Extracellular vesicles derived from different tissues attenuate cardiac dysfunction in murine MI models

Fig. 1. Characterization of cEVs and nEVs. (A) NTA analysis of the diameter of cEVs and nEVs. (B) Representative TEM micrographs of cEVs and nEVs (scale bar = 100 nm). (C) Representative light scattering microscopy (LSM) images of cEVs and nEVs. (D) Protein analysis of the EVs markers (Tsg 101, Alix, and Calnexin). (E) H&E staining of heart tissue from different groups to assess the biosafety of intramyocardial injection of cEVs and nEVs in vivo. Scar bar:100 μm. (F) Relative mRNA expressions of IL-1β, IL-6, TGF-α, and IL-10 in mice hearts among different groups. (*P < 0.05, ***P < 0.001 for MI + cEVs group or MI + nEVs group vs. Sham group; #P < 0.05, ###P < 0.001 for MI + cEVs group or MI + nEVs group vs. PBS group; &&&P < 0.001 for MI + nEVs group vs. MI + cEVs group)

Fig. 2. Intramyocardial injection of cEVs and nEVs improved cardiac function and reduced scar size post-MI. Representative M-mode images (A) and quantification of EF% (B) and FS% (C) of Sham, MI + PBS, MI + cEVs, and MI + nEVs groups at baseline, day 3, day 14, and day 28 post-MI (n = 6). Masson’s trichrome staining of mice hearts in different groups at day 28 post-MI (D) (scale bar = 1 mm, n = 5), and corresponding statistical analysis (E). F. Measurement of the left ventricle wall thickness of the mice hearts in different groups at day 28 post operation. G-I. Protein analysis of collagen I and collagen III in mice hearts at day 28 post-MI (n = 4). J, K. Relative mRNA levels of BNP (J) and ANP (K) in the MI + PBS group, MI + cEVs group, and MI + nEVs group. L. Analysis of the HW/BW (mg/g) in the MI + PBS group, MI + cEVs group, and MI + nEVs group (n = 6). (*P < 0.05, **P < 0.01, ***P < 0.001 for MI + cEVs group or MI + nEVs group vs. PBS group; #P < 0.05 for MI + nEVs group vs. MI + cEVs group)

Fig. 3. Both cEVs and nEVs alleviated inflammatory infiltration into the heart after MI. (A) Representative H&E staining of myocardial sections at 28 days from MI mice treated with PBS, cEVs, or nEVs (scale bar = 100 μm, n = 6). (B) Representative immunofluorescence images of myocardial sections stained with anti-CD45 and anti-F4/80 antibodies at 3 days from MI mice treated with PBS, cEVs, or nEVs (scale bar = 50 μm, n = 5–6). (C) Statistical analysis of CD45+ inflammatory cells (cell counts per field) in the cardiac immunofluorescence images. (D) Statistical analysis of F4/80+ macrophages (cell counts per field) in the cardiac immunofluorescence images. E-H. Relative mRNA expressions of IL-1β (E), IL-6 (F), Arg1 (G), and IL-10 (H) in the hearts of MI mice treated with PBS, cEVs, or nEVs (n = 3). (*P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant)

Fig. 4. Administration of both cEVs and nEVs alleviated cardiomyocytes apoptosis after MI. (A) Representative images of Tunel staining in myocardial sections at 3 days from MI mice treated with PBS, cEVs, or nEVs (scale bar = 50 μm, n = 6). (B) Analysis of Tunel positive cells (cell counts per field) in the cardiac immunofluorescence images. C-E. Protein analysis of Bax, Bcl-2, Cleaved-Caspase 3, and Total-Caspase 3 in mice treated with PBS, cEVs, or nEVs (n = 3). F, G. Apoptosis rate of cardiomyocytes with different treatments detected by flow cytometry (F) and representative analysis (G) (n = 4). (*P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant)

Fig. 5. cEVs and nEVs treatment promoted angiogenesis after MI. (A) Representative images of CD31 staining in myocardial sections at 28 days from MI mice treated with PBS, cEVs, or nEVs (scale bar = 50 μm, n = 6). (B) Quantitative myocardial capillary density in mice hearts at day 28 post-MI. C-F. Relative mRNA expressions of VEGF-A (C), VEGF-B (D), Kdr (E), and Pecam (F) in PBS, cEVs, or nEVs-treated mice hearts at day 28 after MI (n = 3). G. Representative images showing the HUVEC-formed tube structure in different groups (scale bar = 200 μm, n = 5). H-J. Analysis of total tube length (H), number of branches (I), and number of junctions (J) formed by HUVECs in different groups. (*P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant)

Fig. 6. RNA-seq analysis of mRNA profiling in cEVs and nEVs. (A) Pearson’s correlation analysis showing a high degree of similarity between the mRNA expression profiles for the cEVs (n = 3) and nEVs (n = 3) samples. (B) Volcano plot showing differently expressed mRNAs between cEVs (n = 3) and nEVs (n = 3) samples. Red dot represented differently expressed mRNAs with P < 0.05 and |Log2FC|>1, green dot represented mRNAs with P > 0.05 and |Log2FC|>1, blue dot represented mRNAs with P < 0.05 and |Log2FC|<1, grey dot represented mRNAs with not significant. C, D. Gene Ontology (GO) analysis of the overexpressed mRNAs in cEVs (C) or nEVs (D), including biological process (BP), cellular component (CC), and molecular function (MF). E, F. KEGG analysis of the overexpressed mRNAs in cEVs (E) or nEVs (F). NOTEs: mmu01100: metabolic pathways; mmu04530: tight junction; mmu00190: oxidative phosphorylation; mmu05208: chemical carcinogenesis - reactive oxygen species; mmu00561: glycerolipid metabolism; mmu04662: B cell receptor signaling pathway; mmu04611: platelet activation; mmu04670: leukocyte transendothelial migration; mmu04064: NF-kappa B signaling pathway; mmu04660: T cell receptor signaling pathway

Fig. 7. Identification of protective mRNAs in cEVs and nEVs. (A-G). Heat map illustrating the log2 mass abundance values for the mRNA expression enriched in the cEVs and nEVs involved in cardioprotection pathological processes, including antioxidant (A), redox homeostasis (B), glycolysis (C), unfolded protein response (UPR) signaling pathway (D), Ca2+ homeostasis (E), heat shock proteins (HSPs) (F), and AMPK signaling (G). H. PPI network showing the top 10 hub genes involved in the above cardioprotection pathological processes. I. Relative expression of hub top 9 genes (Gapdh was excluded) in cEVs and nEVs. J. Relative expression of the top 9 hub genes (Gapdh was excluded) in mice hearts with different treatments 1 day after MI (n = 4). K. ROS production in mice hearts of different groups 1 day after MI was indicated by DHE staining (scale bar = 50 μm, n = 4). L. ATP content in mice hearts of different groups 1 day after MI (n = 5). (****P < 0.001, ***P < 0.01 for MI + cEVs group or MI + nEVs group vs. MI + PBS group; ###P < 0.01, ##P < 0.05 for MI + nEVs group vs. MI + cEVs group)