Response Of Hepatic Stellate Cells Research Articles

MicroRNA-146a modulates TGF-beta1-induced hepatic stellate cell proliferation by targeting SMAD4

Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. MicroRNAs (miRNAs) have recently been shown to regulate cell proliferation, differentiation, and apoptosis. The involvement of miRNAs and their roles in TGF-β1-induced HSC activation remains largely unknown. Our study found that the expression of miR-146a was downregulated in HSC in response to TGF-β1 stimulation in dose-dependent manner by one-step real-time quantitative PCR. Moreover, we sought to examine whether miR-146a became dysregulated in CCl4-induced hepatic fibrosis in rats. Our study revealed that miR-146a was downregulated in liver fibrotic tissues. In addition, The HSC transfected with miR-146a mimics exhibited attendated TGF-β1-induced α-smooth muscle actin (α-SMA) expression compared with the control. Furthermore, overexpression of miR-146a suppressed TGF-β-induced HSC proliferation, and increased HSC apoptosis. Bioinformatics analyses predict that SMAD4 is the potential target of miR-146a. MiR-146a overexpression in TGF-β1-treated HSC did not decrease target mRNA levels, but significantly reduced target protein expression. These results suggested that miR-146a may function as a novel regulator to modulate HSC activation during TGF-β1 induction by targeting SMAD4.

Anti‐fibrotic activity and enhanced interleukin‐6 production by hepatic stellate cells in response to imatinib mesylate

To examine imatinib mesylate's effects on stellate cell responses in vivo and in vitro. The hepatic stellate cell (HSC) is a key target of anti-fibrotic therapies. Imatinib mesylate is a small molecule receptor tyrosine kinase inhibitor indicated for treatment of chronic myelogenous leukaemia and GI stromal tumours. Because imatinib inhibits β-PDGFR signalling, which stimulates HSC proliferation, we assessed its activity in culture and in vivo, and examined downstream targets in a human stellate cell line (LX-2) using cDNA microarray. Imatinib inhibited proliferation of LX-2 cells (0.5-10 mM) but not primary human stellate cells, with no effect on viability, associated with attenuated β-PDGFR phosphorylation. Mitochondrial activity and superoxide anion production were decreased in response to imatinib. cDNA microarray uncovered up-regulation of 29 genes in response to imatinib, including interleukin-6 (IL-6) mRNA, which was correlated with progressive IL-6 secretion. Imatinib also decreased gene expression of collagen α(1) (I), alpha smooth muscle actin, β-PDGFR, transforming growth factor β receptor type 1, matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2. In vivo, imatinib administered to rats beginning 4 weeks after starting thioacetamide (TAA) led to reduced collagen content, with significant reductions in portal pressure and down-regulation of fibrogenic genes in whole liver. Importantly, hepatic IL-6 mRNA levels were significantly increased in TAA-treated animals receiving imatinib. These findings reinforce the anti-fibrotic activity of imatinib and uncover an unexpected link between inhibition of HSC activation by imatinib and enhanced secretion of IL-6, a regenerative cytokine.

Micro-RNA Profiling in Human Serum Reveals Compartment-Specific Roles of miR-571 and miR-652 in Liver Cirrhosis

