Nintedanib decreases muscle fibrosis and improves muscle function in a murine model of dystrophinopathy

Duchenne muscle dystrophy (DMD) is a genetic disorder characterized by progressive skeletal muscle weakness. Dystrophin deficiency induces instability of the sarcolemma during muscle contraction that leads to muscle necrosis and replacement of muscle by fibro-adipose tissue. Several therapies have been developed to counteract the fibrotic process. We report the effects of nintedanib, a tyrosine kinase inhibitor, in the mdx murine model of DMD. Nintedanib reduced proliferation and migration of human fibroblasts in vitro and decreased the expression of fibrotic genes such as COL1A1, COL3A1, FN1, TGFB1, and PDGFA. We treated seven mdx mice with 60 mg/kg/day nintedanib for 1 month. Electrophysiological studies showed an increase in the amplitude of the motor action potentials and an improvement of the morphology of motor unit potentials in the animals treated. Histological studies demonstrated a significant reduction of the fibrotic areas present in the skeletal muscles. Analysis of mRNA expression from muscles of treated mice showed a reduction in Col1a1, Col3a1, Tgfb1, and Pdgfa. Western blot showed a reduction in the expression of collagen I in skeletal muscles. In conclusion, nintedanib reduced the fibrotic process in a murine model of dystrophinopathy after 1 month of treatment, suggesting its potential use as a therapeutic drug in DMD patients.España, Ministerio de Economía y Competitividad BFU2016-74975-PEspaña, Instituto Ramón y Cajal PI13/0134

Nintedanib Reduces Muscle Fibrosis and Improves Muscle Function of the Alpha-Sarcoglycan-Deficient Mice

Sarcoglycanopathies are a group of recessive limb-girdle muscular dystrophies, characterized by progressive muscle weakness. Sarcoglycan deficiency produces instability of the sarcolemma during muscle contraction, leading to continuous muscle fiber injury eventually producing fiber loss and replacement by fibro-adipose tissue. Therapeutic strategies aiming to reduce fibro-adipose expansion could be effective in muscular dystrophies. We report the positive effect of nintedanib in a murine model of alpha-sarcoglycanopathy. We treated 14 Sgca mice, six weeks old, with nintedanib 50 mg/kg every 12 h for 10 weeks and compared muscle function and histology with 14 Sgca mice treated with vehicle and six wild-type littermate mice. Muscle function was assessed using a treadmill and grip strength. A cardiac evaluation was performed by echocardiography and histological study. Structural analysis of the muscles, including a detailed study of the fibrotic and inflammatory processes, was performed using conventional staining and immunofluorescence. In addition, proteomics and transcriptomics studies were carried out. Nintedanib was well tolerated by the animals treated, although we observed weight loss. Sgca mice treated with nintedanib covered a longer distance on the treadmill, compared with non-treated Sgca mice, and showed higher strength in the grip test. Moreover, nintedanib improved the muscle architecture of treated mice, reducing the degenerative area and the fibrotic reaction that was associated with a reversion of the cytokine expression profile. Nintedanib improved muscle function and muscle architecture by reducing muscle fibrosis and degeneration and reverting the chronic inflammatory environment suggesting that it could be a useful therapy for patients with alpha-sarcoglycanopathy

Platelet Derived Growth Factor-AA Correlates With Muscle Function Tests and Quantitative Muscle Magnetic Resonance in Dystrophinopathies

Introduction: Duchenne (DMD) and Becker (BMD) muscular dystrophy are X-linked muscular disorders produced by mutations in the DMD gene which encodes the protein dystrophin. Both diseases are characterized by progressive involvement of skeletal, cardiac, and respiratory muscles. As new treatment strategies become available, reliable biomarkers and outcome measures that can monitor disease progression are needed for clinical trials.Methods: We collected clinical and functional data and blood samples from 19 DMD patients, 13 BMD patients, and 66 healthy controls (8 pediatric and 58 adult controls), and blood samples from 15 patients with dysferlinopathy (DYSF) and studied the serum concentration of 4 growth factors involved in the process of muscle fibrosis. We correlated the serum concentration of these growth factors with several muscle function tests, spirometry results and fat fraction identified by quantitative Dixon muscle MRI.Results: We found significant differences in the serum concentration of Platelet Derived Growth Factor-AA (PDGF-AA) between DMD patients and pediatric controls, in Connective Tissue Growth Factor (CTGF) between BMD patients and adult controls, and in and Transforming Growth Factor- β1 (TGF-β1) between BMD and DYSF patients. PDGF-AA showed a good correlation with several muscle function tests for both DMD and BMD patients and with thigh fat fraction in BMD patients. Moreover, PDGF-AA levels were increased in muscle biopsies of patients with DMD and BMD as was demonstrated by immunohistochemistry and Real-Time PCR studies.Conclusion: Our study suggests that PDGF-AA should be further investigated in a larger cohort of DMD and BMD patients because it might be a good biomarker candidate to monitor the progression of these diseases

