Properties Of Hepatic Stellate Cells Research Articles

Agalactosyl IgG induces liver fibrogenesis via Fc gamma receptor 3a on human hepatic stellate cells.

The relevance of aberrant serum IgG N-glycosylation in liver fibrosis has been identified; however, its causal effect remains unclear. Because hepatic stellate cells (HSCs) contribute substantially to liver fibrosis, we investigated whether and through which mechanisms IgG N-glycosylation affects the fibrogenic properties of HSCs. Analysis of serum IgG1 N-glycome from 151 patients with chronic hepatitis B or liver cirrhosis revealed a positive correlation between Ishak fibrosis grading and IgG1 with agalactosyl N-glycoforms on the crystallizable fragment (Fc). Fc gamma receptor (FcγR) IIIa was observed in cultured human HSCs and HSCs in human liver tissues, and levels of FcγRIIIa in HSCs correlated with the severity of liver fibrosis. Additionally, agalactosyl IgG treatment caused HSCs to have a fibroblast-like morphology, enhanced migration and invasion capabilities, and enhanced expression of the FcγRIIIa downstream tyrosine-protein kinase SYK. Furthermore, agalactosyl IgG treatment increased fibrogenic factors in HSCs, including transforming growth factor (TGF)-β1, total collagen, platelet-derived growth factor subunit B and its receptors, pro-collagen I-α1, α-smooth muscle actin, and matrix metalloproteinase 9. These effects were more pronounced in HSCs that stably expressed FCGR3A and were reduced in FCGR3A knockout cells. Agalactosyl IgG and TGF-β1 each increased FCGR3A in HSCs. Furthermore, serum TGF-β1 concentrations in patients were positively correlated with agalactosyl IgG1 levels and liver fibrosis severity, indicating a positive feedback loop involving agalactosyl IgG, HSC-FcγRIIIa, and TGF-β1. In conclusion, agalactosyl IgG promotes fibrogenic characteristics in HSCs through FcγRIIIa. © 2024 The Pathological Society of Great Britain and Ireland.

Taurocholic acid promotes hepatic stellate cell activation via S1PR2/p38 MAPK/YAP signaling under cholestatic conditions.

Disrupted bile acid regulation and accumulation in the liver can contribute to progressive liver damage and fibrosis. However, the effects of bile acids on the activation of hepatic stellate cells (HSCs) remain unclear. This study investigated the effects of bile acids on HSC activation during liver fibrosis, and examined the underlying mechanisms. The immortalized HSCs, LX-2 and JS-1cells were used for the in vitro study. in vitro, the adeno-associated viruses adeno-associated virus-sh-S1PR2 and JTE-013 were used to pharmacologically inhibit the activity of S1PR2 in a murine model of fibrosis induced by a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Histological and biochemical analyses were performed to study the involvement of S1PR2 in the regulation of fibrogenic factors as well as the activation properties of HSCs. S1PR2 was the predominant S1PR expressed in HSCs and was upregulated during taurocholic acid (TCA) stimulation and in cholestatic liver fibrosis mice. TCA-induced HSC proliferation, migration and contraction and extracellular matrix protein secretion were inhibited by JTE-013 and a specific shRNA targeting S1PR2 in LX-2 and JS-1 cells. Meanwhile, treatment with JTE-013 or S1PR2 deficiency significantly attenuated liver histopathological injury, collagen accumulation, and the expression of fibrogenesis-associated genes in mice fed a DDC diet. Furthermore, TCAmediated activation of HSCs through S1PR2 was closely related to the yes-associated protein (YAP) signaling pathway via p38 mitogen-activated protein kinase (p38 MAPK). TCA-induced activation of the S1PR2/p38 MAPK/YAP signaling pathways plays a vital role in regulating HSC activation, which might be therapeutically relevant for targeting cholestatic liver fibrosis.

