Myeloid immune cells present abundantly in both ruptured and unruptured brain arteriovenous malformations (bAVMs). The role of central nervous system (CNS) resident and circulating monocyte-derived macrophages in bAVM pathogenesis has not been fully understood. RNA sequencing using cultured cells and bAVM samples revealed that downregulation of activin-like kinase 1 (ALK1) or endoglin (two bAVM causative genes) increased pro-angiogenic, endothelial inflammation and innate immune signaling, which provided endogenous underpinnings of the active inflammation in bAVM. To further understand the role of CNS resident macrophages in bAVM development and hemorrhage, we administrated a colony-stimulating factor 1 receptor (CSF1R) inhibitor to bAVM mice with endothelial Alk1 deletion. Transient depletion of CNS resident macrophages at early stage of bAVM development remarkably mitigated the subsequent phenotype severity of bAVM. This therapeutic effect exhibited a prolonged inhibition of angiogenesis, dysplastic vasculature formation, and infiltration of CNS resident and circulating monocyte-derived macrophages during bAVM development. Transient depletion of CNS resident macrophages also reduced the dysplasia vessels and improved the integrity of endothelial tight junctions in established bAVMs. Administration of CSF1R inhibitor also prevented severe hemorrhage of bAVMs. Thus, endothelial AVM causative gene mutation can activate CNS resident macrophages promoting bAVM progression. CNS resident macrophages could be specific targets to mitigate the development and severity of bAVMs.

Myeloid immune cells are abundant in both ruptured and unruptured brain arteriovenous malformations (bAVMs). The role of central nervous system (CNS) resident and circulating monocyte-derived macrophages in bAVM pathogenesis has not been fully understood. We hypothesize that CNS resident macrophages enhance bAVM development and hemorrhage. RNA sequencing using cultured endothelial cells (ECs) and mouse bAVM samples revealed that downregulation of two bAVM causative genes, activin-like kinase 1 (ALK1) or endoglin, increased inflammation and innate immune signaling. To understand the role of CNS resident macrophages in bAVM development and hemorrhage, we administrated a colony-stimulating factor 1 receptor inhibitor to bAVM mice with brain focal Alk1 deletion. Transient depletion of CNS resident macrophages at an early stage of bAVM development mitigated the phenotype severity of bAVM, including a prolonged inhibition of angiogenesis, dysplastic vasculature formation, and infiltration of CNS resident and circulating monocyte-derived macrophages during bAVM development. Transient depletion of CNS resident macrophages increased EC tight junction protein expression, reduced the number of dysplasia vessels and severe hemorrhage in established bAVMs. Thus, EC AVM causative gene mutation can activate CNS resident macrophages promoting bAVM progression. CNS resident macrophage could be a therapeutic target to mitigate the development and severity of bAVMs.

Endoglin (ENG) mutation causes type 1 hereditary hemorrhagic telangiectasia (HHT1). HHT1 patients have arteriovenous malformations (AVMs) in multiple organs, including the brain. In mice, Eng deletion induced by R26RCreER or SM22αCre leads to AVM development in the brain and other organs. We hypothesized that an increase in Eng- negative ECs will enhance AVM severity. To increase EC Eng deletion, we used a codon-improved cre (icre), which is more potent in recombination of the floxed alleles than the wild-type (WT) cre. R26RCreER;Engf/f mice that have a Rosa promoter driving and tamoxifen (TM)-inducible WT cre expression globally, and PdgfbiCreER;Engf/f mice that have a Pdgfb promoter driving and TM-inducible icre expression in ECs were treated with three intra-peritoneal injections of TM (2.5 mg/25 g of body weight) to delete Eng globally or in the ECs. AAV-VEGF was stereotactically injected into the brain to induce brain focal angiogenesis and brain AVM. We found that icre caused more Eng deletion in the brain, indicated by a lower level of Eng proteins (p < 0.001) and fewer Eng-positive ECs (p = 0.01) than mice with WT cre. Mice with icre-mediated Eng deletion have more abnormal vessels (p = 0.02), CD68+ macrophages (p = 0.002), and hemorrhage (p = 0.04) and less vascular pericyte and smooth muscle coverage than mice with WT cre. In addition, arteriovenous shunts were detected in the intestines of icre mice, a phenotype that has not been detected in WT cre mice before. RNA-seq analysis showed that 8 out of the 10 top upregulated pathways identified by gene ontology (GO) analysis are related to inflammation. Therefore, the increase in Eng deletion in ECs exacerbates AVM severity, which is associated with enhanced inflammation. Strategies that can reduce Eng-negative ECs could be used to develop new therapies to reduce AVM severity for HHT1 patients.

Background:

Increase of collagen I (COL I)/COL III ratio enhances vessel wall stiffness and renders vessels less resistant to blood flow and pressure changes. We hypothesized that COL I/COL III ratio is increased in the brain arteriovenous malformations (bAVMs), which is associated with bAVM hemorrhage.

Method:

Surgically resected human bAVM samples and mouse bAVMs induced in three transgenic mouse lines with activin receptor-like kinase 1 or endoglin deletion in the endothelial cells or in the brain focally. Colony-stimulating factor receptor inhibitor was used to transiently deplete microglia. COL I and COL III levels, hemorrhage, and gene expression in bAVMs were analyzed.

Results:

The COL I and COL III levels and the COL I/COL III ratio were higher on human and mouse bAVMs than controls, which was associated with bAVM hemorrhage. Col I and Col III mRNAs were also increased in mouse bAVMs. The degree of microhemorrhage in mouse bAVMs was positively correlated with Col I/Col III ratios. Transient depletion of microglia reduced Col I/Col III ratio and microhemorrhage in mouse bAVMs.

Conclusion:

COL I/COL III ratio are increased in bAVM vessels, which is associated with bAVM hemorrhage. Depletion of microglia reduces Col I/Col III ratio and hemorrhage in mice bAVMs.

Background: The increase in the collagen I (COL I)/COL III ratio enhances vessel wall stiffness and renders vessels less resistant to blood flow and pressure changes. Activated microglia enhance inflammation-induced fibrosis. Hypotheses: The COL I/COL III ratio in human and mouse brain arteriovenous malformations (bAVMs) is associated with bAVM hemorrhage, and the depletion of microglia decreases the COL I/COL III ratio and hemorrhage. Method: COL I, COL III, and hemorrhages were analyzed in 12 human bAVMs and 6 control brains, and mouse bAVMs induced in three mouse lines with activin receptor-like kinase 1 (n = 7) or endoglin (n = 7) deleted in the endothelial cells or brain focally (n = 5). The controls for the mouse study were no-gene-deleted litter mates. Mouse bAVMs were used to test the relationships between the Col I/Col III ratio and hemorrhage and whether the transient depletion of microglia reduces the Col I/Col III ratio and hemorrhage. Results: The COL I/COL III ratio was higher in the human and mouse bAVMs than in controls. The microhemorrhage in mouse bAVMs was positively correlated with the Col I/Col III ratio. Transient depletion of microglia reduced the Col I/Col III ratio and microhemorrhage. Conclusions: The COL I/COL III ratio in the bAVMs was associated with bAVM hemorrhage. The depletion of microglia reduced the bAVM Col I/Col III ratio and hemorrhage.