S. Martin-Vilchez

Enhanced intrahepatic inducible nitric oxide synthase expression and nitrotyrosine accumulation in primary biliary cirrhosis and autoimmune hepatitis.

BACKGROUND/AIMS Nitrosative stress resulting from increased nitric oxide (NO) synthesis contributes to the pathogenesis of chronic inflammatory diseases, including chronic viral hepatitis. Our goal was to assess the expression of inducible nitric oxide synthase (iNOS) and the formation of nitrotyrosine (NTY), as a marker of nitrosative stress, in liver biopsies from primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) patients. METHODS Intrahepatic expression of iNOS and NTY was measured immunohistochemically and compared to histological scores of the severity of liver disease. RESULTS Hepatocellular iNOS expression was observed in liver sections from PBC patients (with a diffuse lobular distribution) and from AIH patients (marked staining in areas of pronounced inflammation and necrosis), but not in control liver sections, including non-autoimmune cholestatic liver disease. Liver samples from PBC and AIH patients, but not from controls, showed NTY accumulation in clusters of hepatocytes and Kupffer cells. Increased iNOS expression and NTY accumulation correlated with the histological severity of PBC or AIH, especially with the degree of inflammation. CONCLUSIONS Patients with PBC and AIH showed an enhanced intrahepatic iNOS expression and NTY accumulation, related to the histological severity of liver disease, consistent with NO-mediated nitration of hepatocellular proteins contributing to liver damage in both diseases.

The hepatitis B virus X protein induces paracrine activation of human hepatic stellate cells.

Chronic hepatitis B virus (HBV) infection is a major cause of liver fibrosis, eventually leading to cirrhosis and hepatocellular carcinoma. Although the involvement of the X protein of HBV (HBx) in viral replication and tumor development has been extensively studied, little is known about its possible role in the development of fibrosis. In this work we show that expression of HBx in hepatocytes results in paracrine activation and proliferation of hepatic stellate cells (HSCs), the main producers of extracellular matrix proteins in the fibrotic liver. Both human primary HSCs and rat HSCs exposed to conditioned medium from HBx‐expressing hepatocytes showed increased expression of collagen I, connective tissue growth factor, α smooth muscle actin, matrix metalloproteinase‐2, and transforming growth factor‐β (TGF‐β), together with an enhanced proliferation rate. We found that HBx induced TGF‐β secretion in hepatocytes and that the activation of HSCs by conditioned medium from HBx‐expressing hepatocytes was prevented by a neutralizing anti‐TGF‐β antibody, indicating the involvement of this profibrotic factor in the process. Conclusion: Our results propose a direct role for HBx in the development of liver fibrosis by the paracrine activation of stellate cells and reinforce the indication of antiviral treatment in patients with advanced HBV‐related chronic liver disease and persistent liver replication. (HEPATOLOGY 2008.)

The potential of angiogenesis soluble markers in chronic hepatitis C

Angiogenesis, the formation of new vessels, has been reported to play a significant pathogenic role in liver damage–associated hepatitis C virus infection. Most of our current knowledge derives from immunohistochemical studies of hepatic biopsy samples obtained from chronic hepatitis C (CHC) patients. We evaluated whether CHC is associated with elevated serum levels of angiogenesis markers and whether these are modulated by therapy. Vascular endothelial growth factor (VEGF), angiopoietin‐2 (Ang‐2), and soluble Tie‐2 (sTie‐2) were determined in the serum of 36 CHC patients, before and after receiving antiviral combination therapy with pegylated interferon alpha‐2b plus ribavirin, and in 15 healthy controls. CHC patients showed elevated baseline VEGF and Ang‐2 levels. After treatment, both factors were decreased, whereas antiangiogenic sTie‐2 was increased, indicating a shift toward an “anti‐angiogenic” profile of serum markers in CHC patients. In conclusion, this suggests that serum VEGF, Ang‐2, and sTie‐2 levels could be useful as noninvasive, mechanistically based markers of response to therapy and disease progression in CHC. (HEPATOLOGY 2005.)

