RESUMEN
Fibrosis is a common feature of several chronic diseases and is characterized by exacerbated accumulation of ECM. An understanding of the cellular and molecular mechanisms involved in the development of this condition is crucial for designing efficient treatments for those pathologies. Connective tissue growth factor (CTGF/CCN2) is a pleiotropic protein with strong profibrotic activity. In this report, we present experimental evidence showing that ECM stimulates the synthesis of CTGF in response to lysophosphatidic acid (LPA).The integrin/focal adhesion kinase (FAK) signaling pathway mediates this effect, since CTGF expression is abolished by the use of the Arg-Gly-Asp-Ser peptide and also by an inhibitor of FAK autophosphorylation at tyrosine 397. Cilengitide, a specific inhibitor of αv integrins, inhibits the expression of CTGF mediated by LPA or transforming growth factor ß1. We show that ECM obtained from decellularized myofibroblast cultures or derived from activated fibroblasts from muscles of the Duchenne muscular dystrophy mouse model ( mdx) induces the expression of CTGF. This effect is dependent on FAK phosphorylation in response to its activation by integrin. We also found that the fibrotic ECM inhibits skeletal muscle differentiation. This novel regulatory mechanism of CTGF expression could be acting as a positive profibrotic feedback between the ECM and CTGF, revealing a novel concept in the control of fibrosis under chronic damage.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Matriz Extracelular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Quinasa 1 de Adhesión Focal/metabolismo , Integrina alfaV/metabolismo , Lisofosfolípidos/farmacología , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Duchenne/enzimología , Mioblastos/efectos de los fármacos , Animales , Línea Celular , Factor de Crecimiento del Tejido Conjuntivo/genética , Modelos Animales de Enfermedad , Matriz Extracelular/enzimología , Matriz Extracelular/patología , Fibroblastos/enzimología , Fibroblastos/patología , Fibrosis , Integrina alfaV/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Mioblastos/enzimología , Mioblastos/patología , Fosforilación , Transducción de Señal/efectos de los fármacosRESUMEN
INTRODUCTION: Duchenne muscular dystrophy (DMD) is a degenerative disease of skeletal, respiratory, and cardiac muscles caused by defects in the dystrophin gene. More recently, brain involvement has been verified. Mitochondrial dysfunction and oxidative stress may underlie the pathophysiology of DMD. In this study we evaluate Krebs cycle enzymes activity in the cerebral cortex, diaphragm, and quadriceps muscles of mdx mice. METHODS: Cortex, diaphragm, and quadriceps tissues from male dystrophic mdx and control mice were used. RESULTS: We observed increased malate dehydrogenase activity in the cortex; increased malate dehydrogenase and succinate dehydrogenase activities in the diaphragm; and increased citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase activities in the quadriceps of mdx mice. CONCLUSION: This study showed increased activity of Krebs cycle enzymes in cortex, quadriceps, and diaphragm in mdx mice.
Asunto(s)
Citrato (si)-Sintasa/metabolismo , Ciclo del Ácido Cítrico/fisiología , Isocitrato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/metabolismo , Distrofia Muscular de Duchenne/enzimología , Animales , Corteza Cerebral/enzimología , Diafragma/enzimología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Músculo Esquelético/enzimología , Distrofia Muscular de Duchenne/genéticaRESUMEN
The purpose of the present study was to investigate the role of cyclooxygenase-2 (COX-2) expression in fibrotic lesion in mdx mice. A total of six male C57BL/10 mice and six C57BL/10-DMD/mdx were distributed into two groups: control and animals with Duchenne muscular dystrophy (DMD). The medial part of gastrocnemius muscle was evaluated being the specimens stained with hematoxylin and eosin (H&E) and Sirius Red under normal and polarized light to differentiate type I (red and yellow) and III (green) collagen. COX-2 expression was assessed by immunohistochemistry. The results revealed histopathological changes in C57BL/10-DMD/mdx as depicted by regenerating fibers. Sirius Red stain showed a substantial increase in the amount of type I collagen of mdx mice. DMD induced a strong COX-2 immunoexpression in intercellular space. Taken together, our results are consistent with the notion that necrotic and fibrotic lesions are able to increase COX-2 expression in DMD.
Asunto(s)
Ciclooxigenasa 2/biosíntesis , Distrofina/deficiencia , Músculo Esquelético/enzimología , Distrofia Muscular de Duchenne/enzimología , Animales , Ciclooxigenasa 2/metabolismo , Distrofina/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/patologíaRESUMEN
INTRODUCTION: In Duchenne muscular dystrophy and in the mdx mouse, muscle fiber degeneration and subsequent fibrosis lead to cardiorespiratory failure. Previously, we demonstrated that the anti-fibrotic agent suramin was effective in decreasing fibrosis in mdx muscles. In this study, we were interested to see whether suramin could affect metalloproteinases (MMP) and improve the functional activity of the mdx diaphragm muscle. METHODS: Zymography was performed to evaluate MMP-2 and MMP-9 activity. Western blotting was used to analyze the levels of beta-dystroglycan. Muscle function was assessed in hemidiaphragm in vitro preparations. RESULTS: We found that suramin affects metalloproteinase-9 activity and increases beta-dystroglycan. Furthermore, suramin also protects against diaphragm muscle fatigue over time. CONCLUSIONS: These results show the potential benefits of suramin in maintaining the structure of the dystrophin-glycoprotein complex.
