Tyrosol Facilitates Neovascularization by Enhancing Skeletal Muscle Cells Viability and Paracrine Function in Diabetic Hindlimb Ischemia Mice

Figures

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  Figure 1. Tyrosol increases skeletal muscle cells viability by repressing hyperglycemia-induced intracellular ROS level. (A) Chemical structures of tyrosol. (B) Intracellular reactive oxygen species (ROS) levels in C2C12 cells cultured under hyperglycemia and treated with tyrosol, as detected by DCFH-DA staining; left, representative images (scale bars, 200 µm); right, relative fluorescence intensity quantification (n = 6). (C) Total cell number of C2C12 cells cultured under hyperglycemia and treated with tyrosol at the designated time points. (D) Percentage of apoptotic C2C12 cells cultured under hyperglycemia and treated with tyrosol. Cells were labeled Annexin-V-FITC/PI, and the percentage of apoptotic cells was analyzed using FACS; left, representative images; right, average percentage of apoptotic cells (n = 3). (E) Ratio of proliferative cells in C2C12 cells cultured under hyperglycemia and treated with tyrosol. Proliferative cells were examined using EdU-incorporation assay; left, representative images (scale bars, 200 µm); right proliferative cells quantification (n = 6). All experiments were done by subjecting the cells into hypoxia. Cells treated with PBS under normoglycemia were…

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  Figure 2. Tyrosol increases skeletal muscle cells viability under hyperglycemia by inducing HO-1 expression. (A) Protein expression level in C2C12 cells cultured under hyperglycemia and treated with tyrosol, as analyzed by Western blotting using corresponding antibodies; upper panels, representative images; lower panels, quantification results (n = 3). β-Actin was used as loading control. Cells treated with PBS under normoglycemia were used as control. (B) Intracellular ROS levels in C2C12 cells treated with tyrosol and zinc protoporphyrin IX (ZnPP; final concentration, 10 µM), as detected by DCFH-DA staining; upper panels, representative images (scale bars, 200 µm); lower panels, relative fluorescence intensity quantification (n = 6). Cells treated with DMSO under hyperglycemia were used as control. (C) Percentage of apoptotic cells in C2C12 cells cultured under hyperglycemia and treated with tyrosol and ZnPP (final concentration, 10 µM). Cells were labeled with Annexin-V-FITC/PI, and the percentage of apoptotic cells were analyzed using FACS; left, representative images; right, average percentage of apoptotic cells (n = 3). Cells treated with DMSO under hyperglycemia were used as…

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  Figure 3. Tyrosol enhances VEFG-A and PDGF-BB secretion from skeletal muscle cells under hyperglycemia. (A) Protein expression levels of VEGF-A and PDGF-BB in C2C12 cells cultured under hyperglycemia and treated with tyrosol; left, representative images; right, quantification results. (B) Secretion level of VEGF-A in C2C12 cells cultured under hyperglycemia and treated with tyrosol, as determined by ELISA. (C) Secretion level of PDGF-BB in C2C12 cells cultured under hyperglycemia and treated with tyrosol, as determined by ELISA. All experiments were done by subjecting the cells into hypoxia; cells treated with PBS under normoglycemia were used as control. Quantification data were shown as relative to that of control, and expressed as mean ± SD (n = 3). **P < 0.01; Con, normoglycemia; High, hyperglycemia; Tyr, tyrosol.

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  Figure 4. Tyrosol restores migration capability of skeletal muscle cells suppressed by hyperglycemia. (A) Morphological changes of F-actin in C2C12 cells treated with tyrosol and cultured under hyperglycemia; left, representative images (scale bars, 100 µm for upper panels and 50 µm for lower panels; lower panels show the high magnification images of the cropped part in the upper panels); right, quantification analysis of fractal dimension. (B) Migration capability in C2C12 cells cultured under hyperglycemia and treated with tyrosol, as analyzed using transwell chamber assay; left, representative images (scale bars, 100 µm); right, quantification of migrated cells. All experiments were done by subjecting the cells into hypoxia. Quantification data were expressed as mean ± SD (n = 6; **P < 0.01); Con, normoglycemia; High, hyperglycemia; Tyr, tyrosol.

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  Figure 5. Tyrosol-treated skeletal muscle cells’ secretome promotes vascular endothelial and smooth muscle cells proliferation capability suppressed by hyperglycemia. (A–B) Ratio of proliferative cells in HUVECs (A) and MOVAS cells (B) cultured with conditioned media collected from tyrosol-treated C2C12 cells. Proliferative cells were examined using EdU-incorporation assay; left, representative images; right, the quantitative results. (C–D) Ratio of proliferative cells in HUVECs (C) and MOVAS cells (D) cultured with conditioned media collected from tyrosol-treated C2C12 cells and treated with VEGFR inhibitor (Ki8751; final concentration, 2 nM) or PDGFR-β inhibitor (CP868596; final concentration, 0.3 µM). Proliferative cells were examined using EdU-incorporation assay; left, representative images; right, the quantitative results. Scale bars, 200 µm. All experiments were done by subjecting the cells into hypoxia. Quantification data were expressed as mean ± SD (n = 6; *P< 0.05, **P < 0.01); CM-C, conditioned medium derived from C2C12 cells treated with PBS under normoglycemia; CM-H, conditioned medium derived from C2C12 cells treated with PBS under hyperglycemia; CM-H/Tyr, conditioned medium…

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  Figure 6. Tyrosol-treated skeletal muscle cells’ secretome promotes vascular endothelial and smooth muscle cells migration capability suppressed by hyperglycemia. (A) Morphological changes of F-actin in HUVECs cultured with conditioned medium collected from tyrosol-treated C2C12 cells; left, representative images (scale bars, 100 µm for upper panels and 50 µm for lower panels; lower panels show the high-magnification images of the cropped part in the upper panels); right, quantification analysis of fractal dimension. (B) Migration capability in HUVECs cultured with conditioned medium collected from tyrosol-treated C2C12 cells, as analyzed using transwell chamber assay; left, representative images (scale bars, 100 µm); right, the quantitative results. (C) Morphological changes of F-actin in MOVAS cells cultured with conditioned medium collected from tyrosol-treated C2C12 cells; left, representative images (scale bars, 100 µm for upper panels and 50 µm for lower panels; lower panels show the high magnification images of the cropped part in the upper panels); right, quantification analysis of fractal dimension. (D) Migration capability in MOVAS cells cultured with conditioned medium collected…

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  Figure 7. Tyrosol induces mature blood vessels formation and blood perfusion recovery in diabetic HLI model mice. (A–B) Blood perfusion recovery in the ischemic and non-ischemic hindlimb of diabetic HLI model mice injected by tyrosol: representative of Laser Doppler Perfusion Images (A); and quantification of the blood perfusion ratio at day 0 and day 14 (B) (n = 5, P value was analyzed by one-way ANOVA) were shown. (C–D) Immunofluorescence staining of PECAM-1 (green) and α-SMA (red) in the gastrocnemius muscle of the ischemic hindlimbs of diabetic HLI model mice injected by tyrosol at 14 days post-surgery. Representative images (C; scale bars, 200 µm); and quantification results of PECAM-1 and α-SMA-positive areas (D; n = 6) were shown. Tyrosol and PBS were administered by local injection intramuscularly. Quantification data were expressed as mean ± SD (**P < 0.01); Con, mice injected with PBS; Tyr, mice injected with tyrosol.

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  Figure 8. Schematic diagram of the effect of tyrosol in promoting angiogenic capability in diabetic HLI.