Albert Geerts

The hepatic stellate (Ito) cell: its role in human liver disease

The hepatic stellate (Ito) cell lies within the space of Disse and has a variety of functions. Stellate cells store vitamin A in characteristic lipid droplets. In the normal human liver, the cells can be identified by the presence of these lipid droplets; in addition, many stellate cells in the normal liver express a-smooth muscle actin. In acute liver injury, there is an expansion of the stellate cell population with increased α-smooth muscle actin expression; stellate cells appear to play a role in extracellular matrix remodelling after recovery from injury. In chronic liver injury, the stellate cell differentiates into a myofibroblast-like cell with marked expression of α-smooth muscle actin and occasional expression of desmin. Myofibroblast-like cells have a high fibrogenic capacity in the chronically diseased liver and are also involved in matrix degradation. In vitamin A intoxication, hypertrophy and proliferation of the stellate and myofibroblast-like cells may lead to non-cirrhotic portal hypertension, fibrosis and cirrhosis. In liver tumours, myofibroblast-like cells are involved in the capsule formation around the tumour and in the production of extracellular matrix within it. The transition of stellate cells into myofibroblast-like cells is regulated by an intricate network of intercellular communication between stellate cells and activated Kupffer cells, damaged hepatocytes, platelets, endothelial and inflammatory cells, involving cytokines and nonpeptide mediators such as reactive oxygen species, eicosanoids and acetaldehyde. The findings suggest that the stellate cell plays an active role in a number of human liver diseases, with a particular reactivity pattern in fibrotic liver disorders.

The interleukin‐17 pathway is involved in human alcoholic liver disease

Immune dysregulations in alcoholic liver diseases are still unclear, especially regarding alcoholic hepatitis inflammatory burst. Interleukin‐17 (IL‐17) is known to enhance neutrophil recruitment. We studied the IL‐17 pathway in alcoholic cirrhosis and alcoholic hepatitis. Patients with alcoholic liver disease were compared with patients with chronic hepatitis C virus (HCV) infection or autoimmune liver disease and with healthy controls. IL‐17 plasma levels and peripheral blood mononuclear cell secretion were assessed by enzyme‐linked immunosorbent assay (ELISA) and T cell phenotype by flow cytometry. IL‐17 staining and co‐staining with CD3 and myeloperoxidase were performed on liver biopsy specimens. IL‐17 receptor expression was studied on liver biopsies and in human hepatic stellate cells as well as their response to recombinant IL‐17 by chemotaxis assays. IL‐17 plasma levels were dramatically increased in alcoholic liver disease patients. Peripheral blood mononuclear cells of patients with alcoholic liver disease produced higher amounts of IL‐17, and their CD4+ T lymphocytes disclosed an IL‐17–secreting phenotype. In the liver, IL‐17–secreting cells contributed to inflammatory infiltrates in alcoholic cirrhosis, and alcoholic hepatitis foci disclosed many IL‐17+ cells, including T lymphocytes and neutrophils. In alcoholic liver disease, liver IL‐17+ cells infiltrates correlated to model for end‐stage liver disease score, and in alcoholic hepatitis to modified discriminant function. IL‐17 receptor was expressed in alcoholic liver disease by hepatic stellate cells, and these cells recruited neutrophils after IL‐17 stimulation in a dose‐dependent manner through IL‐8 and growth related oncogen α (GRO‐α) secretion in vitro. Conclusion: Human alcoholic liver disease is characterized by the activation of the IL‐17 pathway. In alcoholic hepatitis, liver infiltration with IL‐17–secreting cell infiltrates is a key feature that might contribute to liver neutrophil recruitment. (Clinical trials number NCT00610597). (HEPATOLOGY 2009;49:646–657.)

Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension

Accumulating evidence suggests that stellate cells are involved in the regulation of the liver microcirculation and portal hypertension. Activated hepatic stellate cells have the necessary machinery to contract or relax in response to a number of vasoactive substances. Because stellate cells play a role in both fibrosis and portal hypertension, they are currently regarded as therapeutic targets to prevent and treat the complications of chronic liver disease.

In vitro differentiation of fat-storing cells parallels marked increase of collagen synthesis and secretion

Fat-storing cells were isolated and purified from livers of normal adult rats and maintained in primary culture. By light and electron microscopy it was established that they underwent phenotypic changes into cells with the ultrastructural characteristics of myofibroblasts, between the third and sixth day in culture. These morphological changes were accompanied by a 2-fold increase of L-[3H]proline incorporation into secretory proteins and an 11-fold increase into secreted collagenase-sensitive proteins. In contrast, incorporation into cell layer-associated proteins and into cell layer-associated collagenase-sensitive proteins was not significantly elevated. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with fluorography, demonstrated that the main collagen type secreted by the myofibroblast-like cells was collagen type I. Collagen types III and IV, and fibronectin were present in lesser amounts. The similarity between the well known in vivo alterations of fat-storing cells under pathological conditions and the spontaneous in vitro differentiation described in this study, makes primary cultures of fat-storing cells a valuable tool for studying their role in chronic liver disease.

