SCC064 Sigma-AldrichLX-2 Human Hepatic Stellate Cell Line

In patients with active brucellosis, the liver is frequently affected by histopathologic lesions, such as granulomas, inflammatory infiltrations, and parenchymal necrosis. Herein, we examine some potential mechanisms of liver damage in brucellosis. We demonstrate that Brucella abortus infection inhibits matrix metalloproteinase-9 (MMP-9) secretion and induces collagen deposition and tissue inhibitor of matrix metalloproteinase-1 secretion induced by hepatic stellate cells (LX-2). These phenomena depend on transforming growth factor-β1 induction. In contrast, supernatants from B. abortus-infected hepatocytes and monocytes induce MMP-9 secretion and inhibit collagen deposition in hepatic stellate cells. Yet, if LX-2 cells are infected with B. abortus, the capacity of supernatants from B. abortus-infected hepatocytes and monocytes to induce MMP-9 secretion and inhibit collagen deposition is abrogated. These results indicate that depending on the balance between interacting cells and cytokines of the surrounding milieu, the response of LX-2 cells could be turned into an inflammatory or fibrogenic phenotype. Livers from mice infected with B. abortus displayed a fibrogenic phenotype with patches of collagen deposition and transforming growth factor-β1 induction. This study provides potential mechanisms of liver immune response induced by B. abortus-infected hepatic stellate cells. In addition, these results demonstrate that the cross talk of these cells with hepatocytes and macrophages implements a series of interactions that may contribute to explaining some of mechanisms of liver damage observed in human brucellosis.

The human hepatic cell line LX-2 has been described as tool to study mechanisms of hepatic fibrogenesis and the testing of antifibrotic compounds. It was originally generated by immortalisation with the Simian Vacuolating Virus 40 (SV40) transforming (T) antigen and subsequent propagation in low serum conditions. Although this immortalized line is used in an increasing number of studies, detailed genetic characterisation has been lacking. We here have performed genetic characterisation of the LX-2 cell line and established a single-locus short tandem repeat (STR) profile for the cell line and characterized the LX-2 karyotype by several cytogenetic and molecular cytogenetic techniques. Spectral karyotyping (SKY) revealed a complex karyotype with a set of aberrations consistently present in the metaphases analyses which might serve as cytogenetic markers. In addition, various subclonal and single cell aberrations were detected. Our study provides criteria for genetic authentication of LX-2 and offers insights into the genotype changes which might underlie part of its phenotypic features.

Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up-regulation in fibrotic or steatotic livers, or in culture-activated or ethanol-treated primary mouse HSC is associated with a reciprocal down-regulation of microRNA-214 (miR-214). By using protector or reporter assays to investigate the 3'-untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis-inducing stimuli was due to reduced expression of miR-214 which otherwise inhibited CCN2 expression by directly binding to the CCN2 3'-UTR. Additionally, miR-214 was present in HSC exosomes, which were bi-membrane vesicles, 50-150nm in diameter, negatively charged (-26mV), and positive for CD9. MiR-214 levels in exosomes but not in cell lysates were reduced by pre-treatment of the cells with the exosome inhibitor, GW4869. Co-culture of miR-214-transfected donor HSC with CCN2 3'-UTR luciferase reporter-transfected recipient HSC resulted in miR-214- and exosome-dependent regulation of a wild type CCN2 3'-UTR reporter but not of a mutant CCN2 3'-UTR reporter lacking the miR-214 binding site. Exosomes from HSC were a conduit for uptake of miR-214 by primary mouse hepatocytes. Down-regulation of CCN2 expression by miR-214 also occurred in human LX-2 HSC, consistent with a conserved miR-214 binding site in the human CCN2 3'-UTR. MiR-214 in LX-2 cells was shuttled via exosomes to recipient LX-2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3'-UTR activity or expression of CCN2 downstream targets, including αSMA or collagen. Experimental fibrosis in mice was associated with reduced circulating miR-214 levels. Conclusion: Exosomal transfer of miR-214 is a paradigm for the regulation of CCN2-dependent fibrogenesis and identifies fibrotic pathways as targets of epigenetic regulation by exosomal miRs. (Hepatology 2013;).

