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Background & Aims: The liver is an immunologically-privileged organ. Breakdown of liver immune privilege has been reported in chronic liver disease; however, the role of adaptive immunity in liver injury is poorly defined. Nuclear factor-κB-inducing kinase (NIK) is known to regulate immune tissue development, but its role in maintaining liver homeostasis remains unknown. This study aimed to assess the role of NIK, particularly thymic NIK, in regulating liver adaptive immunity., Methods: NIK was deleted systemically or conditionally using the Cre/loxp system. Cluster of differentiation [CD]4 + or CD8 + T cells were depleted using anti-CD4 or anti-CD8 antibody. Donor bone marrows or thymi were transferred into recipient mice. Immune cells were assessed by immunohistochemistry and flow cytometry., Results: Global, but not liver-specific or hematopoietic lineage cell-specific, deletion of NIK induced fatal liver injury, inflammation, and fibrosis. Likewise, adoptive transfer of NIK-null, but not wild-type, thymi into immune-deficient mice induced liver inflammation, injury, and fibrosis in recipients. Liver inflammation was characterized by a massive expansion of T cells, particularly the CD4 + T cell subpopulation. Depletion of CD4 + , but not CD8 + , T cells fully protected against liver injury, inflammation, and fibrosis in NIK-null mice. NIK deficiency also resulted in inflammation in the lung, kidney, and pancreas, but to a lesser degree relative to the liver., Conclusions: Thymic NIK suppresses development of autoreactive T cells against liver antigens, and NIK deficiency in the thymus results in CD4 + T cell-orchestrated autoimmune hepatitis and liver fibrosis. Thus, thymic NIK is essential for the maintenance of liver immune privilege and liver homeostasis., Lay Summary: We found that global or thymus-specific ablation of the NIK gene results in fatal autoimmune liver disease in mice. NIK-deficient mice develop liver inflammation, injury, and fibrosis. Our findings indicate that thymic NIK is essential for the maintenance of liver integrity and homeostasis., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Liver fibrosis can lead to cirrhosis and hepatocellular carcinoma if not treated in the early stages. The molecular mechanisms of the pathogenesis of hepatic fibrosis remain unclear., Aim: To identify the molecules involved in the pathogenesis of liver fibrosis and to investigate the potential effect and mechanism of Annexin A2 up-regulation during liver fibrosis progression., Methods: Twenty Sprague-Dawley rats were divided into two groups: the carbon tetrachloride (CCl 4 )-induced liver fibrosis group and the normal control group. Hematoxylin and eosin staining or Masson Trichrome staining and enzyme-linked immunosorbent assay were applied to assess the degree of liver damage and fibrosis in rats with CCl 4 -induced liver fibrosis. Liver tissue protein profiles were analyzed using iTRAQ and mass spectrometry. RT-PCR and western blotting analyses were employed to validate differentially expressed proteins. Small interfering RNA-based silencing was performed to study the function of Annexin A2., Results: Twelve weeks after CCl 4 injection, significant body weight changes and liver injury and liver fibrosis were observed in rats. In addition, 130 proteins were differentially expressed in the liver fibrosis group. Overexpression of Annexin A2 was confirmed by RT-PCR and Western blotting analysis. Silencing of Annexin A2 expression in HepG2 and LX-2 cells significantly reduced the secretion of von Willebrand factor (vWF)., Conclusion: Annexin A2 promotes liver fibrosis by mediating vWF secretion, which can be used to mitigate the progression of liver fibrosis., (Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Bile duct ligation (BDL) and subsequent cholestasis are correlated with oxidative stress, hepatocellular injury and fibrosis. Quercetin is a flavonoid with antifibrotic, and hepatoprotective properties. However, the molecular mechanism underlying quercetin-mediated hepatoprotection is not fully understood. The current study was to evaluate mechanisms of hepatoprotective effect of quercetin in BDL rat model., Methods: We divided male Wistar rats into 4 groups (n=8 for each): sham, sham+quercetin (30 mg/kg per day), BDL, and BDL+quercetin (30 mg/kg per day). Four weeks later, the rats were sacrificed, the blood was collected for liver enzyme measurements and liver for the measurement of Rac1, Rac1-GTP and NOX1 mRNA and protein levels by quantitative PCR and Western blotting, respectively., Results: Quercetin significantly alleviated liver injury in BDL rats as evidenced by histology and reduced liver enzymes. Furthermore, the mRNA and protein expression of Rac1, Rac1-GTP and NOX1 were significantly increased in BDL rats compared with those in the sham group (P<0.05); quercetin treatment reversed these variables back toward normal (P<0.05). Another interesting finding was that the antioxidant markers e.g. superoxide dismutase and catalase were elevated in quercetin-treated BDL rats compared to BDL rats (P<0.05)., Conclusion: Quercetin demonstrated hepatoprotective activity against BDL-induced liver injury through increasing antioxidant capacity of the liver tissue, while preventing the production of Rac1, Rac1-GTP and NOX1 proteins.

