MicroRNA Regulation of Macrophage Polarization in Muscle Regeneration

描述(由申请人提供)： 干细胞和组织再生在治疗各种疾病方面具有很大的治疗前景。例如，与目前的应用相关的是，在伊拉克/阿富汗战争退伍军人中观察到的所有战斗伤亡中，肢体损伤占50%-60%，导致大量软组织缺陷(肌肉和皮肤)和高截肢率。骨骼肌虽然是四肢最容易受到缺血性损伤的组织，但由于卫星细胞的存在，它也具有惊人的再生潜力。卫星细胞是驻留在骨骼肌中的干细胞，在肌肉受损的情况下，可以增殖并融合在一起或与受损的肌肉纤维一起再生肌肉；巨噬细胞在这一过程中至关重要。多种类型的巨噬细胞参与再生过程；经典激活(M1)、伤口愈合(M2a)和调节(M2c)巨噬细胞亚群存在于皮肤伤口和再生骨骼肌中；分别促进炎症、组织修复和炎症的消退。然而，microRNAs(MiRNAs)对巨噬细胞特化(极化)的调节以及巨噬细胞亚群对肌肉再生的影响尚未阐明。MiRNAs是一种小的、非编码的RNA，可以抑制基因表达，从而调节包括免疫细胞分化在内的许多过程。更好地了解骨骼肌再生和皮肤创伤愈合的机制，以及巨噬细胞极化对这些事件的影响，可能会导致新的和辅助的肢体保肢治疗。我们的长期目标是确定炎症，包括趋化因子系统，在血管生成、伤口愈合和骨骼肌再生中的影响。我们已经广泛研究了CC趋化因子受体2(CCR2)在肌肉再生中的重要性。CCR2对单核/巨噬细胞的募集和分化至关重要。肌肉损伤后，与野生型(WT)小鼠相比，CCR2-/-小鼠在巨噬细胞募集、血管生成和肌肉再生方面存在损害。重要的是，通过提供WT骨髓，CCR2-/-小鼠的巨噬细胞募集和肌肉再生缺陷可以被逆转，因此，骨髓来源细胞上CCR2的表达对于正常的肌肉再生至关重要，巨噬细胞可能是介导这一结果的骨髓来源细胞。将测试以下3个特定目标：1)确定选定的miRNAs(miR-21和-147)对巨噬细胞极化的体外调节。2)建立巨噬细胞极化过程中特定miRNAs(miR-21和-147)的mRNA靶点；3)确定单个miRNAs对体内巨噬细胞亚群的生物学效应以及创伤愈合过程中的协同生物学事件。通过增加或减少miRNA的表达来调节巨噬细胞的极化，我们寻求改善伤口愈合，并减少许多影响退伍军人群体的疾病中存在的急性和慢性炎症的不利影响。拟议的研究具有创新性，因为它们将有助于确定miRNAs对巨噬细胞极化以及随后的肌肉再生和伤口愈合的贡献。体外和体内研究的结合将共同确定对巨噬细胞极化重要的miRNAs，确定mRNA靶标，并评估对肌肉再生和伤口愈合的任何影响。鉴于锁定核酸修饰的寡核苷酸(LNA-AntimiR)的可用性，即补充特定miRNAs的RNA寡核苷酸，可用于动物以减少miRNA的表达，一种新的治疗药物可能对人类可用。这项研究的意义在于，更好地了解骨骼肌再生和伤口愈合的机制，可以设计出新的初级或辅助治疗方法，利用miRNA改变化合物来改善肢体挽救。

DESCRIPTION (provided by applicant): Stem cells and tissue regeneration have great therapeutic promise for treatment of a wide variety of medical conditions. For example and relevant to the current application, extremity injuries compromise 50-60% of all combat casualties observed in Iraqi/Afghanistan War Veterans resulting in large soft tissue defects (muscle and skin) and high amputation rates. Skeletal muscle, while being the tissue most vulnerable to ischemic damage in the extremities, also has an amazing potential to regenerate due to satellite cells. Satellite cells are stem cells that reside in skeletal muscle and with muscle injury, can proliferate and fuse together or with damaged muscle fibers to regenerate muscle; macrophages are critical to this process. Multiple types of macrophages participate in the regenerative process; classically activated (M1), wound healing (M2a) and regulatory (M2c) macrophage subsets are present in skin wounds and regenerating skeletal muscle; promoting inflammation, tissue repair and resolution of inflammation, respectively. However, the regulation of macrophage specialization (polarization) by microRNAs (miRNAs), and the consequential effects of macrophage subsets on muscle regeneration have not been elucidated. MiRNAs are small, noncoding RNAs that inhibit gene expression, thereby regulating many processes, including immune cell differentiation. A better understanding of the mechanisms of skeletal muscle regeneration and skin wound healing, and the influence of macrophage polarization on these events, could lead to new and adjunct therapies for limb salvage. Our long-term goal is to define the influence of inflammation, including the chemokine system, in angiogenesis, wound healing and skeletal muscle regeneration. We have extensively studied the importance of the CC Chemokine Receptor 2 (CCR2) in muscle regeneration. CCR2 is crucial for monocyte/macrophage recruitment and differentiation. Following muscle injury, CCR2-/- mice have impairments in macrophage recruitment, angiogenesis and muscle regeneration compared to wild type (WT) mice. Importantly, macrophage recruitment and muscle regeneration defects can be reversed in CCR2-/- mice by providing WT bone marrow, thus, CCR2 expression on bone marrow-derived cells is critical for normal muscle regeneration and macrophages are the likely BM-derived cell that mediates this outcome. The following 3 specific aims will be tested: 1) Define the in vitro regulation of macrophage polarization by selected miRNAs (miR-21 and -147). 2) Establish the mRNA targets of selected miRNAs (miR-21 and -147) during macrophage polarization and 3) Determine the biological effects of individual miRNAs on in vivo macrophage subsets and coordinated biological events during wound healing. By modulating macrophage polarization via increased or decreased miRNA expression, we seek to improve wound healing and decrease the adverse affects of acute and chronic inflammation present in many diseases that affect the Veteran population. The proposed studies are innovative because they will help define the contribution of miRNAs to macrophage polarization and subsequent muscle regeneration and wound healing. The combination of in vitro and in vivo studies will collectively identify miRNAs important for macrophage polarization, determine the mRNA targets, and assess any effects on muscle regeneration and wound healing. Given the availability of locked-nucleic-acid-modified oligonucleotide (LNA-antimiR), RNA oligonucleotides complementary to specific miRNAs that can be used in animals to decrease miRNA expression, a new therapeutic class of agents could become available for humans. The significance of this research is that a better understanding of the mechanisms of skeletal muscle regeneration and wound healing could lead to the design of novel primary or adjuvant treatments for improved limb salvage using miRNA- altering compounds.