PGE2 induces apoptosis of hepatic stellate cells and attenuates liver fibrosis in mice by downregulating miR-23a-5p and miR-28a-5p.

MicroRNAs (miRNAs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver diseases, whose end stage is hepatic fibrosis, a major global health burden. Pharmacological strategies for prevention or treatment of hepatic fibrosis are still limited, what makes it necessary to establish a better understanding of the molecular mechanisms underlying its pathogenesis. In this context, we have recently shown that cyclooxygenase-2 (COX-2) expression in hepatocytes restricts activation of hepatic stellate cells (HSCs), a pivotal event in the initiation and progression of hepatic fibrosis. Here, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs on a mouse model of CCl4 and bile duct ligation (BDL)-induced liver fibrosis. Our results provide evidence that COX-2 represses miR-23a-5p and miR-28-5p expression in HSC. The decrease of miR-23a-5p and miR-28-5p expression promotes protection against fibrosis by decreasing the levels of pro-fibrogenic markers α-SMA and COL1A1 and increasing apoptosis of HSC. Moreover, we demonstrate that serum levels of miR-28-5p are decreased in patients with chronic liver disease. These results suggest a protective effect exerted by COX-2-derived prostanoids in the process of hepatofibrogenesis.

Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.

The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.

Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease progression.

Our understanding of the mechanisms by which nonalcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH) is still very limited. Despite the growing number of studies linking the disease with altered serum metabolite levels, an obstacle to the development of metabolome-based NAFLD predictors has been the lack of large cohort data from biopsy-proven patients matched for key metabolic features such as obesity. We studied 467 biopsied individuals with normal liver histology (n=90) or diagnosed with NAFLD (steatosis, n=246; NASH, n=131), randomly divided into estimation (80% of all patients) and validation (20% of all patients) groups. Qualitative determinations of 540 serum metabolite variables were performed using ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). The metabolic profile was dependent on patient body-mass index (BMI), suggesting that the NAFLD pathogenesis mechanism may be quite different depending on an individual's level of obesity. A BMI-stratified multivariate model based on the NAFLD serum metabolic profile was used to separate patients with and without NASH. The area under the receiver operating characteristic curve was 0.87 in the estimation and 0.85 in the validation group. The cutoff (0.54) corresponding to maximum average diagnostic accuracy (0.82) predicted NASH with a sensitivity of 0.71 and a specificity of 0.92 (negative/positive predictive values=0.82/0.84). The present data, indicating that a BMI-dependent serum metabolic profile may be able to reliably distinguish NASH from steatosis patients, have significant implications for the development of NASH biomarkers and potential novel targets for therapeutic intervention.

Endoglina: estructura, funciones biológicas y papel en la fibrogénesis / No disponible

No disponible

Increased intrahepatic and circulating levels of endoglin, a TGF-beta1 co-receptor, in patients with chronic hepatitis C virus infection: relationship to histological and serum markers of hepatic fibrosis.

Endoglin, a transforming growth factor (TGF)-beta1 co-receptor, has been associated with renal and cutaneous fibrosis, as overexpression of this protein has been observed in biopsies from patients with glomerulosclerosis and scleroderma, respectively. Our aim was to evaluate whether endoglin may be associated with hepatic fibrosis featuring chronic hepatitis C virus (HCV) infection. Fifty-two anti-HCV+ patients, five anti-HCV- patients and 27 healthy subjects were studied. Western blot and immunohistochemistry were used to quantify the expression levels of endoglin and TGF-beta1 in liver biopsy samples, and serum concentrations of endoglin and hyaluronic acid were determined by enzyme-linked immunosorbent assays (ELISAs). In patients with advanced fibrosis, intrahepatic expression levels of endoglin and TGF-beta1 were significantly higher than those in patients with early fibrosis (mean: 3- and 5.8-fold, respectively) and normal liver (mean: 3.9- and 12-fold, respectively). Interestingly, activated hepatic stellate cells as well as portal and septal myofibroblasts expressed endoglin. Serum levels of endoglin were also significantly higher in patients with advanced fibrosis than in those with early fibrosis (55.5 +/- 1.6 vs 47.5 +/- 0.9 ng/mL, P < 0.001), showing a positive correlation with serum hyaluronic acid concentrations (r = 0.57, P = 0.01). In conclusion, increased intrahepatic endoglin and TGF-beta1 expression is significantly associated with progressive hepatic fibrosis in chronic HCV infection. Circulating endoglin levels are elevated in HCV patients showing a significant correlation with histological and serum markers of hepatic fibrosis. These data suggest an active role for endoglin in the fibrotic process featuring chronic HCV infection.

Colitis por Cryptosporidium como forma de presentación en un paciente con síndrome de inmunodeficiencia adquirida / Colitis by Cryptosporidium as initial manifestation of acquired immunodeficiency syndrome

No disponible

Association of pretreatment serum interferon gamma inducible protein 10 levels with sustained virological response to peginterferon plus ribavirin therapy in genotype 1 infected patients with chronic hepatitis C.

