RESUMEN

Patients with cirrhosis waiting for liver transplantation (LT) frequently present a nutritional disorder, which represents an independent predictor of morbidity and mortality before and after transplantation. Thus, a proper assessment of the food intake by using different methods, such as food records, food frequency questionnaires, and 24 h recall, should be deemed an important step of the nutritional management of these patients. The available published studies indicate that the daily food intake is inadequate in the majority of waitlisted patients. These findings were confirmed by our experience, showing that the daily intake of total calories, proteins and carbohydrates was inadequate in approximately 85-95% of patients, while that of lipids and simple carbohydrates was inadequate in almost 50% of them. These data highlight the need to implement an effective educational program provided by certified nutritionists or dieticians, who should work in close collaboration with the hepatologist to provide a nutritional intervention tailored to the individual patient requirements.

Asunto(s)

Registros de Dieta , Ingestión de Alimentos , Cirrosis Hepática/cirugía , Trasplante de Hígado , Desnutrición/diagnóstico , Evaluación Nutricional , Estado Nutricional , Valor Nutritivo , Listas de Espera , Anciano , Ingestión de Energía , Femenino , Humanos , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/fisiopatología , Masculino , Desnutrición/fisiopatología , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Ingesta Diaria Recomendada

RESUMEN

BACKGROUND: Sorafenib is the first targeted agent proven to improve survival of patients with advanced hepatocellular carcinoma (HCC) and it has been used in first line treatments with heterogeneous response across patients. Most of the promising agents evaluated in first-line or second-line phase III trials for HCC failed to improve patient survival. The absence of molecular characterisation, including the identification of pathways driving resistance might be responsible for these disappointing results. METHODS: 2D DIGE and MS analyses were used to reveal proteomic signatures resulting from Notch3 inhibition in HepG2 cells, combined with brivanib treatment. The therapeutic potential of Notch3 inhibition combined with brivanib treatment was also demonstrated in a rat model of HCC and in cell lines derived from different human cancers. RESULTS: Using a proteomic approach, we have shown that Notch3 is strongly involved in brivanib resistance through a p53-dependent regulation of enzymes of the tricarboxylic acid (TCA), both in vitro and in vivo. CONCLUSION: We have demonstrated that regulation of the TCA cycle is a common mechanism in different human cancers, suggesting that Notch3 inhibitors combined with brivanib treatment may represent a strong formulation for the treatment of HCC as well as Notch3-driven cancers.

Asunto(s)

Alanina/análogos & derivados , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Receptor Notch3/antagonistas & inhibidores , Triazinas/farmacología , Alanina/farmacología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Resistencia a Antineoplásicos , Electroforesis en Gel de Poliacrilamida , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/terapia , Células MCF-7 , Terapia Molecular Dirigida , Proteómica , ARN Interferente Pequeño/administración & dosificación , Ratas , Ratas Wistar , Receptor Notch3/deficiencia , Receptor Notch3/genética , Electroforesis Bidimensional Diferencial en Gel

RESUMEN

Most hepatocellular carcinomas (HCCs) arise in the context of chronic liver disease and/or cirrhosis. Thus, chemoprevention in individuals at risk represents an important but yet unproven approach. In this study, we investigated the ability of microRNA (miRNA)-based molecules to prevent liver cancer development in a cirrhotic model. To this end, we developed a mouse model able to recapitulate the natural progression from fibrosis to HCC, and then we tested the prophylactic activity of an miRNA-based approach in the model. The experiments were carried out in the TG221 transgenic mouse, characterized by the overexpression of miR-221 in the liver and predisposed to the development of liver tumors. TG221 as well as wild-type mice were exposed to the hepatotoxin carbon tetrachloride (CCl4) to induce chronic liver damage. All mice developed liver cirrhosis, but only TG221 mice developed nodular lesions in 100% of cases within 6 months of age. The spectrum of lesions ranged from dysplastic foci to carcinomas. To investigate miRNA-based prophylactic approaches, anti-miR-221 oligonucleotides or miR-199a-3p mimics were administered to TG221 CCl4-treated mice. Compared to control animals, a significant reduction in number, size, and, most significantly, malignant phenotype of liver nodules was observed, thus demonstrating an important prophylactic action of miRNA-based molecules. In summary, in this article, we not only report a simple model of liver cancer in a cirrhotic background but also provide evidence for a potential miRNA-based approach to reduce the risk of HCC development.