Edaravone protects against liver fibrosis progression via decreasing the IL-1β secretion of macrophages

Edaravone (EDA), a strong novel free radical scavenger, have been demonstrated to exert neurovascular protective effects clinically. Furthermore, EDA can suppress the lung injury, pulmonary fibrosis and skin fibrosis, while the precise effects and mechanisms of EDA on liver injury and fibrosis remain unclear. The effects of EDA on the Thioacetamide (TAA)-induced liver fibrosis were evaluated by sirius red staining, α-SMA immunohistochemistry. The percentages of immune cell subsets were analyzed by flow cytometry. Immunofluorescence assay was performed to identify the fibrotic properties of hepatic stellate cells (HSCs). Western blot and qPCR were used to detect the levels of liver fibrosis-related molecules and IL-1β. EDA displayed a hepatic protective role in TAA-induced chronic liver fibrosis via inhibiting monocyte/macrophages recruitment and IL-1β production of macrophages. Mechanically, EDA inhibited of NF-κB signal pathway and reactive oxygen species (ROS) production in macrophages. Moreover, EDA treatment indirectly suppressed the activation of HSCs by decreasing the IL-1β secretion of macrophages. Together, EDA protects against TAA-induced liver fibrosis via decreasing the IL-1β production of macrophages, thereby providing a feasible solution for clinical treatment of liver fibrosis.

Hydrostatic pressure induces profibrotic properties in hepatic stellate cells via the RhoA/ROCK signaling pathway.

Elevated interstitial fluid hydrostatic pressure is commonly observed in diseased livers. We herein examined the hypothesis that hydrostatic pressure induces hepatic stellate cells to acquire profibrotic properties under pathological conditions. Human hepatic stellate cells were exposed to 50 mmHg pressure for 24 h. Although we observed few changes of cell growth and morphology, PCR array data on the expression of fibrosis‐associated genes suggested the acquisition of profibrotic properties. The exposure of hepatic stellate cells to 50 mmHg pressure for 24 h also significantly enhanced the expression of RhoA, ROCK1, α‐SMA, TGF‐β1, p‐MLC, and p‐Smad2, and this was effectively attenuated by the ROCK inhibitor Y‐27632. Our ex vivo experimental data suggest that elevated interstitial fluid hydrostatic pressure under pathological conditions may promote liver fibrosis by inducing acquisition of profibrotic properties of hepatic stellate cells through the RhoA/ROCK signaling pathway.

Angiotensin receptor blocker alleviates liver fibrosis by altering the mechanotransduction properties of hepatic stellate cells.

Angiotensin receptor blockers have been reported to be beneficial to liver fibrosis, but the relevant molecular and cellular mechanisms remain unclear. We herein investigated whether low-dose angiotensin receptor blocker alleviated liver fibrosis through mechanotransduction regulation. Hydrostatic pressure-induced liver fibrosis model was established in mice by ligating partially the inferior vena cava, and then randomly received a very low dose of losartan (0.5 mg/kg) or placebo treatment for 8 weeks. We found that losartan administration interfered the expression of several mechanotransductive molecules, and effectively alleviated liver fibrosis. Using a commercial device, we further confirmed that ex vivo loading of hepatic stellate cells to 50 mmHg hydrostatic pressure for 24 h significantly upregulated RhoA, ROCK, AT1R, and p-MLC2, which was effectively attenuated by adding 10 nM losartan in medium. Our in vivo and ex vivo experimental data suggest that low-dose angiotensin receptor blockers may alleviate hydrostatic pressure-induced liver fibrosis by altering the mechanotransduction properties of hepatic stellate cells.NEW & NOTEWORTHY Our ex vivo and in vivo experiments clearly indicated that low-dose losartan alleviated liver fibrosis, likely by modulating the mechanotransduction properties of HSCs. Uncovering the biomechanical signaling pathway of ARB treatment on liver fibrosis will be helpful to develop novel molecular targeting therapy for liver diseases.

Irisin Regulates the Functions of Hepatic Stellate Cells.

