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The clinical course of chronic liver diseases is significantly dependent on the progression rate of fibrosis which is the unstructured replacement of injured parenchyma by extracellular matrix. Despite intensive studies, the clinical opportunities for patients with fibrosing liver diseases have not improved. This will be changed by increasing knowledge of new pathogenetic mechanisms, which complement the "canonical principle" of fibrogenesis. The latter is based on the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts induced by hepatocellular injury and consecutive inflammatory mediators such as TGF-β. Stellate cells express a broad spectrum of matrix components. New mechanisms indicate that the heterogeneous pool of (myo-)fibroblasts can be supplemented by epithelial-mesenchymal transition (EMT) from cholangiocytes and potentially also from hepatocytes to fibroblasts, by influx of bone marrow-derived fibrocytes in the damaged liver tissue and by differentiation of a subgroup of monocytes to fibroblasts after homing in the damaged tissue. These processes are regulated by the cytokines TGF-β and BMP-7, chemokines, colony-stimulating factors, metalloproteinases and numerous trapping proteins. They offer innovative diagnostic and therapeutic options. As an example, modulation of TGF-β/BMP-7 ratio changes the rate of EMT, and so the simultaneous determination of these parameters and of the connective tissue growth factor (CTGF) in serum might provide information on fibrogenic activity. Also, proteomic and glycomic approaches of serum are under investigation to set up specific protein profiles in patients with liver fibrosis. The aim of this article is to present the current pathogenetic concepts of liver fibrosis and to discuss established and novel diagnostic approaches to reflect the process of hepatic fibrogenesis in the medical laboratory.

Aim: Several epidemiological studies suggest a beneficial effect of coffee consumption on the formation and progression of fibrogenic diseases, particularly of the liver. Recent data now point to a modulation of transforming growth factor-β (TGF-β) signaling by paraxanthine (1,7-dimethylxanthine [1,7-DMX]), the demethylated primary metabolite of caffeine Methods: Twenty adult Sprague–Dawley rats were bile duct ligated (BDL) or sham operated with or without concomitant oral 1,7-DMX (1 mM) application. Serum was investigated by standard biochemical analysis, in-house connective tissue growth factor (CTGF), enzyme linked immunosorbent assay (ELISA) or liquid chromatography-mass spectrometry analysis. Liver tissue was stained using hematoxylin-eosin (HE) and Sirius-red staining. Whole liver lysates, primary rat hepatocytes (PC) and hepatic stellate cells (HSC) were investigated by CTGF, and total Smad2/3 Western blot analysis, CTGF reporter gene assay or an in-house malondialdehyde ELISA. Results: The in vitro 50% inhibitory dose (ID50) of 1,7-DMX was 0.95 mM by for CTGF promoter activity and protein expression in PC and 1.25 mM for protein expression in HSC. Oral 1,7-DMX application (1 mM) attenuated cholestatic hepatocellular injury in vivo as determined by biochemical serum analysis and reduced intercellular collagen deposition in the cholestatic rat liver (HE- and Sirius-red staining). Western Blot analysis of whole liver lysates revealed a reduction of intrahepatic concentrations of Smad2/3 and CTGF following oral 1,7-DMX intake. However, serum CTGF concentrations were not reduced in 1,7-DMX treated BDL rats. Oral 1,7-DMX furthermore led to a reduction of intrahepatic lipid peroxidation (malondialdehyde concentrations) as markers of oxidative stress in BDL rats. Conclusion: Our pilot study warrants further studies of 1,7-DMX as a potential new drug to fight fibrotic processes, not just of the liver.

Several epidemiological studies suggest that coffee drinking is associated with a slower progression of fibrogenesis in patients with chronic, particularly alcoholic, liver disease. However, a causal, mechanistic explanation was pending. New results indicate that the methylxanthine caffeine, major component of coffee and the most widely consumed pharmacologically active substance in the world, might be responsible for this phenomenon as it inhibits the synthesis of Connective Tissue Growth Factor (CTGF/CCN2) in liver parenchymal and non-parenchymal cells, primarily by inducing degradation of Smad2 (and to a much lesser extent Smad3). In particular paraxanthine has been identified as the most potent inhibitor of CTGF synthesis among the three primary metabolites of caffeine, i.e. paraxanthine, theophylline, and theobromine. CTGF plays a crucial role in the fibrotic remodeling of various organs which has therefore frequently been proposed as therapeutic target in the management of fibrotic disorders. This article summarizes the clinical- epidemiological observations as well as the pathophysiological background and provides suggestions for the therapeutic use of methylxanthine derivatives in the management of fibrotic liver diseases.

