Bone Marrow-Derived Alk1 Mutant Endothelial Cells and Clonally Expanded Somatic Alk1 Mutant Endothelial Cells Contribute to the Development of Brain Arteriovenous Malformations in Mice

Abstract

We have previously demonstrated that deletion of activin receptor-like kinase 1 (Alk1) or endoglin in a fraction of endothelial cells (ECs) induces brain arteriovenous malformations (bAVMs) in adult mice upon angiogenic stimulation. Here, we addressed three related questions: (1) could Alk1^- mutant bone marrow (BM)-derived ECs (BMDECs) cause bAVMs? (2) is Alk1^- ECs clonally expended during bAVM development? and (3) is the number of mutant ECs correlates to bAVM severity? For the first question, we transplanted BM from PdgfbiCreER;Alk1^2f/2f mice (EC-specific tamoxifen-inducible Cre with Alk1-floxed alleles) into wild-type mice, and then induced bAVMs by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor and intra-peritoneal injection of tamoxifen. For the second question, clonal expansion was analyzed using PdgfbiCreER;Alk1^2f/2f;confetti^+/- mice. For the third question, we titrated tamoxifen to limit Alk1 deletion and compared the severity of bAVM in mice treated with low and high tamoxifen doses. We found that wild-type mice with PdgfbiCreER;Alk1^2f/2f BM developed bAVMs upon VEGF stimulation and Alk1 gene deletion in BMDECs. We also observed clusters of ECs expressing the same confetti color within bAVMs and significant proliferation of Alk1^- ECs at early stage of bAVM development, suggesting that Alk1^- ECs clonally expanded by local proliferation. Tamoxifen dose titration revealed a direct correlation between the number of Alk1^- ECs and the burden of dysplastic vessels in bAVMs. These results provide novel insights for the understanding of the mechanism by which a small fraction of Alk1 or endoglin mutant ECs contribute to development of bAVMs.

Keywords: Alk1; Arteriovenous malformation; Bone marrow derived endothelial cells; Clonal expansion; Endothelial cells.

Fig. 1.. Mutation of Alk1 in BMDECs caused bAVMs.

A. Study design. BM collected from PdgfbiCreER;Alk1^2f/2f;Ai14^+/− or EGFP transgenic mice were transplanted to lethally irradiated WT mice. AAV-VEGF (2×10⁹ gcs) was injected to the brains of recipients 4 weeks after BM transplantation followed by TM (2.5 mg/25g of body weight) treatment 2 weeks later when a fraction of BM derived cells had differentiated into ECs in the brain angiogenic region to delete Alk1 gene in BMDECs. AVM phenotype was analyzed 6 weeks after TM treatment by latex cast and histology. B. Image of latex perfused brain sections collected from a WT mouse with EGFP BM (WT+WT BM) and a WT mouse with PdgfbiCreER;Alk1^2f/2f;Ai14^+/− BM (WT+Alk1 BM). Right: Enlarged image of the boxed area in the middle picture shows an AVM nidus. Scale bars: 1 mm for left and middle images, 200 μm for the right image. C. Representative images of brain sections. The ECs were stained by intravascular perfused lectin (green). *: dilated bAVM vessels. Scale bars: 100 μm. D. Quantification of vessel density and Dysplasia Index (number of abnormal vessels/200 vessels). Alk1 BM: WT mice transplanted with PdgfbiCreER;Alk1^2f/2f;Ai14^+/− BM. WT BM: WT mice transplanted with EGFP BM. The numbers of dysplasia index were log transformed because they were not normally distributed. N=6. E. Microscopic image shows many dilated vessels (*) in bAVM lesion. ECs were stained by intravascular perfused lectin (green). Recombined BMDECs expressed red fluorescent Ai14 reporter, therefore, they are yellow in the image. The two images on the right show two large vessels indicate by arrowheads in the left image that have clustered BMDECs (Arrows). Scale bar: 50 μm.

