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Background: The Baveno VI consensus proposed a dual liver stiffness (LS) by transient elastography threshold of <10 and >15 kPa for excluding and diagnosing compensated advanced chronic liver disease (cACLD) in the absence of other clinical signs. Herein, we aimed to validate these criteria in a real-world multicentre study., Methods: We included 5,648 patients (mean age 51 ± 13 years, 53% males) from 10 European liver centres who had a liver biopsy and LS measurement within 6 months. We included patients with chronic hepatitis C (n = 2,913, 52%), non-alcoholic fatty liver disease (NAFLD, n = 1,073, 19%), alcohol-related liver disease (ALD, n = 946, 17%) or chronic hepatitis B (n = 716, 13%). cACLD was defined as fibrosis stage ≥F3., Results: Overall, 3,606 (66%) and 987 (18%) patients had LS <10 and >15 kPa, respectively, while cACLD was histologically confirmed in 1,772 (31%) patients. The cut-offs of <10 and >15 kPa showed 75% sensitivity and 96% specificity to exclude and diagnose cACLD, respectively. Examining the ROC curve, a more optimal dual cut-off at <7 and >12 kPa, with 91% sensitivity and 92% specificity for excluding and diagnosing cACLD (AUC 0.87; 95% CI 0.86-0.88; p <0.001) was derived. Specifically, for ALD and NAFLD, a low cut-off of 8 kPa can be used (sensitivity=93%). For the unclassified patients, we derived a risk model based on common patient characteristics with better discrimination than LS alone (AUC 0.74 vs. 0.69; p <0.001)., Conclusions: Instead of the Baveno VI proposed <10 and >15 kPa dual cut-offs, we found that the <8 kPa (or <7 kPa for viral hepatitis) and >12 kPa dual cut-offs have better diagnostic accuracy in cACLD., Lay Summary: The term compensated advanced chronic liver disease (cACLD) was introduced in 2015 to describe the spectrum of advanced fibrosis and cirrhosis in asymptomatic patients. It was also suggested that cACLD could be diagnosed or ruled out based on specific liver stiffness values, which can be non-invasively measured by transient elastography. Herein, we assessed the suggested cut-off values and identified alternative values that offered better overall accuracy for diagnosing or ruling out cACLD., Competing Interests: Conflicts of interest Victor de Ledinghen reports consultancy for Echosens and SuperSonic Imagine. All other authors have nothing to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The pathogenesis of non-alcoholic fatty liver disease, particularly the mechanisms whereby a minority of patients develop a more severe phenotype characterised by hepatocellular damage, inflammation, and fibrosis is still incompletely understood. Herein, we discuss two pivotal aspects of the pathogenesis of NASH. We first analyse the initial mechanisms responsible for hepatocellular damage and inflammation, which derive from the toxic effects of excess lipids. Accumulating data indicate that the total amount of triglycerides stored in hepatocytes is not the major determinant of lipotoxicity, and that specific lipid classes act as damaging agents on liver cells. In particular, the role of free fatty acids such as palmitic acid, cholesterol, lysophosphatidylcholine and ceramides has recently emerged. These lipotoxic agents affect the cell behaviour via multiple mechanisms, including activation of signalling cascades and death receptors, endoplasmic reticulum stress, modification of mitochondrial function, and oxidative stress. In the second part of this review, the cellular and molecular players involved in the cross-talk between the gut and the liver are considered. These include modifications to the microbiota, which provide signals through the intestine and bacterial products, as well as hormones produced in the bowel that affect metabolism at different levels including the liver. Finally, the activation of nuclear receptors by bile acids is analysed., (Copyright © 2017. Published by Elsevier B.V.)

Background: The accuracy of non-invasive methods for the quantification of liver fibrosis in primary biliary cholangitis (PBC) is still debated., Aims: To determine the histo-morphometric measurement of fibrotic tissue and to explore the possible association between indirect markers (APRI, FORNS, FIB-4, and Lok) and morphometry., Methods: Retrospective analysis of medical data from patients with PBC, on whom needle liver biopsy was performed as part of the diagnostic assessment. One section of each biopsy stained with Sirius red was used for calculating the percentage of collagen. Quantitative measure of fibrotic tissue (fibrosis morphometry) was calculated as a percentage of collagen content by digital image analysis. Morphometry results were divided into four groups reflecting Ludwig's staging and compared with values for indirect serum markers., Results: 50 PBC patients were enrolled (86% females, mean age 57 ± 12.30 years), 19 were Ludwig's stage I (38%), 14 stage II (28%), 12 stage III (24%), and five stage IV (10%). Morphometry results were significantly different among Ludwig stages (p<0.05). No significant differences were found for indirect serum markers. A significant correlation was found between morphometry results and indirect serum markers tested (p<0.05)., Conclusion: In our cohort, the histo-morphometric values of fibrotic tissue increased progressively with Ludwig's stages of PBC, while non-invasive markers did not., (Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Aim: To evaluate the association between liver stiffness (LS) prior to the initiation of dual/triple therapy and viral response., Methods: LS was measured in all patients before treatment was administered. The therapeutic approach was based on hepatic, virological, and immunological evaluations and considered the fact that patients with severe fibrosis (F3) or compensated cirrhosis (F4) in Child-Pugh class A are the primary candidates for triple therapy. In total, 65 hepatitis C virus (HCV) patients were treated with Peg-interferon/ribavirin (Peg-IFN/RBV); 24 patients were classified as genotypes 1/4 (36.92%), and 41 patients were classified as genotypes 2/3 (63.08%) (dual therapy). In addition, 20 HCV treatment-experienced genotype 1 patients were treated with PegIFN-RBV and boceprevir (triple therapy). Wilcoxon rank-sum tests were used to compare the groups., Results: LS significantly differed between dual therapy and triple therapy (P = 0.002). The mean LS value before dual therapy treatment was 8.61 ± 5.79 kPa and was significantly different between patients achieving a sustained virologic response (SVR) 24 weeks after therapy and those who did not (7.23 ± 5.18 kPa vs 11.72 ± 5.99 kPa, respectively, P = 0.0003). The relative risk of non-response to therapy was 4.45 (95%CI: 2.32-8.55). The attributable risk of non-response to therapy was 49%. The mean LS value before triple therapy treatment was 13.29 ± 8.57 kPa and was significantly different between patients achieving and not achieving SVR24 (9.41 ± 5.05 vs 19.11 ± 9.74, respectively; P = 0.008). The relative risk of non-response to therapy was 5.57% (95%CI: 1.50-20.65). The attributable risk of non-response to therapy (70%) was increased compared with dual therapy patients. Pre-treatment stiffness > 12 kPa was significantly associated with non-SVR (P < 0.025) in both groups., Conclusion: Pre-treatment liver stiffness may be useful for predicting the response to treatment in patients treated with either dual or triple anti-HCV therapy.

