Connective tissue growth factor (CTGF/CCN2) in serum is an indicator of fibrogenic progression and malignant transformation in patients with chronic hepatitis B infection.

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-ß, 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.

Connective tissue growth factor reacts as an IL-6/STAT3-regulated hepatic negative acute phase protein.

AIM: 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. METHODS: 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. RESULTS: 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 TGFß1 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. CONCLUSION: 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.

Impact of quality control accepted inter-laboratory variations on calculated Fibrotest/Actitest scores for the non-invasive biochemical assessment of liver fibrosis.

INTRODUCTION: Non-invasive, i.e. serum-based assessment of liver fibrosis is still an important diagnostic challenge although multiple single and multiparametric panels of biomarkers have been suggested. AIM: Two approaches were followed to determine the diagnostic reliability of Fibrotest and Actitest, two commercially distributed non-invasive multiparametric tests for staging and grading of liver fibrosis. METHODS: (i) Haptoglobin, ALT, gamma GT, alpha-2-macroglobulin, apolipoprotein A1 and bilirubin, required for calculation of respective scores, were determined in sera of 4 patients with histologically defined stages of fibrosis (F1-F4) and activities of fibrogenesis (A1-A3). Analytes were determined by 6 quality controlled external laboratories. Inter-laboratory variations of the calculated Fibrotest score for staging and Actitest score for grading (BioPredictive), and their error ratios referred to histologic results were calculated. (ii) The variability of respective Fibrotest/Actitest scores depending on 64 selected combinations of analytes within the accepted ranges of analyte-specific maximum/minimum limits given by the external quality control of the German Association of Clinical Chemistry and Laboratory Medicine (DGKL) was calculated. RESULTS: (i) Fibrotest and Actitest scores were largely reproducible among the different laboratories. However, the error ratio was 77% for all results calculated by both, Fibrotest and Actitest when referred to histologic findings. (ii) Calculated scores of stages varied between F2 (9%), F3 (31%), F3-F4 (6%), and F4 (54%) (Fibrotest), and A1/A2 (48%), A2 (9%), A2-A3 (5%), and A3 (38%) for grades of fibrogenic activity. CONCLUSION: Despite reproducibility of Fibro- and Actitest scores among the six laboratories, large scale investigation displayed high levels of variability depending on inter-laboratory differences that were still in a quality controlled, analytically acceptable range. Calculated scores coincided with histologic findings in less than 25% of all cases. Thus, the diagnostic accuracy of these tests must be considered as low, if histology is accepted as reference standard.

Increased serum xylosyltransferase activity in patients with liver fibrosis.

BACKGROUND: We investigated the xylosyltransferase (XT) activity in the serum of liver fibrotic patients with hepatitis C virus induced liver fibrosis at different stages as determined according to the scoring system of Desmet and Scheuer. METHODS: Measurement of XT activity was performed by liquid chromatography-tandem mass spectrometry. RESULTS: We found that serum XT activity in males (n=59, median+/-SD, 27.2+/-2.8 mU/L, p<0.001) and females (n=54, 23.6+/-3.0 mU/L, p<0.01) with liver fibrosis is significantly elevated in comparison to a corresponding healthy control cohort of males (n=50, 23.9+/-2.8 mU/L) and females (n=52, 21.5+/-3.7 mU/L), respectively. Of note, independent from gender, serum XT activity positively correlated with the stage of fibrosis but declined again in patients with histologically proven cirrhosis. CONCLUSIONS: XT activity is increased in the serum of patients with liver fibrosis at different stages, pointing to a possible pathogenetic role in elevated proteoglycan biosynthesis in fibrotic remodeling of this organ during chronic injury.

BMP-7 as antagonist of organ fibrosis.

Fibrosis is a scarring process that is a common feature of chronic organ injury. It is characterized by elevated activity of transforming growth factor-beta resulting in increased and altered deposition of extracellular matrix and other fibrosis-associated proteins. Recent work has demonstrated that bone morphogenetic protein-7 blocks transforming growth factor-beta signaling. Moreover, member of the CCN family, Endoglin, Sclerostin, Sclerostin domain-containing proteins, Gremlin, Noggin, Chordin, and Kielin/Chordin-like protein influence the biological activity of both cytokines. As a consequence, they modulate cellular proliferation, migration, adhesion and extracellular matrix production. This tight protein network consisting of transforming growth factor-betas, bone morphogenetic proteins and various binding partners includes potential novel molecular targets and biomarkers useful for prognostication, disease monitoring and therapy. We here summarize recent advances in understanding bone morphogenetic protein-7 function and signaling and the current attempts to use this critical modulator as a pharmacological device to reverse transforming growth factor-beta-induced fibrogenesis.

