Activation of endothelial Wnt/β-catenin signaling by protective astrocytes repairs BBB damage in ischemic stroke

Abstract

The role of astrocytes in dysregulation of blood-brain barrier (BBB) function following ischemic stroke is not well understood. Here, we investigate the effects of restoring the repair properties of astrocytes on the BBB after ischemic stroke. Mice deficient for NHE1, a pH-sensitive Na⁺/H⁺ exchanger 1, in astrocytes have reduced BBB permeability after ischemic stroke, increased angiogenesis and cerebral blood flow perfusion, in contrast to wild-type mice. Bulk RNA-sequencing transcriptome analysis of purified astrocytes revealed that ∼177 genes were differentially upregulated in mutant astrocytes, with Wnt7a mRNA among the top genes. Using a Wnt reporter line, we confirmed that the pathway was upregulated in cerebral vessels of mutant mice after ischemic stroke. However, administration of the Wnt/β-catenin inhibitor, XAV-939, blocked the reparative effects of Nhe1-deficient astrocytes. Thus, astrocytes lacking pH-sensitive NHE1 protein are transformed from injurious to "protective" by inducing Wnt production to promote BBB repair after ischemic stroke.

Keywords: Angiogenesis; Astrocytes; Blood-Brain barrier; Na(+)/H(+)exchanger isoform-1; Wnt/β-catenin signaling.

Fig. 1.

Nhe1 Astro-KO mice show increased angiogenesis and improved rCBF. (A) Experimental protocol. Gfap-CreER+/−;Nhe1f/f mice at postnatal days P60–90 were treated with either corn oil or tamoxifen (Tam) in corn oil (75 mg/kg/day, ip) for 5 days. After 30 days, ischemic stroke was induced by tMCAO at P90–120. Measurement of rCBF and biochemical assays were conducted in the CL or IL peri-lesion cortex and striatum. (B and C) Representative confocal images of BrdU+ and Glut1+ cerebral vessels in the peri-lesion areas at 5 or 14 days reperfusion (Rp) were shown and angiogenesis (Brdu+/Glut1+ double positive vessels) was quantified. Data are mean ± SEM, n = 4-5 *p < 0.05 (Student’s t-test). (D and E) Representative images of Laser Speckle analysis of regional cerebral blood flow (rCBF) in the wild-type and Nhe1 Astro-KO mice prior to tMCAO (baseline), at 5 min during tMCAO, at 5 days or 14 days of reperfusion. Black dashed circles indicate the regions of interest for quantification of blood perfusion in MCA regions. Pseudo color scale indicate red as high blood perfusion and blue as low blood perfusion. Summary analysis of rCBF changes as percentage of pre-ischemic baseline in CL and IL hemispheres. Data are mean ± SEM, n = 5. * p < 0.05, ** p < 0.01; *** p < 0.001 (two-way ANOVA followed by Sidak’s multiple comparisons test).

Fig. 2.

Nhe1 Astro-KO mice show reduced transcytosis and paracellular permeability after ischemic stroke. (A) Representative confocal images of alb-Alexa 488 labelled caveolae and other transcytosis vesicles in Glut1+ cerebral vessels of wild-type and Nhe1Astro-KO brains at 48 h Rp. (B) Quantification of Glut1+ endothelial area filled with alb-Alexa 488. Data are mean ± SEM, n = 3–5 animals per time point, *p<0.05; ** p < 0.01; ****p<0.0001 (three-way ANOVA followed by Tukey’s multiple comparisons test). (C) Representative TEM images of cerebral vessels in the peri-infarct areas of wild-type and Nhe1 Astro-KO brains at 24 h Rp. Arrows: endocytotic vesicles in the endothelial lumen. (D) Summary. Data are ± SEM, n = 3, *p < 0.05. (Student’s t-test) (E) Confocal images of biocytin-TMR immunofluorescence in the IL peri-lesion areas. Arrows; increased tracer leakage in the parenchyma and neural cells. Arrow heads; reduced tracer leakage. (F) Bar graph represents the biocytin-TMR average pixel intensity in the IL peri-infarct and CL areas. Data are mean ± SEM, n = 3. * p<0.05; (three-way ANOVA followed by Tukey’s multiple comparisons test).

Fig. 3.

