Reduced expression of integrin alphavbeta8 is associated with brain arteriovenous malformation pathogenesis

Abstract

Brain arteriovenous malformations (BAVMs) are a rare but potentially devastating hemorrhagic disease. Transforming growth factor-beta signaling is required for proper vessel development, and defective transforming growth factor-beta superfamily signaling has been implicated in BAVM pathogenesis. We hypothesized that expression of the transforming growth factor-beta activating integrin, alphavbeta8, is reduced in BAVMs and that decreased beta8 expression leads to defective neoangiogenesis. We determined that beta8 protein expression in perivascular astrocytes was reduced in human BAVM lesional tissue compared with controls and that the angiogenic response to focal vascular endothelial growth factor stimulation in adult mouse brains with local Cre-mediated deletion of itgb8 and smad4 led to vascular dysplasia in newly formed blood vessels. In addition, common genetic variants in ITGB8 were associated with BAVM susceptibility, and ITGB8 genotypes associated with increased risk of BAVMs correlated with decreased beta8 immunostaining in BAVM tissue. These three lines of evidence from human studies and a mouse model suggest that reduced expression of integrin beta8 may be involved in the pathogenesis of sporadic BAVMs.

Figure 1

Integrin subunit β8 and β3 immunostaining in BAVMs relative to control brain samples. β8 immunohistochemical localization (A, D, G, J) was compared with β3 (B, E, H, K) and no primary antibody control staining (C, F, I, L). Shown are representative staining patterns seen in the normal insular cortex (A–C), temporal lobe from a patient with epilepsy (D–F) or from two separate BAVMs (G–L). A and D: Photomicrographs demonstrate the presence of perivascular β8 staining adjacent to small cerebral vessels (V) or in G and J, absence of staining in the perivascular cell processes surrounding the walls of large BAVM vessels (VW) or small perinidal BAVM vessels (v) vessels. Scale bar = 50 μm in A–C, G–L) and 25 μm in D–F. Shown in A and D are photomicrographs of small vessels with perivascular staining tracing the outer wall of the vessel (white arrows), which is absent from BAVM samples (G and J, black arrows). Diffuse β8 immunostaining of the neuropil is shown in both control (A and D) and BAVM (G and J) samples. β8 neuropil staining tended to be lighter in BAVM samples. Little or no staining is observed in samples stained with anti-β3 (B, E, H, K) or no primary antibody controls (C, F, I, L).

Figure 2

Integrin β8 and not β3 subunit immunostaining is reduced in BAVM samples relative to controls. Tissue staining intensity from BAVM (n = 34) and control (n = 15) samples was graded on a scale of 0–3 with diffuse neuropil stain being grade 1, neuropil + cell body staining, grade 2, and neuropil + cell body + perivascular staining grade 3. Shown are bar graphs (±SEM); **P = 0.002.

Figure 3

Adenoviral Cre-mediated deletion of itgb8 in mouse brain. Adenoviral Cre (Ad-Cre) or adenoviral associated virus-LacZ (AAV-LacZ) was stereotactically injected into the basal ganglia of adult male C57BL/6J mice with loxP sites flanking exon 4 of the murine integrin β8 (itgb8) gene. A: The recombined locus was detected by PCR of genomic DNA isolated from the injected (lesional) site or the noninjected (contralateral) site, 3 weeks after injection, using primers designed to flank the upstream and downstream loxP sites. An amplicon of the expected size (340 bp) was detected only in the Ad-Cre injected mouse brains (n = 3) and not in the contralateral hemisphere or in the brains of mice injected with AAV-LacZ. B: qPCR was used to determine the efficacy of Cre-mediated recombination of the itgb8 locus. Total RNA was isolated from the basal ganglia of the ipsilateral injected (lesional) or contralateral noninjected sites and SYBR green PCR was performed. Shown is mean transcript copy number relative to GAPDH. *Ad-Cre lesional versus Ad-Cre contralateral; Ad-Cre versus AAV-LacZ lesional, P < 0.05.