Platelet Derived Growth Factor-AA Correlates With Muscle Function Tests and Quantitative Muscle Magnetic Resonance in Dystrophinopathies

Introduction: Duchenne (DMD) and Becker (BMD) muscular dystrophy are X-linked muscular disorders produced by mutations in the DMD gene which encodes the protein dystrophin. Both diseases are characterized by progressive involvement of skeletal, cardiac, and respiratory muscles. As new treatment strategies become available, reliable biomarkers and outcome measures that can monitor disease progression are needed for clinical trials. Methods: We collected clinical and functional data and blood samples from 19 DMD patients, 13 BMD patients, and 66 healthy controls (8 pediatric and 58 adult controls), and blood samples from 15 patients with dysferlinopathy (DYSF) and studied the serum concentration of 4 growth factors involved in the process of muscle fibrosis. We correlated the serum concentration of these growth factors with several muscle function tests, spirometry results and fat fraction identified by quantitative Dixon muscle MRI. Results: We found significant differences in the serum concentration of Platelet Derived Growth Factor-AA (PDGF-AA) between DMD patients and pediatric controls, in Connective Tissue Growth Factor (CTGF) between BMD patients and adult controls, and in and Transforming Growth Factor- β1 (TGF-β1) between BMD and DYSF patients. PDGF-AA showed a good correlation with several muscle function tests for both DMD and BMD patients and with thigh fat fraction in BMD patients. Moreover, PDGF-AA levels were increased in muscle biopsies of patients with DMD and BMD as was demonstrated by immunohistochemistry and Real-Time PCR studies. Conclusion: Our study suggests that PDGF-AA should be further investigated in a larger cohort of DMD and BMD patients because it might be a good biomarker candidate to monitor the progression of these diseases

BNIP3 Is Involved in Muscle Fiber Atrophy in Late-Onset Pompe Disease Patients

Late-onset Pompe disease (LOPD) is a rare genetic disorder produced by mutations in the GAA gene and is characterized by progressive muscle weakness. LOPD muscle biopsies show accumulation of glycogen along with the autophagic vacuoles associated with atrophic muscle fibers. The expression of molecules related to muscle fiber atrophy in muscle biopsies of LOPD patients was studied using immunofluorescence and real-time PCR. BCL2 and adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a well-known atrogene, was identified as a potential mediator of muscle fiber atrophy in LOPD muscle biopsies. Vacuolated fibers in LOPD patient muscle biopsies were smaller than nonvacuolated fibers and expressed BNIP3. The current data suggested that BNIP3 expression is regulated by inhibition of the AKT-mammalian target of rapamycin pathway, leading to phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) at Ser317 by AMP-activated protein kinase. Myoblasts and myotubes obtained from LOPD patients and age-matched controls were studied to confirm these results using different molecular techniques. Myotubes derived from LOPD patients were likewise smaller and expressed BNIP3. Conclusively, transfection of BNIP3 into control myotubes led to myotube atrophy. These findings suggest a cascade that starts with the inhibition of the AKT-mammalian target of rapamycin pathway and activation of BNIP3 expression, leading to progressive muscle fiber atrophy. These results open the door to potential new treatments targeting BNIP3 to reduce its deleterious effects on muscle fiber atrophy in Pompe disease.Peer reviewe

Papel del factor de crecimiento derivado de plaquetas bb en la distrofia muscular de duchenne en humanos y estudio de nintedanib como tratamiento de un modelo murino de déficit de distrofina

