Ablation of astrocytic laminin impairs vascular smooth muscle cell function and leads to hemorrhagic stroke

Abstract

Astrocytes express laminin and assemble basement membranes (BMs) at their endfeet, which ensheath the cerebrovasculature. The function of astrocytic laminin in cerebrovascular integrity is unknown. We show that ablation of astrocytic laminin by tissue-specific Cre-mediated recombination disrupted endfeet BMs and led to hemorrhage in deep brain regions of adult mice, resembling human hypertensive hemorrhage. The lack of astrocytic laminin led to impaired function of vascular smooth muscle cells (VSMCs), where astrocytes have a closer association with VSMCs in small arterioles, and was associated with hemorrhagic vessels, which exhibited VSMC fragmentation and vascular wall disassembly. Acute disruption of astrocytic laminin in the striatum of adult mice also impaired VSMC function, indicating that laminin is necessary for VSMC maintenance. In vitro, both astrocytes and astrocytic laminin promoted brain VSMC differentiation. These results show that astrocytes regulate VSMCs and vascular integrity in small vessels of deep brain regions. Therefore, astrocytes may be a possible target for hemorrhagic stroke prevention and therapy.

Figure 1.

LNγ1-KO mice present with ICH in adulthood. Brains of 4-mo-old LNγ1-KO (A) and control littermate (C) mice. The LNγ1-KO mouse brain shows ICH in the temporal lobe (A, arrow), whereas the control mouse is normal. Hematoxylin-stained brain sections of LNγ1-KO (B) and control (D) mice. There is evidence of hemorrhage in the LNγ1-KO (B, arrow) but not in the control mouse brain sections. Brains of LNγ1-KO mice were enlarged near sites of hemorrhage, likely due to edema. Analysis of hemorrhages in different brain regions of LNγ1-KO mice showed that most hemorrhages occurred in striatum and thalamus (E; n = 16 mice). CTX, cortex; HPC, hippocampus; HTH, hypothalamus; ST, striatum; THAL, thalamus. Bar, 2 mm.

Figure 2.

Disruption of astrocytic but not endothelial laminins in LNγ1-KO mice. (A) Immunohistochemical analysis showed vascular laminin γ1 is expressed in both control and LNγ1-KO mice (a and b), but its expression level is decreased in LNγ1-KO mice compared with control (a vs. b). Expression levels of laminin α1 and α2 (astrocytic laminins) were dramatically decreased in LNγ1-KO mice (c and e vs. d and f). However, laminin α4 (endothelial) expression remained similar between control and LNγ1-KO mice (g vs. h). Bar, 100 µm. (B) Western blot analysis and quantification of different laminin chain expression levels in the brains of control and LNγ1-KO mice showed lamininγ1, α1, and α2 were significantly decreased in LNγ1-KO mice, but laminin α4 expression was similar between control and KO mice (Student’s t test, n = 7 in each group). n.s: not significant.

Figure 3.

Cerebral vascular wall changes in the striatum of LNγ1-KO mice. (A) Immunohistochemistry analysis revealed expressions of CD31 (endothelial cell marker; Aa and Ab), PDGFR-β (pericyte marker; Ac and Ad), and GFAP (astrocyte marker; Ae and Af) were similar between control and LNγ1-KO mice. However, expression of SMA (smooth muscle cell marker) was decreased in the striata of LNγ1-KO compared with control mice (Ag and Ah). GFAP expression was associated with SMA in the striata of control mice (Ae and Ag, arrows), but minimal SMA expression was observed in the striata of LNγ1-KO mice (Af and Ah). Bar, 100 µm. (B) Western blot analysis and quantification showed CD31, PDGFR-β, and GFAP expressions were not significantly changed in LNγ1-KO mice, but SMA expression was significantly decreased in LNγ1-KO mice compared with control mice (Student’s t test, n = 7 in each group). n.s: not significant.

Figure 4.

Region-specific VSMC contractile protein expression changes in LNγ1-KO mice. (A) Immunohistochemistry showed that SMA (a and c) and SM1 (b and d) expression in cerebral cortex were similar between control and LNγ1-KO mice, but decreased in striatum of LNγ1-KO mice compared with controls (arrows). (B) Western blot analysis and quantification showed that expression levels of SMA and SM1 were significantly decreased in deep cerebral regions of LNγ1-KO mice compared with controls. SMA and SM1 expression in cerebral cortex and hippocampus of LNγ1-KO mice were not significantly changed compared with the same regions of control mice (Student’s t test, n = 7 in each group). n.s: not significant.

Figure 5.

Impaired VSMC differentiation but not cell survival in striatum of LNγ1-KO mice. TUNEL staining showed blood vessel–associated apoptotic cell death was not significantly increased in LNγ1-KO mice compared with controls during development (P0, P14, and P28) and in adulthood (A and B; Student’s t test, n = 5–7 in each group at each time point). (C) Impairment of arterial SMC differentiation. Arteries labeled by EphrinB2 (a and b) expressed high level of SM1 in control (c) but very low level in LNγ1-KO (d) mice. Western blot analysis and quantification of VSMC differentiation protein expression in the striatum of control and LNγ1-KO mice. Expression of SM2, Smoothelin, and SM22 were significantly decreased in the striatum of LNγ1-KO mice compared with control mice (E). Bars: (A) 100 µm; (C) 50 µm.

Figure 6.

