RESUMEN
Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.
Asunto(s)
Carcinoma Hepatocelular , Progresión de la Enfermedad , Elasticidad , Matriz Extracelular , Cirrosis Hepática , Neoplasias Hepáticas , Animales , Humanos , beta Catenina/metabolismo , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proliferación Celular , Colágeno/química , Colágeno/metabolismo , Simulación por Computador , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Matriz Extracelular/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Integrina beta1/metabolismo , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Invasividad Neoplásica , Viscosidad , Proteínas Señalizadoras YAP/metabolismo , Cirrosis Hepática/complicaciones , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patologíaRESUMEN
Fibrosis describes a dysregulated tissue remodelling response to persistent cellular injury and is the final pathological consequence of many chronic diseases that affect the liver, kidney and lung. Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (NOX) enzymes produce reactive oxygen species (ROS) as their primary function. ROS derived from NOX1 and NOX4 are key mediators of liver, kidney and lung fibrosis. Setanaxib (GKT137831) is a first-in-class, dual inhibitor of NOX1/4 and is the first NOX inhibitor to progress to clinical trial investigation. The anti-fibrotic effects of setanaxib in liver, kidney and lung fibrosis are supported by multiple lines of pre-clinical evidence. However, despite advances in our understanding, the precise roles of NOX1/4 in fibrosis require further investigation. Additionally, there is a translational gap between the pre-clinical observations of setanaxib to date and the applicability of these to human patients within a clinical setting. This narrative review critically examines the role of NOX1/4 in liver, kidney and lung fibrosis, alongside the available evidence investigating setanaxib as a therapeutic agent in pre-clinical models of disease. We discuss the potential clinical translatability of this pre-clinical evidence, which provides rationale to explore NOX1/4 inhibition by setanaxib across various fibrotic pathologies in clinical trials involving human patients.
Asunto(s)
Fibrosis Pulmonar , Humanos , NADPH Oxidasa 1 , Especies Reactivas de Oxígeno , Fibrosis Pulmonar/patología , Células Estrelladas Hepáticas , Hígado/patología , NADPH Oxidasas , Riñón/patología , NADPH Oxidasa 4RESUMEN
BACKGROUND AND AIMS: Older patients with obesity/type II diabetes mellitus frequently present with advanced NASH. Whether this is due to specific molecular pathways that accelerate fibrosis during aging is unknown. Activation of the Src homology 2 domain-containing collagen-related (Shc) proteins and redox stress have been recognized in aging; however, their link to NASH has not been explored. APPROACH AND RESULTS: Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation, and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. CONCLUSIONS: Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress and accelerated fibrosis in the aged.
Asunto(s)
Envejecimiento/metabolismo , NADPH Oxidasa 2/fisiología , Enfermedad del Hígado Graso no Alcohólico/etiología , Animales , Hepatocitos/metabolismo , Humanos , Cirrosis Hepática/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/antagonistas & inhibidores , Proteínas Adaptadoras de la Señalización Shc/fisiología , Dominios Homologos srcRESUMEN
Shc expression rises in human nonalcoholic steatohepatitis (NASH) livers, and Shc-deficient mice are protected from NASH-thus Shc inhibition could be a novel therapeutic strategy for NASH. Idebenone was recently identified as the first small-molecule Shc inhibitor drug. We tested idebenone in the fibrotic methionine-choline deficient (MCD) diet and the metabolic fast food diet (FFD) mouse models of NASH. In the fibrotic MCD NASH model, idebenone reduced Shc expression and phosphorylation in peripheral blood mononuclear cells and Shc expression in the liver; decreased serum alanine aminotransferase and aspartate aminotransferase; and attenuated liver fibrosis as observed by quantitative polymerase chain reaction (qPCR) and hydroxyproline quantification. In the metabolic FFD model, idebenone administration improved insulin resistance, and reduced inflammation and fibrosis shown with qPCR, hydroxyproline measurement, and histology. Thus, idebenone ameliorates NASH in two mouse models. As an approved drug with a benign safety profile, Idebenone could be a reasonable human NASH therapy.