La distrofia muscular de Duchenne (DMD) es un tipo de distrofia muscular de origen genético que se caracteriza por una degeneración muscular a causa de la deficiencia distrofina, una proteína del citoesqueleto involucrada en la estabilización del sarcolema. Esta enfermedad es caracterizada por una debilidad muscular rápida y progresiva que aparece a los 5-7 años e involucra los músculos proximales y las extremidades inferiores. El corazón y el sistema respiratorio también están afectados, resultando ser las principales causas de muerte. Desafortunadamente, no existe un tratamiento efectivo descrito hasta la fecha que haya cambiado sustancialmente la historia natural de la enfermedad. Desde el punto de vista fisiopatológico, la pérdida de distrofina lleva a un daño del sarcolema, infiltrados de células inflamatorias y necrosis de fibras musculares reemplazados por tejido adiposo y fibroso. Muchos factores contribuyen a este proceso, pero las vías moleculares relacionadas a la regeneración y la degeneración muscular no son completamente conocidas. Los factores de crecimiento derivados de plaquetas (PDGF) pertenecen a una familia de factores de crecimiento que regulan la proliferación, migración y diferenciación de células mesenquimales. Mientras que la isoforma PDGF-AA está asociada a fibrosis, el papel de la isoforma PDGF-BB en la regeneración muscular en humanos no ha sido estudiado. En este estudio se estudió la expresión de PDGF-BB en muestras de biopsia muscular de controles y pacientes con DMD. Para ello, se realizaron experimentos in vitro para entender los efectos de PDGF-BB en mioblastos, los cuales están involucrados en la patofisiología de las distrofias musculares. Estos resultados se confirmaron in vivo mediante el tratamiento del modelo murino mdx de DMD con inyecciones intramusculares repetidas de PDGF-BB durante 1 mes. Como resultado, se observó que PDGF-BB estaba expresado en las fibras musculares necróticas y regenerativas en muestras de biopsias musculares de pacientes con DMD. In vitro, PDGF-BB activó la proliferación y migración de mioblastos. El análisis de muestras musculares de ratón de animales tratados con PDGF-BB mostró un incremento en la población de células satélite (SCs) y en el número de fibras regenerativas, con una reducción en los infiltrados inflamatorios, en comparación con los animales tratados con vehículo. Basado en estos resultados, sugerimos que PDGF-BB podría jugar un papel protector en las distrofias musculares al mejorar la regeneración muscular a través de la activación de la proliferación y migración de las células satélite. Muchas terapias se han desarrollado para combatir el proceso fibrótico en distrofias musculares y otras enfermedades. En este trabajo se estudiaron los efectos de nintedanib, un triple inhibidor tirosina cinasa, en un modelo murino de DMD, el ratón mdx. Nintedanib redujo la proliferación y migración de fibroblastos humanos in vitro y disminuyó la expresión de genes fibróticos como COL1A1, COL3A1, FN1, TGFB1 y PDGFA. Siete ratones mdx fueron tratados con 60 mg/kg/día con nintedanib durante 1 mes. Estudios electrofisiológicos mostraron un incremento en la amplitud de los potenciales de acción motora y un mejoramiento de la morfología de los potenciales de unidad motora en los animales tratados. Estudios histológicos demostraron una reducción significativa de las áreas fibróticas presentes en los músculos esqueléticos. El análisis de la expresión de mRNA de músculos de ratón tratado indicó una reducción en los genes de Col1a1, Col3a1, Tgfb1 y Pdgfa. El western blot (WB) manifestó una reducción en la expresión de proteína del colágeno I en los músculos tratados. En conclusión, nosotros observamos que nintedanib ralentizó el proceso fibrótico muscular en un modelo murino de distrofinopatía después de 1 mes de tratamiento, sugiriendo su potencial papel como agente terapéutico en pacientes DMD.Duchenne muscular dystrophy (DMD) is genetic disorder characterized by a continuous muscle degeneration produced by the deficiency of dystrophin, which is a cytoskeletal protein involved in the stabilization of the sarcolemma. The disease is characterized by a rapidly progressive muscle weakness that appears at 5-7 years old involving the proximal muscles of the lower limbs. All patients lose the ambulation by the age of 12-15 and are completely dependent of their relatives after the 20 years of age. Cardiac and respiratory involvement is a constant, and they are the main causes of death. Unfortunately, there is not an effective treatment described so far that has changes substantially the natural history of the disease. From a pathophysiological point of view, dystrophin loss leads to sarcolemma damage, infiltrates of inflammatory cells and muscle fiber necrosis and replacement by fat and fibrotic tissue. Although several of the factors that contribute to this process have been revealed, the molecular pathways involved in the regeneration and degeneration of muscle are not completely known. Platelet derived growth factor (PDGF) belongs to a family of growth factors that regulate proliferation, migration, and differentiation of mesenchymal cells. While PDGF-AA isoform is associated to fibrosis, the role of PDGF-BB isoform in muscle regeneration in humans has not been studied. In this study it was analyzed the expression of PDGF-BB in muscle biopsy samples from controls and patients with DMD. Also, it was performed in vitro experiments to understand the effects of PDGF-BB on myoblasts, which are cells involved in the pathophysiology of muscular dystrophies and in vivo, by treating the mdx murine model of DMD with repeated intramuscular injections of PDGF-BB. As a result, it was observed that PDGF-BB was expressed by regenerating and necrotic muscle fibers in muscle biopsy samples from DMD patients. In vitro, PDGF-BB activated proliferation and migration of myoblasts. The analysis of muscles samples from the animals treated with PDGF-BB showed an increase in the population of satellite cells (SCs) and in the number of regenerative fibers, with a decrease in the inflammatory infiltrates, compared with those vehicle-treated mice. Based on these results, we suggest that PDGF-BB may play a protective role in muscular dystrophies by enhancing muscle regeneration through activation of satellite cell proliferation and migration. Several therapies have been developed to counteract the fibrotic process in muscle dystrophies and other diseases. In this study the effects of nintedanib, which is a triple tyrosine kinase inhibitor, in the mdx murine model of DMD were investigated. Nintedanib reduced proliferation and migration of human fibroblasts in vitro and decreased the expression of fibrotic genes such as COL1A1, COL3A1, FN1, TGFB1 and PDGFA. Seven mdx mice were treated with 60 mg/kg/day nintedanib for 1 month. Electrophysiological studies showed an increase in the amplitude of the motor action potentials and an improvement of the morphology of motor unit potentials in the animals treated. Histological studies demonstrated a significant reduction of the fibrotic areas present in the skeletal muscles. Analysis of mRNA expression from muscles of treated mice showed a reduction in Col1a1, Col3a1, Tgfb1, and Pdgfa. Western blot showed a reduction in the expression of collagen I in skeletal muscles. In conclusion, we observed that nintedanib slowed down the process of muscle fibrosis in a murine model of dystrophinopathy after 1 month of treatment, suggesting its potential role as a therapeutic agent in DMD patients

