NINJ1 hypomethylation enhances endothelial dysfunction in brain arteriovenous malformations

Brain arteriovenous malformations (bAVMs) are a major cause of intracranial haemorrhage in children and young adults, yet the molecular basis of endothelial dysfunction and vascular fragility remains incompletely understood. Although somatic activation of the KRAS-MAPK pathway has been implicated in bAVM pathogenesis, the contribution of epigenetic dysregulation has not been systematically defined. We performed genome-wide DNA methylation profiling using the Illumina Infinium MethylationEPIC v2 (935K) array on human bAVM nidus tissues, followed by targeted pyrosequencing and immunofluorescence validation. Functional and mechanistic studies were conducted in human brain microvascular endothelial cells, complemented by transcriptomic analyses and in vivo assessment in an endothelial-specific zebrafish model. Genome-wide methylation analysis revealed a distinct promoter-centric epigenetic landscape in bAVM tissue, with enrichment of genes involved in cell adhesion, angiogenesis and inflammatory signalling. Among these, Nerve Injury-Induced Protein 1 (NINJ1) emerged as a prominently hypomethylated gene with increased endothelial expression in bAVM lesions. In human brain microvascular endothelial cells, NINJ1 overexpression enhanced proliferation, migration and tube formation, whereas NINJ1 knockdown produced opposite effects. DNMT1 silencing upregulated NINJ1 expression and induced angiogenic behaviour, which was abolished by concurrent NINJ1 knockdown. Mechanistically, NINJ1 enhanced KRAS-MAPK/ERK signalling, at least in part by increasing KRAS protein stability. NINJ1 overexpression also increased endothelial permeability and reduced VE-cadherin and ZO-1 expression. In vivo, endothelial-specific overexpression of ninj1 in zebrafish impaired vascular integrity and induced intracranial haemorrhage. These findings identify promoter hypomethylation-associated activation of NINJ1 as a previously unrecognised epigenetic contributor to endothelial dysfunction in bAVM and support a role for NINJ1 as an upstream enhancer of KRAS-MAPK signalling. Epigenetic dysregulation of NINJ1 may therefore contribute to vascular instability and haemorrhage risk in bAVM.