Role of satellite cells in microvascular recovery during skeletal muscle regeneration

卫星细胞在骨骼肌再生过程中微血管恢复中的作用

• 批准号: 10154006
• 负责人: Nicole Lynne Jacobsen
• 金额: $ 6.86万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10154006
• 关键词: Acute; Address; Adult; Affect; Aging; Apoptosis; Attenuated; Basement membrane; Blood Vessels; Blood capillaries; Cell Cycle Progression; Cell Proliferation; Cell Survival; Cells; Conditioned Culture Media; Confocal Microscopy; Cues; Data; Degenerative Disorder; Development; Diffusion; Diphtheria Toxin; Disease; Endothelial Cells; Endothelium; Event; Female; Flow Cytometry; Fluorescence-Activated Cell Sorting; Growth; Homeostasis; Impairment; In Vitro; Inflammatory; Inflammatory Response; Injections; Injury; Intercellular Junctions; Ischemia; KDR gene; Knowledge; Link; Mediating; Metabolic; Microcirculation; Microvascular Permeability; Molecular; Mus; Muscle; Muscle satellite cell; Muscular Dystrophies; Natural regeneration; Nature; Nutrient; Oxygen; Paracrine Communication; Pathway interactions; Perfusion; Pericytes; Permeability; Phenotype; Process; Recovery; Recovery of Function; Regulation; Research Project Grants; Role; Signal Transduction; Site; Skeletal Muscle; Skeletal muscle injury; Structure; Supporting Cell; Testing; Time; Tissues; Transgenic Mice; Trauma; Trauma recovery; Vascular Endothelial Growth Factors; Vascular remodeling; Wild Type Mouse; angiogenesis; barium chloride; base; cadherin 5; cell motility; confocal imaging; density; experimental study; improved; in vivo; inducible gene expression; injured; insight; macromolecule; male; muscle regeneration; myogenesis; novel; novel therapeutics; protein expression; recruit; regeneration function; repaired; response; restoration; satellite cell; stem cells

Project Summary Skeletal muscle has the remarkable ability to repair itself following injury through activation, proliferation, and differentiation of resident stem cells (satellite cells, SCs). Acute injury destroys capillary networks and abolishes perfusion coincident with myofiber degeneration. Although the cellular and molecular events of myofiber regeneration are well defined, little is known of corresponding events in the regenerating microvasculature. To address this gap in knowledge, local injection of the myotoxin BaCl2 is used to initiate degeneration and regeneration in the gluteus maximus muscle (GM) of adult (~4 mo) male and female mice. Surviving endothelial cell (EC) segments sprout within 2-3 days post injury (d PI) then elongate and fuse into new capillary networks that become perfused by 5d PI, which coincides with the initial stages of myofiber regeneration. Despite their intimate association and concurrent activation, crosstalk between myogenesis and microvascular regeneration is poorly understood. The central hypothesis of this project is that paracrine signaling from SC progeny is integral to the regeneration and stabilization of microvascular networks. Transgenic mice in which SCs have been depleted prior to injury, and therefore cannot regenerate myofibers, will be used. Preliminary data show that microvascular density is reduced following injury in the absence of myogenesis when compared to wild-type mice. Thus, confocal microscopy and flow cytometry will be used to determine whether myogenesis controls endothelial tip cell selection, proliferation, and/or survival as the basis for this attenuated angiogenic response after injury (Aim 1). Confocal imaging and immunostaining of whole mount GM for tip cell markers (e.g., VEGFR2) will assess the magnitude of EC sprouting; proliferation of ECs will be analyzed in vivo with EdU. In addition, primary ECs will be isolated from injured GM to determine the extent of endothelial cell cycle progression and apoptosis-mediated vascular pruning using fluorescence activated cell sorting. In Aim 2, the role of myogenesis in the regulation of the endothelial permeability barrier will be tested because the diffusional exchange of oxygen, nutrients, and metabolic byproducts between myofibers and their microvascular supply is integral in restoring and maintaining tissue homeostasis. Because barrier integrity recovers early in regeneration in wild-type mice but remains leaky in the absence of myogenesis, pericyte (PC) integration into the nascent microvascular wall and organization of VE-cadherin based intercellular junctions will be evaluated by immunostaining in whole GM; both components are essential for tight EC-EC junctions. Experiments using conditioned medium from myogenic cells will investigate the direct and indirect regulation of intercellular junctions through paracrine signaling in vitro. Results from these studies will provide critical new insight into how myogenesis affects both the initial microvascular regeneration and long-term stabilization of networks after muscle injury. This project represents a critical step towards developing novel therapies to promote muscle recovery from trauma, ischemia, and disease.

项目摘要 骨骼肌具有通过激活，增殖和 驻留干细胞（卫星细胞，SCS）的分化。急性伤害破坏毛细管网络并废除 灌注与肌纤维变性一致。虽然肌纤维的细胞和分子事件 再生的定义很好，对再生微脉管系统中的相应事件知之甚少。到 在知识方面解决这一差距，局部注入肌毒素BACL2用于启动变性和 成年男性和雌性小鼠的臀大肌（GM）的再生。幸存的内皮 受伤后2-3天内的细胞（EC）段发芽（D PI），然后伸长并融合到新的毛细管网络中 5D PI引起了灌注，这与肌纤维再生的初始阶段相吻合。尽管他们 亲密关联和并发激活，肌发生与微血管再生之间的串扰 理解很差。该项目的中心假设是SC后代的旁分泌信号传导是 微血管网络的再生和稳定不可或缺。 SC具有的转基因小鼠 在受伤之前被耗尽，因此无法再生肌纤维。初步数据显示 与野生型相比，在没有肌发生的情况下损伤后，微血管密度降低了 老鼠。因此，将使用共聚焦显微镜和流式细胞仪来确定肌发生是否控制 内皮尖端细胞的选择，增殖和/或生存，作为这种减毒的血管生成反应的基础 受伤后（目标1）。整个GM的共聚焦成像和尖端细胞标记的免疫染色（例如VEGFR2） 将评估EC发芽的大小； EC的扩散将在体内与EDU分析。此外， 主要EC将从受伤的GM中分离出来，以确定内皮细胞周期进程的程度和 凋亡介导的血管修剪使用荧光激活的细胞分选。在AIM 2中，肌发生的作用 在调节内皮渗透性屏障中，将被测试，因为氧的扩散交换， 肌纤维及其微血管供应之间的营养和代谢副产品在恢复方面是不可或缺的 并保持组织稳态。因为野生型小鼠的再生早期屏障完整性恢复 但是在没有肌发生的情况下，周围（PC）整合到新生的微血管壁中，仍然漏水 以及基于VE-钙粘着蛋白的细胞间结的组织将通过整个GM进行免疫染色来评估； 这两个组件对于紧密的EC-EC连接至关重要。实验使用来自肌原性的条件培养基 细胞将通过体外旁分泌信号传导研究细胞间连接的直接和间接调节。 这些研究的结果将提供有关肌发生如何影响初始的关键新见解 肌肉损伤后网络的微血管再生和长期稳定。这个项目代表 开发新疗法以促进创伤，缺血和疾病的肌肉恢复的关键步骤。