RESUMEN

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, especially in patients not amenable for curative treatments. The multi-kinase inhibitor sorafenib represents the first-line treatment option for advanced HCC; nevertheless, its effectiveness is limited due to tumor heterogeneity as well as innate or acquired drug resistance, raising the need for new therapeutic strategies. MicroRNAs (miRNAs) involvement in treatment response as well as their safety and efficacy in preclinical models and clinical trials have been widely documented in the oncologic field, including HCC. Here, we identified miR-494 upregulation in a subgroup of human and rat HCCs with stem cell-like characteristics, as well as multiple epigenetic mechanisms involved in its aberrant expression in HCC cell lines and patients. Moreover, we identified p27, puma and pten among miR-494 targets, contributing to speed up cell cycle progression, enhance survival potential in stressful conditions and increase invasive and clonogenic capabilities. MiR-494 overexpression increased sorafenib resistance via mTOR pathway activation in HCC cell lines and, in line, high miR-494 levels associated with decreased sorafenib response in two HCC animal models. A sorafenib-combined anti-miR-494-based strategy revealed an enhanced anti-tumor potential with respect to sorafenib-only treatment in our HCC rat model. In conclusion, our findings suggested miR-494 as a possible therapeutic target as well as a candidate biomarker for patient stratification in advanced HCC.

Asunto(s)

Carcinoma Hepatocelular/patología , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Neoplasias Hepáticas/patología , MicroARNs/metabolismo , Antígeno AC133/genética , Antígeno AC133/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Modelos Animales de Enfermedad , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Ratones , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fenotipo , Ratas , Sorafenib/farmacología , Sorafenib/uso terapéutico , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , ADN Metiltransferasa 3B

RESUMEN

BACKGROUND AIMS: Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133+ stem/progenitor cell (SPC) reinfusion in patients with ESLD is feasible and safe and improve, albeit transiently, liver function. However, the mechanism(s) through which BM-derived SPCs may improve liver function are not fully elucidated. METHODS: Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133+ cell therapy. Next, we set up an in vitro model mimicking SPCs/liver microenvironment interaction by culturing granulocyte colony-stimulating factor (G-CSF)-mobilized CD133+and LX-2 hepatic stellate cells. RESULTS: We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133+ SPCs and human hepatic stellate cells LX-2. Human hepatic stellate cells LX-2 showed reduced activation and fibrotic potential. In turn, hepatic stellate cells enhanced the proliferation and survival of CD133+ SPCs as well as their endothelial and hematopoietic function while promoting an anti-inflammatory profile. DISCUSSION: We demonstrated that the interaction between CD133+ SPCs from patients with ESLD and hepatic stellate cells induces significant functional changes in both cellular types that may be instrumental for the improvement of liver function in cirrhotic patients undergoing cell therapy.

Asunto(s)

Antígeno AC133/metabolismo , Enfermedad Hepática en Estado Terminal/terapia , Células Estrelladas Hepáticas/citología , Hígado/citología , Trasplante de Células Madre/métodos , Células Madre/metabolismo , Proliferación Celular , Técnicas de Cocultivo , Enfermedad Hepática en Estado Terminal/patología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Estrelladas Hepáticas/fisiología , Humanos , Hígado/metabolismo , Hígado/patología , Neovascularización Fisiológica , Células Madre/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo

RESUMEN

Decompensated cirrhosis is associated to extensive post-transcriptional changes of human albumin (HA). This study aims to characterize the occurrence of HA homodimerization in a large cohort of patients with decompensated cirrhosis and to evaluate its association with clinical features and prognosis. HA monomeric and dimeric isoforms were identified in peripheral blood by using a HPLC-ESI-MS technique in 123 cirrhotic patients hospitalized for acute decompensation and 50 age- and sex-comparable healthy controls. Clinical and biochemical parameters were recorded and patients followed up to one year. Among the monomeric isoforms identified, the N- and C-terminal truncated and the native HA underwent homodimerization. All three homodimers were significantly more abundant in patients with cirrhosis, acute-on-chronic liver failure and correlate with the prognostic scores. The homodimeric N-terminal truncated isoform was independently associated to disease complications and was able to stratify 1-year survival. As a result of all these changes, the monomeric native HA was significantly decreased in patients with cirrhosis, being also associated with a poorer prognosis. In conclusion homodimerization is a novel described structural alteration of the HA molecule in decompensated cirrhosis and contributes to the progressive reduction of the monomeric native HA, the only isoform provided of structural and functional integrity.