Summary. Clinical and experimental studies have demonstrated that connective-tissue growth factor (CTGF) expression is increased in fibrotic human liver and experimental animal models of liver fibrogenesis. CTGF has been linked to transforming growth factor-beta (TGF-β) pathways in fibroproliferative diseases and specific polymorphisms within the CTGF gene may predispose for fibrosis in systemic sclerosis. As CTGF is detectable in various human fluids (serum, plasma and urine), it may provide information about fibrotic remodelling processes and reflect hepatic TGF-β bioactivity. We established a novel ELISA for the measurement of serum CTGF and tested its clinical value in patients with chronic hepatitis C virus (HCV) infection and chronic liver disease (CLD). HCV infected patients (n = 138) had significantly higher serum CTGF levels than healthy controls. CTGF was linked to the histological degree of liver fibrosis. To expand the results to other aetiologies, a separate cohort of CLD patients (n = 129) was evaluated, showing higher serum CTGF than healthy controls and again an association with advanced stages of liver cirrhosis (Child B and C). Although independent of the underlying aetiology, serum CTGF was most powerful in indicating fibrosis/advanced disease states in HCV-related disorders. The genotyping of six polymorphisms (rs6917644, rs9399005, rs6918698, rs9493150, rs2151532 and rs11966728) covering the CTGF locus in 365 patients suffering from chronic hepatitis C revealed that none of these polymorphisms showed a genotypic or allelic association with the severity of hepatic fibrosis. Taken together, serum CTGF is suitable for determination of hepatic fibrosis and most powerful in patients with chronic HCV infection.

Background: Recently, we identified hepatocytes as the major cellular source of profibrogenic connective tissue growth factor (CTGF/CCN2) in the liver. Based on reports of a hepatoprotective effect of coffee consumption, we were the first to provide evidence that caffeine suppresses transforming growth factor (TGF)-β dependent and -independent CTGF expression in hepatocytes in vitro and in vivo, thus suggesting this xanthine-alkaloid as a potential therapeutic agent. Aim: This study aims at comparing the inhibitory capacities of caffeine and its three demethylated derivates paraxanthine, theophylline and theobromine on CTGF expression in hepatocytes and hepatic stellate cells (HSC). Results: Our data suggest paraxanthine as the most important pharmacological repressor of hepatocellular CTGF expression among the caffeine-derived metabolic methylxanthines with an inhibitory dosage (ID)50 of 1.15 mM, i.e. 3.84-fold lower than what is observed for caffeine. In addition, paraxanthine displayed the least cell toxicity as proven by the water-soluble tetrazolium-1 cell vitality assay. However, caffeine or any of the metabolites did not inhibit CTGF expression in HSC. At the toxicological threshold concentration of 1 mM for paraxanthine, we observed an inhibition of hepatocellular CTGF synthesis by 44%, which was strongly reverted in the presence of the specific competitive cyclic adenosine monophosphate inhibitor Rp-adenosine 3′,5-cyclic monophosphorothioate triethylammonium salt. Furthermore, CTGF protein expression induced by various concentrations of TGF-β (0.13–1 ng/ml) is still reduced by, on average, 27%/45% in the presence of paraxanthine (1.25 mM/2.5 mM). Conclusion: Our data provide an evidence-based suggestion of the caffeine-derived primary metabolite paraxanthine as a potentially powerful antifibrotic drug by its inhibitory effect on (hepatocellular) CTGF synthesis.

Background and aim Chronic hepatitis induced liver fibrogenesis is characterized by epithelial-to-mesenchymal transition of liver parenchymal cells (hepatocytes) to fibroblast (-like cells), i.e. increasing hepatocellular dedifferentiation, ultimatively leading to the development of hepatocellular carcinoma (HCC). Up to now the spectrum of valid serum biomarkers for this process of hepatocellular dedifferentiation is very limited. We therefore investigated the dynamics of alterations in the serum transferrin isoform pattern in the pathogenetic sequence from liver fibrosis to hepatocellular carcinoma, to evaluate the suitability of one of the isoforms as potential biomarker for hepatocellular dedifferentiation in chronic liver disease. Results Our data on 252 patients with hepatitis C virus (HCV) induced fibrogenic liver disease and on 43 patients with HCV induced HCC demonstrate a dynamic alteration of serum % trisialotransferrin levels in the pathogenetic sequence from early stage hepatic fibrosis to fully developed hepatocellular carcinoma, whereas serum % di- and pentasialotransferrin values seem not to be affected. We show that patients with early stage fibrosis (METAVIR stage F1) and weak fibrogenic activitiy (METAVIR grade A1) display significantly lower values of serum % trisialotransferrin compared to healthy controls, and that serum % trisialotransferrin values increased steadily parallel to an increase of fibrotic stage and grade, respectively, while finally exceeding normal values in those patients with hepatocellular carcinoma. Conclusion These findings propose a possible diagnostic value of serum % trisialotransferrin concentrations in the pathogenesis of hepatocellular dedifferentiation and the use of this parameter as possible predictive tumor marker in patients with chronic liver disease. Monitoring the pattern of transferrin bound sialic acid residues may thus be a helpful tool in assessing the risk of malignant degeneration in patients with chronic fibrogenic liver disease.

