RESUMEN
Post-mitotic, non-proliferative dermal fibroblasts have crucial functions in maintenance and restoration of tissue homeostasis. They are involved in essential processes such as wound healing, pigmentation and hair growth, but also tumor development and aging-associated diseases. These processes are energetically highly demanding and error prone when mitochondrial damage occurs. However, mitochondrial function in fibroblasts and the influence of mitochondrial dysfunction on fibroblast-specific demands are still unclear. To address these questions, we created a mouse model in which accelerated cell-specific mitochondrial DNA (mtDNA) damage accumulates. We crossed mice carrying a dominant-negative mutant of the mitochondrial replicative helicase Twinkle (RosaSTOP system) with mice that express fibroblast-specific Cre Recombinase (Collagen1A2 CreERT) which can be activated by Tamoxifen (TwinkleFIBRO). Thus, we are able to induce mtDNA deletions and duplications in specific cells, a process which resembles the physiological aging process in humans, where this damage accumulates in all tissues. Upon proliferation in vitro, Tamoxifen induced Twinkle fibroblasts deplete most of their mitochondrial DNA which, although not disturbing the stoichiometry of the respiratory chain complexes, leads to reduced ROS production and mitochondrial membrane potential as well as an anti-inflammatory and anti-fibrotic profile of the cells. In Sodium Azide treated wildtype fibroblasts, without a functioning respiratory chain, we observe the opposite, a rather pro-inflammatory and pro-fibrotic signature. Upon accumulation of mitochondrial DNA mutations in vivo the TwinkleFIBRO mice are protected from fibrosis development induced by intradermal Bleomycin injections. This is due to dampened differentiation of the dermal fibroblasts into α-smooth-muscle-actin positive myofibroblasts in TwinkleFIBRO mice. We thus provide evidence for striking differences of the impact that mtDNA mutations have in contrast to blunted mitochondrial function in dermal fibroblasts and skin homeostasis. These data contribute to improved understanding of mitochondrial function and dysfunction in skin and provide mechanistic insight into potential targets to treat skin fibrosis in the future.
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Bleomicina , Diferenciación Celular , ADN Mitocondrial , Fibrosis , Mutación , Miofibroblastos , Animales , Bleomicina/efectos adversos , Bleomicina/toxicidad , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Ratones , Miofibroblastos/metabolismo , Miofibroblastos/patología , Miofibroblastos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , ADN Helicasas/genética , ADN Helicasas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Tamoxifeno/farmacología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Modelos Animales de Enfermedad , Especies Reactivas de Oxígeno/metabolismo , Humanos , Piel/patología , Piel/metabolismo , Piel/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Colágeno Tipo IRESUMEN
Magnesium-based biomaterials hold remarkable promise for various clinical applications, offering advantages such as reduced stress-shielding and enhanced bone strengthening and vascular remodeling compared to traditional materials. However, ensuring the quality of preclinical research is crucial for the development of these implants. To achieve implant success, an understanding of the cellular responses post-implantation, proper model selection, and good study design are crucial. There are several challenges to reaching a safe and effective translation of laboratory findings into clinical practice. The utilization of Mg-based biomedical devices eliminates the need for biomaterial removal surgery post-healing and mitigates adverse effects associated with permanent biomaterial implantation. However, the high corrosion rate of Mg-based implants poses challenges such as unexpected degradation, structural failure, hydrogen evolution, alkalization, and cytotoxicity. The biocompatibility and degradability of materials based on magnesium have been studied by many researchers in vitro; however, evaluations addressing the impact of the material in vivo still need to be improved. Several animal models, including rats, rabbits, dogs, and pigs, have been explored to assess the potential of magnesium-based materials. Moreover, strategies such as alloying and coating have been identified to enhance the degradation rate of magnesium-based materials in vivo to transform these challenges into opportunities. This review aims to explore the utilization of Mg implants across various biomedical applications within cellular (in vitro) and animal (in vivo) models.
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Materiales Biocompatibles , Magnesio , Magnesio/química , Animales , Materiales Biocompatibles/química , Humanos , Proyectos de Investigación , Ensayo de Materiales , Corrosión , Prótesis e ImplantesRESUMEN
Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.
