Extracorporeal Shock Wave‐Supported Adipose‐Derived Fresh Stromal Vascular Fraction Preserved Left Ventricular (LV) Function and Inhibited LV Remodeling in Acute Myocardial Infarction in Rat

Figure 1. Surface markers of EPC and MSC and fluorescent intensity of mitotracker staining in H9C2 cells undergoing menadione and SVF treatments. (a–n) Flow cytometry for endothelial progenitor cell (EPC), cardiac stem cell, and mesenchymal stem cell (MSC) surface markers. (o) Flow cytometric analysis (n = 6) showed that CD34 and CD271 were the clearest EPC and MSC surface markers for SVF, respectively. (p, 1–8) Illustrating the immunofluorescent (IF) microscopic finding of the mitotracker stain for identification of mitochondria in H9C2 cells in the normal control group (p, 1 and 2) and AD-SVF treated group (p, 3 and 4). The number of mitotracker emission positive cells (i.e., counted in five randomly chosen fields) was significantly increased after receiving SVF treatment, ∗ vs. †, p < 0.0001 (red symbols). (p, 5–8) Illustrating the IF microscopic finding of the mitotracker stain for identification of mitochondria in H9C2 cells in the menadione-treated group (p, 5 and 6) and menadione + AD-SVF treated group (p, 7 and 8). The number of mitotracker emission positive cells was significantly increased in menadione + SVF treatment than in menadione only, ‡ vs. §, p < 0.001 (green symbols).…

Figure 2. Coculture with SVF reduced 53BP1 nuclear distribution in H9C2 cells undergoing menadione treatment. (a–h) Illustrating the immunofluorescent (IF) microscopic finding for identification of positively stained 53BP1 in nuclei of the H9C2 cell with and without menadione and SVF treatment. The results showed that the 53BP1 protein was enriched in the nuclei of H9C2 cells undergoing menadione treatment as compared with those of the control cells (a vs. e). However, this enhanced expression of 53BP1 protein in nuclei by menadione was remarkably reduced undergoing the SVF treatment (e vs. g). (i) Nuclear fluorescent intensity of 53BP1 was calculated by Image J software from 5 random fields (n = 4/each group). Data were expressed as means ± SEM. ∗∗ indicated p < 0.01. AD-SVF = adipose-derived stromal vascular fraction.

Figure 3. Coculture with SVF decreased XRCC1 nuclear translocation in H9C2 cells undergoing menadione treatment and identification of fusion and fission after menadione treatment. (a–h) Illustrating the immunofluorescent (IF) microscopic finding for identification of positively stained XRCC1 protein in nuclei of the H9C2 cell with and without menadione and SVF treatment. The results showed that XRCC1 protein was notably enriched in the nuclei under menadione treatment (e) as compared with the control cells (a). On the other hand, however, in condition of H9C2 cells cocultured with AD-SVF, the distribution of XRCC1 in cytosol was notably promoted (g). (i) Fluorescent intensity of 53BP1 in nuclei was calculated by Image J software from 5 random fields (n = 4/each group). Analytical result: ∗ vs. other groups with different symbols (†, ‡), p < 0.001. (j) Nuclear area was calculated by Image J software from 5 random fields (n = 4/each group). Analytical result: ∗ vs. other groups with different symbols (†, ‡), p < 0.001. AD-SVF = adipose-derived stromal vascular fraction. (k–n) Illustrating the time courses of menadione induced the process of mitochondrial fusion/fission (from 0, 10, and 30 to…

Figure 4. Condensed collagen deposition area and apoptotic nuclei in LV myocardium by day 42 after AMI induction. (a–e) Sirius stain microscopy (200x, red) to identify the condensed collagen deposition area. (f) Analysis of collagen deposition area, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. (g–k) Immunohistochemistry (IHC, 100x, TUNEL assay) for apoptotic nuclei (gray). (l) Analysis of number of apoptotic nuclei, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). HPF = high-power field; SC = sham control; AMI = acute myocardial infarction; SW = shock wave; SVF = adipose-derived stromal vascular fraction.

Figure 5. Histopathology for infarcted and fibrotic areas in LV myocardium by day 42 after AMI induction. (a–e) H&E staining (100x) to identify the infarct area. (f) Analysis of the infarct area, ∗ vs. other groups with different symbols (‡, ‡, §, ¶) , p < 0.0001. (g–k) Masson's trichrome stain (400x) to identify the fibrotic area in LV myocardium. (l) Analysis of the fibrotic area, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). HPF = high-power field; SC = sham control; AMI = acute myocardial infarction; SW = shock wave; SVF = adipose-derived stromal vascular fraction.