Papel del factor de crecimiento derivado de plaquetas bb en la distrofia muscular de duchenne en humanos y estudio de nintedanib como tratamiento de un modelo murino de déficit de distrofina

La distrofia muscular de Duchenne (DMD) es un tipo de distrofia muscular de origen genético que se caracteriza por una degeneración muscular a causa de la deficiencia distrofina, una proteína del citoesqueleto involucrada en la estabilización del sarcolema. Esta enfermedad es caracterizada por una debilidad muscular rápida y progresiva que aparece a los 5-7 años e involucra los músculos proximales y las extremidades inferiores. El corazón y el sistema respiratorio también están afectados, resultando ser las principales causas de muerte. Desafortunadamente, no existe un tratamiento efectivo descrito hasta la fecha que haya cambiado sustancialmente la historia natural de la enfermedad. Desde el punto de vista fisiopatológico, la pérdida de distrofina lleva a un daño del sarcolema, infiltrados de células inflamatorias y necrosis de fibras musculares reemplazados por tejido adiposo y fibroso. Muchos factores contribuyen a este proceso, pero las vías moleculares relacionadas a la regeneración y la degeneración muscular no son completamente conocidas. Los factores de crecimiento derivados de plaquetas (PDGF) pertenecen a una familia de factores de crecimiento que regulan la proliferación, migración y diferenciación de células mesenquimales. Mientras que la isoforma PDGF-AA está asociada a fibrosis, el papel de la isoforma PDGF-BB en la regeneración muscular en humanos no ha sido estudiado. En este estudio se estudió la expresión de PDGF-BB en muestras de biopsia muscular de controles y pacientes con DMD. Para ello, se realizaron experimentos in vitro para entender los efectos de PDGF-BB en mioblastos, los cuales están involucrados en la patofisiología de las distrofias musculares. Estos resultados se confirmaron in vivo mediante el tratamiento del modelo murino mdx de DMD con inyecciones intramusculares repetidas de PDGF-BB durante 1 mes. Como resultado, se observó que PDGF-BB estaba expresado en las fibras musculares necróticas y regenerativas en muestras de biopsias musculares de pacientes con DMD. In vitro, PDGF-BB activó la proliferación y migración de mioblastos. El análisis de muestras musculares de ratón de animales tratados con PDGF-BB mostró un incremento en la población de células satélite (SCs) y en el número de fibras regenerativas, con una reducción en los infiltrados inflamatorios, en comparación con los animales tratados con vehículo. Basado en estos resultados, sugerimos que PDGF-BB podría jugar un papel protector en las distrofias musculares al mejorar la regeneración muscular a través de la activación de la proliferación y migración de las células satélite. Muchas terapias se han desarrollado para combatir el proceso fibrótico en distrofias musculares y otras enfermedades. En este trabajo se estudiaron los efectos de nintedanib, un triple inhibidor tirosina cinasa, en un modelo murino de DMD, el ratón mdx. Nintedanib redujo la proliferación y migración de fibroblastos humanos in vitro y disminuyó la expresión de genes fibróticos como COL1A1, COL3A1, FN1, TGFB1 y PDGFA. Siete ratones mdx fueron tratados con 60 mg/kg/día con nintedanib durante 1 mes. Estudios electrofisiológicos mostraron un incremento en la amplitud de los potenciales de acción motora y un mejoramiento de la morfología de los potenciales de unidad motora en los animales tratados. Estudios histológicos demostraron una reducción significativa de las áreas fibróticas presentes en los músculos esqueléticos. El análisis de la expresión de mRNA de músculos de ratón tratado indicó una reducción en los genes de Col1a1, Col3a1, Tgfb1 y Pdgfa. El western blot (WB) manifestó una reducción en la expresión de proteína del colágeno I en los músculos tratados. En