Elevating VEGF-A and PDGF-BB secretion by salidroside enhances neoangiogenesis in diabetic hind-limb ischemia

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  Figure 1. Hyperglycemia suppresses angiogenic factors expression in skeletal muscle cells by inducing PHD3 protein accumulation (A–C) The expression levels of angiogenic factors in C2C12 cells cultured under hyperglycemia: (A) mRNA expression levels, as examined by quantitative RT-PCR (qPCR); (B) Protein expression levels, as examined by western blotting; and (C) quantification of protein expression levels. (D, E) Protein expression levels of PHD family in C2C12 cells cultured under hyperglycemia, as determined using western blotting: (D) representative images, and (E) quantification of protein expression levels. (F–H) Expression levels of angiogenic factors in PHD3-silenced C2C12 cells cultured under hyperglycemia: (F) mRNA expression levels, as examined using qPCR; (G) protein expression levels, as determined using western blotting; and (H) quantification of protein expression levels. Cells cultured under low glucose condition were used as control. All experiments were done under hypoxia. β-Actin was used for normalization in qPCR, and as a loading control in western blotting. Quantification data were shown as relative to that of control, and expressed as mean ± S.D. (n = 3 from three…

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  Figure 2. Salidroside restores the expression of skeletal muscle cells angiogenic factors repressed by hyperglycemia (A, B) Protein expression levels of PHD family in the C2C12 cells treated with salidroside and cultured under hyperglycemia, as determined by western blotting: (A) representative images; and (B) quantification of protein expression levels. (C, D) Protein expression level of HIF-1α in C2C12 cells treated with salidroside and cultured under hyperglycemia, as determined by western blotting: (C) representative images; and (D) quantification of protein expression level. (E, F) Protein expression level of HIF-1α in the PHD3-overexpressing C2C12 cells cultured under hyperglycemia and treated with salidroside, as determined by western blotting: (E) representative images; and (F) quantification of protein expression level. (G–I) Expression levels of VEGF-A and PDGF-BB in C2C12 cells treated with salidroside and cultured under hyperglycemia: (G) mRNA expression levels, as analyzed by quantitative RT-PCR (qPCR); (H) protein expression levels, as examined by western blotting; and (I) quantification of protein expression levels. (J) Secretion levels of VEGF-A (left panel) and PDGF-BB…

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  Figure 3. Salidroside restores skeletal muscle cells proliferation and migration potentials suppressed by hyperglycemia (A, B) The percentage of proliferative C2C12 cells treated with salidroside and cultured under hyperglycemia, as analyzed using Ki67 staining: (A) representative images (scale bars: 200 μm); and (B) quantification of the ratio of Ki67 positive cells to DAPI positive cells. (C) The number of C2C12 cells treated with salidroside and cultured under hyperglycemia, as analyzed using crystal violet staining. (D, E) The migration potential of C2C12 cells treated with salidroside and cultured under hyperglycemia, as examined using transwell chamber assay: (D) representative images (scale bars: 100 μm); and (E) quantification of migrated cells. All experiments were done under hypoxia. Data shown are representative from three independent experiments. Quantification data were expressed as mean ± S.D. (n = 6). **P < 0.01; SA: salidroside.

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  Figure 4. Salidroside promotes endothelial cells proliferation and migration potentials via skeletal muscle cells secretome (A, B) The percentage of proliferative HUVECs cultured with indicated conditioned media, as analyzed by Ki67 staining: (A) representative images (scale bars: 200 μm); and (B) quantification of the ratio of Ki67 positive cells to DAPI positive cells. (C) The number of HUVECs cells cultured with indicated conditioned media, as analyzed by crystal violet staining. (D, E) The mobility of HUVECs cultured with indicated conditioned media, as examined by transwell chamber assay: (D) representative images (scale bars: 100 μm); and (E) quantification of migrated cells. (F, G) Morphological changes of F-Actin, as examined by phalloidin staining: (F) representative images (scale bars: 100 μm for upper panels and 50 μm for lower panels), lower panels showed the enlarged images of the cropped part in the upper panels; (G) quantification analysis of fractal dimension. (H) The number of HUVECs cultured with CM-H/SA and indicated inhibitors, as analyzed by crystal violet staining. (I, J) The mobility of HUVECs cultured with CM-H/SA and indicated inhibitors, as examined by transwell…

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  Figure 5. Salidroside promotes smooth muscle cells proliferation and migration potentials via skeletal cells secretome (A, B) The proliferation of MOVAS cells cultured with indicated conditioned media, as analyzed by Ki67 staining: (A) representative images (scale bars: 200 μm); and (B) quantification of the ratio of Ki67 positive cells to DAPI positive cells. (C) The number of MOVAS cells cultured with indicated conditioned media, as analyzed by crystal violet. (D, E) The mobility of MOVAS cells cultured with indicated conditioned media was examined using transwell chamber assay: (D) representative images (scale bars: 100 μm); and (E) quantification of migrated cells. (F, G) Morphological changes of F-Actin, as examined by phalloidin staining: (F) representative images (scale bars: 100 μm for upper panels and 50 μm for lower panels), lower panels showed the enlarged images of the cropped part in the upper panels; (G) quantification analysis of fractal dimension. (H) The number of MOVAS cells cultured with CM-H/SA and indicated inhibitors, as analyzed by crystal violet staining. (I, J) The mobility of MOVAS cells cultured with CM-H/SA and indicated inhibitors, as examined by transwell…

