Empagliflozin-pretreated mesenchymal stem cell-derived extracellular vesicles facilitate cardiac repair via AGGF1

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  Fig. 1. EMPA exhibits pro-proliferative, pro-migratory, and anti-senescence effects on MSCs. (a) Flow cytometry analysis of surface markers on MSCs. (b) CCK-8 assay for cell viability of MSCs treated with different concentrations of EMPA at 24 h and 48 h (n = 3). (c-d) JC-1 fluorescence staining for mitochondrial membrane potential and quantitative analysis of the red/green fluorescence intensity ratio to measure mitochondrial depolarization in MSCs treated with different concentrations of EMPA for 48 h (n = 3) (scale bar = 50 μm). (e, i) EdU immunostaining for cell proliferation markers and quantitative analysis of EdU-positive cell ratio in the Ctrl group and EMPA group (n = 5) (scale bar = 50 μm). (f, j) Scratch assay for cell migration ability and quantitative analysis of migration area in the Ctrl group and EMPA group (n = 5) (scale bar = 500 μm). (g, k) Flow cytometry for cell cycle distribution and quantitative analysis of cell percentages in each phase in the Ctrl group and EMPA group (n = 3). (h, l) β-galactosidase staining for senescence degree and quantitative analysis of cumulative optical density value in the Ctrl group and EMPA group (n = 5) (scale bar = 200 μm). (m)…

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  Fig. 2. Preparation, characterization, and cellular uptake of EVs. (a) Workflow for the preparation of MSC-EVs and EMPA-EVs by differential ultracentrifugation (created with BioGDP.com). (b) Transmission electron microscopy showing that MSC-EVs and EMPA-EVs have similar typical cup-shaped structures (Scale bars: 500 nm (upper), 100 nm (lower)). (c-d) Nanoparticle tracking analysis for particle size and concentration of MSC-EVs and EMPA-EVs. (e-f) Comparisons of protein concentration and the particle-to-protein ratio of MSC-EVs and EMPA-EVs. (g) Western blot for characteristic marker proteins of MSC-EVs and EMPA-EVs (n = 3). (h-j) Dil-EVs were co-cultured with H9c2 cells, HUVECs, and RAW264.7 macrophages for 24 h. Actin-Tracker Green was used to stain the cytoskeleton and DAPI to stain the nucleus, demonstrating successful uptake of EVs by cells (scale bar = 50 μm). Continuous variables are shown as mean ± SEM (ns: Not significant)

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  Fig. 3. EMPA-EVs can inhibit the apoptosis of H9c2 cells and improve their mitochondrial function, while EMPA-EVs can also promote therapeutic angiogenesis. (a-b) Quantitative analysis of apoptosis levels and apoptosis rates of H9c2 cells in the control group, MSC-EVs and EMPA-EVs treatment groups under H/SD conditions by flow cytometry (n = 6). (c, f-g) Western blot and quantitative analysis of apoptosis-related protein expression levels in H9c2 cells of the control group, MSC-EVs and EMPA-EVs treatment groups (n = 3). (d, h) JC-1 fluorescence staining for quantitative analysis of mitochondrial membrane potential and the ratio of red to green fluorescence intensity as a measure of mitochondrial depolarization in the control group, MSC-EVs and EMPA-EVs treatment groups (n = 3) (scale bar = 50 μm). (e, i) TMRE staining for quantitative analysis of mitochondrial function and mean fluorescence intensity in the control group, MSC-EVs and EMPA-EVs treatment groups (DAPI staining for nucleus) (n = 3) (scale bar = 50 μm). (j, m) Quantitative analysis of EdU staining for proliferation cells and the proportion of EdU-positive cells in the control group, MSC-EVs and EMPA-EVs treatment groups under…