Transforming growth factor-β gene expression in normal and fibrotic rat liver

BACKGROUND/AIMS: Transforming growth factor-beta (TGF-beta) is considered to be an important mediator in the development of fibrosis in several chronic liver diseases. To understand the mechanism(s) by which TGF-beta exerts its action(s), we investigated the cellular distribution of TGF-beta(1,2,3) transcripts in normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver. METHODS: Parenchymal, sinusoidal endothelial, Kupffer and stellate cells were isolated and purified. The exact cellular composition of each isolate was determined by transmission electron microscopy. Expression of TGF-beta(1,2,3) transcripts was investigated using Northern hybridization analysis. Hybridization signals were quantified by scanning densitometry and corrected for: (i) differences in extractable RNA per cell type, (ii) signal contribution from contaminating cells, and (iii) differences in loading, capillary transfer and hybridization. RESULTS: In normal liver, TGF-beta1 mRNA was predominantly expressed in Kupffer cells, exhibiting values approximately 9-fold higher than those in stellate cells. No expression was found in endothelial and parenchymal cells. Signals for TGF-beta2 and TGF-beta3 were much weaker when compared to TGF-beta1. In Kupffer cells, the level of TGF-beta2 was approximately 4-fold higher than in stellate cells. Little expression was found in endothelial cells. TGF-beta3 expression could only be detected in stellate cells. TGF-beta2 and TGF-beta3 was not expressed in parenchymal cells. In fibrotic liver, TGF-beta1 mRNA was strongly expressed in all the sinusoidal cells. TGF-beta2 and TGF-beta3 could no longer be detected. When compared to the level of expression in normal stellate cells, the level of TGF-beta1 increased 12-fold in stellate cells from fibrotic livers, and 6-fold in endothelial cells. In Kupffer cells, the level of expression remained unchanged. CONCLUSIONS: (i) In both normal and fibrotic liver, TGF-beta1 is the most abundant isoform, (ii) in normal liver, TGF-beta1 is expressed strongly by Kupffer cells and moderately by stellate cells, TGF-beta2 expression is highest in Kupffer cells, followed by stellate cells and endothelial cells. TGF-beta3 is expressed by stellate cells, (iii) in fibrotic liver, the level of TGF-beta1 expression increases selectively in stellate cells and endothelial cells. This suggests an important role, not only for stellate, but also for endothelial cells in fibrogenesis.

Glutathione levels discriminate between oxidative stress and transforming growth factor-beta signaling in activated rat hepatic stellate cells.

Reactive oxygen species are implicated in the pathogenesis of several diseases, including Alzheimers disease, multiple sclerosis, human immunodeficiency virus, and liver fibrosis. With respect to liver fibrosis, we have investigated differences in antioxidant enzymes expression in stellate cells (SCs) and parenchymal cells from normal and CCl4-treated rat livers. We observed an increase in the expression of catalase in activated SCs. Treatment with transforming growth factor-β (TGF-β) increased the production of H2O2. Treatment with catalase decreased TGF-β expression. Addition of H2O2resulted in increased TGF-β production. 3-Amino-1,2,4-triazole abolished the capacity of SCs to remove H2O2. A paradoxical increase in capacity was observed when the cells were pretreated with diethyl maleate. Treatment with 3-amino-1,2,4-triazole increased TGF-β production. A paradoxical decrease of TGF-β production was observed with diethyl maleate. Treatment of the cells with N-acetylcysteine resulted in increased TGF-β production. TGF-β decreased the capacity of the SCs to remove H2O2. An increase in the capacity to remove H2O2 was observed when TGF-β was removed by neutralizing antibodies. In conclusion, our results suggest: 1) a link between cellular GSH levels and TGF-β production and 2) that cellular GSH levels discriminate whether H2O2 is the result of oxidative stress or acts as second messenger in the TGF-β signal transduction pathway.

The histone-deacetylase inhibitor Trichostatin A blocks proliferation and triggers apoptotic programs in hepatoma cells

BACKGROUND/AIMS Effective treatment for hepatocellular carcinoma is urgently needed. The histone-deacetylase inhibitor Trichostatin A (TSA) was shown to induce apoptosis in non-hepatic cells at submicromolar concentrations. However, the effect of TSA on hepatoma cells is unknown. METHODS The hepatoma cells HepG2, MH1C1, Hepa1-6 and Hep1B as well as human fibroblasts (control cells) were exposed to TSA (10(-6) to 10(-9)M). Cell proliferation was assessed by measuring DNA-synthesis and cell numbers. Apoptosis was quantified by flow cytometry and by the TdT-mediated dUTP nick-end labeling method. Expression patterns of cell cycle- and/or apoptosis-associated p27, p21(cip/waf), bax, bcl-2, cyclin A and (pro)-caspase 3 were studied using quantitative Western blotting. Activation of caspase 3 was analyzed via a colorimetric assay. RESULTS 10(-6)M TSA inhibited DNA-synthesis by 46% (HepG2) to 64% (MH1C1) after 24h, inducing a G(2)/M-phase arrest and apoptosis. TSA increased activation of caspase 3 and expression of cyclin A, p2l(cip/waf), bax and (pro)-caspase 3, while bcl-2 was downregulated. Human fibroblasts remained unaffected. CONCLUSIONS TSA inhibits hepatoma cell growth in vitro, which are otherwise particularly resistant to chemotherapy. Its anti-proliferative activity is paralleled by a comparable rate of apoptosis. TSA may be a promising agent for treatment of hepatocellular carcinoma in vivo.