BackgroundHepatic stellate cells (HSCs) are known to play a fundamental role in the progression of liver fibrosis. Once HSCs are activated, they are involved in proliferation, migration, and contractility which are characteristics of liver fibrogenesis. Recent studies have shown that irisin, a myokine secreted during physical exercise, has a protective effect in various metabolic diseases, especially in renal fibrosis. However, whether irisin is involved in HSC activation and other processes associated with liver fibrosis has not yet been investigated. In this study, we reveal the role of irisin in HSC activation as well as in proliferation, migration, and contractile properties of HSCs in vitro.MethodsLX-2 cells, immortalized human HSCs, were treated with transforming growth factor beta 1 (TGF-β1), a core regulator of HSC fibrosis, with or without irisin, and markers of the aforementioned processes were analyzed. Further, an inflammatory response was stimulated with TGF-β1 and lipopolysaccharide (LPS) in combination with irisin and the expression of cytokines was measured.ResultsRecombinant irisin significantly suppressed the expression of TGF-β1-stimulated fibrosis markers including alpha-smooth muscle actin and collagen type 1 alpha 1 and prevented the TGF-β1-induced proliferation, migration, and contractility of LX-2 cells. Additionally, irisin ameliorated the production of interleukin-6 (IL-6) and IL-1β induced by TGF-β1 and LPS treatments.ConclusionThese findings suggested that irisin potently improved the progression of hepatic fibrosis by regulating HSC activation, proliferation, migration, contractility, and HSC-mediated production of inflammatory cytokine.

Pharmacological Inhibition of Cyclin-Dependent Kinases Triggers Anti-Fibrotic Effects in Hepatic Stellate Cells In Vitro.

Liver fibrosis is a wound healing process in response to chronic liver injury, which is characterized by the accumulation of extracellular collagen produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of normally quiescent HSCs controlled by cyclins and associated cyclin-dependent kinases (Cdks). Cdk2 mediates the entry and progression through S-phase in complex with E-and A-type cyclins. We have demonstrated that cyclin E1 is essential for liver fibrogenesis in mice, but it is not known if this is dependent on Cdk2 or related Cdks. Here, we aimed to evaluate the benefit of the pan-Cdk inhibitor CR8 for treatment of liver fibrosis in vitro. CR8-treatment reduced proliferation and survival in immortalized HSC lines and in addition attenuated pro-fibrotic properties in primary murine HSCs. Importantly, primary murine hepatocytes were much more tolerant against the cytotoxic and anti-proliferative effects of CR8. We identified CR8 dosages mediating anti-fibrotic effects in primary HSCs without affecting cell cycle activity and survival in primary hepatocytes. In conclusion, the pharmacological pan-Cdk inhibitor CR8 restricts the pro-fibrotic properties of HSCs, while preserving proliferation and viability of hepatocytes at least in vitro. Therefore, CR8 and related drugs might be beneficial for the treatment of liver fibrosis.

Canonical hedgehog signalling regulates hepatic stellate cell-mediated angiogenesis in liver fibrosis.

Hepatic stellate cells (HSCs) are liver-specific pericytes regulating angiogenesis during liver fibrosis. We aimed to elucidate the mechanisms by which hedgehog signalling regulated HSC angiogenic properties and to validate the therapeutic implications. Rats and mice were treated with carbon tetrachloride for in vivo evaluation of hepatic angiogenesis and fibrotic injury. Diversified molecular approaches including real-time PCR, Western blot, luciferase reporter assay, chromatin immunoprecipitation, electrophoretic mobility shift assay and co-immunoprecipitation were used to investigate the underlying mechanisms in vitro. Angiogenesis was concomitant with up-regulation of Smoothened (SMO) and hypoxia inducible factor-1α (HIF-1α) in rat fibrotic liver. The SMO inhibitor cyclopamine and Gli1 inhibitor GANT-58 reduced expression of VEGF and angiopoietin 1 in HSCs and suppressed HSC tubulogenesis capacity. HIF-1α inhibitor PX-478 suppressed HSC angiogenic behaviour, and inhibition of hedgehog decreased HIF-1α expression. Furthermore, heat shock protein 90 (HSP90) was characterized as a direct target gene of canonical hedgehog signalling in HSCs. HSP90 inhibitor 17-AAG reduced HSP90 binding to HIF-1α, down-regulated HIF-1α protein abundance and decreased HIF-1α binding to DNA. 17-AAG also abolished 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) (a SMO agonist)-enhanced HSC angiogenic properties. Finally, the natural compound ligustrazine was found to inhibit canonical hedgehog signalling leading to suppressed angiogenic properties of HSCs in vitro and ameliorated liver fibrosis and sinusoidal angiogenesis in mice. We have provided evidence that the canonical hedgehog pathway controlled HSC-mediated liver angiogenesis. Selective inhibition of HSC hedgehog signalling could be a promising therapeutic approach for hepatic fibrosis.