Background & Aims: Alcoholic liver disease (ALD) ranges from fatty liver to inflammation and cirrhosis. miRNA-155 is an important regulator of inflammation. In this study, we describe the in vivo role of miR-155 in ALD., Methods: Wild-type (WT) (C57/BL6J) or miR-155 knockout (KO) and TLR4 KO mice received Lieber DeCarli diet for 5weeks. Some mice received corn oil or CCl4 for 2 or 9weeks., Results: We found that miR-155 KO mice are protected from alcohol-induced steatosis and inflammation. The reduction in alcohol-induced fat accumulation in miR-155 KO mice was associated with increased peroxisome proliferator-activated receptor response element (PPRE) and peroxisome proliferator-activated receptors (PPAR)α (miR-155 target) binding and decreased MCP1 production. Treatment with a miR-155 inhibitor increased PPARγ expression in naïve and alcohol treated RAW macrophages. Alcohol increased lipid metabolism gene expression (FABP4, LXRα, ACC1 and LDLR) in WT mice and this was prevented in KO mice. Alcohol diet caused an increase in the number of CD163(+) CD206(+) infiltrating macrophages and neutrophils in WT mice, which was prevented in miR-155 KO mice. Kupffer cells isolated from miR-155 KO mice exhibited predominance of M2 phenotype when exposed to M1 polarized signals and this was due to increased C/EBPβ. Pro-fibrotic genes were attenuated in miR-155 KO mice after alcohol diet or CCl4 treatment. Compared to WT mice, attenuation in CCl4 induced hydroxyproline and α-SMA was observed in KO mice. Finally, we show TLR4 signaling regulates miR-155 as TLR4 KO mice showed no induction of miR-155 after alcohol diet., Conclusions: Collectively our results demonstrated the role of miR-155 in alcohol-induced steatohepatitis and fibrosis in vivo., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Liver fibrosis is an increasing health concern worldwide and a major risk factor for hepatocellular carcinoma (HCC). Although the involvement of Hedgehog signaling in hepatic fibrosis has been known for some time, the causative role of activated Hedgehog signaling in liver fibrosis has not been verified in vivo., Methods: Using hydrodynamics-based transfection, a transgenic mouse model has been developed that expresses Sonic Hedgehog (SHH), a ligand for Hedgehog signaling, in the liver. Levels of hepatic fibrosis and fibrosis-related gene expression were assessed in the model. Hepatic expression of SHH was induced in a murine model for hepatocellular adenoma (HCA) and tumor development was subsequently investigated., Results: The transgenic mice revealed SHH expression in 2-5% of hepatocytes. Secreted SHH activated Hedgehog signaling in numerous cells of various types in the tissues. Hepatic expression of SHH led to fibrosis, activation of hepatic stellate cells, and an upregulation of various fibrogenic genes. Liver injury and hepatocyte apoptosis were observed in SHH mice. Persistent expression of SHH for up to 13months failed to induce tumors in the liver; however, it promoted liver tumor development induced by other oncogenes. By employing a HCA model induced by P53(R172H) and KRAS(G12D), we found that the SHH expression promoted the transition from HCA to HCC., Conclusions: SHH expression in the liver induces liver fibrosis with concurrent activation of hepatic stellate cells and fibrogenic genes. It can also enhance hepatocarcinogenesis induced by other oncogenes., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: The secretory protein Slit2 and its receptor Robo1 are believed to regulate cell growth and migration. Here, we aimed to determine whether Slit2-Robo1 signaling mediates the pathogenesis of liver fibrosis., Methods: Serum levels of Slit2 in patients with liver fibrosis were determined by ELISA. Liver fibrosis was induced in wild-type (WT), Slit2 transgenic (Slit2-Tg) and Robo1(+/-)Robo2(+/-) double heterozygotes (Robo1/2(+/-)) mice by carbon tetrachloride (CCl4). The functional contributions of Slit2-Robo1 signaling in liver fibrosis and activation of hepatic stellate cells (HSCs) were investigated using primary mouse HSCs and human HSC cell line LX-2., Results: Significantly increased serum Slit2 levels and hepatic expression of Slit2 and Robo1 were observed in patients with liver fibrosis. Compared to WT mice, Slit2-Tg mice were much more vulnerable to CCl4-induced liver injury and more readily develop liver fibrosis. Development of hepatic fibrosis in Slit2-Tg mice was associated with a stronger hepatic expression of collagen I and α-smooth muscle actin (α-SMA). However, liver injury and hepatic expression of collagen I and α-SMA were attenuated in CCl4-treated Robo1/2(+/-) mice in response to CCl4 exposure. In vitro, Robo1 neutralizing antibody R5 and Robo1 siRNA downregulated phosphorylation of Smad2, Smad3, PI3K, and AKT in HSCs independent of TGF-β1. R5 and Robo1 siRNA also inhibited the expression of α-SMA by HSCs. Finally, the protective effect of R5 on the CCl4-induced liver injury and fibrosis was further verified in mice., Conclusions: Slit2-Robo1 signaling promotes liver injury and fibrosis through activation of HSCs., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Hepatic fibrosis, featured by the accumulation of excessive extracellular matrix in liver tissue, is associated with metabolic disease and cancer. Inhalation exposure to airborne particulate matter in fine ranges (PM2.5) correlates with pulmonary dysfunction, cardiovascular disease, and metabolic syndrome. In this study, we investigated the effect and mechanism of PM2.5 exposure on hepatic fibrogenesis., Methods: Both inhalation exposure of mice and in vitro exposure of specialized cells to PM2.5 were performed to elucidate the effect of PM2.5 exposure on hepatic fibrosis. Histological examinations, gene expression analyses, and genetic animal models were utilized to determine the effect and mechanism by which PM2.5 exposure promotes hepatic fibrosis., Results: Inhalation exposure to concentrated ambient PM2.5 induces hepatic fibrosis in mice under the normal chow or high-fat diet. Mice after PM2.5 exposure displayed increased expression of collagens in liver tissues. Exposure to PM2.5 led to activation of the transforming growth factor β-SMAD3 signaling, suppression of peroxisome proliferator-activated receptor γ, and expression of collagens in hepatic stellate cells. NADPH oxidase plays a critical role in PM2.5-induced liver fibrogenesis., Conclusions: Exposure to PM2.5 exerts discernible effects on promoting hepatic fibrogenesis. NADPH oxidase mediates the effects of PM2.5 exposure on promoting hepatic fibrosis., Competing Interests: the authors Z.Z., X.Z., J.W., A.D., H.K., Y.Q., X.X., Y.C., A.W. L.C.C., S.R., Q.S., and K.Z. have nothing to declare., (Copyright © 2015. Published by Elsevier B.V.)

Background & Aims: Common pathogenic steps in liver fibrosis are inflammation and accumulation of extracellular matrix proteins including collagen, which lead to disruption of tissue microarchitecture and liver dysfunction. Adequate fibronectin fibril formation is required for collagen matrix deposition in several cell types in vitro. We therefore hypothesized that preventing fibronectin fibril assembly will result in decreased collagen matrix accumulation, and hence diminish liver injury associated with fibrosis., Methods: In vitro studies on hepatic stellate cells and in vivo studies in mice were performed., Results: In vitro studies on hepatic stellate cells confirmed that a fibronectin assembly inhibitor, pUR4 diminishes the amount of both fibronectin and collagen, accumulating in the extracellular matrix, without affecting their production. Induction of fibrosis using CCl4 or DMN was therefore combined with pUR4-treatment. pUR4 normalized the amount of fibrotic tissue that accumulated with injury, and improved liver function. Specifically, pUR4-treatment decreased collagen accumulation, without changing its mRNA expression. Most interestingly, we did not detect any changes in Kupffer cell numbers (F4/80+) or α-smooth muscle actin expressing hepatic stellate cell numbers. Further, there was no impact on TGF-β or TNF-α. Thus, in line with the in vitro findings, decreased fibrosis is due to inhibition of matrix accumulation and not a direct effect on these cells., Conclusions: In summary, a peptide that blocks fibronectin deposition results in decreased collagen accumulation and improved liver function during liver fibrogenesis. Thus, fibronectin matrix modulation offers a therapeutic benefit in preclinical models of liver fibrosis., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Conversion of hepatic stellate cells (HSCs) into hepatic myofibroblasts is a necessary event during the development of liver fibrosis. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of profibrotic genes, is selectively induced in myofibroblasts from diseased livers. Treatment of HSC with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-azadC), prevented TGF-β1-induced proliferation and alpha-smooth muscle actin (α-SMA) and collagen expression. 5-AzadC also rescued TGF-β1-induced suppression of Smad7 expression which occurs during HSC activation. Similarly, silencing the expression of the DNMT1 gene ameliorated the suppression of Smad7 expression by TGF-β1. In addition, DNMT1 inhibition, by 5-azadC or DNMT1 silencing, prevented the phosphorylation of Smad2 and Smad3. These studies suggest that epigenetic repression of Smad7 promotes the phosphorylation of Smad2 and Smad3 that may be an important molecular mechanism for perpetuated HSC activation and liver fibrosis., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objective: During the neonatal and infancy periods, some chronic liver diseases may lead to progressive hepatic fibrosis, which is a condition that can ultimately result in the loss of organ function and severe portal hypertension necessitating hepatic transplantation. In a previous report, pharmacological interventions were demonstrated to modulate hepatic fibrosis induced by bile duct ligation in young rats. The administration of pentoxifylline or prednisolone, or the combination of both, resulted in reduced fibrogenesis in portal spaces. The objectives of the present study were to evaluate the expression of transforming growth factor β and vascular endothelial growth factor after bile duct ligation in young rats and to assess the effect of those same drugs on cytokine expression., Methods: In this experimental study, 80 young rats (21 or 22 days old) were submitted either to laparotomy and common bile duct ligation or to sham surgery. The animals were allocated into four groups according to surgical procedure, and the following treatments were administered: (1) common bile duct ligation + distilled water, (2) sham surgery + distilled water, (3) common bile duct ligation + pentoxifylline, or (4) common bile duct ligation + prednisolone. After 30 days, a hepatic fragment was collected from each animal for immunohistochemical analysis using monoclonal antibodies against transforming growth factor β and vascular endothelial growth factor. Digital morphometric and statistical analyses were performed., Results: The administration of pentoxifylline reduced the transforming growth factor β-marked area and the amount of transforming growth factor β expressed in liver tissue. This effect was not observed after the administration of prednisolone. There was a significant reduction in vascular endothelial growth factor expression after the administration of either drug compared with the non-treatment group., Conclusions: The administration of pentoxifylline to cholestatic young rats resulted in the diminished expression of transforming growth factor β and vascular endothelial growth factor in liver tissue. The administration of steroids resulted in the diminished expression of vascular endothelial growth factor only. These pathways may be involved in hepatic fibrogenesis in young rats submitted to bile duct ligation and exposed to pentoxifylline or prednisolone.

