Skeletal Muscle and Vascular Remodeling in Peripheral Artery Disease

 描述(申请人提供)：外周动脉疾病(PAD)是人类发病率和死亡率的主要原因。有证据表明，PAD最严重的表现是严重的肢体缺血(CLI)，在临床上有别于更为良性的间歇性跛行综合征(IC)。在小鼠中，发生CLI样组织坏死的倾向与应变有关。已鉴定出敏感株(BALB/c)和耐药株(C57BL/6)，这表明人类也存在类似的遗传易感性。人类遗传学研究已经证实了与PAD的联系，然而，CLI与IC的易感机制仍不清楚。其中一个原因可能是绝大多数研究肢体缺血对组织坏死的易感性的研究都集中在血管系统上。然而，我们发现骨骼肌细胞的反应，特别是骨骼肌祖细胞的反应，是小鼠肢体缺血后组织坏死的关键决定因素，也是人类CLI易感性的关键决定因素。此外，我们的发现提供了一个新的机制模型，解释了已知的PAD调节剂，如血管内皮生长因子，在CLI的发展中所起的作用。在初步研究中，我们建立了一种类似于人类CLI的亚急性肢体缺血导致肌肉坏死的小鼠模型，我们已经证明：1)在该模型中，但不是在急性缺血中，小鼠在非缺血肢体发育出大的、成熟的新生血管；2)这些血管包含共同表达MPC标记Pax7和CD31的细胞，表明MPC可以分化为内皮细胞；3)Pax7+MPC的消融导致显著的组织坏死，即使在具有坏死抗性的小鼠品系中也是如此；4)血管内皮生长因子受体VEGFR-2在MPC上的表达是由缺血诱导的，而不是由缺血引起的；5)血管内皮生长因子诱导MPC的增殖和分化；6)体内MPC中VEGFR-2的缺失导致肌肉再生不足。综上所述，这些发现提出了一种模型，在该模型中，MPC响应于血管内皮生长因子的刺激，结合到新的血管中，以支持组织灌流并保护肌肉细胞免受缺血损伤。此外，血管内皮生长因子促进MPC对肌肉再生的已知直接贡献。因此，我们假设内源性肌祖细胞中的血管内皮生长因子受体信号调节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。为了研究这一假说，我们的具体目的是：1.确定体内新生血管和肌肉再生是否需要MPC-VEGF受体。2.确定MPC介导的体内新生血管是否需要旁分泌的血管内皮生长因子信号。3.确定MPC血管内皮生长因子受体是否在体外对缺血的MPC的增殖、存活和分化是必需的，以及在CLI患者中MPC是否受到类似的影响。

 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.