Fig 2: Plasma-derived EV characterizzation. (A) Plasma derived EV purification protocol. (B) Dynamic light scatter analyses of particle size and concentration of plasma derived EV before (pre-MI) and after (post-MI) myocardial infarction (n=5 repeated measurements of 5 different plasma samples per group), red lines represent standard deviations. (C) Western blot analysis of specific exosomal markers TSG101, CD63 and CD81. (D) Quantification of plasma derived EV cytotoxicity on rat primary neonatal cardiomyocytes. n=4 independent experiments treated with 4 different pools of EV. Left panels are representative images of viability assay for the conditions without EV (w/o EV), EV derived from plasma before (EV pre-MI) and after (EV post-MI) myocardial infarction.Viable cells stain green, dead cells red. All data are presented as mean ± SEM and analyzed by one-way analyses of variance-ANOVA with post-hoc multiple comparisons using the Bonferroni correction (**p < 0.01). Mean, SEM and statistics are reported in full in Table S1.

Fig 3: Systemic inhibition of extracellular vesicles release regulates inflammation in heart after MI. (A) NTA analysis, cumulative curves for concentration and size distribution of plasma-derived EV. EV pre-MI (grey; n=5 repeated measurements of 14 different plasma samples), EV post MI GW4869 (blue; n=5 repeated measurements of 7 different plasma samples), EV post MI vehicle (yellow; n=5 repeated measurements of 7 different plasma samples) and EV post MI from not injected animals (reference line bordeaux). (B) LC-MS/MS quantification of EV ceramide content for EV pre-MI (gray reference line), EV post-MI vehicle (yallow bars) and EV post-MI GW4869 (blue bars), n=6 different plasma samples. All data are presented as mean ± SEM pmol/300ml plasma samples and normalized over pre-MI ceramide levels (fold increase vs EV pre-MI). (C) EV cytokine content analyses. Data are presented as mean fold change (over blank) ± SEM of 4 independent experiments. (D) Western blotting analysis and relative quantification of specific exosomal markers TSG101, CD63 and CD81 and specific inflammatory macrophages markers iNOS and CD68. (E) Representative images and quantification of infiltrated CD68+ (stained in red) macrophages in heart section 24 hrs post-MI (Sham: n=7, GW4869: n=8, vehicle: n=7 heart section/group). (F) Quantification of TNFa in heart tissue 24hrs post-MI (Sham: n=9, GW4869: n=6, vehicle: n=7 heart section/group). All data are presented as mean ± SEM and analyzed by one-way ANOVAs with post-hoc multiple comparisons using the Bonferroni correction (*p < 0.05, **p < 0.01). Mean, SEM and statistics are reported in full in Table S2.

Fig 4: Identification of plasma EVs. (A and B) Observation of EVs by transmission electron microscopy. (A) Scale bar, 500 nm. (B) Scale bar, 100 nm. (C) Nanoparticle tracking analysis. Left, sample EV particle size distribution map; right, display of particles in the nanoparticle tracking analysis detection window. Maximum area, 1,000 nm; minimum area, 5 nm; minimum brightness, 20 nm. (D) Qualitative detection of CD63, TSG101, Alix and CD81 proteins by western blotting. EV, extracellular vesicle; TSG101, tumor susceptibility gene 101.