Asunto(s)
Dieta/efectos adversos , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/etiología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/etiología , Sustancias Protectoras/administración & dosificación , Proteínas Adaptadoras de la Señalización Shc/antagonistas & inhibidores , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de Señal/efectos de los fármacos , Ubiquinona/análogos & derivados , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Deficiencia de Colina/complicaciones , Modelos Animales de Enfermedad , Comida Rápida/efectos adversos , Leucocitos Mononucleares/metabolismo , Hígado/lesiones , Hígado/metabolismo , Cirrosis Hepática/sangre , Cirrosis Hepática/complicaciones , Masculino , Metionina/deficiencia , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Fosforilación/efectos de los fármacos , Terapéutica , Ubiquinona/administración & dosificaciónRESUMEN
BACKGROUND AND AIMS: Cardiovascular disease (CVD) is a leading cause of mortality in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the relationship of leptin-to-adiponectin (L/A) ratio with hepatic steatosis and arterial stiffness in NAFLD. METHODS: The subjects were 871 Japanese adults who participated in a health survey. Dietary intake, body composition, lipid profile, serum interleukin-6 (IL-6), leptin, and adiponectin were analyzed. NAFLD was defined as fatty liver on ultrasonography in the absence of other causes of steatosis. Arterial stiffness was evaluated by the brachial-ankle pulse wave velocity (baPWV). RESULTS: The subjects with NAFLD had a greater body mass index (BMI) and body fat percentage (BFP); a higher intake of daily energy (kcal) and carbohydrates; and a higher prevalence of hypertension, diabetes, and hyperlipidemia. The subjects with NAFLD had higher serum leptin and lower serum adiponectin concentrations and a higher L/A ratio than subjects without NAFLD. The L/A ratio increased with increasing severity of steatosis. The L/A ratio showed positive correlations with BMI and BFP, and a negative correlation with age. Women had higher L/A ratio and BFP levels than men regardless of the presence or absence of NAFLD. There was a weak positive correlation between baPWV and severity of steatosis. BaPWV was strongly correlated with age, while no relation was found between baPWV and L/A ratio. IL-6 level was correlated with baPVW and age, while the correlation between Il and 6 level and L/A ratio was very weak. The L/A ratio was correlated with triglycerides and the ratio of total cholesterol to high-density lipoprotein-cholesterol. CONCLUSION: L/A ratio and arterial stiffness were associated with the severity of steatosis, whereas there was no correlation between L/A ratio and arterial stiffness in NAFLD. These findings suggest that not only leptin and adiponectin but also other factors might be involved in the pathogenesis for atherosclerosis in NAFLD.
Asunto(s)
Adiponectina/sangre , Leptina/sangre , Enfermedad del Hígado Graso no Alcohólico/patología , Rigidez Vascular/fisiología , Índice Tobillo Braquial , Presión Sanguínea/fisiología , Índice de Masa Corporal , Enfermedades Cardiovasculares/mortalidad , Dieta/efectos adversos , Femenino , Humanos , Hipertensión/diagnóstico , Interleucina-6/sangre , Japón , Masculino , Persona de Mediana Edad , Encuestas y CuestionariosRESUMEN
BACKGROUND: An exploratory study was performed to determine the prevalence of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs78409 [G] allele among the Hmong as a risk factor for nonalcoholic fatty liver disease (NAFLD). NAFLD/nonalcoholic steatohepatitis is the world's most common chronic liver disease and is expected to replace viral hepatitis as the leading cause of cirrhosis and potential precursor to hepatocellular carcinoma (HCC). Of all populations in California, the Hmong experience the highest risk of death from HCC and the highest prevalence of metabolic syndrome risk factors among Asians that predispose them to NAFLD. Here a genetic explanation was sought for the high rates of chronic liver disease among the Hmong. The literature pointed to the PNPLA3 rs738409 [G] allele as a potential genetic culprit. METHODS: Cell-free DNA was isolated from 26 serum samples previously collected in community settings. Quantitative polymerase chain reaction-based single-nucleotide polymorphism (SNP) genotyping was performed with a validated TaqMan SNP genotyping assay, and results were analyzed with TaqMan Genotyper software. RESULTS: The PNPLA3 rs738409 [C>G] variant occurred at a frequency of 0.46 (12 of 26; 95% confidence interval, 0.27-0.67). This carrier rate would rank the Hmong as the third highest population in the 1000 Genomes Project. CONCLUSIONS: Although this small sample size limits the generalizability, the high frequency rates of this allele along with the presence of metabolic syndrome risk factors warrant further studies into the etiology of NAFLD among the Hmong. Cancer 2018;124:1583-9. © 2018 American Cancer Society.
