Influence of secretome from porcine cardiosphere-derived cells on porcine macrophage polarization and their possible implications for cardiac remodeling post-myocardial infarction in vitro

Figures

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  FIGURE 1. Workflow of materials and methods. (A) Isolation and bioinformatic analysis of secretome from CDCs. (B) Differentiation and treatment of macrophages with S-CDCs (C) Functional assays with conditioned medium from macrophages: (C1) Interaction with endothelial cells and (C2) interaction with cardiac fibroblasts. CDCs: cardiosphere-derived cells; S-CDCs: secretome released by cardiosphere-derived cells. HUVECs: Human umbilical vein endothelial cells. PCF: porcine cardiac fibroblasts. This figure was created with BioRender.com.

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  FIGURE 2. Analysis of macrophage markers and cytokine expression following S-CDC treatment. Throughout the figure: untreated M1 macrophages (orange), M1 treated with S-CDCs (light orange), M2 macrophages (purple) and untreated M2 treated with S-CDCs (light purple). (A) Percentage of cells positive for SLA-II, CD206, and CD163 markers as measured by flow cytometry (n = 9). (B) Mean fluorescence intensity (MFI) of SLA-II, CD206, CD163 measured by flow cytometry (n = 9). (C) Quantitative PCR analysis of IL10, TNFA, and VEGFA gene expression levels (n = 4). Statistical analysis was conducted using Student’s t-test. Data are presented as mean values, and error bars indicate SEM. Statistical significance is denoted by asterisks: *p < 0.05 **p < 0.005.

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  FIGURE 3. Effect of S-CDCs treated macrophages on HUVECs. For the whole figure: untreated M1 macrophages (orange), M1 treated with S-CDCs (light orange), untreated M2 macrophages (purple) and M2 treated with S-CDCs (light purple). (A) Percentage of wound closure was measured by wound healing assay (n = 4). (B) Migration assay results (n = 4). (C) Tube formation assay measured by tube length pixels (n = 5). Statistical analysis was conducted using Student’s t-test. Data are presented as mean values, and error bars indicate SEM. Statistical significance is denoted by asterisks: *p < 0.05 **p < 0.005.

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  FIGURE 4. Effect of S-CDC-treated macrophages on PCFs. For the figure panels: untreated M1 macrophages (orange), M1 treated with S-CDCs (light orange), untreated M2 macrophages (purple) and M2 treated with S-CDCs (light purple). (A) Percentage of wound closure was measured by wound healing assay (n = 5). (B) Migration assay results (n = 4). (C) Gene expression levels of ACTA2, COL1A1 and COL3A1 measured by qPCR (n = 3). Statistical analysis was conducted using Student’s t-test. Data are presented as mean values, and error bars indicate SEM. Statistical significance is denoted by asterisks: *p < 0.05 **p < 0.005.

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  FIGURE 5. Interaction network between cytokines and macrophages. The network illustrates cytokines known to polarize macrophages towards M1 or M2 phenotypes. Genes encoding cytokines in the S-CDC are highlighted in shades of red, with color intensity representing relative abundance (pale for low abundance to deep red for high abundance). White nodes indicate cytokines not detected in the secretome.