CYP2C8*3 and *4 define CYP2C8 phenotype: An approach with the substrate cinitapride.

Cinitapride is a gastrointestinal prokinetic drug, prescribed for the treatment of functional dyspepsia, and as an adjuvant therapy for gastroesophageal reflux disease. In this study, we aimed to explore the impact of relevant variants in CYP3A4 and CYP2C8 and other pharmacogenes, along with demographic characteristics, on cinitapride pharmacokinetics and safety; and to evaluate the impact of CYP2C8 alleles on the enzyme's function. Twenty-five healthy volunteers participating in a bioequivalence clinical trial consented to participate in the study. Participants were genotyped for 56 variants in 19 genes, including cytochrome P450 (CYP) enzymes (e.g., CYP2C8 or CYP3A4) or transporters (e.g., SLC or ABC), among others. CYP2C8*3 carriers showed a reduction in AUC of 42% and Cmax of 35% compared to *1/*1 subjects (p = 0.003 and p = 0.011, respectively). *4 allele carriers showed a 45% increase in AUC and 63% in Cmax compared to *1/*1 subjects, although these differences did not reach statistical significance. CYP2C8*3 and *4 alleles may be used to infer the following pharmacogenetic phenotypes: ultrarapid (UM) (*3/*3), rapid (RM) (*1/*3), normal (NM) (*1/*1), intermediate (IM) (*1/*4), and poor (PM) metabolizers (*4/*4). In this study, we properly characterized RMs, NMs, and IMs; however, additional studies are required to properly characterize UMs and PMs. These findings should be relevant with respect to cinitapride, but also to numerous CYP2C8 substrates such as imatinib, loperamide, montelukast, ibuprofen, paclitaxel, pioglitazone, repaglinide, or rosiglitazone.

Histone modifications associated with biological drug response in moderate-to-severe psoriasis.

INTRODUCTION: Epigenetic factors play an important role in psoriasis onset and development. Biological drugs are used to treat moderate-to-severe psoriasis patients resistant to conventional systemic drugs. Although they are safe and effective, some patients do not respond to them. Therefore, it is necessary to find biomarkers that could predict response to these therapies. OBJECTIVE: To find epigenetic biomarkers that could predict response to biological drugs (ustekinumab, secukinumab, adalimumab, ixekizumab). MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 39 psoriasis patients treated with biological therapies before and after drug administration and from 42 healthy subjects. Afterwards, histones were extracted from PBMCs. Four histone modifications (H3 and H4 acetylation, H3K4 and H3K27 methylation) were determined by ELISA. Data were analysed by IBM-SPSS v.23. RESULTS AND CONCLUSIONS: Psoriasis patients presented reduced levels of acetylated H3 and H4 and increased levels of methylated H3K4 compared to controls. Non-significant changes were observed after treatment administration in any of the histone modifications analysed. Nevertheless, significant changes in methylated H3K27 were found between responders and non-responders to biological drugs at 3 months. As 28% of these patients also presented psoriatic arthritis (PsA), the former analysis was repeated in the subsets of patients with or without PsA. In patients without PsA, significant changes in methylated H3K4 were found between responders and non-responders to biological drugs at 3 and 6 months. Although further studies should confirm these results, these findings suggest that H3K27 and H3K4 methylation may contribute to patients' response to biological drugs in psoriasis.

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 patient's 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.

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

BACKGROUND & AIMS: 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. METHODS: Ang1 and Ang2 expression was evaluated by western blotting and enzyme-linked immunosorbent assay (ELISA) in liver homogenates of CHC patients (n=47) and uninfected subjects (n=8). Their association with disease progression (according to METAVIR classification) was assessed by Spearman's correlation. Statistical differences among the expression of angiopoietins at different CHC stages were calculated by Mann-Whitney U-test. Finally, the in vitro expression of Angiopoietins in HCV replicons (complete or non-structural subgenomic) and the main signalling pathways involved were also examined. RESULTS: Ang2 levels were significantly higher in the liver of CHC patients compared to controls and significantly correlated with inflammation and fibrosis. Accordingly, an increased expression of Ang2 was found in all HCV replicons tested. Interestingly, the inhibition of MEK and PI3K signalling pathways exerted differential effects on Ang2 expression concerning to the genomic region of HCV. CONCLUSIONS: Hepatitis C virus induces Ang2 expression in hepatocytes through different signalling routes which may lead to the disregulation of vascular homeostasis in the liver. Thus, pharmacologic intervention on Ang2 signalling might constitute an important therapeutic tool.

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.

Angiopoietin-2 Serum Levels Improve Noninvasive Fibrosis Staging in Chronic Hepatitis C: A Fibrogenic-Angiogenic Link.

