Plasma extracellular vesicles from patients with heart failure retain pro-inflammatory features
C Macchi, I Fichtner, M Ruscica, F Tafuri, G Esposito, C Perrino, M Vicenzi, S CarugoAbstract
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. Aims of the present project were: 1) to characterize EVs in HFrEF and HFpEF to investigate their potential as biomarkers; 2) to evaluate how protein EV-cargo can affect the inflammatory milieu in human monocytes and cardiomyocytes.
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 (NTA), transmission electron microscopy (TEM), Western blot (WB), flow cytometry (FACS) and proteomic analyses (targeted and untargeted). Functional assays were performed to assess the impact of patient-derived EVs on a cellular model of monocyte and cardiomyocytes.
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. Compared to CTR, no differences were found in terms EV size and concentration. Among the various EV subpopulations, vesicles originating from monocytes (CD14+), macrophages (CD206+), neutrophils (C66b+), activated endothelial cells (CD62E+), cardiomyocytes (CD172a), CD4+ T lymphocytes, and B lymphocytes (CD19+) were significantly reduced in HF by −58.2%, −82.3%, −25%, −40.3%, −60.4%, −55.2%, and −63.3%, respectively—an effect predominantly driven by EVs from HFpEF patients. Proteomic analysis showed that EVs carried proteins associated with thrombo-inflammation and fibrosis (e.g., PAI-1, vWF, GDF-15, IL-6, and galectin-3). Treatment of human monocytes (THP-1) with HF-derived EVs induced higher expression of pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, and IL-8) compared with cells exposed to EVs from CTR subjects. In human cardiomyocytes (AC16), exposure to HF-derived EVs increased IL-1β expression. These effects were largely driven by EVs isolated 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.