Asunto(s)

Cirrosis Hepática/sangre , Albúmina Sérica Humana/química , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Estimación de Kaplan-Meier , Cirrosis Hepática/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Isoformas de Proteínas/sangre , Isoformas de Proteínas/química , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Índice de Severidad de la Enfermedad , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

Chronic inflammation is a risk factor for the onset of cancer and the regular use of aspirin reduces the risk of cancer development. Here we showed that therapeutic dosages of aspirin counteract the pro-tumorigenic effects of the inflammatory cytokine interleukin(IL)-6 in cancer and non-cancer cell lines, and in mouse liver in vivo. We found that therapeutic dosages of aspirin prevented IL-6 from inducing the down-regulation of p53 expression and the acquisition of the epithelial mesenchymal transition (EMT) phenotypic changes in the cell lines. This was the result of a reduction in c-Myc mRNA transcription which was responsible for a down-regulation of the ribosomal protein S6 expression which, in turn, slowed down the rRNA maturation process, thus reducing the ribosome biogenesis rate. The perturbation of ribosome biogenesis hindered the Mdm2-mediated proteasomal degradation of p53, throughout the ribosomal protein-Mdm2-p53 pathway. P53 stabilization hindered the IL-6 induction of the EMT changes. The same effects were observed in livers from mice stimulated with IL-6 and treated with aspirin. It is worth noting that aspirin down-regulated ribosome biogenesis, stabilized p53 and up-regulated E-cadherin expression in unstimulated control cells also. In conclusion, these data showed that therapeutic dosages of aspirin increase the p53-mediated tumor-suppressor activity of the cells thus being in this way able to reduce the risk of cancer onset, either or not linked to chronic inflammatory processes.

Asunto(s)

Aspirina/farmacología , Interleucina-6/farmacología , Ribosomas/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Células Hep G2 , Humanos , Inflamación , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Neoplasias/inmunología , Neoplasias/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Polimerasa I/metabolismo , ARN Ribosómico/metabolismo , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

Human serum albumin (HSA) is the most abundant plasma protein, endowed with several biological properties unrelated to its oncotic power, such as antioxidant and free-radicals scavenging activities, binding and transport of many endogenous and exogenous substances, and regulation of endothelial function and inflammatory response. These non-oncotic activities are closely connected to the peculiarly dynamic structure of the albumin molecule. HSA undergoes spontaneous structural modifications, mainly by reaction with oxidants and saccharides; however, patients with cirrhosis show extensive post-transcriptional changes at several molecular sites of HSA, the degree of which parallels the severity of the disease. The present work reports the development and application of an innovative LC-MS analytical method for a rapid and reproducible determination of the relative abundance of HSA isoforms in plasma samples from alcoholic hepatitis (AH) patients. A condition of severe oxidative stress, similar to that observed in AH patients, is associated with profound changes in circulating HSA microheterogeneity. More interestingly, the high resolution provided by the analytical platform allowed the monitoring of novel oxidative products of HSA never reported before.

Asunto(s)

Hepatitis Alcohólica/sangre , Espectrometría de Masas/métodos , Plasma/química , Albúmina Sérica/química , Adulto , Anciano , Estudios de Casos y Controles , Cromatografía Liquida/métodos , Humanos , Cirrosis Hepática/sangre , Persona de Mediana Edad , Oxidación-Reducción , Estrés Oxidativo/fisiología , Isoformas de Proteínas/sangre , Isoformas de Proteínas/química , Reproducibilidad de los Resultados , Adulto Joven

Asunto(s)

Insuficiencia Hepática Crónica Agudizada/mortalidad , Infecciones Bacterianas/diagnóstico , Cirrosis Hepática/complicaciones , Animales , Femenino , Humanos , Masculino

