Angiogenesis: From Chronic Liver Inflammation to Hepatocellular Carcinoma

Recently, new information relating to the potential relevance of chronic hepatic inflammation to the development and progression of hepatocellular carcinoma (HCC) has been generated. Persistent hepatocellular injury alters the homeostatic balance within the liver; deregulation of the expression of factors involved in wound healing may lead to the evolution of dysplastic lesions into transformed nodules. Progression of such nodules depends directly on the development and organization of a vascular network, which provides the nutritional and oxygen requirements to an expanding nodular mass. Angiogenic stimulation promotes intense structural and functional changes in liver architecture and physiology, in particular, it facilitates transformation of dysplasia to nodular lesions with carcinogenic potential. HCC depends on the growth and spreading of vessels throughout the tumor. Because these vascular phenomena correlate with disease progression and prognosis, therapeutic strategies are being developed that focus on precluding vascular expansion in these tumors. Accordingly, an in-depth study of factors that promote and support pathological angiogenesis in chronic hepatic diseases may provide insights into methods of preventing the development of HCC and/or stimulating the regression of established HCC

High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods

HepatitisCvirus(HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, in comparisonwith thoseof two commercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing(69%1b,31%1a) in 81 specimensandidentified amixed-subtype infection (1b/3a/1a) in one sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing in all but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in 13 (16%) samples eachandwere unable to identify theHCVgenotype and/or subtype inmore than half of the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methodsandallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect to informing treatment strategies withnewDAA-containing regimens across allHCVsubtypesThis study has been supported by CDTI (Centro para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20110115; MINECO projects SAF 2009-10403; and also by the Spanish Ministry of Health, Instituto de Salud Carlos III (FIS) projects PI10/01505, PI12/01893, and PI13/00456. CIBERehd is funded by the Instituto de Salud Carlos III, Madrid, Spain. Work at CBMSO was supported by grant MINECO-BFU2011-23604, FIPSE, and Fundación Ramón Areces. X. Forns received unrestricted grant support from Roche and has acted as advisor for MSD, Gilead, and Abbvie. M. Alvarez-Tejado, J. Gregori, and J. M. Muñoz work in Roche Diagnostic

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

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.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.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.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

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 (p40 IU/L (p40 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

<div><p>Background</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

The activation and invasive potential of HSC are prevented by a Tie2 neutralizing antibody or by the inhibition of Akt/PI3k and MAPK signaling pathways.

<p>(A) α-SMA expression was assessed by western blotting (1A4, 1∶1000) in 20 µL of Laemmli lysates from HSC exposed during 24 h to different culture conditions (hepatic-derived CM, 0% FBS DMEM or 10% FBS DMEM) in presence (+) or absence (-) anti-Tie2 blocking antibody (AF313, 8 µg/ml). Quantitative analysis of α-SMA/tubulin bands in presence of neutralizing antibody is displayed in the graph as percentage of expression observed without anti-Tie2 (100%) for each experimental condition. Bars show mean +SD of 3 independent experiments. *p<0.05. (B) HSC cells (5×10⁴ cells/100 µL) cultured on upper transwell chambers able to invade collagen under different stimuli (hepatic-derived CM, 0% FBS DMEM or 10% FBS DMEM) with or without AF313 anti-Tie2 antibody (8 µg/ml) dispensed at bottom compartments of transwell are illustrated in the representative epifluorescence pictures after DAPI staining (400x). 5 randomly selected microscope fields were quantified per experiment (3 independent). Bars show the mean +SD average of migrating cells per field from the different experiments. *p<0.05 indicates statistical differences owing to the presence of anti-Tie2 neutralizing antibody. (C) The influence of different CM in presence or absence of LY294002 and PD98059 at 25 µmol/ml each (Akt/PI3k and MAPK inhibitors, respectively) on the expression of Tie2 (AF313, 1∶200) and α-SMA (1A4, 1∶1000) by HSC is illustrated. Respective western blots were analyzed in relation to tubulin to normalize total protein loading and the expression in presence of inhibitors was displayed as percentage of the expression without the respective compound (100%) in the same experimental conditions. Data from 3 experiments in duplicate are shown. *p<0.05. (D) Invasive potential of HSC (5×10⁴ cells/100 µL) under the influence of LY294002 and PD98059 (25 µmol/ml both) at different experimental conditions (hepatic-derived CM, 0% FBS DMEM or 10% FBS DMEM) is shown by fluorescence images (400x). Graph depicts the mean +SD of average migrating HSC/field (5 microscope fields) per experiment (3 independent) of denoted HSC culture conditions. *p<0.05: statistical difference of presence <i>versus</i> absence of inhibitors.</p

Activation of HSC increases Tie2 expression.

<p>(A) Kinetics of Tie2 expression during <i>in vitro</i> activation of HSC. HSC, plated at 50,000 cells/cm2 density and grown in 2% DMEM during 24, 48, 72 and 96 hours, were lysed in Laemmli buffer and loaded in 7% acrylamide gels (20 µL of total protein extract per well). SDS-PAGE resolved proteins were transferred to nitrocellulose membranes and probed with respective antibodies against COL-I (H-197, 1∶2000), Tie2 (AF313, 1∶200) or tubulin (DM1A, 1∶5000). Quantitative analysis of Tie2 or COL-1 chemiluminescence in relation to tubulin (FUJIFILM Science Lab Image Gauge) is shown in respective graphs. *p<0.05 <i>versus</i> 24 h (mean +SD, 3 independent experiments). (B) Expression of HCV proteins by hepatic cell lines (Huh7 or HCV replicons). Protein lysates from hepatic cells (20 µL each) were SDS-PAGE resolved and probed with antibodies against Core (C7–50, 1∶500), NS5A (6F3, 1∶1000) or tubulin (DM1A, 1∶5000). (C) Expression of α-SMA in HSC exposed to conditioned media (CM) from hepatic cell lines (Huh7 and HCV replicons) compared to that expressed by HSC incubated with 0% FBS DMEM. Expression α-SMA by HSC grown in 10% FBS DMEM was used as positive control of HSC activation. CM from hepatic cells, plated at equal densities and cultured during 24 h in 0% FBS DMEM, were used to grow HSC deprived of serum 24 h before. After HSC lysis in Laemmli, equal volumes (20 µL) of protein extracts were analyzed for α-SMA expression by western blotting (1A4, 1∶1000). Tubulin expression (DM1A, 1∶5000) was also evaluated in order to control protein loading and relative α-SMA/tubulin data are shown in the graph (mean +SD of 3 independent experiments). #p<0.05: statistic differences <i>versus</i> cultures in 0% FBS DMEM; *p<0.05: statistic differences <i>versus</i> Huh7. (D) HSC conditioned during different times with media from hepatic cells (Huh7 or HCV replicons) or 10% FBS DMEM. HSC, plated at same density in 0% FBS DMEM 24 h before, were exposed during 24, 48 or 72 h to 10% FBS DMEM or CM from hepatic cell lines and the expression of Tie2 (AF313, 1∶200), normalized with tubulin (DM1A, 1∶5000), was analyzed. Graph shows quantitative densitometric analysis of western blot bands (mean +SD of 3 independent experiments). #p<0.05: statistic differences <i>versus</i> Huh7 at same times; *p<0.05, same CM <i>versus</i> 24 h.</p