Non-invasive biomarkers for monitoring the fibrogenic process in liver: a short survey.

The clinical course of chronic liver diseases is significantly dependent on the progression rate and the extent of fibrosis, i.e. the non-structured replacement of necrotic parenchyma by extracellular matrix. Fibrogenesis, i.e. the development of fibrosis can be regarded as an unlimited wound healing process, which is based on matrix (connective tissue) synthesis in activated hepatic stellate cells, fibroblasts (fibrocytes), hepatocytes and biliary epithelial cells, which are converted to matrix-producing (myo-)fibroblasts by a process defined as epithelial-mesenchymal transition. Blood (non-invasive) biomarkers of fibrogenesis and fibrosis can be divided into class I and class II analytes. Class I biomarkers are those single tests, which are based on the pathophysiology of fibrosis, whereas class II biomarkers are mostly multiparametric algorithms, which have been statistically evaluated with regard to the detection and activity of ongoing fibrosis. Currently available markers fulfil the criteria of ideal clinical-chemical tests only partially, but increased understanding of the complex pathogenesis of fibrosis offers additional ways for pathophysiologically well based serum (plasma) biomarkers. They include TGF-beta-driven marker proteins, bone marrow-derived cells (fibrocytes), and cytokines, which govern pro- and anti-fibrotic activities. Proteomic and glycomic approaches of serum are under investigation to set up specific protein or carbohydrate profiles in patients with liver fibrosis. These and other novel parameters will supplement or eventually replace liver biopsy/histology, high resolution imaging analysis, and elastography for the detection and monitoring of patients at risk of developing liver fibrosis.

Identification of paraxanthine as the most potent caffeine-derived inhibitor of connective tissue growth factor expression in liver parenchymal cells.

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)-beta 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-beta (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.

Secreted cysteine-rich FGF receptor derives from posttranslational processing by furin-like prohormone convertases.

Cysteine-rich FGF receptor (CFR) was originally identified as a FGF2 receptor and found to be identical to Golgi complex-localized glycoprotein-1 (GLG1), also known as MG-160, and to a murine E-selectin ligand-1 (ESL-1). Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the cis-medial Golgi complex, a substantial proportion of CFR is secreted but the underlying mechanism is unknown. CFR contains several possible furin-like proprotein convertase (PC) and matrix metalloproteinase cleavage sites. Cells expressing CFR were treated with the furin protease inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (decCMK) or the MMP-inhibitor GM6001. In the presence of furin-like PC inhibitor, secretion of CFR was almost completely inhibited. Secretion was not affected by the GM6001 inhibitor. The secreted forms were further characterized by creating different mutant CFR proteins with N-terminal and C-terminal tags. Immunoblot analysis and immunofluorescence indicated, that successive endoproteolytical processing of CFR which takes place in the Golgi complex is essential for secretion. Secreted CFR bound to heparan sulphate proteoglycan (HSPG) could trap FGFs and thereby directly competing with tyrosine kinase receptors for FGF binding.

Evaluation of serum percent trisialotransferrin as potential predictive biomarker of hepatocellular dedifferentiation in chronic liver disease.

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.

Inverse association between serum concentrations of actin-free vitamin D-binding protein and the histopathological extent of fibrogenic liver disease or hepatocellular carcinoma.