Figure 6. Identification of cardiac stem cells in LV myocardium by day 42 after AMI induction. (a–e) IHC (400x) to identify c-Kit+ cells (gray). (f) Analysis of number of c-Kit+ cells, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. (g–k) IHC microscopy (400x) for Sca-1+ cells (gray). (l) Analysis of number of Sca-1+ cells, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). HPF = high-power field; SC = sham control; AMI = acute myocardial infarction; SW = shock wave; SVF = adipose-derived stromal vascular fraction.

Figure 7. Expression of endothelial cells in LV myocardium by day 42 after AMI induction. (a–e) Immunofluorescence (IF; 400x) to identify CD31+ cells (green). (f) Analysis of numbers of CD31+ cells, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. (g–k) IF microscopy (400x) for the von Willebrand factor+ (vWF+) cells (green). (l) Analysis for the number of vWF+ cells, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. Red color in (d, e, j, and k) indicates the implanted MSCs in LV myocardium. All statistical analyses were performed by one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). HPF = high-power field; SC = sham control; AMI = acute myocardial infarction; SW = shock wave; SVF = adipose-derived stromal vascular fraction.

Figure 8. Expression of endothelial progenitor cells in LV myocardium by day 42 after AMI induction. (a–e) IF microscopy (400x) to identify CXCR4+ cells (green). (f) Analysis for the number of CXCR4+ cells, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. (g–k) IF microscopy (400x) to identify stromal cell-derived factor- (SDF-) 1α+ cells (green color). (l) Analysis of the number of SDF-1α+ cells, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. Red color in (d, e, j, and k) indicates the implanted MSCs in LV myocardium. All statistical analyses were performed by one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). HPF = high-power field; SC = sham control; AMI = acute myocardial infarction; SW = shock wave; SVF = adipose-derived stromal vascular fraction.

Figure 9. Identification of DNA damage and inflammatory biomarkers in LV myocardium by day 42 after AMI induction. (a–e) IF microscopy (400x) to identify γ-H2AX+ cells (green color). (f) Analysis of the number of γ-H2AX+ cells, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. (g–k) IF microscopy for CD68+ cells (green color). Analysis of number of CD68+ cells, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.0001. Red color in (d, e, j, and k) indicates the implanted MSCs in LV myocardium. All statistical analyses were performed by one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group). HPF = high-power field; SC = sham control; AMI = acute myocardial infarction; SW = shock wave; SVF = adipose-derived stromal vascular fraction.

Figure 10. Protein expression of angiogenesis biomarkers in LV myocardium by day 42 after AMI induction. Protein expression of (a) endothelial nitric oxide synthase (eNOS), (b) CD31, (c) vascular endothelial growth factor (VEGF), (d) CXCR4, and (e) stromal cell-derived factor- (SDF-) 1α, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.001. All statistical analyses were performed by one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; SW = shock wave; SVF = adipose-derived stromal vascular fraction.

Figure 11. Protein expression of inflammation and oxidative stress biomarkers in LV myocardium by day 42 after AMI induction. Protein expressions of (a) matrix metalloproteinase- (MMP-) 9, (b) tumor necrotic factor- (TNF-) α, (c) interleukin- (IL-) 1β, (d) nuclear factor- (NF-) κB, (e) NOX-1, (f) NOX-2, ∗ vs. other groups with different symbols (‡, ‡, §, ¶), p < 0.001, and (g) oxidized protein expression, ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. (Note: left and right lanes shown on the upper panel represent protein molecular weight marker and control oxidized molecular protein standard, respectively). M.W. = molecular weight; DNP = 1-3 dinitrophenylhydrazone. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; SW = shock wave; SVF = adipose-derived stromal vascular fraction.

Figure 12. Protein expressions of energy reservoirs, apoptotic, and fibrosis biomarkers in LV myocardium by day 42 after AMI induction. Protein expression of (a) mitochondrial cytochrome C (mit-Cyt C), (b) peroxisome proliferator activated receptor-γ coactivator 1 α (PGC-1α), (c) cytosolic cytochrome C (cyt-Cyt C), (d) mitochondrial Bax (mit-Bax), (e) cleaved caspase 3 (c-Casp 3), (f) cleaved poly(ADP-ribose) polymerase (c-PARP), (g) Smad3, and (h) transforming growth factor- (TGF-) β, ∗ vs. other groups with different symbols (†, ‡, §, ¶), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (∗, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; ECSW = shock wave; SVF = adipose-derived stromal vascular fraction.