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: 10415837
• 负责人: Nicole Lynne Jacobsen
• 金额: $ 7.17万
• 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/10415837
• 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.

项目概要 骨骼肌在损伤后具有通过激活、增殖和修复而具有卓越的自我修复能力。 常驻干细胞（卫星细胞，SC）的分化。急性损伤会破坏毛细血管网络并废除 灌注与肌纤维变性同时发生。尽管肌纤维的细胞和分子事件 再生已被明确定义，但对再生微脉管系统中的相应事件知之甚少。到 为了解决这一知识空白，局部注射肌毒素 BaCl2 用于引发变性并 成年（约 4 个月）雄性和雌性小鼠臀大肌 (GM) 的再生。存活的内皮细胞 细胞 (EC) 片段在损伤后 (d PI) 2-3 天内发芽，然后伸长并融合成新的毛细血管网络 被 5d PI 灌注，这与肌纤维再生的初始阶段一致。尽管他们的 密切关联和同时激活，肌生成和微血管再生之间的串扰 人们对此知之甚少。该项目的中心假设是来自 SC 后代的旁分泌信号是 微血管网络的再生和稳定不可或缺的部分。 SC 具有的转基因小鼠 受伤前已耗尽，因此无法再生肌纤维，将被使用。初步数据显示 与野生型相比，在没有肌生成的情况下，损伤后微血管密度降低 老鼠。因此，共聚焦显微镜和流式细胞术将用于确定肌生成是否控制 内皮尖端细胞选择、增殖和/或存活作为这种减弱的血管生成反应的基础 受伤后（目标 1）。用于尖端细胞标记物（例如 VEGFR2）的全装 GM 的共聚焦成像和免疫染色 将评估 EC 发芽的程度； ECs 的增殖将使用 EdU 进行体内分析。此外， 原代 EC 将从受损的 GM 中分离出来，以确定内皮细胞周期进展的程度和 使用荧光激活细胞分选进行细胞凋亡介导的血管修剪。在目标 2 中，肌生成的作用 由于氧的扩散交换，内皮通透性屏障的调节将受到测试， 肌纤维及其微血管供应之间的营养物质和代谢副产物对于恢复至关重要 并维持组织稳态。因为野生型小鼠的屏障完整性在再生早期就恢复了 但在没有肌生成、周细胞 (PC) 整合到新生微血管壁的情况下仍然存在渗漏 基于 VE-钙粘蛋白的细胞间连接的组织将通过整个 GM 中的免疫染色进行评估； 这两种成分对于紧密的 EC-EC 连接都是必不可少的。使用来自 myogenic 的条件培养基进行实验 细胞将在体外研究通过旁分泌信号传导对细胞间连接的直接和间接调节。 这些研究的结果将为肌生成如何影响最初的肌生成提供重要的新见解。 肌肉损伤后微血管再生和网络的长期稳定。该项目代表了 开发新疗法以促进肌肉从创伤、缺血和疾病中恢复的关键一步。