RESUMEN
Chronic pancreatitis increases the risk of developing pancreatic cancer through the upregulation of pathways favouring proliferation, fibrosis, and sustained inflammation. We established in previous studies that the ligand tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) signals through its cognate receptor fibroblast growth factor-inducible 14 (Fn14) to regulate these underlying cellular processes in the chronic liver injury niche. However, the role of the TWEAK/Fn14 signalling pathway in pancreatic disease is entirely unknown. An analysis of publicly available datasets demonstrated that the TWEAK receptor Fn14 is upregulated in pancreatitis and pancreatic adenocarcinoma, with single cell RNA sequencing revealing pancreatic ductal cells as the main Fn14 producers. We then used choline-deficient, ethionine-supplemented (CDE) diet feeding of wildtype C57BL/6J and Fn14 knockout littermates to (a) confirm CDE treatment as a suitable model of chronic pancreatitis and (b) to investigate the role of the TWEAK/Fn14 signalling pathway in pancreatic ductal proliferation, as well as fibrotic and inflammatory cell dynamics. Our time course data obtained at three days, three months, and six months of CDE treatment reveal that a lack of TWEAK/Fn14 signalling significantly inhibits the establishment and progression of the tissue microenvironment in CDE-induced chronic pancreatitis, thus proposing the TWEAK/Fn14 pathway as a novel therapeutic target.
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BACKGROUND AND AIM: Dietary fat intake has long been associated with fatty liver. Our study aimed to determine the effect of dietary fats on longitudinal fatty liver index (FLI) trajectories from adolescence to young adulthood. METHODS: Nine hundred eighty-five participants in the Raine Study, Perth, Western Australia, Australia, had cross-sectional assessments at ages 14, 17, 20 and 22 years, during which anthropometric measurements and blood tests were obtained. FLI trajectories were derived from the longitudinal FLI results. Dietary fat intake was measured with a semi-quantitative food frequency questionnaire at 14 years and log multinominal regression analyses were used to estimate relative risks. RESULTS: Three FLI trajectories were identified and labelled as stable-low (79.1%, N = 782), low-to-high (13.9%, N = 132), and stable-high (7%, N = 71). The low-to-high group associated with an increased intake of the long-chain polyunsaturated fatty acids EPA, DPA and DHA (RR 1.27, 95% CI 1.10-1.48) relative to the stable-low group. Compared to the stable-low group, omega-6 and the ratio of omega-6 to omega-3 in the stable-high group were associated with an increased relative risk of 1.34 (95% CI 1.02-1.76) and 1.10 (95% CI 1.03-1.16), respectively. CONCLUSION: For those at high risk of fatty liver in early adolescence, high omega-6 fatty acid intake and a high ratio of omega-6 to omega-3 fatty acids are associated with increased risk of fatty liver. There should be caution in assuming these associations are causal due to possible undetected and underestimated confounding factors.
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Ácidos Grasos Omega-3 , Hígado Graso , Hepatopatías , Adolescente , Humanos , Adulto Joven , Adulto , Estudios de Seguimiento , Estudios Transversales , Grasas de la Dieta , Ácidos Grasos , Ácidos Grasos Omega-6 , Hígado Graso/epidemiologíaRESUMEN
Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.
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Deficiencia de Colina/etiología , Modelos Animales de Enfermedad , Etionina/administración & dosificación , Lesión Pulmonar/etiología , Animales , Proliferación Celular/fisiología , Deficiencia de Colina/metabolismo , Dieta , Suplementos Dietéticos , Hígado/patología , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Parenteral iron is integral in the treatment of anaemia of chronic kidney disease patients on haemodialysis (HD). However, increased liver iron concentration (LIC) can result from such treatment, and this correlates poorly with serum ferritin or transferrin saturation values. It is unclear whether increased cardiac iron concentration also occurs in this setting. We aimed to evaluate the relationship of intravenous iron supplementation to hepatic and cardiac iron deposition in chronic HD subjects. METHODS: A cohort of 10 patients on chronic HD for at least 1 year underwent MRI-based quantitation of hepatic and cardiac iron content to evaluate the relationship between intravenous iron supplements and hepatic and cardiac iron deposition. The results were compared against the cumulative parenteral iron dose and serum iron markers. RESULTS: The median age was 61 years (95% confidence interval (CI) 50-71), HD time 2.5 years (95%CI 2.0-5.3) and cumulative iron dose 4300 mg (95%CI 2110-9045). Hepatic iron concentration was elevated in eight of 10 subjects (median 46 mmol/kg, range 31-76). Cardiac iron levels were within the reference range in all subjects. There was poor correlation between conventional haematinic values and either LIC or cardiac iron levels. None of the study subjects exhibited elevated cardiac iron concentration. CONCLUSION: Whilst HD patients receiving standard parenteral iron therapy have elevated LICs, this is not associated with cardiac iron deposition. Transferrin saturation and serum ferritin levels are poor markers of either liver or cardiac iron deposition in HD subjects.