BACKGROUND AND AIM: Next to its role as a carrier protein for vitamin D and its plasma metabolites, the primary function of vitamin D-binding protein (DBP; Gc-globulin) is to serve and to bind and neutralize extracellular monomeric actin (G-actin) released from necrotic cells, which in its long filamentous form (F-actin) triggers coagulation. We, therefore, investigated the kinetics of serum concentrations of actin-free DBP during the pathogenetic sequence from liver fibrosis to hepatocellular carcinoma (HCC) to initiate a discussion on a hypothetical association of serum concentrations of actin-free DBP and, thus, altered actin clearance in the circulation, and an increased risk of thrombosis in patients with functional liver insufficiency. RESULTS: Our data show that serum levels of actin-free DBP are decreased in patients with liver fibrosis (n=288) and HCC (n=102) compared with healthy controls (n=120 and n=63) and nonliver disease sick (n=312) and that the level of decrease correlates with the severity of disease as determined according to the score by the French METAVIR Cooperative Study Group staging system for hepatic fibrosis or the Edmondson-Steiner's grading system for the assessment of HCC. CONCLUSION: Although further, in particular, longitudinal studies are still necessary to back up our data, the presented findings suggest that decreasing levels of the actin-scavenger DBP could potentially contribute to the thrombophilic predisposition frequently observed in patients with chronic fibrogenic liver disease and HCC.

Connective tissue growth factor is a Smad2 regulated amplifier of transforming growth factor beta actions in hepatocytes--but without modulating bone morphogenetic protein 7 signaling.

UNLABELLED: 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. CONCLUSION: CTGF acts as a Smad2-dependent sensitizer of TGF-beta actions that does not influence BMP7 signaling in hepatocytes.

Expression of E-selectin ligand-1 (CFR/ESL-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis.

Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

Questioning the role of actinfree Gc-Globulin as actin scavenger in neurodegenerative central nervous system disease: relationship to S-100B levels and blood-brain barrier function.

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.

Altered factor VII activating protease expression in murine hepatic fibrosis and its influence on hepatic stellate cells.

BACKGROUND: Platelet-derived growth factor-BB (PDGF-BB) is a profibrotic factor in liver fibrosis through its ability to stimulate hepatic stellate cells (HSC). The liver-derived serine protease factor VII activating protease (FSAP) regulates the activities of PDGF-BB in a cell-specific manner. AIMS: Our aim was to determine the influence of FSAP on the activation of HSC and to analyse the regulation of FSAP in hepatic fibrogenesis. METHODS: The effect of FSAP on PDGF-stimulated p42/p44 mitogen-activated protein kinase (MAPK) activation in primary rat HSC was determined by Western blotting. Migration and proliferation of HSC was evaluated in Boyden chamber experiments and (3)H-thymidine incorporation assays respectively. Expression of FSAP was analysed in a CCl(4) mouse model of liver fibrosis by Western blot, quantitative real-time polymerase chain reaction and immunohistochemistry. RESULTS: FSAP inhibited PDGF-BB-stimulated p42/p44 MAPK phosphorylation, proliferation and migration of HSC. FSAP mRNA expression level was increased 3 h after CCl(4) application and decreased after 18 h and, in established fibrosis, after chronic CCl(4) administration. In parallel, there was a decrease in the circulating FSAP protein in chronic fibrosis. Concurrently, the homogenous hepatic expression pattern of FSAP was disturbed. Immunohistochemistry revealed a decrease of FSAP in hepatocytes in inflammatory and fibrotic lesions. CONCLUSIONS: Our results demonstrate an inhibitory effect of FSAP on PDGF-mediated activation of HSC. In addition, FSAP expression is transiently increased in acute-phase reaction but decreased during chronic fibrogenesis, which in turn may influence PDGF-BB availability and myofibroblast activity.

Elevated concentrations of 15-deoxy-Delta12,14-prostaglandin J2 in chronic liver disease propose therapeutic trials with peroxisome proliferator activated receptor gamma-inducing drugs.

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Connective tissue growth factor: a fibrogenic master switch in fibrotic liver diseases.

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.

Platelet-derived growth factor isoform expression in carbon tetrachloride-induced chronic liver injury.

