Extracellular vesicles in hypertrophic cardiomyopathy patients

Introduction Hypertrophic cardiomyopathy (HCM) is a genetically determined heart disease characterized by marked clinical heterogeneity and limited availability of specific circulating biomarkers that reflect underlying disease mechanisms. This study investigated whether plasma-derived extracellular vesicles (EVs) capture disease-related molecular features in patients with HCM. Methods Patients diagnosed with HCM were consecutively enrolled (December 2023 to October 2025) at the Cardiomyopathy Unit, Italy. Diagnosis was established according to the 2023 ESC guidelines. Sex-matched healthy volunteers (CTRL) with normal ECG and no personal or family history of cardiovascular disease served as controls. We also enrolled patients with hypertensive hypertrophy. EVs were isolated from plasma by size-exclusion chromatography and ultracentrifugation, in accordance with the latest ISEV guidelines (MISEV2023), then characterized using nanoparticles tracking analysis, transmission electron microscopy (TEM), Western blot (WB), flow cytometry (FACS) and proteomic analyses (targeted and untargeted). Results A total of 46 patients with HCM (median age: 61 years [IQR: 50–69.5]), 31 CTRL (median age: 48 years [IQR: 44–56]) and 10 with hypertensive hypertrophy (median age: 55 years [IQR: 46–61])) were enrolled in the study. Pathogenic or likely pathogenic variants were identified in 46.5% of HCM patients, most commonly in the sarcomeric genes MYBPC3 and MYH7. Median EV size and concentrations did not differ significantly among the three groups. Compared with CTRL and hypertensive hypertrophy samples, HCM-derived EVs exhibited a 45% reduction in neutrophil-derived EVs and a 76% reduction in lymphatic endothelial–derived EVs. Conversely, platelet-derived EVs were significantly enriched (+15.6%) in HCM. Enrichment analysis coming from untargeted proteomic analysis identified markers of chronic inflammation, immune cell trafficking and vascular remodeling (Serum amyloid A, α-1-acid glycoprotein and aminopeptidase N). The results of targeted proteomic analysis were used in a logistic regression model to combine the predictive effect of multiple proteins to identify a HCM patient. The highest AUC [0.90 (0.79, 0.98)] was obtained when the following proteins were considered together E-selectin, interleukin-1 receptor type 2, bleomycin hydrolase, epidermal growth factor receptor and platelet glycoprotein VI. Summary/Conclusions The combined subcellular and proteomic characterization of plasma-derived EVs identifies a distinct molecular pattern in HCM, predominantly associated with platelet activation and thrombo-inflammation. These findings provide novel insights into disease-related biological processes and open new avenues for the investigation of EV-mediated mechanisms and biomarker development in HCM.