The possibility that epithelial-mesenchymal transition (EMT) could contribute to hepatic fibrogenesis in chronic liver diseases as reported in other organs, particularly the kidney, reinforced the concept that activated hepatic stellate cells were not the only key players in the hepatic fibrogenic process and that other cell types, either hepatic (i.e. portal fibroblast) or extrahepatic (bone marrow-derived cells and circulating fibrocytes) could contribute to this process. The possibility of the rapid mobilization of a large amount of fibrogenic cells by EMT after liver tissue injury made this phenomenon a relevant and suitable target for anti-fibrogenic strategies. Following an initial enthusiasm for the discovery of this novel pathway in fibrogenesis and the publication of a several highly quoted papers, more recent research has started to cast serious doubts upon the real relevance of this phenomenon in human fibrogenetic disorders. The debate on the authenticity of EMT or at least on its real contribution to the fibrogenic process has become very animated, sometimes reaching levels of "religious" integralism. The overall result is a general confusion on the meaning and on the definition of several key aspects. The aim of this article is to analyze and discuss the evidence supporting or confuting this possibility in order to reach reasonable and useful conclusions., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The hepatic wound-healing response to chronic noxious stimuli may lead to liver fibrosis, a key feature of the preneoplastic cirrhotic liver. Fibrogenic cells activate in response to a variety of cytokines, growth factors, and inflammatory mediators. The involvement of members of the epidermal growth factor family in this process has been suggested. Amphiregulin is an epidermal growth factor receptor (EGFR) ligand specifically induced upon liver injury. We investigated the effects of quercetin on the amphiregulin/EGFR signal and on the activation of downstream pathways leading to cell growth. Rats were divided into 4 groups (8 rats/group): rats subjected to common bile duct ligation (CBDL), Sham (rats subjected to simulated CBDL), quercetin-treated sham, and quercetin-treated CBDL (CBDL-Q). Quercetin (50 mg/kg i.p. injection) was administered daily for 2 wk starting on d 14 after surgery. Overexpression of amphiregulin, EGFR, TNFα, IL-6, TGFβ, platelet-derived growth factor (PDGF), extracellular regulated kinase, protein kinase B (Akt), cycloxygenase (COX)-2, and glioma-associated oncogenes (GLI)-1 and-2 were observed in liver of CBDL rats after 4 wk of bile duct ligation. CBDL-Q rats had a significantly diminished expression of amphiregulin and EGFR compared with untreated CBDL rats. Furthermore, mRNA levels of TNFα, IL-6, TGFβ, and PDGF and the protein content of COX-2, GLI-1, and GLI-2 were significantly lower in CBDL-Q rats than in untreated CBDL rats. The findings indicate that quercetin ameliorated activation of survival pathways and downregulated the expression of genes related to inflammation and precancerous conditions. Suppression of amphiregulin/EGFR signals may contribute to this effect.

Background/aims: The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily, which plays an essential role in the regulation of enterohepatic circulation and lipid homeostasis. Here we investigated whether WAY-362450, a synthetic potent FXR agonist, could protect against non-alcoholic steatohepatitis (NASH) in mice fed a methionine and choline-deficient (MCD) diet., Methods: Male C57BL/6 mice on the MCD diet were treated with or without WAY-362450 (30 mg/kg) for 4 weeks., Results: The elevations of serum ALT and AST activities induced by the MCD diet were decreased with WAY-362450 treatment. In terms of liver histology, while WAY-362450 treatment showed no impact on hepatic triglyceride accumulation, it significantly reduced inflammatory cell infiltration and hepatic fibrosis. The reduction in inflammatory cell infiltration correlated with deceased serum levels of keratinocyte derived chemokine (mKC) and MCP 1 and decreased hepatic gene expression of MCP-1 and VCAM-1. The reduction of hepatic fibrosis by WAY-362450 treatment corresponded to a reduction in hepatic gene expression of fibrosis markers. The positive effects of WAY-362450 were FXR-dependent since no protection was observed in MCD diet-fed FXR deficient mice., Conclusions: These findings demonstrate that FXR agonists may be useful for the treatment of non-alcoholic steatohepatitis.

Background/aims: Septins are ubiquitous and multifunctional scaffold proteins involved in cytoskeletal organization, exocytosis and other cellular processes. We disclose the quiescent hepatic stellate cells (HSCs)-specific expression of a septin subunit Sept4 in the liver, and explore the significance of the septin system in liver fibrosis., Methods: We analyzed the expression of alpha-smooth muscle actin (alpha-SMA), collagens and other markers in primary cultured HSCs derived from wild-type and Sept4(-/-) mice. We compared susceptibility of these mice to liver fibrosis induced by either carbon tetrachloride treatment, bile duct ligation or methionine/choline-deficient diet. Collagen deposition, the principal parameter of liver fibrosis, was quantified both histochemically (Masson's trichrome stain) and biochemically (hydroxyproline content)., Results: In vitro, Sept4 mRNA/protein was remarkably downregulated in HSCs through myofibroblastic transformation. Sept4(-/-) HSCs showed normal morphology and proliferation, while myofibroblastic transformation as monitored by the upregulation of alpha-SMA and collagen was accelerated compared to wild-type HSCs. In vivo, liver fibrosis was consistently more severe in Sept4(-/-) mice than in wild-type littermates in all of the three paradigms of hepatitis/liver fibrosis., Conclusions: These data concordantly indicate that the HSC-specific septin subunit Sept4 and perhaps the septin system are involved in the suppressive modulation of myofibroblastic transformation and fibrogenesis associated with liver diseases.

Background: There is strong evidence demonstrating that coagulation system activation contributes to wound healing and promotes organ fibrosis. Several epidemiological studies have now shown that prothrombotic status, including carriage of the factor (F)V Leiden mutation, is associated with rapid progression of hepatic fibrosis., Objectives: To assess the effect of a procoagulant state on progression of hepatic fibrosis in a controlled environment and to test whether anticoagulation could attenuate fibrogenesis., Methods: We investigated the effects of coagulation status on liver fibrosis development in a mouse model of chronic toxic liver injury. Prothrombotic FV Leiden mutant mice, C57BL/6 control animals and anticoagulated mice were studied after chronic exposure to carbon tetrachloride., Results: Carriage of the FV Leiden mutation caused a significant increase in hepatic fibrosis. Anticoagulation with warfarin significantly reduced fibrosis progression in wild-type mice but was less effective against the profibrotic FV Leiden mutation. Changes in the fibrosis scores were mirrored by changes in liver hydroxyproline content and hepatic stellate cell activation detected by alpha-smooth muscle actin expression., Conclusions: These results demonstrate that coagulation status has a strong influence on hepatic fibrogenesis. It is likely that thrombin signaling through the proteinase-activated receptor 1 (PAR(1)) receptor expressed on hepatic stellate cells is responsible for this relationship. These results represent the first reported use of anticoagulation to slow hepatic fibrogenesis and suggest a potential novel anti-fibrotic therapeutic approach for the future.