Sustained hepatic inflammation is an important factor in progression of chronic liver diseases, including hepatitis C or non-alcoholic steatohepatitis. Liver inflammation is regulated by chemokines, which regulate the migration and activities of hepatocytes, Kupffer cells, hepatic stellate cells, endothelial cells, and circulating immune cells. However, the effects of the different chemokines and their receptors vary during pathogenesis of different liver diseases. During development of chronic viral hepatitis, CCL5 and CXCL10 regulate the cytopathic versus antiviral immune responses of T cells and natural killer cells. During development of nonalcoholic steatohepatitis, CCL2 and its receptor are up-regulated in the liver, where they promote macrophage accumulation, inflammation, fibrosis, and steatosis, as well as in adipose tissue. CCL2 signaling thereby links hepatic and systemic inflammation related to metabolic disorders and insulin resistance. Several chemokine signaling pathways also promote hepatic fibrosis. Recent studies have shown that other chemokines and immune cells have anti-inflammatory and antifibrotic activities. Chemokines and their receptors can also contribute to the pathogenesis of hepatocellular carcinoma, promoting proliferation of cancer cells, the inflammatory microenvironment of the tumor, evasion of the immune response, and angiogenesis. We review the roles of different chemokines in the pathogenesis of liver diseases and their potential use as biomarkers or therapeutic targets., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: n-3 polyunsaturated fatty acids (PUFA) ameliorate fatty liver in experimental models, but their effects on inflammation and fibrosis during steatohepatitis are either controversial or lacking. We compared the effects of supplementation with olive oil (OO) alone or OO and n-3 PUFA on the development and progression of experimental steatohepatitis., Methods: Balb/C mice (≥5 mice/group) were fed a methionine- and choline-deficient (MCD) diet or a control diet for 4 or 8 weeks. At the same time, mice were supplemented with n-3 PUFA (eicosapentaenoic and docosahexahenoic acid, 25 mg together with 75 mg OO), or OO alone (100 mg), two times a week by intragastric gavage., Results: After 8 weeks, mice on MCD/n-3 had higher ALT levels compared to MCD/OO and more severe scores of inflammation, including a significant increase in the number of lipogranulomas (26.4 ± 8.4 vs. 5.1 ± 5 per field, P < 0.001). Intrahepatic expression of TNF-α and CCL2 was higher in MCD/n-3 mice at both time points. In addition, increased expression of the profibrogenic genes TIMP-1 and TGF-β, and more severe histological scores of fibrosis were evident in MCD/n-3 mice. After 8 week of MCD diet, portal pressure was higher in mice receiving n-3 than in those on OO alone (5.1 ± 1.4 vs. 7.0 ± 0.9 mmHg, P < 0.05). Analysis of hepatic fatty acid profile showed that supplementation resulted in effective incorporation of n-3 PUFA., Conclusions: In a murine model of steatohepatitis, supplementation with n-3 PUFA and OO is associated with more severe necro-inflammation and fibrosis than in mice treated with OO only., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Liver stiffness has been suggested as a parameter of fibrosis progression/regression in hepatitis C virus (HCV) patients., Aim: To evaluate stiffness before and after peginterferon-ribavirin treatment., Methods: Stiffness was prospectively measured in 74 HCV patients, 32 genotypes 1/4 (43.25%) and 42 genotypes 2/3 (56.75%), before, at end of treatment, and after 3 years of follow-up (49 patients). On the same study day, 21 patients underwent liver biopsy., Results: In 55 patients with sustained virological response (74.32%), liver stiffness decreased significantly at end of therapy (6.8±4.9kPa) vs. baseline (9.5±6.9kPa, p=0.04). The decrease vs. baseline was maintained in 30 sustained virological response patients after 3 years follow-up (6.8±4.6kPa vs. 10.8±8.5kPa, p=0.0141). No difference was found at end of treatment vs. baseline (10.1±4.7kPa vs. 9.7±4.2kPa, p=0.825) and after 3 years of follow-up vs. baseline (10.2±3.4kPa vs. 9.7±4.2kPa, p=0.765) in null responders. Similar results were found in relapsers at end of treatment vs. baseline (13.7±7.7kPa vs. 15.2±8.2kPa, p=0.74), and after 3 years of follow-up vs. baseline (16.9±10.0kPa vs. 15.2±8.2kPa, p=0.734). Pre-treatment stiffness >12kPa was significantly associated with no SVR (p<0.025), RR=2.44 (95%C.I. 1.17-5.07)., Conclusion: Liver stiffness may be useful to assess long-term antiviral treatment response., (Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Transient elastography (TE) is increasingly employed in clinical practice for the noninvasive detection of tissue fibrosis in patients with chronic liver disease (CLD), and particularly chronic hepatitis C virus (HCV)-related hepatitis. The present study was designed to provide a definitive characterization of the "confounding" increase in liver stiffness (LS) following a standardized meal in a consecutive population of 125 patients with chronic HCV infection at different stages of fibrotic evolution. LS values were obtained after overnight fasting and 15, 30, 45, 60, and 120 minutes following the onset of a standardized liquid meal (400 mL, 600 Kcal, 16.7% protein, 53.8% carbohydrates, 29.5% fat). An evident increase in LS values was observed 15 to 45 minutes after the onset of the meal with return to baseline premeal levels within 120 minutes in all patients. The peak postmeal delta increase in LS was progressively more marked with increasing stages of fibrosis (P < 0.001), becoming maximal in patients with cirrhosis. However, the probability of identifying the Metavir stage of fibrosis, the Child-Pugh class, or the presence/absence of esophageal varices with the postmeal delta increase in LS was inferior to that obtained with baseline LS values., Conclusion: The results of the present study provide definitive evidence of the confounding effect of a meal on the accuracy of LS measurements for the prediction of fibrosis stage in patients with chronic HCV hepatitis and suggest that a fasting period of 120 minutes should be observed before the performance of TE., (Copyright © 2013 American Association for the Study of Liver Diseases.)

Aminotransferase elevation is a frequent cause of consultation for the Hepatologist, in both the outpatient and inpatient settings, but identifying the origin of these biochemical alterations may be challenging. Here we report a case where acute elevation of aminotransferases, associated with abdominal symptoms, was the cause of two hospitalizations in a short period of time. As the patient suffered from type 1 diabetes, celiac disease, and autoimmune thyroiditis, several potential causes of damage could be hypothesized, including celiac hepatitis, fatty liver, and autoimmune hepatitis. A liver biopsy performed in the occasion of the second hospitalization allowed to rule out autoimmune hepatitis and celiac hepatitis, showing mild signs of fatty infiltration. Staining with periodic acid-Schiff with or without diastase showed a marked accumulation of glycogen, indicating the presence of a glycogenic hepatopathy associated with poorly controlled type 1 diabetes. This condition may be a cause of liver damage in patients with type 1 and occasionally type 2 diabetes, but its occurrence is often overlooked. This case report illustrates the fact that glycogenic hepatopathy may relapse, and prompts the clinician to take into account this condition in the differential diagnosis of causes of liver injury.