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Anemia/tratamiento farmacológico , Compuestos Férricos/administración & dosificación , Hematínicos/administración & dosificación , Hígado/metabolismo , Miocardio/metabolismo , Diálisis Renal/efectos adversos , Insuficiencia Renal Crónica/terapia , Administración Intravenosa , Anciano , Anemia/sangre , Anemia/diagnóstico , Anemia/etiología , Biomarcadores/sangre , Femenino , Compuestos Férricos/efectos adversos , Compuestos Férricos/metabolismo , Ferritinas/sangre , Hematínicos/efectos adversos , Hematínicos/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Proyectos Piloto , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/diagnóstico , Factores de Tiempo , Distribución Tisular , Transferrina/metabolismo , Resultado del TratamientoRESUMEN
Complications of end-stage chronic liver disease signify a major cause of mortality worldwide. Irrespective of the underlying cause, most chronic liver diseases are characterized by hepatocellular necrosis, inflammation, fibrosis, and proliferation of liver progenitor cells or ductular reactions. Vast differences exist between experimental models that mimic these processes, and their identification is fundamental for translational research. We compared two common murine models of chronic liver disease: the choline-deficient, ethionine-supplemented (CDE) diet versus thioacetamide (TAA) supplementation. Markers of liver injury, including serum alanine transaminase levels, apoptosis, hepatic fat loading, and oxidative stress, as well as inflammatory, fibrogenic and liver progenitor cell responses, were assessed at days 3, 7, 14, 21, and 42. This study revealed remarkable differences between the models. It identified periportal injury and fibrosis with an early peak and slow normalization of all parameters in the CDE regimen, whereas TAA-treated mice had pericentral patterns of progressive injury and fibrosis, resulting in a more severe hepatic injury phenotype. This study is the first to resolve two different patterns of injury and fibrosis in the CDE and TAA model and to indisputably identify the fibrosis pattern in the TAA model as driven from the pericentral vein region. Our data provide a valuable foundation for future work using the CDE and TAA regimens to model a variety of human chronic liver diseases.
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Modelos Animales de Enfermedad , Hepatocitos/fisiología , Hepatopatías/fisiopatología , Células Madre/fisiología , Animales , Enfermedad Crónica , Hepatocitos/patología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Madre/patologíaRESUMEN
UNLABELLED: Liver progenitor cells (LPCs) are necessary for repair in chronic liver disease because the remaining hepatocytes cannot replicate. However, LPC numbers also correlate with disease severity and hepatocellular carcinoma risk. Thus, the progenitor cell response in diseased liver may be regulated to optimize liver regeneration and minimize the likelihood of tumorigenesis. How this is achieved is currently unknown. Human and mouse diseased liver contain two subpopulations of macrophages with different ontogenetic origins: prenatal yolk sac-derived Kupffer cells and peripheral blood monocyte-derived macrophages. We examined the individual role(s) of Kupffer cells and monocyte-derived macrophages in the induction of LPC proliferation using clodronate liposome deletion of Kupffer cells and adoptive transfer of monocytes, respectively, in the choline-deficient, ethionine-supplemented diet model of liver injury and regeneration. Clodronate liposome treatment reduced initial liver monocyte numbers together with the induction of injury and LPC proliferation. Adoptive transfer of monocytes increased the induction of liver injury, LPC proliferation, and tumor necrosis factor-α production. CONCLUSION: Kupffer cells control the initial accumulation of monocyte-derived macrophages. These infiltrating monocytes are in turn responsible for the induction of liver injury, the increase in tumor necrosis factor-α, and the subsequent proliferation of LPCs.