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Cicatriz , Colágeno , Fibroblastos , Cicatrización de Heridas , Pez Cebra , Humanos , Animales , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Colágeno/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Cicatriz/metabolismo , Cicatriz/patología , Cicatriz/tratamiento farmacológico , Piel/metabolismo , Piel/patología , Piel/efectos de los fármacos , Fibrosis , Péptidos/farmacología , Péptidos/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/patología , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacosRESUMEN
The updated S2k guideline deals with the diagnosis and therapy of localized scleroderma (LoS). LoS represents a spectrum of sclerotic skin diseases in which, depending on the subtype and localisation, structures such as adipose tissue, muscles, joints, and bones may also be affected. Involvement of internal organs or progression to systemic sclerosis does not occur. LoS can be classified into four main forms: limited, generalized, linear, and mixed forms, with some additional subtypes. For cases of limited skin involvement, the guideline primarily recommends therapy with topical corticosteroids. UV therapy can also be recommended. In subtypes with severe skin or musculoskeletal involvement, systemic therapy with methotrexate is recommended. During the active phase of the disease, systemic glucocorticosteroids can be used additionally. In cases of methotrexate and steroid refractory courses, contraindications, or intolerance, mycophenolate mofetil, mycophenolic acid, or abatacept can be considered as second-line systemic therapies. In the case of linear LoS, autologous adipose-derived stem cell transplantation can also be performed for correcting soft tissue defects.
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Fármacos Dermatológicos , Esclerodermia Localizada , Humanos , Metotrexato/uso terapéutico , Esclerodermia Localizada/diagnóstico , Esclerodermia Localizada/terapia , Piel , Fármacos Dermatológicos/uso terapéutico , Ácido Micofenólico/uso terapéuticoRESUMEN
The present review summarizes the beneficial and detrimental roles of reactive oxygen species in myocardial ischemia/reperfusion injury and cardioprotection. In the first part, the continued need for cardioprotection beyond that by rapid reperfusion of acute myocardial infarction is emphasized. Then, pathomechanisms of myocardial ischemia/reperfusion to the myocardium and the coronary circulation and the different modes of cell death in myocardial infarction are characterized. Different mechanical and pharmacological interventions to protect the ischemic/reperfused myocardium in elective percutaneous coronary interventions and coronary artery bypass grafting, in acute myocardial infarction and in cardiotoxicity from cancer therapy are detailed. The second part keeps the focus on ROS providing a comprehensive overview of molecular and cellular mechanisms involved in ischemia/reperfusion injury. Starting from mitochondria as the main sources and targets of ROS in ischemic/reperfused myocardium, a complex network of cellular and extracellular processes is discussed, including relationships with Ca2+ homeostasis, thiol group redox balance, hydrogen sulfide modulation, cross-talk with NAPDH oxidases, exosomes, cytokines and growth factors. While mechanistic insights are needed to improve our current therapeutic approaches, advancements in knowledge of ROS-mediated processes indicate that detrimental facets of oxidative stress are opposed by ROS requirement for physiological and protective reactions. This inevitable contrast is likely to underlie unsuccessful clinical trials and limits the development of novel cardioprotective interventions simply based upon ROS removal.
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Infarto del Miocardio , Daño por Reperfusión Miocárdica , Humanos , Especies Reactivas de Oxígeno/metabolismo , Miocardio/metabolismo , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/metabolismo , Oxidación-ReducciónRESUMEN
BACKGROUND: There is a strong rationale to develop locally-acting surgical treatments for digital ulcers (DUs) in patients with systemic sclerosis (SSc). Our aim was to examine the safety and efficacy of local surgical management for SSc-DU. METHODS: A systematic literature review was carried out until to August 2022 using 7 different databases. Original research studies concerning adult patients with SSc-DUs, and local surgical treatments were analysed using the PICO framework. We included randomized controlled trials, prospective/retrospective studies, and case series (minimum of 3 patients) References were independently screened by two reviewers including assessment of the risk of bias using validated tools. RESULTS: Out of 899, 13eligible articles were included. Autologous fat (adipose tissue AT) grafting was the surgical modality most identified (7 studies, 1 randomized controlled double blinded trial and 6 prospective open-label single arm studies). The healing rate (HR) with autologous fat grafting (4 studies) was 66-100 %. Three studies reported autologous adipose-derived stromal vascular fraction grafting: HR of 32-60 %. Bone marrow derived cell transplantation in a single study showed 100 % healing rate over 4-24 weeks. Surgical sympathectomy was examined in 3 studies, prospective without comparator with a median healing rate of 81 %. Two surgical studies (of direct microsurgical revascularisation and microsurgical arteriolysis) showed 100 % healing of ulcers, with no complications. CONCLUSION: Several surgical approaches for SSc-DUs have demonstrated some degree of safety and effectiveness for DU healing. However, there are significant methodological issues. Future studies are warranted to rigorously investigate surgical interventions for SSc-DUs.