In vivo knockdown of connective tissue growth factor (CTGF/CCN2) was recently shown to attenuate the formation of experimental liver fibrosis. The secreted, cysteine-rich growth factor is proposed to adversely modulate the binding of profibrogenic transforming growth factor beta (TGF-beta) and its natural antagonist bone morphogenetic protein (BMP) to their cognate receptors in several cellular systems, but the functionality of CTGF in modulation of the TGF-beta/BMP signaling pathways is still unknown. This study aims at characterizing a potentially differential modulating role of CTGF on TGF-beta- and BMP7-dependent transactivation of reporter gene [Ad-(CAGA)(12)-MLP-luc, Ad-hCTGF-luc, and Ad-(BRE)(2)-luc reporter gene] expression in rat hepatocytes. In this context, emphasis is also placed on the differential roles of Smad2 and Smad3 in the TGF-beta-dependent transactivation of the endogenous CTGF gene and the CTGF gene reporter, as investigated following adenoviral infection of wild-type and dominant negative Smad2/3 or treatment with the specific inhibitor of Smad3 or ALK5-specific (SB-431542) inhibitor. In this analysis, we found (1) a selective transcriptional activation of the CTGF promoter by Smad2 (but not Smad3); (2) the failure of BMP7 to inhibit the transcriptional activation of the Smad3-selective (CAGA)(12)-luc reporter by TGF-beta, as well as the failure of TGF-beta to inhibit the transcriptional activation of the Smad5-selective (BRE)(2)-luc reporter by BMP7; and (3) the sensitization of hepatocytes toward TGF-beta type I receptor (ALK5)/Smad2 and Smad3-mediated TGF-beta signaling by CTGF, whereas BMP type I receptor (ALK1)/Smad5-mediated BMP7 signaling is not modulated.CTGF acts as a Smad2-dependent sensitizer of TGF-beta actions that does not influence BMP7 signaling in hepatocytes.

Background/Aims: Epidemiological studies suggest that coffee drinking is inversely correlated with the risk of development of liver fibrosis but the molecular basis is unknown. Methods: We investigated the pharmacological mechanisms involved in caffeine-dependent regulation of CTGF expression, an important modulator protein of fibrogenic TGF-b, in rat hepatocytes using Western-blot, co-immunoprecipitations, reporter-gene-assays and ELISAs. Results: It is demonstrated that caffeine, similar to 8-Br-cAMP, suppresses CTGF expression, decreases SMAD2 protein levels and inhibits SMAD1/3-phosphorylation. The SMAD2 level can be restored by a proteasome inhibitor. Additionally, caffeine leads to an up-regulation of PPARc expression, that enhances the inhibitory effect of the natural PPARc agonist 15-PGJ2 on CTGF expression by inducing a dissociation of the SMAD2/3-CBP/p300-transcriptional complex. Conclusions: We show that caffeine strongly down-modulates TGF-b-induced CTGF expression in hepatocytes by stimulation of degradation of the TGF-b effector SMAD 2, inhibition of SMAD3 phosphorylation and up-regulation of the PPARc-receptor. Long-term caffeinization might be an option for anti-fibrotic trials in chronic liver diseases. 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Connective tissue growth factor (CTGF=CCN2), one of six members of cysteine-rich, secreted, heparin-binding proteins with a modular structure, is recognized as an important player in fibrogenic pathways as deduced from findings in non-hepatic tissues and emerging results from liver fibrosis. Collectively, the data show strongly increased expression in fibrosing tissues and transforming growth factor (TGF-beta)-stimulated expression in hepatocytes, biliary epithelial cells and stellate cells. Functional activity as a mediator of fibre-fibre, fibre-matrix and matrix-matrix interactions, as an enhancer of profibrogenic TGF-beta and several secondary effects owing to TGF-beta enhancement, and as a down-modulator of the bioactivity of bone morphogenetic protein-7 has been proposed. By changing the activity ratio of TGF-beta to its antagonist bone-morphogenetic protein-7, CTGF is proposed as a fibrogenic master switch for epithelial-mesenchymal transition. Consequently, knockdown of CTGF considerably attenuates experimental liver fibrosis. The spill-over of CTGF from the liver into the blood stream proposes this protein as a non-invasive reporter of TGF-beta bioactivity in this organ. Indeed, CTGF-levels in sera correlate significantly with fibrogenic activity. The data suggest CTGF as a multifaceted regulatory protein in fibrosis, which offers important translational aspects for diagnosis and follow-up of hepatic fibrogenesis and as a target for therapeutic interventions. In addition, CTGF-promoter polymorphism might be of importance as a prognostic genetic marker to predict the progression of fibrosis.

Background/Aims: Up to now, the effect of activin A on the expression of the important transforming growth factor (TGF)-b downstream modulator connective tissue growth factor (CTGF) is not known, but might be of relevance for the functional effects of this cytokine on several liver cell types. Methods: In this study, activin A-dependent CTGF expression in hepatocytes (PC) primed by exogenous activin A and in PC maintained under complete activin-free culture conditions was analysed by Western blots, metabolic labelling, gene silencing, reverse transcriptasepolymerase chain reaction (RT-PCR)and CTGFreporter gene assays. This studywas supplemented by immunocytochemical staining of activin A and CTGF in PC of injured liver. Results: Using alkaline phosphatase a-alkaline phosphatase staining, it is demonstrated that activin A becomes increasingly detectable during the course of CCl4-liver damage. Addition of activin A to cultured PC induced CTGF protein expression via phosphorylation of Smad2 and Smad3. This induction can be inhibited by the antagonist follistatin and a-activin A antibody respectively. When PC were cultured under serum(i.e. activin A)-free culture conditions, a timedependent increase of activin expression during the course of the culture was proven by RT-PCR. Silencing of inhibin bA gene expression under serum-free conditions by small interfering RNAs greatly suppressed CTGF synthesis and the phosphorylations of Smad2 and Smad3. However, both the extracellularly acting follistatin and the aactivin A antibody couldnot inhibit spontaneous CTGFexpression, which, however, was achieved by the cell-permeable TGF-b Alk4/Alk5 receptor-kinase-inhibitor SB431542. Conclusions: In conclusion, the results point to activin A as an inducer of CTGF synthesis in PC. Intracellular activin A contributes to spontaneous CTGF expression in PC independent of exogenous activin A, which is proposed to occur via Alk4/Alk5-receptors. The findings might be important for many actions of activin A on the liver.

