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Obesity is an epidemic condition linked to cardiovascular disease severity and mortality. Fat localization and type represent cardiovascular risk estimators. Importantly, visceral fat secretes adipokines known to promote low-grade inflammation that, in turn, modulate its secretome and cardiac metabolism. In this regard, IL-33 regulates the functions of various immune cells through ST2 binding and-following its role as an immune sensor to infection and stress-is involved in the pro-fibrotic remodeling of the myocardium. Here we further investigated the IL-33/ST2 effects on cardiac remodeling in obesity, focusing on molecular pathways linking adipose-derived IL-33 to the development of fibrosis or hypertrophy. We analyzed the Zucker Fatty rat model, and we developed in vitro models to mimic the adipose and myocardial relationship. We demonstrated a dysregulation of IL-33/ST2 signaling in both adipose and cardiac tissue, where they affected Epac proteins and myocardial gene expression, linked to pro-fibrotic signatures. In Zucker rats, pro-fibrotic effects were counteracted by ghrelin-induced IL-33 secretion, whose release influenced transcription factor expression and ST2 isoforms balance regulation. Finally, the effect of IL-33 signaling is dependent on several factors, such as cell types' origin and the balancing of ST2 isoforms. Noteworthy, it is reasonable to state that considering IL-33 to have a unique protective role should be considered over-simplistic.
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Interleucina-33 , Obesidad , Receptores de Interleucina-1 , Remodelación Ventricular , Animales , Ratas , Cardiomegalia/genética , Cardiomegalia/metabolismo , Modelos Animales de Enfermedad , Fibrosis/genética , Fibrosis/metabolismo , Ghrelina/genética , Ghrelina/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1/genética , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Miocardio/metabolismo , Obesidad/complicaciones , Obesidad/genética , Obesidad/metabolismo , Ratas Zucker , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Remodelación Ventricular/genética , Remodelación Ventricular/fisiologíaRESUMEN
In Duchenne muscular dystrophy (DMD), sarcolemma fragility and myofiber necrosis produce cellular debris that attract inflammatory cells. Macrophages and T-lymphocytes infiltrate muscles in response to damage-associated molecular pattern signalling and the release of TNF-α, TGF-ß and interleukins prevent skeletal muscle improvement from the inflammation. This immunological scenario was extended by the discovery of a specific response to muscle antigens and a role for regulatory T cells (Tregs) in muscle regeneration. Normally, autoimmunity is avoided by autoreactive T-lymphocyte deletion within thymus, while in the periphery Tregs monitor effector T-cells escaping from central regulatory control. Here, we report impairment of thymus architecture of mdx mice together with decreased expression of ghrelin, autophagy dysfunction and AIRE down-regulation. Transplantation of dystrophic thymus in recipient nude mice determine the up-regulation of inflammatory/fibrotic markers, marked metabolic breakdown that leads to muscle atrophy and loss of force. These results indicate that involution of dystrophic thymus exacerbates muscular dystrophy by altering central immune tolerance.
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Tolerancia Inmunológica/inmunología , Músculo Esquelético/patología , Atrofia Muscular/patología , Distrofia Muscular Animal/patología , Timo/patología , Animales , Autofagia/fisiología , Ghrelina/biosíntesis , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Desnudos , Distrofia Muscular de Duchenne/patología , Linfocitos T/trasplante , Linfocitos T Reguladores/inmunología , Timo/trasplante , Factores de Transcripción/biosíntesis , Proteína AIRERESUMEN
BACKGROUND AND AIMS: Magnesium is a fundamental cation that regulates neuronal transmission, protein synthesis, energy metabolism. Magnesium deficiency mostly affects nervous and cardiovascular systems determining weakness, tremors, seizure and arrhythmias. This condition retains also a role in memory function and neuronal plasticity. Importantly magnesium deficiency could remain latent and asymptomatic resulting a risk factor for cardiovascular disease. In this sense we aim to determine magnesium status in patient presenting cognitive impairment of vascular origin. METHODS: 21 healthy subjects and 27 patients presenting vascular cognitive impairment were included in this study. Both plasma and intraerythrocitary magnesium level were measured to detect magnesium deficiency and cognitive performance was evaluated trough Mini Mental State Evaluation (MMSE). RESULTS: Here we showed that patients presenting vascular cognitive impairment present intraerythrocitary magnesium level lower than age-matched healthy subjects. To note their plasma magnesium resulted within reference limit. CONCLUSION: We suggest that intracellular magnesium laboratory measurement is needed to detect occult magnesium deficiency in population at risk. Magnesium supplementation could represent an adjuvant for healthy aging in high risk population.
