REVIEW Open Access Aberrant repair and - Skeletal Muscle

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REVIEW Open Access Aberrant repair and - Skeletal Muscle

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Mann et al. Skeletal Muscle 2011, 1:21 http://www.skeletalmusclejournal.com/content/1/1/21

Skeletal Muscle

REVIEW

Open Access

Aberrant repair and fibrosis development in skeletal muscle
Christopher J Mann1†, Eusebio Perdiguero1†, Yacine Kharraz1, Susana Aguilar1, Patrizia Pessina1, Antonio L Serrano1† and Pura Muñoz-Cánoves1,2*

Abstract
The repair process of damaged tissue involves the coordinated activities of several cell types in response to local and systemic signals. Following acute tissue injury, infiltrating inflammatory cells and resident stem cells orchestrate their activities to restore tissue homeostasis. However, during chronic tissue damage, such as in muscular dystrophies, the inflammatory-cell infiltration and fibroblast activation persists, while the reparative capacity of stem cells (satellite cells) is attenuated. Abnormal dystrophic muscle repair and its end stage, fibrosis, represent the final common pathway of virtually all chronic neurodegenerative muscular diseases. As our understanding of the pathogenesis of muscle fibrosis has progressed, it has become evident that the muscle provides a useful model for the regulation of tissue repair by the local microenvironment, showing interplay among muscle-specific stem cells, inflammatory cells, fibroblasts and extracellular matrix components of the mammalian wound-healing response. This article reviews the emerging findings of the mechanisms that underlie normal versus aberrant muscle-tissue repair.

Introduction Pathophysiologic fibrosis, which is essentially an excessive accumulation of extracellular matrix (ECM) components, particularly collagen, is the end result of a cascade of events proceeding from tissue injury via inflammation, and resulting in permanent scar formation. Fibrosis can impair tissue function and cause chronic diseases in a large variety of vital organs and tissues, including bone marrow (BM). Despite the diverse range of tissues susceptible to fibrosis, all fibrotic reactions share common cellular and molecular mechanisms, such as cell and tissue degeneration, leukocyte infiltration, persistent inflammation of the tissue, and proliferation of cells with a fibroblast-like phenotype. The interplay and imbalance of different cell types sustains the production of numerous growth factors, proteolytic enzymes, angiogenic factors and fibrogenic cytokines, which together perturb the microenvironment of the damaged tissue, and stimulate the deposition of connective-tissue elements that progressively remodel, destroy and replace the normal tissue architecture. However,
* Correspondence: [email protected] † Contributed equally

despite many common elements, there are also important differences between distinct tissue systems, and the identity of some cellular and soluble factors initiating and contributing to fibrogenic pathways are still unknown. Thus, improving our understanding of the mechanisms, cell types and factors involved in this process is crucial to develop treatment strategies for these diseases.
The muscle tissue microenvironment controls normal repair versus fibrosis development
Muscular dystrophies In skeletal muscle, fibrosis is most often associated with the muscular dystrophies, a clinically and molecularly heterogeneous group of diseases. Phenotypically, these diseases are characterized by inflammation of the muscle tissue and skeletal-muscle wasting, which compromises patient mobility so that affected people become confined to a wheelchair. In the most severe cases, such as Duchenne muscular dystrophy (DMD, caused by the lack of the dystrophin protein), muscle loss and fibrosis also cause premature death through respiratory and cardiac failure [1]. In many dystrophies, including DMD, the mutation affects proteins that form a link between the cytoskeleton and the basal lamina, generally resulting in

© 2011 Mann et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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the disassembly of whole protein complexes. As a result, the sarcolemma becomes fragile, especially during intense contractile activity. In turn, there is focal or diffuse damage to the fiber and increased entry of calcium, although the underlying molecular mechanisms for these effects have not yet been elucidated in detail [2]. Several parallels can also be made between the muscular dystrophies and the idiopathic inflammatory myopathies (IIMs), which share common phenotypic features such as inflammation and muscle weakness, although the underlying causes are different.
In normal muscle repair after acute injury, such as in experimental animals and in humans after sports injuries, damaged or dead fibers are first removed by inflammatory cells, and they are then repaired or replaced by

tissue-resident muscle stem cells known as satellite cells [3]. However, in chronic human diseases such as DMD and many other dystrophies, newly generated fibers are also prone to degeneration because they retain the underlying molecular defect, producing constant cycles of fiber degeneration associated with chronic inflammation (Figure 1) [4]. Until a few years ago, satellite cells were the only known post-natal regenerative cells with myogenic potential. In DMD, this satellite-cell population is either exhausted over time or it loses the capacity to mediate repair, and the muscle tissue is progressively replaced by adipose and fibrotic tissue. Fibrosis and loss of muscle tissue in dystrophies not only reduces motile and contractile functions, but also diminishes the amount of target tissue available for therapeutic

