Introduction Moyamoya disease (MMD) is a malignant cause of childhood stroke characterized by occlusion of the distal internal carotid arteries and branches by neointimal cells staining positive for smooth muscle cell (SMC) markers. ACTA2 encodes smooth muscle α-actin (α-SMA). A subset of ACTA2 mutations that predispose to thoracic aortic aneurysms and dissections including mutations altering arginine 179 are also associated with bilateral MMD-like occlusions. This study aims to elucidate the molecular pathogenesis of this disease to potentially inform the etiology of large vessel disease in stroke patients in the general population. Hypothesis We hypothesize that Acta2SMC-R179C/+ mice have disrupted differentiation of SMCs in cerebrovascular arteries, and thus the cells are primed to proliferate and migrate excessively, leading to MMD-like disease. Methods 
We generated patient-derived ACTA2 R179C and control induced pluripotent stem cell (iPSC) lines, and the Acta2SMC-R179C/+ mouse model, which expresses the Acta2 R179C mutation in SMCs via the SM-22-Cre-Lox system. Phenotypic differences between mutant and WT brains are characterized using micro CT, non-invasive cuff blood pressure measurements, and echocardiography. SMC phenotype is evaluated by protein expression of contractile genes, BrdU ELISA proliferation assay, Transwell migration assay, and immunofluorescence. Results/Anticipated Results: Patient-derived ACTA2 R179C iPSC-derived SMCs from patients show de-differentiation, continued expression of stem cell markers, and increased proliferation and migration. rt-PCR and sequencing of genomic DNA performed in aortic tissue and explanted SMCs confirms the presence of the mutation in the mutant mice and absence in mice with only the floxed allele (WT). Two-dimensional gel confirms presence of mutant α-SMA. Acta2SMC-R179C/+ mice display straightened and narrowed cerebral arteries at 8 weeks of age and hypotension but no aortic disease between 2 and 12 months of age. Explanted SMCs from Acta2SMC-R179C/+ mice exhibit decreased levels of differentiation markers and increased proliferation and migration. Acta2SMC-R179C/+ SMCs also show less robust and disrupted α-SMA filaments. Conclusion Acta2SMC-R179C/+ mice display MMD-like cerebrovascular disease and Acta2SMC-R179C/+ SMCs exhibit stem cell-like characteristics including de-differentiation, increased proliferation and migration.

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