Hepatitis C Virus Nonstructural 3/4A Protein Dampens Inflammation and Contributes to Slow Fibrosis Progression during Chronic Fibrosis In Vivo

HCV infection typically induces liver injury and inflammation, which appears to be responsible for the associated fibrogenesis. To date, the mechanism underlying the different rates of disease progression remains unclear. The aim of the study is to understand the possible role of the HCV non-structural (NS) 3/4A protein in the fibrosis progression. We used NS3/4A-expressing transgenic mice (NS3/4A-Tg) to accomplish the goals of the study. Different stages of liver fibrosis were induced in wild-type and NS3/4A-Tg mice by single carbon tetrachloride (acute) or multiple injections for 4 (intermediate) or 8 (chronic) weeks. Fibrotic parameters, inflammatory responses and hepatocyte turnover were extensively examined. Hepatic expression of HCV NS3/4A did not induce spontaneous liver damage. However, NS3/4A expression exerted contrasting effects during acute and chronic liver damage. During early fibrogenesis and intermediate fibrosis (4 weeks), NS3/4A-Tg mice exhibited enhanced liver damage whereas reduced fibrosis was observed in NS3/4A-Tg during chronic liver fibrosis (8 weeks). Furthermore, attenuated inflammation was observed in NS3/4A-Tg during chronic fibrosis with increase in M2 macrophages, hepatocyte proliferation, decreased hepatocyte apoptosis and decreased ductular reaction. In conclusion, during early fibrogenesis, HCV NS3/4A contributes to liver damage. While, during chronic liver fibrosis, NS3/4A dampens inflammation and induces hepatocyte regeneration thereby contributing to slow fibrosis progression to promote its survival or persistence.     

Subcellular proteome analysis unraveled annexin A2 related to immune liver fibrosis

It is important to study the mechanism of liver fibrogenesis, and find new non‐invasive biomarkers. In this study, we used subcellular proteomic technology to study the plasma membrane (PM) proteins related to immune liver fibrosis and search for new non‐invasive biomarkers. A rat liver fibrosis model was induced by pig serum injection. The liver fibrogenesis from stage (S) S0‐1, S2, S3‐4, and S4 was detected by Masson staining and HE staining in this rat model after 2, 4, 6, and 8 weeks of treatment. The liver PM was enriched and analyzed using subcellular proteomic technology. The differentially expressed proteins were verified by Western blotting, immunohistochemistry, and ELISA. PM with 149‐fold purification was obtained and 22 differentially expressed proteins were identified. Of which, annexin A2 (ANXA2) was detected to be increased obviously in S4 compared with S0‐1, and verified by Western blotting of rat liver tissue and immunohistochemistry of rat and human liver tissue. The expression of ANXA2 in human plasma with S1‐2 was also found to be up‐regulated for 1.4‐fold than that in S0. Furthermore, ANXA2 was detected to translocate from nuclear membrane and cytosol to PM as HBV stimulation through immunocytochemical analysis in vitro. This study identified 22 differentially expressed proteins related to liver fibrosis, and verified a potential biomarker (ANXA2) for non‐invasive diagnosis of immune liver fibrosis. To our knowledge, it was the first time to dynamically study the proteins related to liver fibrosis and select biomarkers for liver fibrosis diagnosis through PM proteome research. J. Cell. Biochem. 110: 219–228, 2010. © 2010 Wiley‐Liss, Inc.     

Hepcidin expression in the liver: relatively low level in patients with chronic hepatitis C

Patients with chronic hepatitis C frequently have serum and hepatic iron overload, but the mechanism is unknown. Recently identified hepcidin, exclusively synthesized in the liver, is thought to be a key regulator for iron homeostasis and is induced by infection and inflammation. This study was conducted to determine the hepatic hepcidin expression levels in patients with various liver diseases. We investigated hepcidin mRNA levels of liver samples by real-time detection-polymerase chain reaction; 56 were hepatitis C virus (HCV) positive, 34 were hepatitis B virus (HBV) positive, and 42 were negative for HCV and HBV (3 cases of auto-immune hepatitis, 7 alcoholic liver disease, 13 primary biliary cirrhosis, 9 nonalcoholic fatty liver disease, and 10 normal liver). We analyzed the relation of hepcidin to clinical, hematological, histological, and etiological findings. Hepcidin expression levels were strongly correlated with serum ferritin (P < 0.0001) and the degree of iron deposit in liver tissues (P < 0.0001). Hepcidin was also correlated with hematological parameters (vs. hemoglobin, P = 0.0073; vs. serum iron, P = 0.0012; vs. transferrin saturation, P < 0.0001) and transaminase levels (P = 0.0013). The hepcidin-to-ferritin ratio was significantly lower in HCV(+) patients than in HBV(+) patients (P = 0.0129) or control subjects (P = 0.0080). In conclusion, hepcidin expression levels in chronic liver diseases were strongly correlated with either the serum ferritin concentration or degree of iron deposits in the liver. When adjusted by either serum ferritin values or hepatic iron scores, hepcidin indices were significantly lower in HCV(+) patients than in HBV(+) patients, suggesting that hepcidin may play a pivotal role in the pathogenesis of iron overload in patients with chronic hepatitis C.     