Figure 1 Extracellular matrix (ECM) deposition in acute and chronic muscle regeneration. Acute injury to healthy muscle produces rapid and controlled inflammation that removes dead and damaged myofibers, and promotes replacement of the injured muscle. However, in conditions of chronic injury, as occurs in the muscular dystrophies, chronic inflammatory events result in the excessive accumulation of ECM components, which inhibit myogenic repair and lead to muscle being replaced by fibrotic/scar tissue. (Top) Tibialis anterior muscles of mice were injected with cardiotoxin and samples were taken at different stages of the regeneration process. A representative sample showing the inflammatory phase, characterized by a transient increase in collagen deposition, and subsequently the resolving phase of healing, with progressive recovery of the normal tissue morphology (hematoxylin and eosin). (Bottom) Evolution of the morphological changes seen in the diaphragm of mdx dystrophic mice with disease progression, leading to heterogeneity in fiber size and increased collagen deposition between the altered myofibers. Bars = 50 μm.

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intervention, or impairs the efficiency of these therapies [5]. Currently there is no effective therapy for DMD despite continuing efforts. The only relatively effective pharmacotherapy for DMD involves corticosteroid administration, which prolongs muscle strength and walking capacity in the early years, but eventually leads to undesirable secondary effects [6]. Furthermore, there is also no effective clinical treatment to combat or attenuate fibrosis in patients with DMD. For these reasons, recent studies using the mdx mouse model of DMD have focused more attention on the cellular and molecular mechanisms underlying fibrosis associated with dystrophin deficiency. Importantly, these studies have tested several pharmacological agents that target muscle fibrosis, and the results strongly suggest that combating the development of fibrosis could ameliorate DMD progression and increase the success of new celland gene-based therapies.
Aging muscle As well as the muscular dystrophies, aging is associated with loss of skeletal-muscle mass and function with concomitant fibrosis and ECM deposition. Age-associated muscle loss (sarcopenia) causes and/or exacerbates agerelated health problems. Therefore, understanding the processes involved is important not only for unraveling the mechanisms of fibrosis, but also for improving quality of life and healthcare for the older person. Sarcopenia seems to occur by mechanisms that partly are unique to it, and partly are common to other forms of atrophy. Some of these may involve changes in soluble effectors, such as altered hormone status, inflammatory factors, and altered caloric and protein intake, perhaps triggered by modifications or decline in the central and peripheral nervous systems. The net consequence of these alterations firstly involves progressive atrophy and loss of individual muscle fibers, associated with concomitant loss of motor units [7]. In addition, there is infiltration of fat and other non-contractile material, which causes a reduction in muscle ‘quality’ [8]. At the ultrastructural level, aging has also been associated with myofibril disarrangements in a dystrophic animal model, and drops in force without alterations in motor protein function as measured by in vitro motility assays [9]. Additional factors associated with DMD and age-associated fibrosis are discussed in further detail below.