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  Fig. 4. EMPA-EVs facilitate the transformation of macrophages into a reparative phenotype. (a-f) Flow cytometry detection and quantitative analysis of the proportion of CD80-positive and CD206-positive cells in RAW264.7 macrophages of the control group, MSC-EVs and EMPA-EVs treatment groups under LPS-induced stimulation (n = 3). (g-k) RT-qPCR detection of the expression of pro-inflammatory and pro-reparative related genes in macrophages of the control group, MSC-EVs and EMPA-EVs treatment groups (n = 3 for each group). (l) Kit colorimetric assay for the detection of the level of NO oxidation products in the cell supernatant of the control group, MSC-EVs and EMPA-EVs treatment groups at 12 h and 24 h (n = 3). (m-o) Western blot detection and quantitative analysis of the protein expression of pro-inflammatory (iNOS) and pro-reparative (ARG-1) related genes in the control group, MSC-EVs and EMPA-EVs treatment groups (n = 3). Continuous variables are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 5. EMPA-EVs reprogram macrophages toward a reparative phenotype in rats. (a)Schematic diagram of the in vivo experimental process (created with BioGDP.com). (b) TTC staining to verify myocardial changes 24 h after myocardial infarction modeling in rats, red represents viable myocardial tissue and grayish-white represents necrotic myocardial tissue. (c) Quantitative analysis of myocardial infarction area in rats of the myocardial infarction group and the sham operation group (n = 3). (d) The optical imaging system in small animals showed the distribution of EVs or Dir-EVs in different groups of rats at different times after surgery. (e)Representative images of immunohistochemistry analysis for IL-1β and CD206 expression at 3 days in different groups and (h-i) quantification analysis of IOD (n = 5) (Scale bar = 50 μm). (f-g, j-k) Representative images of iNOS and CD163 immunofluorescence staining in myocardial tissues of rats from different groups on the 3rd day after surgery and the corresponding quantitative analysis of average fluorescence intensity (n = 5). Continuous variables are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 6. EMPA-EVs reduce infarct size and enhance cardiac function in MI rats. (a) Representative images of the parasternal short-axis view of the left ventricle by echocardiography on the 28th day after surgery in sham-operated, control, MSC-EVs and EMPA-EVs treated groups. (b-c) LVEF and LVFS evaluated by echocardiography in different groups (n = 3). (d, f) Masson staining showing the degree of infarction in the border and infarct zones and quantitative analysis of the infarct area in each group (muscle fibers are red, collagen fibers are blue) (n = 3) (scale bars = 2000 μm, 200 μm). (e, g) Sirius red staining showing the degree of fibrosis in the border and infarct zones and quantitative analysis of collagen fibers in each group (muscle fibers are yellow, collagen fibers are red) (n = 3) (scale bars = 2000 μm, 200 μm). (h) Hematoxylin-Eosin staining showing the degree of inflammatory cell infiltration in the border and infarct zones (n = 3) (scale bar = 500 μm). (i) Hematoxylin-Eosin staining of major organs, including lung, liver, spleen, and kidney tissues from rats in each group (scale bar = 50 μm). Continuous variables are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001,…

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  Fig. 7. Proteomics reveals the key role of AGGF1, and EMPA-EVs regulate macrophage transition through the AGGF1/AKT axis. (a) Heatmap showing the representative protein expression with significant differences between the MSC-EVs and EMPA-EVs groups. (b) Volcano plot showing the significantly differentially expressed proteins between the MSC-EVs and EMPA-EVs groups (threshold: 1.2-fold change, P < 0.05). (c) The biological process enrichment results for up-regulated and overall differentially expressed genes were visualized using a Gene Ontology functional enrichment bubble plot. (x-axis: The fold enrichment of differentially expressed genes, y-axis: The GO term descriptions; the size of the dots in the figure represents the number of differentially expressed proteins enriched, and the color represents the P value). (d-e) Western blot and quantitative analysis confirm that EMPA-EVs are rich in AGGF1 protein expression. (f) RT-qPCR detection of the expression of AGGF1 in control-overexpressing and AGGF1-overexpressing MSCs (n = 3). (g) RT-qPCR detection of the expression of AGGF1 in control-silenced and AGGF1-silenced EMPA-MSCs (n = 3). (h-k) Western blot analysis and quantification of…

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  Fig. 8. EMPA-EVs inhibit cardiomyocyte apoptosis and enhance therapeutic angiogenesis through the AGGF1/AKT axis. (a-d) Quantitative analysis of mitochondrial function and mean fluorescence intensity of H9c2 cells co-cultured with MSC-EVsAGGF1−OE, MSC-EVsNC−OE, EMPA-EV siAGGF1, and EMPA-EVsiNC groups, respectively, as detected by TMRE staining (n = 5) (scale bar = 100 μm). (e-h) Quantitative analysis of cardiomyocyte apoptosis levels and apoptosis rates in each group as detected by flow cytometry (n = 4) (scale bar = 100 μm). (i-k) Western blot and quantitative analysis of AGGF1, apoptosis-related proteins, AKT, and p-AKT protein expression levels in each group (n = 3). (m-p) Tube formation assay to detect the formation of vascular networks and quantitative analysis of tube length in each group after co-culture with MSC-EVsAGGF1−OE, MSC-EVsNC−OE, EMPA-EVs siAGGF1, and EMPA-EVssiNC groups and HUVECs (n = 5) (scale bar = 200 μm). (q-t) EdU staining to detect proliferating endothelial cells in each group and quantitative analysis (n = 4) (scale bar = 50 μm). (u-x) Western blot and quantitative analysis of AGGF1, Bcl-2, AKT, and p-AKT protein expression levels in endothelial cells of each…

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  Fig. 9. AGGF1 mediates the cardioprotective effects of EMPA-EVs in myocardial infarction and workflow diagram of this research. (a) Representative images of echocardiography on the 28th day after surgery in MI + PBS group, EMPA-EVsiNC and EMPA-EVsiAGGF1groups. (b-c) LVEF and LVFS evaluated by echocardiography in different groups (n = 3). (d-e) Masson staining showing the degree of infarction in the border and quantitative analysis of the infarct area in each group (n = 3) (scale bars = 2000 μm). (f) MSC-EVs pretreated with empagliflozin exerted myocardial protective functions after myocardial infarction at least partially by activating the AGGF1/AKT signaling pathway to synergistically inhibit myocardial cell apoptosis, regulate the transformation of macrophages to pro-repair phenotypes, and promote angiogenesis