Basal Lamina Of Muscle Fibers Research Articles

Role of Regulatory T Cells in Skeletal Muscle Regeneration: A Systematic Review.

Muscle injuries are frequent in individuals with genetic myopathies and in athletes. Skeletal muscle regeneration depends on the activation and differentiation of satellite cells present in the basal lamina of muscle fibers. The skeletal muscle environment is critical for repair, metabolic and homeostatic function. Regulatory T cells (Treg) residing within skeletal muscle comprise a distinct and special cell population that modifies the inflammatory environment by secreting cytokines and amphiregulin, an epidermal growth factor receptor (EGFR) ligand that acts directly upon satellite cells, promoting tissue regeneration. This systematic review summarizes the current knowledge regarding the role of Treg in muscle repair and discusses their therapeutic potential in skeletal muscle injuries. A bibliographic search was carried out using the terms Treg and muscle regeneration and repair, covering all articles up to April 2021 indexed in the PubMed and EMBASE databases. The search included only published original research in human and experimental animal models, with further data analysis based on the PICO methodology, following PRISMA definitions and Cochrane guidelines.

The effect of hypoxia on myogenic differentiation and multipotency of the skeletal muscle-derived stem cells in mice

BackgroundSkeletal muscle-derived stem cells (SC) have become a promising approach for investigating myogenic differentiation and optimizing tissue regeneration. Muscle regeneration is performed by SC, a self-renewal cell population underlying the basal lamina of muscle fibers. Here, we examined the impact of hypoxia condition on the regenerative capacity of SC either in their native microenvironment or via isolation in a monolayer culture using ectopic differentiation inductions. Furthermore, the effect of low oxygen tension on myogenic differentiation protocols of the myoblasts cell line C2C12 was examined.MethodsHind limb muscles of wild type mice were processed for both SC/fiber isolation and myoblast extraction using magnetic beads. SC were induced for myogenic, adipogenic and osteogenic commitments under normoxic (21% O2) and hypoxic (3% O2) conditions. SC proliferation and differentiation were evaluated using histological staining, immunohistochemistry, morphometric analysis and RT-qPCR. The data were statistically analyzed using ANOVA.ResultsThe data revealed enhanced SC proliferation and motility following differentiation induction after 48 h under hypoxia. Following myogenic induction, the number of undifferentiated cells positive for Pax7 were increased at 72 h under hypoxia. Hypoxia upregulated MyoD and downregulated Myogenin expression at day-7 post-myogenic induction. Hypoxia promoted both SC adipogenesis and osteogenesis under respective induction as shown by using Oil Red O and Alizarin Red S staining. The expression of adipogenic markers; peroxisome proliferator activated receptor gamma (PPARγ) and fatty acid-binding protein 4 (FABP4) were upregulated under hypoxia up to day 14 compared to normoxic condition. Enhanced osteogenic differentiation was detected under hypoxic condition via upregulation of osteocalcin and osteopontin expression up to day 14 as well as, increased calcium deposition at day 21. Hypoxia exposure increases the number of adipocytes and the size of fat vacuoles per adipocyte compared to normoxic culture. Combining the differentiation medium with dexamethasone under hypoxia improves the efficiency of the myogenic differentiation protocol of C2C12 by increasing the length of the myotubes.ConclusionsHypoxia exposure increases cell resources for clinical applications and promotes SC multipotency and thus beneficial for tissue regeneration.

Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusion.

Cell-cell fusion is a frequent and essential event during development, and its dysregulation causes diseases ranging from infertility to muscle weakness. Fusing cells need to repeatedly remodel their plasma membrane through orchestrated formation and disassembly of actin filaments, but how the dynamic reorganization of the cortical actin cytoskeleton is controlled is still poorly understood. Here, we identified a ubiquitin-dependent toggle switch that establishes reversible actin bundling during mammalian cell fusion. We found that EPS8-IRSp53 complexes stabilize cortical actin bundles at sites of cell contact to promote close membrane alignment. EPS8 monoubiquitylation by CUL3KCTD10 displaces EPS8-IRSp53 from membranes and counteracts actin bundling, a dual activity that restricts actin bundling to allow paired cells to progress with fusion. We conclude that cytoskeletal rearrangements during development are precisely controlled by ubiquitylation, raising the possibility of modulating the efficiency of cell-cell fusion for therapeutic benefit.

Satellite cell-mediated breast muscle regeneration decreases with broiler size

Satellite cells (SCs) reside between the sarcolemma and basal lamina of muscle fibers and are the primary contributor of DNA for post-hatch muscle growth and repair. Alterations in SC content or properties by intrinsic and extrinsic factors can have detrimental effects on muscle health and function, and ultimately meat quality. We hypothesized that disrupted SC homeostasis may account in part for the increased breast myopathies observed in growing broilers. To test this hypothesis, we selected broilers with different body weights at comparable ages and studied SC characteristics in vitro and in vivo. Data shows that SC numbers in the breast muscles decrease (P < 0.001) and their inherent abilities to proliferate and differentiate diminish (P < 0.001) with age and size. Further, when breast muscle is presented with an insult, muscle of larger broilers regenerates more slowly than their smaller, age-matched counterparts arguing that SC quality changes with size and age. Together, our studies show that birds with greater muscle hypertrophy have less SCs with diminished ability to function, and suggest that aggressive selection for breast growth in broilers may exhaust SC pools when birds are grown to heavier processing weights. These findings provide new insights into a possible mechanism leading to breast myopathies in the poultry industry and provide targets for mitigating adverse fresh breast quality.

Role of Myofibrillar Protein Catabolism in Development of Glucocorticoid Myopathy: Aging and Functional Activity Aspects

Muscle weakness in corticosteroid myopathy is mainly the result of the destruction and atrophy of the myofibrillar compartment of fast-twitch muscle fibers. Decrease of titin and myosin, and the ratio of nebulin and MyHC in myopathic muscle, shows that these changes of contractile and elastic proteins are the result of increased catabolism of the abovementioned proteins in skeletal muscle. Slow regeneration of skeletal muscle is in good correlation with a decreased number of satellite cells under the basal lamina of muscle fibers. Aging causes a reduction of AMP-activated protein kinase (AMPK) activity as the result of the reduced function of the mitochondrial compartment. AMPK activity increases as a result of increased functional activity. Resistance exercise causes anabolic and anticatabolic effects in skeletal muscle: muscle fibers experience hypertrophy while higher myofibrillar proteins turn over. These changes are leading to the qualitative remodeling of muscle fibers. As a result of these changes, possible maximal muscle strength is increasing. Endurance exercise improves capillary blood supply, increases mitochondrial biogenesis and muscle oxidative capacity, and causes a faster turnover rate of sarcoplasmic proteins as well as qualitative remodeling of type I and IIA muscle fibers. The combination of resistance and endurance exercise may be the fastest way to prevent or decelerate muscle atrophy due to the anabolic and anticatabolic effects of exercise combined with an increase in oxidative capacity. The aim of the present short review is to assess the role of myofibrillar protein catabolism in the development of glucocorticoid-caused myopathy from aging and physical activity aspects.

Abstract 317: Paracrine Effects of Satellite Cells on Collateral Vessel Formation

Peripheral artery disease is a major health problem in the United States that effects 8.5 million people and can lead to limb pain, decreased mobility, and in severe cases amputation. The ability to form a robust collateral network to restore blood flow and prevent ischemia leads to a better prognosis and restoration of function. The growth of collaterals is a complex process that involves recruitment of various cell types including smooth muscle cells, endothelial cells, and macrophages. Migration and proliferation of these cells are processes regulated by numerous cytokine and paracrine signals. We hypothesize that an important and novel source of these signals is satellite cells. Satellite cells are myogenic progenitor cells that lie below the basal lamina of muscle fibers. In healthy muscle, the cells are quiescent but in response to injury, such as ischemia, they become activated and proliferate. We hypothesized that activated satellite cells produce factors that will influence critical cells for vessel formation in addition to differentiating to repair muscle. To study the paracrine effects of satellite cells on vascular smooth muscle cells, we used a co-culture system with freshly isolated satellite cells from the ischemic leg as the stimulus. We found that satellite cells significantly increased smooth muscle migration 2.5 fold compared to media alone using a modified Boyden chamber assay. BrdU staining to assess proliferation showed modest increases in smooth muscle proliferation (1.3 fold change, p&lt;0.01). Finally, to investigate these paracrine effects in vivo, we delivered alginate encapsulated satellite cells to mice following the hind limb ischemia procedure, which is a model of collateral growth. We found that mice that received the encapsulated satellite cells had significantly improved perfusion as measured by Laser Doppler imaging at day 14 post surgery when compared to empty capsules (perfusion ratio of 0.87 ± 0.04 (cells) vs 0.68 ± 0.07 (empty capsules), p&lt;0.05). This result demonstrates that satellite cells can positively influence collateral growth in vivo. We believe that satellite cells play a critical role in collateral vessel formation and may potentially be a therapeutic strategy for the treatment of peripheral artery disease.

Human Skeletal Muscle–derived CD133+ Cells Form Functional Satellite Cells After Intramuscular Transplantation in Immunodeficient Host Mice

Stem cell therapy is a promising strategy for treatment of muscular dystrophies. In addition to muscle fiber formation, reconstitution of functional stem cell pool by donor cells is vital for long-term treatment. We show here that some CD133(+) cells within human muscle are located underneath the basal lamina of muscle fibers, in the position of the muscle satellite cell. Cultured hCD133(+) cells are heterogeneous and multipotent, capable of forming myotubes and reserve satellite cells in vitro. They contribute to extensive muscle regeneration and satellite cell formation following intramuscular transplantation into irradiated and cryodamaged tibialis anterior muscles of immunodeficient Rag2-/γ chain-/C5-mice. Some donor-derived satellite cells expressed the myogenic regulatory factor MyoD, indicating that they were activated. In addition, when transplanted host muscles were reinjured, there was significantly more newly-regenerated muscle fibers of donor origin in treated than in control, nonreinjured muscles, indicating that hCD133(+) cells had given rise to functional muscle stem cells, which were able to activate in response to injury and contribute to a further round of muscle regeneration. Our findings provide new evidence for the location and characterization of hCD133(+) cells, and highlight that these cells are highly suitable for future clinical application.

Muscle satellite cell heterogeneity and self-renewal.

Adult skeletal muscle possesses extraordinary regeneration capacities. After muscle injury or exercise, large numbers of newly formed muscle fibers are generated within a week as a result of expansion and differentiation of a self-renewing pool of muscle stem cells termed muscle satellite cells. Normally, satellite cells are mitotically quiescent and reside beneath the basal lamina of muscle fibers. Upon regeneration, satellite cells are activated, and give rise to daughter myogenic precursor cells. After several rounds of proliferation, these myogenic precursor cells contribute to the formation of new muscle fibers. During cell division, a minor population of myogenic precursor cells returns to quiescent satellite cells as a self-renewal process. Currently, accumulating evidence has revealed the essential roles of satellite cells in muscle regeneration and the regulatory mechanisms, while it still remains to be elucidated how satellite cell self-renewal is molecularly regulated and how satellite cells are important in aging and diseased muscle. The number of satellite cells is decreased due to the changing niche during ageing, resulting in attenuation of muscle regeneration capacity. Additionally, in Duchenne muscular dystrophy (DMD) patients, the loss of satellite cell regenerative capacity and decreased satellite cell number due to continuous needs for satellite cells lead to progressive muscle weakness with chronic degeneration. Thus, it is necessary to replenish muscle satellite cells continuously. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is crucial for maintenance of satellite cells as a muscle stem cell pool throughout life. In addition, we discuss roles in the stem cell niche for satellite cell maintenance, as well as related cell therapies for approaching treatment of DMD.

