Impaired neurovascular remodeling mediated by Apelin signaling and Cdc42 activity in endothelial Rbpj deficient brain arteriovenous malformation [RNA-seq]

Brain arteriovenous malformations (bAVM) are characterized by enlarged blood vessels, which direct blood through arteriovenous AV 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. 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 ChIP-Seq analyses from isolated brain ECs. Rbpj-deficient (mutant) brain ECs acquired abnormally rounded shape (with no change to cell area), altered basement membrane dynamics, and increased EC density along AV shunts, compared to controls, suggesting impaired remodeling of neonatal brain vasculature. Consistent with impaired EC dynamics, we found increased Cdc42 activity in isolated mutant ECs, suggesting that Rbpj regulates small GTPase-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. ChIP-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 EC morphology and AV 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. Overall design: Differential gene expression anlayses from isolated brain endothelial cells from P7 Cdh5(PAC)-CreERT2; Rbpjflox/WT (control) and Cdh5(PAC)-CreERT2; Rbpjflox/flox (mutant) mice in experimental triplicates (total 6 samples). Each experimental cohort included 13-15 biological samples from >3 independent litters.