Role of satellite cells in microvascular recovery during skeletal muscle regeneration

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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.