Bulk RNAseq analysis of transcriptome changes of wild-type and Nhe1 Astro-KO astrocytes. (A) Schematics of bulk RNAseq analysisof astrocyte isolated from wild-type and Nhe1 Astro-KOischemic brains at 24 h Rp. n=3. (B) Venn diagram depicting differential gene expression in astrocytes from wild-type and Nhe1 Astro-KObrains (log2 fold change 1.2 and FDR p value < 0.05). (C) Heat map and unsupervised hierarchial clustering illustrates up-and down-regulated genes. (D) Volcano plots illustrates the gene expression pattern detected with log2 fold change 1.2 and an adjusted FDR p-value <0.05. (E) Significantly altered canonical pathways by Ingenuity pathway analysis (IPA).

Fig. 4.

Nhe1 Astro-KO brains exhibited increased Wnt7a/7b expression. (A) Representative confocal images of AQP4+ and Wnt7a/7b+ positively stained vessels in wild-type and Nhe1 Astro-KO ischemic brains at 48 h Rp. Arrows: high expression of Wnt7a/7b proteins. Arrowheads: low expression of Wnt7a/7b. (B)Bar graphs show quantification of Wnt7a/7b fluorescence signal intensity. Data are ± SEM, n=6. *p < 0.05 (Student’s t-test). (C) RT-qPCR analysis of changes in expression of Wnt7a, Wnt7b, and β-catenin mRNA in the astrocytes isolated from wild-type and (A) Astro-KO brains at 24 h Rp. Data are mean ± SEM, n=4, *p < 0.05 (by Mann-Whitney Test). Representative confocal images of AQP4+ and Wnt7a/7b+ positively stained vessels in wild-type and Nhe1 Astro-KO ischemic brains at 48 h Rp. Arrows: high expression of Wnt7a/7b proteins. Arrowheads: low expression of Wnt7a/7b. (B)Bar graphs show quantification of Wnt7a/7b fluorescence signal intensity. Data are ± SEM, n=6. *p < 0.05 (Student’s t-test). (C) RT-qPCR analysis of changes in expression of Wnt7a, Wnt7b, and β-catenin mRNA in the astrocytes isolated from wild-type and Nhe1 Astro-KO brains at 24 h Rp. Data are mean ± SEM, n=4, *p < 0.05 (by Mann-Whitney Test).

Fig. 5.

Elevated Wnt reporter transgene expression in wild-type and Nhe1Astro-KO brainsafter ischemic stroke. (A)Breeding scheme for generation of astrocyte specific Nhe1 KO mice containing the TCF/LEF1::H2B-eGFP Wnt reporter transgene. (B) Representative confocal images showing immunofluorescence for eGFP (green) and Glut1 (cerebral vessels; red) in the IL hemispheres of Nhe1 Astro-KO and wild-type reporter mice. (C) Quantification of Wnt reporter activity (eGFP immunofluorescence) in Glut1+ vessels. Data are mean ± SEM, n=4, *p<0.05 (two-way ANOVA for followed by Tukey’s multiple comparisons test). (D) Repre-sentative images of AQP4 and β-catenin staining in the peri-lesion areas of wild-type and Nhe1 Astro-KO brains at 48 h Rp. Arrows: high expression of β-catenin protein. Arrowheads: Low expression of β-catenin. (E) Quantification of β-catenin staining intensity. Data are mean ± SEM, n = 5, *p < 0.05 (two-way ANOVA followed by Tukey’s multiple comparisons test).

Fig. 6.

Pharmacological inhibition of Wnt/β-catenin signaling by β-catenin specific inhibitor XAV-939 abolished the BBB protection in Nhe1Astro-KO brains after ischemic stroke. (A) Representative confocal images showing albumin immunofluorescence staining in ischemic peri-lesion areas of wild-type and Nhe1 Astro-KO brains treated with vehicle and XAV-939 at 24 h Rp. (B) Summary of albumin infiltration. Albumin immunoreactivity was quantified by measuring the fluorescence intensity of images in panel A and B. Data are mean ± SEM, n = 5, *p < 0.05 (three-way ANOVA followed by Newman-Keuls multiple comparisons test). (C) Representative images of DAB-positive red blood cells showing the presence of intracerebral hemorrhages in the IL cortex of XAV-939 treated brains at 24 h Rp. (D) Quantification of the hemorrhagic percent area. Data are mean ± SEM, n = 5, *p < 0.05 (three-way ANOVA followed by Newman-Keuls multiple compari-sons test).

Fig. 7.

Mechanisms of astrocytic NHE1 in BBB damage in ischemic stroke.Ischemia stimulates NHE1 expression and activation in reactive astrocytes. Over-stimulation of NHE1 activity leads to pHi, and ion dysregultion. Selective deletion of astrocytic Nhe1 prevents NHE1 overstimulation, reduces BBB damage, and increases Wnt/β-catenin signaling in ECs, which promotes angiogenesis and improves rCBF.