N-terminal region of gelsolin induces apoptosis of activated hepatic stellate cells by a caspase-dependent mechanism

Activated hepatic stellate cells (HSCs) are the major source for alteration of extracellular matrix in fibrosis and cirrhosis. Conditioned medium (CM) collected from immortalized human hepatocytes (IHH) have earlier been shown to be responsible for apoptosis of HSCs. In this study, we have shown that antibodies raised against a peptide derived from a linear B-cell epitope in the N-terminal region of gelsolin identified a gelsolin fragment in IHH CM. Analysis of activated stellate cell death by CM collected from Huh7 cells transfected with plasmids encoding gelsolin deletion mutants suggested that the N-terminal half of gelsolin contained sequences which were responsible for stellate cell death. Further analysis determined that this activity was restricted to a region encompassing amino acids 1-70 in the gelsolin sequence; antibody directed to an epitope within this region was able to neutralize stellate cell death. Gelsolin modulation of cell death using this fragment involved upregulation of TRAIL-R1 and TRAIL-R2, and involved caspase 3 activation by extrinsic pathway. The apoptotic activity of N-terminal gelsolin fragments was restricted to activated but not quiescent stellate cells indicating its potential application in therapeutic use as an anti-fibrotic agent. Gelsolin fragments encompassing N-terminal regions in polypeptides of different molecular sizes were detected by N-terminal peptide specific antiserum in IHH CM immunoprecipitated with chronically HCV infected patient sera, suggesting the presence of autoantibodies generated against N-terminal gelsolin fragments in patients with chronic liver disease.     

Schistosoma japonicum soluble egg antigens induce apoptosis and inhibit activation of hepatic stellate cells: a possible molecular mechanism

Hepatic stellate cells play a key role in the development of hepatic fibrosis. Activated hepatic stellate cells can be reversed to a quiescent-like state or apoptosis can be induced to reverse fibrosis. Some studies have recently shown that Schistosoma mansoni eggs could suppress the activation of hepatic stellate cells and that soluble egg antigens from schistosome eggs could promote immunocyte apoptosis. Hence, in this study, we attempt to assess the direct effects of Schistosoma japonicum soluble egg antigens on hepatic stellate cell apoptosis, and to explore the mechanism by which the apoptosis of activated hepatic stellate cells can be induced by soluble egg antigens, as well as the mechanism by which hepatic stellate cell activation is inhibited by soluble egg antigens. Here, it was shown that S. japonicum-infected mouse livers had increased apoptosis phenomena and a variability of peroxisome proliferator-activated receptor γ expression. Soluble egg antigens induce morphological changes in the hepatic stellate cell LX-2 cell line, inhibit cell proliferation and induce cell-cycle arrest at the G₁ phase. Soluble egg antigens also induce apoptosis in hepatic stellate cells through the TNF-related apoptosis-inducing ligand/death receptor 5 and caspase-dependent pathways. Additionally, soluble egg antigens could inhibit the activation of hepatic stellate cells through peroxisome proliferator-activated receptor γ and the transforming growth factor β signalling pathways. Therefore, our study provides new insights into the anti-fibrotic effects of S. japonicum soluble egg antigens on hepatic stellate cell apoptosis and the underlying mechanism by which the liver fibrosis could be attenuated by soluble egg antigens.     

Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months. Albumin- and fibrinogen-secreting cells were selected and cloned by limiting dilution to obtain homologous cell populations. The established IHH (immortalized human hepatocyte) cell lines were evaluated for their usefulness in studying the regulation of cell growth and of certain differentiated hepatocyte functions.IHH cells retain several differentiated features of normal hepatocytes. They display albumin secretion at a level comparable to cultured primary human hepatocytes (30 µg albumin/ml per day). A portion of the IHH cells are polarized, forming bile canaliculi-like vacuoles where exogeneous organic anions accumulate. The multidrug resistance (MDR) P-glycoprotein, known to be localized at the canalicular membrane, is also present in these vacuoles. The polarized features allowed the use of IHH cells for the study of localization of the newly characterized multidrug resistance protein MRP1. The homologues of MRP were found in hepatocytes, MRP1 and MRP2 (cMOAT), both functioning in ATP-dependent excretion of anionic conjugates. In differentiated hepatocytes, MRP1 expression is extremely low. In contrast, MRP1 is highly expressed in proliferating IHH cells, where it is localized in lateral membranes. A highly differentiated feature of short-term cultured primary hepatocytes which is not detectable in IHH cells is active uptake of the bile salt taurocholate. Furthermore, IHH cells secrete triglyceride (TG)-rich lipoproteins, apolipoprotein B (0.6 µg/ml per day), and apolipoprotein A-I (1 µg/ml per day). However, they secrete apoB-containing TG-rich lipoproteins mainly in the LDL density range, while short-term cultured primary hepatocytes mainly secrete TG-rich lipoproteins in the VLDL density range.In conclusion, functions that are rapidly lost in short-term hepatocyte cultures are, in general, not displayed by IHH cells. Immortalized human hepatocytes provide a valuable tool for studying the regulation of hepatocyte proliferation-related phenomena.     