conclusión, nosotros observamos que nintedanib ralentizó el proceso fibrótico muscular en un modelo murino de distrofinopatía después de 1 mes de tratamiento, sugiriendo su potencial papel como agente terapéutico en pacientes DMD.Duchenne muscular dystrophy (DMD) is genetic disorder characterized by a continuous muscle degeneration produced by the deficiency of dystrophin, which is a cytoskeletal protein involved in the stabilization of the sarcolemma. The disease is characterized by a rapidly progressive muscle weakness that appears at 5-7 years old involving the proximal muscles of the lower limbs. All patients lose the ambulation by the age of 12-15 and are completely dependent of their relatives after the 20 years of age. Cardiac and respiratory involvement is a constant, and they are the main causes of death. Unfortunately, there is not an effective treatment described so far that has changes substantially the natural history of the disease. From a pathophysiological point of view, dystrophin loss leads to sarcolemma damage, infiltrates of inflammatory cells and muscle fiber necrosis and replacement by fat and fibrotic tissue. Although several of the factors that contribute to this process have been revealed, the molecular pathways involved in the regeneration and degeneration of muscle are not completely known. Platelet derived growth factor (PDGF) belongs to a family of growth factors that regulate proliferation, migration, and differentiation of mesenchymal cells. While PDGF-AA isoform is associated to fibrosis, the role of PDGF-BB isoform in muscle regeneration in humans has not been studied. In this study it was analyzed the expression of PDGF-BB in muscle biopsy samples from controls and patients with DMD. Also, it was performed in vitro experiments to understand the effects of PDGF-BB on myoblasts, which are cells involved in the pathophysiology of muscular dystrophies and in vivo, by treating the mdx murine model of DMD with repeated intramuscular injections of PDGF-BB. As a result, it was observed that PDGF-BB was expressed by regenerating and necrotic muscle fibers in muscle biopsy samples from DMD patients. In vitro, PDGF-BB activated proliferation and migration of myoblasts. The analysis of muscles samples from the animals treated with PDGF-BB showed an increase in the population of satellite cells (SCs) and in the number of regenerative fibers, with a decrease in the inflammatory infiltrates, compared with those vehicle-treated mice. Based on these results, we suggest that PDGF-BB may play a protective role in muscular dystrophies by enhancing muscle regeneration through activation of satellite cell proliferation and migration. Several therapies have been developed to counteract the fibrotic process in muscle dystrophies and other diseases. In this study the effects of nintedanib, which is a triple tyrosine kinase inhibitor, in the mdx murine model of DMD were investigated. Nintedanib reduced proliferation and migration of human fibroblasts in vitro and decreased the expression of fibrotic genes such as COL1A1, COL3A1, FN1, TGFB1 and PDGFA. Seven mdx mice were treated with 60 mg/kg/day nintedanib for 1 month. Electrophysiological studies showed an increase in the amplitude of the motor action potentials and an improvement of the morphology of motor unit potentials in the animals treated. Histological studies demonstrated a significant reduction of the fibrotic areas present in the skeletal muscles. Analysis of mRNA expression from muscles of treated mice showed a reduction in Col1a1, Col3a1, Tgfb1, and Pdgfa. Western blot showed a reduction in the expression of collagen I in skeletal muscles. In conclusion, we observed that nintedanib slowed down the process of muscle fibrosis in a murine model of dystrophinopathy after 1 month of treatment, suggesting its potential role as a therapeutic agent in DMD patients