Hepatocyte growth factor activates endothelial proangiogenic mechanisms relevant in chronic hepatitis C-associated neoangiogenesis

BACKGROUND Angiogenesis occurs in inflamed portal tracts of chronic hepatitis C (CHC) patients. AIMS To characterize this phenomenon, by investigating the molecular mechanisms involved in neovessel formation in the livers of CHC patients and the angiogenic effects of hepatocyte growth factor (HGF) on human endothelial cells. METHODS Vascular endothelial growth factor (VEGF), VE-cadherin and alphavbeta3 integrin were determined in CHC biopsies by Western blot and immunohistochemistry. Effects of HGF on VEGF and cell adhesion molecules expression by cultured human microvascular endothelial cells were evaluated by Western blot, Northern blot or immunofluorescence. HGF effects on cell proliferation were assessed by [(3)H]thymidine incorporation. RESULTS VEGF, VE-cadherin and alphavbeta3 integrin were increased in CHC liver samples. In cultured endothelial cells, HGF transcriptionally increased VEGF expression, an effect which was blocked by an anti-VEGF receptor antibody. HGF transiently decreased VE-cadherin expression and its associated cytoskeleton-linking molecule beta-catenin, thus weakening intercellular contacts. HGF increased alphavbeta3 integrin at focal contacts, and cell proliferation, an effect which was inhibited by an anti-VEGF receptor antibody. CONCLUSIONS Our results show that HGF and VEGF modulate the expression of cell adhesion and migration molecules and induce proliferation in endothelial cells, mechanisms through which these factors may contribute to CHC-associated liver angiogenesis.

Review article: angiogenesis soluble factors as liver disease markers.

Angiogenesis is the formation of new blood vessels from pre‐existing ones; it has been studied at the molecular level in different pathologies and is currently considered a promising novel therapeutic target in cancer. Recently, the use of angiogenesis soluble factors as markers of tumour growth has been investigated. The knowledge gained has led to test their use as therapeutic agents. Additionally, angiogenesis soluble factors could be used for the follow‐up of pathologies that currently require monitoring with invasive techniques, like chronic viral hepatitis or renal and haematological diseases. The different factors have been described in multiple studies. In some cases, such as hepatocellular carcinoma, a potential use as prognostic markers has been suggested.

The molecular and pathophysiological implications of hepatitis B X antigen in chronic hepatitis B virus infection.

Hepatitis B virus is considered one of the most significant environmental carcinogens in humans. Because the mechanisms of HBV replication and the development of hepatocellular carcinoma (HCC) are partially known, HBV‐associated pathogenesis remains a challenge to increase its understanding. Evidence suggests that the regulatory protein hepatitis B virus X (HBx) mediates the establishment and maintenance of the chronic carrier state. HBx is a multifunctional and potentially oncogenic protein that is conserved among mammalian hepadnaviruses; it is produced very early after infection and throughout the chronic phase. HBx exerts its effects by interacting with cellular proteins and activating various signaling pathways. HBx stimulates the transcription of genes that regulate cell growth, apoptosis, and DNA repair. It also interacts with proteasome subunits and affects mitochondrial stability. Moreover, HBx participates in processes that are associated with the progression of chronic liver disease, including angiogenesis and fibrosis. This review discusses the function of HBx in the life cycle of HBV and its contribution to the pathogenesis of HCC. Copyright

Polymorphisms in histone deacetylases improve the predictive value of IL-28B for chronic hepatitis C therapy

Histone deacetylases (HDACs) influence many cellular processes, including the modulation of signal transducer and activator of transcription activity (STAT) in response to interferon (IFN). To identify genetic markers that help optimize the IL-28B prediction of chronic hepatitis C (CHC) sustained virological response (SVR), we evaluated 27 single-nucleotide polymorphisms (SNPs) in HDAC1–11. Three SNPs, rs3778216, rs976552 and rs368328 in HDAC2, HDAC3 and HDAC5, respectively, were independently associated with SVR (P<0.05). The addition of these three HDAC’s SNPs to the IL-28B predictive model (area under the curve (AUC)=0.630) rendered an important improvement of AUC-receiver operating characteristic value (AUC=0.747, P=0.021). Chi-squared Automatic Interaction Detector (CHAID) analysis denoted the significance of the rs3778216 C/C genotype in identifying a group of good responders despite carrying IL-28B T allele (79.2% of SVR), whereas HDAC5 G allele characterized a subgroup with poor response rate (25.5%). However, HDAC3 rs976552 did not display a relevant role for the hierarchical classification of patients. Variables related to SVR in hepatitis C virus genotype 1 (HCV-1) cohort were the same of those obtained for the overall population. Interestingly, in non-HCV-1 patients (n=56) the HDAC2 C/C genotype was the unique predictive variable related to SVR (AUC=0.733, P<0.007). Thus, these preliminary results suggest the potential usefulness of combined IL-28B and HDAC genotyping for the CHC patients’ classification by likelihood of an SVR.