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Comunicación Celular/fisiología , Macrófagos del Hígado/fisiología , Regeneración Hepática/fisiología , Hígado/citología , Monocitos/fisiología , Células Madre/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) and serum 25-hydroxyvitamin D (s25[OH]D) concentrations are both associated with adiposity and insulin resistance (IR) and thus may be pathogenically linked. We aimed to determine the prevalence of vitamin D deficiency in adolescents with NAFLD and to investigate the prospective and cross-sectional associations between s25[OH]D concentrations and NAFLD. METHODS: Participants in the population-based West Australian Pregnancy (Raine) Cohort had seasonally adjusted s25(OH)D concentrations determined at ages 14 and then 17 years. NAFLD was diagnosed at 17 years using liver ultrasonography. Associations were examined after adjusting for potential confounders. Odds ratios (ORs) and confidence intervals (CIs) are reported per standard deviation in s25(OH)D concentrations. RESULTS: NAFLD was present in 16% (156/994) of adolescents. The majority of participants with NAFLD had either insufficient (51%) or deficient (17%) vitamin D status. s25(OH)D concentrations at 17 years were inversely associated with risk of NAFLD (OR 0.74, 95% CI 0.56, 0.97; P = 0.029), after adjusting for sex, race, physical activity, television/computer viewing, body mass index, and IR. The effect of s25(OH)D concentrations at 17 years was minimally affected after further adjusting for s25(OH)D concentrations at 14 years (OR 0.76, 95% CI 0.56, 1.03; P = 0.072). CONCLUSIONS: Lower s25(OH)D concentrations are significantly associated with NAFLD, independent of adiposity and IR. Screening for vitamin D deficiency in adolescents at risk of NAFLD is appropriate, and clinical trials investigating the effect of vitamin D supplementation in the prevention and treatment of NAFLD may be warranted.
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Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/etiología , Deficiencia de Vitamina D/complicaciones , Vitamina D/análogos & derivados , Adiposidad , Adolescente , Biomarcadores/sangre , Estudios de Cohortes , Estudios Transversales , Femenino , Humanos , Resistencia a la Insulina , Masculino , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Prevalencia , Estudios Prospectivos , Riesgo , Vitamina D/administración & dosificación , Vitamina D/sangre , Deficiencia de Vitamina D/diagnóstico , Deficiencia de Vitamina D/epidemiologíaRESUMEN
Chronic intestinal inflammation and high dietary iron are associated with colorectal cancer development. The role of Stat3 activation in iron-induced colonic inflammation and tumorigenesis was investigated in a mouse model of inflammation-associated colorectal cancer. Mice, fed either an iron-supplemented or control diet, were treated with azoxymethane and dextran sodium sulfate (DSS). Intestinal inflammation and tumor development were assessed by endoscopy and histology, gene expression by real-time PCR, Stat3 phosphorylation by immunoblot, cytokines by ELISA and apoptosis by TUNEL assay. Colonic inflammation was more severe in mice fed an iron-supplemented compared with a control diet one week post-DSS treatment, with enhanced colonic IL-6 and IL-11 release and Stat3 phosphorylation. Both IL-6 and ferritin, the iron storage protein, co-localized with macrophages suggesting iron may act directly on IL-6 producing-macrophages. Iron increased DSS-induced colonic epithelial cell proliferation and apoptosis consistent with enhanced mucosal damage. DSS-treated mice developed anemia that was not alleviated by dietary iron supplementation. Six weeks post-DSS treatment, iron-supplemented mice developed more and larger colonic tumors compared with control mice. Intratumoral IL-6 and IL-11 expression increased in DSS-treated mice and IL-6, and possibly IL-11, were enhanced by dietary iron. Gene expression of iron importers, divalent metal transporter 1 and transferrin receptor 1, increased and iron exporter, ferroportin, decreased in colonic tumors suggesting increased iron uptake. Dietary iron and colonic inflammation synergistically activated colonic IL-6/IL-11-Stat3 signaling promoting tumorigenesis. Oral iron therapy may be detrimental in inflammatory bowel disease since it may exacerbate colonic inflammation and increase colorectal cancer risk.
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Colitis/metabolismo , Neoplasias del Colon/metabolismo , Interleucina-11/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Proliferación Celular/efectos de los fármacos , Colitis/inducido químicamente , Colitis/genética , Neoplasias del Colon/genética , Sulfato de Dextran/toxicidad , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica , Immunoblotting , Etiquetado Corte-Fin in Situ , Interleucina-11/genética , Interleucina-6/genética , Hierro de la Dieta/efectos adversos , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients.