Basic knowledge in the interpretation of hepatic imaging is essential for the clinical hepatologist. In recent years, the availability of dynamic imaging studies of the liver using computed tomography or magnetic resonance has led to appreciate the importance of early changes in arterial perfusion for the interpretation of hepatic lesions. Transient Hepatic Parenchymal Enhancement (THPE) is defined as a normal area of liver parenchyma that enhances after injection of contrast agent during the arterial phase of perfusion. Appearance of this sign is mostly associated with a reduction in portal perfusion or with inflammation, and appears in different morphologic patterns. THPE should not be considered a radiological artefact, and its interpretation is essential to avoid misclassification of hepatic lesions that may have clinical significance, such as hepatocellular carcinoma or hepatic metastases. In this short review we provide essential information on the causes, pathophysiology and morphology of THPE, and discuss the relevance of these findings in a clinical perspective., (Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Liver stiffness values were recently proposed to identify patients with methotrexate-induced liver fibrosis. Aim of this study was to assess the clinical and laboratory determinants of the association between liver stiffness, measured by transient elastography, and methotrexate treatment in patients with rheumatoid arthritis in the absence of other factors contributing to liver damage and fibrosis., Methods: 100 patients with rheumatoid arthritis, with a cumulative methotrexate dose ranging from 1530 to 13,000 mg over a mean period of 7.07±3.89 yrs, were retrospectively evaluated., Results: The average liver stiffness value in the whole population was 4.93±1.8 kPa, excluding the presence of significant fibrosis. At univariate analysis, a significant correlation was found between liver stiffness and methotrexate cumulative dose, duration of treatment, alanine transaminases levels, body mass index, gamma glutamyl-transpeptidase and the presence of steatosis. At multivariate analysis, a significant association was detected only between liver stiffness and methotrexate cumulative dose. Out of 11 patients with liver stiffness >7.0 kPa, five were subjected to liver biopsy and mild or moderate perisinusoidal fibrosis was detected in two patients with a cumulative dose >4000 mg and liver stiffness >9 kPa., Conclusions: Chronic methotrexate treatment induces a progressive increase in liver stiffness corresponding to mild or moderate perisinusoidal fibrosis for values >9 kPa., (Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Leptin modulates the angiogenic properties of hepatic stellate cells (HSC), but the molecular mechanisms involved are poorly understood. We investigated the pathways regulating hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in leptin-stimulated myofibroblastic HSC. Exposure to leptin enhanced the phosphorylation of TSC2 on T1462 residues and of p70 S6 kinase and the translational inhibitor 4E-binding protein-1, indicating the ability of leptin to activate the mammalian target of rapamycin (mTOR) pathway. Similar findings were observed when HSC were exposed to PDGF. Both leptin and PDGF increased the expression of HIF-1α and VEGF in HSC. In the presence of rapamycin, a specific mTOR inhibitor, leptin and PDGF were no longer able to activate mTOR, and expression of VEGF was reduced, whereas HIF-1α abundance was not affected. Moreover, knockdown of Raptor, a component of the mTORC1 complex, reduced the ability of leptin to increase VEGF. mTOR was also necessary for leptin- and PDGF-dependent increase in HSC migration. Leptin increased the generation of reactive oxygen species in HSC, which was reduced by NADP(H) oxidase inhibitors. Both N-acetyl cysteine and diphenylene iodonium, a NADP(H) inhibitor, inhibited the expression of HIF-1α and VEGF stimulated by leptin or PDGF. Finally, conditioned media from HSC treated with leptin or PDGF induced tube formation in cultured human umbilical vein endothelial cells. In conclusion, in HSC exposed to leptin or PDGF, increased expression of VEGF requires both activation of mTOR and generation of reactive oxygen species via NADPH-oxidase. Induction of HIF-1α requires NADP(H) oxidase but not mTOR activation.

We have investigated the presence and the possible clinical implications of oxidative stress in children with non-alcoholic fatty liver disease (NAFLD). The present study was an observational study of oxidative stress parameters in the progression of paediatric NAFLD. We observed the role of oxidative stress in children diagnosed with NAFLD by evaluating: serum protein carbonyls, hepatic expression of 8-hydroxy-2-deoxyguanosine (8-OHG), and circulating antibody against malondialdehyde adducted human serum albumin (MDA-HSA). Forty consecutive children with biopsy-proven NAFLD (27 male; 13 female) referred to Bambino Gesù Children's Hospital, Rome, Italy, from January 2007 to April 2008 were included in the study. Serum variations of protein carbonyls, 8-OHG, and circulating antibody against MDA-HSA were evaluated. Elevated protein carbonyls were evident in 33 subjects (83%) irrespective of obesity and insulin resistance. Moreover, liver biopsies of NAFLD patients positive for circulating protein carbonyls also showed a significant increase in the nuclear staining for 8-OHG (p=0.006; 95% CI 3.1-17.7). Anti-MDA-HSA IgG above control threshold was detected in 25 (63%) children. Although protein carbonyl levels were unrelated with disease severity, patients with elevated anti-MDA-HSA IgG had scores for lobular inflammation significantly higher (p=0.019) than subjects with antibodies within the control range, while steatosis, hepatocyte ballooning and fibrosis were similar. High anti-MDA-HSA reactivity was also associated with a 13-fold increased risk (OR=12.9; 95= CI 1.5-113.8; p=0.013) of a NAFLD activity score (NAS) >or=5. These results demonstrate that oxidative stress has an high prevalence in children with NAFLD and is associated with an increased severity of steatohepatitis.

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.

Unlabelled: Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR., Conclusion: Activation of AMPK negatively modulates the activated phenotype of HSCs.

Background/aims: Hepatic stellate cells (HSC) are liver-specific pericytes implicated in liver tissue repair. Activation of signaling pathways in HSC modulates hepatic fibrogenesis, but no information is available on the possible role of ERK5, a member of the mitogen-activated protein kinase family, in this process. In this study, we investigated the role of ERK5 in the biologic responses triggered by platelet-derived growth factor (PDGF) in HSC., Methods: Human HSC were cultured on plastic and studied in their myofibroblast-like phenotype., Results: PDGF-BB rapidly induced ERK5 activation and translocation to the nucleus. EGF and PDGF-DD were also found to activate ERK5. Interfering with Src activation blocked PDGF-BB-dependent ERK5 phosphorylation. To establish the biological significance of ERK5 activation, HSC were transfected with non-targeting siRNA or siRNA targeting ERK5. ERK5 silencing inhibited PDGF-BB-induced cell proliferation, and expression and activation of c-Jun. In contrast, depletion of ERK5 was associated with significantly increased cell migration, both in the presence or absence of PDGF-BB. This effect was associated with a redistribution of focal contacts, and with decreased phosphorylation of FAK, paxillin, and PAK., Conclusions: ERK5 modulates PDGF-dependent biologic activities in human HSC, generating positive signals for cell proliferation downregulating the ability of the cells to migrate.

Pathological angiogenesis is associated with the fibrogenic progression of chronic liver diseases. Experimental data suggest that hypoxia and vascular endothelial growth factor (VEGF) may stimulate proliferation and synthesis of type I collagen in activated, myofibroblast-like rat hepatic stellate cells (HSC/MFs). In this study, we investigated whether hypoxia, recombinant VEGF, or angiopoietin 1 (Ang-1) may affect other crucial profibrogenic features. In human HSC/MFs, which constitutively express VEGF receptor-1 and -2 (VEGFR-1, VEGFR-2) and the Ang-1 receptor Tie-2, exposure to hypoxia, VEGF, or Ang-1 resulted in a Ras/Erk-dependent stimulation of chemokinesis and chemotaxis. Migration of human HSC/MFs under hypoxic conditions involved up-regulation of VEGF-A, Ang-1, and related receptors and was mainly dependent on VEGFR-2 (Flk-1). In specimens from either cirrhotic rat livers or from patients with hepatitis C virus-related cirrhosis, HSC/MFs expressed proangiogenic factors and related receptors in areas of active fibrogenesis (ie, at the leading or lateral edge of developing incomplete fibrotic septa). Data presented herein suggest that VEGF and Ang-1 may contribute to fibrogenesis by acting as hypoxia-inducible, autocrine, and paracrine factors able to recruit myofibroblast-like cells. Moreover, HSC/MFs, in addition to their established profibrogenic role, may also contribute to neoangiogenesis during chronic hepatic wound healing.