Background Gc-globulin or vitamin D binding protein is a highly expressed, multifunctional and polymorphic serum protein, which also serves as the major transporter for vitamin D metabolites in the circulation. The present study was performed to analyze the interaction between gc-globulin of hepatocytes and hepatic stellate cells, the most important fat-/retinol-storing cell type in the liver, which spontaneously transdifferentiates to myofibroblasts in culture. Methods Hepatic stellate cells and hepatocytes were isolated by the pronase/collagenase reperfusion method, hepatocytes by collagenase reperfusion of the organ. Gc-globulin expression was monitored by immunocytochemistry, immunoblotting, RT-PCR, metabolic labelling with [35S]-methionine, and its intracellular binding to α-smooth-muscle actin was investigated by co-immunoprecipitation. Cytoskeletal stainings of gc-globulin and α-smooth-muscle actin in hepatic stellate cells and the identification of the receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2 were performed with confocal immunocytochemistry, immunoblotting and/or FACS-analysis. Results Hepatocytes synthesize and secrete gc-globulin as shown by RT-PCR and [35S]-methionine labelling, which could be suppressed by cycloheximide. Also, a strong signal for gc-globulin was detected in the immunoblot of native hepatic stellate cell lysates. However, no mRNA for gc-globulin was found in this cell type, which suggests no active synthesis by hepatic stellate cells. Hepatic stellate cells were tested positively for the presence of known gc-globulin interacting receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2. Inhibition of the megalin/gp330 receptor by a competitive, neutralizing antibody resulted in decreased intracellular availability of gc-globulin in hepatic stellate cells. The latter effect was enhanced by additional incubation of hepatic stellate cells with EDTA for complexing Ca2+, suggesting a Ca2+-dependent internalization of gc-globulin into hepatic stellate cells via the megalin/gp300 receptor. This was supported by confocal microscopy which showed a co-localization of gc-globulin with the multifunctional megalin/gp330 receptor on this cell type. Inside hepatic stellate cells, a linkage between gc-globulin and α-smooth muscle actin filaments of hepatic stellate cells was detected by immunocytochemistry. Intracellular binding of gc-globulin to α-smooth-muscle actin filaments was confirmed by co-immunoprecipitation. Conclusion We give evidence to the expression of the megalin/gp330 receptor on hepatic stellate cells and that this receptor is involved in the Ca2+-dependent internalization of gc-globulin into hepatic stellate cells, a protein synthesized and secreted into the extracellular space and circulation by hepatocytes. Inside hepatic stellate cells, it co-localizes with and binds to α-smooth muscle actin filaments. Under consideration of the available literature, these findings propose a participation of gc-globulin in hepatic vitamin D metabolism as well as in hepatic stellate cell stability and apoptosis as important mechanisms of liver regeneration.

Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV-family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up-regulated by exogenous TGF-beta. In this study TGF-beta-dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF-beta-free conditions was analysed by Western-blots, metabolic labelling, and CTGF-reporter gene assays. In alkaline phosphatase monoclonal anti-alkaline phosphatase complex (APAAP)-staining of cultured hepatocytes it was demonstrated that latent TGF-beta within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF-beta antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p-Smad2, whereas p-Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF-beta, but calpain inhibitors partially suppressed intracellular TGF-beta activation and subsequently CTGF up-regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF-beta signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4-/Alk 5-specific inhibitor SB-431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF-beta is activated by calpains during culture-induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF-beta. In conclusion, these data are the first to suggest the possibility of intracrine TGF-beta signalling due to calpain-dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF-beta-sensitive reporter gene. This mechanism might be deleterious for keeping long-term hepatocyte cultures due to TGF-beta-induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial-mesenchymal transition of hepatocytes in injured liver.

Background/Aims: Haemoglobin-depleted erythrocyte ghosts have been recommended as vesicle carriers of drugs with hepatotropic properties. However, the influence of liver injury on ghost elimination and targeting has not been reported so far. Methods: Human and rat ghosts were prepared and loaded with model substances, and the basic parameters were characterized. Ghosts were injected intravenously into rats with acute, subacute and chronic liver injuries. Elimination from circulation, organ distribution and cellular targeting was measured. The uptake of ghosts by liver macrophages/Kupffer cells was determined in cell culture. Results: Ghosts are strong hepatotropic carriers with a recovery of 90% in normal liver. Kupffer cells are the almost exclusive target cell type. Hepatotropic properties remain in rats with chronic liver diseases, but are reduced by 60–70% in acute liver damage as a result of decline of phagocytosis of macrophages/Kupffer cells. Although the uptake of ghosts per gram liver tissue in chronic liver injury was also reduced by about 40%, the increase of liver mass and of macrophages/Kupffer cells compensated for the reduced phagocytotic activity. In subacute injury, the uptake per gram liver tissue was only moderately reduced. Conclusion: Drug targeting with ghosts might be feasible in chronic and subacute liver injuries, e.g. fibrogenesis and tumours, because the content of ingested ghosts is released by Kupffer cells into the micro-environment, providing the uptake by and pharmacological effects on adjacent cells.

