Targeting vascular endothelium for muscular dystrophy therapy

摘要 骨骼肌是一种高度有序的复杂组织，它包含几种细胞类型，每种细胞类型相互作用 其他用来维持结构和动态平衡。肌肉卫星细胞(SCs)是一种干细胞群 用于肌肉生长、修复和再生。保持供应链差异化与自主性之间的平衡 肌肉的动态平衡需要更新。自我更新能力的缺陷导致SC数量减少， 导致SC池耗尽和肌肉再生能力降低，这发生在老年人和 病变肌肉，包括杜氏肌营养不良症(DMD)。DMD是一种进行性疾病，在这种疾病中 肌营养不良蛋白的缺失会导致肌膜肌营养不良蛋白桥的缺失。近期工作 证明了DMD模型mdx小鼠内皮细胞的血管生成功能受损。我们演示了增加的 血管密度可以改善与DMD相关的表型。然而，血管生成在心脏疾病中的重要性 DMD的治疗尚未得到很好的解决。最近的一项研究表明，干细胞优先位于 其次是毛细血管内皮细胞(ECs)。然而，SCs和ECs之间的确切关系尚未确定 检查过了。最近，我们建立了一种优化的骨骼肌组织清除方案。我们利用了 SCs和ECs的荧光记者以及组织清除以证明SCs与 三维成像中的毛细血管，提示干细胞的血管旁生态位对干细胞的维持。SCS 血管内皮细胞生长因子和Notch受体的表达 再生过程。因此，我们假设干细胞通过血管内皮生长因子募集毛细血管内皮细胞来建立 在动态平衡和再生期间维持干细胞的血管旁生态位。在这份提案中，我们将确定 在出生后条件基因敲除Flt1和通过 抗Flt1纳米抗体，由骆驼免疫和噬菌体展示技术相结合在MDX中创造 小鼠可增加SC数量，减少病理性内皮细胞到间充质转化(EndMT)，并 改善与DMD相关的表型。因此，我们将阐明调节血管血管的关键分子- 肌肉发生，包括参与SC动态平衡的那些，这是发展治疗的基础 治疗DMD的方法。干细胞和它们的血管旁壁龛是诱导和 维持干细胞种群，以减缓DMD骨骼肌功能的丧失。

ABSTRACT Skeletal muscle is a highly ordered, yet complex tissue which contains several cell types that interact with each other to maintain structure and homeostasis. Muscle satellite cells (SCs) are a stem cell population responsible for muscle growth, repair and regeneration. Maintenance of the balance between SC differentiation and self- renewal is required for muscle homeostasis. A defect in self-renewal ability leads to a decrease in SC number, resulting in depletion of the SC pool and reduced muscle regeneration capacity, which occurs in aged and diseased muscle, including Duchenne muscular dystrophy (DMD). DMD is a progressive disorder in which the absence of the dystrophin protein results in loss of the dystrophin bridge at the muscle membrane. Recent work demonstrates the angiogenic impairment of the ECs in DMD model mdx mice. We demonstrates the increased vascular density can ameliorate phenotypes associated with DMD. However, the importance of angiogenesis in DMD treatment has not yet been well addressed. A recent study demonstrated that SCs are preferentially located next to capillary endothelial cells (ECs). However, the exact relationship between SCs and ECs has yet to be examined. Recently, we established an optimized tissue clearing protocol for skeletal muscle. We utilized fluorescent reporters for SCs and ECs along with tissue clearing to demonstrate the close proximity of SCs to capillaries in 3-dimentional imaging, suggesting the juxtavascular niche of SCs for stem cell maintenance. SCs express vascular endothelial cell growth factor (VEGF) and Notch receptors which is dynamically altered during the regeneration process. Therefore, we hypothesize that SCs recruit capillary ECs via VEGF to establish a juxtavascular niche for SC maintenance during homeostasis and regeneration. In this proposal, we will determine the extent to which an increased vascular niche in postnatal conditional knockout of Flt1 and by blocking Flt1 via anti-Flt1 nanobodies, created by a combination of llama-immunization and phage display technologies, in mdx mice can increase SC number, reduced pathological endothelial-to-mesenchymal transition (EndMT), and ameliorate phenotypes associated with DMD. Consequently, we will elucidate key molecules that regulate angio- myogenesis, including those involved in SC homeostasis, which is fundamental to developing therapeutic approaches to treat DMD. SCs and their juxtavascular niche are potential therapeutic targets to induce and maintain SC populations that slow the loss of skeletal muscle function with DMD.