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Disfunción Cognitiva , Deficiencia de Magnesio , Enfermedades Vasculares , Disfunción Cognitiva/complicaciones , Eritrocitos , Humanos , Deficiencia de Magnesio/complicaciones , Proyectos PilotoRESUMEN
The receptor for advanced glycation end products (RAGE), a well-known player of diabetes mellitus (DM)-related morbidities, was supposed to be involved in coronavirus disease-19 (COVID-19), but no data exist about COVID-19, DM, and the soluble RAGE (sRAGE) forms. We quantified total sRAGE and its forms, the endogenously secretory esRAGE and the membrane-cleaved cRAGE, in COVID-19 patients with and without DM and in healthy individuals to explore how COVID-19 may affect these molecules and their potential role as biomarkers. Circulating sRAGE and esRAGE were quantified by enzyme-linked-immunosorbent assays. cRAGE was obtained by subtracting esRAGE from total sRAGE. sRAGE, esRAGE, cRAGE, and the cRAGE/esRAGE ratio did not differ between DM and non-DM patients and had the same trend when compared to healthy individuals. Levels of total sRAGE, cRAGE, and cRAGE/esRAGE ratio were upregulated, while esRAGE was downregulated. The lack of difference between DM and non-DM COVID-19 patients in the levels of sRAGE and its forms supports the hypothesis that in COVID-19 the RAGE system is modulated regardless of glycemic control. Identifying how sRAGE and its forms associate to COVID-19 prognosis and the potential of RAGE as a therapeutic target to control inflammatory burden seem of relevance to help treatment of COVID-19.
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Background: Coronavirus Disease 2019 (COVID-19)-associated coagulopathy is characterized by a prothrombotic state not yet comprehensively studied. We investigated the coagulation pattern of patients with COVID-19 acute respiratory distress syndrome (ARDS), comparing patients who survived to those who did not. Methods: In this prospective cohort study on 20 COVID-19 ARDS patients, the following biomarkers were measured: thrombin generation (prothrombin fragment 1 + 2 (PF 1 + 2)), fibrinolysis activation (tissue plasminogen activator (tPA)) and inhibition (plasminogen activator inhibitor 2 (PAI-2)), fibrin synthesis (fibrinopeptide A) and fibrinolysis magnitude (plasmin-antiplasmin complex (PAP) and D-dimers). Measurements were done upon intensive care unit (ICU) admission and after 10-14 days. Results: There was increased thrombin generation; modest or null release of t-PA; and increased levels of PAI-2, fibrinopeptide A, PAP and D-dimers. At baseline, nonsurvivors had a significantly (p = 0.014) higher PAI-2/PAP ratio than survivors (109, interquartile range (IQR) 18.1-216, vs. 8.7, IQR 2.9-12.6). At follow-up, thrombin generation was significantly (p = 0.025) reduced in survivors (PF 1 + 2 from 396 pg/mL, IQR 185-585 to 237 pg/mL, IQR 120-393), whereas it increased in nonsurvivors. Fibrinolysis inhibition at follow-up remained stable in survivors and increased in nonsurvivors, leading to a significant (p = 0.026) difference in PAI-2 levels (161 pg/mL, IQR 50-334, vs. 1088 pg/mL, IQR 177-1565). Conclusion: Severe patterns of COVID-19 ARDS are characterized by a thrombin burst and the consequent coagulation activation. Mechanisms of fibrinolysis regulation appear unbalanced toward fibrinolysis inhibition. This pattern ameliorates in survivors, whereas it worsens in nonsurvivors.
