Olav A. Gressner

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‐β)‐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‐β and several secondary effects owing to TGF‐β enhancement, and as a down‐modulator of the bioactivity of bone morphogenetic protein‐7 has been proposed. By changing the activity ratio of TGF‐β 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‐β 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.

Serum resistin levels in critically ill patients are associated with inflammation, organ dysfunction and metabolism and may predict survival of non-septic patients

IntroductionBlood glucose levels and insulin resistance in critically ill patients on admission to intensive care units (ICUs) have been identified as factors influencing mortality. The pathogenesis of insulin resistance (IR) in critically ill patients is complex and not fully understood. Resistin is a hormone mainly derived from macrophages in humans and from adipose tissue in rodents, which regulates glucose metabolism and insulin sensitivity. In non-critically ill patients, resistin was found to be related to impaired glucose tolerance, insulin resistance, metabolic syndrome, obesity and type 2 diabetes. Therefore, resistin might represent a link between inflammation, acute phase response and insulin resistance in critically ill patients. We aimed to examine the correlation of serum resistin concentrations to parameters of inflammation, organ function, metabolism, disease severity and survival in critically ill patients.MethodsOn admission to the Medical ICU, 170 patients (122 with sepsis, 48 without sepsis) were studied prospectively and compared with 60 healthy non-diabetic controls. Clinical data, various laboratory parameters, metabolic and endocrine functions as well as investigational inflammatory cytokine profiles were assessed. Patients were followed for approximately three years.ResultsResistin serum concentrations were significantly elevated in all critical care patients compared with healthy controls, and significantly higher in sepsis than in non-sepsis patients. Serum resistin concentrations were not associated with pre-existing type 2 diabetes or obesity. For all critically ill patients, a correlation to the homeostasis model assessment index of insulin resistance (HOMA-IR) was shown. Serum resistin concentrations were closely correlated to inflammatory parameters such as C-reactive protein, leukocytes, procalcitonin, and cytokines such as IL6 and TNF-α, as well as associated with renal failure and liver synthesis capacity. High resistin levels (> 10 ng/ml) were associated with an unfavourable outcome in non-sepsis patients on ICU and the overall survival.ConclusionsSerum resistin concentrations are elevated in acute inflammation due to sepsis or systemic inflammatory response syndrome (SIRS). The close correlation with other acute phase proteins suggests a predominant, clinically relevant resistin release from macrophages in ICU patients. Moreover, resistin could potentially serve as a prognostic biomarker in non-sepsis critically ill patients.

Activation of hepatic stellate cells is associated with cytokine expression in thioacetamide-induced hepatic fibrosis in mice

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 α-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-1β, IL-6), platelet-derived growth factors (PDGF-B, PDGF-D) , tumor necrosis factor (TNF)-α, and (transforming growth factor (TGF)-β1 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-1β, IL-6, TGF-β1, and PDGF-B (p.m. week 12) whereas TNF-α 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-1β 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.

Pharmacological application of caffeine inhibits TGF-β-stimulated connective tissue growth factor expression in hepatocytes via PPARγ and SMAD2/3-dependent pathways ☆

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-beta, 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 PPARgamma expression, that enhances the inhibitory effect of the natural PPARgamma agonist 15-PGJ(2) on CTGF expression by inducing a dissociation of the SMAD2/3-CBP/p300-transcriptional complex. CONCLUSIONS We show that caffeine strongly down-modulates TGF-beta-induced CTGF expression in hepatocytes by stimulation of degradation of the TGF-beta effector SMAD 2, inhibition of SMAD3 phosphorylation and up-regulation of the PPARgamma-receptor. Long-term caffeinization might be an option for anti-fibrotic trials in chronic liver diseases.

Biomarkers of hepatic fibrosis, fibrogenesis and genetic pre-disposition pending between fiction and reality

•  Introduction •  Pathways, cells and molecular mediators in liver fibrogenesis •  Classification of biomarkers of fibrosis •  Class I biomarkers of fibrosis •  Class II fibrosis biomarkers •  Genetic pre‐disposition biomarkers •  Future developments

Changing the pathogenetic roadmap of liver fibrosis? Where did it start; where will it go?

