The enhanced effect and underlying mechanisms of mesenchymal stem cells with IL-33 overexpression on myocardial infarction

Figures

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  Fig. 1. Isolation and identification of MSCs. a MSC morphology was observed after 4–6 days of culture. Bar, 500 μm (left), 200 μm (right). b Characterization of MSCs by flow cytometry with CD105, CD73, CD90, CD29, CD45, and CD11b/c antibodies (red open histogram). Green open histogram represents the control

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  Fig. 2. Detection of the IL-33 expression level. Real-time PCR was used to determine the expression of IL-33 in MSCs (a), cardiomyocytes (CMs) (b), and cardiac fibroblasts (CFs) (c). *P < 0.05, NS not significant, as indicated, ANOVA. Each experiment was performed three times

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  Fig. 3. Construction and verification of the plasmid including IL-33. a Morphology of MSCs infected with lentivirus observed under an inverted fluorescence microscope (left panel, bright field; right panel, fluorescence field). Bar, 200 μm. Real-time PCR (b) and ELISA (c) were used to determine the expression of IL-33 in MSCs infected with different lentivirus. ***P < 0.001, as indicated, ANOVA. Each experiment was performed three times

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  Fig. 4. Evaluation of survival, proliferation, and the expression level of immunoregulatory molecules of IL33-MSCs. MSCs were transfected with Vector-MSCs or IL33-MSCs for 48 h. Apoptosis was analyzed by Annexin V-PE/7-AAD staining and flow cytometry. A scatter diagram of apoptosis and histogram of apoptosis are shown in a. b, c IL-33 had no influence on MSC proliferation and viability as determined by EdU and CCK 8 respectively (n = 6). d Real-time PCR was used to determine the expression of IDO and COX2 in MSCs transfected with different plasmids (n = 4). e ELISA was used to determine the protein level of pGE2 in MSCs transfected with different plasmids (n = 4). *P < 0.05, ***P < 0.001, as indicated, ANOVA. Each experiment was performed three times

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  Fig. 5. IL33-MSCs alter T cell proliferation and macrophage M1/M2 polarization in vitro. a Proliferation of T cells was measured by EdU+ using flow cytometry. b The percent of CD45+ immune cells used for macrophage polarization analysis. c The flow cytometry plots and analyses of the percentages of CD68+ macrophages and the M1 marker iNOS (d) and M2 marker CD206 (e) in cells co-cultured with different MSCs. *P < 0.05, ***P < 0.001, as indicated, ANOVA. Each experiment was performed three times

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  Fig. 6. Inflammatory level at different time points after MI. Rats were sacrificed at different times following MI surgery. a Flow cytometry analysis of T cell populations and subtype polarization from the spleen and blood 3, 5, 7, and 14 days after MI. Real-time PCR showed transcription factors of T cell subtype (b) and inflammatory factors (c) from the heart border and infarct zone, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001, as indicated, ANOVA. Each experiment was performed three times

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  Fig. 7. Echocardiography and histological analysis of heart function. a Representative echocardiography of rats following the PBS, Vector-MSCs and IL33-MSC injection for 28 days. b Quantitative analysis of the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic diameter (LVEDD), and left ventricular end-systolic diameter (LVESD) in different groups. c Representative Masson trichrome staining of the explanted heart showed a reduced fibrosis level in the IL33-MSC injection group (n = 4). Bar, 1 mm. d Percentage of the fibrotic area calculated and averaged by ImageJ software (n = 4)

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  Fig. 8. Histological examination of the inflammation level. a Fluorescent photographs of surviving MSCs with red fluorescence in vivo. b Representative H&E staining of explanted hearts at 5 days after MI showed a reduced inflammatory level in the IL33-MSCs group (n = 4). Bar, 100 μm. c Representative immunofluorescence staining of macrophages, the M2 marker CD68 (green) and CD206 (red), and the M1 marker CD68 (green) and iNOS (red). Bar, 100 μm. d Real-time PCR analysis of inflammatory factor in infarct zone and bore zone of hearts with different MSC injection. n = 4, *P < 0.05, as indicated, ANOVA