Background/aims: Thromboxane A2 (TXA2) has been suggested to play a significant role in the development of portal hypertension in fibrosis, and Kupffer cell (KC) derived TXA2 has been shown to mediate the hyperresponsiveness of the portal circulation to the vasoconstrictive actions of endothelin-1 (ET-1) during endotoxemia. The aim of this study was to determine whether the double stresses of prefibrotic changes and endotoxemia additively activate KC to increase release of TXA2 in response to ET-1, resulting in elevated portal resistance., Methods: One week Bile duct ligation (BDL) rats and sham-operated controls were subjected to isolated liver perfusions following LPS or saline for 6h. In a separate experiment, KC were isolated from BDL or sham rats and incubated with LPS or saline for 6h before the ET-1 treatment., Results: The double stresses of early fibrosis and LPS resulted in a greater sustained increase in portal pressure in response to ET-1 in BDL rats, and this increase correlated well with the much enhanced release of TXA2 in the perfusate. Media from the cultured KC showed significantly greater TXA2 release in response to ET-1 in BDL group than those in sham group, and LPS exacerbated this effect. Protein levels of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2, and thromboxane synthase were also significantly elevated in KC from BDL rats. ET-1 produced a marked increase in cPLA2 activation as measured by the phosphorylation of cPLA2 in KC of both BDL and sham groups. LPS greatly exacerbated the activation of cPLA2., Conclusions: The data suggest that the double stresses additively activate KC with an upregulation of the key enzymes in the TXA2 biosynthesis and release increased amount of TXA2 via the augmented activation of cPLA2 in response to ET-1, which leads to the increased portal resistance and ultimately hepatic microcirculatory dysfunction.

Background/aims: The portacaval anastamosis (PCA) rat is a model of the nutritional and endocrine consequences of liver cirrhosis. We hypothesized that failure to gain body weight in the PCA rat was the consequence of low testosterone levels and will be reversed by 4-hydroxyandrostenedione, a specific aromatase inhibitor., Methods: Male Sprague-Dawley rats subjected to an end-to-side PCA or sham surgery were administered either 4-hydroxyandrostenedione or vehicle. Food intake, food efficiency, and body weight were measured, animals sacrificed 3 weeks after surgery, visceral organs harvested and plasma sex steroids measured. Hepatic RNA was extracted and dot blots done to quantify the expression of sex hormone dependent enzymes 16alpha hydroxylase and 15beta hydroxylase., Results: Growth, food intake, food efficiency and plasma testosterone were lower and plasma estradiol higher in PCA than sham rats. Hepatic expression of testosterone driven 16alpha hydroxylase was lower and estradiol driven 15beta hydroxylase higher in PCA rats. These alterations were reversed by 4-hydroxyandrostenedione., Conclusions: These observations demonstrate that use of aromatase inhibitor reverses the nutritional and endocrine abnormalities in the PCA rat and suggest that this approach may be useful in cirrhotic patients.

We previously reported that the strain difference in the development of porcine-serum (PS)-induced rat hepatic fibrosis was closely related to the difference in the mode of MHC class-II-related genes expression. This study was carried out to clarify the serological and immunohistochemical changes in this hepatic fibrosis model. Six-week-old male Brown Norway (BN) and Wistar rats were injected with 0.5 ml of sterile PS twice a week for up to 8 weeks. The serum levels of PS-specific IgG1, IgG2a, and IgM were elevated more prominently in BN rats than Wistar rats. In the liver, significant increases in the numbers of PS-, OX-6 (RT1.B)-, CD4-, CD8, ED1-, and ED2-positive cells occurred earlier in BN rats than Wistar rats. At 8 weeks, deposition of PS and immunoglobulins was observed in hepatic fibrous septa and renal glomerular mesangium, and IgG1- and IgG2a-positive cells were found in the white pulp of the spleen. The present results suggest that humoral immunity probably regulated by MHC class II molecules and inflammatory cells may be involved in PS-induced hepatic fibrosis in rats.

Background/aims: Inchin-ko-to (TJ-135) is an herbal medicine used in Japan for treatment of icteric patients with cirrhosis. Its efficacy as an anti-fibrogenic drug was evaluated in relation to stellate cell activation., Methods/results: Liver fibrosis was induced in rats by repeated injections of carbon tetrachloride (CCl4) or pig-serum. Oral administration of TJ-135 improved the mortality of rats given CCl4 with reduced extents of liver necrosis and fibrosis. Similar improvement of liver fibrosis was found in rats given pig-serum showing no liver necrosis. DNA synthesis of stellate cells activated in vitro after isolation from normal rat liver was decreased by culture with TJ-135 in a dose-related manner, accompanied by decreased smooth muscle alpha actin expression and contractility. Such attenuation was not found in the cells cultured with geniposide, an iridoid compound of TJ-135, but genipin, an aglycone of geniposide formed in the gut by action of bacterial flora, markedly decreased stellate cell activation without affecting synthesis of proteins other than collagen., Conclusions: TJ-135 may be useful for treatment of liver fibrosis and portal hypertension through suppression of activated hepatic stellate cell function by genipin, an absorbed form of its component.

Bile duct ligation (BDL) in rats induces portal fibrosis. This process has been linked to changes in the oxidative state of the hepatic cells and in the production of nitric oxide. Our objective was to find possible temporal connections between hepatic redox state, NO synthesis and liver injury. In this work we have characterized hepatic lesions 17 and 31 days after BDL and determined changes in hepatic function, oxidative state, and NO production. We have also analyzed the expression and localization of inducible NO synthase (NOS2) and constitutive NO synthase (NOS3). After 17 and 31 days from ligature, lipid peroxidation is increased and both plasma concentration and biliary excretion of nitrite+nitrate are rised. 17 days after BDL both NOS2 and NOS3 are expressed intensely and in the same regions. 31 days after BDL, the expression of NOS2 remains elevated and is localized mostly in preserved hepatocytes in portal areas and in neighborhoods of centrolobulillar vein. NOS3 is localized in vascular regions of portal spaces and centrolobulillar veins and in preserved sinusoids and although its expression is greater than in control animals (34%), it is clearly lower (50%) than 17 days after BDL. The time after BDL is crucial in the study of NO production, intrahepatic localization of NOS isoforms expression, and cell type involved, since all these parameters change with time. BDL-induced, peroxidation and fibrosis are not ligated by a cause-effect relationship, but rather they both seem to be the consequence of common inductors., (Copyright 2004 Elsevier Inc.)

The role of iron in initiating liver fibrosis in iron overload diseases is not clearly established. Partly, this is due to the lack of suitable animal models that can produce the full liver pathology seen in genetic hemochromatosis. Recent advances in this field have demonstrated that iron may be interacting with other potential liver-damaging agents. The aim of this study was to investigate if feeding with carbonyl iron (CI) facilitates the development of carbon tetrachloride (CCl4)-induced liver fibrosis in the mouse. Mice were given a diet containing 3% CI and treated with CCl4 intraperitoneally twice weekly and 5% alcohol added to the drinking water for 12 weeks. Hepatic iron content increased 15- and 22-fold in animals receiving CI and CI + CCl4. At histological examination, iron-laden hepatocytes were found in CI treated animals, whereas these were absent in animals not exposed to CI. Mice receiving iron-enriched diet alone showed a mild fibrosis. Conversely, a marked collagen deposition was observed in CCl4 and CI + CCl4 groups. In particular, in this latter group, there was evidence of liver cirrhosis. Biochemical evaluation of collagen content substantiated histologic analysis. These results demonstrate that the addition of iron facilitates the development of cirrhosis in animals exposed to subtoxic doses of CCl4. This model may be useful in exploring the pathogenesis of liver cirrhosis. Moreover, its use in genetically altered mouse strains might provide new insight on the role of iron in fibrosis.