Advanced glycation end products induce production of reactive oxygen species via the activation of NADPH oxidase in murine hepatic stellate cells

BACKGROUND & AIMS Advanced glycation end products are known to play an important role in the metabolic syndrome and were recently suggested to contribute to liver fibrosis development. However, little is known about the effect of advanced glycation end products on hepatic stellate cells, the major contributors to liver fibrosis development. We therefore studied the effect of advanced glycation end products on reactive oxygen species generation, a main feature for the activation hepatic stellate cells. METHODS Three different types of advanced glycation end products were generated by BSA incubation with different substrates. The presence of advanced glycation end product receptors was examined by RTq-PCR, immunofluorescence and western blotting. Reactive oxygen species production was measured using DCFH-DA. RESULTS Hepatic stellate cells express five advanced glycation end product receptors: Galectin-3, CD36, SR-AI, SR-BI and RAGE. All receptors, except SR-BI, showed up-regulation during HSC activation. All three advanced glycation end product types induced reactive oxygen species generation. DPI and NSC, a NADPH oxidase and a Rac1 inhibitor respectively, inhibited reactive oxygen species production. Rottlerin, a molecule often used as a PKCdelta inhibitor, also abrogated reactive oxygen species production. SiRNA mediated knockdown of p47(phox), Rac1 and PKCdelta decreased reactive oxygen species production induced by advanced glycation end products, establishing a role for these proteins in reactive oxygen species induction. CONCLUSIONS The demonstration of advanced glycation end product-induced reactive oxygen species generation in hepatic stellate cells unveils a potential new route through which advanced glycation end products induce liver fibrosis in the metabolic syndrome.

Monocyte chemoattractant protein 1 (MCP-1) expression occurs in toxic rat liver injury and human liver disease.

Considerable evidence suggests that monocytes/macrophages play a crucial role in the process of liver injury and repair. Recent investigations have focused on the function of various macrophage‐produced cytokines in liver disease. Much is still unknown, however, about the mechanism of macrophage recruitment and activation during liver disease. To further define this process, the gene expression of the monocyte chemoattractant monocyte chemoattractant protein 1 (MCP‐1) was examined in animal and human liver disease. MCP‐1 mRNA was not found in normal rat liver by Northern blot analysis. After single‐dose treatments with the hepatotoxins carbon tetrachloride and galactosamine, MCP‐1 mRNA was detectable beginning at 2 and 4 h after treatment, respectively, and was expressed continuously until 60–72 h. During chronic carbon tetrachloride administration, MCP‐1 mRNA levels were elevated for the entire 10 weeks of treatment with peak levels of expression occurring early (weeks 1–3) and late (weeks 8–10) in this model. Isolated liver cell fractions from rats treated for 3 weeks with carbon tetrachloride revealed the major cellular source of MCP‐1 mRNA to be fat‐storing or Ito cells, with some expression occurring in the endothelial cell fraction. Studies of potential inducers of hepatic MCP‐1 expression showed that lipopolysaccharide, tumor necrosis factor‐α, and interleukin‐lα and β treatments all led to MCP‐1 expression. Finally, studies of human liver samples revealed MCP‐1 gene expression in nondiseased liver and greatly increased levels in livers from patients with fulminant hepatic failure. These data implicate MCP‐1 from fat‐storing cells as a modulator of the process of liver injury and further support a role for MCP‐1 in the pathogenesis of human disease. J. Leukoc. Biol. 55: 120–126; 1994.

Kupffer cells from CCl4-induced fibrotic livers stimulate proliferation of fat-storing cells.

The interaction between fat-storing cells (FSCs) and Kupffer cells (KCs) in vitro has been studied in an attempt to clarify certain aspects of the pathogenesis of fibrotic process in the liver. FSCs and KCs were isolated from the livers of rats either treated with CCl4 for 6 weeks, or with vitamin A for 6 weeks or from untreated rats by the pronase-collagenase digestion method. FSCs were further purified by centrifugation over a double layered metrizamide gradient, and KCs were separated from other sinusoidal cells by the dish adherence technique. FSCs from CCl4-treated rats divided rapidly, while those from vitamin A-treated rats divided slowly, as compared with untreated rats. Furthermore, the proliferation of FSCs was enhanced in the presence of KCs from CCl4-treated rats, but was slightly suppressed by KCs from normal and vitamin A-treated rats. This enhancement was mediated by a non-dialyzable, soluble factor present in the conditioned medium of KCs from CCl4-treated rats, but was not detected in the conditioned medium of KCs from normal or vitamin A-treated rats. From the present study, a growth factor secreted by KCs from CCl4-treated rats may play an important role in controlling the proliferation of FSCs during the pathogenesis of liver fibrosis.