Exercise-Primed Exosomes in an Injectable Hydrogel Promote Myocardial Repair via Angiogenesis and Ferroptosis Inhibition

Figures

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  Fig. 1. Characterization and distribution of exercise-derived serum exosomes. A, Schematic diagram illustrating the proposed mode of action and therapeutic strategy. B, TEM image showing the typical cup-shaped morphology of isolated exosomes. Scale bar = 100 nm. C, Size distribution profile of exosomes measured by DLS. D, Confocal microscopy image showing the incorporation of exosomes (green) within the hydrogel matrix. Scale bar = 50 μm. E, Cellular uptake of DiO-labeled exosomes (green) by HUVECs in vitro. Nuclei were stained with DAPI (blue). Scale bar = 25 μm. F, Western blot analysis of Exosome positive marker proteins TSG101, CD81, HSP70 and exosome negative marker protein Histone. G, Flowchart for isolating exosomes from the serum of exercise and sedentary mice. H, In vivo distribution and sustained release of exosomes in cardiac tissue of MI-Exos and MI-Exos@Gel-PEG treated mice. I, Quantitative analysis of exosomal fluorescence intensity in heart tissues (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 2. Preparation and characterization of the Gel-PEG hydrogel. A, Schematic illustration of the synthesis process of 4arm-PEG-NHS, 4arm-PEG-NH2, and the resulting Gel-PEG hydrogel. B, Diagram showing the spontaneous hydrogel formation through ester exchange reaction between 4arm-PEG-NHS and 4arm-PEG-NH2 under mixing conditions, and the subsequent encapsulation of exosomes. C, Representative SEM image showing the microporous structure of the fabricated Gel-PEG hydrogel. Scale bar = 5 μm. D, Photographic image of the formed Exos@Gel-PEG hydrogel. E, Strain-dependent oscillatory shear measurements of storage modulus (G′) and loss modulus (G″) for Gel-PEG and Exos@Gel-PEG hydrogels. F, In vitro degradation profiles of Gel-PEG hydrogels under different conditions. G, Compression modulus of Gel-PEG and Exos@Gel-PEG hydrogels. H, Swelling kinetics of Gel-PEG and Exos@Gel-PEG hydrogels in PBS buffer from 0 to 24 h. I, Cumulative release profiles of exosomes within and outside the 28-day hydrogel and PBS.

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  Fig. 3. Metabolomic and proteomic profiling of serum exosomes reveals exercise-induced alterations in mice. A, Metabolites extracted from serum exosomes of mice were subjected to principal component analysis (PCA). The 2D score plot displays metabolic repertoires of Sed and Exe mice. Each point represents an individual sample, and ellipses indicate 95% confidence intervals. B, Volcano plot illustrating the metabolomic differences between groups. The y-axis represents −log10(P-value) and the x-axis represents log2 (fold change). Each point corresponds to a metabolite, with larger points indicating higher variable importance in the projection values. C, Heatmap of differentially abundant metabolites between Sed and Exe groups, showing distinct clustering patterns of exosomal metabolites. D, KEGG pathway enrichment analysis of differentially expressed metabolites, highlighting the most significantly enriched metabolic pathways. E, GO enrichment analysis based on the differentially abundant metabolites, showing key enriched biological processes, molecular functions, and cellular components. F, PCA of the serum exosome proteome from Sed and Exe mice. Each point represents an individual…

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  Fig. 4. Exos@Gel-PEG ameliorates myocardial infarction by attenuating fibrosis and pathological remodeling. A, Schematic diagram illustrating the isolation of serum exosomes from exercised mice and the therapeutic procedure for MI. B, Representative echocardiographic images of hearts from Sham, MI, Exo, Gel-PEG, and Exos@Gel-PEG groups (n = 5). C, Quantitative analysis of EF and FS measured by echocardiography across all experimental groups (n = 5). D, Masson's trichrome staining of cardiac tissue sections showing collagen deposition (blue) and fibrosis extent in each group (n = 6). E, Statistical analysis of cardiomyocyte cross-sectional area based on WGA staining (n = 6). F, Histological evaluation by HE staining and Masson's trichrome staining of myocardial tissues from each group (n = 6). Scale bars = 1 mm (overview), 100 μm (magnified views). G, Representative images of WGA-stained heart sections indicating cardiomyocyte size. Scale bar = 10 μm ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. Data were presented as the mean ± SD and analyzed by using one-way ANOVA, Tukey's multiple comparisons test. (For interpretation of the references to colour in this figure legend, the reader is referred…

