cobra-cardiotoxin-proteins and Fibrosis

A longstanding goal in regenerative medicine is to reconstitute functional tissues or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised.. We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®.. We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.. Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact.

Topics: Animals; Barium Compounds; Chlorides; Cobra Cardiotoxin Proteins; Cold Injury; Cytokines; Elapid Venoms; Fibrosis; Freezing; Green Fluorescent Proteins; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Animal; Muscle Development; Muscle, Skeletal; Myoblasts; Necrosis; Neovascularization, Physiologic; Regeneration; Satellite Cells, Skeletal Muscle; Stem Cells; Vascular Endothelial Growth Factor Receptor-2

Skeletal muscle tissue has an enormous regenerative capacity that is instrumental for a successful defense against muscle injury and wasting. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) exerts therapeutic effects in several muscle pathologies, but its role in damage-induced muscle regeneration is unclear.. Using muscle-specific gain- and loss-of-function models for PGC-1α in combination with the myotoxic agent cardiotoxin (CTX), we explored the role of this transcriptional coactivator in muscle damage and inflammation.. Interestingly, we observed PGC-1α-dependent effects at the early stages of regeneration, in particular regarding macrophage accumulation and polarization from the pro-inflammatory M1 to the anti-inflammatory M2 type, a faster resolution of necrosis and protection against the development of fibrosis after multiple CTX-induced injuries.. PGC-1α exerts beneficial effects on muscle inflammation that might contribute to the therapeutic effects of elevated muscle PGC-1α in different models of muscle wasting.

Topics: Animals; Cobra Cardiotoxin Proteins; Fibrosis; Gene Expression; Hydroxyproline; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Contraction; Muscle, Skeletal; Myositis; Necrosis; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Regeneration

Profibrotic cells that develop upon injury generate permanent scar tissue and impair organ recovery, though their origin and fate are unclear. Here we show that transient expression of ADAM12 (a disintegrin and metalloprotease 12) identifies a distinct proinflammatory subset of platelet-derived growth factor receptor-α-positive stromal cells that are activated upon acute injury in the muscle and dermis. By inducible genetic fate mapping, we demonstrate in vivo that injury-induced ADAM12(+) cells are specific progenitors of a major fraction of collagen-overproducing cells generated during scarring, which are progressively eliminated during healing. Genetic ablation of ADAM12(+) cells, or knockdown of ADAM12, is sufficient to limit generation of profibrotic cells and interstitial collagen accumulation. ADAM12(+) cells induced upon injury are developmentally distinct from muscle and skin lineage cells and are derived from fetal ADAM12(+) cells programmed during vascular wall development. Thus, our data identify injury-activated profibrotic progenitors residing in the perivascular space that can be targeted through ADAM12 to limit tissue scarring.

Topics: Acute Disease; ADAM Proteins; ADAM12 Protein; Adipocytes; Animals; Blood Vessels; Cell Lineage; Cicatrix; Cobra Cardiotoxin Proteins; Collagen; Crosses, Genetic; Dermis; Ear, External; Fibrosis; Freund's Adjuvant; Gene Knockdown Techniques; Genes, Reporter; Leg Injuries; Mice; Mice, Transgenic; Muscle, Skeletal; Myofibroblasts; Parabiosis; Receptor, Platelet-Derived Growth Factor alpha; Specific Pathogen-Free Organisms; Stromal Cells; Wound Healing

Myostatin (MSTN) is a muscle-specific secreted peptide that functions to limit muscle growth through an autocrine regulatory feedback loop. Loss of MSTN activity in cattle, mice, and humans leads to a profound phenotype of muscle overgrowth, associated with more and larger fibers and enhanced regenerative capacity. Deletion of MSTN in the mdx mouse model of Duchenne muscular dystrophy enhances muscle mass and reduces disease severity. In contrast, loss of MSTN activity in the dyW/dyW mouse model of laminin-deficient congenital muscular dystrophy, a much more severe and lethal disease model, does not improve all aspects of muscle pathology. Here we examined disease severity associated with myostatin (mstn-/-) deletion in mice nullizygous for delta-sarcoglycan (scgd-/-), a model of limb-girdle muscular dystrophy. Early loss of MSTN activity achieved either by monoclonal antibody administration or by gene deletion each improved muscle mass, regeneration, and reduced fibrosis in scgd-/- mice. However, antibody-mediated inhibition of MSTN in late-stage dystrophic scgd-/- mice did not improve disease. These findings suggest that MSTN inhibition may benefit muscular dystrophy when instituted early or if disease is relatively mild but that MSTN inhibition in severely affected or late-stage disease may be ineffective.

Topics: Aging; Animals; Body Weight; Cobra Cardiotoxin Proteins; Disease Models, Animal; Fibrosis; Gene Deletion; Genotype; Hydroxyproline; Mice; Mice, Transgenic; Muscular Dystrophies, Limb-Girdle; Myostatin; Time Factors; Transforming Growth Factor beta