Asunto(s)
Asiático/genética , Predisposición Genética a la Enfermedad , Lipasa/genética , Cirrosis Hepática/genética , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , Adulto , Anciano , California/epidemiología , Enfermedad Crónica , Femenino , Estudios de Seguimiento , Genotipo , Humanos , Incidencia , Cirrosis Hepática/epidemiología , Masculino , Persona de Mediana Edad , Pronóstico , Adulto JovenRESUMEN
Macrophage activation is an important feature of primary biliary cholangitis (PBC) pathogenesis and other cholestatic liver diseases. Galectin-3 (Gal3), a pleiotropic lectin, is produced by monocytic cells and macrophages. However, its role in PBC has not been addressed. We hypothesized that Gal3 is a key to induce NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in macrophages and in turn to propagate proinflammatory IL-17 signaling. In liver tissues from patients with PBC and dnTGF-ßRII mice, a model of autoimmune cholangitis, the expression of Gal3, NLRP3, and the adaptor protein adaptor apoptosis-associated speck-like protein was induced, with the downstream activation of caspase-1 and IL-1ß. In wild-type hepatic macrophages, deoxycholic acid induced the association of Gal3 and NLRP3 with direct activation of the inflammasome, resulting in an increase in IL-1ß. Downstream retinoid-related orphan receptor C mRNA, IL-17A, and IL-17F were induced. In Gal3-/- macrophages, no inflammasome activation was detected. To confirm the key role of Gal3 in the pathogenesis of cholestatic liver injury, we generated dnTGF-ßRII/galectin-3-/- (dn/Gal3-/-) mice, which showed impaired inflammasome activation along with significantly improved inflammation and fibrosis. Taken together, our data point to a novel role of Gal3 as an initiator of inflammatory signaling in autoimmune cholangitis, mediating the activation of NLRP3 inflammasome and inducing IL-17 proinflammatory cascades. These studies provide a rationale to target Gal3 in autoimmune cholangitis and potentially other cholestatic diseases.-Tian, J., Yang, G., Chen, H.-Y., Hsu, D. K., Tomilov, A., Olson, K. A., Dehnad, A., Fish, S. R., Cortopassi, G., Zhao, B., Liu, F.-T., Gershwin, M. E., Török, N. J., Jiang, J. X. Galectin-3 regulates inflammasome activation in cholestatic liver injury.
Asunto(s)
Galectina 3/metabolismo , Inflamasomas/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Transducción de Señal/fisiología , Animales , Caspasa 1/metabolismo , Células Cultivadas , Galectina 3/genética , Humanos , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Hígado/lesiones , Activación de Macrófagos/fisiología , Ratones Transgénicos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Reactive oxygen species are implicated in physiological signaling and cell fate decisions. In chronic liver diseases persistent and increased production of oxidative radicals drives a fibrogenic response that is a common feature of disease progression. Despite our understanding the biology of the main prooxidant enzymes, their targets, and antioxidant mechanisms in the liver, there is still lack of knowledge concerning their precise role in the pathogenesis of fibrosis. This review will examine the role of physiological redox signaling in the liver, provide an overview on recent advances in prooxidant and antioxidant pathways that are dysregulated during fibrosis, and highlight possible novel treatment targets.