Papel del factor de crecimiento derivado de plaquetas bb en la distrofia muscular de duchenne en humanos y estudio de nintedanib como tratamiento de un modelo murino de déficit de distrofina /

Departament responsable de la tesi: Departament de Medicina.La distrofia muscular de Duchenne (DMD) es un tipo de distrofia muscular de origen genético que se caracteriza por una degeneración muscular a causa de la deficiencia distrofina, una proteína del citoesqueleto involucrada en la estabilización del sarcolema. Esta enfermedad es caracterizada por una debilidad muscular rápida y progresiva que aparece a los 5-7 años e involucra los músculos proximales y las extremidades inferiores. El corazón y el sistema respiratorio también están afectados, resultando ser las principales causas de muerte. Desafortunadamente, no existe un tratamiento efectivo descrito hasta la fecha que haya cambiado sustancialmente la historia natural de la enfermedad. Desde el punto de vista fisiopatológico, la pérdida de distrofina lleva a un daño del sarcolema, infiltrados de células inflamatorias y necrosis de fibras musculares reemplazados por tejido adiposo y fibroso. Muchos factores contribuyen a este proceso, pero las vías moleculares relacionadas a la regeneración y la degeneración muscular no son completamente conocidas. Los factores de crecimiento derivados de plaquetas (PDGF) pertenecen a una familia de factores de crecimiento que regulan la proliferación, migración y diferenciación de células mesenquimales. Mientras que la isoforma PDGF-AA está asociada a fibrosis, el papel de la isoforma PDGF-BB en la regeneración muscular en humanos no ha sido estudiado. En este estudio se estudió la expresión de PDGF-BB en muestras de biopsia muscular de controles y pacientes con DMD. Para ello, se realizaron experimentos in vitro para entender los efectos de PDGF-BB en mioblastos, los cuales están involucrados en la patofisiología de las distrofias musculares. Estos resultados se confirmaron in vivo mediante el tratamiento del modelo murino mdx de DMD con inyecciones intramusculares repetidas de PDGF-BB durante 1 mes. Como resultado, se observó que PDGF-BB estaba expresado en las fibras musculares necróticas y regenerativas en muestras de biopsias musculares de pacientes con DMD. In vitro, PDGF-BB activó la proliferación y migración de mioblastos. El análisis de muestras musculares de ratón de animales tratados con PDGF-BB mostró un incremento en la población de células satélite (SCs) y en el número de fibras regenerativas, con una reducción en los infiltrados inflamatorios, en comparación con los animales tratados con vehículo. Basado en estos resultados, sugerimos que PDGF-BB podría jugar un papel protector en las distrofias musculares al mejorar la regeneración muscular a través de la activación de la proliferación y migración de las células satélite.Muchas terapias se han desarrollado para combatir el proceso fibrótico en distrofias musculares y otras enfermedades. En este trabajo se estudiaron los efectos de nintedanib, un triple inhibidor tirosina cinasa, en un modelo murino de DMD, el ratón mdx. Nintedanib redujo la proliferación y migración de fibroblastos humanos in vitro y disminuyó la expresión de genes fibróticos como COL1A1, COL3A1, FN1, TGFB1 y PDGFA. Siete ratones mdx fueron tratados con 60 mg/kg/día con nintedanib durante 1 mes. Estudios electrofisiológicos mostraron un incremento en la amplitud de los potenciales de acción motora y un mejoramiento de la morfología de los potenciales de unidad motora en los animales tratados. Estudios histológicos demostraron una reducción significativa de las áreas fibróticas presentes en los músculos esqueléticos. El análisis de la expresión de mRNA de músculos de ratón tratado indicó una reducción en los genes de Col1a1, Col3a1, Tgfb1 y Pdgfa. El western blot (WB) manifestó una reducción en la expresión de proteína del colágeno I en los