Proteomic analysis of hepatic iron overload in mice suggests dysregulation of urea cycle, impairment of fatty acid oxidation, and changes in the methylation cycle

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.     

Accelerated CCl4-induced liver fibrosis in Hjv-/- mice, associated with an oxidative burst and precocious profibrogenic gene expression

Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl₄. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis.     

Pathogenesis of liver fibrosis: role of oxidative stress

In the liver, the progressive accumulation of connective tissue, a complex and dynamic process termed fibrosis, represents a very frequent event following a repeated or chronic insult of sufficient intensity to trigger a "wound healing"-like reaction. The fibrotic process recognises the involvement of various cells and different factors in bringing about an excessive fibrogenesis with disruption of intercellular contacts and interactions and of extracellular matrix composition. However, Kupffer cells, together with recruited mononuclear cells, and hepatic stellate cells are by far the key-players in liver fibrosis. Their cross-talk is triggered and favoured by a series of chemical mediators, with a prominent role played by the transforming growth factor beta. Both expression and synthesis of this inflammatory and pro-fibrogenic cytokine are mainly modulated through redox-sensitive reactions. Further, involvement of reactive oxygen species and lipid peroxidation products can be clearly demonstrated in other fundamental events of hepatic fibrogenesis, like activation and effects of stellate cells, expression of metalloproteinases and of their specific inhibitors. The important outcome of such findings as regards the pathogenesis of liver fibrosis derives from the observation of a consistent and marked oxidative stress condition in many if not all chronic disease processes affecting hepatic tissue. Hence, reactive oxidant species likely contribute to both onset and progression of fibrosis as induced by alcohol, viruses, iron or copper overload, cholestasis, hepatic blood congestion.     

Ferritin and liver fibrosis among patients with chronic hepatitis C virus infection

IntroductionIn chronic hepatitis C virus (HCV) infection there is increased iron absorption leading to iron overload, a fact that may promote ferritin synthesis. Theoretically, increased ferritin should promote ongoing liver fibrosis but disparate results have been described.ObjectiveWe analyze the behavior of iron metabolism- related variables, comparing them with fibrosis and inflammatory activity in liver biopsy in HCV infected patients.Patients and MethodsWe analyzed among 90 HCV patients subjected to liver biopsy prior to antiviral treatment the relationships of serum levels of iron, ferritin, transferrin, transferrin saturation index (TSI) and total iron binding capacity (TIBC) with liver fibrosis and histological severity, assessed by Metavir-f, Metavir-a and Knodell indices, as well as with liver function, and also compared the aforementioned iron metabolism- related variables with 34 controls.ResultsPatients showed higher values of sideremia (T = 2.04; p = 0.044) and transferrin (T = 2.29; p = 0.004) compared with controls; but not ferritin, that was significantly higher among the 33 patients who also consumed alcohol (Z = 2.05; p = 0.041). Most patients showed a well preserved liver function (86 cases, Child A). Patients with Child B or C showed higher ferritin levels (Z = 2.68; p = 0.007) and TSI (Z = 2.41; p = 0.016), but lower transferrin and TIBC (Z = 3.25; p = 0.001) than Child A patients. Transferrin and TIBC were directly related to albumin (ρ = 0.24; p = 0.026), whereas bilirubin showed direct relationships with iron (ρ = 0.25; p = 0.016), TSI (ρ = 0.39; p < 0.001) and ferritin (ρ = 0.36; p < 0.001). Both ferritin (ρ = −0.22; p = 0.04) and TSI (ρ = −0.25; p = 0.016) were related to platelet count. No relationships were observed between iron variables and Knodell index, but serum iron, serum transferrin, and TSI were directly related to Metavir-f score (ρ = 0.28; p = 0.009, ρ = 0.22; p = 0.044, and ρ = 0.22; p = 0.044, in this order).ConclusionAlterations of iron related variables are relatively subtle in our series of 90 well compensated HCV patients. Serum ferritin was not related to liver fibrosis and increases only when alcoholism co-exists with HCV infection.     