Curcumin regulates cell fate and metabolism by inhibiting hedgehog signaling in hepatic stellate cells

Accumulating evidence indicates that Hedgehog (Hh) signaling becomes activated in chronic liver injury and plays a role in the pathogenesis of hepatic fibrosis. Hepatic stellate cells (HSCs) are Hh-responsive cells and activation of the Hh pathway promotes transdifferentiation of HSCs into myofibroblasts. Targeting Hh signaling may be a novel therapeutic strategy for treatment of liver fibrosis. We previously reported that curcumin has potent antifibrotic effects in vivo and in vitro, but the underlying mechanisms are not fully elucidated. This study shows that curcumin downregulated Patched and Smoothened, two key elements in Hh signaling, but restored Hhip expression in rat liver with carbon tetrachloride-induced fibrosis and in cultured HSCs. Curcumin also halted the nuclear translocation, DNA binding, and transcription activity of Gli1. Moreover, the Hh signaling inhibitor cyclopamine, like curcumin, arrested the cell cycle, induced mitochondrial apoptosis, reduced fibrotic gene expression, restored lipid accumulation, and inhibited invasion and migration in HSCs. However, curcumin's effects on cell fate and fibrogenic properties of HSCs were abolished by the Hh pathway agonist SAG. Furthermore, curcumin and cyclopamine decreased intracellular levels of adenosine triphosphate and lactate, and inhibited the expression and/or function of several key molecules controlling glycolysis. However, SAG abrogated the curcumin effects on these parameters of glycolysis. Animal data also showed that curcumin downregulated glycolysis-regulatory proteins in rat fibrotic liver. These aggregated data therefore indicate that curcumin modulated cell fate and metabolism by disrupting the Hh pathway in HSCs, providing novel molecular insights into curcumin reduction of HSC activation.

Liver fibrosis occurs through dysregulation of MyD88‐dependent innate B‐cell activity

Chronic liver disease mediated by activation of hepatic stellate cells (HSCs) leads to liver fibrosis. Here, we postulated that the immune regulatory properties of HSCs might promote the profibrogenic activity of B cells. Fibrosis is completely attenuated in carbon tetrachloride-treated, B cell-deficient µMT mice, showing that B cells are required. The retinoic acid produced by HSCs augmented B-cell survival, plasma cell marker CD138 expression, and immunoglobulin G production. These activities were reversed following addition of the retinoic acid inhibitor LE540. Transcriptional profiling of fibrotic liver B cells revealed increased expression of genes related to activation of nuclear factor κ light chain enhancer of activated B cells, proinflammatory cytokine production, and CD40 signaling, suggesting that these B cells are activated and may be acting as inflammatory cells. Biological validation experiments also revealed increased activation (CD44 and CD86 expression), constitutive immunoglobulin G production, and secretion of the proinflammatory cytokines tumor necrosis factor-α, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α. Likewise, targeted deletion of B-cell-intrinsic myeloid differentiation primary response gene 88 signaling, an innate adaptor with involvement in retinoic acid signaling, resulted in reduced infiltration of migratory CD11c(+) dendritic cells and Ly6C(++) monocytes and, hence, reduced liver pathology. Liver fibrosis occurs through a mechanism of HSC-mediated augmentation of innate B-cell activity. These findings highlight B cells as important "first responders" of the intrahepatic immune environment.

Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells.

Hepatic fibrosis is concomitant with sinusoidal pathological angiogenesis, which has been highlighted as novel therapeutic targets for the treatment of chronic liver disease. Our prior studies have demonstrated that curcumin has potent antifibrotic activity, but the mechanisms remain to be elucidated. The current work demonstrated that curcumin ameliorated fibrotic injury and sinusoidal angiogenesis in rat liver with fibrosis caused by carbon tetrachloride. Curcumin reduced the expression of a number of angiogenic markers in fibrotic liver. Experiments in vitro showed that the viability and vascularization of rat liver sinusoidal endothelial cells and rat aortic ring angiogenesis were not impaired by curcumin. These results indicated that hepatic stellate cells (HSCs) that are characterized as liver-specific pericytes could be potential target cells for curcumin. Further investigations showed that curcumin inhibited VEGF expression in HSCs associated with disrupting platelet-derived growth factor-β receptor (PDGF-βR)/ERK and mTOR pathways. HSC motility and vascularization were also suppressed by curcumin associated with blocking PDGF-βR/focal adhesion kinase/RhoA cascade. Gain- or loss-of-function analyses revealed that activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) was required for curcumin to inhibit angiogenic properties of HSCs. We concluded that curcumin attenuated sinusoidal angiogenesis in liver fibrosis possibly by targeting HSCs via a PPAR-γ activation-dependent mechanism. PPAR-γ could be a target molecule for reducing pathological angiogenesis during liver fibrosis.