Platelet-derived growth factor (PDGF) has an essential role in liver fibrogenesis, as PDGF-B and -D both act as potent mitogens on culture-activated hepatic stellate cells (HSCs). Induction of PDGF receptor type-beta (PDGFR beta) in HSC is well documented in single-dose carbon tetrachloride (CCl(4))-induced acute liver injury. Of the newly discovered isoforms PDGF-C and -D, only PDGF-D shows significant upregulation in bile duct ligation (BDL) models. We have now investigated the expression of PDGF isoforms and receptors in chronic liver injury in vivo after long-term CCl(4) treatment and demonstrated that isolated hepatocytes have the requisite PDGF signaling pathways, both in the naive state and when isolated from CCl(4)-treated rats. In vivo, PDGF gene expression showed upregulation of all PDGF isoforms and receptors, with values peaking at 4 weeks and decreasing to near basal levels by 8 and 12 weeks. Interestingly, PDGF-C increased significantly when compared to BDL-models. PDGF-A, PDGF-C and PDGF receptor type-alpha (PDGFR alpha) correlated closely with inflammation and steatosis. Immunohistochemistry revealed expression of PDGF-B, -C and -D in areas corresponding to centrilobular necrosis, inflammation and fibrosis, whereas PDGF-A localized in regenerative hepatocytes. PDGFR beta was identified along the fibrotic septa, whereas PDGFR alpha showed positive staining in fibrotic septa and regenerative hepatocytes. Despite a significant decline of PDGF isoforms, hepatocyte regeneration peaked at 8 weeks. A marked difference in the degree of fibrosis was observed amongst the individual animals. In summary, PDGF expression in liver damage primarily parallels mesenchymal cell proliferation and extracellular matrix production, rather than hepatocyte regeneration. We conclude that PDGF levels in chronic liver injury peak at 4 weeks after onset of injury, and that the outcome of chronic toxic liver injury strongly depends on the individual capacity for tissue regeneration in the weeks following the peak of PDGF expression.

Transforming growth factor beta 1 gene variants increase transcription and are associated with liver cirrhosis in Chinese.

BACKGROUND AND AIMS: Transforming growth factor beta1 (TGFbeta1) acts as an important profibrogenic cytokine in liver fibrogenesis. The aim of this study was to explore the association between TGFbeta1 gene polymorphisms and liver cirrhosis. METHODS: Totally 118 Chinese suffering from liver cirrhosis induced by HBV infection and 104 healthy controls were recruited. The polymorphisms at positions -988, -800, -509 and codon10, codon25, codon263 of the TGFbeta1 gene were genotyped by ARMS-PCR or LightCycler. Enzyme immunoassay was used for TGFbeta1 measurement. The promoter activities and DNA-binding capacities containing -509C>T were analyzed by reporter gene and EMSA. RESULTS: The allele frequencies of CAT -509 and of T at codon10 were elevated in patients at severe Child-Pugh grade. Elevated concentrations of TGFbeta1 were observed in patients, especially in those with -509CC/CT and codon10 TT/TC. Flanking sequences containing -509C showed higher promoter activities than -509T. EMSA showed one nucleotide change at -509C>T influenced nuclear protein binding to the putative binding site. CONCLUSIONS: The C allele at -509 and the T allele at codon10 could play important roles in progression of liver cirrhosis. The C allele at -509 mediates higher transcriptional activity than the T allele providing a potential explanation for the clinical findings.

Maternal factor V Leiden mutation is associated with HELLP syndrome in Caucasian women.

OBJECTIVE: There is growing evidence that hypertensive pregnancy complications and other adverse pregnancy outcomes are associated with the presence of inherited or acquired thrombophilias. As hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome is one of the most severe forms of pre-eclampsia we aimed to assess the prevalence of the factor V Leiden, the prothrombin 20210G >A mutation and the methylenetetrahydrofolate reductase (MTHFR) 677C >T polymorphism in women with HELLP syndrome and in their fetuses from the same index pregnancy. DESIGN: The study was performed retrospectively in a case-control design. SAMPLE: Seventy-one mother-child pairs with HELLP syndrome and 79 control mother-child pairs with uncomplicated pregnancies were included in the study. METHODS: Genotyping of the three thrombophilic mutations was performed using the LightCycler technology. The chi-squared test was used for statistical analysis. Main outcome measures were maternal and fetal genotypes and their correlation with clinical parameters. RESULTS: Maternal heterozygosity for factor V Leiden was significantly more prevalent in the HELLP group than in controls (OR 4.45, 95% CI 1.31-15.31). No significant association was observed for maternal prothrombin mutation or MTHFR polymorphism (p=0.894, p=0.189, respectively). The fetal genotype was not associated with HELLP syndrome for any of the three mutations investigated. Analysis of gene-gene interactions and genotype-phenotype correlation with respect to clinical parameters and perinatal outcome revealed no further differences. CONCLUSIONS: Our study confirms that women heterozygous for factor V Leiden have an increased risk of developing HELLP syndrome, while the most frequent mutations of the prothrombin and MTHFR gene do not play a major role in the pathogenesis of HELLP syndrome.