Interferon-related genetic markers of necroinflammatory activity in chronic hepatitis C

Introduction Chronic hepatitis C (CHC) is a major cause of liver disease worldwide which often leads to progressive liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma (HCC). CHC displays heterogeneous progression depending on a broad set of factors, some of them intrinsic to each individual such as the patients genetic profile. This study aims to evaluate the contribution of certain genetic variants of crucial interferon alpha and lambda signaling pathways to the hepatic necroinflammatory activity (NIA) grade of CHC patients. Methods NIA was evaluated in 119 CHC patients by METAVIR scale and classified as low (NIA = 0–2, n = 80) or high grade (NIA = 3, n = 39). In a candidate gene approach, 64 SNPs located in 30 different genes related to interferon pathways (IL-28B, IFNAR1-2, JAK-STAT and OAS1-3, among others) were genotyped using the Illumina GoldenGate® Genotyping Assay. Statistical association was determined by logistic regression and expressed as OR and 95% CI. Those SNPs significantly associated were further adjusted by other covariates. Results Seven SNPs located in IL-28B (rs12979860), JAK1 (rs11576173 and rs1497056), TYK2 (rs280519), OAS1 (rs2057778), SOCS1 (rs33932899) and RNASEL (rs3738579) genes were significantly related to severe NIA grade (p<0.05). Regarding to clinical variables, elevated NIA was notably associated with aspartate aminotransferase (AST) serum levels >40 IU/L (p<0.05) but not with other clinical factors. Multivariate logistic regression analysis of these factors reflected that AST (>40 IU/L), TYK2 rs280519 (G allele) and RNASEL rs3738579 (G allele) were factors independently associated with elevated NIA (p<0.05). AST concentration showed a moderate AUC value (AUC = 0.63), similar to TYK2 (rs280519) and RNASEL (rs3738579) SNPs (AUC = 0.61, both) in the ROC_AUC analysis. Interestingly, the model including all significant variables reached a considerable predictive value (AUC = 0.74). Conclusion The identified genetic variants in interferon signaling pathways may constitute useful prognostic markers of CHC progression. Further validation in larger cohorts of patients is needed.

Inhibition of Tyrosine Kinase Receptor Tie2 Reverts HCV-Induced Hepatic Stellate Cell Activation

Background Hepatitis C virus (HCV) infection is a major cause of chronic liver disease (CLD) and is frequently linked to intrahepatic microvascular disorders. Activation of hepatic stellate cells (HSC) is a central event in liver damage, due to their contribution to hepatic renewal and to the development of fibrosis and hepatocarcinoma. During the progression of CLDs, HSC attempt to restore injured tissue by stimulating repair processes, such as fibrosis and angiogenesis. Because HSC express the key vascular receptor Tie2, among other angiogenic receptors and mediators, we analyzed its involvement in the development of CLD. Methods Tie2 expression was monitored in HSC cultures that were exposed to media from HCV-expressing cells (replicons). The effects of Tie2 blockade on HSC activation by either neutralizing antibody or specific signaling inhibitors were also examined. Results Media from HCV-replicons enhanced HSC activation and invasion and upregulated Tie2 expression. Notably, the blockade of Tie2 receptor (by a specific neutralizing antibody) or signaling (by selective AKT and MAPK inhibitors) significantly reduced alpha-smooth muscle actin (α-SMA) expression and the invasive potential of HCV-conditioned HSC. Conclusions These findings ascribe a novel profibrogenic function to Tie2 receptor in the progression of chronic hepatitis C, highlighting the significance of its dysregulation in the evolution of CLDs and its potential as a novel therapeutic target.

Intrahepatic angiopoietin-2 correlates with chronic hepatitis C progression and is induced in hepatitis C virus replicon systems.

Chronic hepatitis C (CHC) is a major cause of cirrhosis and hepatocellular carcinoma and angiogenesis is closely related to the pathogenesis and progression of different chronic liver diseases (CLD). Thus, the intrahepatic expression of angiopoietins 1 and 2 (Ang1 and Ang2), as relevant mediators of pathological angiogenesis in several CLD, was investigated. In addition, the differential influence of structural and non‐structural genomic regions of HCV on the expression of angiopoietins and the possible signalling involved were studied.