Belinda Knight

Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation†

Oval cells are hepatocytic precursors that proliferate in late‐stage cirrhosis and that give rise to a subset of human hepatocellular carcinomas. Although liver regeneration typically occurs through replication of existing hepatocytes, oval cells proliferate only when hepatocyte proliferation is inhibited. Transforming growth factor‐β (TGF‐β) is a key inhibitory cytokine for hepatocytes, both in vitro and in vivo. Because TGF‐β levels are elevated in chronic liver injury when oval cells arise, we hypothesized that oval cells may be less responsive to the growth inhibitory effects of this cytokine. To examine TGF‐β signaling in vivo in oval cells, we analyzed livers of rats fed a choline‐deficient, ethionine‐supplemented (CDE) diet for phospho‐Smad2. Phospho‐Smad2 was detected in more than 80% of hepatocytes, but staining was substantially reduced in oval cells. Ki67 staining, in contrast, was significantly more common in oval cells than hepatocytes. To understand the inverse relationship between TGF‐β signaling and proliferation in oval cells and hepatocytes, we examined TGF‐β signaling in vitro. TGF‐β caused marked growth inhibition in primary hepatocytes and the AML12 hepatocyte cell line. Two oval cell lines, LE/2 and LE/6, were less responsive. The greater sensitivity of the hepatocytes to TGF‐β–induced growth inhibition may result from the absence of Smad6 in these cells. Conclusion: Our results indicate that oval cells, both in vivo and in vitro, are less sensitive to TGF‐β–induced growth inhibition than hepatocytes. These findings further suggest an underlying mechanism for the proliferation of oval cells in an environment inhibitory to hepatocytic proliferation. (HEPATOLOGY 2007;45:31–41.)

Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells

Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte‐mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemochromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor–like weak inducer of apoptosis (TWEAK), is promitotic for LPCs; whether it acts directly is not known. In our murine choline‐deficient, ethionine‐supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor‐inducible 14 (Fn14)] expression in the whole liver is massively upregulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE‐induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2‐week‐CDE‐treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin–positive cells. Stimulation of an Fn14‐positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NFκB) activation and dose‐dependent proliferation, which was diminished after targeting of the p50 NFκB subunit by RNA interference. Conclusion: TWEAK acts directly and stimulates LPC mitosis in an Fn14‐dependent and NFκB‐dependent fashion, and signaling via this pathway mediates the LPC response to CDE‐induced injury and regeneration. (HEPATOLOGY 2010)

Liver inflammation and cytokine production, but not acute phase protein synthesis, accompany the adult liver progenitor (oval) cell response to chronic liver injury

Oval cells are facultative liver progenitor cells, which are invoked during chronic liver injury in order to replenish damaged hepatocytes and bile duct cells. Previous studies have observed inflammation and cytokine production in the liver during chronic injury. Further, it has been proposed that inflammatory growth factors may mediate the proliferation of oval cells during disease progression. We have undertaken a detailed examination of inflammation and cytokine production during a time course of liver injury and repair, invoked by feeding mice a choline‐deficient, ethionine‐supplemented (CDE) diet. We show that immediately following initial liver injury, B220‐expressing leucocytes transiently infiltrate the liver. This inflammatory response occurred immediately before oval cell numbers began to expand in the liver, suggesting that the two events may be linked. Two waves of liver cytokine production were observed during the CDE time course. The first occurred shortly following commencement of the diet, suggesting that it may represent a hepatic acute phase response. However, examination of acute phase marker expression in CDE‐fed mice did not support this hypothesis. The second wave of cytokine expression correlated with the expansion of oval cell numbers in the liver, suggesting that these factors may mediate oval cell proliferation. No inflammatory signalling was detected following withdrawal of the injury stimulus. In summary, our results document a close correlation between inflammation, cytokine production and the expansion of oval cells in the liver during experimental chronic injury.

