Small Extracellular Vesicles from Young Healthy Human Plasma Inhibit Cardiac Fibrosis After Myocardial Infarction via miR-664a-3p Targeting SMAD4

Figures

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  Figure 1. Characterization and internalization of sEVs. (A) Transmission electron microscopy imaging of young healthy human plasma-derived sEVs (scale bar = 100 nm). (B) Particle size distribution analysis using NanoSight tracking analysis. (C) Western blotting analysis of the sEVs-associated markers CD63, CD81 and TSG101. (D) Dil-labeled sEVs derived from human plasma were taken up by CFs (scale bar = 25 µm). (E) Six hours after injecting Dil-labeled sEVs (red) into mouse cardiac tissue, immunofluorescence staining and identification were performed on the heart tissues (n = 5, magnification: 20×; scale bar = 200 μm). Data are presented from three independent experiments.

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  Figure 2. Young-sEVs inhibit cardiac fibrosis after MI. (A) The flowchart of experimental design in vivo. (B) Representative echocardiograms of mouse hearts in each group at 28 days post-MI. (C) Echocardiographic measurements of EF, FS, LVIDs and LVIDd (n = 6). (D) Masson’s trichrome staining of representative cardiac cross-sections and the collagen volume fraction (n = 6, magnification: 20 ×; scale bar = 100 μm). (E) Western blotting analysis and quantification of Col-I, Col-III and α-SMA protein levels in cardiac tissues (n = 3). (F) qRT-PCR analysis of the mRNA expressions of Col-I, Col-III and α-SMA in cardiac tissues (n = 3). Data were presented as mean ± SD. **P < 0.01, ***P < 0.001, compared with the sham group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the MI group. &P < 0.05, &&P < 0.01, &&&P < 0.001, compared with the MI + Old-sEVs group.

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  Figure 3. Young-sEVs inhibit fibrosis of TGF-β1-induced CFs in vitro. (A-C) Proliferation of CFs was assessed using the EdU assay and CCK-8 assay. For the EdU experiment, red fluorescence indicated proliferating cells, while blue fluorescence denoted the cell nuclei. The proportion of EdU-positive CFs and results of CCK-8 assay were shown in panel (scale bar = 100 μm). (D) The Wound healing assay and Transwell assay were used to determine the migration of CFs. The two methods were evaluated after incubation for 24 hours and the relative migration speed were measured (scale bar = 100 μm). (E) Western blotting analysis and quantification of Col-I, Col-III and α-SMA protein levels in CFs. (F) qRT-PCR analysis of the mRNA expressions of Col-I, Col-III and α-SMA in CFs. Data are presented from three independent experiments. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the negative control (NC) group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the TGF-β1 group. &P < 0.05, &&P < 0.01, &&&P < 0.001, compared with the TGF-β1 + Old-sEVs group.

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  Figure 4. Identification of miR-664a-3p as the main effector in Young-sEVs against cardiac fibrosis. (A) Heatmap of upregulated and downregulated miRNAs in Young-sEVs compared to Old-sEVs. Select differentially expressed miRNAs (marked in red) for further validation. (B) Further qRT-PCR validation experiments comparing 10 young healthy individuals with 10 old healthy individuals revealed that 4 out of the top 15 highly expressed miRNAs identified in sequencing showed significantly higher expression levels (n = 10). (C) qRT-PCR verified the expression of the above 4 miRNAs in TGF-β1-induced CFs (n = 3). (D) qRT-PCR was further utilized to confirm the expression of the above 4 miRNAs in cardiac tissue of mice after MI (n = 3). Data were presented as mean ± SD. ***P < 0.001, compared with the negative control (NC) group or sham group or old group. ##P < 0.01, compared with the TGF-β1 group or MI group.

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  Figure 5. The function of miR-664a-3p mimic in inhibiting fibrosis of TGF-β1-induced CFs. (A) The expression of miR-664a-3p in CFs transfected with miR-664a-3p mimic, inhibitor or negative control (NC) was detected by qRT-PCR. (B-D) The proliferation of CFs transfected with the miR-664a-3p mimic was evaluated using EdU and CCK-8 cell proliferation assays. The proportion of EdU-positive CFs and results of CCK-8 assay were shown in panel (scale bar = 100 μm). (E) The migration of CFs transfected with the miR-664a-3p mimic was assessed using the Wound healing assay and Transwell migration assay. The two methods were evaluated after incubation for 24 hours and the relative migration speed were measured (scale bar = 100 μm). (F) Western blotting analysis and quantification of Col-I, Col-III and α-SMA protein levels in CFs after transfection with miR-664a-3p mimic. (G) qRT-PCR analysis of the mRNA expressions of Col-I, Col-III and α-SMA in CFs transfected with miR-664a-3p mimic. Data are presented from three independent experiments. Data were presented as mean ± SD. **P < 0.01, ***P < 0.001, compared with the negative control (NC) group. &P < 0.05, &&P < 0.01, &&&P < 0.001, compared with the…