Background/aims: The aim of this study was to investigate whether in the liver, as in other tissues, there is evidence that angiotensin II, acting via the angiotensin II type 1 receptor (AT1-R), plays a role in fibrogenesis., Methods: Sprague-Dawley rats were divided into three groups; sham, bile duct ligated (BDL) and BDL + AT1-R antagonist, irbesartan. Real time RT-PCR was utilised to assess gene expression of the AT1 receptor, TGF-beta1 and alpha1 (I) collagen in the liver. TGF-beta1 and alpha1 (I) collagen mRNA expression and localisation were also assessed by in situ hybridisation. TGF-beta1 activity was assessed by using the TGF-beta inducible gene product betaig-h3. Fibrosis was assessed by the Knodell scoring system, tissue hydroxyproline content and picro-sirius red staining., Results: Real time RT-PCR revealed that there was a 6-fold up-regulation in AT1 receptor expression in BDL animals compared with shams. This was associated with marked increases in TGF-beta1, betaig-h3 and alpha1 (I) collagen gene expression which were attenuated by AT1-RA treatment. However, AT1-RA therapy produced no significant change in liver histology or hydroxyproline content., Conclusions: These results suggest that in the liver angiotensin II may play an important role in the fibrogenic response to injury. However, whether treatment with an AT1-RA will be of therapeutic benefit remains to be determined.

Background/aims: Hepatic stellate cells (HSC) are involved in the pathogenesis of liver fibrosis; although ET-1 is increased in cirrhosis, its pathophysiological role in fibrogenesis and portal hypertension remains controversial. The aim of this study was to investigate splanchnic hemodynamics and to correlate them with changes in ET-1 expression and HSC activation in bile duct ligated (BDL) rats., Methods/results: Expression of the ET-1 gene was increased early as measured by quantitative reverse transcriptase-polymerase chain reaction (6-fold 3 days after BDL) whereas ET-1 peptide measured by RIA increased significantly only in the late phase (30-fold at 28 days). There was a linear correlation between portal pressure and the amount of ET-1 in the portal vein (r = 0.66; P = 0.003), as well as between ET-1 and the volume fraction of myofibroblasts (r = 0.80, P < 10(-7)) as assessed by morphometry and immunohistochemical staining using alpha-smooth muscle actin., Conclusions: During chronic liver injury activation of HSCs and of preproET-1 mRNA is accentuated in the early phase after BDL. The late increase in ET-1 peptide may indicate that this peptide is only secondarily involved in HSC activation. The correlation between ET-1 in portal vein and portal pressure suggests that ET-1 may play an important role in the development of portal hypertension.

The fibrotic response after diverse forms of injury is characterized by the accumulation of extracellular matrix proteins, proliferation of myofibroblast-like cells, and organ contraction. Myofibroblasts are key effector cells in the development of the fibrotic response. They contribute to fibrosis through both increased cell number (proliferation) and enhanced matrix synthesis. Integrins, a class of cell adhesion molecules, are mediators of cell-extracellular matrix protein interactions that are important in the proliferative and migratory response of cells to matrix proteins. We have previously cloned the human integrin subunit alpha8, documented its high expression in lung tissue, and established it as a receptor for the matrix proteins fibronectin, vitronectin, and tenascin. We now demonstrate that alveolar interstitial cells are the primary cell type expressing alpha8beta1 in the lung parenchyma. Expression of alpha8beta1 is concentrated primarily along the thinned extensions of cells and at the tips of filopodia. Because of its unique distribution in alveolar interstitial cells, we hypothesized that it may play a role in the fibrotic response after injury. In bleomycin-induced pulmonary fibrosis, there is increased expression of alpha8beta1 by interstitial fibroblasts, the majority of which coexpress alpha smooth muscle actin, a marker of tissue myofibroblasts. To establish a more general role for alpha8beta1 during organ fibrosis, we further examined its expression in two rat models of liver fibrosis. During hepatic injury due to either carbon tetrachloride injury or bile duct ligation, we demonstrate de novo expression of alpha8beta1 in activated hepatic stellate cells, the myofibroblast equivalent in liver. Taken together, the data localize alpha8beta1 to myofibroblast-like cells during wound healing and suggest that signal transduction through the alpha8beta1 integrin may contribute to the fibrotic response of organs to injury.

Hepatic fibrosis results from an imbalance between fibrogenesis and fibrolysis in the liver. It remains uninvestigated whether Kupffer cells produce matrix metalloproteinase-13 (MMP-13), which mainly hydrolyzes extracellular matrix (ECM). We sought to determine the role of Kupffer cells in fibrogenesis/fibrolysis. In vivo, we used the rat model of pig serum-induced liver fibrosis. A subset was treated with gadolinium chloride (GdCl(3)), which specifically acts on Kupffer cells. Administration of GdCl(3) remarkably decreased the hydroxyproline content of the liver and increased the expression of MMP-13 mRNA in the liver without a difference in procollagen type I and tissue inhibitors of metalloproteinase-1 (TIMP-1) mRNA expression on Northern blot analysis with the elimination of ED2-positive cells. In vitro, addition of GdCl(3) to isolated Kupffer cells showed increased type I collagen-degrading activity in a dose-dependent manner as well as MMP-13 mRNA expression on Northern blot analysis. It is concluded that Kupffer cells are a major source of MMP-13 and modulation of Kupffer cells by GdCl(3) prevents liver fibrosis with increased expression of MMP-13 mRNA and protein, whereas procollagen type I and TIMP-1 mRNA, which encode two major effectors of fibrogenesis, were unchanged. This is the first report showing that Kupffer cells produce interstitial collagenase (MMP-13) resulting in the reduction of ECM. This discovery may provide new insights into therapy for hepatic fibrosis., (Copyright 2000 Academic Press.)

Background/aims: Plasminogen activators and plasminogen activator inhibitors are important regulators of the balance between the proteolytic and antiproteolytic activities that determine extracellular matrix turnover. We examined the expression of plasminogen activator-plasmin system components in experimental liver fibrosis of rats., Methods: Liver fibrosis was produced in rats by injecting carbon tetrachloride for 6 to 12 weeks. Gene expression for plasminogen activator inhibitor-1 (PAI-1), urokinase and tissue plasminogen activators (uPA and tPA), urokinase plasminogen activator receptor (uPAR), and transforming growth factor-beta1 (TGF-beta1) was examined by Northern analysis. Western analysis was performed to detect protein expression of PAI-1, uPA and uPAR. An immunohistochemical study was performed to detect the localization of PAI-1. Additionally, primary cultured liver cells were examined by Northern and Western analyses for this protein with or without prior incubation with TGF-beta1., Results: At 6 weeks, when fibrosis had occurred, uPA and uPAR mRNAs had increased 2.8-fold and 1.8-fold, respectively; PAI-1 and tPA mRNA levels were unchanged. At the cirrhotic stage (9 to 12 weeks), mRNA levels for PAI-1, uPA, uPAR and tPA were all increased. Western analysis also showed increased uPA and uPAR expressions in fibrotic liver, and increased PAI-1, uPA and uPAR expressions in cirrhotic liver. PAI-1 protein was also demonstrated immunohistochemically along sinusoids, vessels, and bile duct cells of normal and fibrotic liver. In liver cell cultures, Kupffer cells, hepatocytes, and especially stellate cells, expressed PAI-1. Expression was enhanced in stellate cells cultured from fibrotic or cirrhotic liver or stimulated in vitro with TGF-beta1., Conclusion: Though increased uPA and uPAR may act on matrix degradation in fibrotic liver, increased PAI-1 together with uPA, uPAR and tPA are associated with overall inhibition of matrix degradation in cirrhotic liver. Hepatic stellate cells are an important source of PAI-1 during liver fibrosis.