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Química Encefálica/genética , Antígenos de Histocompatibilidad Clase I/fisiología , Sobrecarga de Hierro/genética , Proteínas de la Membrana/fisiología , Transcriptoma/genética , Animales , Suplementos Dietéticos , Hemocromatosis/genética , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Hierro/metabolismo , Hierro de la Dieta/farmacología , Hígado/metabolismo , Masculino , Proteínas de la Membrana/genética , Memoria/fisiología , Ratones , Ratones Noqueados , Análisis por Micromatrices , Trastornos del Movimiento/genética , Plasticidad Neuronal/genética , Proteínas de Hierro no Heme/sangre , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Elementos Reguladores de la Transcripción/genética , Transmisión Sináptica/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Iron (Fe) overload may complicate parenteral Fe therapy used to enhance the efficacy of erythropoietic-stimulating agents in the treatment of anemia of chronic kidney disease. However, serum Fe markers are influenced by inflammation or malignancy and may not accurately reflect the amount of body Fe. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We studied the relationship between parenteral Fe therapy, conventional serum Fe markers, and liver iron concentration (LIC) measured using magnetic resonance R2 relaxometry (FerriScan) in 25 Fe-deficient predialysis chronic kidney disease patients before and 2 and 12 weeks after single high-dose intravenous Fe and in 15 chronic hemodialysis patients with elevated serum ferritin (>500 µg/L). RESULTS: In predialysis patients, there was strong dose dependency between the administered Fe dose and changes in LIC at weeks 2 and 12; however, no dose dependency between Fe dose and changes in ferritin or transferrin saturation (TSAT) were observed. In hemodialysis patients, LIC correlated with the cumulative Fe dose and duration of dialysis but not with current ferritin or TSAT. The cumulative Fe dose remained a significant independent predictor of LIC in a multiple regression model. Two dialysis patients who received >6 g parenteral Fe had substantially elevated LIC >130 µmol/g, which is associated with hemochromatosis. CONCLUSIONS: In Fe-deficient predialysis patients, intravenous Fe therapy is associated with increases in LIC unrelated to changes in conventional Fe markers. In hemodialysis patients, TSAT and ferritin are poor indicators of body Fe load, and some patients have LICs similar to those found in hemochromatosis.
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Ferritinas/sangre , Hierro/uso terapéutico , Enfermedades Renales/sangre , Transferrina/análisis , Adulto , Anciano , Enfermedad Crónica , Estudios Transversales , Femenino , Hemoglobinas/análisis , Humanos , Deficiencias de Hierro , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Diálisis RenalRESUMEN
UNLABELLED: Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte-mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemochromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is promitotic for LPCs; whether it acts directly is not known. In our murine choline-deficient, ethionine-supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor-inducible 14 (Fn14)] expression in the whole liver is massively upregulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE-induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2-week-CDE-treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin-positive cells. Stimulation of an Fn14-positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NFkappaB) activation and dose-dependent proliferation, which was diminished after targeting of the p50 NFkappaB subunit by RNA interference. CONCLUSION: TWEAK acts directly and stimulates LPC mitosis in an Fn14-dependent and NFkappaB-dependent fashion, and signaling via this pathway mediates the LPC response to CDE-induced injury and regeneration.
