to OGD/R, but the expressions of tight junction proteins (Claudin-5 and ZO-1) markedly decreased compared to that of the controls; Furthermore, OGD/R-induced expression of three cell adhesion molecules and the phosphorylation of NF-kB were enhanced by diminishing the levels of KLF4 in the BECs; likewise, the decreased expression of tight junction proteins caused by OGD/R were further augmented by silencing the levels of KLF4 in the BECs. OGD/R signi�cantly increased the mRNA expression of TNF-α in the siRNA-Ctl-treated BECs, but siRNA-mediated knockdown of KLF4 showed no notable effects on the expression of TNF-α following OGD/R.