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Esclerodermia Sistémica , Úlcera Cutánea , Adulto , Humanos , Dedos/cirugía , Estudios Prospectivos , Estudios Retrospectivos , Úlcera Cutánea/etiología , Úlcera Cutánea/cirugía , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/cirugíaRESUMEN
BACKGROUND: Current recommendations on the management of systemic sclerosis (SSc) suggest that autologous hematopoietic stem cell therapy (HSCT) can be a rescue therapy for patients with rapidly progressive SSc. OBJECTIVES: To assess the safety and efficacy of HSCT for patients with SSc and to compare these with non-HSCT patients in a control cohort with adjusted risk factors. METHODS: A retrospective analysis of data from the multicentric German network for systemic scleroderma (DNSS) with 5000 patients with SSc. Control groups consisted of all patients with diffuse cutaneous (dc)-SSc (group A) and an adjusted high-risk cohort of male patients with Scl70-positive dc-SSc (group B). RESULTS: Eighty SSc patients received an HSCT 4.1 ± 4.8 years after SSc diagnosis. Among them, 86.3% had dc-SSc, 43.5% were males, and 71.3% were positive for Scl70 antibodies. The control group A (n=1513) showed a significant underrepresentation of these risk factors for mortality. When the survival of the control group B (n=240) was compared with the HSCT group, a lower mortality of the latter was observed instead. Within 5 years after HSCT, we observed an improvement of the mRSS from 17.6 ± 11.5 to 11.0 ± 8.5 (p=0.001) and a stabilization of the DLCO. We did not see differences in transplant-related mortality between patients who received HSCT within 3 years after SSc diagnosis or later. CONCLUSION: Our analysis of real-life data show that the distribution of risk factors for mortality is critical when HSCT cohorts are compared with non-HSCT control groups.
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Trasplante de Células Madre Hematopoyéticas , Esclerodermia Difusa , Esclerodermia Sistémica , Humanos , Masculino , Femenino , Estudios Retrospectivos , Trasplante Autólogo , Esclerodermia Sistémica/terapia , Sistema de RegistrosRESUMEN
With more than 25 million people affected, heart failure (HF) is a global threat. As energy production pathways are known to play a pivotal role in HF, we sought here to identify key metabolic changes in ischemic- and non-ischemic HF by using a multi-OMICS approach. Serum metabolites and mRNAseq and epigenetic DNA methylation profiles were analyzed from blood and left ventricular heart biopsy specimens of the same individuals. In total we collected serum from n = 82 patients with Dilated Cardiomyopathy (DCM) and n = 51 controls in the screening stage. We identified several metabolites involved in glycolysis and citric acid cycle to be elevated up to 5.7-fold in DCM (p = 1.7 × 10-6). Interestingly, cardiac mRNA and epigenetic changes of genes encoding rate-limiting enzymes of these pathways could also be found and validated in our second stage of metabolite assessment in n = 52 DCM, n = 39 ischemic HF and n = 57 controls. In conclusion, we identified a new set of metabolomic biomarkers for HF. We were able to identify underlying biological cascades that potentially represent suitable intervention targets.
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Biomarcadores/metabolismo , Cardiomiopatía Dilatada/genética , Epigenómica/métodos , Perfilación de la Expresión Génica/métodos , Insuficiencia Cardíaca/genética , Metabolómica/métodos , Adulto , Anciano , Biomarcadores/sangre , Cardiomiopatía Dilatada/diagnóstico , Cardiomiopatía Dilatada/metabolismo , Estudios de Cohortes , Epigénesis Genética , Femenino , Glucólisis/genética , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Análisis de Componente PrincipalRESUMEN
In this study, we investigated the role of cardiomyocyte (CM) and endothelial cell (EC) specific interactions with collagen in the assembly of an operational myocardium in vitro. Engineered cardiac patches represent valuable tools for myocardial repair following infarction and are generally constituted of a suitable biomaterial populated by CMs and supportive cell types. Among those, ECs are required for tissue vascularization and positively modulate CM function. To direct the function of human embryonic stem cell (hESC)-derived CM and EC seeded on biomaterials, we replicated cell-collagen interactions, which regulate cellular behaviour in the native myocardium, using triple-helical peptides (THPs) that are ligands for collagen-binding proteins. THPs enhanced proliferation and activity of CMs and ECs separately and in co-culture, drove CM maturation and enabled coordinated cellular contraction on collagen films. These results highlight the importance of collagen interactions on cellular response and establish THP-functionalized biomaterials as novel tools to produce engineered cardiac tissues.