The characteristic cardiovascular changes in liver cirrhosis are vasodilatation and increased cardiac output. Augmented activity of the vasorelaxant factor, nitric oxide (NO), stimulated by cytokines, have been suggested to play a role in the pathogenesis, but previous studies show conflicting results. We therefore aimed to evaluate the entire pathway from cytokines to the final metabolites, nitrate/nitrite. The levels of serum Tumor Necrosis Factor-alpha (TNFalpha) and nitrate/nitrite (NOx) were measured, and aorta content of inducible (iNOS) and endothelial nitric oxide synthase (eNOS) mRNA and protein were determined by reverse-transcription polymerase chain reaction and Western blotting in rats with cirrhosis due to chronic bile duct ligation and sham-operated controls. Compared to control rats, serum TNFalpha levels were significantly elevated in cirrhotic rats (48.4+/-21.1 vs 16.8+/-9.0 pg/ml, p<0.01); iNOS mRNA was detectable whereas it was absent in controls, and eNOS mRNA levels was significantly higher in aortae of cirrhotic rats. Aortic eNOS protein content was significantly higher in cirrhotic rats, but iNOS protein was undetectable by Western blotting in both groups. Serum NOx concentrations in the cirrhotic group were significantly higher than those in controls (3.5+/-1.0 vs 2.3+/-0.5 microM, p<0.01). These results suggest that NO activity in cirrhosis is increased, and is predominantly due to eNOS since the detectable iNOS mRNA does not seem to be expressed as protein. The increased NOS activity in the arterial system may play a role in the systemic hemodynamic changes occurring in cirrhosis.

Background/aim: The aim of this study was to investigate the effect of interferon gamma on the synthesis of matrix proteins such as collagens with the relation to transforming growth factor-beta1 expression in vivo., Methods: We investigated the effects of interferon gamma in a model of liver fibrosis induced by pig serum in male Wistar rats, which develops fibrosis without an increase in serum alanine aminotransferase (i.e., without hepatocyte injury). Rats were injected with 0.5 ml of pig serum twice a week for 8 weeks with or without 20,000 or 50,000 U of interferon gamma., Results: Interferon gamma at doses up to 50,000 U/day prevented fibrosis, as indicated by reduced hydroxyproline content in the liver. Interferon gamma at 50,000 U/day also reduced expression of type I and III procollagen in the liver. However, the expression of transforming growth factor-beta1 mRNA and protein in the liver was not reduced by interferon gamma. Histologically, interferon gamma at 50,000 U/day also reduced the number of myofibroblast-like cells (activated stellate cells)., Conclusions: These results indicate that interferon gamma can prevent fibrosis by inhibiting the activation and proliferation of stellate cells, resulting in reduced expression of procollagen without affecting transforming growth factor-beta1 expression in pig serum-induced rat liver fibrosis in vivo.

Background/aim: A herbal medicine, Sho-saiko-to (TJ-9), has recently been orally administered to patients with chronic liver disease in Japan and has been reported to inhibit the development of hepatocellular carcinoma. The aim of this study was to investigate whether TJ-9 has an inhibitory effect on the development of preneoplastic lesions and liver fibrosis in rats., Methods: The effects of the TJ-9 were examined using the choline-deficient L-amino acid-defined (CDAA) diet-induced liver fibrosis model in 16-week-old male Wistar rats., Results: TJ-9 (1% w/w) prevented fibrosis, as indicated by reduced hydroxyproline content in the liver and inhibition of the increase in a serum marker of fibrosis (hyaluronic acid), without reducing the increase in serum alanine aminotransferase and aspartate aminotransferase. TJ-9 also reduced the expression of type III procollagen alpha 1 mRNA in the liver, as well as the proliferation of myofibroblast-like cells (activated stellate cells, activated Ito cells). Furthermore, TJ-9 reduced the number of preneoplastic lesions, detected as enzyme-altered (glutathione S-transferase placental form-positive) lesions, in the liver., Conclusions: These results indicate that the herbal medicine Sho-saiko-to (TJ-9) prevents fibrosis as well as preneoplastic lesions, not by inhibiting hepatocyte cell death but by inhibiting the activation of stellate cells, which are considered to be the main collagen-producing cells, leading to a reduction in the development of preneoplastic lesions.

Background/aims: The aim of this study was to investigate the effect and mechanism of fibrosuppression by a newly synthesized prolyl 4-hydroxylase inhibitor [HOE 077, 2, 4-pyridine dicarboxylic acid bis [(2-methoxyethyl) amide]] on pig serum-induced liver fibrosis in the rat., Methods: Male Wistar rats received 0.5 ml of pig serum twice a week for 10 weeks with 0, 100 or 200 ppm of HOE 077. At the end of the experiment, the hydroxyproline content of the liver, and alanine aminotransferase were measured. Histological stains used were HE, azan and a stain for alpha-smooth muscle actin (alpha-SMA). Electron microscopy was also performed. Messenger RNA expressions of type I and III procollagen were examined by Northern blot analysis. alpha-SMA positive cells and fibers with azan staining were assessed as percent area of the tissue specimen, using an image analysis system., Results: Rats that received pig serum for 10 weeks showed an increased liver hydroxyproline content of 318+/-39 microg/g wet weight (n=15). HOE 077 at doses up to 200 ppm significantly (p<0.01) reduced this increase of liver hydroxyproline content (181+/-39 microg/g wet weight, n=15) in accordance with improved histological findings. 200 ppm of HOE 077 significantly reduced mRNA expressions of alpha2(I) (486+/-102 vs 151+/-36, p<0.01) and alpha1(III) (276+/-127 vs 160+/-67, p<0.05) procollagen and percent area of alpha-SMA positive cells (2.94+/-2.14 vs 1.17+/-0.88%). Electron microscopy revealed that 200 ppm of HOE 077 prevented the loss of fat droplets., Conclusions: A prolyl 4-hydroxylase inhibitor (HOE 077) prevented pig serum-induced rat liver fibrosis by inhibiting stellate cell activation.

Background/aims: Pyridinoline, a specific cross-link of mature collagen, increases in liver during fibrogenesis and its hepatic level is related to the degree of reversibility of the fibrotic process. Since pyridinoline is excreted in urine, we have investigated the relationship between its urinary level and liver fibrogenesis in a model of mild and reversible liver fibrosis, murine schistosomiasis., Methods: Pyridinoline was measured by HPLC in urine and in liver of Schistosoma mansoni-infected mice during the acute and the chronic phases of the infection. Collagen deposition was measured colorimetrically. Both the isolated granulomas and the surrounding liver parenchyma were analyzed., Results: In infected mice, pyridinoline increased mainly in the isolated granulomas, corresponding to the fibrotic lesions, and slightly in the surrounding parenchyma. The urinary excretion of pyridinoline increased during liver fibrogenesis and was correlated to the duration of infection (r=0.81) and to the collagen content of granulomas (r=0.81). The treatment of infected mice by praziquantel, an antiparasitic drug, did not lead to significant changes in liver collagen cross-linking by pyridinoline either in granulomas or in parenchyma. The major effect of the drug was targeted at the collagen content of parenchyma, which decreased by 50%, 18 weeks after treatment. The urinary level of pyridinoline of treated mice was negatively correlated to the length of the treatment follow-up (r=-0.76)., Conclusions: The measurement of the urinary excretion of pyridinoline could be helpful to monitor the remodeling of liver extracellular matrix occurring in fibrogenesis and the effect of chemotherapy.

