K. R. Bridle

Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3.

Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non‐alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade.

The Role of Hepatic Stellate Cells and Transforming Growth Factor-β1 in Cystic Fibrosis Liver Disease

Liver disease causes significant morbidity and mortality from multilobular cirrhosis in patients with cystic fibrosis. Abnormal bile transport and biliary fibrosis implicate abnormal biliary physiology in the pathogenesis of cystic fibrosis-associated liver disease (CFLD), yet the mediators linking biliary events to fibrosis remain unknown. Activated hepatic stellate cells (HSCs) are the pre-eminent mediators of fibrosis in a range of hepatic disorders. The dominant stimulus for matrix production by HSCs is the cytokine transforming growth factor (TGF)-beta(1). In CFLD, the role of HSCs and the source of TGF-beta(1) have not been evaluated. Liver biopsy tissue obtained from 38 children with CFLD was analyzed. Activated HSCs, identified by co-localization of procollagen alpha(1)(I) mRNA and alpha-smooth muscle actin, were demonstrated as the cellular source of excess collagen production in the fibrosis surrounding the bile ducts and the advancing edge of scar tissue. TGF-beta protein and TGF-beta(1) mRNA expression were shown to be predominantly expressed by bile duct epithelial cells. TGF-beta(1) expression was significantly correlated with both hepatic fibrosis and the percentage of portal tracts showing histological abnormalities associated with CFLD. This study demonstrates a definitive role for HSCs in fibrogenesis associated with CFLD and establishes a potential mechanism for the induction of HSC collagen gene expression through the production of TGF-beta(1) by bile duct epithelial cells.

Fibrogenesis in pediatric cholestatic liver disease: Role of taurocholate and hepatocyte‐derived monocyte chemotaxis protein‐1 in hepatic stellate cell recruitment

Cholestatic liver diseases, such as cystic fibrosis (CF) liver disease and biliary atresia, predominate as causes of childhood cirrhosis. Despite diverse etiologies, the stereotypic final pathway involves fibrogenesis where hepatic stellate cells (HSCs) are recruited, producing excess collagen which initiates biliary fibrosis. A possible molecular determinant of this recruitment, monocyte chemotaxis protein‐1 (MCP‐1), an HSC‐responsive chemokine, was investigated in CF liver disease and biliary atresia. The bile‐duct‐ligated rat and in vitro coculture models of cholestatic liver injury were used to further explore the role of MCP‐1 in HSC recruitment and proposed mechanism of induction via bile acids. In both CF liver disease and biliary atresia, elevated hepatic MCP‐1 expression predominated in scar margin hepatocytes, closely associated with activated HSCs, and was also expressed in cholangiocytes. Serum MCP‐1 was elevated during early fibrogenesis. Similar observations were made in bile‐duct‐ligated rat liver and serum. Hepatocytes isolated from cholestatic rats secreted increased MCP‐1 which avidly recruited HSCs in coculture. This HSC chemotaxis was markedly inhibited in interventional studies using anti‐MCP‐1 neutralizing antibody. In CF liver disease, biliary MCP‐1 was increased, positively correlating with levels of the hydrophobic bile acid, taurocholate. In cholestatic rats, increased MCP‐1 positively correlated with taurocholate in serum and liver, and negatively correlated in bile. In normal human and rat hepatocytes, taurocholate induced MCP‐1 expression. Conclusion: These observations support the hypothesis that up‐regulation of hepatocyte‐derived MCP‐1, induced by bile acids, results in HSC recruitment in diverse causes of cholestatic liver injury, and is a key early event in liver fibrogenesis in these conditions. Therapies aimed at neutralizing MCP‐1 or bile acids may help reduce fibro‐obliterative liver injury in childhood cholestatic diseases. (HEPATOLOGY 2008.)