of any of these stages can result in unsuccessful muscle regeneration, typically characterized by persistent degeneration of myofibers, inflammation and fibrosis [10-12]. The key events leading to normal and defective/fibrotic muscle repair are shown in Figure 2 and 3, and detailed below.
Immediately after skeletal-muscle injury, cytokines and growth factors are released from both the injured blood vessels and from infiltrating inflammatory cells [13,14]. These factors stimulate the migration of the inflammatory cells to and at the site of injury, and mediate proliferation and cell survival. Invading inflammatory cells are also responsible for phagocytosing any cell debris. The specific influence of many damage signals, growth factors and inflammatory molecules on satellite cells remains unclear [14], but the next crucial stage of repair is the formation of new muscle fibers by these cells. This process begins with their activation, because satellite cells normally lie in a quiescent state beneath the basal lamina of muscle fibers, followed by their extensive proliferation. Some cells undergo self-renewal to replenish the satellite-cell pool, but most become committed and subsequently differentiate. These later myoblasts fuse either to themselves or to the damaged myofibers to replace the lost muscle.
In addition to inflammatory and satellite cells, efficient muscle repair also requires the migration and proliferation of fibroblasts, in order to produce new temporary ECM components, such as collagen types I and III, fibronectin, elastin, proteoglycans, and laminin. These elements serve to stabilize the tissue, and they act as a scaffold for the new fibers. Moreover, the satellite cells also utilize the basement membranes of pre-existing necrotic fibers to ensure the myofiber maintains a similar position. Basement membranes and temporary ECM components are also crucial for guiding the formation of neuromuscular junctions (NMJs) [15]. The formation and degradation of the ECM is mediated by the expression of proteases and their specific inhibitors during tissue repair. ECM degradation also leads to the generation of protein fragments that mediate important biological activities required to facilitate normal tissue repair [16]. Finally, in addition to ECM remodeling, angiogenesis facilitates the development of a new vascular network at the site of injury, while newly formed muscle fibers undergo growth and maturation.

Normal skeletal-muscle repair In nature, survival of an organism can often depend on the ability to rapidly repair damage to muscle from mechanical trauma, exposure to toxins or infections. This rapid resolution of tissue injury requires a sequential and well-orchestrated series of events. Perturbation

Inflammation in efficient muscle repair and fibrosis The first event after muscle damage is the invasion of the injury site by inflammatory cells. There is now a wealth of evidence to suggest that the nature, duration and intensity of the inflammatory response after muscle damage and regeneration can crucially influence the

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Figure 2 Chronic inflammation leads to fibrosis in skeletal-muscle repair. Resident and extravasating peripheral macrophages play an important role in the early stages of muscle repair after acute injury, with pro-inflammatory (M1) macrophages first acting to clear the damage, and anti-inflammatory (M2c) macrophages and alternatively activated macrophages (M2a), implicated in the subsequent resumption of inflammation, extracellular matrix (ECM) deposition and tissue repair. M2c and M2a macrophages release anti-inflammatory cytokines and profibrotic molecules such as transforming growth factor (TGF)-b, which in turn activate fibroblasts in a regulated manner to produce ECM components and ECM-remodeling factors, including autocrine production of TGFb, collagen, fibronectin, serine proteases (such as uPA/plasmin), and metalloproteinases (MMPs) and their inhibitors (TIMPs). However, during chronic tissue damage, as in muscular dystrophies, the increased and persistent presence of macrophages modify the intensity, duration and interactions of these released factors, leading to excessive ECM accumulation and replacement of muscle with fibrotic tissue.

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Inflammation (Acute injury)

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Figure 3 Inflammatory control of skeletal-muscle regeneration. Replacement of damaged muscle fibers is dependent on satellite cells, resident stem cells that are normally quiescent, and are located under the basal lamina of muscle fibers. Tissue damage leads to their activation, proliferation, differentiation and fusion to form new myofibers. However, their capacity to mediate repair is modified by the extent and type of injury, and consequently by their interaction with various cellular and soluble mediators, most importantly with infiltrating macrophages. The proposed paracrine interaction between macrophages and satellite cells is as follows. During the timely, regulated process of regeneration after acute injury (left), pro-inflammatory cytokines released from M1-macrophages may promote satellite-cell proliferation, whereas cytokines released by anti-inflammatory (M2c) and alternatively activated (M2a) macrophages, respectively, may favor their differentiation and fusion. In particular, interleukin (IL)-4 was shown to regulate fusion of myoblasts in vitro and in vivo [129]. It could be expected that, during chronic damage (right), such as in muscular dystrophies, the increased and persistent presence of the distinct macrophage cell types could modify the relative levels and kinetics of these cytokines, resulting in altered satellite-cell functions and aberrant regeneration, with progressive development of fibrosis and fat accumulation, ultimately leading to non-functional muscle tissue.

outcome of muscle repair, or alternatively, fibrosis [12,14,17,18]. For example, interfering with the transient inflammatory response after acute injury may negatively affect the phagocytosis of dead and damaged fibers, thereby impeding the formation of new tissue. By contrast, modulating the chronically high levels of inflammation in dystrophic muscle can be beneficial in reducing

both muscle degeneration and fibrosis, while simultaneously promoting regeneration [19].
These results highlight two key notions: firstly, that some form of inflammatory response is necessary to repair damaged tissues effectively; and secondly, that chronic inflammatory responses drive unrestrained wound healing and fibrosis.