Background/aims: Calcitonin gene-related peptide (CGRP), a potent vasodilator; plays an important role in modulating vascular tone, acting as a noncholinergic nonadrenergic neurotransmitter. The aim of this study was to assess the role of CGRP, present in the vascular system, in the development of the hyperdynamic circulation observed in liver cirrhosis., Methods: Two doses of human alpha-CGRP [8-37], a specific antagonist of CGRP, were administered to cirrhotic and controls rats. Hemodynamics were evaluated using radioactive microspheres in conscious animals. To investigate the arterial depressor effect of exogenous CGRP, we constructed a dose-response curve for mean arterial pressure in cirrhotic and control rats by administering human alpha-CGRP., Results: The administration of high-dose human alpha-CGRP [8-37] (300 nmol.kg body weight-1.min-1) significantly increased both the mean arterial pressure (21 +/- 2 vs. 13 +/- 1%, p < 0.01) and total vascular resistance (76 +/- 5 vs. 54 +/- 5%, p < 0.01) in cirrhotic rats, compared to control rats. The splanchnic hemodynamic effects induced by human alpha-CGRP [8-37] were a significant decrease in percent change of portal venous inflow -42 +/- 3 vs. -33 +/- 3%, p < 0.05) and a significant increase in percent change of splanchnic arterial resistance (110 +/- 9 vs. 76 +/- 5%, p < 0.01) in cirrhotic rats, compared to control rats. Low-dose human alpha-CGRP [8-37] (60 nmol.kg body weight-1. min-1) caused similar hemodynamic changes, but the degree of change was much less than for the high-dose administration. The vascular response to human alpha-CGRP was significantly reduced in cirrhotic rats as compared to controls (ANOVA, p < 0.01). Plasma concentrations of CGRP were significantly elevated in cirrhotic rats., Conclusions: CGRP in the vascular system was involved in the modulation of vasodilatation in rats with liver cirrhosis, as demonstrated by the administration of a selective CGRP antagonist and exogenous CGRP.

Background/aims: The chronic bile duct-ligated rat is used to study hemodynamic changes in cirrhosis but suffers from total biliary obstruction and deep jaundice. The extent of reversibility of hemodynamics and histology following bile flow reconnection is controversial. We aimed to characterize the hemodynamics and histology of bile duct-ligated cirrhotic rats in which bile flow was reconnected by a Roux-en-y choledochojejunostomy., Methods: Operations created four groups: double sham (control), bile duct ligated, and two reconnected groups. Cardiac index and regional blood flows were measured by radioactive microspheres 4 weeks following the last operation in the first three groups and 8 weeks afterwards in the second reconnected group. Liver histology was assessed by a computer-aided scoring program., Results: Cardiac index, mean arterial pressure, and systemic vascular resistance in the reconnected groups were different from bile duct-ligated rats and returned to control values. Portal pressures in the reconnected groups (4-weeks, 10.0 +/- 0.5 and 8-week, 9.7 +/- 0.6 mmHg) were significantly lower than in bile-duct-ligated rats (13.7 +/- 0.6) but remained elevated compared to controls (7.0 +/- 0.3). Portal pressure in the reconnected rats was correlated with cardiac index and mesenteric blood flow, r = 0.66 and r = 0.45, respectively. Liver histology was improved in the reconnected rats, with decreased bile duct proliferation, fibrosis and apoptosis., Conclusions: We conclude that many of the histological features of secondary biliary cirrhosis are reversible after bilioenteric anastomosis. Furthermore, the hyperdynamic circulation is also largely reversible and is related to the degree of portal hypertension.

Background/aims: The aminoterminal procollagen type-III-peptide, which can be released during collagen type III deposition, has been suggested as a serum marker of fibrogenesis in patients with chronic liver disease. However, longitudinal studies correlating procollagen type-III-peptide concentrations in serum with the evolution of liver fibrosis are still needed. The purpose of the present study was to prove the significance of procollagen type-III-peptide concentrations in serum as a non-invasive marker of liver fibrogenesis in an animal model that best resembles progressive human liver fibrosis., Methods: In 88 female Wistar rats the biliary system was occluded by double ligation of the choledochal duct followed by retrograde injection of a mixture of prolamine/ethanole (Ethibloc). Sixteen rats served as controls. Groups of 8-10 rats were sacrificed at days 2, 7, 14, 21, 30, 32, 35, 60 and 90 after bile duct occlusion. In the groups histological staging (fibrosis score), determination of total liver hydroxyproline, measurement of serum procollagen type-III-peptide and routine liver function tests were performed., Results: First histological signs of liver fibrosis were seen as early as 7 days after bile duct occlusion. Progressive fibrosis was paralleled by an increase of serum procollagen type-III-peptide. There was a significant correlation between serum procollagen type-III-peptide and histological stages of fibrosis (r = 0.80; p < 0.0001) as well as between serum procollagen type-III-peptide and hydroxyproline in total liver tissue (r = 0.85; p < 0.0001), Conclusions: These results indicate that: (1) complete bile duct occlusion in rats produces progressive hepatic fibrosis resembling human secondary biliary fibrosis, and (2) procollagen type-III-peptide concentrations in serum reflect ongoing collagen formation in the liver unrelated to serum markers of cholestasis and inflammation.

Background/aims: The hypothesis that chronic alcohol ingestion potentiates iron-associated liver injury was investigated in the 'carbonyl iron-overload rat model'., Methods: Newborn male and female Wistar-Furth rats (seven per group) were used to investigate iron-alcohol interaction over a 26-week period. Groups 1 and 2 were iron loaded from birth, while the others received normal diet. At 10 weeks all rats commenced Lieber-DeCarli liquid diet; additional treatments were: group 1 6 g carbonyl iron/1000 ml diet plus alcohol; group 2 carbonyl iron in the liquid diet; group 3 alcohol in the liquid diet; group 4, the controls, received liquid diet only., Results: This study confirmed our previous observation that iron-loading from birth resulted in grade III-IV siderosis, in both male and female rats, and caused fibrosis associated with periportal macrophages. Alcohol-feeding, in addition to iron-feeding for 26 weeks significantly lowered the hepatic iron concentration in both male and female rats compared to those fed iron only (p < 0.05). Alcohol feeding did increase hepatic fibrosis in the iron-loaded animals. However, serum alanine aminotransferase activity was significantly higher in the iron-alcohol group than in the other groups (p < 0.05)., Conclusions: Thus, contrary to expectation, chronic alcohol feeding failed to potentiate hepatic fibrosis in iron-overloaded rats, although there was rather more hepatocyte necrosis, and the serum alanine aminotransferase activity was significantly higher in the iron-alcohol group than in the other groups.

Rats were malnourished for 12 months with a highly inadequate fat-rich, calorie-sufficient but otherwise poly-deficient liquid diet composed of mashed potatoes with mayonnaise, comparable with the nutritional intake of many chronic alcoholics. When alcohol was incorporated into this diet, administered as whisky in drinking water available ad libitum, the livers of all eight rats showed increased fibrosis and cirrhosis as compared to the livers of the eight non-alcohol-treated, isocalorically fed, paired control rats. Alcohol-treated rats developed fibrosis and cirrhosis on a dietary fat content of 38% of total caloric intake and low blood alcohol levels, ranging from 50 to 126 mg/dl, due to gradual intake over the day and to low absolute intake (mean 11.9 +/- 0.6 g/kg per day). None of the rats died spontaneously. Malnutrition is likely to be an important factor in the development of the fibrosis of alcoholic liver disease, and this rat model may be used to study aspects of the pathogenesis.