Evidence that myofibroblast-like cells are the cellular source of capsular collagen in hepatocellular carcinoma

BACKGROUND/AIMS The prognosis for patients with hepatocellular carcinoma is poor although tumour encapsulation has been associated with improved survival and disease-free rates. While the source of the tumour capsule is unclear, the major role that activated hepatic stellate cells play in the deposition of liver matrix in normal and diseased states suggests the possible involvement of these cells in tumour encapsulation. METHODS Twenty-four liver tumours (seven encapsulated HCC, seven non-encapsulated HCC, 10 colorectal metastases) were studied. Activated hepatic stellate cells were identified by immunohistochemistry for alpha-smooth muscle actin (alpha-SMA) and in situ hybridization for pro-collagen alpha1 (I) mRNA. Collagen deposition was localized using Massons trichrome stain. RESULTS Pro-collagen alpha1 (I) mRNA co-localized to alpha-SMA positive hepatic stellate cells within the region of increased collagen deposition in (i) the tumour capsule of encapsulated HCC, and (ii) the tumour junction of non-encapsulated HCC and colorectal metastasis. In addition, there was marked peritumour expression of alpha-SMA and procollagen alpha1 (I) mRNA, which diminished with distance away from the tumour in all tumour groups. The degree of expression was greatest with encapsulated HCC, less with non-encapsulated HCC and least with colorectal metastasis. This contrasted with the absence of alpha-SMA expression in normal liver from the same patients. Within the tumours, colorectal metastases differed from HCC by demonstrating marked alpha-SMA expression and collagen deposition in the septa. CONCLUSIONS Our findings demonstrate that activated hepatic stellate cells (i) are responsible for increased peritumour collagen production in non-encapsulated HCC and colorectal metastasis, and (ii) may be implicated in tumour capsule formation in HCC and metastasis stroma development. Thus, stellate cells may influence the local hepatic invasion by these tumours.

Repetitive acute pancreatic injury in the mouse induces procollagen alpha1(I) expression colocalized to pancreatic stellate cells.

Pancreatic stellate cells may be a major source of extracellular matrix deposition during injury. This study was undertaken to establish whether pancreatic stellate cells are a source of Type I collagen in vivo and whether they continue to be a source of matrix production in the post-injury fibrotic pancreas. To induce pancreatic fibrogenesis, acute pancreatic injury was induced in mice three times weekly with supraphysiologic doses of cerulein. Animals were treated for 6 weeks and allowed to recover for an additional 6 weeks. Stellate cell activation and pancreatic collagen expression were measured by immunohistochemistry, whole tissue RNA analysis, and in situ hybridization. Histology and digital image analysis demonstrated the development of substantial pancreatic fibrosis after 6 weeks of treatment. During recovery, incomplete resolution of the fibrosis was found. Procollagen α1(I) mRNA increased more than15-fold during treatment and continued to be 5-fold elevated during the post-injury phase. In situ hybridization studies demonstrated that collagen gene expression was colocalized to activated pancreatic stellate cells. Collagen expression and fibrosis persisted in focal areas during recovery. These findings show that pancreatic stellate cells are the major source of collagen during repetitive injury in vivo. Additionally, focal areas of sustained pancreatic fibrogenesis persist after cessation of cerulein treatment, and these areas may contribute to sustained total organ collagen expression in the absence of ongoing injury.

Rapamycin inhibits hepatic fibrosis in rats by attenuating multiple profibrogenic pathways

