Extracorporeal cardiac shock wave therapy modulates post-infarction neovascularization via CAC-mediated pro-angiogenic effects and modulation of the S100A4/CSF2/FOXO1 protein network

Figures

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  Figure 1. Validation of CACs isolation and optimization of shock wave parameters. (A) Isolatio n and culture of Coronary Angiogenic Cells (CACs) (detailed in method 2.2) Left diagram: The primary cells were round and suspended. Picture on the right: The adherent cells were passaged and amplified, showing a typical spindle-shaped and fibroblast-like morphology (ruler = 2 μm). (B) Flow cytometry was used to detect the expression of CACs surface markers CD133 and CD34, and Blank was an unstained blank control; CD133/CD34 was single staining group; CD133-merge/CD34-merge was the staining group, showing that the positive rate of CD133 was 91.6%, and the positive rate of CD34 was 96.8% (detailed in method 2.3). (C) Seismic wave treatment CACs with different energy flux densities were divided into control group (CACs), 0.03,0.06,0.09,0.12,0.15,0.18 mJ/mm2 seismic wave treatment groups (scale=2μm) (detailed in method 2.4). (D) CCK-8 method was used to detect the cell proliferation activity of CACs after shock wave treatment (detailed in method 2.6). Statistical analysis was performed using t-test or one-way analysis of variance combined with Tukey post hoc test. Data were expressed as Mean ± SD, n…

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  Figure 2. In vitro validation of CSWT enhancing the pro-angiogenic function of CACs via upregulation of pro-angiogenic factor expression. (A) Tube formation assay was used to detect the effect of CSWT on the angiogenesis ability of CACs and RUVECs co-culture system. Left: control CACs and RUVEC co-culture (CACs + RUVEC); right: CSWT treated CACs and RUVEC co-culture (CACs + RUVEC + CSWT). The number of vascular nodes (Vascular nodes), the number of vascular networks (Number of meshes) and the length of main branches (master segments length) were counted in the histogram (scale: 2μm) (method 2.8 details). (B) Transwell migration assay was used to detect the effect of CSWT on the migration ability of CACs cells in the co-culture system. Left: control group; right: CSWT treatment group, DAPI staining showed the nucleus (blue) (method 2.7 details) (scale: 2μm). (C) RT-qPCR was used to detect the mRNA expression levels of angiogenesis-related factors VEGF, SDF-1, bFGF and HGF in CACs after CSWT treatment, with GAPDH as an internal reference (method 2.12 details). (D) Western blot was used to detect the protein expression levels of VEGF, HGF, bFGF and SDF-1 in CACs after CSWT treatment, with…

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  Figure 3. Extracorporeal cardiac shock wave therapy remodels post-infarction myocardium in rats. (A) The overall process and intervention plan of animal experiments. The above is a schematic diagram of the surgical steps constructed for the myocardial infarction model; the following was the experimental time axis: MI model was constructed at 0d, and CSWT intervention was performed with parameters of 1 Hz and 0.09 mJ/mm2 from the 5th day after operation, once every 4 days until the 21st day; samples were taken on the 22 nd day (see method 2.1 for details). (B) The weight change curve of mice in each group during the experiment was recorded in weeks (W). (C) TTC staining results and quantitative analysis of infarct size in myocardial tissue of mice in each group. The upper part was the normal control group, the middle part was the MI model group, and the lower part was the MI + CSWT intervention group. The histogram showed the percentage of myocardial infarction area (%) in the three groups, and CSWT intervention could significantly reduce the myocardial infarction area (see method 2.13). Statistical analysis was performed using t-test or one-way analysis of variance combined with Tukey post…