Figure 4

Morphological alterations in VEGF-induced new blood vessels resulting from local deletion of itgb8 or smad4. Digital images display lectin staining of blood vessels (green) around stereotactic injection sites. The insets show enlarged images of capillary formations. Shown in the upper panels are itgb8 fl/fl and on the lower panels smad4 fl/fl mice. The insets show enlarged images of capillaries in AdCre and AAV-VEGF injected brain and normal capillaries in the AAV-VEGF or AdCre plus control vector injected brain. The co-injection strategy with AAV-VEGF, Ad-Cre, or control adenovirus is indicated. Scale bar = 100 μm.

Figure 5

Vascular morphology but not vascular density is affected in VEGF-induced new blood vessel formation resulting from local deletion of itgb8 or smad4. Bar graphs show capillary density (mean vessel counts/field) (A) and the dysplasia index (number of enlarged, irregular capillaries for every 200 capillaries examined) (B) 3 weeks following viral transduction in the basal ganglia of itgb8 fl/fl or smad4 fl/fl mice. Shown is SEM; *P < 0.05.

Figure 6

Adenoviral-Cre mediated deletion of itgb8 results in abrogation of TGF-β activation, in vitro and in vivo. A: Neonatal astrocytes were cultured from itgb8 fl/fl mice (n = 8) and infected with Ad-Cre or Ad-LacZ. After 72 hours the astrocytes were harvested and co-cultured with TGF-β reporter cells (TMLC) in the presence or absence of a pan-TGF-β isoform neutralizing antibody (1D11). Shown are arbitrary luciferase units relative to the 1D11 control; ***P = 0.0003 B–D: Ad-Cre was injected into the basal ganglia of 6 weeks old itgb8 fl/fl mice (n = 2) and after 7 days the brains were harvested, fixed, immunohistochemically stained with anti-pSmad-2 or anti-pSmad-1/5/8 and the area surrounding the needle tip or the corresponding contralateral side were digitally imaged. Microscopic fields (n = 64) from digital images were blindly assessed for nuclear staining. Shown are bar graphs showing quantification of nuclear staining intensity (0–2 scale). Shown are representative fields showing staining in the area of the needle tract (C) compared with the noninjected side (D). Scale bar = 100 μm; *P = 0.031. E: Lysates from the from the basal ganglia of itgb8 fl/fl mice injected with Ad-Cre or Ad-GFP (or the contralateral noninjected basal ganglia) were immunoblotted using anti-pSmad-2 (top panel) or anti-Smad-2/3 (bottom panel). Shown is a representative experiment of 4 with similar results. F: Densitometric analysis of pSmad-2 from Ad-Cre and Ad-GFP injected basal ganglia relative to noninjected basal ganglia. Shown in SEM; *P < 0.05.

Figure 7

Genetic variation in ITGB8 is associated with BAVM risk. A: LD plot of ITGB8 5′ region in Caucasian HapMap samples. In the upper panel, the genomic location on chromosome 7 of ITGB8 (NM 002214) exon 1 (gray shaded box) and intron 1 (black line) is shown. In the lower panel is the LD plot where shading represents pairwise LD between SNPs in terms of r²: black shading = 1 (perfect correlation), white shading = 0 (no correlation), shades of gray = 0 < r² < 1. SNPs identified by Haploview residing in the ITGB8 5′ flanking region, Exon 1 or intron 1 are numbered 1 through 15 and their location indicated by lines extending to the upper panel. Note that SNPs 2 and 3 are not shown since they are monomorphic (ie, minor allele frequency is 0%). Thus, no data exists between SNP1 and 4 and the 5′ boundary of the 4.2-kb LD block remains undefined. ITGB8 haplotype-tagging SNPs were selected using the Tagger algorithm with pairwise selection, minor allele frequency >5%, and r² > 0.8; genotyped SNPs are indicated by large bold font. B: OR and 95% CI for ITGB8 haplotype-tagging SNPs in 194 BAVM cases and 127 healthy controls, all of Caucasian ancestry. Vertical dotted line indicates an OR = 1 (no association). Two SNPs (rs10486391 and rs11982847) were associated with BAVM with 95% CI excluding 1.0.

Figure 8

Decreased integrin β8 immunostaining correlates with ITGB8 genotypes associated with increased risk of BAVMs. Tissue staining intensity was graded on a scale of 0–3. AA (n = 11), AG (n = 12), GG (n = 5). *P = 0.016, AA versus AG+GG.