Asunto(s)
Cirrosis Hepática/metabolismo , Hígado/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Estrelladas Hepáticas/metabolismo , Humanos , Oxidación-Reducción , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND & AIMS: Reactive oxidative species (ROS) are believed to be involved in the progression of nonalcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis. METHODS: Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4(hepKO)) and NOX4(floxp+/+) C57BL/6 mice (controls) were given fast-food diets (supplemented with high-fructose corn syrup) or choline-deficient l-amino acid defined diets to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase, and phospho-eIF-2α kinase-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. RESULTS: Levels of NOX4 were increased in patients with NASH compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of double-stranded RNA-activated protein kinase and phosphorylation of extracellular signal-regulated kinase-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831 had increased insulin sensitivity. CONCLUSIONS: NOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.
Asunto(s)
Hígado Graso/tratamiento farmacológico , Hepatocitos/efectos de los fármacos , Resistencia a la Insulina , Cirrosis Hepática/tratamiento farmacológico , NADPH Oxidasas/metabolismo , NADP/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Biopsia , Dieta/métodos , Modelos Animales de Enfermedad , Hígado Graso/inducido químicamente , Hígado Graso/genética , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Hígado/citología , Hígado/patología , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADP/administración & dosificación , NADPH Oxidasa 4 , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Proteína Fosfatasa 1/metabolismo , Pirazoles/metabolismo , Pirazolonas , Piridinas/metabolismo , Piridonas , Estrés Fisiológico/efectos de los fármacosRESUMEN
There is an urgent need to develop antifibrotic therapies for chronic liver disease, and clarify which endpoints in antifibrotic trials will be acceptable to regulatory agencies. The American Association for the Study of Liver Diseases sponsored an endpoints conference to help accelerate the efficient testing of antifibrotic agents and develop recommendations on clinical trial design for liver fibrosis. In this review, we summarize the salient and novel elements of this conference and provide directions for future clinical trial design. The article follows the structure of the conference and is organized into five areas: (1) antifibrotic trial design; (2) preclinical proof-of-concept studies; (3) pharmacological targets, including rationale and lessons to learn; (4) rational drug design and development; and (5) consensus and recommendations on design of clinical trials in liver fibrosis. Expert overviews and collaborative discussions helped to summarize the key unmet needs and directions for the future, including: (1) greater clarification of at-risk populations and study groups; (2) standardization of all elements of drug discovery and testing; (3) standardization of clinical trial approaches; (4) accelerated development of improved noninvasive markers; and (5) need for exploration of potential off-target toxicities of future antifibrotic drugs.
Asunto(s)
Ensayos Clínicos como Asunto , Cirrosis Hepática/tratamiento farmacológico , Medicamentos bajo Prescripción/farmacología , Chicago , Congresos como Asunto , Diseño de Fármacos , Femenino , Humanos , Cirrosis Hepática/patología , Masculino , Guías de Práctica Clínica como Asunto , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Hepatocellular carcinoma (HCC) carries a poor prognosis with no effective treatment available other than liver transplantation for selected patients. Vascular invasion of HCC is one of the most important negative predictor of survival. As the regulation of invasion of HCC cells is not well understood, our aim was to study the mechanisms by which galectin 3, a ß-galactosidase-binding lectin mediates HCC cell migration. HCC was induced by N-diethylnitrosamine in wild-type and galectin 3(-/-) mice, and tumor formation, histology, and tumor cell invasion were assessed. The galectin 3(-/-) mice developed significantly