Therapeutic revascularization with PDGF-CC in ischemic limbs. (A) RNAse protection analysis, showing that PDGF-Rα expression in the gastrocnemius muscle was decreased at 2 days after femoral artery ligation but restored to normal levels after PDGF-CC treatment. The ratio of the PDGF-Rα levels (arbitrary units), normalized for β-actin levels, is shown. (B and C) PDGF-CC protein treatment increased the PECAM capillary (B) and α-SMA⁺ arteriolar (C) density in the ischemic gastrocnemius muscle. (D and E) PDGF-CC protein treatment decreased muscle necrosis (D) and increased muscle regeneration (E) in the gastrocnemius muscle at 7 days after femoral artery ligation. Areas are percentage of total muscle area. *P < 0.05 (A–E). (F and G) Compared with vehicle (F), PDGF-CC protein treatment increased the density of PECAM vessels in the regenerating areas of the ischemic gastrocnemius muscle (G). No signs of edema, hemorrhage. or fibrosis were observed. (H and I) H&E staining, showing larger areas of regenerating myocytes (small cells with central nuclei) after PDGF-CC treatment (I) than after treatment with vehicle (H). The regions containing regenerating myocytes are surrounded by a dashed black line in both panels. (J–L) Higher magnification of H&E-stained sections of a normal gastrocnemius muscle (J); ischemic muscle, treated with vehicle, containing numerous necrotic ghost myocytes, and few blood vessels (K); ischemic muscle, treated with PDGF-CC, containing numerous regenerating myocytes with a central nucleus and numerous blood vessels (L). Values are mean ± SEM of at least 15 mice. The lumen of the arterioles is filled with dark bismuth gelatin in F–L. Scale bars: 50 μm.