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  Figure 4. Extracorporeal cardiac shock wave therapy modulates CACs activation and promotes neovascularization. (A,B) Immunohistochemical staining was performed to detect the expression levels of VEGF (A) and HGF (B) in rat myocardial tissues of each group. The 15× magnification shows the overall view of myocardial tissue (Scale bar = 500 μm), and the 200× magnification presents the positive expression distribution in the infarcted area (red box), border zone (yellow box), and remote area (green box), respectively (Scale bar = 50 μm) (detailed in Method 2.9). (C,D) Semi-quantitative analysis results of VEGF (C) and HGF (D) protein expression levels (detailed in Method 2.15). (E,F) Immunohistochemical staining was used to detect the expression levels of bFGF (E) and SDF-1 (F) in rat myocardial tissues of each group. The 15× magnification shows the overall view of myocardial tissue (Scale bar = 500 μm), and the 200× magnification presents the positive expression distribution in the infarcted area (red box), border zone (yellow box), and remote area (green box), respectively (Scale bar = 50 μm) (detailed in Method 2.9). (G,H) Semi-quantitative analysis results of bFGF (G) and SDF-1 (H) protein…

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  Figure 5. (A) volcano plot showed the distribution of differentially expressed proteins between the two groups. The abscissa is the difference multiple (log₂FC), and the ordinate was the significant level (-log₁₀P). Red dots represent significantly up-regulated proteins, blue dots represent significantly down-regulated proteins, and gray dots are proteins with no statistical difference. (B) Boxplot shows the expression levels of all significantly different proteins in each group. The upper figure: group A (control group) VS group B (model group), the lower figure: group B (model group) VS group C (CSWT group). The ordinate is the protein quantitative signal value (NPX), and the abscissa is the name of each protein, showing the difference in expression between groups and statistically significant markers. (C) Heatmap cluster analysis of differentially expressed proteins, left figure: group A (control group) VS group B (model group), right figure: group B (model group) VS group C (CSWT group). The color gradient represents the protein expression level (red for high expression, blue for low expression), each row represents a protein, and each column represents a sample, clearly showing the…

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  Figure 6. Olink proteomics analysis reveals key molecules regulating CAC function following in vitro cardiac shock wave treatment. (A) GO function enrichment bubble diagram of differentially expressed proteins. The abscissa is the enrichment factor (Rich Factor, representing the degree of enrichment), and the ordinate is the GO entry (including biological processes, cell components, and molecular functions); the bubble size represented the number of proteins enriched to this item, and the color represented the significant P-value). (B) KEGG pathway enrichment bubble diagram of differentially expressed proteins. The abscissa is the enrichment factor, and the ordinate is the name of the KEGG signaling pathway; the bubble size represented the number of proteins enriched in the pathway, and the color represented the significant P-value. (C) Pearson correlation analysis heat map between differentially expressed proteins. The color gradient represents the correlation coefficient of protein expression (red is positively correlated, blue is negatively correlated, and the circle size and color depth reflect the degree of correlation. (D) Protein-protein interaction (PPI) network of differentially…

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  Figure 7. qPCR validation of key factor expression (as described in methods 2.12). (A) Gene set enrichment analysis (GSEA) of key target proteins. The enrichment intensity (NES value) of S100A4, CSF2 and FOXO1 in different signaling pathways was shown respectively, and the color gradient represented the enrichment significance (P-value). The results showed that the above molecules were significantly enriched in the PI3K-Akt signaling pathway and ECM-receptor interaction pathway. (B) RT-qPCR as used to detect the mRNA expression levels of CSF2, S100A4 and F0XO1 in CACs before and after CSWT treatment, and GAPDH was used as an internal reference. (C) in vivo animal level verification: RT-qPCR was used to detect the mRNA expression levels of CSF2, S100A4 and FOXO1 in myocardial tissue of Control group, MI model group and MI + CSWT intervention group, with GAPDH as internal reference. t-test or one-way analysis of variance combined with Tukey post hoc test was used for statistical analysis. The data were expressed as Mean ± SD, n = 3. ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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  Figure 8. CSWT promotes angiogenesis in CACs by modulating the S100A4/FOXO1/CSF2 network and its associated PI3K-Akt and ECM-receptor interaction pathways.