Angiogenic Gene-Modified Muscle Cells for Enhancement of Tissue Formation

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Abstract
Engineering muscle tissue with inadequate vascularity may lead to fibrosis and loss of muscle function. In this study we combined the isolation and genetic engineering of myoblasts with tissue transplantation in an attempt to create well-vascularized muscle tissue. Myoblasts were obtained from a single explant of adult Lewis rat myofibers and transfected with a bicistronic plasmid encoding vascular endothelial growth factor (VEGF) and green fluorescent protein (GFP) or with a plasmid encoding a nonfunctional VEGF–alkaline phosphatase (AP) fusion protein. VEGF expression and GFP expression in vitro were, respectively, assessed by Western blot analysis ELISA and fluorescence microscopy, showing that the myoblasts were successfully expressing the recombinant proteins. The transfected cells were suspended in collagen type I and injected subcutaneously into nude mice. Analysis of the retrieved engineered muscle tissues by RT-PCR immunostaining and fluorescence showed expression of VEGF and GFP proteins. Immunohistochemical analysis of the muscle tissues 1, 3, and 4 weeks after implantation confirmed the muscle phenotype. Neovascularization and muscle tissue mass significantly increased with functional VEGF-transfected cells compared with nonfunctional VEGF-transfected cells. In conclusion, this study demonstrates that in vivo engineered muscle tissues improve their volumes when VEGF-expressing muscle cells are used.