Supplementary Material for: Minimal Homozygous Endothelial Deletion of Eng with VEGF Stimulation Is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse

<b><i>Background:</i></b> 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 (<i>ENG, </i>HHT1) or activin receptor-like kinase 1 (<i>ALK1, </i>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 <i>Alk1</i> deletion plus vascular endothelial growth factor (VEGF) stimulation. We hypothesized that <i>Eng</i> deletion plus VEGF stimulation would induce a similar degree of cerebrovascular dysplasia as the <i>Alk1</i>-deleted brain. <b><i>Methods:</i></b> 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 <i>Eng</i><sup>2f/2f</sup> (exons 5 and 6 flanked by loxP sequences), <i>Alk1</i><sup>2f/2f</sup> (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. <b><i>Results:</i></b> AAV-VEGF induced a similar degree of angiogenesis in the brain with or without <i>Alk1</i>- or <i>Eng</i>-deletion. Abnormally patterned and dilated dysplastic vessels were found in the viral vector-injected region of <i>Alk1</i><sup>2f/2f</sup> and <i>Eng</i><sup>2f/2f</sup> brain sections, but not in WT. <i>Alk1</i><sup>2f/2f</sup> mice had about 1.8-fold higher dysplasia index than <i>Eng</i><sup>2f/2f</sup> 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 (<i>Alk1</i><sup>2f/2f</sup>: 16% and <i>Eng</i><sup>2f/2f</sup>: 1%), we found that about 8-fold higher dysplasia was induced per copy of <i>Eng</i> deletion (2.5) than that of <i>Alk1</i> deletion (0.3). ENG-negative endothelial cells were detected in the Ad-Cre-treated brain of <i>Eng</i><sup>2f/2f</sup> mice, suggesting homozygous deletion of <i>Eng</i> in the cells. VEGF induced more severe vascular dysplasia in the Ad-Cre-treated brain of <i>Eng</i><sup>2f/2f</sup> mice than that of <i>Eng</i><sup>+/–</sup> mice. <b><i>Conclusions:</i></b> (1) Deletion of <i>Eng</i> induces more severe cerebrovascular dysplasia per copy than that of <i>Alk1</i> upon VEGF stimulation. (2) Homozygous deletion of <i>Eng</i> 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.