Muscle-Resident Stem Cells for Angiogenesis and Vascular Maturation in PAD

描述（由申请人提供）：外周动脉疾病（PAD）引起大量发病率和死亡率，但几乎没有有效的治疗方法。促进血管生成治疗PAD的策略几乎没有成功，这导致了干细胞干预的研究。基于细胞的方法的有益效果通常被认为是由辅助效应产生的，例如局部血管生长因子的分泌，尽管对于缺血肌肉中的常驻肌肉祖细胞是否可以分化成新血管以及如何实现这一目标知之甚少。我们实验室的初步研究表明，共伴侣蛋白Bag3在保护肌肉组织免于缺血中起重要作用。众所周知，Bag3在肌肉中起着重要作用，因为小鼠中Bag3的缺失会导致暴发性肌病，而人类Bag3的突变与遗传性心肌病有关。在确定Bag3是肢体存活（Lsq1）数量性状位点（QTL）的一个组成部分后，我们研究了Bag3在肢体缺血中的作用。除了对肌肉细胞的影响外，我们意外地发现外源性的Bag3与腺相关病毒（AAV6）的肌肉特异性表达诱导了非缺血和缺血肌肉血管生成的急剧增加。此外，肌肉中Bag3的表达显著促进了血管成熟，这一点在平滑肌肌动蛋白（SMA）阳性血管中得到了证明。Bag3诱导Pax7+祖细胞增加，SMA和CD31染色阳性，表明Bag3诱导血管祖细胞增加。肌肉中含有较多的Pax7+祖细胞，Pax7+祖细胞在出生后肌肉增生而非肥大的再生过程中起重要作用。有证据表明Pax7+祖细胞与内皮细胞和周细胞谱系有关。基于这些观察结果，我们假设肌肉中Bag3的表达驱动Pax7+细胞向血管细胞的分化，这可能为PAD的治疗提供内源性血管干细胞的来源。为了验证这一假设，本课题的具体目的是：1)确定外源Bag3在骨骼肌细胞体内或体外表达后Pax7+肌肉祖细胞是否能分化为内皮细胞或内皮周围细胞；2)确定体内骨骼肌表达Bag3后是否需要Pax7+细胞参与血管生成和血管成熟。该探索性R21提案是针对PA-09-248“心脏、肺和血液疾病的定向干细胞分化细胞疗法”而提交的，目的是确定骨骼肌特异性Bag3表达对Pax7+祖细胞分化为血管细胞的影响，作为PAD的潜在治疗方法。完成这一提议的具体目标将为骨骼肌组织中肌肉干细胞分化和血管与肌肉细胞之间的串扰机制提供重要的见解。此外，这些研究可能会导致治疗PAD的有效疗法。

DESCRIPTION (provided by applicant): Peripheral artery disease (PAD) causes substantial morbidity and mortality but has few effective treatments. Strategies to promote angiogenesis for the treatment of PAD have met with little success, which has led to the investigation of stem cell-based interventions. Beneficial effects of cell-based approaches are generally thought to result from accessory effects, such as the local secretion of vascular growth factors, although less is known about whether resident muscle progenitor cells in ischemic muscle can differentiate into new blood vessels and how this can be achieved. Preliminary studies in our lab have demonstrated that the co-chaperone protein Bag3 plays an important role in the protection of muscle tissue from ischemia. Bag3 is known to play an important role in muscle, as loss of Bag3 in mice results in a fulminant myopathy, and mutation in human BAG3 has been linked to inherited cardiomyopathies. We investigated Bag3's effects in limb ischemia after it was identified as a component of the limb survival (Lsq1) quantitative trait locus (QTL). In addition t effects on muscle cells, we unexpectedly found that exogenous muscle-specific expression of Bag3 with adeno-associated virus (AAV6) induced a dramatic increase in angiogenesis in non-ischemic and ischemic muscle. Moreover, Bag3 expression in muscle significantly increased vascular maturation, as evidenced by smooth muscle actin (SMA)-positive vessels. Bag3 induced an increase in Pax7+ progenitor cells, which stained positive for SMA and CD31, suggesting that Bag3 induced an increase in vascular progenitor cells. Muscle contains a relatively large number of Pax7+ progenitor cells, which play an important role in postnatal muscle regeneration through hyperplasia but not hypertrophy. Evidence has linked Pax7+ progenitor cells to both endothelial and pericyte lineages. Based on these observations, we hypothesize that expression of Bag3 in muscle drives differentiation of Pax7+ cells into vascular cells, which may provide a source of endogenous vascular stem cells for the treatment of PAD. To test this hypothesis, the Specific Aims of this proposal are to: 1) Determine whether Pax7+ muscle progenitor cells can differentiate into endothelial or peri- endothelial cells after exogenous expression of Bag3 in skeletal muscle cells in vivo or in vitro; and 2) Determine whether Pax7+ cells are required for angiogenesis and vascular maturation after Bag3 expression in skeletal muscle in vivo. This exploratory R21 proposal is submitted in response to PA-09-248, "Directed Stem Cell Differentiation for Cell-Based Therapies for Heart, Lung, and Blood Diseases", with the goal of determining the effects of skeletal muscle-specific Bag3 expression on the differentiation of Pax7+ progenitor cells into vascular cells as a potential treatment for PAD. Accomplishing the Specific Aims of this proposal will provide important insights into the mechanisms of muscle stem cell differentiation and crosstalk between blood vessels and muscle cells in skeletal muscle tissue. Furthermore, these studies may lead to effective therapies for the treatment of PAD.