RESUMEN

BACKGROUND & AIMS: Patients with cirrhosis present structural changes of human serum albumin (HSA) affecting non-oncotic functions. Ischaemia-modified albumin (IMA), which reflects the capacity to bind cobalt, has been associated to patient mortality during acute-on-chronic liver failure. This study aimed to assess whether circulating IMA is elevated in advanced cirrhosis and its relationship with severity of cirrhosis and specific complications. METHODS: A total of 127 cirrhotic patients hospitalized for an acute complication of the disease and 44 healthy controls were enrolled. Plasma IMA and IMA to albumin ratio (IMAr) were measured with a cobalt-binding assay. HSA isoforms carrying post-transcriptional molecular changes were assessed with HPLC-ESI-MS. The effect of endotoxemia on IMA was evaluated in rats with CCl4 -cirrhosis. RESULTS: IMA/IMAr is significantly higher in cirrhotic patients than in controls, but no correlations were found with prognostic scores. IMA did not correlate with the altered HSA isoforms. Ascites, renal impairment and hepatic encephalopathy did not influence IMA/IMAr levels. In contrast, IMA/IMAr is significantly higher in infected than non-infected patients. ROC curves showed that IMA/IMAr had similar discriminating performances for bacterial infection as C-reactive protein (CRP). Moreover, CRP and IMA were independently associated with bacterial infection. Consistently, endotoxin injection significantly increased IMA in cirrhotic, but not in healthy rats. CONCLUSIONS: IMA is elevated in patients with advanced cirrhosis. The IMA level does not correlate with disease severity scores, but it is specifically associated to bacterial infection, showing a discriminating performance similar to CRP. Further investigations to assess IMA as a novel diagnostic test for bacterial infection are advocated.

Asunto(s)

Insuficiencia Hepática Crónica Agudizada/mortalidad , Infecciones Bacterianas/diagnóstico , Cirrosis Hepática/complicaciones , Anciano , Animales , Biomarcadores/sangre , Proteína C-Reactiva/análisis , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/sangre , Curva ROC , Ratas , Ratas Wistar , Albúmina Sérica , Albúmina Sérica Humana

RESUMEN

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings. Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC-MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity.

Asunto(s)

Biomarcadores/sangre , Biomarcadores/química , Cirrosis Hepática/sangre , Isoformas de Proteínas/sangre , Isoformas de Proteínas/química , Albúmina Sérica/química , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Multimerización de Proteína , Albúmina Sérica/biosíntesis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos

RESUMEN

To successfully target Notch receptors as part of a multidrug anticancer strategy, it will be essential to fully characterize the factors that are modulated by Notch signaling. We recently reported that Notch3 silencing in HCC results in p53 up-regulation in vitro and, therefore, we focused on the mechanisms that associate Notch3 to p53 protein expression. We explored the regulation of p53 by Notch3 signalling in three HCC cell lines HepG2, SNU398 and Hep3B.We found that Notch3 regulates p53 at post-transcriptional level controlling both Cyclin G1 expression and the feed-forward circuit involving p53, miR-221 and MDM2. Moreover, our results were validated in human HCCs and in a rat model of HCC treated with Notch3 siRNAs. Our findings are becoming an exciting area for further in-depth research toward targeted inactivation of Notch3 receptor as a novel therapeutic approach for increasing the drug-sensitivity, and thereby improving the treatment outcome of patients affected by HCC. Indeed, we proved that Notch3 silencing strongly increases the effects of Nutilin-3.With regard to therapeutic implications, Notch3-specific drugs could represent a valuable strategy to limit Notch signaling in the context of hepatocellular carcinoma over-expressing this receptor.

Asunto(s)

Carcinoma Hepatocelular/metabolismo , Ciclina G1/metabolismo , Neoplasias Hepáticas/metabolismo , MicroARNs/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Receptores Notch/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Apoptosis , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Ciclo Celular , Movimiento Celular , Proliferación Celular , Ciclina G1/antagonistas & inhibidores , Ciclina G1/genética , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/genética , Masculino , Mutación/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Notch3 , Receptores Notch/antagonistas & inhibidores , Receptores Notch/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

