Wanqiu Chen

Arteriovenous malformation in the adult mouse brain resembling the human disease

Brain arteriovenous malformations (bAVMs) are an important cause of hemorrhagic stroke. The underlying mechanisms are not clear. No animal model for adult bAVM is available for mechanistic exploration. Patients with hereditary hemorrhagic telangiectasia type 2 (HHT2) with activin receptor‐like kinase 1 (ALK1; ACVRL1) mutations have a higher incidence of bAVM than the general population. We tested the hypothesis that vascular endothelial growth factor (VEGF) stimulation with regional homozygous deletion of Alk1 induces severe dysplasia in the adult mouse brain, akin to human bAVM.

Novel Brain Arteriovenous Malformation Mouse Models for Type 1 Hereditary Hemorrhagic Telangiectasia

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 Eng2fl/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 Eng2fl/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.

De Novo Cerebrovascular Malformation in the Adult Mouse After Endothelial Alk1 Deletion and Angiogenic Stimulation

Background and Purpose— In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor stimulation induces brain AVMs in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal vascular endothelial growth factor stimulation is sufficient to induce brain AVM in the adult mouse. Methods— Focal angiogenesis was induced in the brain of 8-week-old Pdgfb-iCreER;Alk12f/2f mice by injection of adeno-associated viral vectors expressing vascular endothelial growth factor. Two weeks later, EC-Alk1 deletion was induced by tamoxifen treatment. Vascular morphology was analyzed, and EC proliferation and dysplasia index (number of vessels with diameter >15 &mgr;m per 200 vessels) were quantified 10 days after tamoxifen administration. Results— Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of tamoxifen-treated Pdgfb-iCreER;Alk12f/2f mice. Induced brain AVMs were marked by increased dysplasia index (P<0.001) and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs. Conclusions— Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo brain AVM.

Reduced Mural Cell Coverage and Impaired Vessel Integrity After Angiogenic Stimulation in the Alk1-deficient Brain

Objective—Vessels in brain arteriovenous malformations are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of brain arteriovenous malformation than the general population. We tested the hypothesis that vascular endothelial growth factor impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell coverage. Methods and Results—Adult Alk11f/2f mice (loxP sites flanking exons 4–6) and wild-type mice were injected with 2×107 PFU adenovious-cre recombinase and 2×109 genome copies of adeno-associated virus-vascular endothelial growth factor to induce focal homozygous Alk1 deletion (in Alk11f/2f mice) and angiogenesis. Brain vessels were analyzed 8 weeks later. Compared with wild-type mice, the Alk1-deficient brain had more fibrin (99±30×103 pixels/mm2 versus 40±13×103; P=0.001), iron deposition (508±506 pixels/mm2 versus 6±49; P=0.04), and Iba1+ microglia/macrophage infiltration (888±420 Iba1+ cells/mm2 versus 240±104 Iba1+; P=0.001) after vascular endothelial growth factor stimulation. In the angiogenic foci, the Alk1-deficient brain had more &agr;-smooth muscle actin negative vessels (52±9% versus 12±7%, P<0.001), fewer vascular-associated pericytes (503±179/mm2 versus 931±115, P<0.001), and reduced platelet-derived growth factor receptor-&bgr; expression. Conclusion—Reduction of mural cell coverage in response to vascular endothelial growth factor stimulation is a potential mechanism for the impairment of vessel wall integrity in hereditary hemorrhagic telangiectasia type 2-associated brain arteriovenous malformation.

Induction of brain arteriovenous malformation in the adult mouse.

Brain arteriovenous malformations (bAVM) are tangles of abnormal, dilated vessels that directly shunt blood between the arteries and veins. The pathogenesis of bAVM is currently unknown. Patients with hereditary hemorrhagic telangiectasia (HHT) have a higher prevalence of bAVM than the general population. Animal models are important tools for dissecting the disease etiopathogenesis and for testing new therapies. Here, we introduce a method that induces the bAVM phenotype through regional deletion of activin-like kinase 1 (Alk1, the causal gene for HHT2) and vascular endothelial growth factor (VEGF) stimulation.

Thalidomide Reduces Hemorrhage of Brain Arteriovenous Malformations in a Mouse Model

Background and Purpose— Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model. Methods— bAVMs were induced through induction of brain focal activin-like kinase 1 (Alk1, an AVM causative gene) gene deletion and angiogenesis in adult Alk1-floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain. Results— Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68+ cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide. Conclusions— Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression.