RESUMO
Connective tissue growth factor or cellular communication network 2 (CCN2/CTGF) is a matricellular protein member of the CCN family involved in several crucial biological processes. In skeletal muscle, CCN2/CTGF abundance is elevated in human muscle biopsies and/or animal models for diverse neuromuscular pathologies, including muscular dystrophies, neurodegenerative disorders, muscle denervation, and muscle overuse. In this context, CCN2/CTGF is deeply involved in extracellular matrix (ECM) modulation, acting as a strong pro-fibrotic factor that promotes excessive ECM accumulation. Reducing CCN2/CTGF levels or biological activity in pathological conditions can decrease fibrosis, improve muscle architecture and function. In this work, we summarize information about the role of CCN2/CTGF in fibrosis associated with neuromuscular pathologies and the mechanisms and signaling pathways that regulate their expression in skeletal muscle.
RESUMO
Several skeletal muscle diseases are characterized by fibrosis, the excessive accumulation of extracellular matrix. Transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CCN2/CTGF) are two profibrotic factors augmented in fibrotic skeletal muscle, together with signs of reduced vasculature that implies a decrease in oxygen supply. We observed that fibrotic muscles are characterized by the presence of positive nuclei for hypoxia-inducible factor-1α (HIF-1α), a key mediator of the hypoxia response. However, it is not clear how a hypoxic environment could contribute to the fibrotic phenotype in skeletal muscle. We evaluated the role of hypoxia and TGF-ß on CCN2 expression in vitro. Fibroblasts, myoblasts and differentiated myotubes were incubated with TGF-ß1 under hypoxic conditions. Hypoxia and TGF-ß1 induced CCN2 expression synergistically in myotubes but not in fibroblasts or undifferentiated muscle progenitors. This induction requires HIF-1α and the Smad-independent TGF-ß signaling pathway. We performed in vivo experiments using pharmacological stabilization of HIF-1α or hypoxia-induced via hindlimb ischemia together with intramuscular injections of TGF-ß1, and we found increased CCN2 expression. These observations suggest that hypoxic signaling together with TGF-ß signaling, which are both characteristics of a fibrotic skeletal muscle environment, induce the expression of CCN2 in skeletal muscle fibers and myotubes.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Músculo Esquelético/patologia , Fator de Crescimento Transformador beta1/administração & dosagem , Regulação para Cima , Animais , Diferenciação Celular , Hipóxia Celular , Linhagem Celular , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Injeções Intramusculares , Isquemia/etiologia , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Células NIH 3T3 , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset progressive neurodegenerative disease characterized by upper and lower motoneuron degeneration. A total of 20% of familial ALS (fALS) cases are explained by mutations in the superoxide dismutase 1 (SOD1) enzyme. Although more than 20 years have passed since the generation of the first ALS mouse model, the precise molecular mechanisms of ALS pathogenesis remain unknown. CTGF/CCN2 is a matricellular protein with associated fibrotic activity that is up-regulated in several chronic diseases. The inhibition of CTGF/CCN2 with the monoclonal neutralizing antibody FG-3019 reduces fibrosis in several chronic disorders including the mdx mice, a murine model for Duchenne muscular dystrophy (DMD). In this work, we show that there are increased levels of CTGF/CCN2 in skeletal muscle and spinal cord of hSOD1G93A mice. In this scenario, we show evidence that FG-3019 not only reduces fibrosis in skeletal muscle of hSOD1G93A mice, but also improves muscle and locomotor performance. We demonstrate that treatment with FG-3019 reduces muscle atrophy in hSOD1G93A mice. We also found improvement of neuromuscular junction (NMJ) innervation together with a reduction in myelin degeneration in the sciatic nerve, suggesting that alterations in nerve-muscle communication are partially improved in FG-3019-treated hSOD1G93A mice. Moreover, we also found that CTGF/CCN2 is expressed in astrocytes and neurons, predominantly in dorsal areas of spinal cord from symptomatic hSOD1G93A mice. Together, these results reveal that CTGF/CCN2 might be a novel therapeutic target to ameliorate symptoms and improve the quality of life of ALS patients.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Fator de Crescimento do Tecido Conjuntivo/genética , Distrofia Muscular de Duchenne/tratamento farmacológico , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Modelos Animais de Doenças , Fibrose/tratamento farmacológico , Fibrose/genética , Fibrose/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Locomoção/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologiaRESUMO
In Duchenne muscular dystrophy (DMD) and the mdx mouse model, the absence of the cytoskeletal protein dystrophin causes defective anchoring of myofibres to the basal lamina. The resultant myofibre degeneration and necrosis lead to a progressive loss of muscle mass, increased fibrosis and ultimately fatal weakness. Connective tissue growth factor (CTGF/CCN-2) is critically involved in several chronic fibro-degenerative diseases. In DMD, the role of CTGF might extend well beyond replacement fibrosis secondary to loss of muscle fibres, since its overexpression in skeletal muscle could by itself induce a dystrophic phenotype. Using two independent approaches, we here show that mdx mice with reduced CTGF availability do indeed have less severe muscular dystrophy. Mdx mice with hemizygous CTGF deletion (mdx-Ctgf+/-), and mdx mice treated with a neutralizing anti-CTGF monoclonal antibody (FG-3019), performed better in an exercise endurance test, had better muscle strength in isolated muscles and reduced skeletal muscle impairment, apoptotic damage and fibrosis. Transforming growth factor type-ß (TGF-ß), pERK1/2 and p38 signalling remained unaffected during CTGF suppression. Moreover, both mdx-Ctgf+/- and FG-3019 treated mdx mice had improved grafting upon intramuscular injection of dystrophin-positive satellite cells. These findings reveal the potential of targeting CTGF to reduce disease progression and to improve cell therapy in DMD.