1. Volume 15, Issue 9, Pages 1616-1622 (September 2007)
Decorin Gene Transfer Promotes Muscle Cell Differentiation and Muscle Regeneration 
Yong Li, Juan Li, Jinghong Zhu, Bin Sun, Maria Branca, Ying Tang, William Foster, Xiao Xiao, Johnny Huard 
Molecular Therapy 
Volume 15, Issue 9, Pages (September 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Decorin plasmid construction and initial transfection in vitro. (a) The decorin plasmid used for the study contained the full sequence of a mouse decorin gene inserted at the NotI site, which placed it under the control of a cytomegalovirus (CMV) promoter. (b) We transfected the plasmid into 293 cells. We observed decorin expression in both the 293 cells and their supernatant, but not in the control adeno-associated virus (AAV)–transfected (green fluorescent protein, GFP) cells. (c) Western blot analysis also revealed decorin expression in CD clone cells within different time period cultures (lane 2, 24 hours; lane 4, 48 hours), but not in C2C12 (lane 1, 24 hours; lane 3, 48 hours). We used 5 μg of decorin as a positive control (lane 5). GAPDH, glyceraldehyde-3-phosphate dehydrogenase is used as a control. (d) Decorin expression in muscle-derived stem cells (MDSCs) in pellet form was low, but this was not the case in the supernatant. Both MDSCs and their cultured supernatant strongly expressed decorin after mDec-AAV gene transfer. β-actin is used as a control. (e) We did not detect decorin in normal C2C12 cells, but C2C12 cells and their cultured supernatant both expressed decorin after mDec-AAV gene transfer.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Decorin stimulates C2C12 differentiation in vitro. (a) Decorin treatment accelerated the differentiation and fusion of myoblasts (C2C12 cells) compared with non-treated myoblasts (C2C12 cells). (a) The cultures of decorin-treated C2C12 cells contained more myotubes at the 3- and 4-day time points than control cells. (b, c) Similarly, decorin-transfected C2C12 clone cells (CD cells) produced more myotubes than did C2C12 cells, including larger myotubes (containing more than three nuclei) in vitro. Red staining shows myosin heavy chain fluorescence after immunostaining (b).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Decorin gene transfer up-regulates myogenic proteins and p21 and down-regulates myostatin during muscle cell differentiation. (a) Genetic engineering of myoblasts to express decorin influenced the expression of myogenic proteins (including Myf5/6, Myogenin, and MyoD), as shown by western blot results. However, C2C12 cells and CD clone cells expressed comparable levels of desmin. (b) We detected the presence of p21 expressed in CD clone cells. We also detected peroxisome-proliferator-activated receptor-gamma co-activator-1α(PGC-1α) and follistatin expressed in CD, but not C2C12, cells. In addition, CD cells exhibited lower levels of myostatin, a negative regulator of muscle mass. The induction of tumor growth factor (TGF)-β1 auto-expression was also inhibited by decorin over-expression in CD cells. (c) Similar results were obtained by real-time polymerase chain reaction. Lanes 1–6 show results for C2C12 cells exposed to different concentrations of decorin (0, 0.001, 0.01, 0.1, 1.0, and 5.0 ng/ml, respectively). Lane 7 displays the test results for CD cells, which served as the positive control. We did not detect a visible change in the expression of PGC-1α, follistatin, myostatin, or p21 after 12 hours of stimulation with different concentrations of decorin. PGC-1α and follistatin were up-regulated in C2C12 cells in a dose-dependent manner after 18 hours of stimulation with decorin. Myostatin was down-regulated in C2C12 cells in a dose-dependent manner after 24 hours of decorin stimulation. The concentration of p21 did not visibly change after cell stimulation with any experimental concentration of decorin over all time points. With decorin gene transfer, we found that CD cells consistently expressed follistatin, p21, and PGC-1α but were negative for myostatin. Note that β-actin was selected as a positive gene control.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Decorin gene transfer stimulates muscle regeneration in vivo. C2C12 cells regenerated muscle fibers after transplantation into skeletal muscle of MDX/SCID mice, as shown on both (a) LacZ- and (b) dystrophin-expressing myofibers. However, transplantation of CD clone cells, rather than C2C12 cells, resulted in larger muscle fibers in MDX/SCID mice, as shown in some of the (d) LacZ- and (e) dystrophin-positive myofibers. (c) Although there was no significant difference between the number of LacZ-labeled muscle fibers that formed in muscles transplanted with C2C12 or CD cells, (f) the transplantation of CD cells resulted in the regeneration of larger-diameter myofibers (P < 0.01).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
mDec-AAV vector gene therapy in injured muscle prevents fibrosis and promotes muscle regeneration. Decorin-treated muscle exhibits a greater number of regenerating myofibers than the control muscle at all time points (a–d 2 weeks; e–h 4 weeks). (i) The mDec-AAV-injected muscle contained significantly higher numbers of centronucleated (regenerated) myofibers than did control (sham-injected) muscle at 4 weeks after therapy. We also found that decorin gene therapy minimized fibrosis in injured skeletal muscle. We used Masson's trichrome staining to reveal collagen in injured skeletal muscle, the results of which show that (f) mDec-AAV-injected muscle contained significantly less fibrosis in the injured area than did the (h) control muscle at (j) 4 weeks after injury.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Schematic of the potential effect of decorin on muscle healing. Decorin may improve muscle healing through various pathways: inhibition of tumor growth factor (TGF)-β1, up-regulation of follistatin, peroxisome-proliferator-activated receptor-gamma co-activator-1α(PGC-1α), p21, and myogenic genes (such as MyoD), and down-regulation of myostatin expression. MDSCs, muscle-derived stem cells.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions