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.

Figure 1. Dysplastic cerebral vasculature was detected in the Ad-Cre and AAV-VEGF-injected brain of Alk1^2f/2f and Eng^2f/2f mice

(A) Diagram indicates the viral vector injection site (gray square). (B) Representative images of the microfil-perfused brain. Injection of Ad-Cre with AAV-VEGF to the WT brain caused focal normal angiogenesis, while it induced localized dysplastic vessel formation in the Alk1^2f/2f and Eng^2f/2f brain (arrows). (C) Representative images of lectin-perfused brain sections. Dysplastic vessels were observed in the Ad-Cre/AAV-VEGF-injected Alk1^2f/2f and Eng^2f/2f brain (arrows), but not in the similarly treated WT brain. Scale bars are 100 µm (B) and 50 µm (C).

Figure 2. Injection of Ad-Cre and AAV-VEGF increased cerebrovascular density and induced cerebrovascular dysplasia in Alk1^2f/2f and Eng^2f/2f mice

(A) Quantification of vascular density in the brain of WT mice injected with AAV-VEGF or AAV-lacZ. (B) Bar graph shows the mean vascular densities in the brain of WT, Alk1^2f/2f, and Eng^2f/2f mice injected with Ad-Cre and AAV-VEGF. (C) Bar graph shows dysplasia index. LacZ: AAV-lacZ; VEGF: AAV-VEGF; WT, Alk1, and Eng: WT, Alk1^2f/2f, and Eng^2f/2f mice treated with Ad-Cre and AAV-VEGF. *: p < 0.05 and #: p < 0.001. n=6 per group.

Figure 3. Injection of Ad-Cre mediated target gene deletion in the viral vector-injected region of the adult mouse brain

(A) Representative image of Ad-Cre-treated R26R mouse brain sections. The vessels were perfused with lectin and lacZ expression was detected by immunostaining. Deletion of the floxed allele was observed in endothelial cells (arrow). (B) Representative image of Ad-Cre-treated Eng^2f/2f mouse brain sections shows enlarged dysplastic vessels. ENG expression was detected by immunostaining. Hematoxylin was used for counterstaining. A mosaic pattern of ENG expression was detected in the endothelial cells lining this vessel; majority of endothelial cells expressed ENG, two endothelial cells were ENG negative (arrow). Scale bars are 20 µm.

Figure 4. Ad-Cre mediated more effective deletion of the Alk1-floxed allele than Eng-floxed allele in the adult mouse brain

(A) Bar graph shows the amounts of Alk1- and Eng-floxed alleles and Mmp-9 in tail genomic DNA. The mean value of Mmp-9 was set as a value of one. Values of Alk1- and Eng-floxed alleles were presented as the ratios to Mmp-9. (B) Bar graph shows percentage of deleted Alk1- and Eng-floxed alleles in the Ad-Cre-injected brain of Alk^2f/2f and Eng^2f/2f mice. (C) Bar graph shows dysplasia per percentage of gene deletion. n=3 per group in (A) and (B) and n=6 per group in (C).