Hepatic stellate cell transdifferentiation, epithelial‐mesenchymal cell transition, and the ductular reaction each contribute to the development of hepatic fibrosis in cholestatic liver diseases. Inhibitors of mammalian target of rapamycin have antifibrotic properties. We evaluated the hypothesis that the antifibrotic action of rapamycin is due to attenuated myofibroblast proliferation in addition to an inhibitory effect on epithelial‐mesenchymal transition and the ductular reaction. Hepatic fibrosis was induced by bile duct ligation, and rodents received 1.5 mg/kg/day rapamycin by subcutaneous infusion for 21 days. The expression of various markers of hepatic fibrosis, stellate cell transactivation, epithelial‐mesenchymal transition, and the ductular reaction was compared between treated and untreated animals. Hepatic fibrosis, hepatic procollagen type 1 messenger RNA, and alpha‐smooth muscle actin expression were significantly reduced in treated animals. Hepatic stellate cell procollagen expression and proliferation were also reduced by rapamycin. The following markers of epithelial‐mesenchymal transition—vimentin protein expression, S100 calcium binding protein A4 and transforming growth factor beta 1 messenger RNA, and the mothers against decapentaplegic homolog signaling pathway—were all reduced after rapamycin treatment. The intensity of the ductular reaction was reduced by rapamycin as assessed by histopathological scoring and by reduced cytokeratin 19 expression. Rapamycin caused a reduction in hepatic progenitor cell proliferation. Together, these data show that multiple profibrogenic pathways are activated in an animal model of cholestasis and that rapamycin attenuates epithelial‐mesenchymal transition and the ductular reaction as well as hepatic stellate cell activation. Liver Transpl 15:1315–1324, 2009.

Diagnostic imaging and therapeutic application of nanoparticles targeting the liver

Liver diseases, particularly viral hepatitis, cirrhosis and hepatocellular carcinoma, are common in clinical practice with high morbidity and mortality worldwide. Many substances for diagnostic imaging and therapy of liver diseases may have either severe adverse effects or insufficient effectiveness in vivo because of their nonspecific uptake. Therefore, by targeting the delivery of drugs into the liver or specific liver cells, drug efficiency may be largely improved. This review summarizes the up-to-date research progress focusing on nanoparticles targeting the liver for both diagnostic and therapeutic purposes. Targeting strategies, mechanisms of enhanced effects, and clinical applications of nanoparticles are discussed specifically. We believe that new targeting nanotechnology such as nanoprobes for multi-modality imaging and multifunctional nanoparticles would facilitate significant advancements in this active research area in the near future.

Evidence for a sub-morphological inflammatory process in the liver in haemochromatosis

BACKGROUND/AIMS The role of cytokines in hepatic injury has been examined for many liver diseases however little is known of the cytokine involvement in haemochromatosis. The aim of the current study was to examine the hepatic gene expression of potential proinflammatory and profibrogenic cytokines in haemochromatosis. METHODS Interferon-gamma, interleukin-10, transforming growth factor-beta(1) and tumor necrosis factor-alpha mRNA expression was assessed in liver tissue from 20 haemochromatosis patients, eight controls and eight chronic hepatitis C patients. To assess the immunophenotype of the inflammatory infiltrate in haemochromatosis, liver sections were subjected to immunohistochemistry using markers for macrophages (CD68, HAM56, MAC387) or T cells (CD3 and CD45RO). RESULTS Interferon-gamma mRNA was increased in both haemochromatosis (0.29+/-0.08%, P=0.01) and hepatitis C patients (1.02+/-0.32%, P=0.03) compared to controls (0.04+/-0.01%). Interleukin-10 mRNA was significantly decreased in both haemochromatosis and hepatitis C patients (0.01+/-0.003%, P=0.008 and 0.03+/-0.015%, P=0.02, respectively) compared to controls (0.12+/-0.01%). CD3 positive T-cell number was significantly correlated with increasing hepatic iron concentration (r=0.56, P=0.03). CONCLUSIONS This study has demonstrated a distinct pattern of cytokine gene expression in haemochromatosis, which resembles that of inflammatory conditions such as chronic hepatitis C. These factors may play a role in the development of iron-induced hepatic fibrosis in haemochromatosis.

Identification and Characterization of the Hepatic Stellate Cell Transferrin Receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [(3)H]Thymidine incorporation, and real-time RT-PCR for procollagen alpha1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.

Hepcidin regulation in wild-type and Hfe knockout mice in response to alcohol consumption: Evidence for an alcohol-induced hypoxic response

BACKGROUND/AIMS Expression of Hamp1, the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice (Hfe(-/-)). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe(-/-) mice. METHODS Hfe(-/-) and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was measured by western blot. RESULTS Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe(-/-) mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1alpha protein levels were elevated in alcohol-fed wild-type animals compared with controls. CONCLUSION Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.