Skeletal Muscle and Vascular Remodeling in Peripheral Artery Disease

 描述（由申请人提供）：外周动脉疾病（PAD）是人类发病率和死亡率的主要原因。有证据表明，PAD最严重的表现，严重肢体缺血（CLI），在临床上不同于更良性的间歇性跛行（IC）综合征。在小鼠中，发生CLI样组织坏死的倾向是品系依赖性的。敏感（BALB/c）和耐药（C57 BL/6）菌株均已鉴定，表明人类中存在相似的遗传易感性。人类遗传学研究已经证明了与PAD的联系，但是导致CLI与IC的机制仍然未知。其中一个原因可能是，绝大多数研究肢体缺血组织坏死的易感性集中在血管系统。然而，我们发现骨骼肌细胞反应，特别是骨骼肌祖细胞（MPC）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种新的机制模型，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，我们开发了一种亚急性肢体缺血的小鼠模型，该模型导致与患有CLI的人类相似的肌肉坏死，并且我们已经证明：1）在该模型中，但在急性缺血中，小鼠发育出大而成熟的新血管在非缺血肢体中; 2）这些血管含有与CD 31一起共表达MPC标志物Pax 7的细胞，表明MPC可以分化为内皮细胞（EC）; 3）Pax 7 + MPC的消融导致显著的组织坏死，甚至在坏死抗性小鼠品系中也是如此; 4）在坏死抗性而非坏死易感小鼠中，MPC上VEGF受体VEGFR-2的表达由缺血诱导; 5）VEGF诱导MPC增殖和分化;和6）体内MPC中VEGFR-2的缺失导致肌肉再生不足。综上所述，这些发现表明了一种模型，其中MPC响应于VEGF刺激而并入新血管以支持组织灌注并保护肌细胞免受缺血性损伤。此外，VEGF促进MPCs对肌肉再生的已知直接贡献。因此，我们假设内源性肌肉祖细胞中的VEGF受体信号传导介导肢体缺血后骨骼肌新生血管形成和肌纤维再生，以限制肌肉坏死。为了研究这个假设，我们的具体目标是：1。确定MPC VEGF受体是否是体内新血管形成和肌肉再生所必需的。2.确定旁分泌VEGF信号传导是否是MPC介导的体内新生血管形成所必需的。3.确定MPC VEGF受体是否是体外缺血性MPC增殖、存活和分化所必需的，以及MPC在CLI患者中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.