Hepatitis C virus genotype 1a growth and induction of autophagy

We have previously reported that immortalized human hepatocytes (IHH) support the generation of infectious hepatitis C virus (HCV) genotype 1a (clone H77). In the present study, we have investigated the growth of HCV genotype 1a (clone H77) through serial passages and accompanying changes in IHH in response to infection. Eleven serial passages of HCV genotype 1a (clone H77) in IHH were completed. Virus replication was ascertained from the presence of HCV-specific sequences, the detection of core antigen, the virus genome copy number, and the virus titer in IHH culture fluid. Electron microscopy suggested that HCV infection induces autophagic vacuole formation in IHH. Fluorescence microscopy displayed localization of autophagic markers, microtubule-associated protein-1 light chain-3 and Apg5, on the vacuoles of HCV-infected hepatocytes. Taken together, our results suggested that HCV genotype 1a (clone H77) can be serially passaged in IHH and that HCV infection induces an autophagic response in hepatocytes.     

Hepatitis C virus infection induces the beta interferon signaling pathway in immortalized human hepatocytes

Beta interferon (IFN-beta) expression is triggered by double-stranded RNA, a common intermediate in the replication of many viruses including hepatitis C virus (HCV). The recent development of cell culture-grown HCV allowed us to analyze the IFN signaling pathway following virus infection. In this study, we have examined the IFN-beta signaling pathway following infection of immortalized human hepatocytes (IHH) with HCV genotype 1a (clone H77) or 2a (clone JFH1). We observed that IHH possesses a functional Toll-like receptor 3 pathway. HCV infection in IHH enhanced IFN-beta and IFN-stimulated gene 56 (ISG56) promoter activities; however, poly(I-C)-induced IFN-beta and ISG56 expression levels were modestly inhibited upon HCV infection. IHH infected with HCV (genotype 1a or 2a) exhibited various levels of translocation of IRF-3 into the nucleus. The upregulation of endogenous IFN-beta and 2',5'-oligoadenylate synthetase 1 mRNA expression was also observed in HCV-infected IHH. Subsequent studies suggested that HCV infection in IHH enhanced STAT1 and ISG56 protein expression. A functional antiviral response of HCV-infected IHH was observed by the growth-inhibitory role in vesicular stomatitis virus. Together, our results suggested that HCV infection in IHH induces the IFN signaling pathway, which corroborates observations from natural HCV infection in humans.     

Inhibitory effect of soluble PDGF-beta receptor in culture-activated hepatic stellate cells

Following liver injury, hepatic stellate cells undergo phenotypic transformation with acquisition of myofibroblast-like features, characterized by increased cell proliferation, motility, contractility, and extracellular matrix production. Activation of hepatic stellate cells is regulated by several cytokines and growth factors, including platelet-derived growth factor B-chain, a potent mitogen for HSC, overexpressed during hepatic fibrogenesis. This pleiotropic mediator exerts cellular effects by binding to specific receptors, inducing receptor dimerization and tyrosine-autophosphorylation. Activated receptor phosphotyrosines recruit signal transduction molecules, initiating various signaling pathways. We produced a soluble PDGFbeta-receptor (sPDGFRbeta) consisting of an extracellular domain connected to the IgG-Fc part of human immunoglobulin heavy chain. This soluble, chimeric receptor inhibits PDGF signaling and PDGF-induced proliferation in culture-activated hepatic stellate cells. Furthermore, sPDGFR decreased collagen type I (alphaI) mRNA expression and inhibits autocrine-looping in PDGF-BB mRNA production. In summary, sPDGFRbeta clearly shows effective inhibitory properties in early HSC activation, suggesting potential therapeutic impact for anti-PDGF intervention in liver fibrogenesis.     

Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells

BackgroundIntravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs.MethodsHuman AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects.ResultsHSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen–G1, a hAEC-derived factor, significantly decreased TGF-β1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL.ConclusionsHuman AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.     

