PEDF-derived peptide promotes skeletal muscle regeneration through its mitogenic effect on muscle progenitor cells

Abstract

In response injury, intrinsic repair mechanisms are activated in skeletal muscle to replace the damaged muscle fibers with new muscle fibers. The regeneration process starts with the proliferation of satellite cells to give rise to myoblasts, which subsequently differentiate terminally into myofibers. Here, we investigated the promotion effect of pigment epithelial-derived factor (PEDF) on muscle regeneration. We report that PEDF and a synthetic PEDF-derived short peptide (PSP; residues Ser(93)-Leu(112)) induce satellite cell proliferation in vitro and promote muscle regeneration in vivo. Extensively, soleus muscle necrosis was induced in rats by bupivacaine, and an injectable alginate gel was used to release the PSP in the injured muscle. PSP delivery was found to stimulate satellite cell proliferation in damaged muscle and enhance the growth of regenerating myofibers, with complete regeneration of normal muscle mass by 2 wk. In cell culture, PEDF/PSP stimulated C2C12 myoblast proliferation, together with a rise in cyclin D1 expression. PEDF induced the phosphorylation of ERK1/2, Akt, and STAT3 in C2C12 myoblasts. Blocking the activity of ERK, Akt, or STAT3 with pharmacological inhibitors attenuated the effects of PEDF/PSP on the induction of C2C12 cell proliferation and cyclin D1 expression. Moreover, 5-bromo-2'-deoxyuridine pulse-labeling demonstrated that PEDF/PSP stimulated primary rat satellite cell proliferation in myofibers in vitro. In summary, we report for the first time that PSP is capable of promoting the regeneration of skeletal muscle. The signaling mechanism involves the ERK, AKT, and STAT3 pathways. These results show the potential utility of this PEDF peptide for muscle regeneration.

Keywords: myoblast; pigment epithelial-derived factor; satellite cell.

Fig. 1.

Cumulative release in vitro of pigment epithelial-derived factor (PEDF)-derived short peptide from alginate gel in PBS at 37°C. The results are presented as means ± SD for three separate experiments.

Fig. 2.

Immunofluorescence analysis of soleus muscles 4 days after bupivacaine injection and PEDF-derived short peptide (PSP) treatment. A and B: PSP induces muscle satellite cell proliferation in the central area of the damaged soleus muscle. Specimens were assayed by dual-immunofluorescence staining for 5-bromo-2′-deoxyuridine (BrdU, a cell proliferation marker; red) and Pax7 (a muscle satellite cell marker; green). Nuclei were stained by Hoechst 33258 (blue). Pax7 is expressed in the cell nucleus as confirmed by Hoechst 33258 counterstaining. The squares with solid borders show immunostaining for Pax7, BrdU, and nucleus from left to right. Dotted squares show the superimpositions of Pax7, BrdU, and nuclear signals. Arrows indicate Pax7/BrdU double-positive cells. Scale bar: 10 μm. C: variation of the percentages of proliferative Pax7-positive cells among the different treatment groups. Results were evaluated from 6 sections/muscle specimen, with 6 rats in each group. The percentage of BrdU- and Pax7-double positive cells (pale pink) per total Pax7-positive cells was calculated. *P < 0.01 vs. blank. D and E: representative images of three independent experiments show dual-immunofluorescence staining of macrophages by F4/80 (green labeling), BrdU (red labeling), and merged. Insets from left to right denote immunostaining for F4/80, BrdU, and the nucleus. Scale bar: 10 μm. *F4/80-positive macrophages.

Fig. 3.

