The alpha7 integrin subunit in astrocytes promotes endothelial blood-brain barrier integrity

Abstract

The blood-brain barrier (BBB) is a vascular endothelial cell boundary that partitions the circulation from the central nervous system to promote normal brain health. We have a limited understanding of how the BBB is formed during development and maintained in adulthood. We used quantitative transcriptional profiling to investigate whether specific adhesion molecules are involved in BBB functions, with an emphasis on understanding how astrocytes interact with endothelial cells. Our results reveal a striking enrichment of multiple genes encoding laminin subunits as well as the laminin receptor gene Itga7, which encodes the alpha7 integrin subunit, in astrocytes. Genetic ablation of Itga7 in mice led to aberrant BBB permeability and progressive neurological pathologies. Itga7-/- mice also showed a reduction in laminin protein expression in parenchymal basement membranes. Blood vessels in the Itga7-/- brain showed separation from surrounding astrocytes and had reduced expression of the tight junction proteins claudin 5 and ZO-1. We propose that the alpha7 integrin subunit in astrocytes via adhesion to laminins promotes endothelial cell junction integrity, all of which is required to properly form and maintain a functional BBB.

Keywords: Angiogenesis; Astrocyte; Blood vessel; Brain microenvironment; Extracellular matrix; Megalencephalic leukoencephalopathy with subcortical cysts 1; Pericyte; Vascular smooth muscle cell.

Fig. 1.

Itga7 is expressed in perivascular astrocytes of the postnatal mouse brain. (A) Schematic showing the experimental strategy for isolating and characterizing genes expressed in perivascular astrocytes (Mlc1⁺) and non-perivascular astrocyte fractions (Mlc1⁻) from the postnatal brain. P30 brains were dissected from GLAST-DsRed/+;Mlc1-EGFP/+ double-heterozygous mice and single-positive (DsRed⁺) non-perivascular astrocytes and double-positive (DsRed⁺/EGFP⁺) perivascular astrocytes were fractionated by live cell sorting. (B) There is significantly enriched expression of Itga7 mRNA and the laminin mRNAs Lama2, Lama3 and Lama5 in Mlc1⁺ perivascular astrocytes in comparison with Mlc1⁻ non-perivascular astrocyte fractions. **P<0.01, ***P<0.001 (unpaired Student's t-test; n=4). A.U., arbitrary units. These data are from a prior RNA-sequencing effort (Yosef and McCarty, 2020). (C-H) Sagittal sections from P60 Itga7^+/+ control (C,E,G) and Itga7^−/− mutant (D,F,H) mouse brains through the cerebral cortex (C,D), thalamus (E,F) and cerebellum (G,H) were analyzed by double immunofluorescence using antibodies directed against β-galactosidase (green) and GFAP (red). Itga7^−/− mice displayed early-stage brain vascular phenotypes. Bottom panels are higher magnification images of the boxed areas in the merged panels. In comparison with the control, note the expression of β-galactosidase in astrocytes of the Itga7^−/− mutant mouse brain (arrows in D,F,H). Images are representative of n=10 brain samples per genotype. Scale bars: 50 µm (upper panels); 20 µm (bottom panels).

Fig. 2.

