Figure 7

MP depletion affects pro-angiogenic factor production and TGF-β/SMAD signaling during muscle healing. (a–c) TA muscles were collected from sham and Cll-injected mice 5, 7, 10 and 15 days after CTX injection. Muscles were totally lysated and processed for RNA extraction. Real-time PCR analyses for mRNA expression of HIF1-α, Ang-I, Ang-II, PDGF-α, PDGF-β, VEGF-β (a), MMP-2, MMP-13, MMP-14 (b), TGF-β and Snail (c) were performed. Results were normalized to 28S mRNA levels and expressed as relative fold changes compared with sham-treated mice 5 days after damage. The data are expressed as means±S.E.M. (n=5). Statistically significant differences are indicated (sham versus Cll *P<0.05). Western blot analysis of TGF-β expression was performed on total muscle lysates from sham (lanes 1, 3 and 5) and Cll (lanes 2, 4 and 6) treated mice, 5 days (lanes 1 and 2), 7 days (lanes 3 and 4) and 10 days (lanes 5 and 6) after CTX injection. Results are representative of three independent experiments. Day 5 Cll versus sham fold change 5.4; day 7 Cll versus sham fold change 2.8. (d) Representative images of cross-sections triceps muscle immunostained for EYFP (green) and phospoho-SMAD (red) from sham- and Cll-treated Cdh5-CreER^T2;R26R-EYFP mice 7 days after CTX. Arrows point to EYFP⁺ cells showing nuclear SMAD2/3P signal. Magnification × 40. Scale bar: 50 μm. (e) Quantification of EYFP⁺phospho-SMAD2⁺ cells measured as the percentage of positive cells among the total EYFP⁺ cells per square millimeter in immunostained triceps muscle sections from sham- and Cll-treated Cdh5-CreER^T2; R26R-EYFP mice, 7 days after CTX injection. Data are expressed as means±S.E.M. (n=3). Statistically significant differences are indicated (sham versus Cll *P<0.05)