This study aimed to investigate the association between HBV mutations and intrahepatic HBV status and to determine the clinical features and the contribution of HBV mutations to postoperative prognosis for hepatocellular carcinoma (HCC) patients with HBsAg (+) in China. Using TaqMan fluorescent real-time PCR, HBV covalently closed circular DNA (cccDNA) and total DNA (tDNA) were both quantified in 106 pairs of tumor tissues (TT) and adjacent non-tumor tissues (ANTT) obtained from HCC patients who received no antiviral treatment before surgical treatment. The prevalence of 19 hot spot mutations was evaluated by Sanger sequencing. HBV cccDNA and tDNA were lower in TT than in ANTT. The loads of cccDNA and tDNA in ANTT were associated with serum HBV DNA and HBeAg. Three Pre-S mutants (A2962G and C2964A in Pre-S1 and C105T in Pre-S2) were associated with higher tumor cccDNA levels (P < 0.05), and A2962G/C2964A mutants were associated with higher AFP levels. This would assist to disclose the virological features, to implement a more clinically personalized therapy and to improve the prognosis of HBV-related HCC patients.

Still a challenging medical problem is the non-invasive monitoring of patients with a variety of chronic liver diseases being on risk to develop fibrosis, cirrhosis, and, finally, primary liver cell carcinoma. Previously, we have shown that CTGF/CCN2, a down-stream mediator of TGF-β, in serum might be a promising non-invasive biomarker of fibrosis, which is extended in the following study to cirrhosis and liver cell carcinoma. Healthy individuals (n=56), as well as fibrotic (n=77), cirrhotic (n=17), and HCC-patients (n=72) with chronic hepatitis B (HBV) infection, clinically, biochemically and histopathologically well characterized and classified, were included for the measurements of CTGF-concentrations in serum using a newly developed CTGF-enzyme immunoassay. A statistical significant increase of the mean serum CTGF-concentrations was associated with different stages of fibrosis, ranging from 15.9 μg/L (S0), 20.3 μg/L (S1/2) to 36.9 μg/L (S3/4). The highest CTGF-concentrations were measured in cirrhotic patients (43.6 μg/L), compared to healthy subjects (17.7 μg/L), followed by a decrease in cirrhotic HCC-patients (38.5 μg/L; p=0.001). Of note, HCC patients without underlying cirrhosis (n=8) had CTGF levels (13.5±13.2 μg/L) comparable to those in healthy controls. No statistical relation between CTGF levels and parameters of liver injury (e.g. AST, ALT) was noticed, but CTGF levels are correlated negatively with serum albumin levels (p=0.007) and platelet counts (p=0.0032), respectively. The latter was negatively correlated with the stage of fibrosis (p=0.025). In HCC patients, CTGF concentrations decreased with tumor progression and size, with lower levels in TNM stage II (30.5 μg/L) and stage III (33.6 μg/L) compared to TNM stage I (41.6 μg/L). Our data suggest a valuable diagnostic impact of CTGF in serum for the follow-up of patients suffering from chronic liver diseases developing fibrosis, cirrhosis and finally HCC. CTGF serum levels in HCC are most likely due to underlying fibrosis/cirrhosis but not due to malignancy per se.

While autocrine stimulation of cells by Activin A and/or its family member transforming growth factor β (TGF-β) is a phenomenon observed in a variety of cell types, little is known of putative intracellular signaling loops of these cytokines. Intracellular actions of several peptide hormones, growth factors, as well as of extracellular signaling enzymes and DNA-binding proteins, either within target cells or within their cells of synthesis have been shown. Although these intracrine moieties are structurally diverse, they share certain characteristics of synthesis and function. Depending on the cell type, there are reports of stimulatory as well as inhibitory mechanisms induced by such intracrine mechanisms, and this also accounts for transforming growth factor β (TGF-β), whereas only stimulatory intracrine signaling of Activin A could be demonstrated so far. Stimulatory intracrine signaling loops of TGF-β were shown following calpain-dependent intracellular proteolytic activation of the latent cytokine in hepatocytes under cellular stress conditions of this cytokine, leading to transcriptional activation of connective tissue growth factor (CTGF/CCN2) as a representative TGF-β-sensitive reporter gene. In contrast to TGF-β, increasing intrahepatocellular concentrations of Activin A are not the result of release from an intracellularly deposited latent complex, but of active de novo synthesis. The stimulatory intracrine signaling pathways of both, TGF-β and Activin A, are proposed to occur via Alk4/Alk5 receptors and Smad2, whereas additional activation of Smad3 only seems to be involved in intracrine Activin A signaling. However, intracrine TGF-β signaling may itself also be inhibitory as active TGF-β is also able to bind to intracellular TGF-β type II receptor, resulting in a ligand-induced impediment in receptor trafficking to the cell surface. Whether stimulatory or inhibitory modulation of the TGF-β pathway takes place seems to depend on the cell type and environmental conditions. Future studies are necessary at this point.