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Pentraxin 3 (PTX3) is a main component of the innate immune system by inducing complement pathway activation, acting as an inflammatory mediator, coordinating the functions of macrophages/dendritic cells and promoting apoptosis/necrosis. Additionally, it has been found in fibrotic regions co-localizing with collagen. In this work, we wanted to investigate the predictive role of PTX3 in myocardial damage and fibrosis of Duchenne muscular dystrophy (DMD). DMD is an X-linked recessive disease caused by mutations of the dystrophin gene that affects muscular functions and strength and accompanying dilated cardiomyopathy. Here, we expound the correlation of PTX3 cardiac expression with age and Toll-like receptors (TLRs)/interleukin-1 receptor (IL-1R)-MyD88 inflammatory markers and its modulation by the so-called alarmins IL-33, high-mobility group box 1 (HMGB1), and S100ß. These findings suggest that cardiac levels of PTX3 might have prognostic value and potential in guiding therapy for DMD cardiomyopathy.
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Epicardial adipose tissue (EAT) has the unique property to release mediators that nourish the heart in healthy conditions, an effect that becomes detrimental when volume expands and proinflammatory cytokines start to be produced. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a proinflammatory mediator involved in atherosclerosis, is also produced by visceral fat. Due to the correlation of inflammation with PCSK9 and EAT enlargement, we evaluated whether PCSK9 was expressed in EAT and associated with EAT inflammation and volume. EAT samples were isolated during surgery. EAT thickness was measured by echocardiography. A microarray was used to explore EAT transcriptoma. The PCSK9 protein levels were measured by Western Blot in EAT and ELISA in plasma. PCSK9 was expressed at both the gene and protein levels in EAT. We found a positive association with EAT thickness and local proinflammatory mediators, in particular, chemokines for monocytes and lymphocytes. No association was found with the circulating PCSK9 level. The expression of PCSK9 in EAT argues that PCSK9 is part of the EAT secretome and EAT inflammation is associated with local PCSK9 expression, regardless of circulating PCSK9 levels. Whether reducing EAT inflammation or PCSK9 local levels may have beneficial effects on EAT metabolism and cardiovascular risk needs further investigations.
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Tejido Adiposo/metabolismo , Inflamación/metabolismo , Pericardio/metabolismo , Proproteína Convertasa 9/metabolismo , Anciano , Antropometría , Índice de Masa Corporal , Estudios de Casos y Controles , Quimiocinas/metabolismo , Enfermedad de la Arteria Coronaria/complicaciones , Femenino , Enfermedades de las Válvulas Cardíacas/complicaciones , Humanos , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Análisis por Matrices de Proteínas , RiesgoRESUMEN
A decline in metabolic health may take place before observing any alteration in the levels of the traditional metabolic markers. New indicators of metabolic derangement are therefore compelling. Irisin is a myokine with important metabolic functions. The role of irisin as a metabolic biomarker in humans has not been fully established yet. We quantified plasma irisin and esRAGE in 106 apparently healthy individuals and we performed a cluster analysis to evaluate their associations with metabolic profile. Plasma levels of various traditional markers of metabolic risk (i.e., glucose and lipid levels) were all within the ranges of normality. We identified two clusters of individuals. Compared to cluster 2, individuals in cluster 1 had higher irisin levels, a metabolic profile shifted toward the limits of the reference ranges and lower esRAGE levels. The traditional metabolic blood tests seem not to be enough to identify a metabolic decline early. Irisin increase and esRAGE decrease may reflect a metabolic derangement at the beginning of its development. The role of these molecules as early biomarkers of decline of metabolic health seems an interesting topic to be further explored.
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Duchenne muscular dystrophy (DMD) is a debilitating fatal X-linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store-operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti-IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase (SERCA) and removal of intracellular Ca2+ . Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD.