Maturation of regenerated muscle fibers is enhanced by PSP/alginate gel. A: representative photomicrographs of hematoxylin and eosin (H&E)-stained cross sections of bupivacaine-injured soleus muscles at day 14 after intramuscular treatment with alginate blank, 29-mer bolus, and alginate mixture of the PSP (29-mer or 20-mer), respectively. Normal naïve soleus muscle is included for comparison. B: quantitative analyses of centrally nucleated muscle fibers in the central zone of bupivacaine-injured soleus muscles treated as indicated. The results were evaluated from 6 sections/soleus muscle section, with 6 rats in each group. *P < 0.0001 vs. blank. C: quantification of the variation of muscle fiber size. Muscle fibers were classified by their minimal Feret's diameter. Values represent relative numbers of fibers in a given diameter class. Values given represent the average percent +SE from at least 4 rats from each treatment group. The fiber size distribution of the PSP 20-mer/alginate group differs significantly from the blank group in fiber Feret's classes 10–15, 15–20, and 20–25 μm.

Fig. 4.

PEDF and the PSP 20-mer enhance proliferation of C₂C₁₂ myoblasts. A: cell proliferation was determined by BrdU labeling (red) for 6 h before detection by immunofluorescence microscopy (original magnification, ×400). Cell nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, blue). A representative image from three independent experiments is shown. B: quantitative analyses of BrdU-positive cells among different treatments showing the proliferation index (%). Ten randomly selected fields from each treatment were photographed, and the percentage of BrdU-positive cells/total cells was calculated. *P < 0.01 vs. solvent-treated cells.

Fig. 5.

PEDF induces phosphorylation of ERK1/2, p38 MAPK, STAT3, and Akt in C₂C₁₂ myoblasts in a time-dependent fashion. A and B: cells were exposed to PEDF for the time periods indicated. Immunoblotting was performed to detect the active phosphorylated forms (top) and the unphosphorylated forms (bottom) by reincubation with the antibodies indicated. Representative blots and densitometric analysis are shown from three independent experiments. *P < 0.05 vs. untreated cells (time 0). C: PEDF induces the activation of multiple signaling in C₂C₁₂ myoblasts. Cells were pretreated with PD-98059 (10 μM; ERK inhibitor), SB-203580 (10 μM; p38 MAPK inhibitor), 50 μM STAT3 inhibitor or LY-294002 (10 μM; Akt inhibitor) for 1 h and then treated with PEDF for 5 min to detect phosphor (p)-Elk and p-ATF-2 or treated with PEDF for 24 h to detect Socs3 and survivin by Western blotting with antibodies as indicated. The intensities of p-Elk, p-ATF-2, and Socs3/survivin on the immunoblots were determined by densitometry and normalized to Elk-1, ATF-2, and β-actin, respectively. The experiment was repeated three times. *P < 0.001 vs. solvent-treated cells. **P < 0.005 vs. PEDF-treated cells.

Fig. 6.

ERK, STAT3, and Akt inhibitor prevent PEDF-induced C₂C₁₂ cell proliferation. Inhibitor treatments are described in the legend of Fig. 5C and were followed by PEDF treatment for a further 24 h. A: cell proliferation was determined by BrdU labeling for 6 h as described in the legend of Fig. 4. **P < 0.005 vs. PEDF-treated cells. B: Western blot analysis of the expression of cyclin D1 in C₂C₁₂ cells treated as described above. Representative blots and densitometric analysis with the SD from three independent experiments are shown. *P < 0.01 vs. solvent-treated cells. **P < 0.05 vs. PEDF-treated cells.

Fig. 7.

PSP induces proliferation of Pax7-positive satellite cells in vitro. Myofibers and associated satellite cells were isolated from rat EDL muscle and incubated for 24 h with low-serum medium supplemented with 50 nM 20-mer and 10 μM BrdU and 1 μM of the inhibitors indicated, as described in materials and methods. Satellite cells (Pax7) and BrdU were detected by immunofluorescence microscopy. A: representative image from three independent experiments is shown. B: 20 randomly selected fields in each treatment were photographed, and the percentage of BrdU- and Pax7-double positive cells/total Pax7-positive cells was calculated. *P < 0.001 vs. untreated cells. **P < 0.005 vs. 20-mer-treated cells.