Adequate post-ischemic reperfusion of the mouse brain requires endothelial NFAT5

Abstract

Severity and outcome of strokes following cerebral hypoperfusion are significantly influenced by stress responses of the blood vessels. In this context, brain endothelial cells (BEC) regulate inflammation, angiogenesis and the vascular resistance to rapidly restore perfusion. Despite the relevance of these responses for infarct volume and tissue recovery, their transcriptional control in BEC is not well characterized. We revealed that oxygen and nutrient-deprived BEC activate nuclear factor of activated T-cells 5 (NFAT5)-a transcription factor that adjusts the cellular transcriptome to cope with environmental stressors. We hypothesized that NFAT5 controls the expression of genes regulating the response of BEC in the ischemic brain. The functional relevance of NFAT5 was assessed in mice, allowing the conditional EC-specific knock-out of Nfat5 (Nfat5^(EC)-/-). Cerebral ischemia was induced by transient middle cerebral artery occlusion (MCAO) followed reperfusion up to 28 days. While loss of endothelial Nfat5 did not evoke any phenotypic abnormalities in mice under control conditions, infarct volumes, neurological deficits and the degree of brain atrophy were significantly pronounced following MCAO as compared to control animals (Nfat5^fl/fl). In contrast, MCAO-induced edema formation, inflammatory processes and angiogenesis were not altered in Nfat5^(EC)-/- mice. RNAseq analyses of cultured BEC suggested that loss of NFAT5 impairs the expression of Kcnj2 encoding a potassium channel that may affect reperfusion. In fact, lower levels of KCNJ2 were detected in arterial endothelial cells of Nfat5^(EC)-/- versus Nfat5^fl/fl mice. Laser speckle contrast imaging of the brain revealed an impaired perfusion recovery in Nfat5^(EC)-/- versus Nfat5^fl/fl mice after MCAO.Collectively, NFAT5 in arterial BEC is required for an adequate reperfusion response after brain ischemia that is presumably dependent on the maintenance of Kcnj2 expression. Consequently, impairment of the protective role of endothelial NFAT5 results in enlarged infarct sizes and more severe functional deficits of brain functions.

Keywords: Endothelial cells; Ischemic stroke; KCNJ2; NFAT5; Reperfusion.

Fig. 1

Ischemic stress increases the expression of NFAT5 in cerebral endothelial cells. Endothelial cells derived from microvessels prepared from brains of adult Nfat5^fl/fl mice were exposed to OGD conditions (glucose-free aCSF; 1% O₂) for 6 or 12 h. Cells exposed to aCSF (+ Glc) under normoxic conditions for 12 h are used as control. A Real-time RT-PCR was applied to determine Nfat5 transcript levels. Values are normalized to Actb and expressed as fold change of control (n = 4 per group; One-way ANOVA with Holm-Sidak's multiple comparisons test; * p < 0.05, *** p < 0.001). B Nuclear NFAT protein abundance was quantified by immunofluorescence staining (n = 3–4 per group; One-way ANOVA with Holm-Sidak's multiple comparisons test; *** p < 0.001). Representative microphotographs: NFAT5 (red), DAPI (blue). Scale bar: 50 µm. C Nfat5.^fl/fl mice underwent 45 min of MCAO followed by 24 h reperfusion. Sham-operated mice served as control. NFAT5 protein abundance in brain endothelial cells was determined by co-immunofluorescence staining (n = 3–4 per group; unpaired two-tailed Student's t test; * p < 0.05). Representative microphotographs: NFAT5 (green), CD31 (red), DAPI (blue). Arrows point at NFAT5 in nuclei of EC. The arrowhead tags a non-endothelial cell nucleus. The dotted line marks the border between ischemic and non-ischemic areas as evidenced by the lack of NFAT5 in the nuclei of non-endothelial cells (scale bar: 20 µm)

Fig. 2

Endothelial cell-specific knockout of Nfat5 worsens brain tissue damage and sensorimotor impairment in mice upon acute ischemic stroke. A Nfat5^(EC)−/− and Nfat5^fl/fl mice were subjected to 45 min of MCAO followed by 24 h reperfusion. Sham-operated mice served as control. Infarct and edema volume were determined by cresyl violet staining (n = 5–10 (♀)/n = 4–15 (♂) per group; Two-way ANOVA with Holm-Sidak's multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001). B Neurological function was assessed using the modified neurological severity score (median with interquartile range; n = 5–10 (♀)/n = 4–10 (♂) per group; Wilcoxon matched-pairs signed-rank test (pre- vs post-MCAO), Mann–Whitney U rank-sum test (Nfat5^(EC)−/− vs Nfat5.^fl/fl); * p < 0.05, ** p < 0.01, *** p < 0.001). C Motor function was evaluated by using the Rotarod performance test (n = 5–10 (♀)/n = 4–10 (♂) per group; Two-way ANOVA with Holm-Sidak's multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001)

