Journal: International Journal of Surgery (London, England)

Article Title: Shock wave-pretreated ADMSCs of cell-sheet scaffold (CSS) patched on the left ventricular wall (LVW) inhibited LVW remodeling in mini-pig MI: role of CSS on counteracting Laplace’s Law of LVW stress – experimental study

doi: 10.1097/JS9.0000000000002119

Figure Lengend Snippet: Effect of SW and ADMSCs on alleviating inflammation, oxidative stress and apoptosis. (A) Western blot result of MMP-9, * versus other groups with different symbols (†, ‡, §), P <0.0001. (B) Western blot result of interleukin (IL)-6, * versus other groups with different symbols (†, ‡, δ), P <0.0001. (C) Western blot result of COX-2, * versus other groups with different symbols (†, ‡, §), P <0.0001. (D) Western blot result of interferon (IFN)-γ, * versus other groups with different symbols (†, ‡, §), P <0.0001. (E) Western blot result of NOX-1, * versus other groups with different symbols (†, ‡, §), P <0.0001. (F) Western blot result of NOX-2, * versus other groups with different symbols (†, ‡, §), P <0.0001. (G) Flow cytometric assessment of early (AN-V + /PI − ) apoptosis, * versus other groups with different symbols (†, ‡, §), P <0.0001. (H) Flow cytometric assessment of late (AN-V + /PI + ) apoptosis, * versus other groups with different symbols (†, ‡, §), P <0.0001. (I-1) Number of M1 surface expression in Raw 264.7 Cells, * versus other groups with different symbols (†, ‡, §), P <0.0001. (I-2) Number of M2 surface expression in Raw 264.7 Cells, * versus other groups with different symbols (†, ‡, §), P <0.0001. (I-3) Ratio of M2/M1, * versus other groups with different symbols (†, ‡, §), P <0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple-comparison post hoc test ( n = 6 for each group). Symbols (*, †, ‡, §) express significance for each other (at 0.05 level). ADMSCs, adipose-derived mesenchymal stem cells; LPS, lipopolysaccharide; SW, shock wave.

Article Snippet: The membranes were incubated with the indicated primary antibodies [vascular endothelial growth factor (VEGF; 1:1000, Abcam), stromal cell-derived factor-1α (SDF-1α; 1:1000, Cell Signaling), C-X-C chemokine receptor type 4 (CXCR4; 1:1000, Abcam), angioetin-1 (1:1000, Abcam), bone morphogenetic protein-2 (BMP-2; 1:1000, Abcam), phosphorylated Smad1/5 (1:1000, Cell Signaling), von Willebrand factor (vWF; 1:1000, Abcam), endothelial nitric oxide synthase (eNOS; 1:1000, Abcam), angiopoietin-1 (1:1000, Abcam), cytosolic cytochrome (1:5000, BD), mitochondrial cytochrome C (1:5000, BD), γ-H2AX (1:1000, Cell Signaling), phosphorylated Smad3 (1:1000, Cell Signaling), transforming growth factor β (TGF)-β (1:1000, Abcam), complex I (1:5000, Abcam), complex II (1:5000, Abcam), complex III (1:5000, Abcam), complex IV (1:5000, Abcam), complex V (1:5000, Abcam), matrix metalloproteinase-9 (MMP-9; 1:1000, Abcam), interleukin-6 (IL-6; 1:1000, Abcam), cyclooxygenase-2 ([COX-2; 1:1000, Abcam), interferon gamma (IFN-γ; 1:1000, Abcam), NADPH oxidase (NOX-1; 1:1000, Sigma), NOX-2 (1:1000, Sigma), mitochondria respiratory chain (MRC I; 1:5000, Abcam), MRC II (1:5000, Abcam), MRC III (1:5000, Abcam), and MRC IV (1:5000, Abcam)] for 1 h at room temperature.

Techniques: Western Blot, Expressing, Comparison, Derivative Assay

Journal: International Journal of Surgery (London, England)

Article Title: Shock wave-pretreated ADMSCs of cell-sheet scaffold (CSS) patched on the left ventricular wall (LVW) inhibited LVW remodeling in mini-pig MI: role of CSS on counteracting Laplace’s Law of LVW stress – experimental study

doi: 10.1097/JS9.0000000000002119

Figure Lengend Snippet: SW treatment augmented cell proliferation ability, number of mitochondrial cytochrome C, and angiogenesis biomarkers in ADMSCs. (A) Cell viability at 24 h, * versus †, P <0.0001. (B) Cell viability at 48 h, ‡ versus §, P <0.0001. (C) Cell viability at 72 h, ¶ versus δ, P <0.00001. Note that the repeated measures ANOVA was adopted to analyze cell variability at different time points. (D1–D3 and E1–E3) Showing the immunofluorescent microscopic finding (400×) for identifying the expression of mitochondrial cytochrome C (red-green color in the merged picture). (D1 and E1) Indicated the cytochrome C stain (green color); (D2 and E2) Indicated mitochondrial stain (red color); (D3 and E3) Indicated the merged picture of A and B (red-green color). (F) The percentage (%) of positively stained cytochrome C in mitochondria, * versus †, P <0.0001. (G) Western blot analysis of VEGF, * versus †, P <0.0001. (H) Western blot analysis of SDF-1α, * versus †, P <0.0001. n =6 in each group. (I and J) Demonstrating the IF microscopic finding (400×) for identifying the cellular expression of Ki68 (green color). (K) Analytical result of the number of Ki67+ cells, * versus †, P <0.0001. n =4 in each group.

