Inhibitory effect of human interferon-beta-1a on activated rat and human hepatic stellate cells

Background and Aims: Hepatic stellate cells (HSC) are the primary cell type mediating hepatic fibrosis. Although known for its antiviral effects, the inhibitory effects of interferon‐beta (IFN‐β) on HSC treatment have not yet been established. Methods: Both human and rat activated HSC cell lines were incubated with increasing concentrations of recombinant human IFN‐β1a (rhIFN‐β1a) for 24, 48 or 72 h. The effects of rhIFN‐β1a on α‐smooth muscle actin (α‐SMA), collagen types I and III, transforming growth factor‐β1 (TGF‐β1), platelet‐derived growth factor‐BB (PDGF‐BB), and mothers against decapentaplegic homolog (Smad4, Smad7) expression in HSC were examined using Western blotting and immunocytochemistry. Proliferation of HSC was evaluated via bromodeoxyuridine assay. Results: rhIFN‐β1a treatment had a dose‐dependent, inhibitory effect on α‐SMA and collagen type I protein expression. In addition, rhIFN‐β1a decreased the expression of collagen type III, TGF‐β1, PDGF‐BB and Smad4 protein expression in HSC compared with untreated cells. We also observed increased Smad7 protein expression and decreased proliferation in rhIFN‐β1a‐treated HSC. Conclusions: Our data suggest that rhIFN‐β1a treatment decreased α‐SMA and collagen expression and inhibited the activation of HSC through the inhibition of the TGF‐β and PDGF pathways.     

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.     

Platelet-derived adenosine 5'-triphosphate suppresses activation of human hepatic stellate cell: In vitro study

Aim: Activated hepatic stellate cells (HSC) play a critical role in liver fibrosis. Suppressing abnormal function of HSC or reversion from activated to quiescent form is a hopeful treatment for liver cirrhosis. The interaction between platelets and HSC remains unknown although platelets go through hepatic sinusoids surrounded by HSC. This study aimed at clarifying the hypothesis that platelets control activation of HSC. Methods: We used human platelets, platelet extracts, and primary or immortalized human HSC. We examined the effect of platelets on the activation, DNA synthesis, type I collagen production, and fibrosis-relating gene expressions of HSC. We investigated what suppressed activation of HSC within platelets and examined the mechanism of controlling activation in vitro. Results: Platelets and platelet extracts suppressed activation of HSC. Platelets decreased type I collagen production without affecting DNA synthesis. Platelets increased the expression of matrix metallopeptidase 1. As platelet extracts co-cultured with an enzyme of degrading adenosine 5'-triphosphate (ATP) suppressed activation, we detected adenine nucleotides within platelets or on their surfaces and confirmed the degradation of adenine nucleotides by HSC and the production of adenosine. Adenosine and platelets increased the intracellular cyclic adenosine 5'-monophosphate (cAMP), which is important in quiescent HSC. A great amount of adenosine and ATP also suppressed activation of HSC. Conclusion: Activation of human HSC is suppressed by human platelets or platelet-derived ATP via adenosine-cAMP signaling pathway in vitro. Therefore, platelets have the possibility to be used in the treatment of liver cirrhosis.     

Effects of Ginkgo biloba extract on cell proliferation, cytokines and extracellular matrix of hepatic stellate cells.

