Novel brain arteriovenous malformation mouse models for type 1 hereditary hemorrhagic telangiectasia

Abstract

Endoglin (ENG) is a causative gene of type 1 hereditary hemorrhagic telangiectasia (HHT1). HHT1 patients have a higher prevalence of brain arteriovenous malformation (AVM) than the general population and patients with other HHT subtypes. The pathogenesis of brain AVM in HHT1 patients is currently unknown and no specific medical therapy is available to treat patients. Proper animal models are crucial for identifying the underlying mechanisms for brain AVM development and for testing new therapies. However, creating HHT1 brain AVM models has been quite challenging because of difficulties related to deleting Eng-floxed sequence in Eng(2fl/2fl) mice. To create an HHT1 brain AVM mouse model, we used several Cre transgenic mouse lines to delete Eng in different cell-types in Eng(2fl/2fl) mice: R26CreER (all cell types after tamoxifen treatment), SM22α-Cre (smooth muscle and endothelial cell) and LysM-Cre (lysozyme M-positive macrophage). An adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) was injected into the brain to induce focal angiogenesis. We found that SM22α-Cre-mediated Eng deletion in the embryo caused AVMs in the postnatal brain, spinal cord, and intestines. Induction of Eng deletion in adult mice using R26CreER plus local VEGF stimulation induced the brain AVM phenotype. In both models, Eng-null endothelial cells were detected in the brain AVM lesions, and formed mosaicism with wildtype endothelial cells. However, LysM-Cre-mediated Eng deletion in the embryo did not cause AVM in the postnatal brain even after VEGF stimulation. In this study, we report two novel HHT1 brain AVM models that mimic many phenotypes of human brain AVM and can thus be used for studying brain AVM pathogenesis and testing new therapies. Further, our data indicate that macrophage Eng deletion is insufficient and that endothelial Eng homozygous deletion is required for HHT1 brain AVM development.

Figure 1. Developmental onset AVM in the postnatal brain of Eng^2fl/2fl;SM22α-Cre mice.

Representative images of latex dye casting show the cerebrovasculature in the brain of 5-week-old (A) Eng^2fl/2fl, (B) Eng^+/2fl;SM22α-Cre, and (C) Eng^2fl/2fl;SM22α-Cre mice. D–F: Enlarged images of dotted boxes shown in A–C after the brain tissue was cleared in organic solvent. The latex-perfused vasculature inside the brain can be observed. Tangled and dilated vessels were detected only in the brain of Eng^2fl/2fl;SM22α-Cre mice. Scale bars: 1 mm in A–C and 500 µm in D–F. G: Survival curve of Eng^2fl/2fl;SM22α-Cre mice. There was no difference between male (M) and female (F) mice (P = 0.47).

Figure 2. Adult onset brain AVM in Eng^2fl/2fl;R26CreER mice after TM and VEGF treatment.

A: Coronal view of latex-perfused adult Eng^2fl/2fl;R26CreER brain showing no AVM phenotype 8 weeks after TM treatment. B: AVM phenotype (white arrow) developed in the brain of Eng^2fl/2fl;R26CreER mice 8 weeks after intrabrain injection of AAV-VEGF and intraperitoneal injection of TM. C: An enlarged image of the AVM lesion. Latex-perfused veins are clearly shown at the top and bottom of the lesion (black arrows). D: Enlarged vessels observed in a 50 µm frozen section that cuts through the AVM lesion shown in C. E: Quantification of dysplasia index. Data: mean ± SD. *: p<0.05. n = 6 per group. Scale bars: 1 mm in A and B and 500 µm in C and D.

Figure 3. AVM phenotype was not detected in the brain angiogenic region of Eng^2fl/2fl;LysM-Cre mice.

Representative images of the latex-casted cerebrovasculature in (A) Eng^2fl/2fl and (B) Eng^2fl/2fl;LysMCre adult mice 8 weeks after focal VEGF stimulation. C and D: High magnification view of the angiogenic foci (arrows) shown in A and B. Latex was present in arteries only. No vein and enlarged and tangled vessels were detected. Scale bars: 1 mm in A and B and 500 µm in C and D.

Figure 4. ENG-null endothelial cells in dysplastic vessels.

Representative images of lectin-stained brain sections from (A) AVM lesion of 5-week-old Eng^2fl/2fl;SM22α-Cre, (B) VEGF-induced angiogenic focus of TM-treated adult Eng^2fl/2fl;R26CreER, and (C) VEGF-stimulated angiogenic focus of adult Eng^2fl/2fl;LysM-Cre mice. ENG expression in (D) Eng^2fl/2fl;SM22α-Cre, (E) Eng^2fl/2fl;R26CreER, and (F) Eng^2fl/2fl;LysM-Cre brain. G-I: Enlarged images of the dotted boxes shown in D-F. Arrows indicate ENG-negative endothelial cells. Scale bars: 100 µm in A–F and 10 µm in G–I.

Figure 5. Macrophage infiltration and microhemorrhage in brain AVM lesions of Eng^2fl/2fl;SM22αCre and Eng^2fl/2fl;R26CreER mice.

Dysplastic vessels were present in the brain of (A) Eng^2fl/2fl;SM22α-Cre and (B) Eng^2fl/2fl;R26CreER mice. Endothelial cells were visualized by immunostaining using an antibody specific to CD31 (an endothelial cell-specific marker). C and D: Macrophages were detected by immunostaining using a CD68-specific antibody. The nuclei were stained by DAPI. E and F: Macrophages localized around large dysplastic vessels. G and H: Iron deposits, an indicator of hemorrhage, identified by Prussian blue. Scale bar: 100 µm.