THE ROLE OF EXTRACELLULAR VESICLES IN HEART DISEASES

Background and Aim: Heart diseases, including hypertrophic cardiomyopathy (HCM) and heart failure (HF), remain among the leading causes of morbidity and mortality worldwide. Epidemiological data indicate that their burden will continue to rise in the coming years, with HF representing an expanding global health concern. Meanwhile, the prevalence of HCM is increasing as a result of improved diagnostic methods and the widespread adoption of genetic screening. Despite significant advances in diagnostics and therapeutics, there remains a pressing need for non-invasive biomarkers capable of capturing the molecular complexity of these diseases, predicting disease progression, and guiding personalized treatment strategies. EVs, nanosized membrane-bound particles released by all cell types, have emerged as promising biomarkers due to their ability to carry molecular cargo reflective of the state of their cellular origin. This thesis investigates the potential of plasma-derived EVs as biomarkers in heart diseases. Specifically, it aims (1) to characterize EV profiles in patients with HCM in comparison to healthy controls, and (2) to evaluate EV features in patients with HF with reduced (HFrEF) and preserved ejection fractions (HFpEF), focusing on their potential utility in phenotypic refinement, and comparison with healthy controls. By analyzing these two pathologically distinct cardiac conditions, this project also seeks to determine whether differences in EV characteristics – such as size, concentration, protein content, and subcellular origin – are consistently detectable across diseases, and whether EVs hold comparable predictive value despite the divergent underlying mechanisms. Methods: This was a single-center, prospective study involving clinically characterized cohorts of healthy individuals and patients with a diagnosis of HCM, HFrEF, and HFpEF. HCM diagnosis followed the 2023 ESC criteria, including cardiac imaging, clinical assessment, and genetic screening using next-generation sequencing of 54 sarcomere-related genes. Echocardiographic imaging was performed to assess structural and functional parameters in all groups. EVs were isolated from platelet-poor plasma using differential centrifugation and characterized using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western blotting (WB), and flow cytometry (FACS). FACS analysis included the use of (6)-carboxyfluorescein diacetate N-succinimidyl ester staining to gate intact EVs and immunophenotyping for surface markers (e.g. CD9, CD63 for tetraspanins; CD14 for monocytes; CD206 for macrophages; CD202b for endothelial cells; CD172a for cardiomyocytes; CD66b for neutrophils; CD41a for total platelets). For targeted (Olink) and untargeted (HPLC-MS/MS) proteomic profiling, EVs were further purified by size exclusion chromatography. Selected protein markers of fibrosis and remodeling (e.g., Galectin-3 – Gal-3; Vascular Endothelial Growth Factor C – VEGF-C; Chitinase-3-like Protein 1 – YKL-40; Procollagen Type I N-Terminal Propeptide – PINP) were quantified in plasma of the cohorts using ELISA. THP-1 cells, an immortalized human monocytic cell line, were employed in vitro to assess the molecular effects, specifically from an inflammatory standpoint, of plasma-derived EVs of the HF cohort in comparison with EVs of healthy controls. Results and discussion: A total of 46 patients with hypertrophic cardiomyopathy (HCM), 39 with heart failure (HF; 26 HFrEF and 13 HFpEF), and 31 healthy controls were enrolled. All underwent comprehensive clinical