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Células Madre Embrionarias Humanas , Ingeniería de Tejidos , Diferenciación Celular , Células Endoteliales , Humanos , Miocitos Cardíacos , PéptidosRESUMEN
Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis. This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFß, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing. Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.
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Colágeno Tipo XII/genética , Piel/crecimiento & desarrollo , Factor de Crecimiento Transformador beta/genética , Cicatrización de Heridas/genética , Animales , Colágeno Tipo I/genética , Matriz Extracelular , Fibroblastos/metabolismo , Fibroblastos/patología , Homeostasis/genética , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Noqueados/genética , Miofibroblastos/metabolismo , Piel/parasitologíaRESUMEN
Many mitochondrial metabolites and bioactive molecules contain two carboxylic acid moieties that make them unable to cross biological membranes. Hence, there is considerable interest in facilitating the uptake of these molecules into cells and mitochondria to modify or report on their function. Conjugation to the triphenylphosphonium (TPP) lipophilic cation is widely used to deliver molecules selectively to mitochondria in response to the membrane potential. However, permanent attachment to the cation can disrupt the biological function of small dicarboxylates. Here, we have developed a strategy using TPP to release dicarboxylates selectively within mitochondria. For this, the dicarboxylate is attached to a TPP compound via a single ester bond, which is then cleaved by intramitochondrial esterase activity, releasing the dicarboxylate within the organelle. Leaving the second carboxylic acid free also means mitochondrial uptake is dependent on the pH gradient across the inner membrane. To assess this strategy, we synthesized a range of TPP monoesters of the model dicarboxylate, malonate. We then tested their mitochondrial accumulation and ability to deliver malonate to isolated mitochondria and to cells, in vitro and in vivo. A TPP-malonate monoester compound, TPP11-malonate, in which the dicarboxylate group was attached to the TPP compound via a hydrophobic undecyl link, was most effective at releasing malonate within mitochondria in cells and in vivo. Therefore, we have developed a TPP-monoester platform that enables the selective release of bioactive dicarboxylates within mitochondria.
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Ácidos Carboxílicos/química , Cationes/química , Mitocondrias/efectos de los fármacos , Animales , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Ésteres/química , Femenino , Células HeLa , Compuestos Heterocíclicos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Malonatos/química , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Compuestos Organofosforados/química , Ratas , Ratas WistarRESUMEN
Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.
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Mitocondrias/metabolismo , Compuestos de Sulfhidrilo/química , Animales , Cromatografía Líquida de Alta Presión , Dinitroclorobenceno/análisis , Dinitroclorobenceno/química , Dinitroclorobenceno/metabolismo , Dinitroclorobenceno/farmacología , Glutatión/química , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Células Hep G2 , Humanos , Hígado/química , Hígado/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Oxidación-Reducción , Especies Reactivas de Oxígeno/química , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masas en Tándem , Tiorredoxinas/antagonistas & inhibidores , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
PURPOSE: Systemic sclerosis (SSc) is characterized by autoimmunity, vasculopathy and fibrosis. Fibrosis is due to an activation of fibroblasts by the transforming growth factor-ß (TGF-ß). This study investigates the proteomic response of SSc fibroblasts to TGF-ß. EXPERIMENTAL DESIGN: Skin fibroblasts from diffuse SSc patients and healthy controls (HC) are cultured with or without TGF-ß. Two-dimensional differential in-gel electrophoresis and mass spectrometry (MS) combined with Ingenuity Pathway analysis (IPA) and Panther/David software analyze proteins differentially expressed between groups. Real-time cell analyzer (RTCA) assesses fibroblast proliferation and viability. RESULTS: Two-hundred-and-seventy-nine proteins are differentially expressed between groups. Principal component analysis shows significant differences between groups. IPA shows specific process networks such as actin cytoskeleton and integrin signaling. Panther and David software show predominant biological processes such as cellular and metabolic processes. TGF-ß enhances protein synthesis and protein pathways. IPA and RTCA suggest the involvement of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3 kinase (Pi3K). CONCLUSIONS AND CLINICAL RELEVANCE: That the proteome of fibroblasts differs between SSc patients and HC is confirmed, and it is demonstrated that fibroblasts exacerbate their proteomic phenotype upon stimulation with TGF-ß. EGFR and Pi3K are highlighted as proteins of interest in SSc fibroblasts.