Cadmium induces mitochondria-dependent apoptosis of normal human hepatocytes

The heavy metal cadmium, an environmental pollutant, has been widely demonstrated to be toxic, in particular for liver. In murines, cadmium induces apoptosis of hepatocytes and hepatomas. In human cells, apoptosis induced by cadmium has been exclusively demonstrated in tumoral cell lines. Nothing was known in normal liver, in vitro or in vivo. In the present study, we examined the effects of cadmium in nonmalignant human hepatocytes. For that purpose, we investigated whether cadmium was able to induce apoptosis of normal human hepatocytes (NHH) in primary culture and of a SV40-immortalized human hepatocyte (IHH) cell line. Treatment of IHH and NHH with cadmium induced the presence of a sub-G₁ population at 10 and 100 μmol/L, respectively. DAPI staining of both cell types treated with cadmium 100 μmol/L revealed the induction of nuclear apoptotic bodies, supporting the hypothesis of apoptosis. In IHH and NHH, cadmium 100 μmol/L induced PARP cleavage into a 85 kDa fragment. In order to investigate the involvement of mitochondria in cadmium-induced apoptosis, we measured the mitochondrial membrane potential (Δ_Ψm). We observed that in IHH and NHH, cadmium 100 μmol/L induced a decrease of Δ_Ψm. As expected, cadmium under the same conditions enhanced caspase-9 and caspase-3 activities. In addition, cadmium from 1 to 100 μmol/L induced the expression of p53 and phosphorylation of its Ser15 in IHH and NHH. In conclusion, we showed in this study that human hepatocytes were sensitive to cadmium and apoptosis induced at concentrations suggested in the literature to inhibit p53 DNA-binding and DNA repair.     

Extracellular vesicles derived from fat-laden hepatocytes undergoing chemical hypoxia promote a pro-fibrotic phenotype in hepatic stellate cells

BackgroundThe transition from steatosis to non-alcoholic steatohepatitis (NASH) is a key issue in non-alcoholic fatty liver disease (NAFLD). Observations in patients with obstructive sleep apnea syndrome (OSAS) suggest that hypoxia contributes to progression to NASH and liver fibrosis, and the release of extracellular vesicles (EVs) by injured hepatocytes has been implicated in NAFLD progression.AimTo evaluate the effects of hypoxia on hepatic pro-fibrotic response and EV release in experimental NAFLD and to assess cellular crosstalk between hepatocytes and human hepatic stellate cells (LX-2).MethodsHepG2 cells were treated with fatty acids and subjected to chemically induced hypoxia using the hypoxia-inducible factor 1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Lipid droplets, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic-associated genes were assessed. EVs were isolated by ultracentrifugation. LX-2 cells were treated with EVs from hepatocytes. The CDAA-fed mouse model was used to assess the effects of intermittent hypoxia (IH) in experimental NASH.ResultsChemical hypoxia increased steatosis, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic gene expressions in fat-laden HepG2 cells. Chemical hypoxia also increased the release of EVs from HepG2 cells. Treatment of LX2 cells with EVs from fat-laden HepG2 cells undergoing chemical hypoxia increased expression pro-fibrotic markers. CDAA-fed animals exposed to IH exhibited increased portal inflammation and fibrosis that correlated with an increase in circulating EVs.ConclusionChemical hypoxia promotes hepatocellular damage and pro-inflammatory and pro-fibrotic signaling in steatotic hepatocytes both in vitro and in vivo. EVs from fat-laden hepatocytes undergoing chemical hypoxia evoke pro-fibrotic responses in LX-2 cells.     

Anti-fibrotic effect of thymoquinone on hepatic stellate cells

Hepatic stellate cells (HSCs) are the major cell type involved in the production of extracellular matrix in liver. After liver injury, HSCs undergo transdifferentiation process from quiescent state to activated state, which plays an important role in liver fibrosis. Previous studies have shown that thymoquinone (TQ) might have protective effect against liver fibrosis in animal models; however, the underlying mechanism of action is not fully understood. The aim of this study is to examine whether TQ has any direct effect on HSCs. Our results showed that pretreatment of mice with TQ has protective effect against CCl₄-induced liver injury compared to control group (untreated), which is consistent with previous studies. Moreover, our in vivo study showed that COL1A1 and α-SMA mRNA levels were significantly downregulated by TQ treatment. Similarly, in vitro study confirmed that TQ downregulated COL1A1, COL3A1 and α-SMA mRNA levels in activated rat HSCs and LX2 cells, an immortalized human hepatic stellate cell line. Pretreatment with TQ also inhibited the LPS-induced proinflammatory response in LX2 cells as demonstrated by reduced mRNA expression of IL-6 and MCP-1. Mechanistically, inactivation of NF-κB pathway is likely to play a role in the TQ-mediated inhibition of proinflammatory response in HSCs. Finally, we have shown that TQ inhibited the culture-triggered transdifferentiation of freshly isolated rat HSCs as shown by significant downregulation of mRNA expression of several fibrosis-related genes. In conclusion, our study suggests that TQ has a direct effect on HSCs, which may contribute to its overall antifibrotic effect.     