Scientists at the National Institutes of Health have reported that increased coffee consumption is associated with a slower progression of fibrogenesis in patients with chronic and particularly alcoholic liver disease and a reduced incidence of heptocellular carcinoma. However, a causal mechanistic explanation was pending. New results indicate that the methylxanthine caffeine--a major component of coffee and the most widely consumed pharmacologically active substance in the world--might be responsible for this phenomenon, because it inhibits the synthesis of connective tissue growth factor (CTGF/CCN2) in liver parenchymal and nonparenchymal cells, primarily by inducing degradation of Smad2 (and to a much lesser extent Smad3) and thus impairment of transforming growth factor beta (TGF-beta) signaling. CTGF and TGF-beta play crucial roles in the fibrotic remodeling of various organs, and, ultimately, carcinogenesis. This article summarizes the clinical-epidemiological observations as well as the pathophysiological background and provides suggestions for the therapeutic use of (methyl)xanthine derivatives in the management of fibro-/carcinogenic (liver) diseases.

Recently, variation of gene copy numbers was recognized as a novel type of common genetic diversity, but its impact on viral hepatitis is unknown. Here, we determine the influence of copy number variation on the susceptibility and disease severity in hepatitis C virus (HCV) infection, investigating copy number variants (CNVs) of the chemokine CCL3L1 gene, which encodes a potent CCR5 ligand.CNVs were determined in 254 patients with chronic hepatitis C, 144 HCV/HIV co-infected patients, and 210 HCV negative controls, using quality-controlled real-time fluorescent dye-labeled quantitative PCR. Liver biopsies were obtained from HCV infected patients.Copy numbers of the CCL3L1 gene range from 0 to 12 (mean 2.7+/-1.4 copies). Patients with two or less copies are over-represented in the HCV infected cohort compared to HCV negative controls (odds ratio [OR] 1.54; p=0.02). CCL3L1 copies are shifted to lower numbers in HCV infected patients (means 2.6 vs. 2.9 in controls; p=0.011). HCV/HIV co-infected patients carry even lower CCL3L1 copy numbers compared to controls (means 2.2 vs. 2.9; p0.001), with a higher proportion of patients possessing two or less copies (OR=3.42; p0.001). No association was detected between CCL3L1 copy numbers and histological grades of inflammation or stages of fibrosis.Lower CCL3L1 gene copy number compared to the population median is associated with chronic hepatitis C. Copy number variation of host genes represents a novel class of genetic diversity associated with viral hepatitis.

Current knowledge confers a crucial role to connective tissue growth factor (CTGF/CCN2) in hepatic fibrogenesis. Hepatocytes are likely to be the major cellular source of CTGF in the liver in which CTGF is sensitively upregulated by TGF-beta. Recently, we demonstrated that the methylxanthine derivate caffeine leads to an upregulation of peroxisome proliferator activated receptor gamma (PPARgamma) expression in hepatocytes, thus sensitizing these cells to the well-known inhibitory effect of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ(2)) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se; its physiological ligand 15-d-PGJ(2) is required to exert an inhibitory effect on transforming growth factor-beta (TGF-beta) target genes such as CTGF.This study compared serum concentrations of 15-d-PGJ(2) in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non-liver disease (NLD) sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (HCC) (n=43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPARgamma-inducing (i.e. CTGF inhibitory) drugs such as caffeine.The presented data showed that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2+/-5.9 microg/L) displayed strikingly higher serum concentrations of 15-d-PGJ(2) than healthy probands (mean 2.3+/-1.0) and Caucasian patients with NLD (mean 2.7+/-1.4 microg/L). Similar results were found in Chinese patients with fully developed HCC (mean 1.3+/-0.7 microg/L) compared with Chinese healthy controls (mean 0.4+/-0.2 microg/L).In conclusion, our data thus proposed an increased suitability of these patient groups for therapeutic approaches with drugs inducing PPARgamma expression, such as methylxanthine derivates.

Introduction Preliminary studies report on significantly higher levels of the major cytoskeleton protein actin in CSF of patients with neurodegenerative conditions and that the dynamics of these levels obviously correlates with disease progression and clinical disability. One of the primary functions of actinfree Gc-Globulin is to bind and neutralize extracellular monomeric actin, released into the circulation by necrotic or ruptured cells, and thus ameliorating the clinical outcome in situations of severe organ damage. Aim and methods This is the first study to investigate actinfree Gc-Globulin and S100-B levels (as reliable marker of neurodegeneration) in paired CSF and serum samples of patients with multietiological CNS diseases. Results 42% of all patients with CNS disease displayed serum concentrations of actinfree Gc-Globulin above the established reference range. CSF concentrations of actinfree Gc-Globulin and S100-B were positively correlated with the severity of blood–brain barrier (BBB) dysfunction. Furthermore, patients with severe BBB dysfunction presented a higher percentage of intrathecal synthesis of actinfree Gc-Globulin compared to patients with mild to moderate dysfunction and to patients with normal BBB function. Representative longitudinal data from selected patients demonstrated an inverse behaviour of actinfree Gc-Globulin and S100-B CSF concentrations, suggesting a consumption of the actin scavenger capacity of Gc-Globulin in times of increased neuronal damage. This presumption was supported by the fact that those conditions associated with a severe neuronal damage, in particular CNS trauma, and highest S100-B concentrations simultaneously displayed lowest actinfree Gc-Globulin levels, and thus residual actin binding capacity of Gc-Globulin. Conclusion In summary, our data propose a function of actinfree Gc-Globulin also in the clearance of actin filaments from CSF of patients with neuronal damage. However, active recruitment of hepatic derived actinfree Gc-Globulin to the site of CNS injury is not observed. Much more, BBB leakage enables extraneuronally synthesized actinfree Gc-Globulin to extent its scavenger capacity for actin also to the subarachnoidal space. Furthermore, intrathecal synthesis of actinfree Gc-Globulin seems to be increased in patients with severe neurodegeneration.

