Rbpj Deficiency Disrupts Vascular Remodeling via Abnormal Apelin and Cdc42 (Cell Division Cycle 42) Activity in Brain Arteriovenous Malformation.

BACKGROUND: Brain arteriovenous malformations (bAVM) are characterized by enlarged blood vessels, which direct blood through arteriovenous shunts, bypassing the artery-capillary-vein network and disrupting blood flow. Clinically, bAVM treatments are invasive and not routinely applicable. There is critical need to understand mechanisms of bAVM pathologies and develop pharmacological therapies. METHODS: We used an in vivo mouse model of Rbpj-mediated bAVM, which develops pathologies in the early postnatal period and an siRNA in vitro system to knockdown RBPJ in human brain microvascular endothelial cells (ECs). To understand molecular events regulated by endothelial Rbpj, we conducted RNA-Seq and chromatin immunoprecipitation-Seq analyses from isolated brain ECs. RESULTS: Rbpj-deficient (mutant) brain ECs acquired abnormally rounded shape (with no change to cell area), altered basement membrane dynamics, and increased endothelial cell density along arteriovenous shunts, compared to controls, suggesting impaired remodeling of neonatal brain vasculature. Consistent with impaired endothelial cell dynamics, we found increased Cdc42 (cell division cycle 42) activity in isolated mutant ECs, suggesting that Rbpj regulates small GTPase (guanosine triphosphate hydrolase)-mediated cellular functions in brain ECs. siRNA-treated, RBPJ-deficient human brain ECs displayed increased Cdc42 activity, disrupted cell polarity and focal adhesion properties, and impaired migration in vitro. RNA-Seq analysis from isolated brain ECs identified differentially expressed genes in mutants, including Apelin, which encodes a ligand for G protein-coupled receptor signaling known to influence small GTPase activity. Chromatin immunoprecipitation-Seq analysis revealed chromatin loci occupied by Rbpj in brain ECs that corresponded to G-protein and Apelin signaling molecules. In vivo administration of a competitive peptide antagonist against the Apelin receptor (Aplnr/Apj) attenuated Cdc42 activity and restored endothelial cell morphology and arteriovenous connection diameter in Rbpj-mutant brain vessels. CONCLUSIONS: Our data suggest that endothelial Rbpj promotes rearrangement of brain ECs during cerebrovascular remodeling, through Apelin/Apj-mediated small GTPase activity, and prevents bAVM. By inhibiting Apelin/Apj signaling in vivo, we demonstrated pharmacological prevention of Rbpj-mediated bAVM.

Pericytes and vascular smooth muscle cells in central nervous system arteriovenous malformations.

Previously considered passive support cells, mural cells-pericytes and vascular smooth muscle cells-have started to garner more attention in disease research, as more subclassifications, based on morphology, gene expression, and function, have been discovered. Central nervous system (CNS) arteriovenous malformations (AVMs) represent a neurovascular disorder in which mural cells have been shown to be affected, both in animal models and in human patients. To study consequences to mural cells in the context of AVMs, various animal models have been developed to mimic and predict human AVM pathologies. A key takeaway from recently published work is that AVMs and mural cells are heterogeneous in their molecular, cellular, and functional characteristics. In this review, we summarize the observed perturbations to mural cells in human CNS AVM samples and CNS AVM animal models, and we discuss various potential mechanisms relating mural cell pathologies to AVMs.

Pathological pericyte expansion and impaired endothelial cell-pericyte communication in endothelial Rbpj deficient brain arteriovenous malformation.

Pericytes, like vascular smooth muscle cells, are perivascular cells closely associated with blood vessels throughout the body. Pericytes are necessary for vascular development and homeostasis, with particularly critical roles in the brain, where they are involved in regulating cerebral blood flow and establishing the blood-brain barrier. A role for pericytes during neurovascular disease pathogenesis is less clear-while some studies associate decreased pericyte coverage with select neurovascular diseases, others suggest increased pericyte infiltration in response to hypoxia or traumatic brain injury. Here, we used an endothelial loss-of-function Recombination signal binding protein for immunoglobulin kappa J region (Rbpj)/Notch mediated mouse model of brain arteriovenous malformation (AVM) to investigate effects on pericytes during neurovascular disease pathogenesis. We tested the hypothesis that pericyte expansion, via morphological changes, and Platelet-derived growth factor B/Platelet-derived growth factor receptor ß (Pdgf-B/Pdgfrß)-dependent endothelial cell-pericyte communication are affected, during the pathogenesis of Rbpj mediated brain AVM in mice. Our data show that pericyte coverage of vascular endothelium expanded pathologically, to maintain coverage of vascular abnormalities in brain and retina, following endothelial deletion of Rbpj. In Rbpj-mutant brain, pericyte expansion was likely attributed to cytoplasmic process extension and not to increased pericyte proliferation. Despite expanding overall area of vessel coverage, pericytes from Rbpj-mutant brains showed decreased expression of Pdgfrß, Neural (N)-cadherin, and cluster of differentiation (CD)146, as compared to controls, which likely affected Pdgf-B/Pdgfrß-dependent communication and appositional associations between endothelial cells and pericytes in Rbpj-mutant brain microvessels. By contrast, and perhaps by compensatory mechanism, endothelial cells showed increased expression of N-cadherin. Our data identify cellular and molecular effects on brain pericytes, following endothelial deletion of Rbpj, and suggest pericytes as potential therapeutic targets for Rbpj/Notch related brain AVM.