A subset of Itga7^−/− mice develop progressive BBB defects and die prematurely by P60. (A) Representative images of P60 Itga7^−/− and Itga7^+/+ control littermates showing differences in body size as well as hydrocephalus in the mutant animal. About 15% of total Itga7^−/− mutants exhibited these early-onset brain pathologies. (B,C) P60 wild-type control (n=3) and Itga7^−/− mice (n=3) with brain phenotypes were cardiac-perfused with fixative and whole brains were dissected (top) and sliced along the midline (bottom). Note the hemorrhage and brain ventricle enlargement associated with hydrocephalus in the Itga7^−/− brain, highlighted by Hematoxylin and Eosin staining of paraffin-embedded brain sections in C (asterisk). (D,E) P30 Itga7^+/+ control (D) and Itga7^−/− mutant (E) mice (n=3 mice per genotype) were cardiac-perfused with 10 kDa FITC-Dextran and sagittal brain slices were analyzed for Dextran distribution. The Itga7^−/− animals used for this experiment had obvious edema and hydrocephalus. Representative images are shown through the cerebral cortices of control and mutants (n=4 selected fields per brain). In comparison with the control samples, note the increased extravasation of FITC-Dextran across the BBB and into the parenchyma in the Itga7^−/− mutant brain. Scale bars: 50 µm. (F) Quantification of FITC-Dextran extravasation in Itga7^+/+ control and Itga7^−/− mutant cerebral cortices (n=3 mice per genotype). ****P<0.0001 (unpaired Student's t-test). (G,H) P30 Itga7^+/+ control (G) and Itga7^−/− mutant (H) mice (n=3 mice per genotype) were cardiac-perfused with 4% PFA/PBS and brains were dissected, sliced along the midline, and sagittal sections were analyzed for mouse serum albumin distribution by immunofluorescent labeling with anti-serum albumin (white) and anti-CD31 (red). The Itga7^−/− animals used for this experiment had obvious edema and hydrocephalus. Shown are representative images through the cerebral cortices of control and mutants (n=3-4 fields per genotype). In comparison with the Itga7^+/+ control samples, note the increased extravasation of mouse serum albumin across the BBB and into the cortical parenchyma in the Itga7^−/− mutant brain. Scale bars: 50 µm. For these studies, Itga7^−/− mice with early-onset brain phenotypes were analyzed. (I) Quantification of mouse serum albumin extravasation in wild-type control and Itga7^−/− cerebral cortices (n=3 mice per genotype). ***P<0.001 (unpaired Student's t-test). A.U., arbitrary units.

Fig. 3.

Perivascular gliosis in the Itga7^−/− mouse brain. (A-F) Sagittal sections from P30 Itga7^+/+ control (A,C,E) and Itga7^−/− mutant (B,D,F) mouse brains (n=3 brains per genotype) through the cortex (A,B), thalamus (C,D) and cerebellum (E,F) were analyzed by double immunofluorescence using antibodies directed against GFAP (green) and CD31 (red). At least n=3 microscopic fields were analyzed per mouse brain. In comparison with the control brain sections, note the expression of GFAP levels in astrocytes of the Itga7^−/− mutant mouse brain, indicative of reactive astrogliosis. The Itga7^−/− animals used for this experiment had obvious edema and hydrocephalus. Scale bars: 20 µm. (G) Quantification of GFAP⁺ reactive astrogliosis in the wild-type or Itga7^−/− cerebral cortex (n=3 mice per genotype). ****P<0.0001 (unpaired Student's t-test). For GFAP quantification, n=3-4 microscopic images per mouse cortex were analyzed. (H-M) Sagittal sections from P30 Itga7^+/+ control (H,J,L) or Itga7^−/− mutant (I,K,M) mouse brains (n=3 brains per genotype) through the cortex (H,I), thalamus (J,K) and cerebellum (L,M) were analyzed by double immunofluorescence staining using antibodies directed against Iba1 (green) and CD31 (red). The Itga7^−/− animals used for this experiment had obvious edema and hydrocephalus. In comparison with the control, note the enhanced expression of Iba1 and altered morphologies of increasing population of perivascular microglia in the Itga7^−/− mouse brain, indicative of reactive microgliosis. Scale bars: 20 µm. (N) Quantification of reactive Iba1⁺ reactive microgliosis in the Itga7^+/+ control or Itga7^−/− mutant cerebral cortices (n=3 mice per genotype). ****P<0.0001 (unpaired Student's t-test). For Iba1 quantification, n=4 fields per mouse cortex were analyzed. A.U., arbitrary units.

Fig. 4.

Ultrastructural defects in Itga7^−/− brain neurovascular units. (A-D) Brain sections from P50 Itga7^+/+ control (A,B) and Itga7^−/− mutant (C,D) mice (n=2 brains per genotype) were analyzed using transmission electron microscopy. The Itga7^−/− animals used for this experiment did not have obvious neurological phenotypes. Boxed areas in A and C are shown at higher magnification in B and D, respectively. In comparison with control cerebral cortex (A,B), note the abnormal blood vessels with labeled endothelial cells (EC) that are surrounded by reactive astrocyte end feet (labeled with red ‘a’) in the Itga7^−/− mutant brain (C,D). Nearby astrocyte cell bodies are also visible (labeled with red ‘A’). Cortical blood vessels in Itga7^−/− mutant brains also show separation from the surrounding brain parenchyma (red arrows in D). Scale bars: 6 µm (A,C); 2 µm (B,D).