UNLABELLED: Beside the regulation of fluid distribution, human serum albumin (HSA) carries other activities, such as binding, transport, and detoxification of many molecules. In patients with cirrhosis, HSA exhibits posttranscriptional alterations that likely affect its functions. This study aimed at identifying the structural HSA alterations occurring in cirrhosis and determining their relationship with specific clinical complications and patient survival. One hundred sixty-eight patients with cirrhosis, 35 with stable conditions and 133 hospitalized for acute clinical complications, and 94 healthy controls were enrolled. Posttranscriptional HSA molecular changes were identified and quantified by using a high-performance liquid chromatography/electrospray ionization mass spectrometry technique. Clinical and biochemical parameters were also recorded and hospitalized patients were followed for up to 1 year. Seven HSA isoforms carrying one or more posttranscriptional changes were identified. Altered HSA isoforms were significantly more represented in patients than in healthy controls. Conversely, the native, unchanged HSA isoform was significantly reduced in cirrhosis. Native HSA and most altered isoforms correlated with both Child-Pugh and Model for End-Stage Liver Disease scores. In hospitalized patients, oxidized and N-terminal truncated isoforms were independently associated with ascites, renal impairment, and bacterial infection. Finally, the native HSA and cysteinylated/N-terminal truncated isoforms were predictors of 1-year survival, with greater prognostic accuracy than total serum albumin concentration. CONCLUSIONS: Extensive posttranscriptional changes of HSA, involving several molecular sites and increasing in parallel with disease severity, occur in patients with cirrhosis. Altered isoforms are independently associated with specific clinical complications, whereas the residual, native HSA isoform independently predicts patient survival. These findings support the concept of the "effective albumin concentration," which implies that the global HSA function is related not only to its serum concentration, but also to the preservation of its structural integrity.

Asunto(s)

Albúminas/metabolismo , Cirrosis Hepática/metabolismo , Modificación Traduccional de las Proteínas , Adulto , Anciano , Estudios de Casos y Controles , Complicaciones de la Diabetes/metabolismo , Femenino , Voluntarios Sanos , Humanos , Italia/epidemiología , Cirrosis Hepática/complicaciones , Cirrosis Hepática/mortalidad , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/metabolismo , Procesamiento Postranscripcional del ARN

RESUMEN

Several lines of evidence suggest that novel pharmacological approaches aimed at converting white adipose tissue (WAT) into brown adipose tissue (BAT) may represent an effective therapeutic strategy for obesity and related disorders. ((18))F-fluorodeoxyglucose ((18)F-FDG) is the only positron emission tomography (PET) tracer commonly used to study BAT function, and so far no functional tools have been described to investigate in vivo white-to-brown fat conversion. In this report, we show that the PET tracer (11)C-meta-hydroxyephedrine ((11)C-MHED, a norepinephrine analogue) is a useful tool to investigate the sympathetic nervous system (SNS) activity in BAT of lean and dietary obese mice. Moreover, we demonstrate that (11)C-MHED is a specific marker of the SNS-mediated thermogenesis in typical BAT depots, and that this tracer can detect in vivo WAT to BAT conversion.

RESUMEN

Endogenous cannabinoids (EC) are ubiquitous lipid signalling molecules providing different central and peripheral effects that are mediated mostly by the specific receptors CB1 and CB2. The EC system is highly upregulated during chronic liver disease and consistent experimental and clinical findings indicate that it plays a role in the pathogenesis of liver fibrosis and fatty liver disease associated with obesity, alcohol abuse and hepatitis C. Furthermore, a considerable number of studies have shown that EC and their receptors contribute to the pathogenesis of the cardio-circulatory disturbances occurring in advanced cirrhosis, such as portal hypertension, hyperdynamic circulatory syndrome and cirrhotic cardiomyopathy. More recently, the EC system has been implicated in the development of ascites, hepatic encephalopathy and the inflammatory response related to bacterial infection. Rimonabant, a selective CB1 antagonist, was the first drug acting on the EC system approved for the treatment of obesity. Unfortunately, it has been withdrawn from the market because of its neuropsychiatric side effects. Compounds able to target selectively the peripheral CB1 receptors are under evaluation. In addition, molecules stimulating CB2 receptor or modulating the activity of enzymes implicated in EC metabolism are promising areas of pharmacological research. Liver cirrhosis and the related complications represent an important target for the clinical application of these compounds.