evaluation. Specifically: In HCM, left ventricular geometry was markedly increased compared with controls. Interventricular Septal Thickness in Diastole (IVSd) was 16 vs 8 mm, 2.0-fold; Posterior Wall Thickness in Diastole (PWTd) 11 vs 8 mm, 1.38-fold; Maximum Wall Thickness (MWT) 17 vs 9 mm, 1.86-fold; Left Ventricular Mass index 121 vs 60.8 g/m2, ≈2.0-fold), while Left Ventricular Ejection Fraction (LVEF) was mildly reduced (58% vs 62%; −4 pp) and E/e′ was higher (8.5 vs 5.5; 1.55-fold), indicating preserved systolic but impaired diastolic function. ELISA analysis revealed significantly higher circulating Gal-3 (7.09 vs 5.89 ng/mL; +1.20 ng/mL; 1.20-fold; p=0.0286) and VEGF-C (396.6 vs 223.3 pg/mL; +173.3 pg/mL; 1.78-fold; p=0.0108) and lower PINP (17.58 vs 18.87 ng/mL; −1.29 ng/mL; 0.93-fold; p=0.0498) levels in HCM compared with controls, reflecting enhanced fibrotic and angiogenic signaling. Characterization of plasma-derived EVs by NTA showed no significant differences in size (181.5 vs 180.6 nm; p=0.9508) or concentration (3.13×109 vs 3.26×109 particles/mL; p=0.8432), and EV subpopulation analysis similarly revealed no significant differences across any tested surface marker. However, proteomic profiling of HCM-derived EVs identified upregulation of PAI-1 and CD62P (P-selectin) (both p < 0.01), indicating platelet activation and enrichment of thrombosis-related pathways. Plasma proteomics further confirmed increased levels of NT-proBNP, vWF, tPA, SPD, CD163, and GDF-15 (p < 0.05–0.0001), clustering within hemostasis and fibrinolytic networks. Collectively, these findings suggest that circulating EVs in HCM carry molecular signatures consistent with myocardial remodeling, endothelial activation, and pro-thrombotic processes. In HF, the mean EF was 30% in HFrEF, 58% in HFpEF, and 62% in controls. Gal-3 levels were significantly higher in HF compared with controls (8.90 vs 5.95 ng/mL; +2.95 ng/mL; 1.50-fold; p=0.0007), accompanied by increased VEGF-C and YKL-40 and unchanged PINP levels. EVs were significantly larger in HF compared to controls (202.2 vs 180.6 nm; +21.6 nm; 1.12-fold; p<0.0001) across both phenotypes (HFrEF 203.5 nm; HFpEF 202.5 nm), with a non-significant reduction in total EV concentration (2.40×109 vs 3.26×109 particles/mL; −26%; p=0.0659). EV subpopulations were globally reduced, including CD14+ monocyte (0.11 vs 0.71 EVs/μL; 0.15-fold; p=0.0021), CD206+ macrophage (0.008 vs 0.03; 0.27-fold; p=0.0010), CD202b+ endothelial (0.43 vs 1.50; 0.29-fold; p=0.0006), CD62E+ activated-endothelial (0.56 vs 1.35; 0.41-fold; p=0.0093), CD172a+ cardiomyocyte (0.18 vs 1.04; 0.17-fold; p=0.0003), CD66b+ neutrophil (0.23 vs 0.64; 0.36- fold; p=0.0111), and CD41a+ platelet-derived EVs (0.59 vs 7.16; 0.08-fold; p=0.0207), whereas CD4+ T-helper EVs were significantly elevated (rEF 0.21 vs 0.06 EVs/μL; 3.5- fold; p=0.0003; pEF 0.16 vs 0.06; 2.7-fold; p=0.0056). Functionally, EVs from HF patients induced a robust inflammatory response in THP-1 monocytes, upregulating IL-1α (+373%; ~4.7-fold), IL-1β (+160%; ~2.6-fold), and IL-6 (+151%; ~2.5-fold) compared with control EVs, with even stronger effects observed for HFpEF-derived EVs (IL-1α +530%, ~6.3- fold; IL-1β +290%, ~3.9-fold; IL-6 +330%, ~4.3-fold). These results demonstrate that HF- derived EVs reflect the inflammatory and immune activation characteristic of both HFrEF and HFpEF, particularly accentuated in HFpEF. Together, the results support the inclusion of EV analysis as a complementary tool for diagnosis, risk stratification, and personalized disease profiling in cardiomyopathies and heart failure syndromes.