Unlabelled: Measurement of hepatic venous pressure gradient (HVPG) is a standard method for the assessment of portal pressure and correlates with the occurrence of its complications. Liver stiffness measurement (LSM) has been proposed as a noninvasive technique for the prediction of the complications of cirrhosis. In this study, we evaluated the ability of LSM to predict severe portal hypertension compared with that of HVPG in 61 consecutive patients with HCV-related chronic liver disease. A strong relationship between LSM and HVPG measurements was found in the overall population (r=0.81, P<0.0001). However, although the correlation was excellent for HVPG values less than 10 or 12 mm Hg (r=0.81, P=0.0003 and r=0.91, P<0.0001, respectively), linear regression analysis was not optimal for HVPG values>or=10 mm Hg (r2=0.35, P<0.0001) or>or=12 mm Hg (r2=0.17, P=0.02). The AUROC for the prediction of HVPG>or=10 and >or=12 mm Hg were 0.99 and 0.92, respectively and at LSM cutoff values of 13.6 kPa and 17.6 kPa, sensitivity was 97% and 94%, respectively. In patients with cirrhosis, LSM positively correlated with the presence of esophageal varices (P=0.002), although no correlation between LSM and esophageal varices size was detected. The area under the ROC for the prediction of EV was 0.76 and at a LSM cutoff value of 17.6 kPa sensitivity was 90%., Conclusion: LSM represents a non-invasive tool for the identification of chronic liver disease patients with clinically significant or severe portal hypertension and could be employed for screening patients to be subjected to standard investigations including upper GI endoscopy and hemodynamic studies.

Obesity and insulin resistance accelerate the progression of fibrosis during chronic liver disease. Resistin antagonizes insulin action in rodents, but its role in humans is still controversial. The aims of this study were to investigate resistin expression in human liver and to evaluate whether resistin may affect the biology of activated human hepatic stellate cells (HSCs), key modulators of hepatic fibrogenesis. Resistin gene expression was low in normal human liver but was increased in conditions of severe fibrosis. Up-regulation of resistin during chronic liver damage was confirmed by immunohistochemistry. In a group of patients with alcoholic hepatitis, resistin expression correlated with inflammation and fibrosis, suggesting a possible action on HSCs. Exposure of cultured HSCs to recombinant resistin resulted in increased expression of the proinflammatory chemokines monocyte chemoattractant protein-1 and interleukin-8, through activation of nuclear factor (NF)-kappaB. Resistin induced a rapid increase in intracellular calcium concentration, mainly through calcium release from intracellular inositol triphosphate-sensitive pools. The intracellular calcium chelator BAPTA-AM blocked resistin-induced NF-kappaB activation and monocyte chemoattractant protein-1 expression. In conclusion, this study shows a role for resistin as an intrahepatic cytokine exerting proinflammatory actions in HSCs, via a Ca2+/NF-kappaB-dependent pathway and suggests involvement of this adipokine in the pathophysiology of liver fibrosis.

Background and Aims: Activated myofibroblast-like cells, originating from hepatic stellate cells (HSC/MFs) or other cellular sources, play a key profibrogenic role in chronic liver diseases (CLDs) that, as suggested by studies in animal models or rat HSC/MFs, may be modulated by reactive oxygen intermediates (ROI). In this study, human HSC/MFs, exposed to different levels of superoxide anion (O(2)(.-)) and, for comparison, hydrogen peroxide (H(2)O(2)), were analysed in terms of cytotoxicity, proliferative response, and migration., Methods: Cultured human HSC/MFs were exposed to controlled O(2)(.-) generation by hypoxanthine/xanthine oxidase systems or to a range of H(2)O(2) concentrations. Induction of cell death, proliferation, and migration were investigated using morphology, molecular biology, and biochemical techniques., Results: Human HSC/MFs were shown to be extremely resistant to induction of cell death by O(2)(.-) and only high rates of O(2)(.-) generation induced either necrotic or apoptotic cell death. Non-cytotoxic low levels of O(2)(.-), able to upregulate procollagen type I expression (but not tissue inhibitor of metalloproteinase 1 and 2), stimulated migration of human HSC/MFs in a Ras/extracellular regulated kinase (ERK) dependent, antioxidant sensitive way, without affecting basal or platelet derived growth factor (PDGF) stimulated cell proliferation. Non-cytotoxic levels of H(2)O(2) did not affect Ras/ERK or proliferative response. A high rate of O(2)(.-) generation or elevated levels of H(2)O(2 )induced cytoskeletal alterations, block in motility, and inhibition of PDGF dependent DNA synthesis., Conclusions: Low non-cytotoxic levels of extracellularly generated O(2)(.-) may stimulate selected profibrogenic responses in human HSC/MFs without affecting proliferation.

Leptin upregulates collagen expression in hepatic stellate cells (HSCs), but the possible modulation of other actions has not been elucidated. The aim of this study was to investigate the expression and function of leptin receptors (ObR) in human HSCs and the biological actions regulated by leptin. Exposure of HSCs to leptin resulted in upregulation of monocyte chemoattractant protein 1 (MCP-1) expression. Leptin also increased gene expression of the proangiogenic cytokines vascular endothelial growth factor (VEGF) and angiopoietin-1, and VEGF was also upregulated at the protein level. Activated HSCs express ObRb and possibly other ObR isoforms. Exposure to leptin increased the tyrosine kinase activity of ObR immunoprecipitates and resulted in activation of signal transducer and activator of transcription 3. Several signaling pathways were activated by leptin in HSCs, including extracellular-signal-regulated kinase, Akt, and nuclear factor kappaB, the latter being relevant for chemokine expression. Leptin also increased the abundance of hypoxia-inducible factor 1alpha, which regulates angiogenic gene expression, in an extracellular-signal-regulated kinase- and phoshatidylinositol 3-kinase-dependent fashion. In vivo, leptin administration induced higher MCP-1 expression and more severe inflammation in mice after acute liver injury. Conversely, in leptin-deficient mice, the increase in MCP-1 messenger RNA and mononuclear infiltration was less marked than in wild-type littermates. Finally, ObR expression colocalized with VEGF and alpha-smooth muscle actin after induction of fibrosis in rats. In conclusion, ObR activation in HSCs leads to increased expression of proinflammatory and proangiogenic cytokines, indicating a complex role for leptin in the regulation of the liver wound-healing response.

A 28-year-old female developed multidrug-resistant (MDR) tuberculous lymphadenitis following a trip to India. She was initially treated with a four-drug regimen of first-line anti-tuberculosis medications, but when sensitivities indicated resistance to isoniazid and rifampin, her regimen was altered to ciprofloxacin (CFX), pyrazinamide (PZA) and ethambutol. She subsequently developed a rash, flu-like symptoms and fever, which progressed to acute hepatic necrosis despite discontinuation of medication. The clinical presentation and subsequent investigations suggested a hypersensitivity reaction, possibly related to the quinolone. The patient subsequently had an orthoptic liver transplant; second-line anti-tuberculosis medications were restarted to which she responded clinically and radiologically. Our findings raise the possibility that the CFX and PZA combination was responsible for the hepatic necrosis. The patient also illustrates that active, even MDR tuberculosis is not a contraindication to hepatic transplant.