Background and AimsMicro-RNAs (miRNAs) have recently emerged as crucial modulators of molecular processes involved in chronic liver diseases. The few miRNAs with previously proposed roles in liver cirrhosis were identified in screening approaches on liver parenchyma, mostly in rodent models. Therefore, in the present study we performed a systematic screening approach in order to identify miRNAs with altered levels in the serum of patients with chronic liver disease and liver cirrhosis.MethodsWe performed a systematic, array-based miRNA expression analysis on serum samples from patients with liver cirrhosis. In functional experiments we evaluated the relationship between alterations of miRNA serum levels and their role in distinct cellular compartments involved in hepatic cirrhosis.ResultsThe array analysis and the subsequent confirmation by qPCR in a larger patient cohort identified significant alterations in serum levels of miR-513-3p, miR-571 and miR-652, three previously uncharacterized miRNAs, in patients with alcoholic or hepatitis C induced liver cirrhosis. Of these, miR-571 serum levels closely correlated with disease stages, thus revealing potential as a novel biomarker for hepatic cirrhosis. Further analysis revealed that up-regulation of miR-571 in serum reflected a concordant regulation in cirrhotic liver tissue. In isolated primary human liver cells, miR-571 was up-regulated in human hepatocytes and hepatic stellate cells in response to the pro-fibrogenic cytokine TGF-β. In contrast, alterations in serum levels of miR-652 were stage-independent, reflecting a concordant down-regulation of this miRNA in circulating monocytes of patients with liver cirrhosis, which was inducible by proinflammatory stimuli like bacterial lipopolysaccharide.ConclusionAlterations of miR571 and miR-652 serum levels in patients with chronic liver disease reflect their putative roles in the mediation of fibrogenic and inflammatory processes in distinct cellular compartments involved in the pathogenesis of liver cirrhosis.

N-Acetylglucosaminyltransferase V regulates TGF-β response in hepatic stellate cells and the progression of steatohepatitis

N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in tumor metastasis and carcinogenesis. Although the expression of GnT-V is induced in chronic liver diseases, the biological meaning of GnT-V in the diseases remains unknown. The aim of this study was to investigate the effects of GnT-V on the progression of chronic hepatitis, using GnT-V transgenic (Tg) mice fed a high fat and high cholesterol (HFHC) diet, an experimental model of murine steatohepatitis. Although enhanced hepatic lymphocytes infiltration and fibrosis were observed in wild-type (WT) mice fed the HFHC diet, they were dramatically prevented in Tg mice. In addition, the gene expression of inflammatory Th1 cytokines in the liver was significantly decreased in Tg mice than WT mice. Inhibition of liver fibrosis was due to the dysfunction of hepatic stellate cells (HSCs), which play pivotal roles in liver fibrosis through the production of transforming growth factor (TGF)-β1. Although TGF-β1 signaling was enhanced in Tg mouse-derived HSCs (Tg-HSCs) compared with WT mouse-derived HSCs (WT-HSCs), collagen expression was significantly reduced in Tg-HSCs. As a result from DNA microarray, cyclooxygenase-2 (COX2) expression, known as a negative feedback signal for TGF-β1, was significantly elevated in Tg-HSCs compared with WT-HSCs. Prostaglandin E2 (PGE2), the product of COX2, production was also significantly elevated in Tg-HSCs. COX2 inhibition by celecoxib decreased PGE2 and increased collagen expression in Tg-HSCs. In conclusion, GnT-V prevented steatohepatitis progression through modulating lymphocyte and HSC functions.

MicroRNA are Central Players in Anti- and Profibrotic Gene Regulation during Liver Fibrosis

MicroRNA (miRNA) are small non-coding RNA molecules that posttranscriptionally effect mRNA stability and translation by targeting the 3′-untranslated region (3′-UTR) of various transcripts. Thus, dysregulation of miRNA affects a wide range of cellular processes such as cell proliferation and differentiation involved in organ remodeling processes. Divergent miRNA patterns were observed during chronic liver diseases of various etiologies. Chronic liver diseases result in uncontrolled scar formation ending up in liver fibrosis or even cirrhosis. Since it has been shown that miR-29 dysregulation is involved in synthesis of extracellular matrix proteins, miR-29 is of special interest. The importance of miR-29 in hepatic collagen homeostasis is underlined by in vivo data showing that experimental severe fibrosis is associated with a prominent miR-29 decrease. The loss of miR-29 is due to the response of hepatic stellate cells to exposure to the profibrogenic mediators TGF-β and PDGF-BB. Several putative binding sites for the Smad proteins and the Ap1 complex are located in the miR-29 promoter, which are suggested to mediate miR-29 decrease in fibrosis. Other miRNA are highly increased after profibrogenic stimulation, such as miR-21. miR-21 is transcriptionally upregulated in response to Smad-3 rather than Smad-2 activation after TGF-β stimulation. In addition, TGF-β promotes miR-21 expression by formation of a microprocessor complex containing Smad proteins. Elevated miR-21 may then act as a profibrogenic miRNA by its repression of the TGF-β inhibitory Smad-7 protein.

