Improved Muscle Regeneration by Anti-miRNA Alteration in Macrophage Polarization

描述(由申请者提供)：申请者的职业目标是成为一名外科医生兼科学家，研究再生医学的转化研究。一个由不同背景的导师组成的团队的全面指导计划将确保申请者的职业发展。建议的研究将探讨microRNAs(MiRNAs)在体外对巨噬细胞极化的调节以及在骨骼肌损伤、巨噬细胞募集、血管生成和肌肉再生过程中的动态组织事件中的作用。巨噬细胞可以促进伤口的成功愈合和组织重塑；然而，巨噬细胞也可以增加组织损伤。因此，最初的巨噬细胞极化状态可能包含病原体，清除坏死的细胞碎片，并启动祖细胞增殖；然而，随后的抗炎巨噬细胞极化状态通过血管生成、纤维化形成和祖细胞分化来协调组织愈合以化解炎症。极化状态的不同可能解释了巨噬细胞在体内作为伤口愈合的正或负调节因子所起的有争议的作用。我们对巨噬细胞群体的理解--即经典激活的(M1)巨噬细胞和交替激活的(M2)巨噬细胞--源于体外研究。几种分类系统表明，M2群体可进一步细分为创伤修复巨噬细胞(M2a)、集中于Th2的巨噬细胞(M2b)和调节性巨噬细胞(M2c)。然而，体内巨噬细胞的这些极化状态并不明显，经常被描述为亚型的混合。我们的工作假设是，在这个连续体中的进展反映了miRNA对巨噬细胞基因表达的动态调节；选择miRNA的时间操作可以用于促进肌肉再生和减轻促炎巨噬细胞的额外组织损伤。这将通过以下具体目标来解决：1)建立骨髓来源的巨噬细胞暴露于极化细胞因子后的miRNA表达模式；2)确定改变特定miRNAs水平对体外巨噬细胞极化的影响；以及3)建立特定的抗miRNAs对体内巨噬细胞极化、血管生成和肌肉再生的影响。这些研究将提供分子生物学、动物模型、流式细胞术、组织形态计量学、免疫组织化学、生物信息学和细胞培养方面的培训。这些研究具有创新性，因为调节巨噬细胞极化的miRNAs尚未得到严格的探索。这项拟议的研究具有重要意义，因为它将提供关于miRNA对巨噬细胞极化状态的调节以及这些状态在急性组织损伤和修复中的后果的信息。这些发现将影响治疗靶点的开发，以减少巨噬细胞在组织修复和再生中的不良贡献，不仅在肌肉再生方面，而且在涉及巨噬细胞的许多其他急性和慢性炎症性疾病中也是如此。

DESCRIPTION (provided by applicant): 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.