Background/aims: 4-Hydroxynonenal (HNE) is a putative pro-fibrogenic product of oxidative stress able to elicit apoptosis and cytotoxicity in several cell types. This study has been performed to evaluate its 'in vivo' levels in injured liver and whether HNE may induce apoptosis and/or affect selected phenotypic responses in activated human hepatic stellate cells (HSC/MF)., Methods/results: During the development of acute liver injury induced by CCl(4), liver tissue HNE levels were in the range 0.5-10 microM, as shown by high performance liquid chromatography analysis. Cultured human HSC/MF, developed cytotoxicity only if exposed to very high HNE concentrations (25-50 microM) without any sign of induction of classic, caspase-dependent apoptosis, as assessed by evaluating morphology and biochemical parameters of cell death. HNE, at non-cytotoxic doses, up-regulated procollagen type I and tissue inhibitor of metalloproteinases-1 gene expression and/or protein synthesis without significantly affecting chemotaxis (wound healing and haptotaxis assay), matrix metalloproteinases 1 and 2 mRNA expression and activity as well as basal DNA synthesis., Conclusions: HNE, at concentrations compatible with those detected in vivo, does not elicit HSC/MF classic apoptosis but, rather, may act as a potent pro-fibrogenic stimulus for the expression of genes involved in excess extracellular matrix deposition and proposed as survival signals for HSC/MF.

Background & Aims: Several lines of evidence indicate that aldosterone antagonists may exert direct antifibrogenic effects. The aim of this study was to evaluate the possible direct antifibrogenic effects of canrenone, the active metabolite of spironolactone, in activated human hepatic stellate cells., Methods: The effects of canrenone were assessed on platelet-derived growth factor-induced mitogenic and chemotactic effects and the increased de novo synthesis of different extracellular matrix components induced by transforming growth factor-beta1., Results: Canrenone dose-dependently reduced platelet-derived growth factor-induced cell proliferation and motility. This effect was not associated with either changes in the phosphorylation of platelet-derived growth factor receptor and phospholipase C gamma or in the activation of the Ras/extracellular signal-regulated kinase pathway, whereas it was accompanied by a dose-dependent inhibition of platelet-derived growth factor-induced phosphatidylinositol 3-kinase activity. In addition, canrenone inhibited the activity of the Na(+)/H(+) exchanger 1 induced by platelet-derived growth factor. The effect of canrenone on Na(+)/H(+) exchanger 1 activity was reproduced by phosphatidylinositol 3-kinase inhibitors, thus supporting an inhibitory action of canrenone on phosphatidylinositol 3-kinase activity. To further address this possibility, the action of canrenone was compared with that of 2 established Na(+)/H(+) exchanger 1 inhibitors: ethylisopropylamiloride and cariporide. Whereas ethylisopropylamiloride was able to inhibit platelet-derived growth factor-induced phosphatidylinositol 3-kinase activity, cariporide was without any effect. Both compounds reproduced the effects of canrenone on platelet-derived growth factor-induced mitogenesis and chemotaxis. Finally, canrenone was able to reduce transforming growth factor-beta1-induced de novo synthesis of procollagen type I/IV and fibronectin and thrombin-induced hepatic stellate cell contraction., Conclusions: These results indicate that canrenone may be active as an antifibrogenic drug.

Following acute or chronic liver tissue damage, hepatic stellate cells (HSCs) undergo a process of activation toward a phenotype characterized by increased proliferation, motility, contractility, and synthesis of extracellular matrix components. Activation of HSCs is regulated by several soluble factors, including growth factors, cytokines, chemokines, and products of oxidative stress, as well as by extensive changes in the composition and organization of the ECM. Different groups of soluble factors may be classified according to their prevalent biological effect: (a) factors promoting HSC proliferation and/or migration (i.e., platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor-1); (b) factors promoting fibrillar ECM accumulation, particularly transforming growth factor-beta1; (c) factors with a prevalent contractile effect on HSCs, such as endothelin-1, thrombin, angiotensin-II and vasopressin, although all these agents also may promote HSC proliferation; (d) proinflammatory cytokines and chemokines; and (e) cytokines with a prominent antiinflammatory/antifibrogenic activity, such as interleukin-10 and interferon-gamma. Additional important issues are represented by the relationship between cytokine and integrin signaling, and by the effects of oxidative stress-related molecules on cytokine signaling. In the past decade the major intracellular signaling pathways elicited by these factors in HSCs have been greatly elucidated.

Activated hepatic stellate cells (HSC) regulate the liver "wound-healing" response through expression of chemokines, including monocyte chemoattractant protein-1 (MCP-1), which participate in the formation of the inflammatory infiltrate during liver injury. Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid into prostaglandins, which may contribute to the inflammatory response. In this study, we investigated the effects of COX inhibitors on the expression of MCP-1 in cultured HSC. Pretreatment of HSC with nonspecific COX inhibitors such as indomethacin or ibuprofen markedly reduced the expression of MCP-1 caused by exposure to tumor necrosis factor alpha (TNF-alpha) or interleukin-1alpha (IL-1alpha). NS-398, a specific COX-2 inhibitor, also resulted in a dose-dependent inhibition of MCP-1 gene and protein expression. These effects were dependent on reduced MCP-1 transcription, as established using a reporter plasmid. In contrast, the up-regulation of MCP-1 expression caused by interferon gamma (IFN-gamma) was not sensitive to COX inhibitors. Quiescent HSC did not show detectable expression of COX-2, which became evident after activation in culture, and while TNF-alpha and IL-1alpha markedly increased the expression of COX-2, IFN-gamma did not have any effects. Pretreatment of HSC with the stable cyclic adenosine monophosphate (cAMP) analog, 8-bromo cAMP, reverted the effects of the COX-2 inhibitor, but not of a nuclear factor-kappaB (NF-kappaB) inhibitor, demonstrating that prostaglandins modulate MCP-1 expression via production of cAMP. On the other hand, the action of NF-kappaB inhibitors was negligible in IFN-gamma-stimulated cells. These findings indicate that cross-talk between cytokines and a prostaglandin-cAMP pathway differentially regulates the proinflammatory potential of HSC, contributing to the modulation of liver tissue inflammation.

Background & Aims: Nitrovasodilators have been proposed for the treatment of portal hypertension alone or in combination with beta-blockers. In addition to their vasodilatory properties, nitric oxide (NO) donors may exert direct antifibrogenic properties. We evaluated the effect of nitroglycerin (NTG) and S-nitroso-N-acetyl penicillamine (SNAP) on the mitogenic and chemotactic properties of platelet-derived growth factor (PDGF)-BB and the modulation of the relative intracellular signaling pathways in fully activated human hepatic stellate cells (HSCs), a cell type that plays an active role in liver fibrogenesis and portal hypertension., Methods & Results: Both NTG and SNAP induced a dose-dependent decrease in PDGF-induced DNA synthesis and cell migration, which was associated with a decrease in PDGF-induced intracellular Ca(2+) increase and extracellular signal-regulated kinase (ERK) activity. These effects were not related to activation of the classic soluble guanylate cyclase (sGC)/guanosine 3',5'-cyclic monophosphate pathway; accordingly, Western blot analysis of HSC lysates revealed the absence of the alpha(1)beta(1) ubiquitous subunits of sGC, whereas they were detectable in quiescent HSCs, freshly isolated from normal human liver. Conversely, both NTG and SNAP induced a more than 10-20-fold increase in prostaglandin E(2) in cell supernatants within 1 minute, associated with an increase in intracellular adenosine 3',5'-cyclic monophosphate levels. Accordingly, the inhibitory effects of NO donors on PDGF action and signaling were eliminated after preincubation with ibuprofen., Conclusions: These results suggest that NO donors may exert a direct antifibrogenic action by inhibiting proliferation, motility, and contractility of HSCs in addition to a reduction of fibrillar extracellular matrix accumulation.