BACKGROUND: Increased serum and intrahepatic interferon gamma inducible protein 10 (IP-10) levels in patients with chronic hepatitis C (CHC) have been described. AIM: To analyse the possible association of serum IP-10 levels with different outcomes to antiviral therapy. PATIENTS: A total of 137 CHC patients treated with peginterferon plus ribavirin. METHODS: Serum IP-10 levels were determined by enzyme linked immunosorbent assay before therapy, after 12 weeks of treatment, and 24 weeks after cessation of therapy. Variables significantly associated with a sustained virological response (SVR) on univariate analysis were included in a multivariate logistic regression model. RESULTS: Pretreatment serum IP-10 levels in patients with SVR were significantly lower than in non-responders (NR) (332.4 (222.1) v 476.8 (305.3) pg/ml, respectively; p=0.004). Serum IP-10 concentrations significantly decreased in patients with SVR (pretreatment: 332.4 (222.1) pg/ml; post-treatment: 170.2 (140.1) pg/ml; p<0.001) but not in NR (pretreatment: 476.8 (305.3) pg/ml; post treatment: 387.3 (268.1) pg/ml; p=0.06). By multivariate analysis, non-1 genotype (odds ratio (OR) 3.5 (95% confidence interval (CI) 1.1-10.4); p=0.003) and low viral load at baseline (OR 0.34 (95% CI 0.14-0.79); p=0.01) were independent predictors of SVR in all patients. When multivariate analysis was restricted to patients with genotype 1, only baseline viral load (OR 0.38 (95% CI 0.155-0.96); p=0.04) and pretreatment serum IP-10 levels (OR 0.99 (95% CI 0.996-0.999); p=0.03) were identified as predictive factors of SVR. CONCLUSION: Pretreatment serum IP-10 behaves as a predictive factor of SVR to peginterferon plus ribavirin therapy in genotype 1 infected patients.

Gene expression profile of T-cell-specific chemokines in human hepatocyte-derived cells: evidence for a synergistic inducer effect of cytokines and hepatitis C virus proteins.

Increased levels of chemokines (CK) in chronic hepatitis C virus (HCV) infection have been found. Given that NS5A and core can function as transcriptional transactivators, we aimed to determine whether these HCV proteins might induce CK gene expression in human hepatocyte-derived cells. We assessed (i) regulated upon activation, normal T cells expressed and activated (RANTES), interferon gamma-inducible protein-10 (IP-10), and monokine induced by interferon-gamma (MIG) mRNA levels in NS5A and core stably transfected Chang liver (CHL) cells, stimulated or not with a cytokine mixture (CM), by reverse transcriptase-polymerase chain reaction (RT-PCR) and (ii) quantitative enzyme-linked immunosorbent assay (ELISA) measurements of these CK in the supernatants of CHL cells. Induction of RANTES transcripts in resting HCV-transfected cells was clearly observed, being augmented fourfold in resting NS5A-transfected cells and threefold in resting core-transfected cells over that in resting mock-transfected (control) cells, as well as to a similar extent in CM-stimulated NS5A- and core-transfected cells. Increased RANTES secretion followed the same pattern observed for mRNA expression. Both IP-10 and MIG, such as mRNA and protein levels, were undetectable in resting HCV-transfected and -untransfected cells, whereas IP-10 and MIG mRNA expression was increased by seven- and fivefold in CM-stimulated NS5A-transfected cells and by 10- and 3.5-fold in CM-stimulated core-transfected cells, respectively, above that in CM-stimulated control cells. IP-10 and MIG secretion was enhanced by 2.6- and threefold in CM-stimulated NS5A-transfected cells and by 3.6-fold and 3.7-fold in CM-stimulated core-transfected cells, respectively over that in CM-stimulated control cells. These results demonstrate that NS5A and core proteins, alone or by the synergistic effect of cytokines, are capable of upregulating RANTES, IP-10 and MIG gene expression in cultured human hepatocyte-derived cells.

[Autoimmune hepatitis in patients with a diagnosis of multiple sclerosis]. / Hepatitis autoinmune en pacientes con diagnóstico de esclerosis múltiple.

Autoimmune hepatitis (AIH) is a chronic necroinflammatory liver disorder associated with hypergammaglobulinemia and circulating autoantibodies. Two patients previously diagnosed with multiple sclerosis who developed AIH are reported. One patient showed acute presentation with fulminant hepatic failure requiring liver transplantation. Serum autoantibodies were negative in both patients but a characteristic clinical course in the first patient as well as the hepatic histological features with typical pathological changes of AIH in both patients and a score compatible with AIH established the diagnosis.

