Skeletal Muscle and Vascular Remodeling in Peripheral Artery Disease

描述（由申请人提供）：外周动脉疾病（PAD）是人类发病和死亡的主要原因。有证据表明，PAD最严重的表现，即重度肢体缺血（CLI），在临床上与更为良性的间歇性跛行（IC）不同。在小鼠中，发生cli样组织坏死的倾向是品系依赖性的。已鉴定出易感（BALB/c）和耐药（C57BL/6）菌株，提示人类存在类似的遗传易感性。人类遗传学研究已经证明了与PAD的联系，然而，CLI和IC的易感性机制仍然未知。其中一个原因可能是绝大多数检查肢体缺血组织坏死易感性的研究都集中在脉管系统上。然而，我们发现骨骼肌细胞的反应，特别是骨骼肌祖细胞（MPCs）的反应，是小鼠肢体缺血后组织坏死和人类对CLI易感性的关键决定因素。此外，我们的研究结果提供了一个新的机制模型，解释了已知的PAD调节剂（如VEGF）在CLI发展中的作用。在初步研究中，我们已经建立了一种亚急性肢体缺血导致肌肉坏死的小鼠模型，类似于人类的CLI，我们已经证明：1)在这个模型中，而不是在急性缺血中，小鼠在非缺血肢体中发育了大而成熟的新血管；2)这些血管中含有MPC标志物Pax7和CD31共同表达的细胞，表明MPC可以分化为内皮细胞（ECs）；3)即使在抗坏死小鼠品系中，Pax7+ MPCs的消融也会导致剧烈的组织坏死；4)缺血可诱导抗坏死小鼠而非坏死易感小鼠MPCs上VEGF受体VEGFR-2的表达；5) VEGF诱导MPC增殖分化；6)体内MPCs中VEGFR-2的缺失导致肌肉再生不足。综上所述，这些发现提示了一个模型，在该模型中，MPCs响应VEGF刺激，并入新血管，以支持组织灌注并保护肌肉细胞免受缺血性损伤。此外，VEGF促进MPCs对肌肉再生的直接贡献。因此，我们假设内源性肌肉祖细胞中的VEGF受体信号介导肢体缺血后骨骼肌新生血管和肌纤维再生，以限制肌肉坏死。为了研究这一假设，我们的具体目标是：确定MPC VEGF受体在体内新生血管和肌肉再生中是否必需。2. 确定在体内mpc介导的新生血管形成是否需要旁分泌VEGF信号。3. 确定MPC VEGF受体是否需要缺血MPC的增殖、存活和体外分化，以及MPC是否在CLI患者中受到类似的影响。

DESCRIPTION (provided by applicant): Peripheral artery disease (PAD) is a major cause of human morbidity and mortality. Evidence suggests that the most severe manifestation of PAD, critical limb ischemia (CLI), is clinically distinct from the more benign syndrome of intermittent claudication (IC). In mice, the propensity to develop CLI-like tissue necrosis is strain- dependent. Both susceptible (BALB/c) and resistant (C57BL/6) strains have been identified, suggesting that a similar genetic susceptibility exists in humans. Human genetic studies have demonstrated linkages to PAD, however the mechanisms that predispose to CLI vs. IC remain unknown. One reason for this may be that the vast majority of studies examining susceptibility to tissue necrosis in limb ischemia have focused on the vasculature. However, we have found that the skeletal muscle cell response, particularly that of skeletal muscle progenitor cells (MPCs), is a key determinant of tissue necrosis after limb ischemia in mice and susceptibility to CLI in humans. Moreover, our findings provide a novel mechanistic model that accounts for the role of known modulators of PAD, such as VEGF, in the development of CLI. In preliminary studies, we have developed a murine model of subacute limb ischemia that leads to muscle necrosis similar to that seen in humans with CLI, and we have demonstrated that: 1) in this model, but not in acute ischemia, mice develop large, mature neovessels in the non-ischemic limb; 2) these vessels contain cells that co-express the MPC marker Pax7 together with CD31, suggesting that MPCs can differentiate into endothelial cells (ECs); 3) ablation of Pax7+ MPCs results in dramatic tissue necrosis, even in necrosis-resistant mouse strains; 4) expression of the VEGF receptor VEGFR-2 on MPCs is induced by ischemia in necrosis-resistant but not necrosis-susceptible mice; 5) VEGF induces MPC proliferation and differentiation; and 6) loss of VEGFR-2 in MPCs in vivo results in deficient muscle regeneration. Taken together, these findings suggest a model in which MPCs, in response to VEGF stimulation, incorporate into new blood vessels to support tissue perfusion and protect muscle cells from ischemic injury. In addition, VEGF promotes MPCs' known direct contribution to muscle regeneration. Thus, we hypothesize that VEGF receptor signaling in endogenous muscle progenitor cells mediates both skeletal muscle neovascularization and myofiber regeneration after limb ischemia in order to limit muscle necrosis. To investigate this hypothesis, our Specific Aims are to: 1. Determine if MPC VEGF receptors are required for neovascularization and muscle regeneration in vivo. 2. Determine if paracrine VEGF signaling is required for MPC-mediated neovascularization in vivo. 3. Determine if MPC VEGF receptors are required for ischemic MPC proliferation, survival, and differentiation in vitro, and if MPCs are similarly affected in patients with CLI.