Fig. 5.

Reduced laminin protein expression in vascular basement membranes of Itga7^−/− brains. (A-D) Coronal brain sections through the cerebellum (A,B) and thalamus (C,D) of P60 Itga7^+/+ control (A,C) or Itga7^−/− mutant (B,D) mice (n=3 per genotype) were fluorescently labeled with anti-laminin-111 (red) and anti-CD31 (cyan) antibodies. The Itga7^−/− animals used for this experiment did not display obvious brain pathologies. There is reduced expression of laminin protein in the vascular basement membranes of Itga7^−/− mutant brains. Scale bars: 50 µm. (E) Quantification of laminin protein expression levels in Itga7^+/+ control and Itga7^−/− mutant (n=3 mice per genotype) regions from the cerebellum and thalamus. ****P<0.0001 (unpaired Student's t-test). For laminin quantification, n=4 images per mouse brain region were analyzed. (F-I) Sagittal brain sections from 1-year-old Itga7^+/+ control (F,H) and Itga7^−/− mutant (G,I) mice (n=3 brains per genotype) were labeled with anti-laminin-111 (red) in combination with anti-CD31 (cyan) antibodies. In comparison with Itga7^+/+ controls, note the reduced laminin-111 expression in vascular basement membranes of the cortex (F,G) and cerebellum (H,I) in Itga7^−/− mutant brains. Scale bars: 50 µm. A.U., arbitrary units.

Fig. 6.

Analysis of Itga7-dependent ECM adhesion and signaling in cultured brain astrocytes. (A,B) Cells from P5 Itga7^+/+ control (A) and Itga7^−/− mutant (B) brains (n=3 mice per genotype) were cultured on dishes coated with laminin-111. Fixed cells were labeled with anti-GFAP (green) or anti-nestin (red) antibodies in combination with phalloidin (white) to visualize F-actin. Shown are representative low-power fields from control and mutant cultures. Note that nearly 100% of cultured cells express GFAP and/or nestin. Scale bars: 50 µm. (C) Astrocytes were cultured from brains of Itga7^+/+ control and Itga7^−/− mutant mice. Detergent-soluble cell lysates were immunoblotted using antibodies directed against α7 integrin, β1 integrin and β-galactosidase. Note the expression of α7 and β1 integrin proteins in wild-type lysates; in contrast, there is lack of α7 expression in Itga7^−/− lysates that correlates with increased expression of β-galactosidase due to lacZ insertion in the Itga7 locus. (D) Anti-α7 immunoprecipitated fractions from Itga7^+/+ control and Itga7^−/− brain lysates were immunoblotted with anti-α7 antibodies. In comparison with Itga7^+/+ control cells, note the absence of α7 integrin protein in Itga7^−/− mutant cell samples. (E) Astrocytes cultured from brains of Itga7^+/+ control and Itga7^−/− mutant mice were surface-labeled with NHS-biotin and detergent-soluble lysates were immunoprecipitated with the indicated anti-integrin antibodies. Note the absence of the α7β1 integrin heterodimer in Itga7^−/− lysates, whereas the expression of other integrin heterodimers is not affected. MWM, molecular weight marker. (F) Astrocytes (n=3 samples per genotype) were added to tissue culture wells coated with the indicated ECM proteins and cell adhesion was quantified after 2 h. Note that loss of the α7 integrin expression leads to ECM adhesion defects on laminin-111 substrates. Itga7-dependent defects in adhesion to collagens I and IV as well as fibrinogen are also detected. *P<0.05, **P<0.01 (unpaired Student's t-test). (G) Itga7^+/+ and Itga7^−/− astrocytes (n=3 cell samples per genotype) were analyzed for growth and viability in vitro. Note that Itga7^−/− cells show reduced growth and survival in comparison with Itga7^+/+ control cells. Differences between groups were analyzed using two-way ANOVA and Tukey post-hoc analysis (n=3, mean±s.e.m., *P<0.05, **P<0.01, ****P<0.0001). (H) Primary astrocytes were cultured from P5 Itga7^+/+ control and Itga7^−/− mutant mice and detergent-soluble lysates were immunoblotted with an anti-laminin α1 antibody. Note the α7 integrin-dependent reductions in laminin protein expression in cultured astrocytes. The ∼100 kDa band is a fragment of the full-length laminin protein, likely owing to proteolytic processing and/or degradation during sample preparation. A.U., arbitrary units; n.s., not significant.