Nitric Oxide-independent Activation of Soluble Guanylate Cyclase by BAY 60-2770 in Experimental Liver Fibrosis

Liver cirrhosis is a chronic disease with high mortality rate and need for effective pharmacological intervention. The fibrotic remodelling of liver tissue is crucially dependent on hepatic stellate cell activation. Activation of hepatic stellate cells is reduced by an increase in cyclic guanosine monophosphate (cGMP). Stable cGMP analogues also reduce the contractile response of hepatic stellate cells. However, cGMP production is downregulated in the cirrhotic liver due to the reduced activity of the endothelial nitric oxide synthase. Here we report that the novel activator of soluble guanylate cyclase (sGC), BAY 60-2770 (4-({(4-carboxybutyl) [2- (5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl]methoxy}phenyl)ethyl] amino}methyl)benzoic acid), which increases the activity of sGC in a nitric oxide-independent manner, attenuates liver fibrosis in two rat models. The compound was studied in the pig serum model and the carbon tetrachloride model. Fibrosis was assessed by estimating the increase in fibrous collagen by micromorphometry of histological sections stained with Sirius Red/Fast Green and by measuring total hepatic collagen. BAY 60-2770, on a recombinant sGC reporter cell line, stimulated the luminescence signals with an EC50 value of 5.4 +/- 1.2 nmol/L. In the presence of [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 pmol/L) the EC50 was shifted to 0.39 +/- 0.11 nmol/L. In both fibrosis models, once daily oral administration of BAY 60-2770 concomittantly with the fibrotic stimulus prevented 60-75% of fibrosis, the lowest effective dose being 0.1 mg/kg in the pig serum model and 0.3 mg/kg in the carbon tetrachloride model. The treatment was well tolerated by all animals. The doses used were devoid of any significant influence on systemic blood pressure. Nitric oxide-independent activation of sGC might be an innovative therapeutic approach for the treatment of liver fibrosis of necro-inflammatory and immunological origin.

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

BackgroundIn chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC.MethodologyHSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3′-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting.Principal FindingsThe 3′-UTR of the collagen-1 and −4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis.ConclusionsUpregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.

RANTES antagonism: A promising approach to treat chronic liver diseases

Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl(4)) or feeding on a methionine and choline-deficient (MCD) diet. In these models, Ccl5(-/-) mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.

SPARC downregulation attenuates the profibrogenic response of hepatic stellate cells induced by TGF-β1 and PDGF.

Liver fibrosis is an active process that involves changes in cell-cell and cell-extracellular matrix (ECM) interaction. Secreted protein, acidic and rich in cysteine (SPARC) is an ECM protein with many biological functions that is overexpressed in cirrhotic livers and upregulated in activated hepatic stellate cells (aHSCs). We have recently shown that SPARC downregulation ameliorates liver fibrosis in vivo. To uncover the cellular mechanisms involved, we have specifically knocked down SPARC in two aHSC lines [the CFSC-2G (rat) and the LX-2 (human)] and in primary cultured rat aHSCs. Transient downregulation of SPARC in hepatic stellate cells (HSCs) did not affect their proliferation and had only minor effects on apoptosis. However, SPARC knockdown increased HSC adhesion to fibronectin and significantly decreased their migration toward PDFG-BB and TGF-β(1). Interestingly, TGF-β(1) secretion by HSCs was reduced following SPARC small interfering RNA (siRNA) treatment, and preincubation with TGF-β(1) restored the migratory capacity of SPARC siRNA-treated cells through mechanisms partially independent from TGF-β(1)-mediated induction of SPARC expression; thus SPARC knockdown seems to exert its effects on HSCs partially through modulation of TGF-β(1) expression levels. Importantly, collagen-I mRNA expression was reduced in SPARC siRNA-transfected HSCs. Consistent with previous results, SPARC knockdown in aHSCs was associated with altered F-actin expression patterns and deregulation of key ECM and cell adhesion molecules, i.e., downregulation of N-cadherin and upregulation of E-cadherin. Our data together suggest that the upregulation of SPARC previously reported for aHSCs partially mediates profibrogenic activities of TGF-β(1) and PDGF-BB and identify SPARC as a potential therapeutic target for liver fibrosis.