F-actin cytoskeleton reorganization is associated with hepatic stellate cell activation

The activation of hepatic stellate cells (HSCs) is involved in the development of hepatic fibrosis. Previous studies have indicated that the acquisition of certain properties by activated HSCs is highly dependent on the reorganization of the actin cytoskeleton. However, direct evidence showing that the reorganization of the actin cytoskeleton is responsible for HSC activation is lacking. The aim of the present study was to investigate the role of cytoskeletal reorganization during HSC activation and to clarify the underlying mechanism. HSC-T6 cells were treated either with the F-actin stabilizer jasplakinolide (Jas) or the depolymerizer cytochalasin D (Cyto D). The actin cytoskeleton was evaluated via assessment of stress fiber formation. Furthermore, the activation properties of HSCs, including proliferation, adhesion, migration and the expression of α-smooth muscle actin (α-SMA) and collagen 1, were investigated in vitro. The results showed that Jas and Cyto D affected the actin distribution in HSC-T6 cells. Treatment with Jas resulted in thick actin bundles and a patchy appearance in the cytoplasm in HSC-T6 cells. In parallel, polymerization of actin microfilaments induced by Jas upregulated the expression of α-SMA and collagen 1, and also enhanced the migration and adhesion properties of HSC-T6 cells. Furthermore, the activation of HSC-T6 cells induced by the reorganization of the actin cytoskeleton was associated with the p38 mitogen-activated protein kinase (p38 MAPK) pathway. In conclusion, the present study suggests that the reorganization of the F-actin cytoskeleton is associated with HSC activation and that the p38 MAPK pathway is involved in this process. The inhibition of F-actin reorganization may thus be a potential key factor or molecular target for the control of liver fibrosis or cirrhosis.

Hepatic stellate cells promote tumor progression by enhancement of immunosuppressive cells in an orthotopic liver tumor mouse model

The immunosuppressive properties of hepatic stellate cells (HSCs) contribute to the occurrence and development of hepatocellular carcinoma (HCC). The accumulation of cells with immune suppressive activities, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) is a key mechanism for tumor immune evasion. However, the impact of HSCs on immune cell populations in tumor-bearing hosts is unclear. In this study, we established an orthotopic liver tumor mouse model for studying the complex tumor–host interactions in HCC. The activated HSCs promoted HCC growth not only induced tumor angiogenesis and lymphangiogenesis, but also significantly increased the suppressive immune cell population of Tregs and MDSCs in the spleen, bone marrow, and tumor tissues of the tumor-bearing mice. Murine HCC cell line H22-activated HSCs also expanded the expression of Tregs and MDSCs in vitro. In conclusion, our study suggests a novel role for HSCs in the HCC microenvironment. HSCs can promote HCC progression by enhancement of the immunosuppressive cell population. Targeting HSCs, which is a new concept in adjuvant immunotherapy, may be introduced in the near future to improve the outcome of patients with HCC.

Mammalian target of rapamycin mediates the angiogenic effects of leptin in human hepatic stellate cells