Asunto(s)

Antagonistas de Receptores de Cannabinoides/uso terapéutico , Endocannabinoides/metabolismo , Regulación de la Expresión Génica/fisiología , Cirrosis Hepática/fisiopatología , Receptores de Cannabinoides/metabolismo , Transducción de Señal/fisiología , Humanos , Modelos Biológicos , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Rimonabant

RESUMEN

BACKGROUND AND PURPOSE: ß(3) -Adrenoceptors participate in the regulation of vascular tone in physiological and pathological conditions. We aimed to assess the effect of pharmacological modulation of ß(3) -adrenoceptors on portal pressure (PP) and systemic haemodynamics and their expression in the liver and mesenteric vessels of cirrhotic rats. EXPERIMENTAL APPROACH: PP, central venous pressure (CVP) and systemic haemodynamics were invasively assessed in control and CCl(4) -treated cirrhotic rats before and during infusion of the selective ß(3) -adrenoceptor agonist, SR58611A. Tissue samples were also collected from liver, heart, portal vein and mesenteric artery for immunohistochemistry and molecular biology analysis. The effect of SR58611A on isolated portal vein was assessed. KEY RESULTS: At baseline, cirrhotic rats showed portal hypertension, reduced CVP and hyperdynamic circulation. SR58611A induced a significant, dose-dependent decrease in PP in cirrhotic rats, but not in controls. Although both groups manifested a dose-dependent reduction in mean arterial pressure, this effect was associated with decreased cardiac index (CI) and unchanged indicized peripheral vascular resistance (PVRI) in cirrhotic rats and increased CI and decreased PVRI in control animals. Pretreatment with the selective ß(3) -adrenoceptor antagonist SR59230 prevented all SR58611A-induced changes in cirrhotic rats. SR58611A concentration-dependently relaxed portal vein in cirrhotic rats to a significantly greater extent than in healthy rats; pretreatment with SR59230A completely prevented SR58611A-induced cirrhotic portal vein relaxation. Finally, ß(3) -adrenoceptors were identified in the liver, heart and portal vein of cirrhotic and control animals; their expression was increased in cirrhotic rats. CONCLUSIONS AND IMPLICATIONS: ß(3) -Adrenoceptors are altered in portal hypertension of experimental cirrhosis and may represent a novel therapeutic target.

Asunto(s)

Antagonistas de Receptores Adrenérgicos beta 3/farmacología , Hipertensión Portal/fisiopatología , Cirrosis Hepática/fisiopatología , Receptores Adrenérgicos beta 3/fisiología , Tetrahidronaftalenos/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Hemodinámica , Técnicas In Vitro , Hígado/metabolismo , Masculino , Miocardio/metabolismo , Vena Porta/fisiología , Ratas , Ratas Wistar , Vasodilatación/efectos de los fármacos

RESUMEN

PURPOSE: Hypothermic machine perfusion systems seem more effective than the current static storage to prevent cold ischemic liver injury. Thus, we test an innovative hyperbaric hypothermic machine perfusion (HHMP), which combines hyperbaric oxygenation of the preservation solution and continuous perfusion of the graft. METHODS: Rat livers were preserved with Celsior solution according to 4 different modalities: normobaric static preservation; hyperbaric static preservation at 2 atmosphere absolute (ATA); normobaric dynamic preservation, with continuous perfusion; hyperbaric dynamic preservation, with continuous perfusion at 2 ATA. After 24 h cold preservation, we assessed different parameters. RESULTS: Compared to baseline, livers preserved with the current static storage showed severe ultrastructural damage, glycogen depletion and an increased oxidative stress. Normobaric perfused livers showed improved hepatocyte ultrastructure and ameliorated glycogen stores, but they still suffered a significant oxidative damage. The addition of hyperbaric oxygen produces an extra benefit by improving oxidative injury and by inducing endothelial NO synthase (eNOS) gene expression. CONCLUSIONS: Preservation by means of the present innovative HHMP reduced the liver injury occurring after the current static cold storage by lowering glycogen depletion and oxidative damage. Interestingly, only the use of hyperbaric oxygen was associated to a blunted oxidative stress and an increased eNOS gene expression.