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Regeneración Hepática , Mitosis , Células Madre/fisiología , Factores de Necrosis Tumoral/fisiología , Animales , Proliferación Celular , Deficiencia de Colina/metabolismo , Colágeno/metabolismo , Citocina TWEAK , Etionina/farmacología , Hígado/efectos de los fármacos , Hígado/lesiones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitógenos/farmacología , FN-kappa B/agonistas , FN-kappa B/genética , FN-kappa B/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Proteínas Recombinantes/farmacología , Células Madre/efectos de los fármacos , Receptor de TWEAK , Factores de Necrosis Tumoral/farmacologíaRESUMEN
UNLABELLED: Lymphotoxin-beta (LTbeta) is a proinflammatory cytokine and a member of the tumor necrosis factor (TNF) superfamily known for its role in mediating lymph node development and homeostasis. Our recent studies suggest a role for LTbeta in mediating the pathogenesis of human chronic liver disease. We hypothesize that LTbeta co-ordinates the wound healing response in liver injury via direct effects on hepatic stellate cells. This study used the choline-deficient, ethionine-supplemented (CDE) dietary model of chronic liver injury, which induces inflammation, liver progenitor cell proliferation, and portal fibrosis, to assess (1) the cellular expression of LTbeta, and (2) the role of LTbeta receptor (LTbetaR) in mediating wound healing, in LTbetaR(-/-) versus wild-type mice. In addition, primary isolates of hepatic stellate cells were treated with LTbetaR-ligands LTbeta and LTbeta-related inducible ligand competing for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT), and mediators of hepatic stellate cell function and fibrogenesis were assessed. LTbeta was localized to progenitor cells immediately adjacent to activated hepatic stellate cells in the periportal region of the liver in wild-type mice fed the CDE diet. LTbetaR(-/-) mice fed the CDE diet showed significantly reduced fibrosis and a dysregulated immune response. LTbetaR was demonstrated on isolated hepatic stellate cells, which when stimulated by LTbeta and LIGHT, activated the nuclear factor kappa B (NF-kappaB) signaling pathway. Neither LTbeta nor LIGHT had any effect on alpha-smooth muscle actin, tissue inhibitor of metalloproteinase 1, transforming growth factor beta, or procollagen alpha(1)(I) expression; however, leukocyte recruitment-associated factors intercellular adhesion molecule 1 and regulated upon activation T cells expressed and secreted (RANTES) were markedly up-regulated. RANTES caused the chemotaxis of a liver progenitor cell line expressing CCR5. CONCLUSION: This study suggests that LTbetaR on hepatic stellate cells may be involved in paracrine signaling with nearby LTbeta-expressing liver progenitor cells mediating recruitment of progenitor cells, hepatic stellate cells, and leukocytes required for wound healing and regeneration during chronic liver injury.
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Células Estrelladas Hepáticas/fisiología , Hígado/lesiones , Heterotrímero de Linfotoxina alfa1 y beta2/fisiología , Receptor beta de Linfotoxina/fisiología , Cicatrización de Heridas/fisiología , Animales , Línea Celular , Proliferación Celular , Células Cultivadas , Deficiencia de Colina/fisiopatología , Colágeno Tipo I/biosíntesis , Modelos Animales de Enfermedad , Etionina/fisiología , Regeneración Hepática/fisiología , Heterotrímero de Linfotoxina alfa1 y beta2/biosíntesis , Masculino , Ratones , RatasRESUMEN
BACKGROUND & AIMS: Numerous studies have linked the proliferation of liver progenitor cells (LPCs) during chronic liver disease to the risk for development of hepatocellular carcinoma. Thus, selective inhibition of LPC growth during preneoplastic injury may prevent or delay the onset of liver cancer. Rats carrying a germ-line mutation in c-kit have an impaired LPC response to liver injury. Therefore, we hypothesized that the c-kit inhibitor imatinib mesylate (IM) would suppress LPC growth and, therefore, may exert antitumorigenic effects in the liver. METHODS: Expression of IM target proteins was examined in chronically injured rodent and human livers. The effect of IM was examined in vitro using LPC lines and in vivo in mice fed a choline-deficient, ethionine-supplemented (CDE) diet. Livers were examined following short-term (up to 1 month) or long-term (up to 14 months) feeding of CDE diet and drug treatments. RESULTS: C-kit was significantly up-regulated in chronic injury and expressed by LPCs. IM was antiproliferative to LPC lines, and knockdown of c-kit reduced this response. IM treatment inhibited the LPCs response and early fibrogenesis induced by a short-term CDE diet. On the longer term, IM treatment reduced the extent of fibrosis and significantly inhibited tumor formation. CONCLUSIONS: Tyrosine kinase inhibitors, such as IM, may be suited for the prevention of hepatocellular carcinoma in the setting of chronic liver injury via antiproliferative effects on c-kit-expressing LPCs.