Adult stem cells derived from skeletal muscle — biology and potential

AbstractSkeletal muscle contains at least two distinct populations of adult stem cells — satellite cells and multipotent muscle-derived stem cells. Monopotential satellite cells are located under the basal lamina of muscle fibers. They are capable of giving rise only to cells of myogenic lineage, which play an important role in the processes of muscle regeneration. Multipotent muscle-derived stem cells are considered to be predecessors of the satellite cells. Under proper conditions, both in vitro and in vivo, they undergo myogenic, cardiogenic, chondrogenic, osteogenic and adipogenic differentiation. The main purpose of the present article is to summarize current information about adult stem cells derived from skeletal muscle, and to discuss their isolation and in vitro expansion techniques, biological properties, as well as their potential for regenerative medicine.

Digenic mutational inheritance of the integrin alpha 7 and the myosin heavy chain 7B genes causes congenital myopathy with left ventricular non-compact cardiomyopathy

BackgroundWe report an Italian family in which the proband showed a severe phenotype characterized by the association of congenital fiber type disproportion (CFTD) with a left ventricular non-compaction cardiomyopathy (LVNC). This study was focused on the identification of the responsible gene/s.Methods and resultsUsing the whole-exome sequencing approach, we identified the proband homozygous missense mutations in two genes, the myosin heavy chain 7B (MYH7B) and the integrin alpha 7 (ITGA7). Both genes are expressed in heart and muscle tissues, and both mutations were predicted to be deleterious and were not found in the healthy population.The R890C mutation in the MYH7B gene segregated with the LVNC phenotype in the examined family. It was also found in one unrelated patient affected by LVNC, confirming a causative role in cardiomyopathy.The E882K mutation in the ITGA7 gene, a key component of the basal lamina of muscle fibers, was found only in the proband, suggesting a role in CFTD.ConclusionsThis study identifies two novel disease genes. Mutation in MYH7B causes a classical LVNC phenotype, whereas mutation in ITGA7 causes CFTD. Both phenotypes represent alterations of skeletal and cardiac muscle maturation and are usually not severe. The severe phenotype of the proband is most likely due to a synergic effect of these two mutations.This study provides new insights into the genetics underlying Mendelian traits and demonstrates a role for digenic inheritance in complex phenotypes.

Preparation of Adult Mouse Muscle Stem Cells

Muscle stem cells are adult stem cells responsible for muscle development, growth and regeneration. Current knowledge suggests those cells are heterogeneous population shared their position between the sarcolemma and basal lamina of muscle fibers. This protocol describes the technique to dissociate and collect the stem cells from skeletal muscle of adult mice, and separate them from other cells found in muscle (e.g. fat, connective tissue). To purify and preserve those myofiber associated muscle stem cells, we use two steps of enzyme digestion followed by cell pre-plating procedures.

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: Extracellular matrix in skeletal muscle development and meat quality1

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: Extracellular matrix in skeletal muscle development and meat quality1

Mononucleated Cells to Regenerate Skeletal Muscle Syncytial Tissues

Skeletal muscle is one of the most plastic tissues of vertebrates since it may able upon exercises to double in size due to a physiological hypertrophy. Despite the fact that it is mainly a syncytial tissue, it contains a relevant number of mononucleated cells that can be involved in its homeostasis and repair. Although the mononuclear cell types with the highest myogenic potential are the satellite cells located underneath the basal lamina of muscle fibres, other interstitial cells have been shown to contribute to muscle regeneration. Adding complexity to this scenario is the fact that several authors revealed myogenic potential in pluripotent stem cells, which can be generated from patient somatic cells and eventually manipulated to correct the genetic defect. Notwithstanding the copiousness of myogenic cell types, their use in ex vivo cell therapies for muscular degenerative diseases is still questionable. However, new discovers on their biological properties have advanced our comprehension in handling myogenic stem cells significantly. In this review, we will focus on the myogenic potential of multi- and pluri-potent stem cells and their use in preclinical and clinical studies. New insights from direct reprogramming and epigenetic signalling to generate myogenic stem cells are also considered.