Background & Aims: Proliferation and migration of hepatic stellate cells (HSCs) and expression of chemokines are involved in the pathogenesis of liver inflammation and fibrogenesis. Peroxisome proliferator-activated receptor (PPAR)-gamma is a receptor transcription factor that controls growth and differentiation in different tissues. We explored the effects of PPAR-gamma agonists on the biological actions of cultured human HSCs., Methods: HSCs were isolated from normal human liver tissue and used in their myofibroblast-like phenotype or immediately after isolation. Activation of PPAR-gamma was induced with 15-deoxy-Delta(12, 14)-prostaglandin J(2) or with troglitazone., Results: PPAR-gamma agonists dose-dependently inhibited HSC proliferation and chemotaxis induced by platelet-derived growth factor. This effect was independent of changes in postreceptor signaling or expression of c-fos and c-myc and was associated with inhibition of cell cycle progression beyond the G(1) phase. Activation of PPAR-gamma also resulted in a complete inhibition of the expression of monocyte chemotactic protein 1 at the gene and protein levels. Comparison of quiescent and culture-activated HSCs revealed a marked decrease in PPAR-gamma expression in activated cells., Conclusions: Activation of PPAR-gamma modulates profibrogenic and proinflammatory actions in HSCs. Reduced PPAR-gamma expression may contribute to confer an activated phenotype to HSCs.

Background/aim: Several studies have shown that proliferation of hepatic stellate cells is stimulated by insulin-like growth factor-I. The aim of this study was to investigate the effect of insulin-like growth factor-I on human hepatic stellate cells chemotaxis and the intracellular pathways involved in both mitogenic and chemotactic effects., Methods/results: Insulin-like growth factor-I, at the concentration of 100 ng/ml, was able to induce a 2- to 3-fold increase in human hepatic stellate cells migration in a modified Boyden chamber system. This effect was associated with a marked activation of phosphatidylinositol 3-kinase by insulin-like growth factor-I, as evaluated by measurement of phosphatidylinositol 3-kinase activity in phosphotyrosine immunoprecipitates In order to establish a functional link between these observations, we then performed experiments employing two selective phosphatidylinositol 3-kinase inhibitors, namely wortmannin and LY294002. These compounds blocked activation of phosphatidylinositol 3-kinase and inhibited insulin-like growth factor-I-induced hepatic stellate cells migration. Since phosphatidylinositol 3-kinase activation has been shown to be necessary for platelet-derived growth factor-induced mitogenesis in hepatic stellate cells, we verified the effects of phosphatidylinositol 3-kinase inhibition on insulin-like growth factor-I-induced DNA synthesis. Incubation with either wortmannin or LY294002, dose-dependently reduced the mitogenic potential of insulin-like growth factor-I. Since phosphatidylinositol 3-kinase is involved, at least in part, in the activation of the Ras/extracellular signal-regulated kinase pathway in hepatic stellate cells, the role of extracellular signal-regulated kinase activation in mediating the biological effects of insulin-like growth factor-I was explored. Insulin-like growth factor-I induced mitogenesis and chemotaxis were markedly reduced by pre-incubation of hepatic stellate cells with PD-98059, a selective inhibitor of MEK., Conclusions: Activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase is required for both insulin-like growth factor-I-dependent hepatic stellate cells proliferation and chemotaxis. Insulin-like growth factor-I, together with other soluble mediators, may contribute to the hepatic wound-healing response by modulating hepatic stellate cells migration and proliferation.

Upon liver injury, hepatic stellate cells (HSC) show increased proliferation, motility, and extracellular matrix (ECM) production. The extracellular signal-regulated kinases (ERK) control different functions in a cell-specific manner. In this study, we evaluated the role of ERK activation in cultured HSC stimulated with platelet-derived growth factor (PDGF) and after induction of liver injury in vivo. HSC were isolated from normal human liver tissue, cultured on plastic, and used in their myofibroblast-like phenotype. In in vivo experiments, HSC were isolated from normal rats or at different time points after a single intragastric administration of CCl(4). Nontoxic concentrations of PD98059, a specific inhibitor of ERK activation, reduced PDGF-induced activation of ERK in a dose-dependent fashion. Suppression of ERK activation was associated with complete inhibition of HSC proliferation and with a 57% reduction in chemotaxis. In the presence of the ERK inhibitor, binding of the AP-1 complex and of STAT1 to the related regulatory elements was inhibited. The inhibition of the DNA binding activity of STAT1 was mediated by a reduction in PDGF-induced tyrosine phosphorylation. Expression of c-fos in response to PDGF was also reduced, but not suppressed, by treatment with PD98059. In HSC isolated from CCl(4)-treated rats, ERK activity increased as early as 6 hours following liver damage, and declined thereafter. The results of this study indicate that ERK activation regulates proliferation and chemotaxis of HSC, and modulates nuclear signaling. Acute liver damage in vivo leads to activation of ERK in HSC.

Background/aims: Chronic cholestasis stimulates a fibroductular reaction which may progress to secondary biliary fibrosis and cirrhosis. Since platelet-derived growth factor has been indicated as a major fibrogenic factor in chronic liver disease, we analyzed its expression and that of its receptor beta subunit in a rat model of chronic cholestasis., Methods: Liver tissue samples collected at 7, 10, 21, and 28 days after induction of cholestasis obtained by bile duct ligation, were analyzed by immunohistochemistry, in situ hybridization and RNase protection assay for the expression of platelet-derived growth factor (PDGF)-B chain and receptor beta subunit. Furthermore, the expression of PDGF-B chain mRNA was analyzed in highly purified cholangiocytes from normal and cholestatic rat liver., Results: In cholestatic liver, platelet-derived growth factor-BB and B chain mRNA expression increased up to 4 weeks in epithelial cells of proliferating bile ducts, and periductular mesenchymal cells. The increased expression of PDGF-B chain mRNA was confirmed in highly purified cholangiocytes obtained from normal and cholestatic rat liver. The expression of the receptor beta subunit progressively increased after induction of cholestasis and was mainly localized to desmin-positive periductular hepatic stellate cells., Conclusions: These data suggest that platelet-derived growth factor-B chain can be synthesized by cholangiocytes during chronic cholestasis. The presence of its receptor on periductular hepatic stellate cells raises the possibility that, in this experimental setting, this cytokine might contribute to fibrogenesis in vivo.