Leptin modulates the angiogenic properties of hepatic stellate cells (HSC), but the molecular mechanisms involved are poorly understood. We investigated the pathways regulating hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in leptin-stimulated myofibroblastic HSC. Exposure to leptin enhanced the phosphorylation of TSC2 on T1462 residues and of p70 S6 kinase and the translational inhibitor 4E-binding protein-1, indicating the ability of leptin to activate the mammalian target of rapamycin (mTOR) pathway. Similar findings were observed when HSC were exposed to PDGF. Both leptin and PDGF increased the expression of HIF-1α and VEGF in HSC. In the presence of rapamycin, a specific mTOR inhibitor, leptin and PDGF were no longer able to activate mTOR, and expression of VEGF was reduced, whereas HIF-1α abundance was not affected. Moreover, knockdown of Raptor, a component of the mTORC1 complex, reduced the ability of leptin to increase VEGF. mTOR was also necessary for leptin- and PDGF-dependent increase in HSC migration. Leptin increased the generation of reactive oxygen species in HSC, which was reduced by NADP(H) oxidase inhibitors. Both N-acetyl cysteine and diphenylene iodonium, a NADP(H) inhibitor, inhibited the expression of HIF-1α and VEGF stimulated by leptin or PDGF. Finally, conditioned media from HSC treated with leptin or PDGF induced tube formation in cultured human umbilical vein endothelial cells. In conclusion, in HSC exposed to leptin or PDGF, increased expression of VEGF requires both activation of mTOR and generation of reactive oxygen species via NADPH-oxidase. Induction of HIF-1α requires NADP(H) oxidase but not mTOR activation.

Preservation of basal AcSDKP attenuates carbon tetrachloride-induced fibrosis in the rat liver

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous tetrapeptide which has antifibrogenic effects at physiological concentrations in various tissues. AcSDKP is produced locally in the liver, however, little is known about its biological effect in this organ. We hypothesize that basal levels of endogenous AcSDKP decrease during the development of liver fibrosis and preservation of basal AcSDKP attenuates liver fibrosis. Endogenous levels of AcSDKP in the liver were measured by enzyme immunoassay after 2, 6, and 10 weeks of carbon tetrachloride (CCl(4))-induced liver fibrosis in rats. Subcutaneous osmotic pump infusion of vehicle or AcSDKP (800 microg/kg/day) was administered to CCl(4)-treated rats for 8 weeks to study the effect of exogenous AcSDKP on liver fibrosis. The effect of AcSDKP on profibrogenic properties of hepatic stellate cells was studied in vitro. Endogenous AcSDKP was significantly decreased in the liver of CCl(4)-treated rats. Chronic AcSDKP infusion preserved basal levels of AcSDKP and reduced liver injury, inflammation, fibrosis, and profibrogenic transforming growth factor-beta signaling. This was demonstrated by decreased aminotransferase serum levels, CD45 positive cells, collagen accumulation, alpha-smooth muscle actin positivity, transforming growth factor-beta1, phosphorylated Smad2/3 protein, increased bone morphogenetic protein-7, and phosphorylated Smad1/5/8. Further, AcSDKP exerts antifibrogenic effects on hepatic stellate cells (HSCs) by downregulation of HSC activation in vitro. Maintaining physiological levels of AcSDKP is critical in negatively regulating the development of fibrosis in chronic liver injury. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.

Hepatic Stellate Cells and Fibrogenesis in Hepatitis C Virus Infection: An Ultrastructural Insight

An ultrastructural quantitative assessment of hepatic stellate cells (HSCs) was made in relation to hepatic fibrosis, apoptotic cellular changes, intracellular fat deposition, circulating inflammatory cells in the sinusoids, and the necroinflammatory activity in liver specimens of 33 patients proven to be positive for hepatitis C virus (HCV)-RNA by polymerase chain reaction with the intention that electron microscopy may throw more light on the role of HSCs in the complicated process of fibrogenesis. A detailed review concerning these parameters and observed evidence suggesting the potential properties of HSCs to recycle cellular debris into collagen fibers are reported.

Hepatocarcinoma cells stimulate the growth, migration and expression of pro-angiogenic genes in human hepatic stellate cells