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The inflammatory response: role of different macrophage
populations The earliest phases of tissue repair are generally characterized by local activation of the innate immune system, even though the original immunogenic stimuli are not always known (see below) [20]. Macrophages have a prominent role in the innate immune response to infection and/or tissue injury, because of their ability to phagocytose particles such as bacteria or cellular debris, and to secrete pro-inflammatory cytokines [13]. Recent studies have shown that resident macrophages in the muscle epimysium/perimysium orchestrate the innate immune response to injury, which is linked to adaptive immunity through inflammatory dendritic cells [21]. In addition to tissue-resident macrophages, invasion of the site of damage involves both polymorphonuclear leukocytes (for example, neutrophils) and blood-derived monocytes, which also differentiate into macrophages [17]. In some tissues, other inflammatory-cell types such as mast cells and T cells also play a key role in repair and fibrogenesis, although to date there are only limited studies into the role of these cells in muscle repair and DMD [22].
The principal inflammatory cells present in injured muscle are monocytes and macrophages [17]. In regenerating and dystrophic muscle, these serve to clear myofiber debris and in part, they modulate regeneration by secreting cytokines. An important development in our understanding of muscle repair and fibrosis was the demonstration that a heterogeneous population of macrophages exists in regenerating muscle after injury, exhibiting opposing activities (either pro-inflammatory or anti-inflammatory) and different kinetics [23]. A nomenclature for polarized macrophages has been proposed [24,25] and they are now referred to as classically and alternatively activated macrophages, or M1 and M2 macrophages, respectively (Figure 2, Figure 3).
Classically activated (M1) or pro-inflammatory macrophages, arise from exposure to the T-helper (Th)1 cytokines interferon-(IFN)g and tumor necrosis factor (TNF)-a, in addition to lipopolysaccharide (LPS) or endotoxin [24-26]. M1 macrophages play a key role in acute inflammatory processes, and they are therefore considered to be the prototypic macrophage. They are found during the early stages after muscle damage in association with recruited monocytes, and they participate in the processing and presentation of antigens, and in the phagocytic removal of necrotic material. M1 macrophages also produce high levels of pro-inflammatory cytokines, such as TNFa and interleukin (IL)-1b and IL-12. In addition, they can be induced to express nitric oxide synthase (iNOS; also known as NOS2), which is required to efficiently metabolize L-arginine to generate the large amounts of NO involved in killing intracellular pathogens.

The population of M2 macrophages is more complex than that of the M1 macrophages, and it is currently divided into distinct subtypes, reflecting different functional specializations. M2a macrophages or strictly speaking, alternatively activated macrophages, are activated by the Th2 cytokines IL-4 and IL-13, and are most commonly associated with tissue repair, wound healing and fibrosis. M2c macrophages are considered to be anti-inflammatory, because they play a key role in deactivating the M1 phenotype and they promote the proliferation of non-myeloid cells. M2c macrophages release anti-inflammatory cytokines, and they are primed by IL-10. Thus, classically activated M1 macrophages are usually found in the early stages after muscle injury, closely followed by M2c macrophages [23]. M2a macrophages are abundant in advanced stages of the tissuerepair process [12], and have been found in fibrotic muscle of mdx mice [27,28] (Figure 4C).
From muscle injury to the chronic inflammatory response and pathological muscle fibrosis Alterations in the intensity or duration of macrophage responses can have profound effects on muscle regeneration and fibrosis. One example of this was the deletion of IL-10 in mdx mice, which increased muscle damage and reduced muscle strength, due to an imbalance between M1 and M2 macrophages [29]. Similarly, persistence of M1 macrophages has been proposed to have pathological consequences in chronic inflammatory myopathies (see below). In terms of tissue fibrosis, M2a macrophages are generally considered the most important [12]. They express specific cell surface markers such as the mannose receptor CD206 and the type II IL-1 decoy receptor, in addition to releasing a range of regulatory cytokines such as IL-10 and the soluble IL-1 receptor antagonist (IL-1Ra) as well as many pro-fibrotic molecules such as transforming growth factor (TGF)-b, fibronectin, proline, several types of tissue inhibitor of matrix metalloproteinases (TIMP) and chemokine (C-C motif) ligand (CCL)17. CCL17 in particular has been shown to enhance fibrosis in several mouse models of pulmonary disease by binding to CC chemokine receptor (CCR)4 [30]. An additional reason for the ability of M2 macrophages to neutralize the M1 pro-inflammatory response is their high level of expression of arginase (ARG)1, which directly competes with M1-associated inducible nitric oxide synthase (iNOS) for L-arginine [31] (see below and Figure 4C).
Several groups have used different in vitro and in vivo animal models in attempts to unravel the role of macrophages in myogenesis, muscle repair, fibrosis, and the development and treatment of DMD. In vitro, proinflammatory macrophages have a positive influence on myoblast proliferation while repressing myoblast

