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

Abstract

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.

Common and Distinctive Pathogenetic Features of Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia 1 and Hereditary Hemorrhagic Telangiectasia 2 Animal Models—Brief Report

“…It might be rooted in the difference of the recombination efficiency of each gene by Cre. 15 Alternatively, ECs deficient in each gene may respond differently to environmental cues generated by the wound.…”

Endovascular Management of Cerebral Arteriovenous Malformations in Pregnancy: Two Case Reports and a Review of the Literature

“…This vascular lesion is prevalent in approximately 0.01% of the general population, affecting both men and women at about the same rate, and is quite severe with detrimental complications [1][2][3][4][5]. It has been stated that the development of an AVM might be the effect of one or several triggers on a gene defect of the post-capillary endothelium [6,7]. Others have proposed the idea that an AVM might arise through a pathological hemodynamic response to exogenous factors, such as trauma [2].…”

Angiogenic Stimuli and Endoglin Absence Induces Brain Arteriovenous Malformations: Are Local Endoglin Deletion and Angiogenesis the ‘Second Hit’ That Is Necessary for Arteriovenous Malformations Formation in HHT-1?

“…In addition, the study of Choi et al [5] gives a new support to the concept that HHT-related AVMs formation requires a second hit, probably an angiogenic stimulus. These studies also reveal that complete deletion of endoglin in a small number of endothelial cells is sufficient to cause brain AVMs after angiogenic stimulation.…”

“…In the present issue of Cerebrovascular Diseases , Choi et al [5] assessed the relative roles of endoglin and ALK-1 insufficiency in brain malformations induced by the local deletion of endoglin or ALK-1 using the Cre/loxP system in mice and the injection into the basal ganglia of adenoviral vectors with cytomegalovirus promoter-driving Cre recombinase and the simultaneous activation of focal angiogenesis by injecting adeno-associated viral vectors with cytomegalovirus promoter-driving VEGF. Using this ingenious approach, they obtained focal areas of irregularly dilated vessels, arteriovenous shunting, and inflammatory cell infiltration, aspects resembling human brain AVM [5].…”

“…Using this ingenious approach, they obtained focal areas of irregularly dilated vessels, arteriovenous shunting, and inflammatory cell infiltration, aspects resembling human brain AVM [5]. Interestingly, there were more dysplastic vessels formed per copy of endoglin deletion than that of ALK-1, thus suggesting that more severe brain dysplasia is associated to endoglin insufficiency.…”