Asunto(s)

Oxigenoterapia Hiperbárica/métodos , Hipotermia Inducida/métodos , Hígado , Preservación de Órganos/métodos , Animales , Disacáridos/farmacología , Electrólitos/farmacología , Regulación Enzimológica de la Expresión Génica , Glutamatos/farmacología , Glutatión/metabolismo , Glutatión/farmacología , Disulfuro de Glutatión/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Histidina/farmacología , Hipotermia Inducida/instrumentación , Glucógeno Hepático/metabolismo , Manitol/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Soluciones Preservantes de Órganos/farmacología , Estrés Oxidativo/efectos de los fármacos , Perfusión , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Compuestos de Sulfhidrilo/metabolismo

RESUMEN

UNLABELLED: MicroRNA-221 (miR-221) is one of the most frequently and consistently up-regulated microRNAs (miRNAs) in human cancer. It has been hypothesized that miR-221 may act as a tumor promoter. To demonstrate this, we developed a transgenic (TG) mouse model that exhibits an inappropriate overexpression of miR-221 in the liver. Immunoblotting and immunostaining confirmed a concomitant down-regulation of miR-221 target proteins. This TG model is characterized by the emergence of spontaneous nodular liver lesions in approximately 50% of male mice and by a strong acceleration of tumor development in 100% of mice treated with diethylnitrosamine. Similarly to human hepatocellular carcinoma, tumors are characterized by a further increase in miR-221 expression and a concomitant inhibition of its target protein-coding genes (i.e., cyclin-dependent kinase inhibitor [Cdkn]1b/p27, Cdkn1c/p57, and B-cell lymphoma 2-modifying factor). To validate the tumor-promoting effect of miR-221, we showed that in vivo delivery of anti-miR-221 oligonucleotides leads to a significant reduction of the number and size of tumor nodules. CONCLUSIONS: This study not only establishes that miR-221 can promote liver tumorigenicity, but it also establishes a valuable animal model to perform preclinical investigations for the use of anti-miRNA approaches aimed at liver cancer therapy.

Asunto(s)

Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , MicroARNs/fisiología , Animales , Masculino , Ratones , Ratones Transgénicos

RESUMEN

The endocannabinoid system is involved in the pathogenesis of liver fibrosis. Although many substances have been proved to reduce fibrosis in experimental models of chronic liver injury, most of them appear to be effective only if given as a prophylactic or early treatment. This study aimed to explore the effect of pharmacological antagonism of the endocannabinoid cannabinoid type 1 (CB1) receptor started after the stage of full-blown cirrhosis had been reached. Wistar-Han rats with carbon tetrachloride (CCl(4))-induced cirrhosis were randomized to receive the CB1 receptor antagonist Rimonabant (10 mg/kg/day) or the vehicle for 2 weeks. Age-matched healthy rats served as controls. Liver fibrosis was assessed using Sirius red staining, hydroxyproline concentration and α-smooth muscle actin expression. Hepatic gene expression of mediators of fibrogenesis and inflammation were evaluated by real-time PCR. We also assessed the hepatic expression of CB1 and CB2 receptors and that of the enzymes implicated in the endocannabinoid metabolism. Fibrosis was significantly reduced in rats treated with Rimonabant compared with rats receiving the vehicle. CB1 receptor antagonism limited the gene upregulation of fibrogenic and inflammatory mediators occurring in untreated cirrhotic rats. CB1 and CB2 receptor expression was increased in cirrhotic animals. Interestingly, pharmacological CB1 receptor antagonism was associated with a further induction of the CB2 receptor expression. Regression of fibrosis can be achieved by pharmacological blockade of the CB1 receptor even when started in an advanced stage of the disease. This effect is associated with the suppression of pro-fibrogenic and inflammatory mediators and may have been indirectly favoured by the induction of CB2 receptor expression.

Asunto(s)

Cirrosis Hepática Experimental/tratamiento farmacológico , Piperidinas/uso terapéutico , Pirazoles/uso terapéutico , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Animales , Tetracloruro de Carbono , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Matriz Extracelular/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática Experimental/inducido químicamente , Masculino , Piperidinas/farmacología , Pirazoles/farmacología , Ratas , Ratas Wistar , Inducción de Remisión , Rimonabant

RESUMEN

The endocannabinoids are endogenous mediators implicated in many physiologic and pathophysiologic processes. It has been recently shown that they contribute to the pathogenesis of liver fibrosis and hemodynamic alterations of cirrhosis. The pharmacological modulation of the endocannnabinoid system represents a potential target for the treatment of liver diseases.

Asunto(s)

Antagonistas de Receptores de Cannabinoides , Cirrosis Hepática/metabolismo , Sodio/metabolismo , Hemodinámica , Humanos , Cirrosis Hepática/complicaciones , Cirrosis Hepática/fisiopatología , Desequilibrio Hidroelectrolítico/complicaciones