músculos tratados. En conclusión, nosotros observamos que nintedanib ralentizó el proceso fibrótico muscular en un modelo murino de distrofinopatía después de 1 mes de tratamiento, sugiriendo su potencial papel como agente terapéutico en pacientes DMD.Duchenne muscular dystrophy (DMD) is genetic disorder characterized by a continuous muscle degeneration produced by the deficiency of dystrophin, which is a cytoskeletal protein involved in the stabilization of the sarcolemma. The disease is characterized by a rapidly progressive muscle weakness that appears at 5-7 years old involving the proximal muscles of the lower limbs. All patients lose the ambulation by the age of 12-15 and are completely dependent of their relatives after the 20 years of age. Cardiac and respiratory involvement is a constant, and they are the main causes of death. Unfortunately, there is not an effective treatment described so far that has changes substantially the natural history of the disease. From a pathophysiological point of view, dystrophin loss leads to sarcolemma damage, infiltrates of inflammatory cells and muscle fiber necrosis and replacement by fat and fibrotic tissue. Although several of the factors that contribute to this process have been revealed, the molecular pathways involved in the regeneration and degeneration of muscle are not completely known. Platelet derived growth factor (PDGF) belongs to a family of growth factors that regulate proliferation, migration, and differentiation of mesenchymal cells. While PDGF-AA isoform is associated to fibrosis, the role of PDGF-BB isoform in muscle regeneration in humans has not been studied. In this study it was analyzed the expression of PDGF-BB in muscle biopsy samples from controls and patients with DMD. Also, it was performed in vitro experiments to understand the effects of PDGF-BB on myoblasts, which are cells involved in the pathophysiology of muscular dystrophies and in vivo, by treating the mdx murine model of DMD with repeated intramuscular injections of PDGF-BB. As a result, it was observed that PDGF-BB was expressed by regenerating and necrotic muscle fibers in muscle biopsy samples from DMD patients. In vitro, PDGF-BB activated proliferation and migration of myoblasts. The analysis of muscles samples from the animals treated with PDGF-BB showed an increase in the population of satellite cells (SCs) and in the number of regenerative fibers, with a decrease in the inflammatory infiltrates, compared with those vehicle-treated mice. Based on these results, we suggest that PDGF-BB may play a protective role in muscular dystrophies by enhancing muscle regeneration through activation of satellite cell proliferation and migration. Several therapies have been developed to counteract the fibrotic process in muscle dystrophies and other diseases. In this study the effects of nintedanib, which is a triple tyrosine kinase inhibitor, in the mdx murine model of DMD were investigated. Nintedanib reduced proliferation and migration of human fibroblasts in vitro and decreased the expression of fibrotic genes such as COL1A1, COL3A1, FN1, TGFB1 and PDGFA. Seven mdx mice were treated with 60 mg/kg/day nintedanib for 1 month. Electrophysiological studies showed an increase in the amplitude of the motor action potentials and an improvement of the morphology of motor unit potentials in the animals treated. Histological studies demonstrated a significant reduction of the fibrotic areas present in the skeletal muscles. Analysis of mRNA expression from muscles of treated mice showed a reduction in Col1a1, Col3a1, Tgfb1, and Pdgfa. Western blot showed a reduction in the expression of collagen I in skeletal muscles. In conclusion, we observed that nintedanib slowed down the process of muscle fibrosis in a murine model of dystrophinopathy after 1 month of treatment, suggesting its potential role as a therapeutic agent in DMD patients

RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells : implications for Duchenne muscular dystrophy

The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) induces muscle fibre and replacement by fibro-adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been studied, pathways activated by PDGF-AA have not been described so far. Our aim was to study the molecular role of PDGF-AA in the fibrotic process of DMD. Skeletal muscle fibro-adipogenic progenitor cells (FAPs) from three DMD treated with PDGF-AA at 50 ng/mL were analysed by quantitative mass spectrometry-based proteomics. Western-blot, immunofluorescence, and G-LISA were used to confirm the mass spectrometry results. We evaluated the effects of PDGF-AA on the activation of RhoA pathway using two inhibitors, C3-exoenzyme and fasudil. Cell proliferation and migration were determined by BrdU and migration assay. Actin reorganization and collagen synthesis were measured by phalloidin staining and Sircol assay, respectively. In an in vivo proof of concept study, we treated dba/2J-mdx mice with fasudil for 6 weeks. Muscle strength was assessed with the grip strength. Immunofluorescence and flow cytometry analyses were used to study fibrotic and inflammatory markers in muscle tissue. Mass spectrometry revealed that RhoA pathway proteins were up-regulated in treated compared with non-treated DMD FAPs (n = 3, mean age = 8 ± 1.15 years old). Validation of proteomic data showed that Arhgef2 expression was significantly increased in DMD muscles compared with healthy controls by a 7.7-fold increase (n = 2, mean age = 8 ± 1.14 years old). In vitro studies showed that RhoA/ROCK2 pathway was significantly activated by PDGF-AA (n = 3, 1.88-fold increase, P < 0.01) and both C3-exoenzyme and fasudil blocked that activation (n = 3, P < 0.05 and P < 0.001, respectively). The activation of RhoA pathway by PDGF-AA promoted a significant increase in proliferation and migration of FAPs (n = 3, P < 0.001), while C3-exoenzyme and fasudil inhibited FAPs proliferation at 72 h and migration at 48 and 72 h (n = 3, P < 0.001). In vivo studies showed that fasudil improved muscle function (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, 1.76-fold increase, P < 0.013), and histological studies demonstrated a 23% reduction of collagen-I expression area (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, P < 0.01). Our results suggest that PDGF-AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies

Nintedanib decreases muscle fibrosis and improves muscle function in a murine model of dystrophinopathy

Duchenne muscle dystrophy (DMD) is a genetic disorder characterized by progressive skeletal muscle weakness. Dystrophin deficiency induces instability of the sarcolemma during muscle contraction that leads to muscle necrosis and replacement of muscle by fibro-adipose tissue. Several therapies have been developed to counteract the fibrotic process. We report the effects of nintedanib, a tyrosine kinase inhibitor, in the mdx murine model of DMD. Nintedanib reduced proliferation and migration of human fibroblasts in vitro and decreased the expression of fibrotic genes such as COL1A1, COL3A1, FN1, TGFB1, and PDGFA. We treated seven mdx mice with 60 mg/kg/day nintedanib for 1 month. Electrophysiological studies showed an increase in the amplitude of the motor action potentials and an improvement of the morphology of motor unit potentials in the animals treated. Histological studies demonstrated a significant reduction of the fibrotic areas present in the skeletal muscles. Analysis of mRNA expression from muscles of treated mice showed a reduction in Col1a1, Col3a1, Tgfb1, and Pdgfa. Western blot showed a reduction in the expression of collagen I in skeletal muscles. In conclusion, nintedanib reduced the fibrotic process in a murine model of dystrophinopathy after 1 month of treatment, suggesting its potential use as a therapeutic drug in DMD patients