We reasoned that the cardiovascular miRNAs present in the PF could, at least in part, be included in exosomal cargos. Nanosight LM10 nanoparticle tracking analyses (NTA) revealed the presence of exosome-sized (30–120 nm) particles in both the PF and plasma, which we analyzed as a reference (Figures 2A and 2B; the rectangles surround exosome-sized particles). Next, exosomes were enriched from the PF and plasma. The quality of the exosome preparations was validated by western blotting for a panel of exosomes antigens (ALIX, CD63, FLOT1, and EPCAM) (Figure 2C) and by transmission electron microscopy (TEM) using gold particles conjugated with an anti-CD63 antibody (Figure 2D) to confirm the exosome identity. The miRNAs already detected in the total PF where also measurable in the PF exosomes. Moreover, for the majority of the individual miRNAs, the expressional differences appreciated comparing whole PF versus whole plasma were confirmed when looking at the exosomal components of the two fluids (Figure 3A). This set of results suggests that miRNAs produced by cells of the heart and heart vessels are, at least in part, transported to the PF via exosomes. Next, to investigate if the miRNAs are differently distributed between the exosomal and non-exosomal fractions of the PF, we calculated the PF exosome/whole PF concentration ratios of the individual miRNAs. The PF exosome/total PF ratios were highly variable between miRNAs (Figure 3B), thus suggesting (1) a non-random incorporation of the miRNAs in the PF exosomes and/or (2) that miRNAs are released by cells via different mechanisms.Figure 2PF Contains Nanoparticles of the Size and Antigenic Profile of Exosomes(A and B) Nanoparticle tracking analysis (NTA) was used to quantify the concentration (A) and size distribution (B) of particles in the total PF and plasma. The dotted rectangles evidence exosome-sized vesicles (30–120 nm). (C and D) PF and plasma exosome preparations were positive for the exosomal markers ALIX, FLOT1, EpCAM and CD63. Transmission electron microscopy (TEM) using a gold particle-conjugated anti-CD63 antibody further confirmed exosome identity. In the bottom right corner of (D), next to CD63-immunoreactive exosomes, is shown a larger unreactive cytoplasmic vesicle as negative control (the scale bar represents 100 nm). Unpaired two-tailed Student’s t test was applied. All values are mean + SEM; n = 5.Figure 3Human PF Exosomes Carry Cardiovascular miRNAs(A) miRNA level normalized to the spike-in cel-miR-39. (B) PF exosome/total PF ratios calculated for each miRNA. All values are mean + SEM. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus plasma (unpaired Student’s t test); n = 5.