Fig. 2.. Alk1^−/− EC clonally expanded in bAVM.

A. Design. B. Confocal images show clusters of ECs expressing same confetti colors in bAVM lesions. Vessels were stained blue using an anti-CD31 antibody. Enlarged images of the rectangle regions in the left pictures show a cluster of RFP⁺ ECs and a cluster of GFP⁺ ECs in groups of abnormal vessels. C. Mouse brain with Alk1 deleted in ECs without VEGF stimulation. D. WT mouse brain with VEGF stimulation. Enlarged images are the squared regions in D and E showing individual Confetti⁺ ECs scattered in vessels. Scale bars: 50 μm.

Fig. 3.. Equal numbers of Alk1⁺ and Alk1⁻ ECs were proliferating in the established bAVMs.

A. Design. B. Images of bAVM sections. The nuclei of ECs were stained by an Erg antibody (red). Proliferating cells were stained by a Ki67 antibody (green). Alk1 expression was stained by an Alk1 antibody (purple). Arrow: a Ki67⁺ Alk1⁺ EC. Arrowhead: a Ki67⁺ Alk1⁻ EC. Scale bars=50 μm. B. Quantification of Ki67⁺ Alk1⁻ and Alk1⁺ ECs. N=6.

Fig. 4:. Increase of TM dose increased Ai14⁺ and decreased Alk1⁺ ECs.

A. Design. TM 0.01/1.25: mice treated with 0.01 or 1.25 mg/25g of body weight TM. Day 22: samples were collected 22 days after intra-brain injection of AAV-VEGF from mice treated with high dose TM (1.25 mg/25g); Day 28: samples were collected 28 days after intra-brain injection of AAV-VEGF from mice treated with the low dose TM (0.01 mg/25g). B. Representative images of brain sections collected from lectin perfused mice. ECs were stained by intravascular perfused lectin (green). Cre activation in ECs was indicated by the expression of Ai14 reporter (red). Arrows indicate dysplastic vessels. Scale bars: 50 μm. The right-side pictures show enlarged images of abnormal vessels indicated by arrows in the pictures on their left side. C. Quantification of Ai14⁺ ECs in the bAVM lesions. D. Representative images of brain sections co-stained with anti-CD31 (green, ECs) and anti-Alk1 (red) antibodies. Arrows indicate abnormal vessels. Scale bar: 80 μm. The right-side pictures show enlarged images of abnormal vessels indicated by arrows in the pictures on their left side. E. Quantification of Alk1⁺ ECs. WT: corn oil treated mice; 0.01 or 1.25: mice treated with 0.01 mg/25g or 1.25 mg/25g TM. N=6.

Fig. 5.. Reduction of the TM dose reduced the number of dysplastic vessels in bAVM lesions.

A. Representative images of brain sections. ECs were stained by intravascular perfused lectin (green). Arrows indicate dysplastic vessels. Scale bar: 80 μm. B. Quantification of the Dysplasia Index. C. Quantification of vessel density. Vehicle: corn oil treated mice; 0.01 or 1.25: mice treated with 0.01 mg/25g or 1.25mg/25g TM. N=7 for the Vehicle group; N=6 for the 0.01 and 1.25 TM groups.

Fig. 6.. Reduction of TM dose reduced the number of AV shunts in the intestines and mouse mortality.

A. Representative images show AV shunts in the intestines. The vessels were casted with latex dye (blue). Latex dye entered some veins of mice treated with 0.01 and 1.25 mg/25g TM after being injected into intra-left cardiac ventricles. Arrows indicate an artery (A) and a vein (V). Scale bar: 1 mm. B. Quantification of AV shunts. 0.01 and 1.25: mice treated with 0.01 or 1.25 mg/25g TM. Vehicle: mice treated with corn oil. C. The survival curve. Mice were treated with one i.p. injection of 1.25, 0.75, 0.3, or 0.1 mg/25g of body weight TM. N=6.