Caveolin-1 Regulates Perivascular Aquaporin-4 Expression After Cerebral Ischemia

Abstract

Edema is a hallmark of many brain disorders including stroke. During vasogenic edema, blood-brain barrier (BBB) permeability increases, contributing to the entry of plasma proteins followed by water. Caveolae and caveolin-1 (Cav-1) are involved in these BBB permeability changes. The expression of the aquaporin-4 (AQP4) water channel relates to brain swelling, however, its regulation is poorly understood. Here we tested whether Cav-1 regulates AQP4 expression in the perivascular region after brain ischemia in mice. We showed that Cav-1 knockout mice had enhanced hemispheric swelling and decreased perivascular AQP4 expression in perilesional and contralateral cortical regions compared to wild-type. Glial fibrillary acidic protein-positive astrocytes displayed less branching and ramification in Cav-1 knockout mice compared to wild-type animals. There was a positive correlation between the area of perivascular AQP4-immunolabelling and branch length of Glial fibrillary acidic protein-positive astrocytes in wild-type mice, not seen in Cav-1 knockout mice. In summary, we show for the first time that loss of Cav-1 results in decreased AQP4 expression and impaired perivascular AQP4 covering after cerebral ischemia associated with altered reactive astrocyte morphology and enhanced brain swelling. Therapeutic approaches targeting Cav-1 may provide new opportunities for improving stroke outcome.

Significance statement: Severe brain edema worsens outcome in stroke patients. Available treatments for stroke-related edema are not efficient and molecular and cellular mechanisms are poorly understood. Cellular water channels, aquaporins (AQPs), are mainly expressed in astrocytes in the brain and play a key role in water movements and cerebral edema, while endothelial caveolins have been suggested to play a role in vasogenic edema. Here we used an integrative approach to study possible interaction between AQP4 and caveolin-1 (Cav-1) after stroke. Absence of Cav-1 was associated with perivascular changes in AQP4 expression and enhanced brain swelling at 3 days after cerebral ischemia. The present work indicates a direct or indirect effect of Cav-1 on perivascular AQP4, which may lead to novel edema therapy.

Keywords: aquaporin (AQP)-4; astrocyctes; caveolin-1 (CAV1); endfeet; recovery; stroke.

FIGURE 1

Immunostaining of coronal slices of WT and Cav-1 KO mice after sham surgery and at 6 and 72 h after MCAO: (A) MAP-2 expression (red) showing the lesion outlined by a white dotted line. (B) GFAP expression (gray) showing the extent of the astrocyte scar highlighted by yellow arrows. (C) AQP4 expression (green); the contralateral hemisphere area is delineated by a white dotted line relative to the ipsilateral hemisphere to appreciate the swelling and the white arrowhead points to the shift of the midline due to swelling. Red arrows highlight AQP4 staining in the cortical perilesion. Scale bar = 1 mm. (D) Brain swelling at 7 days after MCAO in WT and Cav-1 KO mice. (E) Immuno-staining at ×63 magnification illustrating the relationship between AQP4 (green), vessels labeled by CD31 (red) and reactive astrocytes (gray), scale bar = 10 μm.