Increased intrahepatic cyclooxygenase 2, matrix metalloproteinase 2, and matrix metalloproteinase 9 expression is associated with progressive liver disease in chronic hepatitis C virus infection: role of viral core and NS5A proteins.

BACKGROUND: Cyclooxygenase 2 (COX-2) and matrix metalloproteinases (MMPs) have been implicated in tissue injury and fibrogenesis in animal models but little is known regarding their role in hepatitis C virus (HCV) related liver disease in humans. AIMS: To characterise the intrahepatic expression pattern of COX-2 and MMPs in chronic HCV infection and determine whether HCV core and NS5A proteins could promote their expression in cultured hepatocyte derived cell lines. PATIENTS: Thirty two anti-HCV+ and 10 anti-HCV- patients were studied. METHODS: Western blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunohistochemistry were used to assess the expression pattern of COX-2 and MMPs in liver biopsy samples from all patients. COX-2 gene expression and MMP-9 protein levels were also determined by immunoblot, RT-PCR, and luciferase assays in core and NS5A transfected hepatocyte derived cells. RESULTS: The intrahepatic expression level of COX-2, MMP-2, and MMP-9 was significantly higher in HCV+ than in HCV- patients, increasing with the fibrotic stage of liver disease. We further demonstrated that COX-2 mRNA, protein, and activity were induced in resting and activated core and NS5A transfectants. Both viral proteins induced transcriptional activity of the COX-2 gene promoter whereas core, but not NS5A, exerted an inducer effect on MMP-9 protein levels in cultured hepatocyte derived cells. CONCLUSIONS: Intrahepatic COX-2, MMP-2, and MMP-9 overexpression is associated with progressive hepatic fibrosis in chronic HCV infection, suggesting their pathogenic role in fibrogenesis. HCV core and NS5A proteins were able to upregulate COX-2 and MMP-9 gene expression in hepatocyte derived cells, providing a potential mechanism for hepatic fibrosis during chronic HCV infection.

Increased circulating and intrahepatic T-cell-specific chemokines in chronic hepatitis C: relationship with the type of virological response to peginterferon plus ribavirin combination therapy.

AIMS: To determine the serum and intrahepatic levels of T-helper-1-associated chemokines in patients with chronic hepatitis C before, during and after peginterferon plus ribavirin combination therapy and to search for correlations with baseline characteristics of hepatitis C virus-related chronic liver disease and type of therapeutic response. METHODS: Serum chemokine levels were determined by enzyme-linked immunosorbent assays and intrahepatic chemokine messenger RNA and protein levels were tested by ribonuclease protection assay and immunohistochemistry. RESULTS: Serum and intrahepatic chemokine levels were elevated in all patients with chronic hepatitis C and showed a marked decrease in patients who obtained a virological response vs. non-responders. Increased serum interferon-gamma-inducible protein-10 levels at baseline in genotype 1-infected patients were significantly associated with greater degrees of intrahepatic inflammation and fibrosis (P = 0.0046 and P = 0.02, respectively) and with virological non-response (P = 0.01). In patients with genotype 1, basal serum interferon-gamma-inducible protein-10 levels greater than 299 pg/mL identified 80% of non-responders and lower than 299 pg/mL identified 63% of responders. CONCLUSIONS: Circulating and intrahepatic T-helper-1-associated chemokines are abnormally elevated in patients with chronic hepatitis C. Increased baseline serum interferon-gamma-inducible protein-10 levels in genotype 1-infected patients are associated with virological non-response to peginterferon plus ribavirin combination therapy.

Papel de las quimioquinas en la patogenia de las enfermedades hepáticas / Role of chemokines in the pathogenesis of liver diseases

Objetivo: las quimioquinas son pequeñas proteínas quimiotácticas que son importantes en la patogenia de algunas enfermedades inflamatorias pero se desconoce su papel en la hepatitis crónica C. Material y métodos: hemos realizado una revisión de las publicaciones sobre quimioquinas y enfermedades hepáticas, con especial énfasis en hepatitis C, así como un estudio del patrón de expresión de ciertas quimioquinas CXC y CC y de sus receptores en el tejido hepático y en la sangre periférica de pacientes con hepatitis crónica C. Resultados: existen evidencias en la literatura de que diferentes quimioquinas CXC y CC se expresan intensamente en el hígado de estos pacientes. Nosotros hemos confirmado estos datos y además hemos observado que la mayoría de los linfocitos T intrahepáticos de estos pacientes expresaban sus receptores específicos. También hemos comprobado que el nivel de expresión de quimioquinas como Mig y RANTES correlacionaba con el grado de actividad inflamatoria de la hepatitis crónica. Conclusiones: todos estos datos sugieren que en el compartimento intrahepático de los pacientes con hepatitis crónica C la compleja red de interacciones entre las quimioquinas CXC y CC y sus receptores específicos juegan un papel relevante en la patogenia de un gran número de eventos patológicos que caracterizan a la hepatitis crónica C, por lo que acciones encaminadas a bloquear, inducir o modificar de alguna manera la señalización intrahepática de las quimioquinas podrían tener una importante repercusión en la historia natural de estas enfermedades del hígado y, por lo tanto, implicaciones terapéuticas. (AU)

Role of chemokines in the pathogenesis of liver diseases.

