Immunolabeling of TGF-β during Muscle Regeneration
Muscle regeneration after injury (as irradiation) is of great importance. However, the molecular and cellular mechanisms are still unclear. Cytokines are believed to play fundamental role in the different stages of muscle regeneration. They are secreted by many cell populations, but the predominant producers are macrophages and helper T cells. On the other hand, it has been shown that adipose tissue derived stromal/stem cell (ASC) injection could improve muscle regeneration. Stem cells probably induce the coordinated modulations of gene expression in different macrophage cells. Therefore, we investigated the patterns and timing of changes in gene expression of different cytokines occurring upon stem cells loading. Muscle regeneration was studied in an irradiated muscle of minipig animal model in presence or absence of ASC treatment (irradiated and treated with ASCs, IRR+ASC; irradiated not-treated with ASCs, IRR; and non-irradiated no-IRR). We characterized macrophage populations by immunolabeling in the different conditions. In our study, we found mostly M2 and a few M1 macrophages in the IRR+ASC samples. However, only few M2b macrophages were noticed in the IRR muscles. In addition, we found intensive fibrosis in the IRR samples. With in situ hybridization and immunolabeling, we analyzed the cytokine expression of the different macrophages and we showed that M2d macrophage are the most abundant in the IRR+ASC samples. By in situ hybridization, strong expression of the transforming growth factor β (TGF-β) was observed in the IRR+ASC but very week in the IRR samples. But when we analyzed TGF-β level with immunolabeling the expression was very different: many M2 macrophages showed week expression in IRR+ASC and few cells expressing stronger level in IRR muscles. Therefore, we investigated the MMP expressions in the different muscles. Our data showed that the M2 macrophages of the IRR+ASC muscle expressed MMP2 proteins. Our working hypothesis is that MMP2 expression of the M2 macrophages can decrease fibrosis in the IRR+ASC muscle by capturing TGF-β.
Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1316436Procedia APA BibTeX Chicago EndNote Harvard JSON MLA RIS XML ISO 690 PDF Downloads 405
 Arnold L. et al., (2007) Inflammatory monocytes recruited after skeletal muscle injury switch into anti-inflammatory macrophages to support myogenesis. J Exp Med. 204: 1057–1069.
 Bornemann A. et al., (1999) Satellite Cells as Players and Targets in Normal and Diseased Muscle. Neuropediatrics 30: 167-175
 Cantini M. et al., (1994) Macrophages regulate proliferation and differentiation of satellite cells. Biochem Biophys Res Commun. 202: 1688-1696.
 Cantini M. and Carraro U., (1995) Macrophage-released factor stimulates selectively myogenic cells in primary muscle culture. J Neuropathol Exp Neurol. 54: 121-8.
 Chen L. et al., (2008) Paracrine Factors of Mesenchymal Stem Cells Recruit Macrophages and Endothelial Lineage Cells and Enhance Wound Healing. Plos ONE 3: e1886.
 Delaney K. et al., (2016) The role of TGF-1 during skeletal muscle regeneration. Cell Biol Int doi: 10/1002/cbin.10725.
 Ferrante C.J. And Leibovich (2012) Regulation of Macrophage Polarization and Wound Healing. Adv In Wound Care 1: 10-16.
 Liu X. et al., (2017) Macrophage depletion impairs skeletal muscle regeneration: The roles of regulatory factors for muscle regeneration. Cell Biol Int. 3: 228-238.
 Martinez F.O. and Gordon S., (2014) The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. Mar 3;6:13. doi: 10.12703/P6-13. eCollection 2014.
 Mu X. et al., (2010) Relaxin Regulates MMP Expression and Promotes Satellite Cell Mobilization During Muscle Healing in Both Young and Aged Mice. Am J Pathol. 177: 2399–2410.
 Philippou A. et al., (2012) Cytokines in muscle damage. Adv Clin Chem. 58: 49-87.
 Riccobono D.et al., (2016) Contribution of INTRAMUSCULAR Autologous Adipose Tissue-Derived Stem Cell Injections to Treat Cutaneous Radiation Syndrome: Preliminary Results. Health Phys. 111: 117-126.
 Roszer T. (2015) Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms. Mediators of Inflammation Volume 2015 (2015), Article ID 816460, 16 pages.
 Schiaffino S. et al., (2016) Regulatory T cells and skeletal muscle regeneration. FEBS J. Aug 1. doi: 10.1111/febs.13827.
 Tidball J.G. and Villalta S.A., (2010) Regulatory interactions between muscle and the immune system during muscle regeneration. Am J Physiol Regul Integr Comp Physiol. 298: 1173–1187.
 Tidball J.G et al., (2014) Shared signaling systems in myeloid cell-mediated muscle regeneration. Development. 141: 1184–1196.
 Vedeswari C.P. et al., (2009) In vivo autofluorescence characteristics of pre- and post-treated oral submucous fibrosis: A pilot study Indian J Dent Res. 20: 261-267.
 Villalta S.A. et al., (2009) Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy. Hum Mol Genet. 18: 482–496.