De novo cerebrovascular malformation in the adult mouse after endothelial Alk1 deletion and angiogenic stimulation

Abstract

Background and purpose: In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor stimulation induces brain AVMs in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal vascular endothelial growth factor stimulation is sufficient to induce brain AVM in the adult mouse.

Methods: Focal angiogenesis was induced in the brain of 8-week-old Pdgfb-iCreER;Alk1(2f/2f) mice by injection of adeno-associated viral vectors expressing vascular endothelial growth factor. Two weeks later, EC-Alk1 deletion was induced by tamoxifen treatment. Vascular morphology was analyzed, and EC proliferation and dysplasia index (number of vessels with diameter>15 μm per 200 vessels) were quantified 10 days after tamoxifen administration.

Results: Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of tamoxifen-treated Pdgfb-iCreER;Alk1(2f/2f) mice. Induced brain AVMs were marked by increased dysplasia index (P<0.001) and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs.

Conclusions: Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo brain AVM.

Keywords: AVM (arteriovenous malformation) intracranial; models, animal; telangiectasia, hereditary hemorrhagic, type 2.

Figure 1. Design and groups

(A) Design: Adult mice were injected with AAV-VEGF or AAV-LacZ into the brain. Two weeks later, TM was injected i.p. to induce Alk1 gene deletion. Samples were collected 10 days later. To track proliferating cells, BrdU was injected i.p. daily starting on the day of TM injection and continuing for 10 days. (B) Groups. iECKO: inducible EC Alk1 knockout. Twelve mice per group: 6 for latex perfusion and 6 for immunostaining.

Figure 2. Malformed vessels in the brain of Alk1^iECKO+VEGF mice

(A) Latex-casted vessels and CD31-stained brain sections. Arrows indicate dilated dysplasia vessels. Scale bar: 1 mm (upper panel) and 100 μm (lower panel). (B) Enlarged image shows tortuous and dilated abnormal vessels. Scale bar: 100 μm. (C) Quantifications of dysplasia index.

Figure 3. Increased EC proliferation in the angiogenic foci of Alk1^iECKO+VEGF mice

(A) BrdU⁺ (red) EC nuclei (ERG⁺, green) in the angiogenic foci of WT+VEGF and Alk1^iECKO+VEGF mice. Scale bar: 100 μm. (B) Quantification of ERG⁺/BrdU⁺ ECs. (C) Proliferating ECs (Ki67⁺ or BrdU⁺) are clustered on the dysplastic vessels (outlined by dotted lines in the images on the left and indicated by arrows in the images on the right). Scale bar: 100 μm. (D) Confocal image showing colocalization of BrdU (red) and ERG (green, top), and ki67 (red) and CD31 (green, bottom) positive staining. The nuclei were counterstained with DAPI (blue). Scale bar: 20 μm.