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Músculo Esquelético/crecimiento & desarrollo , Distrofia Muscular de Duchenne/tratamiento farmacológico , Receptor IGF Tipo 2/antagonistas & inhibidores , Regeneración , Animales , Sitios de Unión , Niño , Humanos , Ratones , Ratones Endogámicos mdx , Mioblastos , Adulto JovenRESUMEN
Background: Nutritional compounds can exert both anti-inflammatory and anti-oxidant effects. Since these events exacerbate the pathophysiology of muscular dystrophies, we investigated nutraceutical supplementation as an adjuvant therapy in dystrophic patients, to low costs and easy route of administration. Moreover, this treatment could represent an alternative therapeutic strategy for dystrophic patients who do not respond to corticosteroid treatment. Objective: A 24 weeks randomized double-blind placebo-controlled clinical study was aimed at evaluating the safety and efficacy of daily oral administration of flavonoids- and omega3-based natural supplement (FLAVOMEGA) in patients affected by muscular dystrophy with recognized muscle inflammation. Design: We screened 60 patients diagnosed for Duchenne (DMD), Facioscapulohumeral (FSHD), and Limb Girdle Muscular Dystrophy (LGMD). Using a computer-generated random allocation sequence, we stratified patients in a 2:1:1 ratio (DMD:FSHD:LGMD) to one of two treatment groups: continuous FLAVOMEGA, continuous placebo. Of 29 patients included, only 24 completed the study: 15 were given FLAVOMEGA, 14 placebo. Results: FLAVOMEGA was well tolerated with no reported adverse events. Significant treatment differences in the change from baseline in 6 min walk distance (6MWD; secondary efficacy endpoint) (P = 0.033) and in isokinetic knee extension (P = 0.039) (primary efficacy endpoint) were observed in LGMD and FSHD subjects. Serum CK levels (secondary efficacy endpoint) decreased in all FLAVOMEGA treated groups with significant difference in DMD subjects (P = 0.039). Conclusions: Although the small number of patients and the wide range of disease severity among patients reduced statistical significance, we obtained an optimal profile of safety and tolerability for the compound, showing valuable data of efficacy in primary and secondary endpoints. Trial registration number: NCT03317171 Retrospectively registered 25/10/2017.
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Creatina Quinasa/sangre , Polimiositis/diagnóstico , Electromiografía , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Masculino , Persona de Mediana Edad , Fuerza Muscular , Músculo Esquelético/patología , Mioglobina/sangre , Polimiositis/tratamiento farmacológico , Esteroides/uso terapéuticoRESUMEN
Patients affected by Duchenne muscular dystrophy (DMD) develop a progressive dilated cardiomyopathy characterized by inflammatory cell infiltration, necrosis, and cardiac fibrosis. Standard treatments consider the use of ß-blockers and angiotensin-converting enzyme inhibitors that are symptomatic and unspecific toward DMD disease. Medications that target DMD cardiac fibrosis are in the early stages of development. We found immunoproteasome dysregulation in affected hearts of mdx mice (murine animal model of DMD) and cardiomyocytes derived from induced pluripotent stem cells of patients with DMD. Interestingly, immunoproteasome inhibition ameliorated cardiomyopathy in mdx mice and reduced the development of cardiac fibrosis. Establishing the immunoproteasome inhibition-dependent cardioprotective role suggests the possibility of modulating the immunoproteasome as new and clinically relevant treatment to rescue dilated cardiomyopathy in patients with DMD.
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Cardiomiopatías , Distrofia Muscular de Duchenne , Miocitos Cardíacos , Complejo de la Endopetidasa Proteasomal/inmunología , Animales , Cardiomiopatías/inmunología , Cardiomiopatías/patología , Fibrosis , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/patología , Masculino , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/inmunología , Distrofia Muscular de Duchenne/patología , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/patologíaRESUMEN
OBJECTIVE: The aim of this study was to evaluate the usefulness of magnetic resonance imaging (MRI) in detecting the progression of Duchenne muscular dystrophy (DMD) by quantification of fat infiltration (FI) and muscle volume index (MVI, a residual-to-total muscle volume ratio). METHODS: Twenty-six patients (baseline age: 5-12 years) with genetically proven DMD were longitudinally analyzed with lower limb 3T MRI, force measurements, and functional tests (Gowers, 10-m time, North Star Ambulatory Assessment, 6-min walking test). Five age-matched controls were also examined, with a total of 85 MRI studies. Semiquantitative (scores) and quantitative MRI (qMRI) analyses (signal intensity ratio - SIR, lower limb MVI, and individual muscle MVI) were carried out. Permutation and regression analyses according to both age and functional test-outcomes were calculated. Age-related quantitative reference curves of SIRs and MVIs were generated. RESULTS: FI was present on glutei and adductor magnus in all patients since the age of 5, with a proximal-to-distal progression and selective sparing of sartorius and gracilis. Patients' qMRI measures were significantly different from controls' and among age classes. qMRI were more sensitive than force measurements and functional tests in assessing disease progression, allowing quantification also after loss of ambulation. Age-related curves with percentile values were calculated for SIRs and MVIs, to provide a reference background for future experimental therapy trials. SIRs and MVIs significantly correlated with all clinical measures, and could reliably predict functional outcomes and loss of ambulation. INTERPRETATIONS: qMRI-based indexes are sensitive measures that can track the progression of DMD and represent a valuable tool for follow-up and clinical studies.