BACKGROUND/AIMS: Hepatic fibrosis is the common wound-healing response to chronic liver injury. Ginkgo biloba extract (GbE) has been indicated to reverse hepatic fibrosis and exhibit therapeutic effects both in vitro and in vivo. This study aimed to investigate the underlying mechanism of GbE using HSC-T6 cells, a subline of hepatic stellate cells (HSC) as a model. METHODS: HSC-T6 cells were seeded into six-well plates and allowed to attach overnight. After exposure to different concentrations of GbE761 for 24 or 48 h, cell cycle analysis, semiquantitative RT-PCR, Western blotting analysis and analysis of ECM secretion were performed. RESULTS: It was revealed that GbE (1, 10, 100, 500 mg/l) suppressed HSC proliferation and caused G0/G1 phase arrest in a concentration-dependent manner. RT-PCR and Western blot assays were applied to detect the decline of transforming growth factor beta1(TGF-beta1) and connective tissue growth factor (CTGF) in both mRNA and protein levels after GbE treatment in HSC-T6 cells for 24 or 48 h. Meanwhile, GbE inhibited the synthesis of type I and type III collagens. Secretion of some extracellular matrix (ECM) proteins, such as type III procollagen (PC III), type IV collagen (collagen IV), laminin (LN), hyaluronic acid (HA), were all decreased in supernatant of GbE treated HSC cells. CONCLUSIONS: Our results suggest that GbE confers its anti-fibrosis effects through inhibiting HSC proliferation, reducing TGF-beta1 and CTGF expression and consequently suppressing the collagen production and ECM secretion.     

Melatonin suppresses activation of hepatic stellate cells through RORα‐mediated inhibition of 5‐lipoxygenase

Liver fibrosis is scar tissue resulting from an uncontrolled wound‐healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture‐activated rat HSC. Melatonin dose‐dependently suppressed the expression of HSC activation markers Col1a1 and alpha‐smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor‐related orphan receptor‐alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5‐lipoxygenase (5‐LO). The pharmacological 5‐LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα‐mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.     

Role of hepatic stellate cells on graft injury after small-for-size liver transplantation

Background and Aim: Small‐for‐size grafts are prone to mechanical injury and a series of chemical injuries that are related to hemodynamic force. Hepatic stellate cells activate and trans‐differentiate into contractile myofibroblast‐like cells during liver injury. However, the role of hepatic stellate cells on sinusoidal microcirculation is unknown with small‐for‐size grafts. Methods: Thirty‐five percent of small‐for‐size liver transplantation was performed with rats as donors and recipients. Endothelin‐1 levels as well as hepatic stellate cells activation‐related protein expression, endothelin‐1 receptors, and ultrastructural changes were examined. The cellular localizations of two types of endothelin‐1 receptors were detected. Furthermore, liver function and sinusoidal microcirculation were analyzed using two different selective antagonists of endothelin‐1 receptor. Results: Intragraft expression of hepatic stellate cells activation‐related protein such as desmin, crystallin‐B and smooth muscle α‐actin was upregulated as well as serum endothelin‐1 levels and intragraft expression of the two endothelin receptors. The antagonist to endothelin‐1 A receptor not to the endothelin‐1 B receptor could attenuate microcirculatory disturbance and improve liver function. Conclusions: Small‐for‐size liver transplantation displayed increased hepatic stellate cells activation and high level of endothelin‐1 binding to upregulation of endothelin‐1 A receptor on hepatic stellate cells, which contracted hepatic sinusoid inducing graft injury manifested as reduction of sinusoidal perfusion rate and elevation of sinusoidal blood flow.     

Inhibitory effect of oestradiol on activation of rat hepatic stellate cells in vivo and in vitro

Background—Hepatic stellate cellsplay a key role in the pathogenesis of hepatic fibrosis.
Aims—To examine the inhibitoryeffect of oestradiol on stellate cell activation.
Methods—In vivo, hepatic fibrosiswas induced in rats by dimethylnitrosamine or pig serum. In vitro, ratstellate cells were activated by contact with plastic dishes resultingin their transformation into myofibroblast-like cells.
Results—In the dimethylnitrosamineand pig serum models, treatment with oestradiol at gestation relateddoses resulted in a dose dependent suppression of hepatic fibrosis withrestored content of hepatic retinyl palmitate, reduced collagencontent, lower areas of stellate cells which express α smooth muscleactin (α-SMA) and desmin, and lower procollagen type I and III mRNA levels in the liver. In cultured stellate cells, oestradiol inhibited type I collagen production, α-SMA expression, and cell proliferation. These findings suggest that oestradiol is a potent inhibitor of stellate cell transformation.
Conclusion—The antifibrogenic roleof oestradiol in the liver may contribute to the sex associateddifferences in the progression from hepatic fibrosis to cirrhosis.