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Figure 4 Inflammatory and fibrotic traits in dystrophic muscle of patients with DMD and mdx mice. (A) Fibrin(ogen) accumulates in muscles of patients with DMD and in aged mdx mice. Immunohistochemistry for fibrin(ogen) (brown) in muscle biopsies of (top) patients with DMD and healthy subjects and in (bottom) wild-type (WT) and mdx diaphragms. (B) Increased fibrosis, fibroblast number and TGFb signaling in dystrophic muscles. Staining for collagen deposition (Sirius red) and immunohistochemistry for fibroblast-specific protein (FSP)-1 and P-Smad2 was performed on muscle biopsies taken from patients with DMD and healthy subjects. (C) Presence of alternatively activated macrophages in diaphragm muscle of mdx mice. Cells double-positive for CD206 (red) and Arginase I (green) in mdx diaphragm are shown by immunofluorescence with specific antibodies. The relative increase in the number of these cells in the mdx diaphragm over time is shown. Bars = 50 μm.

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differentiation, whereas anti-inflammatory macrophages stimulate both myoblast differentiation and fusion. Importantly, in vivo depletion of blood monocytes, from which M1 and M2 macrophages probably arise, was shown to have negative effects on the muscle-repair process after injury [23]. Conversely, BM transplantation experiments have revealed an important role for the CC chemokine receptor 2/monocyte chemotactic protein-1 (CCR2/MCP-1) ligand, and for the proteolytic activity of urokinase plasminogen activator (uPA)/plasmin in skeletal-muscle repair by regulating the recruitment of BMderived macrophages into injured muscle [32-37].
Several studies of macrophage depletion or impaired macrophage recruitment have revealed crucial functions of macrophages in the regulation of fibrogenesis in dystrophic muscle, and the potential for therapeutic intervention [19,35,37,38]. Similar benefits on the dystrophic phenotype in mdx mice were reported after using a variety of anti-inflammatory agents acting on cytokines such as TNFa and on their cellular receptors, or on other major pro-inflammatory mediators such as nuclear factor (NF)-B [39-41]. Additionally, the presence of alternatively activated M2a macrophages was shown to increase progressively with age in the diaphragms of fibrotic mdx mice [27] (Figure 4C). Similarly, fibrinogen depletion in mdx mice diminished fibrosis, concomitant with a significant decrease in the number of M2a macrophages in the diaphragm. In patients with DMD, fibrosis has also been associated with increased numbers of alternatively activated macrophages [42]. Overall, these studies show that appropriate modulation of macrophage activity might ameliorate the progression of dystrophy.
A potentially important link between arginine metabolism by M2a macrophages and abnormal repair or fibrosis development has recently been shown by several groups. In one study, deletion of two c-AMP response element (CREB)-binding sites from the C/EBP-b promoter specifically impaired M2 but not M1 gene expression, interfering with the later stages of injury-induced muscle regeneration. Mutant mice were able to remove necrotic tissue from injured muscle, but they exhibited severe defects in myofiber regeneration [43]. Mutation of the C/EBP-b promoter also reduced ARG1 expression in macrophages, which was hypothesized to reroute arginine metabolism away from arginase-mediated polyamine synthesis toward iNOS-mediated NO production, which was previously shown to promote the degradation of the key myogenic transcription factor MyoD [44]. Indeed, similar shifts in macrophage polarization and macrophage competition for arginine metabolism were seen to influence the severity of the muscle pathology in mdx dystrophic mice [28]. In another study [14], Th2 cytokines increased the expression and activity of