The pathophysiological mechanisms of thioacetamide (TAA)-induced hepatic fibrogenesis are not yet fully understood. In particular, the role of hepatic stellate cells (HSCs) remains unclear. We therefore examined proliferation and transdifferentiation of HSC as well as the underlying molecular mechanisms in TAA-induced fibrosis. Hepatic fibrogenesis was induced in mice by addition of TAA to drinking water. Liver damage was determined by assessment of alanine aminotransferase and aspartate aminotransferase levels, and measurement of collagen deposition. Additionally, expression patterns of alpha-smooth muscle actin, glial fibrillary acidic protein (GFAP, specific hepatic biomarker for HSC), cysteine- and glycine-rich protein 2 (CRP2, specific marker of HSC transdifferentiation), tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-9 (MMP-9), interleukins (IL-1beta, IL-6), platelet-derived growth factors (PDGF-B, PDGF-D) , tumor necrosis factor (TNF)-alpha, and (transforming growth factor (TGF)-beta1 were assessed by real-time PCR. Transcription of GFAP and CRP2 were transiently upregulated during TAA-induced fibrogenesis (punctum maxima (p.m.) week 10 for GFAP and week 14 for CRP2). Similar transient expression patterns were demonstrated for IL-1beta, IL-6, TGF-beta1, and PDGF-B (p.m. week 12) whereas TNF-alpha and PDGF-D continuously increased with ongoing liver injury. In particular, not only neutrophil granulocytes, but also macrophages and leukocytes served as a major source for MMP-9 expression. GFAP and CRP2 expression patterns demonstrated transiently increased HSC-activation during TAA-induced hepatic fibrogenesis. The rate of increase of transcription of GFAP correlated best with PDGF-B, whereas CRP2 levels correlated with PDGF-B, PDGF-D, and IL-1beta expression. This study demonstrates for the first time that transiently increased activation patterns of HSC are observed in toxically induced hepatic fibrosis. Thus, TAA in drinking water is an effective and elegant model to induce reproducible states of liver fibrosis without parenchymal damage in mice.

Background Immune paralysis of phagocytic cells due to excess of the complement activation product C5a has been proposed as a critical pathomechanism in sepsis. In vitro studies suggest an interaction of C5a with Group-specific globulin (Gc-globulin). Study objectives To examine the predictive value of serum concentrations of both, C5a and actin-free Gc-globulin, and their ratio for prognosis (mortality) of critically ill patients. Patients 154 critically ill (septic and non-septic) adult patients admitted to a Medical ICU and 38 healthy controls. Measurements Actin-free Gc-globulin and C5a were measured on ICU admission, alongside extensive laboratory, clinical and prospective outcome measures. Results Actin-free Gc-globulin and C5a serum concentrations were significantly reduced in critically ill patients compared with healthy controls. C5a levels, but not actin-free Gc-globulin, were significantly lower in patients with sepsis ( n = 112) than in critically ill patients without sepsis ( n = 42). C5a serum level was a prognostic parameter in patients with sepsis: High C5a levels were associated with increased mortality (at ICU and during follow-up). Although C5a and actin-free Gc-globulin were positively correlated, increasing serum concentrations of actin-free Gc-globulin did not enhance the C5a dependent effects in terms of prognosis or mortality in septic patients. Conclusions Investigation for C5a and/or actin-free Gc-globulin serum levels upon admission to the ICU may be helpful diagnostic tools. In patients with sepsis, C5a levels are an independent predictor of prognosis. However, different to pre-existing in vitro data, a clinically relevant interaction between actin-free Gc-globulin and C5a in terms of prognosis in severe inflammatory conditions is not given.

Background/Aims Connective tissue growth factor (CTGF/CCN2) has been implicated in the pathogenesis of hepatic fibrosis and suggested as a downstream mediator of the fibrogenic master cytokine TGF-β. Methods We investigated the effect of TGF-β1 on CTGF/CCN2 expression in cultured rat hepatic stellate cells and hepatocytes by means of Western and Northern blotting, immunocytochemistry, reporter gene analysis, and metabolic labelling. Results We found that the expression of CTGF/CCN2 in hepatic stellate cells is (i) only marginally (if at all) stimulated by TGF-β and by a constitutively active type I TGF-β receptor, (ii) independent from Smad2/3 phosphorylation, (iii) not reduced by TGF-β1 antagonists or ALK5-receptor inhibitors and (iv) not upregulated during transdifferentiation to myofibroblasts in culture. However, expression and secretion of CTGF/CCN2 in cultured hepatocytes increased spontaneously during culture and was strongly stimulated by TGF-β1. In bile-duct ligated and CCl 4 -treated rat livers, a strong CTGF/CCN2 expression in hepatocytes was noticed. Endothelin-1 stimulated CTGF/CCN2 expression in stellate cells but not in hepatocytes. Pathway specific signalling inhibitors point to the involvement of non-Smad signalling cascades but their contribution to CTGF/CCN2 regulation is different in both cell types. Conclusions The results do not reveal a relevant interrelation between TGF-β function and CTGF/CCN2 expression in hepatic stellate cells, which is in contrast to hepatocytes.