OBJECTIVE: while chemokines appear to be important in certain inflammatory disorders, little is known about the role of these proteins in chronic hepatitis C. MATERIAL AND METHODS: firstly, an extensive review of the expression pattern of chemokines in liver diseases with special emphasis in hepatitis C has been performed. Then, expression of selected CXC and CC chemokines and their receptors was assessed by immunohistochemistry and flow cytometry in a number of patients with chronic hepatitis C. RESULTS: evidence that certain CXC and CC chemokines are markedly expressed in chronic hepatitis C has been recently reported. In agreement with this, we have shown that the majority of liver-derived T lymphocytes from patients with chronic hepatitis C expressed CXCR3 and CCR5 chemokine receptors. In addition, an intense intrahepatic expression of their respective ligands, Mig and RANTES, was detected in the same patients studied. Furthermore, an association between the intrahepatic chemokine expression level and the inflammatory activity of chronic hepatitis C was found. CONCLUSIONS: all of these findings strongly suggest that intra hepatic chemokine signaling could play a key role regulating significant pathological events during chronic hepatitis C, opening new avenues for therapeutic interventions based on chemokines activities.

Hepatitis B virus X protein transactivates inducible nitric oxide synthase gene promoter through the proximal nuclear factor kappaB-binding site: evidence that cytoplasmic location of X protein is essential for gene transactivation.

Nitric oxide appears to play a central role in the pathogenesis of many inflammatory disorders. We have previously shown that there is an enhanced intrahepatic expression of the inducible nitric oxide synthase (iNOS) gene during chronic hepatitis B virus infection, and that viral X protein (HBx) transcriptionally activates this cellular gene, but the molecular basis for this activation remains to be defined. We aimed to explore the involvement of different cis-acting elements of the human iNOS promoter in the HBx-mediated up-regulation as well as the effect of the intracellular distribution of the HBx on its transacting function. Cotransfection of human hepatocyte-derived cells with wild-type or mutated iNOS promoter and with an HBx expression vector showed that functional inactivation of the proximal nuclear factor kappaB (NF-kappaB)-binding site of the iNOS promoter markedly reduced the HBx-mediated transcriptional activity. Mobility shift assays showed increased DNA-protein complexes comprising mainly the p50 and p65 members of NF-kappaB family in the nuclear extracts from HBx-transfected cells. Transient transfection experiments with HBx-expressing plasmids containing distinct cellular localization sequences showed that cytoplasmic location of this viral protein activated the iNOS promoter but when HBx was targeted to the nucleus, the HBx-induced luciferase activity was almost completely abrogated. In conclusion, cytoplasmic location of HBx protein is essential for the transcriptional activation of the iNOS gene through the nuclear translocation of p50-p65 heterodimers.

S-Adenosylmethionine modulates inducible nitric oxide synthase gene expression in rat liver and isolated hepatocytes.

BACKGROUND/AIMS: Hepatocellular availability of S-adenosylmethionine, the principal biological methyl donor, is compromised in situations of liver damage. S-Adenosylmethionine administration alleviates experimental liver injury and increases survival in cirrhotic patients. The mechanisms behind these beneficial effects of S-adenosylmethionine are not completely known. An inflammatory component is common to many of the pathological conditions in which S-adenosylmethionine grants protection to the liver. This notion led us to study the effect of S-adenosylmethionine administration on hepatic nitric oxide synthase-2 induction in response to bacterial lipopolysaccharide and proinflammatory cytokines. METHODS: The effect of S-adenosylmethionine on nitric oxide synthase-2 expression was assessed in rats challenged with bacterial lipopolysaccharide and in isolated rat hepatocytes treated with proinflammatory cytokines. Interactions between S-adenosylmethionine and cytokines on nuclear factor kappa B activation and nitric oxide synthase-2 promoter transactivation were studied in isolated rat hepatocytes and HepG2 cells, respectively. RESULTS: S-Adenosylmethionine attenuated the induction of nitric oxide synthase-2 in the liver of lipopolysaccharide-treated rats and in cytokine-treated hepatocytes. S-Adenosylmethionine accelerated the resynthesis of inhibitor kappa B alpha, blunted the activation of nuclear factor kappa B and reduced the transactivation of nitric oxide synthase-2 promoter. CONCLUSIONS: Our findings indicate that the hepatoprotective actions of S-adenosylmethionine may be mediated in part through the modulation of nitric oxide production.