VEGF alleviates lower limb ischemia in diabetic mice by altering muscle fiber types

Lower limb ischemia caused by diabetic foot (DF) is one of the most serious complications of diabetes. The therapeutic role of VEGF in DF is well documented. However, the mechanism for action of VEGF is still not clear. The present study aimed to explore the effects of VEGF-mediated skeletal muscle fiber type switch in angiogenesis and the treatment of DF. C57BL/6 mice housed in cages equipped with a voluntary running wheel were used to access VEGF protein level and citrate synthase activity (by ELISA) as well as muscle fiber type changes (by immunofluorescence) in the gastrocnemius muscle. C57BL/6 mice were fed on a high-fat diet for 6 weeks and then injected with streptozocin to induce diabetic lower limb ischemia model. Control adenovirus (Ad-GFP) or Ad-VEGF-GFP were then injected into the left gastrocnemius of the ischemic diabetic mice. Blood flow perfusion was examined by laser Doppler imaging at 1, 3, 7 and 14 days after adenovirus transduction. On day 14, all mice were anesthetized and sacrificed. VEGF expression levels, citrate synthase activity and muscle fiber type changes in the gastrocnemius muscle were assayed by ELISA and immunofluorescence analysis of myosin heavy chain IIa (MHCIIa) expression, respectively. Transwell assays were performed to determine whether VEGF-treated C2C12 myotubes played a role on tubule formation and migration of HUVECs. It was found that VEGF levels and citrate synthase activity were upregulated after voluntary exercise, along with the increased frequency of oxidized muscle fibers. Notably, adenovirus-mediated VEGF overexpression in the muscle also increased the frequency of oxidized (MHCIIa-positive) muscle fibers, enhanced citrate synthase activity and ameliorated lower limb ischemia in diabetic mice. VEGF treatment enhanced the phosphorylation of PI3K, Akt and AMPK (assayed by western blotting), as well as glucose consumption and metabolism (assayed by western blotting and glucose uptake assay), in the C2C12 myotubes. Interestingly, VEGF-treated C2C12 myotubes promoted the migration and tubule formation of HUVEC cells. The present findings suggest that skeletal muscle fiber conversion might be a potential approach for VEGF-mediated angiogenesis and disease treatment, which may provide new options for the prevention and treatment of DF.