arginase by M2 macrophages in mdx mice, with intriguing differences in the effects of arginase-2 deletion in different muscles. Indeed, although fibrosis is reduced in quadriceps and diaphragm of mdx mice lacking arginase-2, it seems that it is not the case for the soleus, the cardiac muscle and the longissimus dorsi. More importantly from the clinical perspective, long-term dietary supplementation with arginine increased skeletal and cardiac muscle fibrosis in dystrophic mice, in contrast to the reported benefits from short-term supplementation, thus suggesting caution is needed regarding dietary arginine supplementation for patients with DMD [45]. Taken together, these studies indicate the crucial roles of macrophage polarization in both muscle repair and fibrogenesis, particularly in dystrophic muscle.
Aberrant repair and fibrosis in IIMs In addition to the muscular dystrophies, there is another group of chronic muscle disorders, collectively known as myositis or the IIMs [46]. Phenotypically, IIMs are characterized by muscle weakness, poor endurance, and ongoing regeneration of the muscle tissue. Within the muscle, the presence of inflammatory infiltrates composed largely of macrophages, T cells and dendritic cells correlates with immune-mediated loss of muscle fibers and an inability to resolve the regeneration process effectively. Although not fully characterized, an important feature of these diseases is the persistence of proinflammatory M1 macrophages and associated cytokines such as IL-1, IL-15 and TNFa in the tissue, and an apparent inability to switch to anti-inflammatory M2 macrophages. Hence, the most effective treatments to date have proven to be glucocorticoids and other nonsteroidal anti-inflammatory agents (NSAIDs) such as ibuprofen and aspirin. However, one problem with these drugs has been their ability to promote fiber atrophy [47], in addition to their negative effect on prostaglandin (PG) synthesis via interference with cyclooxygenase (COX) gene functions. PGs have been shown to play various roles in many stages of myogenesis, and are secreted from regenerating muscle [48,49]. Interestingly, macrophage-derived TGFb1 has been shown to induce PGE2 expression from myoblasts via a COX-2-dependent mechanism, which in turn diminishes TGFb1 expression, forming an important feedback loop that can control inflammation and fibrosis development [49]. One other limitation of the drugs used to treat IIMs is that they do not produce complete recovery, nor do they address the underlying defects, which in most cases are multifactorial and not fully defined [50]. Nonetheless, the IIMs represent an important group of diseases to help us understand the complicated role of proinflammatory cells and cytokines in orchestrating normal versus aberrant muscle repair.

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The role of other immune-cell types in muscle repair and
fibrosis Macrophages are not the only cells in the immune system known to play a role in muscle repair and dystrophy. Tissue repair and fibrosis is also tightly regulated by the Th cell response. Like macrophages, T lymphocytes can differentiate into different functional types, which are named Th1 and Th2 cells, and which orchestrate the host response by generating distinct cytokine profiles [26]. CD4+ Th1 cells produce cytokines that promote cell-mediated immunity, including IFN-g, TNFa, IL-12 and IL-2, all of which have been found to be anti-fibrotic cytokines. By contrast, CD4+ Th2 cells promote humoral immunity, and produce the pro-fibrotic cytokines IL-4, IL-5, IL-6 and IL-13. Th1 cytokines inhibit the development of Th2 cells, and conversely, Th2 cytokines inhibit the development of Th1 cells. Clearly, alterations or imbalances in these pathways have the potential to skew repair towards anti- or profibrotic pathways, as witnessed by the importance of Th2 cytokines in the development of liver fibrogenesis [12]. T-cell-produced cytokines also regulate muscle degeneration and repair. For example, loss of uPA proteolytic activity in transgenic knockout mice reduces macrophage and T-lymphocyte infiltration of injured muscle, in association with more persistent myofiber degeneration [33]. Moreover, scid/mdx mice, which are deficient in functional T and B lymphocytes, develop much less diaphragm fibrosis at 1 year of age, concomitant with a decrease in activated TGFb in skeletal muscle, compared with normal mdx mice [51]. In nu/nu/ mdx mice, (immunodeficient nude mice in the mdx background) the lack of functional T cells alone was associated with less diaphragm fibrosis at 3 months, supporting the pathogenic role for T cells in mdx muscle, and revealing this lymphocyte subclass to be an important source of TGFb1 [52].
A specific subpopulation of T cells expressing the Vb8.1/8.2 T-cell receptor (TCR) was recently identified and shown to be enriched in mdx muscle. These T cells produce high levels of osteopontin, a cytokine that promotes immune-cell migration and survival [53], and osteopontin levels are increased in patients with DMD and in mdx mice after disease onset. Importantly, loss of osteopontin in mdx double-mutant mice diminishes the infiltration of natural killer T-cell (NKT)-like cells, which express both T and NK cell markers and neutrophils, in addition to reducing the levels of TGFb. These results correlated well with improvements in muscle strength and reduced diaphragmatic and cardiac fibrosis [53]. Not all studies have produced such definitive results, and the implication of lymphocytes and their subtypes in muscle repair and fibrosis clearly requires further study. For example, thymectomy at 1 month of age induces near-