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Fibroblastos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteómica , Esclerodermia Sistémica/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Receptores ErbB/metabolismo , Femenino , Fibroblastos/patología , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Esclerodermia Sistémica/patología , Electroforesis Bidimensional Diferencial en GelRESUMEN
Mitochondrial reactive oxygen species (ROS) production is a tightly regulated redox signal that transmits information from the organelle to the cell. Other mitochondrial signals, such as ATP, are sensed by enzymes, including the key metabolic sensor and regulator, AMP-activated protein kinase (AMPK). AMPK responds to the cellular ATP/AMP and ATP/ADP ratios by matching mitochondrial ATP production to demand. Previous reports proposed that AMPK activity also responds to ROS, by ROS acting on redox-sensitive cysteine residues (Cys-299/Cys-304) on the AMPK α subunit. This suggests an appealing model in which mitochondria fine-tune AMPK activity by both adenine nucleotide-dependent mechanisms and by redox signals. Here we assessed whether physiological levels of ROS directly alter AMPK activity. To this end we added exogenous hydrogen peroxide (H2O2) to cells and utilized the mitochondria-targeted redox cycler MitoParaquat to generate ROS within mitochondria without disrupting oxidative phosphorylation. Mitochondrial and cytosolic thiol oxidation was assessed by measuring peroxiredoxin dimerization and by redox-sensitive fluorescent proteins. Replacing the putative redox-active cysteine residues on AMPK α1 with alanines did not alter the response of AMPK to H2O2 In parallel with measurements of AMPK activity, we measured the cell ATP/ADP ratio. This allowed us to separate the effects on AMPK activity due to ROS production from those caused by changes in this ratio. We conclude that AMPK activity in response to redox changes is not due to direct action on AMPK itself, but is a secondary consequence of redox effects on other processes, such as mitochondrial ATP production.
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Proteínas Quinasas Activadas por AMP/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Activación Enzimática , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones , Mitocondrias/genética , Fibras Musculares Esqueléticas/enzimología , Fibras Musculares Esqueléticas/metabolismo , Oxidación-ReducciónRESUMEN
TGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer.
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Autofagia/fisiología , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Transporte Biológico/fisiología , Proteínas Portadoras/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo , Fibrosis/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , RatonesRESUMEN
Idiopathic inflammatory myopathies (IIMs) encompass a heterogeneous group of rare autoimmune diseases characterised by muscle weakness and inflammation, but in antisynthetase syndrome arthritis and interstitial lung disease are more frequent and often inaugurate the disease. Clinical practice guidelines (CPGs) have been proposed for IIMs, but they are sparse and heterogeneous. This work aimed at identifying: i) current available CPGs for IIMs, ii) patients ' and clinicians' unmet needs not covered by CPGs. It has been performed in the framework of the European Reference Network on rare and complex connective tissue and musculoskeletal diseases (ReCONNET), a network of centre of expertise and patients funded by the European Union's Health Programme. Fourteen original CPGs were identified, notably recommending that: i) extra-muscular involvements should be assessed; ii) corticosteroids and methotrexate or azathioprine are first-line therapies of IIMs. ii) IVIG is a treatment of resistant-DM that may be also used in other resistant-IIMs; iii) physical therapy and sun protection (in DM patients) are part of the treatment; v) tumour screening for patients with DM include imaging of chest, abdomen, pelvis and breast (in woman) along with colonoscopy (in patients over 50 years); vi) disease activity and damages should be monitor using standardised and validated tools. Yet, only half of these CPGs were evidence-based. Crucial unmet needs were identified both by patients and clinicians. In particular, there was a lack of large multidisciplinary working group and of patients ' preferences. The following fields were not or inappropriately targeted: diagnosis; management of extra-muscular involvements other than skin; co-morbidities and severe manifestations.
RESUMEN
Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2--dependent S-nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2- in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-nitrosation during anoxia alone or by NO2- during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2- exposure.