Background: Increased expression of monocyte chemotactic protein-1 (MCP-1) has been indicated as a mechanism underlying leukocyte recruitment after liver injury. In this study we examined the temporal relationship between MCP-1 expression and the appearance of monocyte infiltration during acute liver injury. In addition, we tested the effects of vitamin E, a well known antioxidant, on these parameters. Rats were intoxicated with a single intragastric administration of CCl4 with or without pretreatment with vitamin E (atocopherol)., Methods: Monocyte chemotactic protein-1 expression was analyzed by northern blotting and in situ hybridization and monocyte infiltration was determined by ED-1 immunostaining. The results were quantitated by computerized image analysis. Expression of MCP-1 mRNA was significantly increased as early as 12 hours following injury, and progressively increased thereafter. In contrast, a significant increase in the number of ED-1 positive cells, an index of monocyte infiltration, was observed only 24 and 48 hours after injury., Results: Vitamin E markedly reduced MCP-1 expression at the mRNA and protein levels, and caused a significant reduction in the number of monocyte/macrophages, indicating a role for oxidative stress in the induction of MCP-1 expression in vivo. Accordingly, in cultured hepatic stellate cells, different oxidative stress-related molecules increased MCP-1 mRNA., Conclusions: These data suggest the existence of a direct relationship between MCP-1 expression and monocyte infiltration after acute liver injury, and that preventing the generation of oxidative stress-related molecules results in decreased expression and release of this chemokine.

Following liver injury, hepatic stellate cells (HSC) undergo proliferation and migrate into damaged areas in response to chemotactic factors. HSC have been shown to regulate leukocyte trafficking by secreting monocyte chemotactic protein-1 (MCP-1), a chemokine that recruits monocytes and lymphocytes. In this study, we explored whether MCP-1 exerts biological actions on HSC. HSC were isolated from normal human livers, cultured on plastic, and studied in their myofibroblast-like phenotype, and three different cells lines were used. Chemotaxis was measured in modified Boyden chambers. Phosphatidylinositol 3-kinase (PI 3-K) was assayed on phosphotyrosine immunoprecipitates. Exposure of HSC to MCP-1 stimulated migration of HSC in a dose-dependent fashion. Maximal stimulation was obtained with 250 ng/mL MCP-1, which resulted in a 3- to 4-fold stimulation of cell migration. Checkerboard analysis showed that the increase in cell migration was almost completely a result of chemotaxis rather than chemokinesis. In contrast, in quiescent HSC, MCP-1 did not exert any effect on cell migration. In leukocytes, MCP-1 activates the pertussis toxin-sensitive CCR2 receptor. However, transcripts for CCR2 could not be shown in HSC, and pertussis toxin only modestly inhibited MCP-1-induced migration. Exposure of HSC to MCP-1 was associated with an increase in cytosolic calcium concentration, PI 3-K activity, protein tyrosine phosphorylation. Blocking calcium influx or pretreatment of HSC with the PI 3-K inhibitor wortmannin markedly reduced cell migration. This study shows, for the first time, a potential direct profibrogenic action of MCP-1 via HSC chemotaxis. MCP-1-dependent signals in these cells are not transduced by CCR2 and may be mediated by alternative chemokine receptors. (HEPATOLOGY 1999;29:140-148.)

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

Hepatic stellate cells (HSC) are presently regarded as one of the key cell types involved in the progression of liver fibrosis and in the related pathophysiological and clinical complications. Following acute or chronic liver tissue damage, HSC undergo a process of activation towards a phenotype characterised by increased proliferation, motility, contractility and synthesis of extracellular matrix (ECM) components. Several factors have been shown to play a key role in the promotion of the full-blown picture of activated HSC. These include extensive changes in the composition and organisation of the ECM, the secretion of several growth factors, cytokines, chemokines, products of oxidative stress and other soluble factors. It is evident that each cellular response to extracellular stimuli must be framed in a scenario where different forces modulate one another and result in a prevalent biological effect. Along these lines, the identification and characterisation of intracellular signalling pathways activated by different stimuli in HSC represent a mandatory step. In this review article we have made an attempt to summarise recent acquisitions to our knowledge of the involvement of different intracellular signalling pathways in key aspects of HSC biology.

Thrombin is generated during tissue damage in several organs, including the liver, and participates in the process of tissue repair through proteolytic activation of a specific thrombin receptor (TR). The aim of this study was to investigate TR expression in human liver by immunohistochemistry and in situ hybridization. In normal liver, immunostaining for TR was present in the endothelial lining of the hepatic sinusoids. During chronic hepatitis, several cells expressing the TR were detected in the inflammatory infiltrate of portal tracts. In cirrhosis with chronic active hepatitis, expression of the TR was also present in mesenchymal cells of fibrous septa. TR expression was markedly up-regulated during fulminant hepatitis, with the highest expression in mesenchymal cells in areas of regeneration. Up-regulation of TR expression was associated with increased levels of TR messenger RNA (mRNA), as assessed by in situ hybridization and RNAse protection assay of liver RNA. Immunostaining of serial sections using specific cellular markers showed that different nonparenchymal cells contribute to TR expression during liver injury. TR expression was also shown in cultured human hepatic stellate cells, with increasing signal comparing activated versus quiescent cells. Because thrombin is rapidly generated after tissue damage, regulated TR expression may be involved in tissue remodeling and/or scarring during liver damage.

We investigated whether activation of integrin receptors could modulate the expression of monocyte chemotactic protein-1 (MCP-1) in human hepatic stellate cells (HSC), mesenchymal cells responsible for extracellular matrix synthesis within the liver. When compared to non-adherent cells, HSC plated on collagen types I or IV, or fibronectin, showed increased MCP-1 gene expression and protein secretion in the conditioned medium. Increased MCP-1 secretion was also observed when cells were plated on dishes coated with a monoclonal antibody directed against the beta1-integrin subunit, demonstrating that ligation of beta1-integrins is sufficient to stimulate MCP-1 expression. Conversely, integrin-independent cell adhesion on poly-L-lysine did not modify MCP-1 secretion. Disruption of the actin cytoskeleton by cytochalasin D blocked the collagen-dependent increase in MCP-1 secretion. Chemotactic assay of HSC-conditioned medium showed that HSC plated on collagen secrete higher amounts of chemotactic factors for lymphomonocytes, and that MCP-1 accounts for the great majority of this effect. These findings indicate a novel mechanism of MCP-1 regulation possibly relevant in those conditions where HSC interact with an altered extracellular matrix.

Background & Aims: Platelet-derived growth factor (PDGF) is the most potent mitogen for hepatic stellate cells (HSCs) in vitro. The aim of this study was to investigate the role of phosphatidylinositol 3-kinase (PI 3-K) activation in mediating the biological effects of PDGF on cultured HSCs and its involvement in vivo., Methods: HSCs were isolated from normal human livers. PI 3-K was assayed on phosphotyrosine or PDGF-receptor immunoprecipitates by in vitro kinase assay., Results: Incubation of HSCs with PDGF caused a time-dependent increase in PI 3-K activity. Immunoprecipitation of PDGF-alpha and -beta receptors showed that both subunits associate with active PI 3-K in PDGF-stimulated HSCs. Wortmannin, a specific PI 3-K inhibitor, dose-dependently blocked PI 3-K activity induced by PDGF and inhibited DNA synthesis. PDGF (homodimer)-BB also stimulated HSC chemotaxis, which was inhibited by pretreatment with wortmannin. To explore the potential role of PI 3-K in vivo, liver homogenates from rats treated with CCl4 and from control rats were immunoprecipitated with anti-PDGF-beta-receptor antibodies. Liver injury was associated with increased PDGF-beta-receptor autophosphorylation, and greater PI 3-K activity associated with the receptor itself., Conclusions: This study shows that in cultured HSCs, PI 3-K activation is necessary for both mitogenesis and chemotaxis induced by PDGF and that this pathway is up-regulated during liver injury in vivo.