Article Snippet: The membranes were incubated with the indicated primary antibodies [vascular endothelial growth factor (VEGF; 1:1000, Abcam), stromal cell-derived factor-1α (SDF-1α; 1:1000, Cell Signaling), C-X-C chemokine receptor type 4 (CXCR4; 1:1000, Abcam), angioetin-1 (1:1000, Abcam), bone morphogenetic protein-2 (BMP-2; 1:1000, Abcam), phosphorylated Smad1/5 (1:1000, Cell Signaling), von Willebrand factor (vWF; 1:1000, Abcam), endothelial nitric oxide synthase (eNOS; 1:1000, Abcam), angiopoietin-1 (1:1000, Abcam), cytosolic cytochrome (1:5000, BD), mitochondrial cytochrome C (1:5000, BD), γ-H2AX (1:1000, Cell Signaling), phosphorylated Smad3 (1:1000, Cell Signaling), transforming growth factor β (TGF)-β (1:1000, Abcam), complex I (1:5000, Abcam), complex II (1:5000, Abcam), complex III (1:5000, Abcam), complex IV (1:5000, Abcam), complex V (1:5000, Abcam), matrix metalloproteinase-9 (MMP-9; 1:1000, Abcam), interleukin-6 (IL-6; 1:1000, Abcam), cyclooxygenase-2 ([COX-2; 1:1000, Abcam), interferon gamma (IFN-γ; 1:1000, Abcam), NADPH oxidase (NOX-1; 1:1000, Sigma), NOX-2 (1:1000, Sigma), mitochondria respiratory chain (MRC I; 1:5000, Abcam), MRC II (1:5000, Abcam), MRC III (1:5000, Abcam), and MRC IV (1:5000, Abcam)] for 1 h at room temperature.

Techniques: Expressing, Staining, Western Blot

Journal: International Journal of Surgery (London, England)

Article Title: Shock wave-pretreated ADMSCs of cell-sheet scaffold (CSS) patched on the left ventricular wall (LVW) inhibited LVW remodeling in mini-pig MI: role of CSS on counteracting Laplace’s Law of LVW stress – experimental study

doi: 10.1097/JS9.0000000000002119

Figure Lengend Snippet: Protein levels of angiogenesis factors and integrity of endothelial cell biomarkers in the peri-infarcted area by 180th day after AMI induction. (A) Protein level of SDF-1α, * versus other groups with different symbols (†, ‡, §), P <0.0001. (B) Protein level of CXCR4, * versus other groups with different symbols (†, ‡, §), P <0.0001. (C) Protein level of VEGF, * versus other groups with different symbols (†, ‡, §), P <0.0001. (D) Protein level of angiopoetin-1, * versus other groups with different symbols (†, ‡, §), P <0.0001. (E) Protein level of von Willebrand factor (vWF), * versus other groups with different symbols (†, ‡, §), P <0.0001. (F) Protein level of eNOS, * versus other groups with different symbols (†, ‡, §), P <0.0001. n =5 for each group. Symbols (*, †, ‡, §) express significance (at 0.05 level).

Article Snippet: The membranes were incubated with the indicated primary antibodies [vascular endothelial growth factor (VEGF; 1:1000, Abcam), stromal cell-derived factor-1α (SDF-1α; 1:1000, Cell Signaling), C-X-C chemokine receptor type 4 (CXCR4; 1:1000, Abcam), angioetin-1 (1:1000, Abcam), bone morphogenetic protein-2 (BMP-2; 1:1000, Abcam), phosphorylated Smad1/5 (1:1000, Cell Signaling), von Willebrand factor (vWF; 1:1000, Abcam), endothelial nitric oxide synthase (eNOS; 1:1000, Abcam), angiopoietin-1 (1:1000, Abcam), cytosolic cytochrome (1:5000, BD), mitochondrial cytochrome C (1:5000, BD), γ-H2AX (1:1000, Cell Signaling), phosphorylated Smad3 (1:1000, Cell Signaling), transforming growth factor β (TGF)-β (1:1000, Abcam), complex I (1:5000, Abcam), complex II (1:5000, Abcam), complex III (1:5000, Abcam), complex IV (1:5000, Abcam), complex V (1:5000, Abcam), matrix metalloproteinase-9 (MMP-9; 1:1000, Abcam), interleukin-6 (IL-6; 1:1000, Abcam), cyclooxygenase-2 ([COX-2; 1:1000, Abcam), interferon gamma (IFN-γ; 1:1000, Abcam), NADPH oxidase (NOX-1; 1:1000, Sigma), NOX-2 (1:1000, Sigma), mitochondria respiratory chain (MRC I; 1:5000, Abcam), MRC II (1:5000, Abcam), MRC III (1:5000, Abcam), and MRC IV (1:5000, Abcam)] for 1 h at room temperature.