Asunto(s)
Diafragma/metabolismo , Distroglicanos/metabolismo , Distrofina/deficiencia , Metaloproteinasa 9 de la Matriz/metabolismo , Suramina/farmacología , Animales , Diafragma/efectos de los fármacos , Distroglicanos/biosíntesis , Distrofina/genética , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Femenino , Fibrosis , Masculino , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Fatiga Muscular/efectos de los fármacos , Fatiga Muscular/fisiología , Distrofia Muscular de Duchenne/enzimología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Commonly used in clinical practice, glutamic oxalacetic (GOT) and glutamic piruvic (GPT) transaminases are produced in various body tissues, including striated muscle, so their blood elevation is not due exclusively to liver disease. The objective of this study is to demonstrate the correlation between elevated creatinkinase (CK) and transaminases in patients with diagnosis of Duchenne muscular dystrophy (DMD), the most frequent neuromuscular disease in children. Patients and Method: Assessment in 61 children with diagnosis of DMD of CK, AST and ALT levels, and their correlation. Results: Aill patients had increase of CK ( = 13.363 IU/L), AST ( = 203 lU/L) and ALT ( = 194 IU/L) above normal values. The increase of transaminases related directly with the increase of CK. Conclusion: Patients with DMD have increased transaminases, so it is necessary to include this diagnostic possibility in a child with hypertransaminemia, prior to performing liver biopsy.
Las transaminasas que comúnmente se utilizan en clínica, glutámico oxalacética (GOT) y glutámico pirúvica (GPT) son producidas en varios tejidos del organismo entre los cuales se cuenta el músculo estriado, por lo que la elevación de transaminasas en sangre no es producida exclusivamente por enfermedades hepáticas. Objetivo: Demostrar la correlación entre el alza de la creatinkinasa (CK) y transaminasas en pacientes con el diagnóstico de distrofia muscular de Duchenne (DMD), la enfermedad neuromuscular más frecuente en niños. Pacientes y Método: Evaluación en 61 niños con diagnóstico de DMD de los niveles de CK, GOT y GPT y la relación entre ellos. Resultados: Todos los pacientes presentaron aumento de CK ( = 13.363 IU/L), GOT ( = 203 IU/L) y GPT ( = 194 IU/L) sobre los valores normales. El aumento de transaminasas se relacionó en forma directa con aumento de CK. Conclusiones: Los pacientes con DMD presentan transaminasas aumentadas, por lo que es necesario incluir esta posibilidad diagnóstica en niños con hipertransaminasemia, previo a realizar biopsia hepática.
Asunto(s)
Humanos , Masculino , Niño , Creatina Quinasa/sangre , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/sangre , Transaminasas/sangre , Creatina Quinasa/análisis , Distrofia Muscular de Duchenne/enzimología , Valores de Referencia , Transaminasas/análisisRESUMEN
In this study we investigated energy metabolism in the mdx mouse brain.To this end, prefrontal cortex, cerebellum, hippocampus, striatum, and cortex were analyzed. There was a decrease in Complex I but not in Complex II activity in all structures. There was an increase in Complex III activity in striatum and a decrease in Complex IV activity in prefrontal cortex and striatum. Mitochondrial creatine kinase activity was increased in hippocampus, prefrontal cortex, cortex, and striatum. Our results indicate that there is energy metabolism dysfunction in the mdx mouse brain.
Asunto(s)
Encéfalo/enzimología , Creatina Quinasa/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Distrofia Muscular de Duchenne/enzimología , Animales , Encéfalo/patología , Cerebelo/enzimología , Cerebelo/patología , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Cuerpo Estriado/enzimología , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Metabolismo Energético/fisiología , Hipocampo/enzimología , Hipocampo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/patología , Corteza Prefrontal/enzimología , Corteza Prefrontal/patologíaRESUMEN
The distribution of nitric oxide synthase at the neuromuscular junction (NMJ) of normal, denervated and mdx mice was studied using a specific antibody against the neuronal isoform of nitric oxide synthase (nNOS). Fluorescence confocal microscopy demonstrated that nNOS immunoreactivity was localised mainly in the sarcolemma and presynaptic region covering acetylcholine receptor branches. The expression of presynaptic nNOS was greatly reduced in dystrophin-deficient muscles. In normal denervated muscles, nNOS was still present in the presynaptic region and there were no qualitative changes in the expression of this protein. These results suggest that the presynaptic distribution of nNOS is associated with terminal Schwann cells. The relationship between nNOS and the presynaptic components of the neuromuscular junction may open new perspectives for improving our understanding of the pathogenesis of dystrophic muscles.