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 骨骼肌在损伤后具有显著的自我修复能力，通过激活、增殖和 常驻干细胞(卫星细胞，SCs)的分化。急性损伤破坏毛细血管网络并废除 血流灌注与肌纤维变性一致。尽管肌纤维的细胞和分子事件 再生是明确的，但对再生微血管系统中的相应事件知之甚少。至 为了解决这一认识上的差距，局部注射肌毒素BaCl2用于启动退变和 成年(~4mo)雌雄小鼠臀大肌(GM)的再生。存活的内皮细胞 细胞(EC)段在损伤后2-3天内萌发，然后伸长并融合成新的毛细血管网络 由5d PI灌流，这与肌纤维再生的初始阶段一致。尽管他们 肌肉发生和微血管再生之间的密切联系和同步激活 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 对于微血管网络的再生和稳定是不可或缺的。干细胞在其体内的转基因小鼠 在受伤前已经耗尽，因此不能再生肌纤维，将被使用。初步数据显示 与野生型相比，在没有肌肉发生的情况下，微血管密度在损伤后减少 老鼠。因此，将使用共聚焦显微镜和流式细胞术来确定肌发生是否控制 内皮末端细胞的选择、增殖和/或存活是这种减弱的血管生成反应的基础 受伤后(目标1)。TIP细胞标记物(如VEGFR2)的整体GM共聚焦成像和免疫染色 将评估EC发芽的大小；ECs的增殖将用EDU在体内进行分析。此外, 原代内皮细胞将从受损的GM中分离出来，以确定内皮细胞周期进展的程度和 利用荧光激活细胞分选技术进行的细胞凋亡介导的血管修剪。在目标2中，肌肉发生的作用 在调节血管内皮细胞通透性屏障方面将进行测试，因为氧的扩散交换， 营养物质和肌纤维及其微血管供应之间的代谢副产物是恢复 以及维持组织的动态平衡。因为野生型小鼠的屏障完整性在再生早期就会恢复 但在缺乏肌肉生成、周细胞(PC)整合到新生微血管壁的情况下仍是渗漏的 以VE-钙粘附素为基础的细胞间连接的组织将通过整个GM的免疫染色来评估； 这两个组件对于紧密的EC-EC连接都是必不可少的。肌源性条件培养液的实验研究 细胞将在体外通过旁分泌信号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关键的新见解，了解肌肉发生如何影响最初的 肌肉损伤后微血管再生和网络的长期稳定。这个项目代表着一个 朝着开发新疗法的关键一步，以促进肌肉从创伤、缺血和疾病中恢复。