Activated hepatic stellate cells (HSC) and other fibrogenic cell types are frequently found around hepatocellular carcinoma. It is unknown whether hepatocarcinoma cells regulate the biological functions of HSC. This study aimed to investigate the paracrine effects of hepatocarcinoma cells on human HSC using a co-culture system. Huh7 or HepG2 cells, human hepatocarcinoma cell lines, were co-cultured with primary human HSC. Intracellular calcium mobilization, proliferation, migration, expression of pro-angiogenic and fibrogenic genes, smooth muscle alpha-actin (alpha-SMA) protein expression, inflammatory properties (nuclear factor kappa B activation and interleukin 8 secretion) and intracellular signalling pathways (AKT and ERK) were analysed in HSC. Culture of HSC with Huh7 cells for 24 h stimulated HSC proliferation, migration and expression of pro-angiogenic genes. The migration effect was corroborated with HepG2 cells. The effects of Huh7 cells on cell proliferation and migration were mediated mainly by PI3K/AKT activation. Moreover, Huh7 cells reduced the expression of genes involved in fibrogenesis, while they did not modify the inflammatory properties of HSC. The expression of alpha-SMA was induced by Huh7 cells. Because hepatitis C virus (HCV) infection is a major cause of hepatocarcinoma, we next investigated whether these effects are regulated by the expression of HCV in hepatocarcinoma cells. Expression of a subgenomic replicon expressing HCV nonstructural proteins (NS3-NS5) in Huh7 cells did not affect paracrine actions in HSC (cell proliferation and migration). These results suggested that there is a cross-talk between hepatocarcinoma cells and HSC. Activated HSC may be stimulated by cancer cells to accumulate and express angiogenic genes.

Ghrelin attenuates hepatocellular injury and liver fibrogenesis in rodents and influences fibrosis progression in humans

There are no effective antifibrotic therapies for patients with liver diseases. We performed an experimental and translational study to investigate whether ghrelin, an orexigenic hormone with pleiotropic properties, modulates liver fibrogenesis. Recombinant ghrelin was administered to rats with chronic (bile duct ligation) and acute (carbon tetrachloride) liver injury. Hepatic gene expression was analyzed by way of microarray analysis and quantitative polymerase chain reaction. The hepatic response to chronic injury was also evaluated in wild-type and ghrelin-deficient mice. Primary human hepatic stellate cells were used to study the effects of ghrelin in vitro. Ghrelin hepatic gene expression and serum levels were assessed in patients with chronic liver diseases. Ghrelin gene polymorphisms were analyzed in patients with chronic hepatitis C. Recombinant ghrelin treatment reduced the fibrogenic response, decreased liver injury and myofibroblast accumulation, and attenuated the altered gene expression profile in bile duct-ligated rats. Moreover, ghrelin reduced the fibrogenic properties of hepatic stellate cells. Ghrelin also protected rats from acute liver injury and reduced the extent of oxidative stress and inflammation. Ghrelin-deficient mice developed exacerbated hepatic fibrosis and liver damage after chronic injury. In patients with chronic liver diseases, ghrelin serum levels decreased in those with advanced fibrosis, and ghrelin gene hepatic expression correlated with expression of fibrogenic genes. In patients with chronic hepatitis C, polymorphisms of the ghrelin gene (-994CT and -604GA) influenced the progression of liver fibrosis. Ghrelin exerts antifibrotic effects in the liver and may represent a novel antifibrotic therapy.

Effects of upregulated expression of microRNA-16 on biological properties of culture-activated hepatic stellate cells

In our previous studies, we identified miR-16 as being downregulated during activation of hepatic stellate cells (HSCs) by microarray hybridization. However, the roles and related mechanisms of miR-16 in HSCs are not understood. In this study, The miRNA RNAi technique was used to analyze the effects of miR-16 on biological properties of HSCs in vitro. The lentiviral vector encoding miR-16 was constructed and transfected. Furthermore, the expression level of miR-16 was measured by real-time PCR. Cellular growth and proliferation capacity were assayed using the cell counting kit-8 (CCK-8). The apoptosis rate and cell-cycle distribution were measured by flow cytometry. Cell morphological characteristics were identified by phase-contrast microscopy, fluorescence microscopy and electron microscopy. The underlying mechanisms related to the changes in biological properties were assessed. The identity of the recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. Virus titer was 10(8) > ifu/m. Restoring the intracellular miRNAs by miR-16 administration greatly reduced the expression levels of cyclin D1 (CD1). Cell-cycle arrest and typical features of apoptosis were detected in activated HSCs treated with pLV-miR-16. Our results indicate that transduction of miR-16 offers a feasible approach to significantly inhibit HSC proliferation and increase the apoptosis index. Thus, targeted transfer of miR-16 into HSC may be useful for the treatment of hepatic fibrosis.

Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells

Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.