complete post-natal depletion of circulating T cells in mdx mice and. when followed by anti-CD4 and/or antiCD8 antibody treatment, failed to improve diaphragm fibrosis at 6 months of age in mdx mice [51,54,55]. In another study, M2 macrophages were shown to influence CD4+ Th cells, because ARG1-expressing macrophages suppressed Th2 cytokine-driven inflammation and fibrosis in the liver induced by Schistosoma mansoni infection [26]. Because these data demonstrate the complexity of the mechanisms regulating inflammation and fibrosis development, further studies are clearly necessary to determine whether distinct types of Th responses and macrophage subtypes operate in dystrophic muscle. and how they mediate their interactions.
Fibroblasts, the collagen-producing cells in skeletal muscle When tissue is damaged, fibroblasts migrate into the wound and begin to produce and remodel the ECM in response to pro-fibrotic cytokines such as TGFb. Stromal fibroblasts produce cytokines, growth factors and proteases that trigger and uphold acute and chronic inflammatory/pro-fibrotic conditions. Indeed, although the fibroblast is necessary and fundamental to tissue homeostasis and normal wound repair, it is also a crucial intermediate in chronic fibrotic diseases, in which persistent inflammation is widely accepted to provoke dysregulated fibroblast activity. Notably, one limitation that has hindered studies of fibrotic disease has been the lack of good genetic markers to label fibroblasts. It is well established that in non-muscle systems. activated fibroblasts may be identified by their increased proliferation, migratory ability, enhanced contractility. and increased expression of vimentin and, in particular, asmooth-muscle actin (aSMA), a contractile protein of stress fibers. These fibers are connected to the ECM through specialized structures called ‘mature’ and ‘super-mature’ focal adhesions, and through intercellular gap and adherent junctions. As a result, when aSMA stress fibers contract, they exert mechanical tension on the ECM, which in turn provides a mechanically resistant support, hence the name ‘myofibroblast’. These cells are associated with tissue repair and fibrosis in many tissues and organs, including muscle, skin, liver, lung, bone and cartilage [56]. However, despite their relevance in these diseases, it remains unclear whether myofibroblasts really do exist in fibrotic skeletal muscle, or whether they are instead mature fibroblasts actively producing ECM components. One reason for this controversy is that classic markers such as vimentin or aSMA are also expressed by myoblasts, albeit at lower levels than fibroblasts. However, a recent study has identified the transcription factor Tcf4 as a potentially important marker of fibroblasts in muscle, although follow-up studies are still needed to

