Isabella Fichtner (1), Chiara Macchi (2), Francesco Tafuri (2), Alessandra S Rizzuto (2), Giovanni Esposito (3), Cinzia Perrino (3), Marco Vicenzi (2), Stefano Carugo (2), Alberto Corsini (2), Massimiliano Ruscica (2)

(1) università degli studi di Milano.
(2) Università degli studi di Milano.
(3) Università degli studi di Napoli-Federico II

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Introduction: Heart failure (HF) is a clinical syndrome involving structural and/or functional cardiac abnormalities, classified as reduced (HFrEF) or preserved (HFpEF) based on the ejection fraction percentage of the left ventricle. As extracellular vesicles (EVs) reflect onset and severity of cardiac diseases, they attract interest as potential liquid biopsies. Aim of the present project was to characterize EVs in HFrEF and HFpEF, investigating their potential as biomarkers and discriminating factors in HFrEF and HFpEF with a particular focus on the underlying molecular mechanisms.

Methods: The study included 39 HF patients (13 HFpEF and 26 HFrEF) and 31 volunteers (CTR). 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) and flow cytometry (FACS). Functional assays were performed to assess the impact of patient-derived EVs on a cellular model of monocyte.

Results: Diagnosis of HF relied on echocardiographic (e.g. E/e' ratio) and biochemical parameters (e.g. NT-proBNP). Isolation of EV was confirmed by FACS and WB analyses (CD63, CD9, CD81, Alix and β1 integrin), while integrity by TEM. EV size was increased in HF (nm: 202 vs 181). Among different subpopulations of EVs, those from monocytes (CD14+), macrophages (CD206), neutrophils (C66b+), endothelial cells (Cd202b+), activated endothelial cells (CD62E+), cardiomyocytes (CD172a), platelets (CD41a), B lymphocytes (CD19) were significantly reduced in HF. Conversely, EVs released by T helper lymphocytes (CD4) were significantly increased in HF patients when compared to controls. Treatment of monocytes (THP-1) with EVs derived from HF patients led to an increased expression of proinflammatory cytokines (i.e. IL-1alfa, IL-1beta, IL-6 and IL-8), when compared to cells treated with EVs isolated from CTR subjects. This change was mostly driven by EVs derived from HFpEF patients.

Summary/Conclusions: EVs derived from HF patients exhibit a distinct profile that reflects the hemodynamic characteristics of the condition and possess proinflammatory properties.