Myocardial Infarction Treatment with a Composite Hydrogel Containing Metformin-Induced Vesicles of Adipose-Derived Stem Cells

Figures

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  Fig. 1. Preparation and characterization of the cECM based Hydrogels. (A) Diagram illustrating the decellularization process of heart tissue and aortic adventitia from porcine. (B) DAPI, HE, picrosirius red, and Alcian blue staining of native and decellularized heart tissue and aortic adventitia. (C) Quantitative analysis of collagen content in the heart and aortic adventitia before and after decellularization. (D and E) Residual dsDNA quantification in native and decellularized heart and aortic adventitia. ∗P < 0.05 vs Native. n = 3. Native: fresh tissue; Decellularized: decellularized tissue; cECM: cardiac decellularized extracellular matrix; Adv: aortic adventitia decellularized extracellular matrix. n = 3. (F) Gross observation of the hydrogels after crosslinking.

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  Fig. 2. Cell survival states on the cECM based hydrogels. (A) Hemolysis test. (B) Cell viability of HUVECs treated with different hydrogels. (C) Cell viability of HUVECs after 1 day, 3 days, and 5 days. (D and E) Calcein-AM/PI staining and quantification of HUVECs after different treatments. (F and G) Calcein-AM/PI staining and quantification of NRVMs after different treatments. cECM, cardiac extracellular decellularized matrix; Adv, aortic adventitia decellularized matrix; CA, hybrid hydrogel of cECM and Adv at 1:1 ratio. Data are expressed as the mean ± SD (n = 3; ∗vs. control group: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; # vs. all other groups: #P < 0.05, ##P < 0.01, ###P < 0.001).

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  Fig. 3. Two-dimensional culture of the HUVECs and NRVMs on the cECM-based hydrogels. (A) Typical fluorescence microscopy images of HUVECs on the hydrogels. (B and C) qRT-PCR of VEGF and FGF-2. (D) Quantitative data for branching length, branches, and nodes after culturing on different hydrogels (n = 3). (E) Immunostaining of connexin 43 (Cx43, in red), along with quantitative data (F) (n = 3). Data are expressed as the mean ± SD (n = 3; ∗vs. control group: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; # vs. all other groups: #P < 0.05, ##P < 0.01; ns: indicates no statistically significant difference).

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  Fig. 4. Preparation and characterization of the EVs derived from the metformin-treated ADSCs. (A) Effect of different metformin concentrations on ADSCs. (B and C) Western blot analysis of CD63 and quantitative data for various concentrations of metformin (n = 3). (D and E) Fluorescence staining of CD63 (scale bars: 50 μm) and semi-quantitative analysis. (F) Western blot analysis of HSP70, CD63, TSG101, and Calnexin. Quantitative data (G) are presented as fold change relative to the EVs control group, with the mean value of the EV group set to 1 (n = 3). (H and I) TEM image of EVs (scale bar: 200 nm) and quantitative analysis of EV numbers. (J and K) NTA analysis of EVs and particle concentrations. N = 3 were analyzed and are shown here. (L) Images of PKH26- labeled Met-EVs uptake by HUVECs and NRVMs, red indicating PKH26 and green phalloidin indicating F-actin (scale bars: 20 μm). EVs derived from ADSCs were referred to as EVs, and EVs from metformin-treated ADSCs were referred to as Met-EVs. DAPI: 4′,6-diamidino-2- phenylindole. (n = 3; ∗vs. 0 mmol/L group or EVs: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; # vs. all other groups: #P < 0.05).

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  Fig. 5. Met-EVs enhancing the pro-angiogenic effects of the HUVECs and mitigating the cardiomyocyte apoptosis after the OGD injury. (A and B) Representative images and quantification of EdU staining in HUVECs. (C) CCK-8 assay of EVs improved the viability of NRVMs after OGD injury. Transwell migration assays (scale bar: 50 μm) (D and E), and representative images and quantitative analysis of tube formation assay on Matrigel (scale bar: 50 μm) (F, G, H, and I) in HUVECs (n = 3). (J and K) JC-1 staining (n = 3): red indicating JC-1 aggregates and green indicating JC-1 monomers. Representative immunoblot (L) for apoptosis-related markers (caspase 3 and cleaved caspase 3) in NRVMs after OGD injury and quantitative analysis (M) (n = 3; ∗vs. 0 mmol/L group or control group: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; # vs. all other groups: #P < 0.05, ##P < 0.01, ###P < 0.001).

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  Fig. 6. Effects of the CA + Met-EVs on the post-MI cardiac functions. (A) Establishment of the MI rat model and intrapericardial (iPC) injection. (B) Representative M-mode short-axis echocardiographic image on Day 28. (C) Corresponding quantitative analyses of LVEF, LVFS, LVIDD, and LVIDS. All data are expressed as mean ± SD (n = 6). (D and E) Masson staining and quantitative assessment of both the infarct wall thickness and the infarct area (%) (bar = 2 mm). (F and G) WGA staining and quantitative analysis of the cross-sectional area of cardiomyocytes (scale bar = 20 μm). (n = 6; # vs. all other groups: #P < 0.05, ##P < 0.01, ###P < 0.001).

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  Fig. 7. Gel + Met-EVs reducing cardiac apoptosis and promoting angiogenesis after MI. (A and B) TUNEL staining and quantitative analysis of TUNEL-positive cells (scale bar = 20 μm). (C and D) Representative immunohistochemical images of Cleaved-caspase 3 (bar = 20 μm) and quantitative analysis of positive cell numbers. (E and F) Representative fluorescence images of α-SMA (bar = 50 μm) and the corresponding statistical analysis of the number of α-SMA-positive vessels. (G and H) Representative fluorescence images of CD31 (bar = 50 μm) and corresponding statistical analysis of CD31-positive vessel numbers (n = 6; # vs. all other groups: #P < 0.05, ##P < 0.01, ###P < 0.001).

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  Fig. 8. Met-EVs ameliorating the apoptosis by activating the PI3K/AKT signaling pathway. (A) Principal component analysis (PCA) of EVs. (B) Volcano plot. (C) GO enrichment analysis. (D) KEGG pathway analysis. (E) Cell viability of NRVMs after OGD injury (n = 6). (F) mRNA levels of C1qtnf1 (n = 3). (G) Western blot analysis of C1QTNF1. (H) TUNEL staining of NRVMs. (I) Expression levels of proteins (cleaved-caspase 3, caspase 3, PI3K, p-PI3K, AKT, and p-AKT) were assessed using western blotting. (J) Corresponding quantitative analysis of TUNEL staining, scale = 50 μm (n = 3). (K) Corresponding quantitative analysis of western blotting (n = 3). Data are expressed as the mean ± SD (n = 3; ∗vs. NS group or Control group: ∗P < 0.05, ∗∗∗P < 0.001; # vs. all other groups: #P < 0.05, ##P < 0.01, ###P < 0.001).

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  Fig. 9. A schematic diagram shows that the Met-EVs -loaded CA hydrogel mediates the biological performance after MI, meanwhile protecting the cardiomyocytes from apoptosis and promoting angiogenesis.