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Duchenne muscular dystrophy is an inherited fatal genetic disease characterized by mutations in dystrophin gene, causing membrane fragility leading to myofiber necrosis and inflammatory cell recruitment in dystrophic muscles. The resulting environment enriched in proinflammatory cytokines, like IFN-γ and TNF-α, determines the transformation of myofiber constitutive proteasome into the immunoproteasome, a multisubunit complex involved in the activation of cell-mediate immunity. This event has a fundamental role in producing peptides for antigen presentation by MHC class I, for the immune response and also for cytokine production and T-cell differentiation. Here, we characterized for the first time the presence of T-lymphocytes activated against revertant dystrophin epitopes, in the animal model of Duchenne muscular dystrophy, the mdx mice. Moreover, we specifically blocked i-proteasome subunit LMP7, which was up-regulated in dystrophic skeletal muscles, and we demonstrated the rescue of the dystrophin expression and the amelioration of the dystrophic phenotype. The i-proteasome blocking lowered myofiber MHC class I expression and self-antigen presentation to T cells, thus reducing the specific antidystrophin T cell response, the muscular cell infiltrate, and proinflammatory cytokine production, together with muscle force recovery. We suggest that i-proteasome inhibition should be considered as new promising therapeutic approach for Duchenne muscular dystrophy pathology.
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Inmunoproteínas/antagonistas & inhibidores , Distrofia Muscular de Duchenne/tratamiento farmacológico , Inhibidores de Proteasoma/administración & dosificación , Linfocitos T/inmunología , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Terapia Genética , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/inmunología , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiologíaRESUMEN
Duchenne muscular dystrophy is the most common genetic muscular dystrophy. It is caused by mutations in the dystrophin gene, leading to absence of muscular dystrophin and to progressive degeneration of skeletal muscle. We have demonstrated that the exon skipping method safely and efficiently brings to the expression of a functional dystrophin in dystrophic CD133+ cells injected scid/mdx mice. Golden Retriever muscular dystrophic (GRMD) dogs represent the best preclinical model of Duchenne muscular dystrophy, mimicking the human pathology in genotypic and phenotypic aspects. Here, we assess the capacity of intra-arterial delivered autologous engineered canine CD133+ cells of restoring dystrophin expression in Golden Retriever muscular dystrophy. This is the first demonstration of five-year follow up study, showing initial clinical amelioration followed by stabilization in mild and severe affected Golden Retriever muscular dystrophy dogs. The occurrence of T-cell response in three Golden Retriever muscular dystrophy dogs, consistent with a memory response boosted by the exon skipped-dystrophin protein, suggests an adaptive immune response against dystrophin.
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Antígeno AC133/metabolismo , Inmunidad Adaptativa , Distrofia Muscular Animal/terapia , Trasplante de Células Madre/métodos , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Perros , Estudios de Seguimiento , Humanos , Distrofia Muscular Animal/inmunología , Células Madre/metabolismo , Trasplante Autólogo , Resultado del TratamientoRESUMEN
Myofibrillar myopathies (MFMs) are genetically heterogeneous dystrophies characterized by the disintegration of Z-disks and myofibrils and are associated with mutations in genes encoding Z-disk or Z-disk-related proteins. The c.626 C > T (p.P209L) mutation in the BAG3 gene has been described as causative of a subtype of MFM. We report a sporadic case of a 26-year-old Italian woman, affected by MFM with axonal neuropathy, cardiomyopathy, rigid spine, who carries the c.626 C > T mutation in the BAG3 gene. The patient and her non-consanguineous healthy parents and brother were studied with whole exome sequencing (WES) to further investigate the genetic basis of this complex phenotype. In the patient, we found that the BAG3 mutation is associated with variants in the NRAP and FHL1 genes that encode muscle-specific, LIM domain containing proteins. Quantitative real time PCR, immunohistochemistry and Western blot analysis of the patient's muscular biopsy showed the absence of NRAP expression and FHL1 accumulation in aggregates in the affected skeletal muscle tissue. Molecular dynamic analysis of the mutated FHL1 domain showed a modification in its surface charge, which could affect its capability to bind its target proteins. To our knowledge this is the first study reporting, in a BAG3 MFM, the simultaneous presence of genetic variants in the BAG3 and FHL1 genes (previously described as independently associated with MFMs) and linking the NRAP gene to MFM for the first time.