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  Fig. 5. Exos@Gel-PEG promotes post-infarction angiogenesis. A, Schematic illustration of the proposed mechanism whereby Exos@Gel-PEG enhances vascular endothelial cell regeneration and functional revascularization following myocardial infarction. B, Representative immunofluorescence images of heart sections from the MI, Exo, Gel-PEG, and Exos@Gel-PEG groups. Nuclei were stained with DAPI (blue), endothelial cells were labeled with CD31 (green), and cardiomyocytes were marked with cTnT (red). Scale bar = 50 μm. The upper and lower panels show the border zone and the infarct zone, respectively. C, Quantitative analysis of CD31+ and cTnT+ area in the border zone, presented as fold change relative to the MI group (n = 5). D, Quantitative analysis of CD31+ and cTnT+ area in the infarct zone, expressed as fold change relative to the MI group (n = 5). E, Western blot analysis of p-eNOS, eNOS, p-AKT, and AKT expression in cardiac tissues from each treatment group. F, Densitometric quantification of p-eNOS/eNOS and p-AKT/AKT ratios (n = 3). ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. Data were presented as the mean ± SD and analyzed by using one-way ANOVA, Tukey's multiple comparisons test. (For…

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  Fig. 6. Exos@Gel-PEG promotes endothelial cell proliferation and differentiation. A, Representative immunofluorescence images of EdU staining in HUVECs from Control, Exo, Gel-PEG, Exos@Gel-PEG and Exos@Gel-PEG + L-NAME. Nuclei were counterstained with DAPI (n = 3). Scale bar = 30 μm. B, Representative images of scratch wound healing assay (upper panel), transwell migration assay (middle panel), and aortic ring sprouting assay (lower panel) in HUVECs treated under different conditions. Scale bar = 100 μm. C, Quantitative analysis of the percentage of EdU-positive cells in each group. D, Quantification and relative distance of HUVECs wound healing (n = 5). E, Statistical analysis of the number of migrated cells in the transwell migration assay (n = 3). ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. Data were presented as the mean ± SD and analyzed by using one-way ANOVA, Tukey's multiple comparisons test.

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  Fig. 7. Exos@Gel-PEG inhibits cardiomyocyte ferroptosis after myocardial infarction. A, Schematic diagram illustrating the proposed mechanism by which Exos@Gel-PEG attenuates ferroptosis in cardiomyocytes. B, Representative images of high-sensitivity Prussian blue staining for iron deposition in cardiac tissues from MI, Exo, Gel-PEG, and Exos@Gel-PEG groups. Scale bar = 50 μm. The upper and lower panels show the border zone and the infarct zone, respectively. C, Transmission electron microscopy (TEM) images of cardiomyocytes in the border zone from Sham, MI, Exo, Gel-PEG, and Exos@Gel-PEG groups. Scale bar = 1 μm. D, Quantitative analysis of cardiac iron content, expressed as fold change relative to the Sham group (n = 6). E, Quantitative analysis of cardiac malondialdehyde level, expressed as fold change relative to the Sham group (n = 6). F, Western blot analysis of xCT, GPX4, FTH1, and GAPDH protein expression in cardiac tissues. G-I, Densitometric quantification of xCT/GAPDH (G), GPX4/GAPDH (H), and FTH1/GAPDH (I) ratios (n = 3). ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. Data were presented as the mean ± SD and analyzed by using one-way ANOVA, Tukey's multiple comparisons test. (For…