The biological actions mediated by Ang are transduced by the Mas receptor. Through pharmacologic and genetic evidence, we have determined that the positive effects of Ang- observed in dystrophic mice are associated with the reduction of TGF-b-Smad-dependent signaling through the Mas receptor. We found that infusion or oral administration of Ang- in mdx mice normalized skeletal muscle architecture, decreased local fibrosis, and NVP-BEZ235 improved muscle function in vitro and in vivo. Consistent with these findings, mdx mice infused with Mas antagonist or deficient for the Mas receptor show worse muscle architecture, increased fibrosis, and increased muscle weakness compared with mdx dystrophic mice. Ang- is mainly produced by the action of the enzyme angiotensin-converting enzyme 2, a pleiotropic monocarboxypeptidase capable of metabolizing a range of peptide substrates, including Ang I, Ang II, des-Arg9-bradykinin, apelin13, and opioids. Ang-, the major enzymatic product of ACE2, has been shown to reduce Ang II-induced cardiac hypertrophy and remodeling and pressure overload-induced heart failure. Because evidence supporting the beneficial effects of Ang- is accumulating, ACE2 is beginning to be considered as a critical pharmacological target with enormous therapeutic projections. In this study we investigated the presence, activity, and localization of ACE2 in wt and mdx skeletal muscle and in a model of fibrosis induced by chronic damage. All the dystrophic muscles studied showed higher ACE2 activity than wt muscle, and ACE2 was localized mainly at the sarcolemma and to a lesser extent in interstitial cells. Furthermore, we evaluated the effect of ACE2 overexpression in mdx muscle and demonstrated a reduction in fibrosis, one of the features of dystrophic muscles together with fewer inflammatory cells infiltrating the tissue. Inflammation is a hallmark of dystrophic muscle and is associated with fibrotic accumulation. It has been shown that ACE2 deficiency induces inflammation. Since the expression of hACE2 was highest at day 5 and we also observed lower levels of collagen I and fibronectin at this time point, we evaluated the effect of the overexpression of ACE2 on inflammatory cells by examining the number of cells positive for CD68 in the muscle sections. Figure 6A shows a great number of macrophages in the TA after Ad-GFP injection. However, the number of macrophages LY2109761 present near the injection site was obviously lower in TA injected with Ad-hACE2. Quantitative analysis of the number of macrophages versus the distance from the injection site showed that the number of M1 macrophages near or far from the Ad-hACE2 injection site was lower than in the Ad-GFP injected muscle. As a control, Figure 6C shows that the adenoviral injections did not affect the number of eosinophils present 5 days after injection. These results suggest that ACE2 might improve the dystrophic muscle phenotype by decreasing the number of inflammatory cells that infiltrate the muscle. We have detected a clear correlation among ACE2 levels and degree of fibrosis. Since Ang- infusion reduces fibrosis we asked whether the oppositive is true, we evaluated whether the levels of ACE2 are modulated as a result of peptide infusion. Immunolocalization studies showed that the amount of ACE2 in mdx GAST was significantly lower in muscle infused with Ang- compared to non-infused mdx tissue.