Objectives: To investigate the role of Gc-globulin and C5 gene variants as co-factors in the regulation of profibrogenic C5 serum activities. Design: Retrospective clinical investigation with 100 healthy probands. Genomic DNA was isolated from whole blood and examined for the human C5 htSNPs rs17611 and rs2300929 . Actin-free Gc-globulin-, total Gc-globulin- and total C5-concentrations in serum were measured using ELISA assays; C5 activities in serum were determined using radial immunodiffusion. Results: C5 serum concentrations were significantly elevated in individuals carrying at least one profibrogenic allele of the C5 htSNP rs17611 , but no association between C5 htSNPs and C5 serum activities was detected, albeit C5 activities correlated positively with C5 concentrations in serum. However, C5 activities were also positively correlated with total and actin-free Gc-globulin concentrations. Conclusion: Our findings indicate that C5 gene variants and Gc-globulin levels co-define the proinflammatory and profibrogenic effects of C5 in patients at-risk for progression of liver fibrosis.

Background: Gc-globulin (vitamin D–binding protein) appears to have important functions in addition to its role as a carrier of vitamin D.Approach: We reviewed recent studies focusing on the pathophysiologic functions and clinical significance of Gc-globulin.Results: Serum concentrations of Gc-globulin, as determined by immunoassay techniques, are decreased in severe injury. The extent of the decrease may have prognostic significance for patient outcomes. Clinical studies and animal models have shown that Gc-globulin has an important role in the clearance of procoagulant actin from the circulation after its release during cell necrosis and tissue injury. Gc-globulin has other potential roles in responses to acute tissue injury through conversion to a macrophage-activating factor, neutrophil chemotactic activity, and enhancement of C5a-mediated signaling.Conclusion: Considering the important physiologic roles of Gc-globulin in responses to tissue injury, such as clearance of actin, measurement of Gc-globulin may have value in directing the care of patients in many clinical disorders.

AIM: To study the expression of cystatin C (CysC), its regulation by transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF) and the potential interference of CysC with TGF-β1 signaling in this special cell type. METHODS: We evaluated the CysC expression in cultured, profibrogenic hepatic stellate cells and trans-differentiated myofibroblasts by Northern and Western blotting and confocal laser scanning microscopy. RESULTS: CysC was increased significantly in the course of trans-differentiation. Both TGF-β1 and PDGF-BB suppressed CysC expression. Furthermore, CysC secretion was induced by the treatment with TGF-β1. Although CysC induced an increased binding affinity of TGF-β receptor type III (beta-glycan) as assessed by chemical cross-linking with [125I]-TGF-β1, it did not modulate TGF-β1 signal transduction as shown by evaluating the Smad2/3 phosphorylation status and [CAGA]-MLP-luciferase reporter gene assay. Interestingly, the shedding of type III TGF-β receptor beta-glycan was reduced in CysC-treated cells. Our data indicated that CysC expression was upregulated during trans-differentiation. CONCLUSION: Increased CysC levels in the serum of patients suffering from liver diseases are at least partially due to a higher expression in activated hepatic stellate cells. Furthermore, TGF-β1 influences the secretion of CysC, highlighting a potentially important role of cysteine proteases in the progression of hepatic fibrogenesis.

To investigate the mechanisms involved in a possible modulator role of interleukin (IL)-6 signalling on CYR61-CTGF-NOV (CCN) 2/connective tissue growth factor (CTGF) expression in hepatocytes (PC) and to look for a relation between serum concentrations of these two parameters in patients with acute inflammation.Expression of CCN2/CTGF, p-STAT3, p-Smad3/1 and p-Smad2 was examined in primary freshly isolated rat or cryo-preserved human PC exposed to various stimuli by Western blotting, electrophoretic mobility shift assay (EMSA), reporter-gene-assays and reverse-transcriptase polymerase chain reaction.IL-6 strongly down-regulated CCN2/CTGF protein and mRNA expression in PC, enhanceable by extracellular presence of the soluble IL-6 receptor gp80, and supported by an inverse relation between IL-6 and CCN2/CTGF concentrations in patients' sera. The inhibition of TGFβ1 driven CCN2/CTGF expression by IL-6 did not involve a modulation of Smad2 (and Smad1/3) signalling. However, the STAT3 SH2 domain binding peptide, a selective inhibitor of STAT3 DNA binding activity, counteracted the inhibitory effect of IL-6 on CCN2/CTGF expression much more pronounced than pyrrolidine-dithiocarbamate, an inhibitor primarily of STAT3 phosphorylation. An EMSA confirmed STAT3 binding to the proposed proximal STAT binding site in the CCN2/CTGF promoter.CCN2/CTGF is identified as a hepatocellular negative acute phase protein which is down-regulated by IL-6 via the STAT3 pathway through interaction on the DNA binding level.