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  Figure 6. Salidroside enhances endothelial cells proliferation and migration potentials by suppressing hyperglycemia-induced skeletal muscle cells PHD3 accumulation (A, B) The proliferation of HUVECs cultured with indicated conditioned media, as analyzed by Ki67 staining: (A) representative images (scale bars: 200 μm); and (B) quantification of the ratio of Ki67 positive cells to DAPI positive cells. (C, D) The mobility of HUVECs cultured with indicated conditioned media, as examined by transwell chamber assay: (C) representative images (scale bars: 100 μm); and (D) quantification of migrated cells. (E, F) Morphological changes of F-Actin, as determined by phalloidin staining: (E) representative images (scale bars: 100 μm for upper panels and 50 μm for lower panels), lower panels showed the enlarged images of the cropped part in the upper panels; (F) quantification analysis of fractal dimension. Conditioned media were collected from C2C12 cells transfected with pcCon or pcPHD3, treated with PBS or salidroside and cultured under hyperglycemia. All experiments were done under hypoxia. Data shown are representative from three independent experiments. Quantification data were expressed as mean…

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  Figure 7. Salidroside enhances smooth muscle cells proliferation and migration potentials by suppressing hyperglycemia-induced skeletal muscle cells PHD3 accumulation (A, B) The proliferation of MOVAS cells cultured with indicated conditioned media, as analyzed by Ki67 staining: (A) representative images (scale bars: 200 μm); and (B) quantification of the ratio of Ki67 positive cells to DAPI positive cells. (C, D) The mobility of MOVAS cells cultured with conditioned media, as examined by transwell chamber assay: (C) representative images (scale bars: 100 μm); and (D) quantification of migrated cells. (E, F) Morphological changes of F-Actin, as examined using phalloidin staining: (E) representative images (scale bars: 100 μm for upper panels and 50 μm for lower panels), lower panels showed the enlarged images of the cropped part in the upper panels; (F) quantification analysis of fractal dimension. Conditioned media were collected from C2C12 cells transfected with pcCon or pcPHD3, treated with PBS or salidroside and cultured under hyperglycemia. All experiments were done under hypoxia. Data shown are representative from three independent experiments. Quantification data were expressed as…

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  Figure 8. Salidroside promotes blood perfusion recovery by inducing neoangiogenesis in diabetic HLI model mice (A) Representative of Laser Doppler Perfusion Images showing blood perfusion in the ischemic and non-ischemic hind limb of diabetic HLI model mice treated with PBS (upper panels) or salidroside (lower panels). (B) The blood perfusion ratio of ischemic hind limb to non-ischemic hind limb. Data were shown as mean ± S.D. (n = 7 per group). (C) Assessment test of the ischemic hind limb morphologies of diabetic HLI model mice treated by salidroside or PBS at indicated times (n = 7 per group, 0 = no difference from the non-ischemic hind limb, 1 = mild discoloration, 2 = moderate discoloration, 3 = severe discoloration, subcutaneous tissue loss, or necrosis, and 4 = amputation). (D, E) Immunohistochemistry against PECAM-1 (green) and α-SMA (red) in the gastrocnemius muscle of the ischemic hind limbs of diabetic HLI model mice treated by either salidroside or PBS at 21 days post-surgery: (D) representative images (scale bars: 100 μm); (E) quantification of PECAM-1 (left panel) and α-SMA positive (right panel) areas. Data shown are representative from three independent experiments.…

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  Figure 9. Salidroside promotes blood perfusion recovery by suppressing PHD3 and promoting angiogenic factors expression in the gastrocnemius muscle of diabetic HLI model mice (A–C) The expression levels of PHD family in the gastrocnemius muscle of the ischemic hind limb of diabetic HLI model mice treated with salidroside or PBS: (A) mRNA expression levels, as examined by quantitative RT-PCR (qPCR); (B) representative images of protein expression levels, as examined by western blotting; and (C) quantification of protein expression levels. (D–F) The expression levels of VEGF-A and PDGF-BB in the gastrocnemius muscle of the ischemic hind limb of diabetic HLI model mice treated with salidroside or PBS: (D) mRNA expression levels, as examined by qPCR; (E) representative images of protein expression levels, as examined by western blotting; and (F) quantification of protein expression levels. β-Actin was used for normalization in qPCR, and as a loading control in western blotting. Quantification data were shown as relative to that of control, and expressed as mean ± S.D. (n = 3 from three independent experiments). NS: not significant; **P < 0.01; Con: animals treated with PBS; SA: animals treated…