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validate its utility [57]. Nonetheless, to remain consistent with the literature, we use the term ‘myofibroblast’ here in the muscle context.
There are many possible origins of myofibroblasts. Resident fibroblasts that differentiate in response to specific effectors are considered to be the main myofibroblast progenitor in most tissues. Alternatively, myofibroblasts may arrive by the influx of circulating BM-derived cells expressing CD34, CD45 and collagen I (cells known as fibrocytes), which undergo further reprogramming in the damaged area. Finally, it has been shown in organs such as lung, kidney and liver that there is a significant number of myofibroblasts derived from parenchymal epithelial cells through the mechanism of epithelial to mesenchymal transition (EMT). Thus, transdifferentiation of distinct cell types into myofibroblasts can potentially account for persistent ECM deposition in chronically damaged tissues. Understanding the origin of myofibroblasts is thus of great importance to develop new approaches to combat the fibrotic process seen in diverse diseases.
Although fibroblasts are the major collagen-producing cells, myofiber-associated satellite cells and C2C12 myoblasts have also been shown to express significant levels of interstitial collagens I and III, which diminish during the process of differentiation [58]. Whereas collagen I can markedly suppress differentiation of C2C12 cells, collagen III expression is retained in aged mdx myogenic cells. This suggests that conversion of myoblasts into myofibroblasts with increasing age may occur via positive feedback [58]. Collagen modifications, such as non-enzymatically regulated crosslinking to produce advanced glycation end (AGE) products, also increase the stiffness of muscle connective tissue, thereby contributing to impaired muscle function in the older person [59]. Several recent studies in other models have also investigated the induction of fibroblastic phenotypes in myogenic cells. In one case, TGFb was able to induce Smad-dependent upregulation of sphingosine kinase SK) 1 in C2C12 myoblasts, whereas pharmacological or small interfering (si)RNA-mediated inhibition of SK1 prevented TGFb from inducing fibrotic markers. Rho/ Rho kinase signaling also appeared to be implicated in the TGFb-mediated transition of myoblasts into myofibroblasts downstream of SK1 activation [60]. Similarly, downregulation of Notch2 expression has also been linked to non-muscle fibrotic tissue and TGFb-dependent induction of myofibroblast markers in C2C12 myoblasts. Overexpression of active Notch2 in C2C12 cells prevents TGFb from inducing the expression of aSMA and collagen I, whereas more surprisingly, transient knockdown of Notch2 by siRNA in cultured myoblasts results in the differentiation of C2C12 myoblasts into myofibroblastic cells that express fibrotic markers such

as aSMA and collagen I, even in the absence of TGFb. Finally, Notch2 can inhibit the differentiation of myoblasts into myofibroblasts by directly counter-regulating Notch3 and limiting its expression [61].
Fibrogenesis in aging muscle The Notch pathway has also been strongly implicated in aging-associated fibrosis. For example, analysis of the microniche of aged murine muscle stem cells found high levels of TGFb and its activated effector Smad3 in both differentiated muscle fibers and satellite cells, which was reciprocal to the levels of active Notch, which is more abundant in the young microniche [62]. Increased levels of activated Smad3 levels in aged muscles attenuate their regenerative capacity by binding to the promoters and stimulating the expression of several cyclin-dependent kinase (CDK) inhibitors (for example,, p15, p16, p21 and p27), negative regulators of cell-cycle progression. Importantly, this imbalance of TGFb/ pSmad3-Notch could be restored by forced activation of Notch. Similar scenarios of reduced Notch activation and increased TGFb/pSmad3 signaling have been reported recently in aged human muscles [63].
In additional studies of aged muscle, the fate of muscle stem-cell progeny was reported to be controlled by an interaction between the Wnt and Notch pathways in which glycogen synthase kinase (GSK)3b plays an important role [64]. The mammalian ortholog of the Drosophila transcriptional coactivator Legless, BCL9/9-2, was also shown in this study to be necessary for activation of the canonical Wnt pathway in adult myogenic progenitors, and for their Wnt-mediated commitment to differentiation and effective muscle regeneration [64]. However, whether GSK3b and/or BCL9 mediate Wntinduced cell-fate changes from myogenic to fibrogenic lineages in resting satellite cells awaits further validation. An earlier study by the same group had already highlighted the role of the canonical Wnt pathway in ageassociated fibrosis, linking increased collagen deposition in aged regenerating muscles to a greater percentage of fibrogenic cells arising from the conversion of myogenic into non-myogenic cells [65]. This fibrogenic conversion could be abrogated experimentally by treating mice with Wnt inhibitors. Wnt3A stimulation negatively modulated cell proliferation in young regenerating muscles, augmenting fibrosis. Thus, aging was associated with alterations in the systemic environment, and because these effects were reversible, this work provides the strategic basis for interventions aimed at improving tissue repair and at reducing fibrosis in pathological conditions.
Fibrogenesis versus adipogenesis in muscle repair It is known that when regeneration fails, the fibrotic scar is infiltrated with adipocytes (fatty degeneration) in
FibrosisMuscleCellsDmdMacrophages