Keywords:hepatic stellate cells; hepatic fibrosis; oestradiol; α smooth muscle actin; retinyl palmitate

     

High glucose stimulates hepatic stellate cells to proliferate and to produce collagen through free radical production and activation of mitogen-activated protein kinase.

BACKGROUND: Nonalcoholic steatohepatitis is a clinicopathologic condition that may progress to liver fibrosis. Hyperglycemia is supposed to be one of the factors inducing hepatic fibrogenesis, but the mechanism has not been fully clarified. Oxidative stress is increasingly found in patients with diabetes/hyperglycemia in which conditions reactive oxygen species (ROS) are produced. METHODS: We performed experiments using hepatic stellate cells (HSCs) in culture in order to confirm the effect of high glucose concentrations on cell proliferation, type I collagen production, ROS production and activation of mitogen-activated protein (MAP) kinase pathway. RESULTS: High glucose stimulated cell growth of HSCs and up-regulated the levels of activated/phosphorylated extracellular signal-regulated kinase 1/2 and free radical production in HSCs. The MAP kinase phosphorylation and cell proliferation were suppressed by diphenylene iodonium chloride, an NADPH oxidase inhibitor, and by calphostin C, a protein kinase C (PKC)-specific inhibitor. Increased type I collagen mRNA and protein levels were also observed in HSCs at high glucose concentrations. CONCLUSIONS: Our findings indicate that high glucose concentrations may stimulate ROS production through PKC-dependent activation of NADPH oxidase, and induce MAP kinase phosphorylation subsequent to proliferation and type I collagen production by HSCs.     

Effect of IL-4 and IL-13 on collagen production in cultured LI90 human hepatic stellate cells.

BACKGROUND: Recently, it has been reported that interleukin 4 (IL-4) and 13 (IL-13) directly activate fibroblasts and promote fibrosis. In the process of hepatic fibrosis, the effects of these cytokines on hepatic stellate cells (HSCs) are not well known. METHODS: We evaluated the effects of IL-4 and IL-13 on the collagen production and the proliferation of LI90, a hepatic stellate cell line. We also examined whether interferon (IFN) interferes with the expression of collagen, since IFN has been reported to clinically suppress hepatic fibrosis. RESULTS: The receptor complex for IL-4 and IL-13 was IL-4Ralpha/IL-13Ralpha1 on LI90 cells, and the phosphorylation of Stat6 was induced by IL-4 and IL-13. The treatment of LI90 cells with IL-4 or IL-13 increased the production of collagen I protein levels by nearly three times in comparison with untreated cells. Collagen mRNA levels were increased roughly 10-fold by IL-4 and 100-fold by IL-13. Interestingly, BrdU incorporation in LI90 cells was decreased by IL-4 or IL-13 treatment. Furthermore, induction of collagen I production by these cytokines was blocked by IFNalpha or IFNbeta treatment, although neither treatment alone suppressed collagen production. CONCLUSIONS: Our data suggested that IL-4 and IL-13 directly affected HSCs by increasing collagen production and suppressing cell proliferation. The anti-fibrogenetic effect of IFN may be due in part to the blockade of IL-4 and IL-13 stimulation of HSCs.     