Tripartite motif protein 52 (TRIM52) promoted fibrosis in LX‐2 cells through PPM1A‐mediated Smad2/3 pathway

To investigate the roles of tripartite motif containing 52 (TRIM52) in human hepatic fibrosis in vitro, human hepatic stellate cell line LX‐2 cells were transfected with hepatitis B virus (HBV) replicon to establish HBV‐induced fibrosis in LX‐2 cells, and then treated with small interfering RNA‐mediated knockdown of TRIM52 (siTRIM52). LX‐2 cells without HBV replicon transfection were treated with lentiviruses‐mediated overexpression of TRIM52 and phosphatase magnesium dependent 1A (PPM1A). Fibrosis response of LX‐2 cells were assessed by the production of hydroxyproline (Hyp) and collagen I/III, as well as protein levels of α‐smooth muscle actin (α‐SMA). PPM1A and phosphorylated (p)‐Smad2/3 were measured to assess the mechanism. The correlation between TRIM52 and PPM1A was determined using co‐immunoprecipitation, and whether and how TRIM52 regulated the degradation of PPM1A were determined by ubiquitination assay. Our data confirmed HBV‐induced fibrogenesis of LX‐2 cells, as evidenced by significant increase in Hyp and collagen I/III and α‐SMA, which was associated with reduction of PPM1A and elevation of transforming growth factor‐β (TGF‐β), p‐Smad2/3, and p‐Smad3L. However, those changes induced by HBV were significantly attenuated with additional siTRIM52 treatment. Similar to HBV, overexpression of TRIM52 exerted promoted effect in the fibrosis of LX‐2 cells. Interestingly, TRIM52 induced the fibrogenesis of LX‐2 cells and the activation of TGF‐β/Smad pathway were significantly reversed by PPM1A overexpression. Furthermore, our data confirmed TRIM52 as a deubiquitinase that influenced the accumulation of PPM1A protein, and subsequently regulated the fibrogenesis of LX‐2 cells. TRIM52 was a fibrosis promoter in hepatic fibrosis in vitro, likely through PPM1A‐mediated TGF‐β/Smad pathway.     

HSCs play a distinct role in different phases of oval cell-mediated liver regeneration

Hepatic stem cell niche plays an important role in hepatic oval cell-mediated liver regeneration. As a component of hepatic stem cell niche, the role of hepatic stellate cells (HSCs) in oval cell proliferation needs further studies. In the present study, we isolated HSCs from rats at indicated time point after partial hepatectomy (PH) in 2-acetylaminofluorene/PH oval cell proliferation model. Conditional medium (CM) from HSCs were collected to detect their effects on proliferation and the mitogen-activated protein kinase pathway activation of two oval cell lines. We found that CM collected from HSCs at early phase of liver regeneration (4 and 9?days group) contained high levels of hepatocyte growth factor (HGF) and stimulated oval cell proliferation via extracellular signal-regulated kinase and p38 pathway. CM collected from HSCs at terminal phase of liver regeneration (12 and 15?days group) contained high levels of transforming growth factor (TGF)-β1, which suppressed DNA synthesis of oval cells. The shift between these two distinct effects depended on the balance between HGF and TGF-β1 secreted by HSCs. Our study demonstrated that HSCs acted as a positive regulator at the early phase and a negative regulator at the terminal phase of the oval cell-mediated liver regeneration. Copyright ? 2012 John Wiley & Sons, Ltd.     

TWEAK promotes hepatic stellate cell migration through activating EGFR/Src and PI3K/AKT pathways