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Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/genética , Proteínas Musculares/genética , Miopatías Estructurales Congénitas/genética , Adulto , Exoma , Femenino , Humanos , Italia , TransfecciónRESUMEN
Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder characterized by muscle wasting and premature death. The defective gene is dystrophin, a structural protein, absence of which causes membrane fragility and myofiber necrosis. Several lines of evidence showed that in adult DMD patients dystrophin is involved in signaling pathways that regulate calcium homeostasis and differentiation programs. However, secondary aspects of the disease, such as inflammation and fibrosis development, might represent a bias in the analysis. Because fetal muscle is not influenced by gravity and does not suffer from mechanical load and/or inflammation, we investigated 12-week-old fetal DMD skeletal muscles, highlighting for the first time early alterations in signaling pathways mediated by the absence of dystrophin itself. We found that PLC/IP3/IP3R/Ryr1/Ca(2+) signaling is widely active in fetal DMD skeletal muscles and, through the calcium-dependent PKCα protein, exerts a fundamental regulatory role in delaying myogenesis and in myofiber commitment. These data provide new insights into the origin of DMD pathology during muscle development.
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Señalización del Calcio , Feto/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Desarrollo de Músculos , Músculo Esquelético/embriología , Distrofia Muscular de Duchenne/embriología , Distrofia Muscular de Duchenne/metabolismo , Animales , Biomarcadores/metabolismo , Biopsia , Calcio/metabolismo , Canales de Calcio/metabolismo , Feto/patología , Regulación del Desarrollo de la Expresión Génica , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/patología , Factor de Transcripción PAX7/metabolismo , Proteína Quinasa C-alfa/metabolismoRESUMEN
Duchenne muscular dystrophy (DMD) is characterized by the loss of a functional dystrophin protein; the muscles of DMD patients progressively degenerate as a result of mechanical stress during contractions, and the condition eventually leads to premature death. By means antisense oligonucleotides (AONs), it is possible to modulate pre-mRNA splicing eliminating mutated exons and restoring dystrophin open reading frame. To overcome the hurdles in using AONs for therapeutic interventions, we exerted engineered human DMD stem cells with a lentivirus, which permanently and efficiently delivered the cloned AONs. Here we describe for the first time the exosome-mediated release of AONs from engineered human DMD CD133+ stem cells allowing the rescue of murine dystrophin expression. Finally, upon release, AONs could be internalized by host cells suggesting a potential role of exosomes acting as vesicular carriers for DMD gene therapy.
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Distrofina/genética , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Células Madre/citología , Animales , Efecto Espectador/fisiología , Células Cultivadas , Distrofina/biosíntesis , Exones/genética , Humanos , Ratones , Ratones SCID , Músculo Esquelético/patología , Oligonucleótidos Antisentido/genética , Empalme del ARN/genéticaRESUMEN
Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, is characterized by muscular wasting caused by dystrophin deficiency that ultimately ends in force reduction and premature death. In addition to primary genetic defect, several mechanisms contribute to DMD pathogenesis. Recently, antioxidant supplementation was shown to be effective in the treatment of multiple diseases including muscular dystrophy. Different mechanisms were hypothesized such as reduced hydroxyl radicals, nuclear factor-κB deactivation, and NO protection from inactivation. Following these promising evidences, we investigated the effect of the administration of a mix of dietary natural polyphenols (ProAbe) on dystrophic mdx mice in terms of muscular architecture and functionality. We observed a reduction of muscle fibrosis deposition and myofiber necrosis together with an amelioration of vascularization. More importantly, the recovery of the morphological features of dystrophic muscle leads to an improvement of the endurance of treated dystrophic mice. Our data confirmed that ProAbe-based diet may represent a strategy to coadjuvate the treatment of DMD.