Ablation of astrocytic laminin in adult mice disrupts VSMC contractile protein expression. Adenovirus expressing Cre recombinase under the control of GFAP promoter (ad-pGFAP-Cre) was injected into the striata of control or fLAMγ1 mice. 7 d after ad-pGFAP-Cre injection, both SMA (B and D) and SM1 (G and I) were dramatically decreased in fLAMγ1 mice (D and I) when compared with control (B and G). Blood vessels were visualized by CD31 staining (A, C, F, and H). Large caliber blood vessels express SMA (A and B, arrows) and SM1 (F and G, arrows) in control mice, whereas expression of SMA (C and D, arrows) and SM1 (H and I, arrows) in fLAMγ1 mice was decreased. The decreases in SMA and SM1 in fLAMγ1 mice after ad-pGFAP-Cre injection were statistically significant when compared to control by Student’s t test (E and J). Bar, 100 µm.

Figure 7.

Astrocytes and astrocytic laminins promote BVSMC differentiation in vitro. (A) Western blot analysis showed BVSMCs cultured on plates coated with laminin-111 and/or -211 had increased SMA expression compared with cells cultured on PLL-coated plates by ANOVA (n = 18 in each group). (B) BVSMCs cultured on plates coated with laminin-111 had significantly more SMA expression that cells cultured on collagen type IV–coated plates by Student’s t test (n = 18 in each group). (C) BVSMCs co-cultured with astrocytes had significant increases in SMA expression compared with controls and is astrocyte number dependent by ANOVA, whereas astrocytes did not express SMA (n = 18 in each group). (D) Immunocytochemistry showed BVSMCs co-cultured with astrocytes had obvious SMA expression and astrocytes were GFAP positive (a), whereas BVSMCs cultured alone only maintained a basal low level SMA expression and there were no GFAP-positive cells (b). Bar, 100 µm.

Figure 8.

Region-specific vascular changes in LNγ1-KO mice. (A and B) Relationship between astrocytes (A), VSMC (S), and pia meninges (*) in small arterioles in striatum of control mice; there are some regions in which the BM of VSMC and astrocytes are fused together (arrow). (B) Higher magnification of the boxed area in A showed details of the BM of VSMC (arrowhead) and astrocytes (arrow) fused together and astrocyte showed close contact with VSMC (open arrowhead). In some regions the pia meninges (*) lied between VSMC and astrocytes. (C and D) Relationship between VSMC and astrocytes in striatum of LNγ1-KO mice. Astrocytic endfeet detached from the wall of blood vessel. (D) Higher magnification of boxed area in C showed that ablation of astrocytic laminin γ1 disrupted astrocytic endfeet BM formation (arrow) as well as the contact between astrocytes and VSMC (the space between them). However, SMC BMs still exist (arrowhead). (E and F) Relationship between VSMCs, astrocytes, and pia meninges in cerebral cortex in control mice. Pia meninges are found between VSMCs and astrocytes, and there is no direct contact between VSMCs and astrocytes. (F) Higher magnification in boxed area in E showed that the BMs of VSMCs and astrocytes were always separated, and the pia meninges are between them. (G and H) Relationship between VSMCs and astrocytes in cerebral cortex of LNγ1-KO mice. VSMC were covered by pia meninges (*). (H) Higher magnification of the boxed area in G shows that ablation of astrocytic laminin γ1 disrupted astrocytic endfeet BM formation (arrow), but the VSMCs were covered by pia meninges (*) and the BM of VSMCs was intact (arrowhead). (I) Quantification of EM micrographs (n = 7 mice per genotype) revealed that VSMC/astrocytic endfeet direct contact was dramatically decreased in the striatum in LNγ1-KO mice (black bar) compared with controls (white bar). There were dramatically more VSMCs showing direct contact with astrocytic endfeet in striatum than in cerebral cortex in control mice (I). There was no significant difference in percentages of VSMCs showing direct contact with astrocytic endfeet between control and LNγ1-KO cortex (I). There were significantly more VSMCs showing abnormal morphology in the striatum in LNγ1-KO (black bar) than in control mice (white bar), but there was no such difference between control and LNγ1-KO cortex (J). All statistical analyses were by Student’s t test. A, astrocytes; CTX, cortex; E, endothelium; n.s: not significant; S, smooth muscle cell; ST, striatum. Bars in A, C, E, and G are the same and is shown in A; Bars in B, D, F, and H are the same and is shown in B.

Figure 9.

Disruption of astrocytic laminin impaired VSMC differentiation and led to ICH. (A) Blood vessels were visualized by BSL (a and b). Laminin α2 was expressed in these blood vessels in control (c) but not LNγ1-KO (d) mice. Laminin α2 was coexpressed with SMA in large caliber blood vessels in control mice (e), but this correlation was absent in LNγ1-KO mice (f). (B) In control mouse striatum hemoglobin was not detected around large caliber blood vessels expressing SM1 (arrow). In LNγ1-KO mice, however, hemorrhagic regions (stained by hemoglobin, arrowheads) was associated with large caliber blood vessels expressing little SM1 (arrow, a representative image). (C) A ruptured blood vessel in LNγ1-KO mice (a) was identified, which shows that the wall of the vessel was disassembled and smooth muscle cells were fragmented (arrowheads). (b) Higher magnification of boxed area in panel a showed the details of fragmented smooth muscle cells (arrowhead). COL4, collagen type IV; Hb, hemoglobin. Bars: (A) 100 µm; (B) 50 µm; (Ca) 2 µm; (Cb) 0.5 µm.