FIGURE 2

(A) High magnification images of AQP4 on vessels stained by CD31 at 72 h post-MCAO (×63 magnification) in the striatum. (B) High magnification images of AQP4 on vessels stained with CD31 at 72 h post-MCAO (×63 magnification) in the cortex. (C) Analysis of the length of AQP4-immunolabeled vessel-like structures with Fiji vascular density plug-in in WT and Cav-1 KO after MCAO and in sham animals; n = 3 areas per animal with three animals per group. Black and gray plots refer to WT and Cav-1 KO animals, respectively. Significant differences between WT and Cav-1 KO animals are noted as *p < 0.05, **p < 0.01, ***p < 0.005 and ****p < 0.001. WT-Sham compared to WT-Ipsi: 95% CI [−0.4949 to −0.1666], p < 0.0001, WT-Sham compared to WT-Contra: 95% CI [−0.3330 to −0.004702], p = 0.04, KO-Sham compared to KO-Ipsi: 95% CI [−0.3355 to −0.007202], p = 0.0354, WT-Contra compared to KO-Contra: 95% CI [0.1888 to 0.5171], p < 0.0001, and WT-Ipsi compared to KO-Ipsi: 95% CI [0.05398 to 0.3822], p = 0.0027. (D) Analysis of the density of AQP4-immunolabeled vessel-like structures assessed by the same Fiji plug-in. WT-Sham compared to WT-Ipsi: 95% CI [−1.985 to −0.09168], p = 0.0229, WT-Contra compared to KO-Contra: 95% CI [0.5432 to 2.436], p = 0.0002, KO-Contra compared to KO-Ipsi: 95% CI [−2.594 to −0.7009], p < 0.0001. (E) Analysis of the coverage of AQP4 on vessels measured as the ratio between the density of AQP-4 vessel-like pattern and CD31-labeled vessels. WT-Sham compared to WT-Ipsi: 95% CI [−73.27 to −1.223], p = 0.0384, WT-Contra compared to KO-Contra: 95% CI [3.157 to 75.20], p = 0.0247, WT-Ipsi compared to KO-Ipsi: 95% CI [3.509 to 75.55], p = 0.0228. Contra: contralateral hemisphere to the lesion, Ipsi: ipsilateral hemisphere to the lesion. Scale bar = 20 μm. Comparisons were carried out by one-way ANOVA with Tukey’s multiple comparisons post-test.

FIGURE 3

(A) Immunofluorescence staining with AQP4 (green) and GFAP (gray) in WT and Cav-1 KO mice at 72 h post-MCAO (63× magnification). AQP4 co-localized with GFAP-positive astrocyte end-feet. Scale bar = 20 μm. (B) Single-channel confocal microscopy ROIs obtained from 40× magnification images, illustrating the overview of a GFAP-positive astrocyte morphology and its skeletonization. (C) Number of GFAP-positive reactive astrocytes. WT-Sham compared to WT-Ipsi: 95% CI: [−13.42 to −5.027], p < 0.0001, WT-Sham compared to WT-Contra: 95% CI [0.4716 to 8.862], p = 0.0212, WT-Sham compared to KO-Sham: 95% CI: [3.805 to 12.20], p < 0.0001, WT-Ipsi compared to WT-Contra: 95% CI [−18.08 to −9.694], p < 0.0001, and WT-Ipsi compared to KO-Ipsi: 95% CI [10.92 to 19.31], p < 0.0001. (D) Average branch length of GFAP-positive astrocytes. WT-Sham compared to WT-Ipsi: 95% CI [−2.455 to −1.005], p < 0.0001, WT-Sham compared to WT-Contra: 95% CI [−1.648 to −0.1988], p = 0.0054, WT-Ipsi compared to WT-Contra: 95% CI: [−1.531 to −0.08208], p = 0.0210, and WT-Ipsi compared to KO-Ipsi: 95% CI [0.5799 to 2.029], p < 0.0001. Comparisons were carried out by one-way ANOVA with Tukey’s multiple comparisons post-test. (E) The positive correlation between the length of AQP4-immunolabeled vessel-like structures and the maximal branch length of GFAP-immunolabeled astrocytes in WT mice (Pearson correlation coefficient = 0.451, p = 0.024) and Cav-1 KO mice (Pearson correlation coefficient = −0.443, p = 0.021).

FIGURE 4

After transient filament MCAO (left), Cav 1 KO mice had larger lesions, enhanced cerebral edema and higher hemispheric swelling (bottom right) compared to WT mice (top right). At the level of the NVU (center images), the absence of caveolae* and Cav-1 correlated with altered astroglial reaction and expression of AQP4 on astrocytic end-feet in Cav-1 KO mice (bottom center) compared to WT mice (top center).