Identification of TAF1, HNF4A,and CALM2 as potential therapeutic target genes for liver fibrosis

The molecular mechanism of liver fibrosis caused by hepatitis C virus (HCV) is not clear. The aim of this study is to understand the molecular mechanism of liver fibrosis induced by HCV and to identify potential therapeutic targets for hepatic fibrosis. We analyzed gene expression patterns between high liver fibrosis and low liver fibrosis samples, and identified genes related to liver fibrosis. We identified TAF1, HNF4A, and CALM2 were related to the development of liver fibrosis. HNF4A is important for hepatic fibrogenesis, and upregulation of HNF4A is an ideal choice for treating liver fibrosis. The gene expression of CALM2 is significantly lower in liver fibrosis samples than nonfibrotic samples. TAF1 may serve as a biomarker for liver fibrosis. The results were further validated by an independent data set GSE84044. In summary, our study described changes in the gene expression during the occurrence and development of liver fibrosis. The TAF1, HNF4A, and CALM2 may serve as novel targets for the treatment of liver fibrosis.     

Local but not systemic administration of mesenchymal stromal cells ameliorates fibrogenesis in regenerating livers

Chronic liver injury leads to the accumulation of myofibroblasts resulting in increased collagen deposition and hepatic fibrogenesis. Treatments specifically targeting fibrogenesis are not yet available. Mesenchymal stromal cells (MSCs) are fibroblast‐like stromal (stem) cells, which stimulate tissue regeneration and modulate immune responses. In the present study we assessed whether liver fibrosis and cirrhosis can be reversed by treatment with MSCs or fibroblasts concomitant to partial hepatectomy (pHx)‐induced liver regeneration. After carbon tetrachloride‐induced fibrosis and cirrhosis, mice underwent a pHx and received either systemically or locally MSCs in one of the two remaining fibrotic/cirrhotic liver lobes. Eight days after treatment, liver fibrogenesis was evaluated by Sirius‐red staining for collagen deposition. A significant reduction of collagen content in the locally treated lobes of the regenerated fibrotic and cirrhotic livers was observed in mice that received high dose MSCs. In the non‐MSC‐treated counterpart liver lobes no changes in collagen deposition were observed. Local fibroblast administration or intravenous administration of MSCs did not ameliorate fibrosis. To conclude, local administration of MSCs after pHx, in contrast to fibroblasts, results in a dose‐dependent on‐site reduction of collagen deposition in mouse models for liver fibrosis and cirrhosis.     

Iron Regulator Hepcidin Exhibits Antiviral Activity against Hepatitis C Virus

Hepatitis C viral infection affects 170 million people worldwide. It causes serious chronic liver diseases. HCV infection has been implicated in iron accumulation in the liver and iron overload has been shown to be a potential cofactor for HCV associated hepatocellular carcinoma progression. The underlying mechanisms are not understood. Human hepcidin, a 25 amino acid peptide mainly produced by hepatocytes, is a key regulator of iron metabolism. Alteration of hepcidin expression levels has been reported in the setting of chronic HCV infection and hepatocellular carcinoma. In this study, we aim to examine the interactions between HCV infection and hepcidin expression in liver cells. We found that hepcidin expression was suppressed in HCV infected cells. The suppressive effect appears to be regulated by histone acetylation but not DNA methylation. Moreover, we found that hepcidin had a direct antiviral activity against HCV replication in cell culture. The antiviral effect is associated with STAT3 activation. In conclusion, hepcidin can induce intracellular antiviral state while HCV has a strategy to suppress hepcidin expression. This may be a novel mechanism by which HCV circumvents hepatic innate antiviral defense.     

Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus

Hepatitis C virus (HCV) is a major cause of viral hepatitis that can progress to hepatic fibrosis, steatosis, hepatocellular carcinoma, and liver failure. HCV infection is characterized by a systemic oxidative stress that is most likely caused by a combination of chronic inflammation, iron overload, liver damage, and proteins encoded by HCV. The increased generation of reactive oxygen and nitrogen species, together with the decreased antioxidant defense, promotes the development and progression of hepatic and extrahepatic complications of HCV infection. This review discusses the possible mechanisms of HCV-induced oxidative stress and its role in HCV pathogenesis.     