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Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Neoplasias Hepáticas Experimentales/patología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/farmacología , Células Madre/efectos de los fármacos , Animales , Benzamidas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Colina/administración & dosificación , Dieta , Relación Dosis-Respuesta a Droga , Etionina/administración & dosificación , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/patología , Hepatocitos/patología , Humanos , Mesilato de Imatinib , Técnicas In Vitro , Hígado/efectos de los fármacos , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células Madre/patologíaRESUMEN
BACKGROUND/AIMS: Several previous studies have suggested that interferon gamma (IFNgamma) may play a key role during hepatic progenitor cell (HPC) mediated liver regeneration. However to date, no studies have directly tested the ability of IFNgamma to mediate the HPC response in an in vivo model. METHODS/RESULTS: Administration of IFNgamma to mice receiving a choline deficient, ethionine (CDE) supplemented diet to induce chronic injury resulted in an augmented HPC response. This was accompanied by increased inflammation, altered cytokine expression and hepatic fibrosis. Serum alanine aminotransferase activity, hepatocyte apoptosis and Bak staining were significantly increased in IFNgamma-treated, CDE-fed mice, demonstrating that liver damage was exacerbated in these animals. Administration of IFNgamma to control diet fed mice did not induce liver damage, however it did stimulate hepatic inflammation. CONCLUSIONS: Our results suggest that IFNgamma increases the HPC response to injury by stimulating hepatic inflammation and aggravating liver damage. This is accompanied by an increase in hepatic fibrogenesis, supporting previous reports which suggest that the HPC response may drive fibrogenesis during chronic liver injury.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Interferón gamma/efectos adversos , Cirrosis Hepática/inducido químicamente , Células Madre/efectos de los fármacos , Animales , Enfermedad Crónica , Dieta , Modelos Animales de Enfermedad , Fibrinógeno/biosíntesis , Hepatocitos/citología , Inflamación/inducido químicamente , Interferón gamma/administración & dosificación , Ratones , Proteínas RecombinantesRESUMEN
Oval cells have great potential for use in cell therapy to treat liver disease, however this cannot be achieved until the factors which govern their proliferation and differentiation are better understood. We describe a method to establish primary cultures of murine oval cells, and the derivation of two novel lines from these. Primary cultures from the livers of wildtype or TAT-GRE lacZ transgenic mice subjected to a choline-deficient, ethionine-supplemented diet comprised up to 80% oval cells at day 7 based on A6 or CK19 staining. Cell lines were clonally derived, which underwent spontaneous immortalisation following prolonged maintenance in culture. Immunostaining and RT-PCR demonstrated they express hepatocytic and biliary markers and they were therefore termed "bipotential murine oval liver" (BMOL) cells. Under proliferating culture conditions, BMOL or BMOL-TAT cells abundantly expressed oval cell and biliary markers, whereas mature hepatocytic markers were upregulated when the growth conditions were changed to facilitate differentiation. Hepatic differentiation of BMOL-TAT cells could be traced by measuring the expression of their lacZ transgene, which is driven by a promoter element from tyrosine aminotransferase (TAT), a marker of adult hepatocytes. Interestingly, haematopoietic markers were upregulated in superconfluent cultures, indicating a possible multipotentiality. None of the cell lines grew in semi-solid agar, nor did they form tumours in nude mice, suggesting they are non-tumourigenic. These novel murine oval cell lines, together with a reliable method for isolation and culture of primary oval cells, will provide a useful tool for investigating the contribution of oval cells to liver regeneration.
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Etionina/administración & dosificación , Alimentos Formulados , Hígado/citología , Células Madre/citología , Animales , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación/metabolismo , Cadherinas/análisis , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Separación Celular/métodos , Colina/administración & dosificación , Conexinas/genética , Factor de Transcripción GATA2/genética , Expresión Génica/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Inmunohistoquímica , Queratina-19/análisis , Queratina-19/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Antígenos Thy-1/genética , Tirosina Transaminasa/genética , Tirosina Transaminasa/metabolismoRESUMEN
BACKGROUND/AIMS: Liver regeneration following chronic injury is associated with inflammation, the proliferation of liver progenitor (oval) cells and fibrosis. Previous studies identified interferon-gamma as a key mediator of oval cell proliferation. Interferon-gamma is known to regulate Th1 cell activities during immune challenge. Therefore, we hypothesised that progenitor cell-mediated regeneration is associated with a Th1 immune response. METHODS: C57Bl/6 (normal Th1 response) and BALB/c mice (deficient in Th1 signalling) were placed on a carcinogenic diet to induce liver injury, progenitor cell proliferation and fibrosis. RESULTS: Serum transaminases and mortality were elevated in BALB/c mice fed the diet. Proliferation of liver progenitor cells was significantly attenuated in BALB/c animals. The pattern of cytokine expression and inflammation differed between strains. Liver fibrosis and hepatic stellate cell activation were significantly inhibited in BALB/c mice compared to C57Bl/6. In addition, interferon-gamma knockout mice also showed reduced fibrosis compared to wild type. These findings are in contrast to published results, in which interferon-gamma is shown to be anti-fibrogenic. CONCLUSIONS: Our data demonstrate that the hepatic progenitor cell response to a CDE diet is inhibited in mice lacking Th1 immune signalling and further show that this inhibition is associated with reduced liver fibrosis.
