MMD is a rare cerebrovascular disorder characterized by slow and progressive steno-occlusive of the bilateral internal carotid arteries, accompanied by the formation of hazy collateral vessels. Whether MMD is an immune-related disease is a matter of controversy. Our previous results indicate aberrant expression levels of immune-related proteins in the serum-derived extracellular vesicles (EVs) from ischemic and hemorrhagic MMD patients. The immunological mechanisms underlying the pathogenesis of early (Suzuki I-III) and late (Suzuki IV-VI) stages MMD remain elusive.

A total of 20 age-matched healthy control donors, 40 early-stage MMD patients, and 36 late-stage MMD patients were enrolled, with no complications. Tandem Mass Tag-labeled quantitative proteomics was performed on serum-derived extracellular vesicles extracted from adult patients diagnosed with early-stage and late-stage ischemic MMD and healthy controls.

Proteomics analysis results identified 261 differentially expressed proteins (DEPs) in EVs between early- and late-stage MMD, among which 129 proteins were stage-specific compared with healthy controls. Bioinformatics analysis of the 129 DEPs indicated dysregulation of neutrophil degranulation, including 30 DEPs enriched in the Biological Process Gene Ontology with MPO as a hub protein. Elisa assay identified upregulation of MPO expression in early-stage MMD, and downregulation in late-stage. Proteome analysis in bEnd.3 suggested EV-induced protein O-linked glycosylation and mitochondrial electron transport chain damage in endothelial cells of early-stage MMD.

MPO encapsulated in serum-derived EVs may potentially identify early- and late-stage MMD. EVs mediate endothelial cell fate possibly by altering protein O-linked glycosylation levels and inducing mitochondrial dysfunction.