Antifibrotic effects of magnesium lithospermate B on hepatic stellate cells and thioacetamide-induced cirrhotic rats

Magnesium lithospermate B (MLB) is one of the major active components of Salvia miltiorrhizae. The anti-oxidative effects of Salvia miltiorrhizae have been previously reported. The aim of this study was to investigate the effect of purified MLB on hepatic fibrosis in rats and on the fibrogenic responses in hepatic stellate cells (HSCs). Hepatic fibrosis was induced in rats by intraperitoneal thioacetamide (TAA) injections over a period of 8 or 12 weeks. MLB was orally administered daily by gavage tube. Serum AST and ALT levels in TAA+ MLB group were significantly lower than those in TAA only group at week 8. Hepatic fibrosis was significantly attenuated in TAA+MLB group than in TAA only group at week 8 or 12. Activation of HSCs was also decreased in TAA+MLB group as compared to TAA only group. Hepatic mRNA expression of α-smooth muscle actin (α-SMA), TGF-β1, and collagen α1(I) was significantly decreased in TAA+MLB group as compared to TAA only group. Incubation with HSCs and MLB (>or=100 μM) for up to 48 h showed no cytotoxicity. MLB suppressed PDGF-induced HSC proliferation. MLB inhibited NF-ΚB transcriptional activation and monocyte chemotactic protein 1 (MCP-1) production in HSCs. MLB strongly suppressed H(2)O(2)-induced reactive oxygen species (ROS) generation in HSCs, and MLB inhibited type I collagen secretion in HSCs. We concluded that MLB has potent antifibrotic effect in TAA-treated cirrhotic rats, and inhibits fibrogenic responses in HSCs. These data suggest that MLB has potential as a novel therapy for hepatic fibrosis.

The role and mechanism of NADPH oxidase in leptin-induced reactive oxygen species production in hepatic stellate cells