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Modelos Animales de Enfermedad , Mitocondrias Cardíacas/metabolismo , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Nitritos/metabolismo , Procesamiento Proteico-Postraduccional , Regulación hacia Arriba , Marcadores de Afinidad/metabolismo , Animales , Cardiotónicos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cisteína/metabolismo , Femenino , Corazón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Isquemia Miocárdica/tratamiento farmacológico , Nitratos/farmacología , Nitritos/farmacología , Nitrosación/efectos de los fármacos , Compuestos de Potasio/farmacología , Proteómica/métodos , Ratas Wistar , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Integrins are transmembrane receptors composed of one α subunit and one ß subunit and are involved in cellular growth, differentiation, and apoptosis. The collagen-binding integrins α1ß1 and α2ß1 have been shown to regulate wound and tumor vascularization by different mechanisms. In this study, we assessed wound and tumor vascularization in mice with genetic ablation of both integrin subunits α1 and α2, which resulted in loss of integrins α1ß1 and α2ß1. Wound angiogenesis was investigated in excisional wounds that were inflicted on the back skin of control and mice lacking integrin α1ß1 and α2ß1. Mutant mice displayed reduced wound angiogenesis, which correlated with decreased macrophage numbers at 3 and 7 days after injury, and showed significantly attenuated vascularization of sponge implants. Angiogenesis induced by tumors arising from intradermal injection of B16 F1 melanoma cells was also reduced in comparison to controls 7 days after injection. This reduction in angiogenesis correlated with increased levels and activity of circulating matrix metalloproteinase 9 and elevated angiostatin levels in plasma of mutant mice, which reduced endothelial cell proliferation. Ex vivo mutant aortic ring explants developed significantly fewer and thinner aortic sprouts with fewer branch points than controls because of impaired endothelial cell proliferation. In conclusion, the loss of integrins α1ß1 and α2ß1 in mice results in reduced wound and tumor angiogenesis by cell-autonomous and extrinsic mechanisms.
Asunto(s)
Integrina alfa1beta1/metabolismo , Integrina alfa2beta1/metabolismo , Neoplasias/irrigación sanguínea , Cicatrización de Heridas/fisiología , Heridas y Lesiones/patología , Animales , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Integrina alfa1beta1/genética , Integrina alfa2beta1/genética , Melanoma/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/etiología , Neoplasias/patología , Neovascularización Patológica , Piel/irrigación sanguínea , Piel/lesiones , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/irrigación sanguínea , Heridas y Lesiones/etiologíaRESUMEN
Activation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-ß induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways.
Asunto(s)
Cicatriz/etiología , Colágeno/metabolismo , Péptidos y Proteínas de Señalización Intercelular/fisiología , Macrófagos/metabolismo , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Cicatrización de Heridas/fisiología , Animales , Cicatriz/metabolismo , Cicatriz/patología , Técnicas de Cocultivo , Dermatitis/metabolismo , Dermatitis/patología , Fibroblastos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Interleucinas/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Microfibrillas/metabolismo , Microfibrillas/ultraestructura , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/biosíntesis , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Receptores de Superficie Celular/deficiencia , Esclerodermia Localizada/metabolismo , Esclerodermia Localizada/patología , Piel/lesiones , Piel/metabolismo , Piel/patologíaRESUMEN
Gut ischemia and reperfusion (IR), e.g. in small bowel transplantation or during resuscitation, may result in severe impairment of the intestinal microcirculation. Potential sequelae are mucosal damage, loss of intestinal barrier function, bacterial translocation, systemic inflammation, multiple organ failure and death. We hypothesized a protective role for extracellular adenosine signalling in intestinal IR injury. Using intravital microscopy we investigated the effects of the adenosine receptor (AR) agonist NECA (5'-N-ethyl carboxamide adenosine) on leukocyte-endothelial interactions and capillary perfusion in the intestinal microcirculation following intestinal IR. Six groups of Lewis rats (n = 44) were studied: control, NECA (5'-N-ethyl carboxamide adenosine), IR (30 minutes of intestinal ischemia, 2 hours of reperfusion), IR + NECA, IR + NECA + MRS1754 (A(2B)AR antagonist), IR + NECA + DPCPX (A(1)AR antagonist). All substances were administered i.v. immediately after declamping of the superior mesenteric artery. Intravital microscopy was performed after 2 hours of reperfusion. Following IR we observed a significant increase of leukocyte adhesion in the intestinal submucosal venules and a reduced capillary perfusion within the muscular layers. NECA reduced leukocyte activation and improved capillary perfusion significantly. Administration of A(2B)AR antagonist completely reversed the NECA effect, whereas A(1)AR inhibition only partially abolished the action of NECA. The data support the hypothesis that adenosine signalling is involved in intestinal IR injury. A(2B)AR may be more important than A(1)AR because A(2B)AR inhibition by MRS1754 completely reversed the effect of the adenosine receptor agonist NECA.