185th ENMC International Workshop: Stem/precursor cells as a therapeutic strategy for muscular dystrophies 3–5 June 2011, Naarden, The Netherlands

The 185th ENMC Workshop on stem/precursor cells as a therapeutic strategy for muscular dystrophies was held at Naarden, The Netherlands, on 3–5 June 2011. Sixteen participants from seven countries (United Kingdom, The Netherlands, France, Germany, Italy, Spain and Singapore) with expertise in satellite cells, muscle stem cells, muscle development, muscular dystrophies, clinical trials and patients’ perspectives, attended. Although skeletal muscle is largely a post-mitotic tissue, it regenerates efficiently following injury. Satellite cells, quiescent cells under the basal lamina of muscle fibres, become activated, proliferate to give a pool of myoblasts that can repair and regenerate muscle fibres [1]. In muscular dystrophies such as Duchenne muscular dystrophy (DMD), satellite cells contribute to muscle fibre regeneration, but soon become exhausted and muscle fibres are lost and replaced by fibrous connective tissue and fat (reviewed [2]). Stem/precursor cells have long been considered as a possible therapy of muscular dystrophies, since work in the late 1980s on myoblast transplantation in the dystrophindeficient mdx mouse model of DMD [3,4]. However, early clinical trials on intra-muscular myoblast transplantation were disappointing, the main problems being low efficiency of donor-derived muscle regeneration and lack of migration of donor cells within transplanted muscle [5]. Nevertheless, lessons learnt from these trials may be used as a basis of future trials. In planning a stem cell clinical trial, decisions have to be made about ethical implications, age of participants, scaling up of cell preparations, testing of cells and criteria used to assess safety and efficacy.

Origin and hierarchy of basal lamina-forming and -non-forming myogenic cells in mouse skeletal muscle in relation to adhesive capacity and Pax7 expression in vitro

As a novel approach to distinguish skeletal myogenic cell populations, basal lamina (BL) formation of myogenic cells was examined in the mouse compensatory enlarged plantaris muscles in vivo and in fiber-bundle cultures in vitro. MyoD(+) myogenic cells located inside the regenerative muscle fiber BL were laminin(-) but interstitial MyoD(+) cells were laminin(+). This was also confirmed by electron microscopy as structural BL formation. Similar trends were observed in the fiber-bundle cultures including satellite cells and interstitial myogenic cells and laminin(+) myogenic cells predominantly showed non-adhesive (non-Ad) behavior with Pax7(-), whereas laminin(-) cells were adhesive (Ad) with Pax7(+). Moreover, non-Ad/laminin(+) and Ad/laminin(-) myotubes were also observed and the former type showed spontaneous contractions, while the latter type did not. The origin and hierarchy of Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells were also examined using skeletal muscle interstitium-derived CD34(+)/45(-) (Sk-34) and CD34(-)/45(-) (Sk-DN) multipotent stem cells, which were composed of non-committed myogenic cells with a few (<1%) Pax7(+) cells in the Sk-DN cells at fresh isolation. Both cell types were separated by Ad/non-Ad capacity in repetitive culture. As expected, both Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells consistently appeared in the Ad and non-Ad cell culture. However, Ad/Pax7(+)/laminin(-) cells were repeatedly detected in the non-Ad cell culture, while the opposite phenomenon did not occur. This indicates that the source of non-Ad/ Pax7(-)/laminin(+) myogenic cells was present in the Sk-34 and Sk-DN stem cells and they were able to produce Ad/ Pax7(+)/ laminin(-) myogenic cells during myogenesis as primary myoblasts and situated hierarchically upstream of the latter cells.

166th ENMC International Workshop on Collagen type VI-related Myopathies, 22–24 May 2009, Naarden, The Netherlands

Twenty-four clinicians, basic scientists and representatives of a patient advocacy organisation and pharmaceutical companies from 8 countries (Australia, England, France, Germany, Italy, Switzerland, Turkey, USA) gathered in Naarden, The Netherlands, to discuss the group of neuromuscular disorders caused by the deficiency of collagen type VI (ColVI), collectively termed ColVI-related myopathies. This meeting focused on three main areas: (1) the great heterogeneity observed at the genetic and clinical levels and the resulting complexity of the molecular diagnosis; (2) the pathophysiological mechanisms linking mutations in the COL6A genes to muscle pathology; (3) the current strategies for therapeutic interventions which have led to the design of upcoming clinical trials. Two sessions were also dedicated to existing and future databases, and to outcome measures.

