Kruppel-like factor 2 protects against ischemic stroke by regulating endothelial blood brain barrier function

Abstract

During an ischemic stroke normal brain endothelial function is perturbed, resulting in blood brain barrier (BBB) breakdown with subsequent infiltration of activated inflammatory blood cells, ultimately leading to neuronal cell death. Kruppel-like factor 2 (KLF2) is regulated by flow, is highly expressed in vascular endothelial cells (ECs), and serves as a key molecular switch regulating endothelial function and promoting vascular health. In this study we sought to determine the role of KLF2 in cerebrovascular function and the pathogenesis of ischemic stroke. Transient middle cerebral artery occlusion was performed in KLF2-deficient (KLF2(-/-)), KLF2 overexpressing (KLF2(tg)), and control mice, and stroke volume was analyzed. BBB function was assessed in vivo by real-time neuroimaging using positron emission tomography and Evan's blue dye assay. KLF2(-/-) mice exhibited significantly larger strokes and impairment in BBB function. In contrast, KLF2(tg) mice were protected against ischemic stroke and demonstrated preserved BBB function. In concordance, gain- and loss-of-function studies in primary brain microvascular ECs using transwell assays revealed KLF2 to be BBB protective. Mechanistically, KLF2 was demonstrated, both in vitro and in vivo, to regulate the critical BBB tight junction factor occludin. These data are first to identify endothelial KLF2 as a key regulator of the BBB and a novel neuroprotective factor in ischemic stroke.

Fig. 1.

Kruppel-like factor 2 (KLF2) is expressed in brain macrovascular and microvascular endothelial cells (ECs). Green fluorescent protein (GFP) and CD31 immunofluorescence staining of brain cerebral cortex sections from KLF2 GFP fusion mice are shown. A: cerebral macrovascular middle cerebral artery (MCA). B: cerebral microvasculature (n = 6–9/group). Representative results shown.

Fig. 2.

Cerebrovascular anatomy and basic physiology is unchanged in KLF2-deficient and KLF2 overexpressing mice. A: India ink staining of brains from KLF2-deficient (K2^−/−), control, and KLF2 overexpressing (K2^tg) mice. Representative results shown (n = 4 to 5/group). B: assessment of mean arterial blood pressure (BP), heart rate (HR), pH, pCO₂, and pO₂ in K2^−/−, K2^tg, and control mice (n = 4 to 5/group). P = nonsignificant for statistical comparisons between all groups. Bpm, beats/min.

Fig. 3.

KLF2-deficient mice have increased stroke volume and blood brain barrier (BBB) permeability. A: 2,3,5-Triphenyltetrazolium (TTC) staining of KLF2-deficient (K2^−/−) and control brains after 1 h transient middle cerebral artery occlusion (tMCAO) and 48 h reperfusion. Representative results shown. B: quantitative analysis of stroke volume (n = 10–20/group). *P = 0.001 vs. control. C: quantitative positron emission tomography (PET) assessment of radiotracer uptake in K2^−/− and control brains at baseline and after stereotactic injection of TNF-α into the cerebral cortex (n = 2–4/group). D: quantification of Evan's blue dye (EBD) permeability in K2^−/− and control brains after sham or stereotactic injection of TNF-α (n = 3/group). *P = 0.0015; #P = 0.002.

Fig. 4.

Generation of KLF2 overexpressing mouse. A: schematic diagram of the targeted (ROSA26) locus and Cre recombined locus for the generation of the KLF2^tg mouse. UbiC, Ubiquitin promoter; HA, hemagglutinin. B: HA and CD31 immunofluorescence staining of brain cerebral cortex sections from KLF2^tg mice (n = 6). Representative results shown.

Fig. 5.

KLF2 overexpressing mice have decreased stroke volume and BBB permeability. A: TTC staining of KLF2 overexpressing (K2tg) and control brains after 1 h tMCAO and 48 h reperfusion. Representative results shown. B: quantitative analysis of stroke volume (n = 8 to 9/group). *P = 0.004 vs. control. C: quantitative PET assessment of radiotracer uptake in K2^tg and control brains at baseline and after stereotactic injection of TNF-α into the cerebral cortex (n = 3/group). D: quantification of EBD permeability in K2^tg and control brains after sham or stereotactic injection of TNF-α (n = 3/group). *P = 0.03; #P = 0.003.

Fig. 6.

KLF2 regulates BBB permeability and occludin expression in brain microvascular ECs. A: transwell assays quantifying permeability of FITC dextran across primary human brain microvascular ECs infected with KLF2 (Ad-KLF2) or control (Ad-GFP) adenovirus at baseline and after 30 min oxygen glucose deprivation (OGD) followed by 2 h of normal media (n = 8/group). *P = 0.005. B: Transwell assays as in A using cells transfected with small interfering (si)-RNA (si-Control or si-KLF2) assessed at baseline (n = 8/group). *P < 0.0005 vs. si-Control. C: quantitative RT-PCR analysis of tight junction factors in human brain microvascular ECs infected with control (Ad-GFP) and KLF2 (Ad-KLF2) adenovirus (n = 3/group). *P < 0.0001, #P < 0.005, **P < 0.01, ##P < 0.05. D: quantitative RT-PCR analysis of tight junction factors in human brain microvascular ECs infected with si-RNA (si-Control or siKLF2) (n = 3 to 4/group). *P < 0.0001, #P = 0.01, **P = 0.01, ##P < 0.0001. E: Western blot analysis of occludin in human brain microvascular ECs infected with Ad-GFP or Ad-KLF2. Quantification of Western blot is shown. *P < 0.0001. F: transient transfection of 293T cells with occludin promoter luciferase reporter and increasing concentrations of KLF2 plasmid (n = 3/group). *P < 0.005, #P < 0.0005 vs. 0 μg. Representative results of 3 independent experiments shown.

Fig. 7.

KLF2 regulates occludin expression in mouse brain. A: quantitative RT-PCR analysis of KLF2 and occludin in whole brain tissue from control and K2^−/− mice (n = 4–7/group). *P < 0.0001, #P = 0.01. B: quantitative RT-PCR analysis of KLF2 and occludin in whole brain tissue from control and K2^tg mice (n = 3 to 4/group). *P = 0.001, #P < 0.05. C: CD31 and occludin immunofluorescence staining of brain cerebral cortex sections from K2^−/−, K2^tg, and control mice. Representative results shown (n = 3 to 4/group).