Lymphotoxin‐β receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury

Lymphotoxin‐beta (LTβ) is a proinflammatory cytokine and a member of the tumor necrosis factor (TNF) superfamily known for its role in mediating lymph node development and homeostasis. Our recent studies suggest a role for LTβ in mediating the pathogenesis of human chronic liver disease. We hypothesize that LTβ co‐ordinates the wound healing response in liver injury via direct effects on hepatic stellate cells. This study used the choline‐deficient, ethionine‐supplemented (CDE) dietary model of chronic liver injury, which induces inflammation, liver progenitor cell proliferation, and portal fibrosis, to assess (1) the cellular expression of LTβ, and (2) the role of LTβ receptor (LTβR) in mediating wound healing, in LTβR−/− versus wild‐type mice. In addition, primary isolates of hepatic stellate cells were treated with LTβR‐ligands LTβ and LTβ‐related inducible ligand competing for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT), and mediators of hepatic stellate cell function and fibrogenesis were assessed. LTβ was localized to progenitor cells immediately adjacent to activated hepatic stellate cells in the periportal region of the liver in wild‐type mice fed the CDE diet. LTβR−/− mice fed the CDE diet showed significantly reduced fibrosis and a dysregulated immune response. LTβR was demonstrated on isolated hepatic stellate cells, which when stimulated by LTβ and LIGHT, activated the nuclear factor kappa B (NF‐κB) signaling pathway. Neither LTβ nor LIGHT had any effect on alpha‐smooth muscle actin, tissue inhibitor of metalloproteinase 1, transforming growth factor beta, or procollagen α1(I) expression; however, leukocyte recruitment‐associated factors intercellular adhesion molecule 1 and regulated upon activation T cells expressed and secreted (RANTES) were markedly up‐regulated. RANTES caused the chemotaxis of a liver progenitor cell line expressing CCR5. Conclusion: This study suggests that LTβR on hepatic stellate cells may be involved in paracrine signaling with nearby LTβ‐expressing liver progenitor cells mediating recruitment of progenitor cells, hepatic stellate cells, and leukocytes required for wound healing and regeneration during chronic liver injury. (HEPATOLOGY 2009;49:227–239.)

C-kit Inhibition by Imatinib Mesylate Attenuates Progenitor Cell Expansion and Inhibits Liver Tumor Formation in Mice

BACKGROUND & AIMS Numerous studies have linked the proliferation of liver progenitor cells (LPCs) during chronic liver disease to the risk for development of hepatocellular carcinoma. Thus, selective inhibition of LPC growth during preneoplastic injury may prevent or delay the onset of liver cancer. Rats carrying a germ-line mutation in c-kit have an impaired LPC response to liver injury. Therefore, we hypothesized that the c-kit inhibitor imatinib mesylate (IM) would suppress LPC growth and, therefore, may exert antitumorigenic effects in the liver. METHODS Expression of IM target proteins was examined in chronically injured rodent and human livers. The effect of IM was examined in vitro using LPC lines and in vivo in mice fed a choline-deficient, ethionine-supplemented (CDE) diet. Livers were examined following short-term (up to 1 month) or long-term (up to 14 months) feeding of CDE diet and drug treatments. RESULTS C-kit was significantly up-regulated in chronic injury and expressed by LPCs. IM was antiproliferative to LPC lines, and knockdown of c-kit reduced this response. IM treatment inhibited the LPCs response and early fibrogenesis induced by a short-term CDE diet. On the longer term, IM treatment reduced the extent of fibrosis and significantly inhibited tumor formation. CONCLUSIONS Tyrosine kinase inhibitors, such as IM, may be suited for the prevention of hepatocellular carcinoma in the setting of chronic liver injury via antiproliferative effects on c-kit-expressing LPCs.

Antiproliferative effects of interferon alpha on hepatic progenitor cells in vitro and in vivo

Hepatic progenitor cells (called oval cells in rodents) proliferate during chronic liver injury. They have been suggested as targets of malignant transformation in chronic liver diseases, including chronic hepatitis C. Interferon alpha therapy reduces the risk of hepatocellular carcinoma (HCC) in chronic hepatitis C regardless of viral clearance. The aim of this study was to determine whether interferon alpha could reduce the risk of HCC by modifying preneoplastic events in the hepatic progenitor cell population. Pre‐ and post‐treatment liver biopsies were evaluated for changes in the hepatic progenitor cell population in 16 patients with non‐responding chronic hepatitis C. Interferon alpha–based treatment significantly reduced the numbers of c‐kit–positive hepatic progenitor cells by 50%. To determine the mechanism of cell number reduction, the effects of interferon alpha on murine hepatic progenitor cells were studied in vitro. MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) proliferation assay and proliferating cell nuclear antigen staining showed that interferon alpha had a dose‐dependent, anti‐proliferative effect. Interferon alpha stimulated hepatocytic and biliary differentiation of the oval cell lines reflected by increased expression of albumin and cytokeratin19 accompanied by decreased expression of alphafetoprotein and Thy‐1. To validate these results in vivo, mice were placed on the choline‐deficient, ethionine‐supplemented diet to induce liver injury and oval cell proliferation and treated with pegylated interferon alpha 2b for 2 weeks. This resulted in a significant four‐fold reduction in the number of oval cells (P < .05). In conclusion, interferon alpha–based treatment reduced the number of hepatic progenitor cells in chronic liver injury by modulating apoptosis, proliferation, and differentiation. (HEPATOLOGY 2006;43:1074–1083.)