Erythrocyte and hepatocyte plasma membranes derived from CCl4-cirrhotic and bile duct ligated rats were studied. Six groups of animals were used: Group 1 received CCl4 for 8 weeks. Animals in group 2 received CCl4 for 12 weeks. Group 3 consisted in animals that received CCl4 for 8 weeks and then only vehicle for 4 weeks more. Group 4 was the control which received only vehicle. In group 5, obstructive jaundice was induced by bile duct ligation (BDL); animals in group 6 were operated without ligation of the bile duct (sham operated). The activity of Na+/K+ and Ca2+ dependent ATPases, as well as the cholesterol/phospholipid ratio (CH/PL) were determined in both, erythrocyte and hepatocyte plasma membranes. In the CCl4 groups, Na+/K+ and Ca(2+)-ATPases activity was decreased about 60-70% while the CH/PL ratio was increased significatively in both, erythrocyte and hepatocyte plasma membranes. Discontinuation of CCl4 treatment reverted the enzymatic and lipidic alterations to normal in both cell types. ATPases activity and CH/PL ratio in erythrocyte resembled to those of hepatocytes in BDL rats. A strong correlation (p < 0.005) was found when erythrocyte ATPases activity and CH/PL ratio were compared with those of hepatocytes. Moreover, histopathological analysis of liver sections correlated well with both, erythrocyte and hepatocyte biochemical findings. We concluded that determination of ATPases activity and CH/PL ratio in erythrocyte membranes could be a simple, safe and useful marker of cell liver damage in CCl4-induced liver cirrhosis and biliary obstruction in the rat.

Micronodular cirrhosis was induced in male SUAH substrain Wistar rats by combined phenobarbitone and carbon tetrachloride treatment. Both pituitary and serum concentrations of growth hormone were significantly reduced in cirrhotic rats compared with age-related untreated rats or those treated only with phenobarbitone. Ultrastructurally growth hormone-secreting cells (somatotrophs) of pituitaries of cirrhotic rats appeared relatively inactive, having few hormone-containing granules, sparse rough endoplasmic reticulum, and small nuclei with areas of condensed chromatin. The cells themselves were smaller than similar cells of untreated rats with a reduced cytoplasmic area. In addition immunocytochemistry of pituitaries at light microscope level, using sheep anti-rat growth hormone antibody, showed that somatotrophs of cirrhotic rats were more heteromorphic and disorganized than those in controls. There was marked development of the folliculo-stellate cell system in pituitaries of cirrhotic rats, the cells were enlarged with distinct golgi, and numerous microvilli were projecting into dilated follicular lumena.

This study reports the findings of hepatic fibrosis and the accumulation of iron in the livers of 12 gerbils. The primary lesion was a haemorrhagic necrosis of the liver that was identical to that produced experimentally in the gerbil by administration of E. coli endotoxin lipopolysaccharide. The resulting extravasation of blood caused focal histiocytic reactions. The number of lesions increased with age, eventually resulting in a micronodular cirrhosis after 9 to 12 months owing to repeated episodes of endotoxin-induced haemorrhages in the liver. The accumulation of iron occurred in perisinusoidal cells, Kupffer cells and hepatocytes. The perisinusoidal cells were responsible for the subsequent hepatic fibrosis. The fibrosis associated with this condition appears to result from iron accumulation in the liver, following haemorrhage caused by endotoxin lipopolysaccharide. The gerbil is the first recorded rodent species to develop hepatic fibrosis in response to hepatic iron overload.

Hepatic fibrosis represents an important stage in the progression of chronic liver disease to cirrhosis. In the present paper we have investigated whether capsaicin-sensitive neuropeptide-containing sensory neurons may participate in the development of liver fibrosis. The expression of hepatic fibrosis induced by common bile duct obstruction has been studied both in capsaicin- and vehicle-treated rats. Common bile duct-induced liver fibrosis was less marked in capsaicin-treated rats than in vehicle-treated rats. Diffuse alterations of liver parenchyma structure with marked collagen deposition and nodular regeneration occurred 8 weeks after common bile duct ligation in vehicle-treated animals, while none of the capsaicin-treated rats exhibited the formation of complete connective septa altering the parenchyma architecture. Both vehicle- and capsaicin-treated rats showed an increasing number of desmin-positive cells in the perivenular zone, but the density of these cells was lower in treated animals than in untreated rats. The hydroxyproline content of the liver increased after common bile duct ligation in a time-dependent manner. Eight weeks after bile duct obstruction vehicle-treated rats showed a 7-fold increase of liver collagen content in comparison to normal animals. This enhancement was about 3.5-fold in capsaicin-treated rats. These findings raise the possibility that the peripheral release of neuropeptides stored in sensory nerves might participate in the development of liver fibrosis following common bile duct obstruction.

Previous experiments showed that the presence of high levels of acute phase reactants (APR) enhance CCl4-induced liver fibrosis in the rat. A high correlation was found between the degree of fibrosis and alpha 2-macroglobulin of the rat (alpha 2-macrofetoprotein, alpha M-FP) used for monitoring the acute phase response. This acute phase reaction was provoked by epinephrine just before CCl4 treatment was started. In the present study we analyzed the effect of APR by repeating these experiments and estimating liver neutral collagenase with a synthetic substrate and endogenous collagen as a substrate, and liver prolyl-4-hydroxylase. A strong depression of liver collagenase activity was found in rats with a preceding acute phase reaction contrary to the rats that underwent CCl4 treatment only. A high level of alpha M-FP correlated negatively with collagenase activity. Also in vitro alpha M-FP proved to inhibit collagenase activity. Prolyl-4-hydroxylase was increased in the rats during acute phase reaction and correlated highly and positively with alpha M-FP, haptoglobin, and ceruloplasmin. Thus high levels of APR promote development of CCl4-induced fibrosis, partly by anticollagenase activity and partly because of enhancement of prolyl-4-hydroxylase activity. The latter phenomenon can also be explained by the presence of APR, but this has to be proved.

Hepatic lipid peroxidation was shown to be stimulated in the livers of cholestatic rats with increased hydroxyproline levels. In another group, cholestatic rats were fed with a copper-supplemented diet to increase hepatic copper levels. Although liver copper concentrations increased about 16-fold in copper supplemented cholestatic rats compared to normally fed cholestatic rats, no change was observed either in hepatic lipid peroxidation or in hydroxyproline levels.

To clarify the process of free and small peptide-bound hydroxyproline synthesis in hepatic fibrogenesis, we measured the in vitro synthesis of [14C]hydroxyproline in the 67% ethanol soluble fraction in rat liver slices, together with hepatic protein-bound [14C]hydroxyproline synthesis. In control rat liver, the amount of free and small peptide-bound [14C]hydroxyproline synthesized was 13.1 +/- 2.6 10(-4) x dpm/g liver/3 hr. In the CCl4-treated rat liver, where the hepatic hydroxyproline content was increased 4.6-fold, the protein-bound [14C]hydroxyproline synthesis was significantly increased 1.5-fold, but free and small peptide-bound [14C]hydroxyproline synthesis was decreased into 70%. There was a significant inverse correlation between free and small peptide-bound [14C]hydroxyproline synthesis, and hepatic hydroxyproline content. These results suggest that the combination of an increase in collagen synthesis and a decrease in free and small peptide-bound [14C]hydroxyproline synthesis contributes to rapid accumulation of collagen in hepatic fibrosis.

Liver fibrosis and extracellular matrix play a central role in liver function impairment. Little information is available on the dynamic aspects and the natural history of fibroplasia, even if there is growing evidence that extracellular matrix accumulation (collagen I, III, IV, fibronectin, laminin, proteoglycans, etc.) is not to be considered only a passive structural support for damaged hepatic tissue, but may actively modulate liver cell behaviour. Clinicians need to date liver fibrosis and to monitor connective tissue synthesis and degradation, but attempts to develop reliable serological markers for collagen metabolism are hampered by the absence of a well defined golden standard to validate them. Nevertheless, serum type III aminoterminal procollagen peptide, at the moment, seems to be the most acceptable parameter of fibrogenesis. The data concerning the mechanisms of collagen production-degradation are becoming so precise and numerous that even if they have not, to date, led to 'routine' advantages for patients, they will end up becoming important tools in the clinical practice and management of liver fibrosis.