Fig. 7.

Itga7-dependent defects in expression of BBB junctional proteins in vascular endothelial cells. (A) Experimental schema for fractionating CD31⁺ vascular endothelial cells from P365 Itga7^+/+ control and Itga7^−/− mutant adult brains (n=3 brains per genotype). Whole brains were digested enzymatically to generate a single-cell suspension and vascular endothelial cells (EC) were sorted using anti-CD31-conjugated magnetic beads. (B) Expression levels of different vascular endothelial cell junctional proteins were determined by immunoblotting detergent-soluble lysates prepared from CD31⁺ cells fractionated from one-year-old Itga7^+/+ control and Itga7^−/− mutant brains. In comparison with controls, note the reduced expression of claudin 5, ZO-1 and VE-cadherin in vascular endothelial cells isolated from Itga7^−/− mutant brains. (C) Quantification of VE-cadherin, ZO-1 and claudin 5 junctional protein levels as determined by laser-scanning densitometry of immunoblots from fractionated brain endothelial cell lysates. **P<0.01, ****P<0.0001 (unpaired Student's t-test). All immunoblot experiments were performed with n=3 different cell lysates. (D,E) Sagittal sections through one-year-old (P365) Itga7^+/+ control (D) and Itga7^−/− mutant (E) cerebral cortices were labeled with anti-claudin 5 (green) and anti-CD31 (red) antibodies. In comparison with Itga7^+/+ control brains, which express claudin 5 in vascular endothelial cells (arrows in D), note the reduced claudin 5 expression in vascular endothelial cells in the Itga7^−/− mutant cerebral cortex (asterisks in E). Scale bars: 20 µm. (F) Quantification of claudin 5 protein levels in wild-type or Itga7^−/− cerebral cortex, as determined by double immunofluorescence staining with anti-claudin 5 and anti-CD31 antibodies. ***P<0.001 (unpaired Student's t-test). For immunofluorescence quantification, n=4 fields per mouse cortex were analyzed. (G,H) Sagittal sections through one-year-old (P365) Itga7^+/+ control (G) and Itga7^−/− mutant (H) cerebellar regions were labeled with anti-claudin 5 (green) and anti-CD31 (red) antibodies. In comparison with Itga7^+/+ control brains, which express claudin 5 in vascular endothelial cells (arrows in G), note the reduced claudin 5 expression in vascular endothelial cells in the Itga7^−/− mutant cerebellum (asterisks in H). Scale bars: 20 µm. (I) Quantification of claudin 5 protein levels in wild-type or Itga7^−/− cerebellum, as determined by double immunofluorescence staining with anti-claudin 5 and anti-CD31 antibodies. **P<0.01 (unpaired Student's t-test). For claudin 5 quantification, n=4 images per mouse cerebellum were analyzed. A.U., arbitrary units.

Fig. 8.

A model for astrocyte stabilization of the BBB via α7 integrin-mediated adhesion to laminins in the vascular basement membrane. (A) In the healthy mammalian brain, astrocytes juxtapose the abluminal surface of cerebral blood vessels and mediate cell-cell contact and communication events with endothelial cells of the neurovascular unit. (B) Perivascular astrocytes utilize α7β1 integrin to adhere to laminins and possibly other ECM ligands in the vascular basement membrane surrounding cerebral blood vessels. These ECM adhesion functions for α7β1 integrin mediate normal cell–cell contact and communication with brain endothelial cells and pericytes to maintain neurovascular unit homeostasis. (C) Genetic ablation of Itga7 leads to defective adhesion between perivascular astroglia and laminins as well as reduced laminin protein levels, resulting in diminished expression of proteins in vascular endothelial cell junctions. This pathological adhesion and signaling results in defective cell–cell communication in the neurovascular unit, leading to defective BBB maturation and stability. Created with BioRender.com.