The pathophysiology of liver injury has attracted the interest of experimentalists and clinicians over many centuries. With the discovery of liver‐specific pericytes – formerly called fat‐storing cells, Ito‐cells, lipocytes, and currently designated as hepatic stellate cells (HSC) – the insight into the cellular and molecular pathobiology of liver fibrosis has evolved and the pivotal role of HSC as a precursor cell‐type for extracellular matrix–producing myofibroblasts has been established. Although activation and transdifferentiation of HSC to myofibroblasts is still regarded as the pathogenetic key mechanism of fibrogenesis, recent studies point to a prominent heterogeneity of the origin of myofibroblasts. Currently, the generation of matrix‐synthesizing fibroblasts by epithelial–mesenchymal transition, by influx of bone marrow–derived fibrocytes into damaged liver tissue, and by differentiation of circulating monocytes to fibroblasts after homing in the injured liver are discussed as important complementary mechanisms to enlarge the pool of (myo‐)fibroblasts in the fibrosing liver. Among the molecular mediators, transforming growth factor‐beta (TGF‐β) plays a central role, which is controlled by the bone‐morphogenetic protein (BMP)‐7, an important antagonist of TGF‐β action. The newly discovered pathways supplement the linear concept of HSC activation to myofibroblasts, point to fibrosis as a systemic response involving extrahepatic organs and reactions, add further evidence to a more or less uniform concept of organ fibrosis in general (e.g. liver, lung, kidney), and offer innovative approaches for the development of non‐invasive biomarkers and antifibrotic trials.

Activation of TGF‐β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

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‐β. In this study TGF‐β‐dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF‐β‐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‐β within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF‐β 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‐β, but calpain inhibitors partially suppressed intracellular TGF‐β activation and subsequently CTGF up‐regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF‐β 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‐β 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‐β. In conclusion, these data are the first to suggest the possibility of intracrine TGF‐β signalling due to calpain‐dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF‐β‐sensitive reporter gene. This mechanism might be deleterious for keeping long‐term hepatocyte cultures due to TGF‐β‐induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial‐mesenchymal transition of hepatocytes in injured liver.

Validation of connective tissue growth factor (CTGF/CCN2) and its gene polymorphisms as noninvasive biomarkers for the assessment of liver fibrosis

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.

Connective tissue growth factor is a Smad2 regulated amplifier of transforming growth factor β actions in hepatocytes—But without modulating bone morphogenetic protein 7 signaling†

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 β (TGF‐β) 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‐β/BMP signaling pathways is still unknown. This study aims at characterizing a potentially differential modulating role of CTGF on TGF‐β– 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‐β–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‐β, as well as the failure of TGF‐β to inhibit the transcriptional activation of the Smad5‐selective (BRE)2‐luc reporter by BMP7; and (3) the sensitization of hepatocytes toward TGF‐β type I receptor (ALK5)/Smad2 and Smad3‐mediated TGF‐β 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‐β actions that does not influence BMP7 signaling in hepatocytes. (HEPATOLOGY 2009.)

Genetic Determinants in Hepatic Fibrosis: From Experimental Models to Fibrogenic Gene Signatures in Humans

Hepatic fibrosis, or scarring of the liver, is a nonspecific reaction to chronic liver injury. Hepatic fibrosis is commonly caused by exogenous factors such as viral hepatitis or alcohol abuse, but recent studies also indicate a genetic predisposition. Although some patients who have chronic liver diseases show only minor morphologic and functional alterations of the liver and are characterized by slow progression of disease with mild clinical symptoms, others develop pronounced hepatic fibrosis rapidly, culminating in cirrhosis, liver failure, or hepatocellular carcinoma, respectively. These well known differences in progression of hepatic fibrosis persist when controlling for age (at infection), gender, and exogenous factors in multivariate analysis, indicating that genetic factors might play important roles in the modulation of hepatic fibrosis and contribute to the variability in fibrosis progression. This review summarizes genetic determinants in hepatic fibrosis.