Activated human hepatic stellate cells (HSCs) showed enhanced ability of migration compared with quiescent HSCs, which is pivotal in liver fibrogenesis. The aim of the present study was to investigate the effects of tumor necrosis factor‐like weak inducer of apoptosis (TWEAK) on the migration of activated HSCs and to explore the relevant potential mechanisms. Human HSCs LX‐2 cells were cultured with TWEAK. TNFRSF12A‐downexpressing lentiviruses were used to infect LX‐2 cells. The specific matrix metalloproteinases inhibitor BB94, the Src family kinase inhibitor, Dasatinib, and the specific inhibitor of phosphoinositide 3‐kinase (PI3K), LY294002 were used to treat LX‐2 cells combined with TWEAK. Cell migration and invasion was tested by the transwell assay. The expression of EGFR/Src, PI3K/AKT, and matrix metallopeptidase 9 (MMP9) was identified by real‐time polymerase chain reaction or western blotting. The result showed TWEAK promoted HSC migration and collagen production. BB94 significantly attenuated the migration of LX‐2 induced by TWEAK. Dasatinib inhibited the ability of cell migration stimulated by TWEAK. TWEAK upregulated the phosphorylation of epidermal growth factor receptor (EGFR) and Src. The phosphorylation of PI3K and AKT was significantly activated by TWEAK stimulation. Inhibition of PI3K/AKT reduced the expression of MMP9 induced by TWEAK. The present study, for the first time, demonstrated that TWEAK promoted HSC migration through the activation of EGFR/Src and PI3K/AKT pathways, and showed a novel potential mechanism of HSC migration regulated by TWEAK.     

HSCs play a distinct role in different phases of oval cell‐mediated liver regeneration

Hepatic stem cell niche plays an important role in hepatic oval cell‐mediated liver regeneration. As a component of hepatic stem cell niche, the role of hepatic stellate cells (HSCs) in oval cell proliferation needs further studies. In the present study, we isolated HSCs from rats at indicated time point after partial hepatectomy (PH) in 2‐acetylaminofluorene/PH oval cell proliferation model. Conditional medium (CM) from HSCs were collected to detect their effects on proliferation and the mitogen‐activated protein kinase pathway activation of two oval cell lines. We found that CM collected from HSCs at early phase of liver regeneration (4 and 9 days group) contained high levels of hepatocyte growth factor (HGF) and stimulated oval cell proliferation via extracellular signal‐regulated kinase and p38 pathway. CM collected from HSCs at terminal phase of liver regeneration (12 and 15 days group) contained high levels of transforming growth factor (TGF)‐β1, which suppressed DNA synthesis of oval cells. The shift between these two distinct effects depended on the balance between HGF and TGF‐β1 secreted by HSCs. Our study demonstrated that HSCs acted as a positive regulator at the early phase and a negative regulator at the terminal phase of the oval cell‐mediated liver regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

3,4-Methylenedioxymethamphetamine ("Ecstasy") induces apoptosis of cultured rat liver cells

"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) has been shown to be hepatotoxic for human users, but molecular mechanisms involved in this effect remained poorly understood. MDMA-induced cell damage is related to programmed cell death in serotonergic and dopaminergic neurons. However, until now there has been no evidence of apoptosis induced by MDMA in liver cells. Here we demonstrate that exposure to MDMA caused apoptosis of freshly isolated rat hepatocytes and of a cell line of hepatic stellate cells (HSC), as shown by chromatin condensation of the nuclei and accumulation of oligonucleosomal fragments in the cytoplasm. In both cell types, apoptosis correlated with decreased levels of bcl-x(L), release of cytochrome c from the mitochondria and activation of caspase 3. In HSC, but not in hepatocytes, MDMA induced poly(ADP-ribose)polymerase (PARP) proteolysis. These results suggest that apoptosis of liver cells could be involved in the hepatotoxicity of MDMA.     

Sphingosine 1-phosphate regulates regeneration and fibrosis after liver injury via sphingosine 1-phosphate receptor 2

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, stimulates proliferation and contractility in hepatic stellate cells, the principal matrix-producing cells in the liver, and inhibits proliferation via S1P receptor 2 (S1P(2)) in hepatocytes in rats in vitro. A potential role of S1P and S1P(2) in liver regeneration and fibrosis was examined in S1P(2)-deficient mice. Nuclear 5-bromo-2'-deoxy-uridine labeling, proliferating cell nuclear antigen (PCNA) staining in hepatocytes, and the ratio of liver weight to body weight were enhanced at 48 h in S1P(2)-deficient mice after a single carbon tetrachloride (CCl(4)) injection. After dimethylnitrosamine (DMN) administration with a lethal dose, PCNA staining in hepatocytes was enhanced at 48 h and survival rate was higher in S1P(2)-deficient mice. Serum aminotransferase level was unaltered in those mice compared with wild-type mice in both CCl(4)- and DMN-induced liver injury, suggesting that S1P(2) inactivation accelerated regeneration not as a response to enhanced liver damage. After chronic CCl(4) administration, fibrosis was less apparent, with reduced expression of smooth-muscle alpha-actin-positive cells in the livers of S1P(2)-deficient mice, suggesting that S1P(2) inactivation ameliorated CCl(4)-induced fibrosis due to the decreased accumulation of hepatic stellate cells. Thus, S1P plays a significant role in regeneration and fibrosis after liver injury via S1P(2).