Plasma-derived small extracellular vesicle miR-660-5p as a predictor for carotid plaque vulnerability and postoperative major adverse cardiovascular event risk

Building on our previous isolation of plaque-derived small extracellular vesicles (sEVs) from carotid atherosclerotic stenosis (CAS) patients, this study aimed to identify plasma sEV-encapsulated miRNAs that reflect plaque vulnerability and predict clinical outcomes, based on differentially expressed miRNAs identified in sEVs from stable versus vulnerable plaques. CAS patients were retrospectively enrolled into four complementary cohorts: (1) a sequencing cohort (n=12) for miRNA profiling of plaque-derived sEVs; (2) a discovery cohort (n=62) to identify candidate plasma-derived sEV miRNAs associated with plaque vulnerability; and (3) validation cohort 1 (n=180; endarterectomy cases) and (4) validation cohort 2 (n=326; stenting cases) for validating the diagnostic efficacy of the identified plasma-derived sEV miRNAs. Candidate miRNAs were quantified using a High-throughput nano-bio chip integrated system (HNCIB). Their associations with plaque vulnerability and 3-year post-endarterectomy major adverse cardiovascular events (MACE) were analyzed. MiRNA sequencing revealed significant upregulation of miR-660-5p in vulnerable plaque-derived sEVs. Its expression levels showed a significant positive correlation between matched plaque- and plasma-derived sEVs from the same patient. Moreover, plasma-derived sEV miR-660-5p levels were independently associated with carotid plaque vulnerability in both validation cohorts, defined either by histopathological criteria (endarterectomy cases) or the clinically validated Plaque-Reporting and Data System (RADS) criteria (stenting cases). Additionally, elevated plasma-derived sEV miR-660-5p levels were also associated with a higher risk of 3-year post-endarterectomy MACE. Plasma-derived sEV miR-660-5p represents a promising non-invasive biomarker for assessing carotid plaque vulnerability and predicting 3-year MACE risk following carotid endarterectomy, offering potential for improved risk stratification and therapeutic targeting. • Plasma-derived sEV miR-660-5p served as an independent predictor of carotid plaque vulnerability. • Plasma-derived sEV miR-660-5p was associated with a higher risk of 3-year major adverse cardiovascular events following endarterectomy.