TNF/LTα double knockout mice display abnormal inflammatory and regenerative responses to acute and chronic liver injury

Following acute liver injury, hepatocytes divide to facilitate regeneration. However, during chronic injury, hepatocyte proliferation is typically blocked and repair is mediated through liver progenitor (oval) cells. Signalling of the p55 tumour necrosis factor (TNF) receptor is central to these processes. Two ligands for p55 are known: TNF and lymphotoxin-alpha (LTα). However, one study suggests that another exists that mediates liver injury following viral challenge. We have therefore investigated whether ligands other than TNF and LTα are required for liver regeneration following either acute or chronic injury. Wild-type and double TNF/LTα knockout (TNF−/−LTα−/−) mice were subjected to either partial hepatectomy (PHx) or a choline-deficient ethionine-supplemented (CDE) diet. Proliferating hepatocytes, oval cells and inflammatory cells were identified and quantified in liver sections by immunohistochemistry. Liver inflammatory cells were characterised by cell surface antigen expression. Liver damage and mortality were monitored. Both hepatocyte and oval cell proliferation was reduced in TNF−/−LTα−/− mice. Lymphocyte clusters were evident in all TNF−/−LTα−/− livers and were heterogeneous, comprising B and T lymphocytes. PHx evoked liver inflammation in TNF−/−LTα−/− but not wild-type mice, whereas no difference was apparent between genotypes in CDE experiments. Thus, TNF/LTα signalling mediates liver regeneration involving both hepatocytes and progenitor cells. The hyper-inflammatory response following PHx in TNF−/−LTα−/− animals, which is absent following CDE-induced injury, demonstrates that the two forms of liver injury evoke discrete inflammatory responses and provides a model in which such differences can be examined further.

Lymphotoxin-beta production following bile duct ligation: possible role for Kupffer cells.

Background and Aims:  Lymphotoxin‐β (LT‐β) may play a role in the pathogenesis of chronic liver injury. The aim of this study was to determine in an animal model of bile duct ligation liver injury whether LT‐β expression is induced and whether Kupffer cells are an intrahepatic source of LT‐β.

Bone marrow cells play only a very minor role in chronic liver regeneration induced by a choline-deficient, ethionine-supplemented diet

Liver progenitor (oval) cells have enormous potential in the treatment of patients with liver disease using a cell therapy approach, but their use is limited by their scarcity and the number of donor livers from which they can be derived. Bone marrow may be a suitable source. Previously the derivation of oval cells from bone marrow was examined in rodents using hepatotoxins and partial hepatectomy to create liver damage. These protocols induce oval cell proliferation; however, they do not produce the disease conditions that occur in humans. In this study we have used the choline-deficient, ethionine-supplemented (CDE) diet (which causes fatty liver) and viral hepatitis as models of chronic injury to evaluate the contribution of bone marrow cells to oval cells under conditions that closely mimic human liver disease pathophysiology. Following transplantation of lacZ-transgenic bone marrow cells into congenic mice, liver injury was induced and the movement of bone marrow cells to the liver monitored. Bone marrow-derived oval cells were observed in response to the CDE diet and viral injury but represented a minor fraction (0-1.6%) of the oval cell compartment, regardless of injury severity. In all situations only rare, individual bone marrow-derived oval cells were observed. We hypothesized that the bone marrow cells may replenish oval cells that are expended by protracted liver injury and regeneration; however, experiments involving a subsequent episode of chronic liver injury failed to induce proliferation of the bone marrow-derived oval cells that appeared as a result of the first episode. Bone marrow-derived hepatocytes were also observed in all injury models and controls at a frequency unrelated to that of oval cells. We conclude that during viral-and steatosis-induced liver disease the contribution of bone marrow cells to hepatocytes, either via oval cells or by independent mechanisms, is minimal and that the majority of oval cells responding to this injury are sourced from the liver.