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  Figure 6. The miR-664a-3p inhibitor further aggravates the fibrosis of TGF-β1-induced CFs. (A-C) The proliferation of CFs transfected with the miR-664a-3p inhibitor was evaluated using EdU and CCK-8 cell proliferation assays. The proportion of EdU-positive CFs and results of CCK-8 assay were shown in panel (scale bar = 100 μm). (D) The migration of CFs transfected with the miR-664a-3p inhibitor was assessed using the Wound healing assay and Transwell migration assay. The two methods were evaluated after incubation for 24 hours and the relative migration speed were measured (scale bar = 100 μm). (E) Western blotting analysis and quantification of Col-I, Col-III and α-SMA protein levels in CFs after transfection with miR-664a-3p inhibitor. (F) qRT-PCR analysis of the mRNA expressions of Col-I, Col-III and α-SMA in CFs transfected with miR-664a-3p inhibitor. Data are presented from three independent experiments. Data were presented as mean ± SD. **P < 0.01, ***P < 0.001, compared with the negative control (NC) group. &&P < 0.01, &&&P < 0.001, compared with the TGF-β1 + NC inhibitor group.

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  Figure 7. The suppression of miR-664a-3p expression in Young-sEVs abolishes its function in inhibiting cardiac fibrosis in vivo. (A) Representative echocardiograms of mouse hearts in each group at 28 days post-MI. (B) Echocardiographic measurements of EF, FS, LVIDs and LVIDd (n = 6). (C) Masson’s trichrome staining of representative cardiac cross-sections and the collagen volume fraction (n = 6, magnification: 20 ×; scale bar = 100 μm). (D) Western blotting analysis and quantification of Col-I, Col-III and α-SMA protein levels in cardiac tissues (n = 3). (E) qRT-PCR analysis of the mRNA expressions of Col-I, Col-III and α-SMA in cardiac tissues (n = 3). Data were presented as mean ± SD. **P < 0.01, ***P < 0.001, compared with the sham group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the MI group. &P < 0.05, &&&P < 0.001, compared with the MI + NC inhibitor-Young-sEVs group.

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  Figure 8. Continued.

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  Figure 8. SMAD4 is identified as the target of miR-664a-3p. (A) Potential targets of miR-664a-3p in the TargetScan 8.0, miRBase, and miRWalk databases. (B) The expression of SMAD4 mRNA in CFs after transfection with miR-664a-3p mimic, inhibitor, or their respective controls. (C) The protein expression levels of SMAD4 in CFs after transfection with miR-664a-3p mimic, inhibitor, or their respective controls. (D) Diagram showing the predicted binding sites of miR-664a-3p on SMAD4 3′-UTR, with the sequences marked in red indicating mutations in the miR-664a-3p matching sequence. (E) After co-transfecting CF cells with mimic-NC, miR-664a-3p mimic, inhibitor-NC, or miR-664a-3p inhibitor and plasmids containing either wild-type or mutant-type 3′-UTR of SMAD4, luciferase activity was detected using a dual-luciferase reporter assay system. (F) Western blotting analysis and quantification of SMAD4 protein levels in TGF-β1-induced CFs treated with Young-sEVs (n = 3). (G) The expression of SMAD4 mRNA in TGF-β1-induced CFs treated with Young-sEVs by qRT-PCR analysis (n = 3). (H) Western blotting analysis and quantification of SMAD4 protein levels in mouse cardiac tissue of post-MI treated with…

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  Figure 9. Continued.

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  Figure 9. The SMAD4 protein can block the anti-fibrotic effect of miR-664a-3p. (A) Western blotting analysis for the SMAD4 protein expression using different amounts of transfected plasmid DNA. (B-D) The proliferation of CFs transfected with the SMAD4 overexpression plasmid was evaluated using EdU and CCK-8 cell proliferation assays. The proportion of EdU-positive CFs and results of CCK-8 assay were shown in panel (scale bar = 100 μm). (E) The migration of CFs transfected with the SMAD4 overexpression plasmid was assessed using the Wound healing assay and Transwell migration assay. The two methods were evaluated after incubation for 24 hours and the relative migration speed were measured (scale bar = 100 μm). (F) Western blotting analysis and quantification of Col-I, Col-III and α-SMA protein levels in CFs after transfection with the SMAD4 overexpression plasmid. (G) qRT-PCR analysis of the mRNA expressions of Col-I, Col-III and α-SMA in CFs transfected with the SMAD4 overexpression plasmid. Data are presented from three independent experiments. Data were presented as mean ± SD. **P < 0.01, ***P < 0.001, compared with the negative control (NC) group. #P < 0.05, ##P < 0.01, ###P < 0.001,…