To investigate whether or not NADPH oxidase (NOX) participates in leptin-induced reactive oxygen species (ROS) production in hepatic stellate cells (HSC) and to explore the possible mechanism. HSC-T6 cells (rat hepatic stellate cells line) were divided into nine groups: Group1: leptin (100 ng/ml) treated; Group2-6: leptin treated together with inhibitors that block different ROS-producing systems: diphenylene-iodonium (DPI) (20 micromol/L), Rotenone (20 micromol/L), Metyrapone (250 micromol/L), Allopurinol (100 micromol/L) and Indomethacin(100 micromol/L); Group7: leptin treated together with Janus kinase (JAK) inhibitor AG490 50 micromol/L; Group8: normal control group (treated DMEM with 0.1% DMSO); Group9: negative control group (untreated). Intracellular ROS levels were measured with dichlorodihydrofluorescein diacetate (DCFH-DA) dye assay by Fluorescence microscope and/or flow cytometry. NOX activity was analyzed by using spectrophotometer to calculate the absorbance of NADPH. The mRNA levels of Rac1 and p22Phox were evaluated by RT-PCR. (1) Leptin increased significantly the ROS production as compared to normal control group (92.91+/-4.19 vs.27.56+/-6.27, P<0.01) in HSC-T6 cells. Both the NADPH oxidase inhibitor DPI and AG490 (50 micromol/L) blocked the ROS production, inhibitors of other ROS producing systems had no significant effect on ROS production induced by lepin (P is more than 0.05). (2) Leptin treated HSC-T6 cells for 1 hour up-regulated the NOX activity significantly compared with that in normal control group [(1.90+/-0.22) pmol.min(-1).mg(-1) vs. (0.76+/-0.06) pmol.min(-1).mg(-1), P<0.05]. Furthermore, the NOX activity increased after being treated with leptin for 12 hours and 24 hours than being treated for 1 hour. Leptin-induced up-regulation of NOX activity was inhibited by pretreatment with DPI or AG490. (3) The RT-PCR results indicated that mRNA expressions of Rac1 and p22Phox in HSC-T6 cells with 12 hours of leptin stimulation increased significantly as compared with normal control group (0.41+/-0.13 vs 0.14+/-0.08, 0.45+/-0.12 vs 0.20+/-0.08, all P<0.05), while the DPI and AG490 had no effect on the mRNA expressions of Rac1 and p22Phox. NOX is the main cellular source of the reactive oxygen species (ROS) generated by HSCs in response to leptin stimulation. The mechanism is probably that leptin can directly activate NOX through JAK signal transduction and hence induce the expression of NOX subunit to promote the activity of NOX which generates considerable ROS in HSC.

Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH-related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild-type (WT) mice, Patched-deficient (Ptc(+/-) ) (overly active Hh signaling) mice, and OPN-deficient mice before and after feeding methionine and choline-deficient (MCD) diets to induce NASH-related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc(+/-) mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN-deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up-regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up-regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte-derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). OPN is Hh-regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH.

Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice

Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl4) or feeding on a methionine and choline-deficient (MCD) diet. In these models, Ccl5-/- mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.

Interleukin-27 acts on hepatic stellate cells and induces signal transducer and activator of transcription 1-dependent responses

BackgroundInterleukin (IL)-27 is a cytokine belonging to the IL-6/IL-12 cytokine family that is secreted by activated macrophages and dendritic cells and which strongly acts on T-cells and cells of the innate immune system. Not much is known about possible effects of IL-27 on other cell types. It signals via the common IL-6-type-cytokine receptor chain gp130 and the IL-27-specific chain WSX-1. We previously described that IL-27 also stimulates hepatoma cells and primary hepatocytes. The aim of this study was to investigate whether IL-27 would also act on hepatic stellate cells (HSC), the second most abundant hepatic cell type, which would demonstrate a more general role of this cytokine in the liver.ResultsUsing a human HSC line and primary rat HSC we investigated the signalling characteristics of IL-27 in these cells. We show that IL-27 activates signal transducer and activator of transcription (STAT) 1 and to a minor extent STAT3 in a human HSC cell line and that it leads to the induction of STAT1 target genes such as interferon response factor-1, myxovirus resistance A and STAT1 itself. Similarly we find that IL-27 also elicits STAT1-dependent responses in primary rat HSC.ConclusionsWe provide the first evidence for a function of IL-27 in HSC and show that its responses resemble Interferon-γ-like functions in these cells. Our data suggests that IL-27 may play an important role in the context of liver inflammation by acting on the different liver cell types.