smaller tumor burden with a less invasive phenotype than the wild-type animals. Galectin 3 was upregulated in the wild-type HCC tumor tissue, but not in the surrounding parenchyma. Galectin 3 expression in HCC was induced by NF-κB transactivation as determined by chromatin immunoprecipitation assays. In vitro studies assessed the pro-migratory effects of galectin 3. The migration of hepatoma cells was significantly decreased after transfection by the galectin 3 siRNA and also after using the Rho kinase inhibitor Y-27632. The reorganization of the actin cytoskeleton, RhoA GTPase activity and the phosphorylation of MLC2 (myosin light chain 2) were decreased in the galectin 3 siRNA-transfected cells. In addition, in vitro and in vivo evidence showed that galectin 3 deficiency reduced hepatoma cell proliferation and increased their apoptosis rate. In conclusion, galectin 3 is an important lectin that is induced in HCC cells, and promotes hepatoma cell motility and invasion by an autocrine pathway. Targeting galectin 3 therefore could be an important novel treatment strategy to halt disease progression.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Galectina 3/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Miosinas Cardíacas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Galectina 3/antagonistas & inhibidores , Galectina 3/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/química , Invasividad Neoplásica , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/agonistas , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
UNLABELLED: Advanced glycation endproducts (AGEs) accumulate in patients with diabetes, yet the link between AGEs and inflammatory and fibrogenic activity in nonalcoholic steatohepatitis (NASH) has not been explored. Tumor necrosis factor alpha (TNF-α)-converting enzyme (TACE) is at the center of inflammatory processes. Because the main natural regulator of TACE activity is the tissue inhibitor of metalloproteinase 3 (Timp3), we hypothesized that AGEs induce TACE through nicotinamide adenine dinucleotide phosphate reduced oxidase 2 (NOX2); and the down-regulation of Sirtuin 1 (Sirt1)/Timp3 pathways mediate fibrogenic activity in NASH. The role of NOX2, Sirt1, Timp3, and TACE was evaluated in choline-deficient L-amino acid defined (CDAA) or Western diet (WD)-fed wild-type (WT) and NOX2(-/-) mice. To restore Timp3, mice were injected with adenovirus (Ad)-Timp3. Sirt1 and Timp3 expressions were studied in livers from NASH patients, and we found that their levels were significantly lower than in healthy controls. In WT mice on the CDAA or WD, Sirt1 and Timp3 expressions were lower, whereas production of reactive oxidative species and TACE activity significantly increased with an increase in active TNF-α production as well as induction of fibrogenic transcripts. Ad-Timp3 injection resulted in a significant decline in TACE activity, procollagen α1 (I), alpha smooth muscle actin (α-SMA) and transforming growth factor beta (TGF-ß) expression. NOX2(-/-) mice on the CDAA or WD had no significant change in Sirt1, Timp3, and TACE activity or the fibrosis markers assessed. In vitro, AGE exposure decreased Sirt1 and Timp3 in hepatic stellate cells by a NOX2-dependent pathway, and TACE was induced after exposure to AGEs. CONCLUSION: TACE activation is central to the pathogenesis of NASH and is mediated by AGEs through NOX2 induction and down-regulation of Sirt1/Timp3 pathways.
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Proteínas ADAM/metabolismo , Hígado Graso/complicaciones , Hígado Graso/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Proteína ADAM17 , Actinas/metabolismo , Animales , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Productos Finales de Glicación Avanzada/farmacología , Humanos , Técnicas In Vitro , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Enfermedad del Hígado Graso no Alcohólico , Ratas Sprague-Dawley , Transducción de Señal/inmunología , Sirtuina 1/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
UNLABELLED: Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson's disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels. CONCLUSION: Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD.