Fig. 3

Endothelial cell-specific knockout of Nfat5 worsens brain tissue loss and sensorimotor impairment in mice during the chronic phase of ischemic stroke. A Nfat5^(EC)−/− and Nfat5^fl/fl mice were subjected to 45 min of MCAO followed by 28 d reperfusion. Sham-operated mice served as control. Cerebral atrophy was assessed by morphovolumetric analysis of cresyl violet-stained brain tissue sections (n = 5–6 per group; Two-way ANOVA with Holm-Sidak's multiple comparisons test; * p < 0.05, *** p < 0.001). B Neurological function was assessed using the modified neurological severity score (median with interquartile range; n = 5–6 per group; Kruskal–Wallis test with Dunn's multiple comparisons test (pre- vs post-MCAO), Mann–Whitney U rank-sum test (Nfat5^(EC)−/− vs Nfat5^fl/fl); * p < 0.05, ** p < 0.01). (C) Motor function was evaluated by using the Rotarod performance test (n = 5–6 per group; One-way repeated measures ANOVA with Holm-Sidak's multiple comparisons test (pre- vs post-MCAO), unpaired two-tailed Student's t test (Nfat5^(EC)−/− vs Nfat5.^fl/fl); * p < 0.05, *** p < 0.001)

Fig. 4

Nfat5 knockout decreases expression of Kcnj2 in OGD-exposed BEC. Primary cultures of Nfat5^fl/fl BEC were exposed to 1 µM 4-hydroxytamoxifen (Nfat5^−/−) or solvent for 3 d followed by a recovery period of 3 d. BEC were exposed to OGD conditions (glucose-free aCSF; 1% O₂) for 12 h. Cells exposed to aCSF (+ Glc) under normoxic conditions for 12 h are used as control (CTR). RNA isolated from BEC of two experimental approaches (performed in duplicate) was subjected to RNAseq analysis. A Venn diagram-based analysis to identify NFAT5-dependent differentially expressed genes (DEGs) in OGD-exposed BEC (located in the intersection). The table lists the DEGs meeting our selection criteria (fold change—FC(log2) < − 2 in at least one approach). B Venn diagram-based analysis to identify NFAT5-dependent differentially expressed genes (DEGs) in OGD-exposed BEC, which were also significantly upregulated in OGD-Nfat5^fl/fl versus CTR-Nfat5^fl/fl BEC (located in the intersection). The table lists the DEGs meeting our selection criteria (mean FC(log2) of all experimental approaches (OGD-Nfat5^−/− versus OGD-Nfat5^fl/fl) < − 2 and mean FC(log2) (OGD-Nfat5^fl/fl versus CTR-Nfat5^fl/fl) > 0.5). Nfat5 expression is shown as reference. (C) Control of the expression of Nfat5 and Kcnj2 in BEC by real-time RT-PCR. Values are normalized to Actb and expressed as fold change of Nfat5.^fl/fl control (n = 4 per group; One-way ANOVA with Holm-Sidak's multiple comparisons test; * p < 0.05; *** p < 0.001)

Fig. 5

Automated immunofluorescence analysis of Kir2.1 (in vivo). Immunofluorescence-based detection of the endothelial cell marker PECAM1 (CD31) and Kir2.1 in brain arteries A and capillaries B of Nfat5^fl/fl and Nfat5^(EC)−/− mice (sham treatment). Images were processed and automatically analyzed by TissueFAXS (TissuGnostics, see Supplement S11 and S12 for details). Detection of CD31^high arterial and capillary structures in defined regions of interest (ROI) is followed by assessment of the corresponding Kir2.1-associated fluorescence. The data of representative ROIs (C: artery, D: capillaries) are plotted as scattergram (y-axis: Kir2.1 immunofluorescence level, x-axis: CD31 level). The mean Kir2.1 fluorescence values determined in all corresponding ROIs of individual mouse brains are summarized as bar graphs (n = 5 per group, unpaired two-tailed Student's t test (Nfat5^(EC)−/− vs Nfat5.^fl/fl); * p < 0.05)

Fig. 6

Endothelial cell-specific knockout of Nfat5 critically affects the reperfusion of the MCA-supplied brain territory. Nfat5^(EC)−/− and Nfat5.^fl/fl mice were subjected to 45 min of MCAO followed by 3 h reperfusion. Cortical blood flow (CBF) was measured on the skull of living mice using high-resolution laser speckle contrast imaging (LSCI). Representative LSCI perfusion images of the cortical surface at 30 min MCAO and 3 h upon reperfusion. Color bar code: The colors from blue to red represent the blood flow velocity from lower to higher. CBF is given in arbitrary units (a.u.). ROIs across the ipsilateral cerebral cortex were defined according the following CBF thresholds: strongly hypoperfused area (CBF < 40% of mean contralateral cerebral cortex during MCAO, CBF↓↓↓), moderately hypoperfused region (CBF between 40 and 70% of mean contralateral cerebral cortex during MCAO, CBF↓) (n = 6 per group; unpaired two-tailed Student's t test; * p < 0.05)