Genetic Labeling Does Not Detect Epithelial-to-Mesenchymal Transition of Cholangiocytes in Liver Fibrosis in Mice

Chronic injury changes the fate of certain cellular populations, inducing epithelial cells to generate fibroblasts by epithelial-to-mesenchymal transition (EMT) and mesenchymal cells to generate epithelial cells by mesenchymal-to-epithelial transition (MET). Although contribution of EMT/MET to embryogenesis, renal fibrosis, and lung fibrosis is well documented, role of EMT/MET in liver fibrosis is unclear. We determined whether cytokeratin-19 positive (K19(+)) cholangiocytes give rise to myofibroblasts (EMT) and/or whether glial fibrillary acidic protein positive (GFAP(+)) hepatic stellate cells (HSCs) can express epithelial markers (MET) in response to experimental liver injury. EMT was studied with Cre-loxP system to map cell fate of K19(+) cholangiocytes in K19(YFP) or fibroblast-specific protein-1 (FSP-1)(YFP) mice, generated by crossing tamoxifen-inducible K19(CreERT) mice or FSP-1(Cre) mice with Rosa26(f/f-YFP) mice. MET of GFAP(+) HSCs was studied in GFAP(GFP) mice. Mice were subjected to bile duct ligation or CCl(4)-liver injury, and livers were analyzed for expression of mesodermal and epithelial markers. On Cre-loxP recombination, >40% of genetically labeled K19(+) cholangiocytes expressed yellow fluorescent protein (YFP). All mice developed liver fibrosis. However, specific immunostaining of K19(YFP) cholangiocytes showed no expression of EMT markers alpha-smooth muscle actin, desmin, or FSP-1. Moreover, cells genetically labeled by FSP-1(YFP) expression did not coexpress cholangiocyte markers K19 or E-cadherin. Genetically labeled GFAP(GFP) HSCs did not express epithelial or liver progenitor markers in response to liver injury. EMT of cholangiocytes identified by genetic labeling does not contribute to hepatic fibrosis in mice. Likewise, GFAP(Cre)-labeled HSCs showed no coexpression of epithelial markers, providing no evidence for MET in HSCs in response to fibrogenic liver injury.

The hepatic vagus nerve stimulates hepatic stellate cell proliferation in rat acute hepatitis via muscarinic receptor type 2

Background & aims: We have previously shown that the hepatic vagus nerve stimulates the activation of hepatic progenitor cells (HPC), via muscarinic acetylcholine receptor type 3. Given the coproliferation of HPC and hepatic stellate cells (HSC) in acute hepatitis, we determined whether HSC proliferation is also modulated by vagal activity. Methods: We induced acute hepatitis in Wistar rats by injection of galactosamine and lipopolysaccharides. Hepatitis was preceded by hepatic branch vagotomy or sham vagotomy, by electrical stimulation or sham stimulation and by muscarinic receptor antagonist atropine, nicotinic receptor antagonist mecamylamine or saline injection. Rats were sacrificed after 12 and 48 h and HSC numbers were quantified on immunohistochemical stainings. Furthermore, we performed reverse transcriptase-polymerase chain reaction with receptor-specific primers on total RNA from isolated HSC and determined the in vitro proliferation of HSC in response to acetylcholine, atropine and mecamylamine. Results: HSC numbers were significantly lower after vagotomy than after sham vagotomy. Conversely, more HSC were seen after electrical stimulation than after sham stimulation. Atropine resulted in less HSC than saline at both time points, while mecamylamine treatment only diminished HSC after 12 h, suggesting a predominant involvement of muscarinic receptors. Moreover, HSC express muscarinic receptor type 2 mRNA and protein, as well as nicotinic receptor α1, α5, β1 and vasoactive intestinal peptide receptor 1 mRNA. Furthermore, acetylcholine enhanced the in vitro proliferation of HSC, which was inhibited by atropine, but not by mecamylamine. Conclusions: We show here that the hepatic vagus nerve stimulates HSC proliferation, most likely through binding of acetylcholine on muscarinic receptor type 2.