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Metilación de ADN/efectos de los fármacos , Hígado/metabolismo , Metionina/metabolismo , Adenosilhomocisteinasa/antagonistas & inhibidores , Adenosilhomocisteinasa/metabolismo , Animales , Betaína/metabolismo , Betaína/farmacología , Cobre/metabolismo , Cobre/farmacología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Estrés del Retículo Endoplásmico , Epigénesis Genética/efectos de los fármacos , Degeneración Hepatolenticular/metabolismo , Degeneración Hepatolenticular/patología , Inflamación/metabolismo , Ratones , Ratones Endogámicos C3H , Penicilamina/farmacología , S-Adenosilhomocisteína/metabolismo , ADN Metiltransferasa 3BRESUMEN
Metabolic dysfunction-associated liver disease (MASLD) and steatohepatitis (MASH) are becoming the most common causes of chronic liver disease in the United States and worldwide due to the obesity and diabetes epidemics. It is estimated that by 2030 close to 100 million people might be affected and patients with type 2 diabetes are especially at high risk. Twenty to 30% of patients with MASLD can progress to MASH, which is characterized by steatosis, necroinflammation, hepatocyte ballooning, and in advanced cases, fibrosis progressing to cirrhosis. Clinically, it is recognized that disease progression in diabetic patients is accelerated and the role of various genetic and epigenetic factors, as well as cell-matrix interactions in fibrosis and stromal remodeling, have recently been recognized. While there has been great progress in drug development and clinical trials for MASLD/MASH, the complexity of these pathways highlights the need to improve diagnosis/early detection and develop more successful antifibrotic therapies that not only prevent but reverse fibrosis.
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Diabetes Mellitus Tipo 2 , Cirrosis Hepática , Humanos , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Progresión de la Enfermedad , Animales , Matriz Extracelular/metabolismo , Hígado Graso/metabolismo , Hígado Graso/etiologíaRESUMEN
BACKGROUND & AIMS: Src homology and collagen (Shc) proteins are major adapters to extracellular signals, however, the regulatory role of Shc isoforms in sterile inflammatory responses in alcoholic hepatitis (AH) has not been fully investigated. We hypothesized that in an isoform-specific manner Shc modulates pre-apoptotic signals, calreticulin (CRT) membrane exposure, and recruitment of inflammatory cells. METHODS: Liver biopsy samples from patients with AH vs healthy subjects were studied for Shc expression using DNA microarray data and immunohistochemistry. Shc knockdown (hypomorph) and age-matched wild-type mice were pair-fed according to the chronic-plus-binge alcohol diet. To analyze hepatocyte-specific effects, adeno-associated virus 8-thyroxine binding globulin-Cre (hepatocyte-specific Shc knockout)-mediated deletion was performed in flox/flox Shc mice. Lipid peroxidation, proinflammatory signals, redox radicals, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio, as well as cleaved caspase 8, B-cell-receptor-associated protein 31 (BAP31), Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist killer (Bak), were assessed in vivo. CRT translocation was studied in ethanol-exposed p46ShcáºSH2-transfected hepatocytes by membrane biotinylation in conjunction with phosphorylated-eukaryotic initiation factor 2 alpha, BAP31, caspase 8, and Bax/Bak. The effects of idebenone, a novel Shc inhibitor, was studied in alcohol/pair-fed mice. RESULTS: Shc was significantly induced in patients with AH (P < .01). Alanine aminotransferase, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratios, production of redox radicals, and lipid peroxidation improved (P < .05), and interleukin 1ß, monocyte chemoattractant protein 1, and C-X-C chemokine ligand 10 were reduced in Shc knockdown and hepatocyte-specific Shc knockout mice. In vivo, Shc-dependent induction, and, in hepatocytes, a p46Shc-dependent increase in pre-apoptotic proteins Bax/Bak, caspase 8, BAP31 cleavage, and membrane translocation of CRT/endoplasmic reticulum-resident protein 57 were seen. Idebenone protected against alcohol-mediated liver injury. CONCLUSIONS: Alcohol induces p46Shc-dependent activation of pre-apoptotic pathways and translocation of CRT to the membrane, where it acts as a damage-associated molecular pattern, instigating immunogenicity. Shc inhibition could be a novel treatment strategy in AH.