1. It has been proposed that pentoxifylline (PTF) acts an antifibrogenic agent by reducing the synthesis of extracellular matrix components, and this possibility has been confirmed in animal models of hepatic fibrosis. In this study the effects of PTF on the proliferation of extracellular matrix producing cells induced by platelet-derived growth factor (PDGF) were evaluated. The study was performed on hepatic stellate cells, currently indicated as the major source of extracellular matrix in fibrotic liver. 2. PTF caused a dose-dependent reduction of PDGF-induced mitogenesis with an IC50 of 170 microM, identical to the EC50 for the increase in intracellular cyclic AMP levels. Preincubation with PTF did not affect either PDGF-receptor autophosphorylation or phosphotidylinositol 3-kinase activity, whereas it markedly reduced PDGF-stimulated extracellular signal-regulated kinase (ERK) activity and ERK isoform phosphorylation. PTF also reduced PDGF-induced c-fos mRNA expression, which is dependent on activation of the RAS/ERK pathway. In addition, the PDGF-induced increase in cytsolic-free calcium was almost completely prevented by pretreating the cells with PTF. 3. The results of the present study indicate that PTF, in addition to its effect on collagen deposition and degradation, may exert an antifibrogenic effect by reducing the PDGF-induced proliferation of extracellular matrix producing cells. This effect appears to be mediated by a reduction of PDGF-stimulated ERK activity as well as of other intracellular signalling pathways such as the PDGF-induced elevation of cytosolic-free calcium.

4-Hydroxy-2,3-nonenal (HNE) has been shown to induce procollagen type I gene expression and synthesis in hepatic stellate cells (HSC), i.e. the cells responsible for deposition of collagen and other extracellular matrix proteins in fibrotic liver. Here we report that the stimulatory effect of HNE mostly depends on the contemporary presence of the hydroxyl group in position C4 and of the double bond between position C2 and C3 since equimolar concentrations of 2,3-nonenal as well as of nonenal did not procollagen type I synthesis either at mRNA or at protein levels. Accordingly to this concept, all the other 4-hydroxy-2,3-alkenals of different chain length tested on cultured human HSC (4-hydroxy-2,3-hexenal, 4-hydroxy-2,3-octenal and 4-hydroxy-2,3-undecenal) strongly induced procollagen type I gene expression and synthesis. The stimulatory effect of 4-hydroxy-2,3-alkenals may depend on the well known ability of these aldehydes to react with either SH-groups or NH2-groups of functional proteins.

Transforming growth factor-beta (TGF-beta) stimulates the accumulation of extracellular matrix in renal and hepatic disease. Kidney glomerular mesangial cells (GMC) and liver fat-storing cells (FSC) produce latent of inactive TGF-beta. In this study, we characterized the latent TGF-beta complexes secreted by these cells. Human FSC produce a single latent TGF-beta complex, predominantly of the TGF-beta 1 isoform, whereas GMC secrete multiple complexes of latent TGF-beta, containing beta 1 and beta 2 isoforms. At least four forms were identified in GMC using ion exchange chromatography, including a peak not previously described in other cell types which eluted at 0.12 M NaCl, and predominantly of the beta 2 isoform. Both cell types secrete the latent TGF-beta 1 binding protein of 190 kDa, as part of a high molecular weight TGF-beta complex. Epidermal growth factor stimulates the secretion of latent TGF-beta and latent TGF-beta binding protein in both cell types. Secretion of latent TGF-beta in both cell types was found to be associated with secretion of decorin. This study shows that vascular pericytes from the kidney and the liver have distinctly different profiles of latent TGF-beta complexes, with GMC secreting a unique form of latent TGF-beta 2. The regulatory effect of epidermal growth factor and platelet-derived growth factor has potential implication for the pathophysiology of liver regeneration and chronic liver and kidney diseases.

Phosphatidylinositol 3-kinase (PI 3-K) is a lipid and protein kinase which associates with the activated platelet-derived growth factor (PDGF) receptor and other tyrosine kinases. We studied the effects of wortmannin, a selective inhibitor of PI 3-K, on the activation of extracellular-signal regulated kinase (ERK) by PDGF in cultured hepatic stellate cells, mesenchymal cells responsible for extracellular matrix synthesis within the liver. Incubation with 100 nM wortmannin, a dose which almost completely blocks PI 3-K, resulted in 50% reduction of ERK activity. Direct inhibition of ERK by wortmannin could not be considered responsible for this effect, since wortmannin did not inhibit ERK activity in vitro. Rather, inhibition of PI 3-K acts on the kinase cascade that leads to ERK activation, since PDGF-dependent phosphorylation of ERK was found to be reduced after incubation with wortmannin. Wortmannin also inhibited the increase in c-fos mRNA induced by PDGF, which is dependent on ERK activation. The results of this study show that in hepatic stellate cells PI 3-K is involved in ERK activation, although it is not necessary. These data indicate cross-talk between PI 3-K and the Ras/ERK pathway in PDGF-stimulated cells.

Liver fat-storing cells (FSC) proliferate and secrete extracellular matrix in experimental models of liver injury. In this study, we determined if thrombin, a serine protease produced during acute and chronic tissue injury, modulates the functions of FSC. Thrombin stimulated DNA synthesis and proliferation of FSC, as assessed by [3H]-thymidine incorporation assay and measurement of cell number, respectively. Thrombin also increased the secretion of monocyte chemotactic protein-1 (MCP-1) in a time- and dose-dependent fashion. The effect of thrombin on both DNA synthesis and MCP-1 secretion was neutralized by pretreatment of thrombin with hirudin. The increased MCP-1 secretion was associated with increased steady-state levels of MCP-1 messenger RNA. Pretreatment of FSC with 5 mumol/retinol for 48 hours inhibited the mitogenic effects of thrombin but not the induction of MCP-1 secretion. FSC express specific transcripts encoding for the human thrombin receptor, as shown by Northern blot analysis of poly(A)+ RNA. Proteolytic activation of the thrombin receptor results in the formation of a new N-terminus that functions as a tethered ligand. We studied the effects of a thrombin receptor activating peptide (TRAP) corresponding to the newly formed N-terminus on FSC. TRAP mimicked the effects of thrombin on [3H]-thymidine incorporation, MCP-1 secretion, and MCP-1 gene expression. This study suggest that thrombin may be involved in modulating FSC proliferation and monocyte chemotaxis during human liver disease, through proteolytic activation of its receptor.

Activated liver fat-storing cells (FSC) are known to play a key role in the development of liver fibrosis. An important element in FSC activation process is the increased expression of receptors for platelet-derived growth factor (PDGF), a potent mitogen for FSC. The aim of the present study was to evaluate the expression PDGF-receptor alpha and beta subunits in cultured human FSC and their regulation induced by transforming growth factor-beta 1 (TGF-beta), a cytokine potentially involved in an autocrine loop. TGF-beta induced a significant increase of the mitogenic effect of PDGF-BB and did not affect the mitogenicity of PDGF-AA and PDGF-AB, suggesting a selective action of the PDGF-receptor-beta subunit. This hypothesis was confirmed by regulation experiments showing selective and time-dependent upregulation of the messenger (m)RNA encoding for the PDGF-receptor-beta subunit and the relative protein induced by TGF-beta. In addition, binding studies showed a parallel increase of PDGF-BB binding sites after incubation of human FSC with TGF-beta. These studies provide evidence for an additional mechanism leading to the perpetuation of FSC activation and proliferation and contribute to a better understanding of the role of TGF-beta and PDGF in the development of liver fibrosis.