AIMS: Accurate liver fibrosis staging is crucial for the management of chronic hepatitis C (CHC). The invasiveness and cost burden of liver biopsy have driven the search for new noninvasive biomarkers of fibrosis. Based on the link between serum angiopoietin-1 and 2 levels and CHC progression, we aimed to determine the value of these angiogenic factors as noninvasive biomarkers of liver fibrosis. METHODS: Serum levels of angiopoietin-1 and -2 were measured by ELISA in 108 CHC patients who underwent pretreatment liver biopsy. The correlation between angiopoietins and clinical and demographic variables with liver fibrosis was analyzed by univariate regression. Significant factors were then subjected to multivariate analysis, from which we constructed a novel noninvasive liver fibrosis index (AngioScore), whose performance was validated in an independent series of 71 CHC patients. The accuracy of this model was compared with other documented fibrosis algorithms by De Long test. RESULTS: Angiopoietins correlated significantly with hepatic fibrosis; however, only angiopoietin-2 was retained in the final model, which also included age, platelets, AST, INR, and GGT. The model was validated and behaved considerably better than other fibrosis indices in discriminating all, significant, moderate and severe liver fibrosis (0.886, 0.920, 0.923). Using clinically relevant cutoffs, we classified CHC patients by discarding significant fibrosis and diagnosing moderate and severe fibrosis with greater accuracy, sensitivity, and specificity. CONCLUSIONS: Our novel noninvasive liver fibrosis model, based on serum angiopoietin-2 levels, outperforms other indices and should help substantially in managing CHC and monitoring long-term follow-up prognosis.

Preliminary evidence of sustained expression of angiopoietin-2 during monocyte differentiation in chronic hepatitis C.

BACKGROUND: Monocytes are essential precursors of antigen-presenting cells, such as macrophages and dendritic cells, and contribute to the pathogenesis of chronic inflammatory diseases and cancer. AIMS: As Tie2-expressing monocytes (TEMs) are increased in the peripheral blood of patients with chronic hepatitis C (CHC), we aimed to examine the expression of Tie2 and angiopoietins (Ang1 and Ang2) during monocyte differentiation and maturation in CHC. METHODS: The expression of Tie2, CD11b, CD80, CD83, CD86 and MHC-II was measured by flow cytometry in peripheral blood monocytes and monocytes-derived cells (Mo-DCs) from nine healthy subjects and eight CHC patients whose HCV infection was unresolved after combination therapy. Ang1 and Ang2 levels were measured in cellular supernatants by ELISA. RESULTS: Mo-DCs from CHC patients expressed differential patterns of maturation markers compared with controls--primarily with regard to CD80. Tie2 was downregulated during monocyte differentiation in controls and CHC patients, whereas Ang1 expression was constant. However, Ang2 levels fell significantly during the differentiation of control monocytes, in contrast with those from CHC patients in whom Ang2 expression remained stable throughout differentiation. CONCLUSIONS: Altered expression of the Ang/Tie2 system in monocytes and Mo-DCs from CHC patients might account for the inflammatory and angiogenic disorders that are related to CHC. An increased understanding of Ang/Tie2 system regulation might be helpful in designing strategies to prevent CHC progression.

[Effects of hepatitis B virus X protein on chronic hepatitis B pathophysiology]. / Mecanismos de acción y efectos de la proteína X (HBx) en la patogenia de la hepatitis crónica B.

The infection by hepatitis B virus often promotes chronic liver inflammation which progresses to cirrhosis and hepatocellular carcinoma in a high percentage of patients. The persistent activation of the immune system causes an incessant liver damage, which fosters a disorganized stimulation of tissue repair and remodelling phenomena. In turn, the viral protein X (HBx) is essential for virus replication and therefore for the maintenance of chronic infection. However, the important oncogenic potential of HBx seems to reside, on one hand, in its ability to integrate into cellular DNA and, additionally, in the transactivation of different cellular signaling pathways involved in cell growth regulation, apoptosis and DNA repair. HBx also interacts with proteasome subunits and notably affects mitochondrial electric potential, thus altering cellular calcium homeostasis. Finally, this review discusses the pathogenic role of HBx in the progression of chronic hepatitis B through its effects on angiogenic, fibrogenic and oncogenic processes.

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.

Expression of pituitary tumor-transforming gene 1 (PTTG1)/securin in hepatitis B virus (HBV)-associated liver diseases: evidence for an HBV X protein-mediated inhibition of PTTG1 ubiquitination and degradation.