Activated hepatic stellate cells participate in liver fibrosis in a patient with transfusional iron overload

We describe liver fibrosis caused by iron overload after a long history of blood transfusion in a patient with chronic renal failure. Pertinent laboratory data were: serum (s)-Fe 148 μg/dl; unsaturated iron binding capacity (UIBC) 14 μg/dl; s-ferritin 9350 ng/ml; human leukocyte antigen (HLA) A2, A24, B39, B55, Cw1, Cw7. Computed tomography revealed a high density in the liver, and laparoscopy revealed a brown liver. Liver histology showed bridging fibrosis from portal tracts. A heavy iron deposit was seen in Kupffer cells as well as in hepatocytes surrounded by fibrosis around the portal tracts. Immunocytochemistry revealed α-smooth muscle actin in many stellate cells distributed along the fibrotic area, and electron microscopy revealed infiltrating myofibroblastic stellate cells coexisting with collagen fibers around degenerated hepatocytes containing iron deposits. The findings are consistent with the notion that stellate cells play an important role in liver fibrogenesis in both genetic and transfusional iron overload hemochromatosis. Received Aug. 15, 1997; accepted Feb. 27, 1998     

Characteristics of the cell proliferation profile of activated rat hepatic stellate cells in vitro in contrast to their fibrogenesis activity

Purpose. Alterations in the kinetics of hepatic stellate cells (HSCs) after the cells are activated once have not been well documented. We investigated the characteristic profiles of cell proliferation of once-activated HSCs in contrast to the in fibrogenesis activity. Methods. HSCs from male Wistar rats were submitted to primary culture for 14 days and to secondary culture for 7 days. The potential for cell proliferation was evaluated by the number of the cells in G2/M phase, based on flow cytometric analysis of the cell cycle. The fibrogenesis activity was assessed by Northern blot analysis of the expression of type I and type III procollagen mRNA. Results. The number of HSCs in G2/M phase was maintained at a low level in primary culture after 6 days, while a significantly (P < 0.05) elevated number of HSCs in G2/M phase was observed on days 3 to 4. In secondary culture, the number of HSCs in G2/M phase was also consecutively maintained at a decreased level. By contrast, HSCs showed progressively increased type I and type III procollagen mRNA expression during the experimental periods of primary culture. Conclusions. These results clearly demonstrated consecutively decreased proliferative activity, evaluated by the potential for cell mitogenesis, in once-activated HSCs, in contrast to their progressively increased fibrogenesis activity. Received: April 12, 2000 / Accepted: December 22, 2000     

Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model,and suppresses fibrogenic response induced by transforming growth factor‐β1 in a cultured stellate cell line

Aim: The aim of this study was to investigate the preventive actions of bezafibrate against non‐alcoholic steatohepatitis (NASH), the activation of hepatic stellate cells (HSC), and fibrogenesis by using a model of NASH and an in vitro model. Methods: Male KK‐A^y/TaJcl (KK‐A^y) mice were fed a methionine and choline‐deficient (MCD) diet or a MCD diet containing bezafibrate or pioglitazone for 7 weeks, after which biochemical parameters, pathological changes, and hepatic mRNA levels were assessed. An in vitro HSC model was designed by using a previously described RI‐T cell line stimulated by transforming growth factor‐β1 (TGF‐β1). Results: MCD diet‐fed KK‐A^y mice developed hepatic steatosis, oxidative stress, inflammation, and hepatic fibrosis. Bezafibrate markedly decreased the hepatic content of triglyceride accumulation of fatty droplets within hepatocytes, and increased the expression of hepatic fatty acid β‐oxidative genes in MCD diet‐fed KK‐A^y mice. Bezafibrate markedly inhibited the increases in the plasma alanine aminotransferase level and hepatic content of thiobarbituric acid‐reactive substances in this model. Moreover, it dramatically reduced hepatic inflammatory changes and fibrosis concomitantly with marked reductions in the mRNA levels for inflammatory cytokine, chemokine, and profibrogenic genes. Importantly, both bezafibrate and pioglitazone markedly reduced the mRNA levels of profibrogenic and fibrogenic genes in TGF‐β1‐stimulated cells. Conclusion: Bezafibrate improved hepatic steatosis and potently prevented inflammation, oxidative stress, HSC activation, and fibrogenesis in the liver. Moreover, this study was the first to demonstrate that bezafibrate directly inhibits hepatic fibrogenic response induced by TGF‐β1 in vitro. Hence bezafibrate may be a new therapeutic strategy against NASH and hepatic fibrosis.