Toll-Like Receptor 9 Promotes Steatohepatitis by Induction of Interleukin-1β in Mice

Development of nonalcoholic steatohepatitis (NASH) involves the innate immune system and is mediated by Kupffer cells and hepatic stellate cells (HSCs). Toll-like receptor 9 (TLR9) is a pattern recognition receptor that recognizes bacteria-derived cytosine phosphate guanine (CpG)-containing DNA and activates innate immunity. We investigated the role of TLR9 signaling and the inflammatory cytokine interleukin-1beta (IL-1beta) in steatohepatitis, fibrosis, and insulin resistance. Wild-type (WT), TLR9(-/-), IL-1 receptor (IL-1R)(-/-), and MyD88(-/-) mice were fed a choline-deficient amino acid-defined (CDAA) diet for 22 weeks and then assessed for steatohepatitis, fibrosis, and insulin resistance. Lipid accumulation and cell death were assessed in isolated hepatocytes. Kupffer cells and HSCs were isolated to assess inflammatory and fibrogenic responses, respectively. The CDAA diet induced NASH in WT mice, characterized by steatosis, inflammation, fibrosis, and insulin resistance. TLR9(-/-) mice showed less steatohepatitis and liver fibrosis than WT mice. Among inflammatory cytokines, IL-1beta production was suppressed in TLR9(-/-) mice. Kupffer cells produced IL-1beta in response to CpG oligodeoxynucleotide. IL-1beta but not CpG-oligodeoxynucleotides, increased lipid accumulation in hepatocytes. Lipid accumulation in hepatocytes led to nuclear factor-kappaB inactivation, resulting in cell death in response to IL-1beta. IL-1beta induced fibrogenic responses in HSCs, including secretion of tissue inhibitor of metalloproteinase-1. IL-1R(-/-) mice had reduced steatohepatitis and fibrosis, compared with WT mice. Mice deficient in MyD88, an adaptor molecule for TLR9 and IL-1R signaling, also had reduced steatohepatitis and fibrosis. TLR9(-/-), IL-1R(-/-), and MyD88(-/-) mice had less insulin resistance than WT mice on the CDAA diet. In a mouse model of NASH, TLR9 signaling induces production of IL-1beta by Kupffer cells, leading to steatosis, inflammation, and fibrosis.

Reduced nicotinamide adenine dinucleotide phosphate oxidase mediates fibrotic and inflammatory effects of leptin on hepatic stellate cells

Although leptin induces fibrotic activity in hepatic stellate cells (HSCs), the mechanisms are not entirely understood. To investigate the potential role of reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH) and reactive oxygen species (ROS) in leptin signaling in HSCs, we analyzed leptin-induced intracellular signaling pathways in primary wild-type (WT), p47(phox(-/-) ), and signal transducer and activator of transcription protein 3 (STAT3)-deleted HSCs. Leptin-stimulated ROS production was attenuated in human and mouse HSCs by the NADPH oxidase inhibitor diphenylene-iodonium (DPI) and in HSCs lacking the NADPH component p47(phox). Leptin-induced phosphorylation of extracellular signal-regulated kinase (ERK) and AKT, but not of STAT3, was blocked by NADPH oxidase inhibition. Moreover, leptin-induced ROS production was inhibited by the Janus kinase (JAK) inhibitor, AG490, but normal ROS production was observed in STAT3-deleted HSCs. Pharmacologic or genetic inhibition of NADPH in HSCs not only resulted in a reduction of leptin-mediated HSC proliferation but also reduced the leptin-mediated up-regulation of the fibrogenic markers collagen alpha1(I) and alpha-smooth muscle actin and of the inflammatory mediators monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), and macrophage inflammatory protein 2 (MIP-2). In vivo, leptin enhanced chemokine expression induced by chemokine (C-C motif) ligand 4 (CCl(4)) in WT mice, but a blunted response was observed in p47(phox-/-) mice. In conclusion, NADPH oxidase is a crucial mediator of proliferative, fibrogenic, and inflammatory actions of leptin. Leptin-induced NADPH oxidase acts downstream of JAK activation but is independent of STAT3. Our results, in conjunction with previous studies on angiotensin II and platelet-derived growth factor (PDGF), place NADPH in the center of the fibrogenic signaling response in HSCs and demonstrate its potential role as a pharmacological target for antifibrotic therapies.