Techniques:

Journal: International Journal of Surgery (London, England)

Article Title: Shock wave-pretreated ADMSCs of cell-sheet scaffold (CSS) patched on the left ventricular wall (LVW) inhibited LVW remodeling in mini-pig MI: role of CSS on counteracting Laplace’s Law of LVW stress – experimental study

doi: 10.1097/JS9.0000000000002119

Figure Lengend Snippet: qPCR analyses of inflammation, oxidative stress, apoptosis, and anti-apoptosis in the peri-infarcted area by 180th day and proposed mechanism of SW-pretreated ADMSCs seeded in CSS patched on LV wall for attenuating LVWSF and improving heart function in MI pig. (A1) Relative gene expression of interleukin (IL)-1β, * versus other groups with different symbols (†, ‡, §), P <0.0001. (A2) Relative gene expression of IL-6, * versus other groups with different symbols (†, ‡, §), P <0.0001. (A3) Relative gene expression of tumor necrosis factor (TNF)-α, * versus other groups with different symbols (†, ‡, §), P <0.0001. (B1) Relative gene expressions of NOX-1, * versus other groups with different symbols (†, ‡, §), P <0.0001. (B2) Relative gene expression of NO-4, * versus other groups with different symbols (†, ‡, §), P <0.0001. (B3) Relative gene expression of FMO2 (flavin-containing dimethylaniline monooxygenase 2), * versus other groups with different symbols (†, ‡, §), P <0.0001. (C1) Relative gene expression of Bim, * versus other groups with different symbols (†, ‡, §), P <0.0001. (D1) Relative gene expression of Bcl-2, * versus other groups with different symbols (†, ‡, §), P <0.0001. (D2) Relative gene expression of Bcl-xL, * versus other groups with different symbols (†, ‡, §), P <0.0001. n = 5 for each group. ADMSCs, adipose-derived mesenchymal stem cells; CSS, cell-sheet scaffold; LV, left ventricular; LVWSF, LV wall stress force; MI, myocardial infarction; TSPG, temperature-sensitive plasticity gel; SW, shock wave. (E) Illustrating during suturing of SSC on LV wall. White arrows indicate the CSS with white color. (F) Illustrating the complete suture and the suture lines (green arrowheads) for firmly fixing the CSS on the LV-free wall can be clearly identified. (G) Demonstrating the gross anatomical morphology of the heart by the 180th day after IM induction. The CSS on the completely absorbed LV-free wall can be clearly identified.

Article Snippet: The membranes were incubated with the indicated primary antibodies [vascular endothelial growth factor (VEGF; 1:1000, Abcam), stromal cell-derived factor-1α (SDF-1α; 1:1000, Cell Signaling), C-X-C chemokine receptor type 4 (CXCR4; 1:1000, Abcam), angioetin-1 (1:1000, Abcam), bone morphogenetic protein-2 (BMP-2; 1:1000, Abcam), phosphorylated Smad1/5 (1:1000, Cell Signaling), von Willebrand factor (vWF; 1:1000, Abcam), endothelial nitric oxide synthase (eNOS; 1:1000, Abcam), angiopoietin-1 (1:1000, Abcam), cytosolic cytochrome (1:5000, BD), mitochondrial cytochrome C (1:5000, BD), γ-H2AX (1:1000, Cell Signaling), phosphorylated Smad3 (1:1000, Cell Signaling), transforming growth factor β (TGF)-β (1:1000, Abcam), complex I (1:5000, Abcam), complex II (1:5000, Abcam), complex III (1:5000, Abcam), complex IV (1:5000, Abcam), complex V (1:5000, Abcam), matrix metalloproteinase-9 (MMP-9; 1:1000, Abcam), interleukin-6 (IL-6; 1:1000, Abcam), cyclooxygenase-2 ([COX-2; 1:1000, Abcam), interferon gamma (IFN-γ; 1:1000, Abcam), NADPH oxidase (NOX-1; 1:1000, Sigma), NOX-2 (1:1000, Sigma), mitochondria respiratory chain (MRC I; 1:5000, Abcam), MRC II (1:5000, Abcam), MRC III (1:5000, Abcam), and MRC IV (1:5000, Abcam)] for 1 h at room temperature.

Techniques: Expressing, Derivative Assay