Impaired neurovascular remodeling mediated by Apelin signaling and Cdc42 activity in endothelial Rbpj deficient brain arteriovenous malformation [ChIP-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. Chromatin immunoprecipitation was performed against Rbpj (Cell Signaling 1:50, #5313) from brain endothelial cells (ECs) isolated from P7 FVB/NJ mice (The Jackson Laboratories Strain 001800). Pooled isolated brain ECs from 15 biological samples per ChIP cohort were crosslinked at a final concentration of 1% formaldehyde for 10 min at RT, and crosslinking reaction quenched with glycine solution, for 5 min at RT. Nuclei from lysed cell were isolated and chromatin was digested around nucleosomal boundaries using Micrococcal nuclease (Cell Signaling). Additionally, MNase digested DNA was sonicated with a probe sonicator (20 kHz, Fisherbrand) for three sets of 10 sec pulses, at 20% amplitude. Sheared DNA fragments were electrophoresed on a 1.5% agarose gel to check DNA fragment size, ideally between 150- 800 base pairs for IP. 5 μg chromatin preparation was used for immunoprecipitation reaction overnight with rotation, at 4°C. Protein-G agarose beads (Active Motif) were added for 2 hrs with rotation, at 4°C to pulldown chromatin-Rbpj IP reaction. Bound DNA was eluted from antibody-Protein G beads and formaldehyde crosslinks were reversed at 65°C in buffer containing 5 M NaCl, 0.5 M EDTA and Proteinase K. Recovered nucleotides were treated with RNase A, and DNA was purified using silica membrane spin columns (Cell Signaling). IP-DNA and 2% Input samples were paired-end sequenced in duplicates on a NovaSeq 6000 platform.