Functional linkage of cirrhosis‐predictive single nucleotide polymorphisms of toll‐like receptor 4 to hepatic stellate cell responses†‡

In a recent study, a single nucleotide polymorphism (SNP) of the Toll-like receptor 4 (TLR4) gene (c.1196C>T [rs4986791, p.T399I]) emerged as conferring protection from fibrosis progression compared to a major, wild-type (WT) CC allele (p.T399). The present study examined the functional linkage of this SNP, along with another common, highly cosegregated TLR4 SNP (c.896A>G [rs4986790, p.D299G]), to hepatic stellate cell (HSC) responses. Both HSCs from TLR4(-/-) mice and a human HSC line (LX-2) reconstituted with either TLR4 D299G and/or T399I complementary DNAs were hyporesponsive to lipopolysaccharide (LPS) stimulation compared to those expressing WT TLR4, as assessed by the expression and secretion of LPS-induced inflammatory and chemotactic cytokines (i.e., monocyte chemoattractant protein-1, interleukin-6), down-regulation of bone morphogenic protein and the activin membrane-bound inhibitor expression (an inhibitory transforming growth factor beta pseudoreceptor), and activation of a nuclear factor kappaB (NF-kappaB)-responsive luciferase reporter. In addition, spontaneous apoptosis, as well as apoptosis induced by pathway inhibitors of NF-kappaB, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase were greatly increased in HSCs from either TLR4(-/-) or myeloid differentiation factor 88(-/-) (a TLR adaptor protein) mice, as well as in murine HSCs expressing D299G and/or T399I SNPs; increased apoptosis in these lines was accompanied by decreased phospho-ERK and Bcl-2. TLR4 D299G and T399I SNPs that are associated with protection from hepatic fibrosis reduce TLR4-mediated inflammatory and fibrogenic signaling and lower the apoptotic threshold of activated HSCs. These findings provide a mechanistic link that explains how specific TLR4 SNPs may regulate the risk of fibrosis progression.

Smad7 Attenuate Inducible Collagen Production in Hepatic Stellate Cells involving the regulation of P21 and P27

An inhibitory Smad7 emerges as a potential therapeutic candidate to intervene the hepatic fibrosis. Given that Smad7 functions through antagonizing the transforming growth factor (TGF)‐beta1 signaling, Smad7 over‐expression might enhance the proliferation of Hepatic Stellate Cells (HSCs). However, our recent monitor show that Smad7 surprisingly promoted apoptosis in these cells. Here we report that such inhibitory machinery of Smad7 during the TGFbeta1‐derived process is coupled to the activity of cyclin‐dependent kinase inhibitors p21 and p27. Initially, Primary isolated HSCs were transiently transfected with an adenoviruses encoding Smad7 gene. Real‐time PCR and western blot analysis was confirmed that the overexpression of Smad7 substantially attenuated TGFbeta1 induced collagen I mRNA and protein levels in HSCs. In parallel, flow cytometric monitoring was recorded that Smad7 gene transfer dramatically enhanced but not abolished the apoptosis in these cells subjected to TGFbeta1. Interestingly, Smad7 induced apoptosis in HSCs was found to be both p21 and p27 linkage by using a specific small interfering RNA technique. In the present of TGFbeta1 was found to stimulate an obvious elevation in p21 protein and mildly increase in p27. However, it was found that Smad7 significantly induced p27 whereas caused a substantial decrease in p21 levels in these cells. Furthermore, silencing of p27 by specific siRNA was found to synergy with the inhibitory effect of Smad7 on the inducible collagen production in these cells. Collectively, our finds indicate the important roles of p21 and p27 in the inhibitory Smad7 working pattern in HSCs in response to TGFbeta1 and thereby provide a novel intracellular basis for associated drug development in the future. (Supported by Guangxi NSF Grants 0135035)