Improved Muscle Regeneration by Anti-Mirna Alteration in Macrophage Polarization

The applicant's career goal is to become a surgeon-scientist studying translational research in regenerative medicine. A comprehensive mentoring plan with a team of mentors of diverse backgrounds will ensure the applicant's career development. The proposed studies will address the role of microRNAs (miRNAs) on the regulation of macrophage polarization in vitro and in the dynamic tissue events during skeletal muscle injury, macrophage recruitment, angiogenesis, and muscle regeneration. Macrophages promote successful wound healing and tissue remodeling; however, macrophages can also contribute to increased tissue injury. Thus, an initial macrophage polarization state may contain pathogens, remove necrotic cell debris, and initiate progenitor cell proliferation; however, a subsequent anti-inflammatory macrophage polarization state coordinates tissue healing via angiogenesis, fibrogenesis, and progenitor cell differentiation to resolve inflammation. Differences in polarization states may explain the controversial role of macrophages as positive or negative modulators of wound healing in vivo. Our understanding of macrophage populations - i.e., classically activated (M1) and alternatively activated (M2) macrophages - is derived from in vitro studies. Several classification systems suggest that the M2 population can be further subdivided into wound repair macrophages (M2a), Th2 focused macrophages (M2b), and regulatory macrophages (M2c). However, these states of macrophage polarization in vivo are not distinct and a blending of subtypes is often described. Our working hypothesis is that progression through this continuum reflects dynamic miRNA regulation of macrophage gene expression; the temporal manipulation of select miRNA can be used to improve muscle regeneration and to mitigate the additional tissue injury of pro-inflammatory macrophages. This will be addressed by the following specific aims: 1) establish miRNA expression patterns of bone marrow-derived macrophages in vitro following exposure to polarizing cytokines; 2) determine the effects of altering the levels of specific miRNAs on macrophage polarization in vitro; and 3) establish the effects of specific anti-miRNAs on in vivo macrophage polarization, angiogenesis, and muscle regeneration. These studies will provide training in molecular biology, animal models, flow cytometry, histomorphometry, immunohistochemistry, bioinformatics, and cell culture. These studies are innovative because miRNAs that regulate macrophage polarization have not been rigorously explored. The proposed research is significant because it will provide information regarding miRNA regulation of macrophage polarization states as well as the consequences of these states in acute tissue injury and repair. These findings will have impact on the development of therapeutic targets to reduce the undesirable contribution of macrophages in tissue repair and regeneration, not only in muscle regeneration, but also in numerous other acute and chronic inflammatory diseases involving macrophages. PUBLIC HEALTH RELEVANCE: Leg and arm wounds, with large muscle defects and high amputation rates, are common in trauma victims and especially in injured soldiers from the Iraqi/Afghanistan war; new treatments to replace the missing muscle and blood vessels are needed to decrease amputation rates and improve limb function. White blood cells, especially macrophages, are important in regenerating damaged muscle, but these same cells may also contribute to tissue damage. The planned studies will characterize how different types of macrophages are regulated and to change macrophages to prevent increased muscle injury.