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Hepatitis Alcohólica , Ratones , Animales , Proteína X Asociada a bcl-2 , Caspasa 8 , Calreticulina , NAD , Ratones Noqueados , Etanol , Inflamación , ColágenoRESUMEN
Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a ß-galactoside-binding lectin, is a regulator of the phagocytic process. In this study, our aim was to study the mechanism by which extracellular galectin-3 modulates HSC phagocytosis and activation. The role of galectin-3 in engulfment was evaluated by phagocytosis and integrin binding assays in primary HSC. Galectin-3 expression was studied by real-time PCR and enzyme-linked immunosorbent assay, and in vivo studies were done in wild-type and galectin-3(-/-) mice. We found that HSC from galectin-3(-/-) mice displayed decreased phagocytic activity, expression of transforming growth factor-ß1, and procollagen α1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the α(v)ß(3) heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin α(v)ß(3) resulted in decreased phagocytosis. Galectin-3 expression and release were induced in active HSC engulfing apoptotic cells, and this was mediated by the nuclear factor-κB signaling. The upregulation of galectin-3 in active HSC was further confirmed in vivo in bile duct-ligated (BDL) rats. Galectin-3(-/-) mice displayed significantly decreased fibrosis, with reduced expression of α-smooth muscle actin and procollagen α1(I) following BDL. In summary, extracellular galectin-3 plays a key role in liver fibrosis by mediating HSC phagocytosis, activation, and subsequent autocrine and paracrine signaling by a feedforward mechanism.
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Galectina 3/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Fagocitosis/fisiología , Animales , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Galectina 3/genética , Células Estrelladas Hepáticas/patología , Hígado/patología , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal/genéticaRESUMEN
UNLABELLED: Among multiple isoforms of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase expressed in the liver, the phagocytic NOX2 isoform in hepatic stellate cells (HSCs) has been demonstrated to play a key role in liver fibrogenesis. The aim of this study was to clarify the role of NOX1, a nonphagocytic form of NADPH oxidase, in the development of fibrosis using Nox1-deficient mice (Nox1KO). Liver injury and fibrosis were induced by bile duct ligation (BDL) and carbon tetrachloride in Nox1KO and wildtype littermate mice (WT). Primary HSCs were isolated to characterize the NOX1-induced signaling cascade involved in liver fibrogenesis. Following BDL, a time-dependent increase in NOX1 messenger RNA (mRNA) was demonstrated in WT liver. Compared with those in WT, levels of collagen-1α mRNA and hydroxyproline were significantly suppressed in Nox1KO with a reduced number of activated HSCs and less severe fibrotic lesions. The expression levels of α-smooth muscle actin, a marker of HSCs activation, were similar in cultured HSCs isolated from both genotypes. However, cell proliferation was significantly attenuated in HSCs isolated from Nox1KO. In these cells, the expression of p27(kip1) , a cell cycle suppressor, was significantly up-regulated. Concomitantly, a significant reduction in phosphorylated forms of Akt and forkhead box O (FOXO) 4, a downstream effector of Akt that regulates the transcription of p27(kip1) gene, was demonstrated in Nox1KO. Finally, the level of the oxidized inactivated form of phosphatase and tensin homolog (PTEN), a negative regulator of PI3K/Akt pathway, was significantly attenuated in HSCs of Nox1KO. CONCLUSION: These findings indicate that reactive oxygen species derived from NOX1/NADPH oxidase oxidize and inactivate PTEN to positively regulate the Akt/FOXO4/p27(kip1) signaling pathway. NOX1 may thus promote proliferation of HSCs and accelerate the development of fibrosis following BDL-induced liver injury.
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Proliferación Celular , Células Estrelladas Hepáticas/patología , Cirrosis Hepática Experimental/etiología , NADH NADPH Oxidorreductasas/fisiología , NADP/fisiología , Animales , Tetracloruro de Carbono/toxicidad , Proteínas de Ciclo Celular , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Factores de Transcripción Forkhead/fisiología , Ligadura , Cirrosis Hepática Experimental/patología , Ratones , NADPH Oxidasa 1 , Fosfohidrolasa PTEN/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called "chicken wire," which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson's trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis. The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.