Minimal homozygous endothelial deletion of Eng with VEGF stimulation is sufficient to cause cerebrovascular dysplasia in the adult mouse

Background: Brain arteriovenous malformations (bAVMs) represent a high risk for hemorrhagic stroke, leading to significant neurological morbidity and mortality in young adults. The etiopathogenesis of bAVM remains unclear. Research progress has been hampered by the lack of animal models. Hereditary Hemorrhagic Telangiectasia (HHT) patients with haploinsufficiency of endoglin (ENG, HHT1) or activin receptor-like kinase 1 (ALK1, HHT2) have a higher incidence of bAVM than the general population. We previously induced cerebrovascular dysplasia in the adult mouse that resembles human bAVM through Alk1 deletion plus vascular endothelial growth factor (VEGF) stimulation. We hypothesized that Eng deletion plus VEGF stimulation would induce a similar degree of cerebrovascular dysplasia as the Alk1-deleted brain.

Methods: Ad-Cre (an adenoviral vector expressing Cre recombinase) and AAV-VEGF (an adeno-associated viral vector expressing VEGF) were co-injected into the basal ganglia of 8- to 10-week-old Eng(2f/2f) (exons 5 and 6 flanked by loxP sequences), Alk1(2f/2f) (exons 4-6 flanked by loxP sequences) and wild-type (WT) mice. Vascular density, dysplasia index, and gene deletion efficiency were analyzed 8 weeks later.

Results: AAV-VEGF induced a similar degree of angiogenesis in the brain with or without Alk1- or Eng-deletion. Abnormally patterned and dilated dysplastic vessels were found in the viral vector-injected region of Alk1(2f/2f) and Eng(2f/2f) brain sections, but not in WT. Alk1(2f/2f) mice had about 1.8-fold higher dysplasia index than Eng(2f/2f) mice (4.6 ± 1.9 vs. 2.5 ± 1.1, p < 0.05). However, after normalization of the dysplasia index with the gene deletion efficiency (Alk1(2f/2f): 16% and Eng(2f/2f): 1%), we found that about 8-fold higher dysplasia was induced per copy of Eng deletion (2.5) than that of Alk1 deletion (0.3). ENG-negative endothelial cells were detected in the Ad-Cre-treated brain of Eng(2f/2f) mice, suggesting homozygous deletion of Eng in the cells. VEGF induced more severe vascular dysplasia in the Ad-Cre-treated brain of Eng(2f/2f) mice than that of Eng(+/-) mice.

Conclusions: (1) Deletion of Eng induces more severe cerebrovascular dysplasia per copy than that of Alk1 upon VEGF stimulation. (2) Homozygous deletion of Eng with angiogenic stimulation may be a promising strategy for development of a bAVM mouse model. (3) The endothelial cells that have homozygous causal gene deletion in AVM could be crucial for lesion development.