Integrin β8 Deletion Enhances Vascular Dysplasia and Hemorrhage in the Brain of Adult Alk1 Heterozygous Mice

Abstract

Brain arteriovenous malformation (bAVM), characterized by tangled dysplastic vessels, is an important cause of intracranial hemorrhage in young adults, and its pathogenesis and progression are not fully understood. Patients with haploinsufficiency of transforming growth factor-β (TGF-β) receptors, activin receptor-like kinase 1 (ALK1) or endoglin (ENG) have a higher incidence of bAVM than the general population. However, bAVM does not develop effectively in mice with the same haploinsufficiency. The expression of integrin β8 subunit (ITGB8), another member in the TGF-β superfamily, is reduced in sporadic human bAVM. Brain angiogenic stimulation results at the capillary level of vascular malformation in adult Alk1 haploinsufficient (Alk1 ^+/- ) mice. We hypothesized that deletion of Itgb8 enhances bAVM development in adult Alk1 ^+/- mice. An adenoviral vector expressing Cre recombinase (Ad-Cre) was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brain of Alk1 ^+/-;Itgb8-floxed mice to induce focal Itgb8 gene deletion and angiogenesis. We showed that compared with Alk ^+/- mice (4.75 ± 1.38/mm²), the Alk1 ^+/-;Itgb8-deficient mice had more dysplastic vessels in the angiogenic foci (7.14 ± 0.68/mm², P = 0.003). More severe hemorrhage was associated with dysplastic vessels in the brain of Itgb8-deleted Alk1 ^+/- , as evidenced by larger Prussian blue-positive areas (1278 ± 373 pixels/mm² vs. Alk1 ^+/- : 320 ± 104 pixels/mm²; P = 0.028). These data indicate that both Itgb8 and Alk1 are important in maintaining normal cerebral angiogenesis in response to VEGF. Itgb8 deficiency enhances the formation of dysplastic vessels and hemorrhage in Alk1 ^+/- mice.

Keywords: Activin receptor-like kinase 1; Brain arteriovenous malformations; Brain hemorrhage; Integrin β8; Mouse model.

Fig. 1. Representative images of Microfil-cast brain

a. Itgb8-deleted Alk1^+/− brain with AAV-VEGF injection. b. Alk1^+/− brain with AAV-VEGF injection. c. Itgb8-deleted Alk1^+/− brain with AAV-LacZ injection. d. Alk1^+/− brain with AAV-LacZ injection. No tangled abnormal vessel was detected. Increased vascular density (white arrow) was observed around the AAV-VEGF injected sites (a & b) Itgb8-deleted Alk1^+/− brain showed significant hemorrhage (red arrow) around the AAV-VEGF injection site. Scale bar=500μm.

Fig. 2. Deletion of Itgb8 increases vascular dysplasia in the angiogenic foci of Alk1^+/− brain

a. Representative lectin-stained sections. Vessels are labeled by green fluorescence. White arrows indicate dysplastic vessels. The insets show enlarged images of dysplastic vessels. Scale bar= 50μm. b. Quantification of vessel density. c. Quantification of dysplasia index. Error bars indicate SD. VEGF: AAV-VEGF treatment. Cre: Ad-Cre treatment. *P<0.0001 versus brain injected with AAV-LacZ and Ad-Cre; §P=0.003 versus brain injected with AAV-VEGF and Ad-GFP; # P<0.05 versus brain injected with AAV-LacZ and Ad-GFP.

Fig. 3. Deletion of Itgb8 increases hemorrhage in the angiogenic foci in Alk1^+/− brain

a. Representative images of Prussian blue stained sections. The iron depositions are stained blue. Scale bar=100μm. b. Quantification of Prussian blue-positive area. VEGF: AAV-VEGF treatment. Cre: Ad-Cre treatment. * P=0.008 and ** P=0.003 versus brain injected with AAV-LacZ and Ad-Cre or Ad-GFP; # P =0.028 versus brain injected with AAV-VEGF and Ad-GFP.

Fig. 4. Correlation between Prussian blue-positive area and vascular features

a. Positive correlation of Prussian blue-positive area with vessel density (R²=0.56, P<0.001). b. Positive correlation of Prussian blue-positive area with vascular dysplasia (R²=0.53, P=0.001).

Fig. 5. Summary of vascular phenotypes in the Alk1-deficient brain and depiction of underlying mechanisms

a. Cerebrovascular phenotype of Alk1 and/or Itgb8 knock-out (KO) mice in the brain angiogenic foci. Homozygous Alk1 KO in the brain causes microscopic vascular dysplasia and macroscopic tangled vessels with arteriovenous (A-V) shunt [23]; heterozygous Alk1 KO or homozygous Itgb8 KO results in microscopic level of vascular dysplasia only. Heterozygous Alk1 KO plus Itgb8 homozygous deletion cause microscopic vascular dysplasia and hemorrhage. Alk1^2f/2f: exons 4–6 of both alleles of Alk1 gene are flanked by loxP sides and are deleted when Cre is expressed [23, 47]; Alk1 ^+/−: one allele of Alk1 is deleted [12]; Itgb8 ^2f/2f: exon 4 of both alleles of Itgb8 gene is flanked by loxP sides and is deleted when Cre is expressed [13]. b. Depiction of the mechanisms of Alk1 and Itgb8 deletion in the vascular malformation and hemorrhage. Normal cerebrovascularture is covered with mural cells (including pericytes and smooth muscle cells, top panel). Alk1 deletion in endothelial cells (reduced color intensity, bottom panel) reduces vascular mural cell coverage [46], and Itgb8 deletion in astrocytes (white color) results in abnormal sprouting [42]. Therefore, deletion of Itgb8 in the Alk1^+/− brain enhances vascular dysplasia and hemorrhage.