CHEMOKINES AND IMMUNE CELLS IN HIND LIMB ISCHEMIA

描述（由申请人提供）：肌肉再生和血管生成是四肢创伤性和/或缺血性损伤后保肢的重要组成部分。我们已经证明，敲除小鼠缺乏单核细胞趋化蛋白-1（MCP-1）或其特异性受体，CC趋化因子受体2（CCR 2），在缺血性或毒性损伤后，巨噬细胞募集和肌肉再生受损。此外，CCR 2-/-小鼠表现出再生肌肉中脂肪细胞积累增加。用野生型（WT）BM（辐射嵌合体）替换CCR 2-/-小鼠的骨髓（BM）导致早期巨噬细胞募集和正常肌肉再生增加（即，概括了WT小鼠的表型）。此外，用CCR 2-/- BM替代WT小鼠的BM导致巨噬细胞募集减少和肌肉再生受损（即，概括了CCR 2-/-小鼠的表型）。这表明BM衍生的细胞调节肌肉的愈合反应，并且巨噬细胞可能是介导CCR 2-/-小鼠中受损的肌肉再生的BM衍生的细胞类型。进一步的研究显示，与WT小鼠相比，CCR 2-/-小鼠的血管生成受损，同时组织血管内皮生长因子（VEGF）降低。有趣的是，在CCR 2-/-和WT小鼠中，VEGF恢复到基线水平与最大毛细血管密度的发展相关。虽然CCR 2-/-小鼠中受损的肌肉再生可归因于BM衍生的细胞，但尚未研究BM衍生的细胞与宿主衍生的细胞在血管生成方面的作用。我们的长期目标是确定炎症，包括趋化因子系统，在血管生成和骨骼肌再生的影响。以下3个具体目标将检验以下总体假设：BM衍生细胞（尤其是单核细胞/巨噬细胞）的募集和活化对于血管生成（损伤后骨骼肌再生的关键组成部分）至关重要。1)确定在损伤后BM衍生的细胞与非BM衍生的细胞中CCR 2表达对骨骼肌中血管生成的贡献，2）确定MCP-1/CCR 2轴对血管生成的离体和体内影响，和3）确定在肌肉损伤之前选择性和完全的单核细胞/巨噬细胞消融的作用，并在损伤后炎症和血管生成的组织修复的整个时间过程中持续。拟议的研究是创新的，因为它们将有助于确定BM衍生细胞对血管生成的贡献。这项研究的意义在于，更好地了解骨骼肌再生和血管生成的机制，可以设计新的主要或辅助治疗方法，以改善肢体挽救和组织工程。公共卫生相关性：腿部和手臂伤口，大肌肉缺损和截肢率高，是常见的创伤受害者，特别是在受伤的士兵从伊拉克/阿富汗战争;新的治疗方法，以取代失踪的肌肉，以减少截肢率和改善肢体功能。我们的研究研究了制造新肌肉（包括血管）所需的多种细胞之间的复杂关系。更好地了解新血管如何帮助肌肉从损伤中恢复可能会导致新的治疗方法，包括组织工程策略，以帮助患者从这些毁灭性的损伤中恢复。

DESCRIPTION (provided by applicant): Muscle regeneration and angiogenesis are important components of limb salvage following traumatic and/or ischemic injury to the extremities. We have demonstrated that knockout mice lacking either monocyte chemotactic protein-1 (MCP-1) or its specific receptor, the CC Chemokine Receptor 2 (CCR2), have impairments in macrophage recruitment and muscle regeneration following ischemic or toxic injury. In addition, CCR2-/- mice exhibit increased adipocyte accumulation in regenerated muscle. Bone marrow (BM) replacement of CCR2 -/- mice with wild type (WT) BM (radiation chimeras) led to increased early macrophage recruitment and normal muscle regeneration (i.e., recapitulated the phenotype of WT mice). Furthermore, replacing the BM of WT mice with CCR2 -/- BM resulted in decreased macrophage recruitment and impaired muscle regeneration (i.e., recapitulated the phenotype of CCR2 -/- mice). This suggests that BM-derived cells modulate the healing responses of muscle and that macrophages are the likely BM-derived cell type that mediates the impaired muscle regeneration in CCR2 -/- mice. Further studies revealed impaired angiogenesis in CCR2 -/- mice in conjunction with decreased tissue vascular endothelial growth factor (VEGF) compared to WT mice. Interestingly, restoration of VEGF to baseline levels was associated with the development of maximal capillary density in both CCR2 -/- and WT mice. While impaired muscle regeneration in CCR2 -/- mice is attributable to a BM-derived cell, the effects of BM-derived vs. host-derived cells have not been studied in regards to angiogenesis. Our long-term goal is to define the influence of inflammation, including the chemokine system, in angiogenesis and skeletal muscle regeneration. The following 3 specific aims will test the overall hypothesis that the recruitment and activation of BM-derived cells, especially monocytes/macrophages, are essential for angiogenesis, a critical component in skeletal muscle regeneration after injury. 1) Determine the contribution of CCR2 expression in BM-derived vs. non-BM- derived cells on angiogenesis in skeletal muscle after injury, 2) Determine the ex vivo and in vivo influence of the MCP-1/CCR2 axis on angiogenesis and 3) Define the effects of selective and complete monocyte/macrophage ablation prior to muscle injury and sustained throughout the time course of tissue repair on inflammation and angiogenesis after injury. The proposed studies are innovative because they will help define the contribution of BM-derived cells to angiogenesis. The significance of this research is that a better understanding of the mechanisms of skeletal muscle regeneration and angiogenesis could lead to the design of novel primary or adjuvant treatments for improved limb salvage and tissue engineering. PUBLICE 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 are needed to decrease amputation rates and improve limb function. Our research studies the complex relationships between the multiple cells that are needed to make new muscle, including blood vessels. A better understanding of how new blood vessels help muscle recover from injury could lead to new therapies, including tissue engineering strategies, to help patients recover from these devastating injuries.