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Deficiencia de Colina/metabolismo , Etionina/administración & dosificación , Cirrosis Hepática/prevención & control , Animales , Apoptosis , Proliferación Celular , Deficiencia de Colina/patología , Citocinas/biosíntesis , Suplementos Dietéticos , Hepatocitos/metabolismo , Hepatocitos/patología , Interferón gamma/deficiencia , Interferón gamma/genética , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Regeneración Hepática/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de la Especie , Células Madre/metabolismo , Células Madre/patología , Células TH1/fisiologíaRESUMEN
Hepatic progenitor cells (called oval cells in rodents) proliferate during chronic liver injury. They have been suggested as targets of malignant transformation in chronic liver diseases, including chronic hepatitis C. Interferon alpha therapy reduces the risk of hepatocellular carcinoma (HCC) in chronic hepatitis C regardless of viral clearance. The aim of this study was to determine whether interferon alpha could reduce the risk of HCC by modifying preneoplastic events in the hepatic progenitor cell population. Pre- and post-treatment liver biopsies were evaluated for changes in t he hepaticprogenitor cell population in 16 patients with non-responding chronic hepatitis C Interferon alpha-based treatment significantly reduced the numbers of c-kit-positive hepatic progenitor cells by 50%. To determine the mechanism of cell number reduction, the effects of interferon alpha on murinehepatic progenitor cells were studied in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay and proliferating cell nuclear antigen staining showed that interferon alpha had a dose-dependent, anti-proliferative effect Interferon alpha stimulated hepatocytic and biliary differentiation of the oval cell lines reflected by increased expression of albumin and cytokeratin19 accompanied by decreased expression of alphafetoprotein and Thy-1. To validatethese results in vivo, mice were placed on the choline-deficient, ethionine-supplemented diet to induce liver injury and oval cell proliferation and treated with pegylated interferon alpha 2b for 2 weeks. This resulted in a significant four-fold reduction in the number of oval cells (P < .05). In conclusion, interferon alpha-based treatment reduced the number of hepatic progenitor cells in chronic liver injury by modulating apoptosis, proliferation, and differentiation. Supplementay material for this article can
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Antivirales/farmacología , Proliferación Celular/efectos de los fármacos , Interferón-alfa/farmacología , Hígado/citología , Hígado/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Antivirales/uso terapéutico , Células Cultivadas , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Polietilenglicoles , Proteínas RecombinantesRESUMEN
Oval cell proliferation precedes neoplasia in many rodent models of hepatocellular carcinoma and prevention of this proliferative response can reduce the risk of subsequent carcinoma. This study aimed to determine whether a selective cyclo-oxygenase-2 (COX-2) inhibitor, SC-236, affects (i) the oval cell response to liver injury in a mouse model of hepatocarcinogenesis and (ii) an oval cell line. Four-week-old mice were fed either normal chow or a choline deficient, ethionine supplemented (CDE) diet in the presence or absence of SC-236. Liver histology and oval cell numbers were determined after 2, 4, 12 and 52 weeks of treatment. Oval cells were scored using morphological criteria and positive immuno-staining for the M(2)-isozyme of pyruvate kinase (M2PK) or A6. An immortalized oval cell line (PIL-2) was used to study the in vitro effects of SC-236 on oval cell proliferation, apoptosis and Akt phosphorylation. The percentage of M2PK-positive oval cells and COX-2-positive cells was reduced by 80% and 45%, respectively, in CDE-fed mice receiving SC-236 compared with CDE-fed animals not receiving SC-236. Some M2PK-positive oval cells were also COX-2 positive. The percentage of A6-positive cells was not affected by SC-236 administration to CDE-fed mice. Administration of SC-236 increased apoptosis as evidenced by a 73% increase in the number of TUNEL-positive cells at 2 weeks in CDE-fed mice. Primary oval cells and PIL-2 cells expressed COX-2. In vitro treatment of PIL-2 cells with SC-236 resulted in a dose-dependent preferential death of A6-negative cells. Administration of 25 and 50 microM Prostaglandin E(2) partially attenuated SC-236 induced cell death by 25%. In vitro oval cell death was associated with apoptosis and a 70% reduction in Akt phosphorylation. These results suggest that the SC-236 induced reduction of M2PK-positive oval cell numbers may be due to COX-2 dependent inhibition of Akt phosphorylation and induction of apoptosis.
