Increased plasma fibronectin mirrors intimal phenotypic switching of vascular smooth muscle cells in moyamoya arteriopathy

Moyamoya disease (MMD) is a rare cerebrovascular disorder characterized by progressive stenosis of large intracranial arteries and formation of fragile collateral vessels that can be triggered by a broad range of genetic and immune factors. Central to MMD pathology is excessive proliferation of vascular smooth muscle cells (VSMCs) in the intima of affected arteries associated with contractile-to-synthetic phenotypic switching, but the underlying molecular mechanisms remain unclear. To identify dysregulated pathways we studied a cohort of 12 patients with well-documented MMD, including one post-mortem autopsy case, using a multi-omics approach combining whole exome sequencing with plasma proteomics. In addition, we conducted an in-depth spatial proteomics analysis of an occluded artery retrieved post-mortem from one idiopathic patient, combining targeted antibody-based imaging with laser capture microdissection coupled to mass spectrometry. Genetic predispositions for MMD was found in 8 out of 12 patients (67%), including three patients with variants in the major susceptibility gene RNF213 and five with varying underlying genetic conditions (trisomy 21, pathogenic variants in ACTA2, SAMHD1, NFIA). Artery spatial proteomics revealed phenotypic switching of vascular smooth muscle cells associated with infiltration of these cells in the intima, including loss of contractile and gain of synthetic marker proteins. Most notably, increased expression of cellular fibronectin in the occluded lesion was associated with increased levels in patients plasma, providing a rational for cellular fibronectin as potential tissue leakage biomarker for moyamoya disease. Finally, infiltration of macrophages and antigen-presenting cells in the intima pointed to a role for inflammatory signals in disease progression. Together, our data provide an unprecedented spatial view on protein changes in an occluded moyamoya artery, revealing intimal infiltration of synthetic vascular smooth muscle cells and antigen-presenting immune cells as key pathological findings, opening novel avenues for future diagnosis and research.