Elevated hepatic iron: A confounding factor in chronic hepatitis C

Historically, iron overload in the liver has been associated with the genetic disorders hereditary hemochromatosis and thalassemia and with unusual dietary habits. More recently, elevated hepatic iron levels also have been observed in chronic hepatitis C virus (HCV) infection. Iron overload in the liver causes many changes including induction of oxidative stress, damage to lysosomes and mitochondria, altered oxidant defense systems and stimulation of hepatocyte proliferation. Chronic HCV infection causes numerous pathogenic changes in the liver including induction of endoplasmic reticulum stress, the unfolded protein response, oxidative stress, mitochondrial dysfunction and altered growth control. Understanding the molecular and cellular changes that could occur in a liver which has elevated hepatic iron levels and in which HCV replication and gene expression are ongoing has clinical relevance and represents an area of research in need of further investigation.     

Effects of retinoic acid on the development of liver fibrosis produced by carbon tetrachloride in mice

The role of retinoic acid (RA) in liver fibrogenesis was previously studied in cultured hepatic stellate cells (HSCs). RA suppresses the expression of alpha2(I) collagen by means of the activities of specific nuclear receptors RARalpha, RXRbeta and their coregulators. In this study, the effects of RA in fibrogenesis were examined in carbon tetrachloride (CCl4) induced liver fibrosis in mice. Mice were treated with CCl4 or RA and CCl4, along side control groups, for 12weeks. RA reduced the amount of histologically detectable fibrosis produced by CCl4. This was accompanied by a attenuation of the CCl4 induced increase in alpha2(I) collagen mRNA and a lower (2-fold versus 3-fold) increase in liver hydroxyproline. Furthermore, RA reduced the levels of 3-nitrotyrosine (3-NT) protein adducts and thiobarbituric acid (TBA) reactive substance (TBARS) in the liver, which are formed as results of oxidative stress induced by CCl4 treatment. These in vivo findings support our previous in vitro studies in cultured HSC of the inhibitory effect of RA on type I collagen expression. The data also provide evidence that RA reduces CCl4 induced oxidative stress in liver, suggesting that the anti-fibrotic role of RA is not limited to the inhibition of type I collagen expression.     

Hepatic injury in chronic iron overload. Role of lipid peroxidation

In both hereditary hemochromatosis and in the various forms of secondary hemochromatosis, there is a pathologic expansion of body iron stores due mainly to an increase in absorption of dietary iron. Excess deposition of iron in the parenchymal tissues of several organs (e.g. liver, heart, pancreas, joints, endocrine glands) results in cell injury and functional insufficiency. In the liver, the major pathological manifestations of chronic iron overload are fibrosis and ultimately cirrhosis. Evidence for hepatotoxicity due to iron has been provided by several clinical studies, however the specific pathophysiologic mechanisms for hepatocellular injury and hepatic fibrosis in chronic iron overload are poorly understood. The postulated mechanisms of liver injury in chronic iron overload include (a) increased lysosomal membrane fragility, perhaps mediated by iron-induced lipid peroxidation, (b) peroxidative damage to mitochondria and microsomes resulting in organelle dysfunction, (c) a direct effect of iron on collagen biosynthesis and (d) a combination of all of the above.     

大鼠肝纤维化过程中组织金属蛋白酶抑制因子I基因的原位表达

为了解组织金属蛋白酶抑制因子１（ＴＩＭＰ－１）基因在实验性肝纤维化形成中的作用,我们应用地高辛原位杂交技术对大鼠肝组织在四氯化碳（ＣＣｌ４）诱发肝纤维化形成过程中ＴＩＭＰ－１ｍＲ－ＮＡ的表达进行了研究。结果表明,ＣＣｌ４肝损伤早期（４周）肝组织中间质细胞（血管及窦内皮细胞及贮脂细胞）中显示ＴＩＭＰ－１的过度表达;ＣＣｌ４肝纤维化早期（８周）及肝纤维化晚期（１２周）,肝组织中间质细胞的ＴＩＭＰ－１表达持续维持在高水平。结果提示,肝间质细胞（内皮细胞及贮脂细胞）是肝内ＴＩＭＰ－１的主要来源细胞;ＴＩＭＰ－１的异常表达是大鼠肝纤维化过程中较早出现的分子变化,与肝纤维化发生有关;纤维化晚期持续高表达的ＴＩＭＰ－１通过抑制胶原酶而在肝纤维化持续进展中起重要作用。