Chronic infection with hepatitis B virus (HBV) is strongly associated with hepatocellular carcinoma (HCC), and the viral HBx protein plays a crucial role in the pathogenesis of liver tumors. Because the protooncogene pituitary tumor-transforming gene 1 (PTTG1) is overexpressed in HCC, we investigated the regulation of this protein by HBx. We analyzed PTTG1 expression levels in liver biopsies from patients chronically infected with HBV, presenting different disease stages, and from HBx transgenic mice. PTTG1 was undetectable in biopsies from chronic hepatitis B patients or from normal mouse livers. In contrast, hyperplastic livers from transgenic mice and biopsies from patients with cirrhosis, presented PTTG1 expression which was found mainly in HBx-expressing hepatocytes. PTTG1 staining was further increased in HCC specimens. Experiments in vitro revealed that HBx induced a marked accumulation of PTTG1 protein without affecting its messenger RNA levels. HBx expression promoted the inhibition of PTTG1 ubiquitination, which in turn impaired its degradation by the proteasome. Glutathione S-transferase pull-down and co-immunoprecipitation experiments demonstrated that the interaction between PTTG1 and the Skp1-Cul1-F-box ubiquitin ligase complex (SCF) was partially disrupted, possibly through a mechanism involving protein-protein interactions of HBx with PTTG1 and/or SCF. Furthermore, confocal analysis revealed that HBx colocalized with PTTG1 and Cul1. We propose that HBx promotes an abnormal accumulation of PTTG1, which may provide new insights into the molecular mechanisms of HBV-related pathogenesis of progressive liver disease leading to HCC development.

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

UNLABELLED: 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, alpha smooth muscle actin, matrix metalloproteinase-2, and transforming growth factor-beta (TGF-beta), together with an enhanced proliferation rate. We found that HBx induced TGF-beta secretion in hepatocytes and that the activation of HSCs by conditioned medium from HBx-expressing hepatocytes was prevented by a neutralizing anti-TGF-beta 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.

Hepatitis B virus promotes angiopoietin-2 expression in liver tissue: role of HBV x protein.

The progression of chronic hepatitis B (CHB) is related to fibrosis and to the emergence of intrahepatic anomalous vascular structures. Hepatitis B virus (HBV) X protein transactivator (HBx) may play a significant role in both processes. To analyze how HBV induces vascular growth and remodeling in vivo, we assessed the expression of angiopoietin-2 (Ang2) in liver biopsies from CHB patients by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry because of the relevant role of Ang2 in vascular development, remodeling, and tumor promotion. In addition, we analyzed the influence of HBx in the expression of Ang2 in HBx-expressing hepatocyte cell lines and in hepatic stellate cells stimulated with conditional medium from HBx-hepatocytes. Ang2 expression was clearly up-regulated at both mRNA and protein levels in the liver of CHB patients, showing an intense staining of inflammatory infiltrates and vascular structures at inflamed portal areas. HBx-expressing hepatocytes and stimulated stellate cells showed a significant induction of Ang2 expression. PI3K inhibitor and antioxidants repressed the 64-kd Ang2 form but further enhanced the inflammation-related 50-kd molecular species. Therefore, HBx could account for the induction of Ang2 observed in CHB, especially the 50-kd form, contributing to pathological angiogenesis and hepatocellular carcinoma progression.

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.

Evidence of angiogenesis in primary biliary cirrhosis: an immunohistochemical descriptive study.

BACKGROUND/AIMS: The intrahepatic inflammatory process occurring during primary biliary cirrhosis contributes to bile duct destruction, but the cellular and molecular pathways involved are largely unknown. Furthermore, additional pathogenetic mechanisms may exist. We aimed at evaluating the cellular infiltrate phenotype; the expression of lymphocyte activation, antigen recognition and cell-adhesion molecules; the occurrence of hepatic angiogenesis and the molecules involved. METHODS: Immunohistochemical investigations were performed in frozen liver biopsy sections from primary biliary cirrhosis patients. RESULTS: CD8+ and CD69+ T cells were predominant in inflammatory infiltrates around damaged cholangiocytes; beta2-microglobulin conformational epitope and intercellular adhesion molecule-1 expression were enhanced in bile ducts and hepatocytes. Inflamed portal areas showed vascular cell adhesion molecule-1 up-regulation; formation of tubule-like structures (neovessels) by endothelial cells expressing vascular endothelial-cadherin and CD-31; vascular endothelial growth factor expression in surrounding sinusoidal endothelial cells; and enhanced expression of angiopoietins 1 and 2, their receptor Tie-2 and endoglin, suggesting their involvement in new vascular structure formation. CONCLUSIONS: The inflammatory infiltrate in primary biliary cirrhosis shows an increased reactivity for lymphocyte activation, antigen recognition and cell- and vascular-adhesion molecules. Additionally, intrahepatic angiogenesis occurs, involving vascular endothelial growth factor, angiopoietins 1 and 2, Tie-2 and endoglin in neovessel formation.

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.