Intra-Cardiac Release of Extracellular Vesicles Shapes Inflammation Following Myocardial Infarction

Abstract

Rationale: A rapid and massive influx of inflammatory cells occurs into ischemic area after myocardial infarction (MI), resulting in local release of cytokines and growth factors. Yet, the mechanisms regulating their production are not fully explored. The release of extracellular vesicles (EVs) in the interstitial space curbs important biological functions, including inflammation, and influences the development of cardiovascular diseases. To date, there is no evidence for in situ release of cardiac EVs after MI.

Objective: The present study tested the hypothesis that local EV generation in the infarcted heart coordinates cardiac inflammation after MI.

Methods and results: Coronary artery ligation in mice transiently increases EV levels in the left ventricle when compared with sham animals. EVs from infarcted hearts were characterized as large vesicles (252±18 nm) expressing cardiomyocyte and endothelial markers and small EVs (118±4 nm) harboring exosomal markers, such as CD (cluster of differentiation) 63 and CD9. Cardiac large EVs generated after MI, but not small EVs or sham EVs, increased the release of IL (interleukin)-6, CCL (chemokine ligand) 2, and CCL7 from fluorescence-activated cell-sorted Ly6C⁺ cardiac monocytes. EVs of similar diameter were also isolated from fragments of interventricular septum obtained from patients undergoing aortic valve replacement, thus supporting the clinical relevance of our findings in mice.

Conclusions: The present study demonstrates that acute MI transiently increases the generation of cardiac EVs characterized as both exosomes and microvesicles, originating mainly from cardiomyocytes and endothelial cells. EVs accumulating in the ischemic myocardium are rapidly taken up by infiltrating monocytes and regulate local inflammatory responses.

Keywords: exosomes; humans; inflammation; myocardial infarction; myocytes, cardiac.

Figure 1.

Cardiac extracellular vesicles (EVs) are transiently released in response to myocardial infarction (MI) in mice and originate mainly from cardiomyocyte. A, Typical representative electronic microscopy of large EVs (lEVs). B, Representative size distribution analysis by tunable resistive pulse sensing (TRPS) of lEVs from ischemic and sham murine cardiac tissue 24 h postligation. C, lEVs release during 72 h after the onset of ischemia in sham (open circles) and MI mice (dark circles). D, Typical representative electronic microscopy of small EVs (sEVs). E, Representative size distribution analysis by TRPS of sEVs from ischemic and sham murine cardiac tissue 24 h postligation. F, sEVs release during 72 h after the onset of ischemia in shams (open circles) and MI mice (dark circles). G, Cellular origin of lEVs isolated from infarcted hearts 24 h postligation. H, Representative Western blots for Hsc70 and troponin T in ischemic left ventricle (LV) in lEV and sEVs derived from sham and MI. I, Representative Western blots for Hsc70, CD63, and CD9 in ischemic LV and sEVs. J, CD63 levels measured using integrated magnetic–electrochemical exosome and normalized to EV concentrations per tissue weight. Data are mean±SEM. n=4 to 7 animals per time point. ND indicates not detectable. **P<0.01 vs corresponding sham, *P<0.05 vs sham, †P<0.05 vs lEVs.

Figure 2.

Cardiac extracellular vesicles (EVs) are engulfed by monocytes and shape the inflammatory response. A–C, Infiltration of neutrophils (Neutro; A), monocytes (Mono; B), and lymphocytes (Lympho; C) in sham (open circles) and myocardial infarction mice (dark circles) for 72 h after coronary artery ligation. Data are mean±SEM. n=4 to 7 animals per time point. **P<0.01 vs corresponding sham-operated mice at the same time point. D, Representative images of engulfment of GFP⁺ material by infiltrating immune cells (CD45⁺/CD11b⁺ leukocytes were gated on F4/80⁻ monocytes and then further stratified by Ly6G and Ly6C expression) and (E) corresponding quantification. Data are mean±SEM. n=4 animals. *P<0.05 vs GFP+ cardiac neutrophils, ††P<0.01 vs GFP+ cardiac T lymphocytes. F–H, IL-6, CCL2, and CCL7 release by monocytes stimulated with cardiac-derived large (lEVs) or small EVs (sEVs). Data are mean±SEM. n=4 to 11 per experimental conditions. *P<0.05, **P<0.01 vs saline control.

Figure 3.

Intracardiac extracellular vesicles (EVs) in human myomectomy biopsies. A, Typical representative electronic microscopy of cardiac EVs from human biopsies indicating the presence of large (lEVs) and small EVs (sEVs). B, Representative size distribution analysis by tunable resistive pulse sensing (TRPS) of lEVs (in white) and sEVS (in grey). C, M-mode analysis of both lEVs and sEVs. D, EV concentration determined by TRPS. Data are mean±SEM. n=4 samples per fraction. *P<0.05 vs lEVs.