Autosomal recessive Bethlem myopathy

Bethlem myopathy is a well-defined clinical entity among collagen VI disorders, featuring proximal muscle weakness and contractures of the fingers, wrists, and ankles. It is an early-onset, slowly progressive, and relatively mild disease, invariably associated to date with heterozygous dominant mutations in the 3 collagen VI genes. We have characterized the clinical, laboratory, and genetic features of autosomal recessive Bethlem myopathy in 2 unrelated patients. This study is based on clinical, histochemical, immunocytochemical, and electron microscope evaluation of the muscle and dermal fibroblasts, CT imaging of the muscles, and biochemical and molecular analysis. Both patients carry a truncating COL6A2 mutation (Q819X; R366X) associated with missense changes in the partnering allele lying within the C2 domain of the alpha2(VI) chain (D871N; R843W-R830Q). They show decreased amounts of collagen VI in the basal lamina of muscle fibers and in dermal fibroblast cultures and altered behavior of collagen VI tetramers. Biochemical studies supported the pathogenic effect of identified amino acid substitutions, which involve strictly conserved residues. The reported patients illustrate the occurrence of Bethlem myopathy with a recessive mode of inheritance. This observation completes the hereditary pattern in collagen VI myopathies with both Ullrich congenital muscular dystrophy and Bethlem myopathy underlined by either recessive or dominant effecting mutations. This finding has relevant implications for genetic counseling and molecular characterization of patients with Bethlem myopathy, as well as for genotype-phenotype correlations in collagen VI disorders.

A novel staining method for quantification and 3D visualisation of capillaries and muscle fibres.

The aim of this study was to introduce a combined fluorescent staining that clearly demonstrates capillaries and distinguishes them from the basal lamina of muscle fibres in skeletal muscle tissue. The triple staining with CD31, Griffonia (Bandeira) simplicifolia lectin (GSL I) and laminin efficiently distinguishes vascular endothelium from the basal lamina of skeletal muscle fibres in physiological and pathological conditions. The presented triple staining method has several advantages, which facilitate quantitative analysis of the capillary network, and its relation to individual muscle fibres.

A synaptic nidogen: Developmental regulation and role of nidogen-2 at the neuromuscular junction

BackgroundThe skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.ResultsIn adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen-2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.ConclusionAll four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.

Connective tissue growth factor is overexpressed in muscles of human muscular dystrophy

The detailed process of how dystrophic muscles are replaced by fibrotic tissues is unknown. In the present study, the immunolocalization and mRNA expression of connective tissue growth factor (CTGF) in muscles from normal and dystrophic human muscles were examined with the goal of elucidating the pathophysiological function of CTGF in muscular dystrophy. Biopsies of frozen muscle from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, congenital muscular dystrophy, spinal muscular atrophy, congenital myopathy were analyzed using anti-CTGF polyclonal antibody. Reverse transcription-polymerase chain reaction (RT-PCR) was also performed to evaluate the expression of CTGF mRNA in dystrophic muscles. In normal muscle, neuromuscular junctions and vessels were CTGF-immunopositive, which suggests a physiological role for CTGF in these sites. In dystrophic muscle, CTGF immunoreactivity was localized to muscle fiber basal lamina, regenerating fibers, and the interstitium. Triple immunolabeling revealed that activated fibroblasts were immunopositive for CTGF and transforming growth factor-beta1 (TGF-beta1). RT-PCR analysis revealed increased levels of CTGF mRNA in the muscles of DMD patients. Co-localization of TGF-beta1 and CTGF in activated fibroblasts suggests that CTGF expression is regulated by TGF-beta1 through a paracrine/autocrine mechanism. In conclusion, TGF-beta1-CTGF pathway may play a role in the fibrosis that is commonly observed in muscular dystrophy.