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Antimetabolitos/administración & dosificación , Deficiencia de Colina , Inhibidores de la Ciclooxigenasa 2/farmacología , Modelos Animales de Enfermedad , Etionina/administración & dosificación , Hepatopatías/prevención & control , Hígado/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclooxigenasa 2/química , Ciclooxigenasa 2/metabolismo , Dieta , Suplementos Dietéticos , Hígado/lesiones , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazoles/farmacología , Piruvato Quinasa/metabolismo , Células Madre/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Oval cells are facultative liver progenitor cells, which are invoked during chronic liver injury in order to replenish damaged hepatocytes and bile duct cells. Previous studies have observed inflammation and cytokine production in the liver during chronic injury. Further, it has been proposed that inflammatory growth factors may mediate the proliferation of oval cells during disease progression. We have undertaken a detailed examination of inflammation and cytokine production during a time course of liver injury and repair, invoked by feeding mice a choline-deficient, ethionine-supplemented (CDE) diet. We show that immediately following initial liver injury, B220-expressing leucocytes transiently infiltrate the liver. This inflammatory response occurred immediately before oval cell numbers began to expand in the liver, suggesting that the two events may be linked. Two waves of liver cytokine production were observed during the CDE time course. The first occurred shortly following commencement of the diet, suggesting that it may represent a hepatic acute phase response. However, examination of acute phase marker expression in CDE-fed mice did not support this hypothesis. The second wave of cytokine expression correlated with the expansion of oval cell numbers in the liver, suggesting that these factors may mediate oval cell proliferation. No inflammatory signalling was detected following withdrawal of the injury stimulus. In summary, our results document a close correlation between inflammation, cytokine production and the expansion of oval cells in the liver during experimental chronic injury.
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Citocinas/biosíntesis , Regulación de la Expresión Génica , Hígado/metabolismo , Hígado/patología , Proteínas de Fase Aguda/biosíntesis , Proteínas de Fase Aguda/genética , Animales , Apoptosis/efectos de los fármacos , Aspartato Aminotransferasas/sangre , Linfocitos B/citología , Recuento de Células , Proliferación Celular/efectos de los fármacos , Deficiencia de Colina/sangre , Deficiencia de Colina/metabolismo , Deficiencia de Colina/patología , Citocinas/genética , Citocinas/inmunología , Etionina/farmacología , Hepatitis/metabolismo , Hepatitis/patología , Hígado/lesiones , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/análisis , ARN Mensajero/genética , Linfocitos T/citología , Factores de TiempoRESUMEN
Multifaceted evidence links the development of liver tumours to the activation and proliferation of adult liver progenitor (oval) cells during the early stages of chronic liver injury. The aim of this study was to examine the role of the peroxisome proliferator activated receptors (PPARs): PPARalpha, delta and gamma, in mediating the behaviour of liver progenitor cells during pre-neoplastic disease and to investigate their potential as therapeutic targets for the treatment of chronic liver injury. We observed increased liver expression of PPARalpha and gamma in concert with expanding oval cell numbers during the first 21 days following commencement of the choline deficient, ethionine supplemented (CDE) dietary model of carcinogenic liver injury in mice. Both primary and immortalized liver progenitor cells were found to express PPARalpha, delta and gamma, but not gamma2, the alternate splice form of PPARgamma. WY14643 (PPARalpha agonist), GW501516 (PPARdelta agonist) and ciglitazone (PPARgamma agonist) were tested for their ability to modulate the behaviour of p53-immortalized liver (PIL) progenitor cell lines in vitro. Both PPARdelta and gamma agonists induced dose-dependent growth inhibition and apoptosis of PIL cells. In contrast, the PPARalpha agonist had no effect on PIL cell growth. None of the drugs affected the maturation of PIL cells along either the hepatocytic or biliary lineages, as judged by their patterns of hepatic gene expression prior to and following treatment. Administration of the PPARgamma agonist ciglitazone to mice fed with the CDE diet for 14 days resulted in a significantly diminished oval cell response and decreased fibrosis compared with those receiving placebo. In contrast, GW501516 did not affect oval cell numbers or liver fibrosis, but inhibited CDE-induced hepatic steatosis. In summary, PPARgamma agonists reduce oval cell proliferation and fibrosis during chronic liver injury and may be useful in the prevention of hepatocellular carcinoma.