Figure 2
Pro-angiogenic genes are upregulated during skeletal muscle regeneration. (a) IF on TA cross-sections from 2-month-old Cdh5-CreERT2; R26R-NGZ mice before and 5, 7 and 15 days after CTX damage. Representative images of sections immunostained for CD31 (red) overlayed to the nuclear X-Gal staining (converted in pseudo fluorescent green signal), showing colocalization of labeled cells with CD31. Magnification × 20. Scale bar: 50 μm. (b) TA muscles from 2-month-old Cdh5-CreERT2; R26R-EYFP mice were collected immediately before and 1, 3, 7, 10 and 15 days after injection of CTX. Total muscle lysates were collected and processed for RNA and protein extraction. Real-time PCR analyses for HIF1-α, MMP-2, MMP-13, MMP-14, VEGF-β, PDGF-α, PDGF-β, TGF-β, Ang-I and Ang-II mRNA expression were performed. Results were normalized to 28S mRNA levels and expressed as relative fold changes compared with undamaged muscles (NT). The data are expressed as means±S.E.M. (n=6). Statistically significant differences are indicated (*P<0.05 versus NT). Western blot analysis of HIF1-α expression on total healthy muscle (NT, lane 1) and at different time point after CTX injection (days 1 to 15, lanes 2 to 6) was performed. Results are representative of three independent experiments. (c) Quadriceps and TA muscles from 2-month-old Cdh5-CreERT2; R26R-EYFP mice were collected 1, 3, 5, 7 and 10 days after injection of CTX. Muscles were digested to isolate CD11b+ cells by magnetic bead sorting and processed for RNA and protein extraction. Real-time PCR analyses for HIF1-α, MMP-2, MMP-13, MMP-14, VEGF-β, PDGF-α, PDGF-β, TGF-β, Ang-I and Ang-II mRNA expression were performed. Results were normalized to 28S mRNA levels and expressed as relative fold changes compared with CD11b+ cells retrieved from peripheral blood (circ. CD11b+). The data are expressed as means±S.E.M. (n=6). Statistically significant differences are indicated (*P<0.05 versus circ. CD11b+). Time course changes in the expression of HIF1-α were evaluated by western blot analysis on muscle-infiltrating CD11b+ cells at different time points of CTX injection. Results are representative of two independent preparations
