Therapeutic potential of extracellular vesicles derived from cardiac progenitor cells in rodent models of chemotherapy-induced cardiomyopathy

Figures

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  Figure 1. Rodent CCM model experimental protocols. (A) Mice were subjected to 3 weekly intraperitoneal injections of doxorubicin (DOX) (cumulative dose = 12 mg/kg). Sham-operated mice underwent isotonic buffer injections without DOX treatment.Three equal doses of EV-CPC (total dose of 30E+9 particles) were given intravenously (IV) by the retro-orbital sinus (n = 30) according to 3 slightly different time frames: starting 15 days after the last Dox injection and performed every 4-5 days (one series) or every 3-4 days (one series) or starting the day after the last Dox injection and performed every 4-5 days (one series). A placebo control group underwent injections of isotonic buffer according to the same timing and delivery protocol. Cardiac MRI and EKG measurements were performed to assess cardiac function. Baseline values correspond to the functional measurements which were taken in 5 healthy mice at the onset of the protocol. (B) Wistar female rats received 5 IP injections of DOX (3mg/kg each; total cumulative dose 15mg/kg) followed by 3 intravenous injections of GMP-EV (100E+9/injection, one every 2/3 days; n = 12). Control rats were placebo-injected with saline (n = 11) or sham-treated…

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  Figure 2. EV-CPC characterization. (A) Cryo-TEM resolution of EV-CPC lipid bilayer membranes. Scale bar = 100 μm. (B) Size distribution of isolated particles by NTA analysis measured on 5 different preparations of EV-CPC. (C) FACS detection of tetraspanin expression on EV-CPC by MACSPlex analysis compared to a negative control, i.e., the CD31 endothelial marker (in technical duplicates; median +/− IQR). (D) Extract from the absolute quantification of proteomic analysis. SD: standard deviation; NTA: Nanoparticle Tracking Analysis; Cryo-TEM: immuno-gold labelling and cryo-transmission electron microscopy; CD: cluster of differentiation; MW: molecular weight.

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  Figure 3. EV-CPC treatment improves heart contractility of CCM mice and decrease adverse remodeling. Survival Kaplan Meier curves with Log rank analysis test performed (A) at 3 weeks after the beginning of DOX treatment and (B) at end-of-study. MRI evaluation of cardiac parameters (C-F): (C) CS measured by MRI (Mean + / − SEM) at the basal global, (D) basal endocardial and (E) basal epicardial levels (Sham: n = 14; DOX+Placebo: n = 14, DOX+EV-CPC: n = 11); and (F) GLS (Median +/− IQR) as a percent change from baseline (some mice, two in sham group and one in each EV or placebo group, could not be included into GLS datasets due to unavailable 2-chamber views in MRI: Sham: n = 14; DOX+Placebo: n = 14, DOX+EV-CPC: n = 11). Relative mRNA expression in mouse hearts of Gal3 (G; Mean +/− SEM), Myh6/Myh7 (H; Median +/− IQR) and NPPA (I; Median +/− IQR): these data integrated a supplemental series of Sham and DOX+placebo animals exclusively used for PCR assessment. *p ≤ 0.05; ***p ≤ 0.001. CS, circumferential strain (%); GLS, Global longitudinal strain; Gal 3, galectin 3; Myh6/Myh7 ratio, myosin heavy chain α/myosin heavy chain β; NPPA, Atrial natriuretic peptide.

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  Figure 4. GMP-EV treatment moderates left ventricular dilatation upon rats doxorubicin exposure. (A) LVESV and (B) LVEDV measured by echocardiography and expressed as a percent change (Median+/−IQR) from day 10 (post-DOX administration). Sham: n = 6; DOX+Placebo: n = 11, DOX+GMP-EV: n = 12. *p ≤ 0.05; (Kruskal Wallis with Dunn's correction test). LV-ESV/LV-EDV, left ventricular-systolic/diastolic function.