Hepatitis C virus core protein (HCVcp), which is secreted by infected cells, is reported as an immunomodulator in immune cells. However, the effects of HCVcp on hepatic stellate cells (HSCs), the key cells in liver fibrosis, still remain unclear. In this study, we investigated the effects of HCVcp on obese receptor (ObR) related downstream signaling pathways and fibrogenic gene expression in HSCs. LX-2, a human HSC line, was incubated with HCVcp. Inhibitors and short interfering RNAs were used to interrogate the mechanisms of HCVcp action on HSCs. HCVcp (20-100 ng/ml) concentration-dependently stimulated α-smooth muscle actin (α-SMA) protein expression and mRNA expression of α-SMA, procollagen α2(I) and TGF-β1 genes, with a plateau of 220% of controls at 100 ng/ml. HCVcp induced mRNA and protein expression of ObR. Blocking of Ob-Rb with a neutralizing antibody inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and AMPKα stimulated by HCVcp. Furthermore, knockdown of Ob-Rb down-regulated HCVcp-induced STAT3, AKT, and AMPKα phosphorylation, and reversed HCVcp-suppressed mRNA expression of matrix metalloproteinase (MMP)-1, peroxisome proliferator-activated receptor (PPAR)γ and sterol regulatory element binding protein-1c (SREBP-1c) genes. AMPKα signaling blockade reversed HCVcp-suppressed SREBP-1c mRNA expression. HCVcp stimulated reactive oxygen species formation and gp91(phox) (a component of NADPH oxidase) protein expression, together with AKT phosphorylation, leading to suppression of PPARγ and SREBP-1c genes. Our results provide a new finding that HCVcp induced ObR-dependent Janus Kinase (JAK) 2-STAT3, AMPKα, and AKT signaling pathways and modulated downstream fibrogenetic gene expression in HSCs.

Hsp47 is a collagen-specific molecular chaperone, whose activity has been implicated in liver fibrosis. In this study, we showed that TRAIL treatment inhibited Hsp47 expression in dose- and time-dependent manners, subsequently leading to the decrease of collagen production in activated human hepatic stellate LX-2 cells. Overexpression of Hsp47 in LX-2 cells acquired resistance for TRAIL-induced collagen reduction and conversely, siRNA suppression of Hsp47 enhanced the decrease of collagen production due to TRAIL treatment. Moreover, we found that Hsp47 expression was under the transcriptional control of heat shock factor (HSF) 1 which is highly located on nucleus in activated human hepatic stellate LX-2 cells. Treatment of LX-2 cells with TRAIL decreased the active trimer formation of HSF1, increased the dephosphorylation of HSF1 (Ser(230)), and enhanced the translocation of HSF1 into cytosol. The accumulated HSF1 in cytosol led to downregulation of Hsp47 expression, resulting in the reduction of collagen production. Consistently, HSF1 silencing by siRNA prevented Hsp47 induction and subsequent collagen production, whereas overexpression of HSF1 restored the expression level of Hsp47 as well as collagen production in response to TRAIL treatment in LX-2 cells. Taken together, our data suggested that TRAIL induced HSF1 inactivation, consequently leading to the suppression of Hsp47-dependent collagen production in activated human hepatic stellate cells. Therefore, this study suggests that TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis.

Peritumoral activated hepatic stellate cells (HSCs) are versatile myofibroblast-like cells closely related with hepatocellular carcinoma (HCC) progression. So far, comprehensive comparison of gene expression of human HSCs during hepatocarcinogenesis is scanty. Therefore, we identified the phenotypic and genomic characteristics of peritumoral HSCs to explore the valuable information on the prognosis and therapeutic targets of HBV related HCC.

Interleukin-21 (IL-21) is involved in effective primary hepatic immune response against hepatitis B virus (HBV) and profibrotic function. However, the role of IL-21 in HBV-associated liver cirrhosis is poorly understood. This study aimed to investigate the role of IL-21 in HBV-associated liver cirrhosis and possible mechanisms.

Septin4, a member of polymerizing GTP-binding proteins family, is reported to be involved in cytoskeletal organization in mitosis, apoptosis, fibrosis, and other cellular processes. Since various Septin4 expression patterns were reported in different diseases, this study aimed to investigate Septin4 expression in human LX-2 cell line stimulated by lipopolysaccharides (LPS) and attempted to clarify the relationship between Septin4 and hepatic inflammatory injury and fibrosis. In this subject, human stellate cell line LX-2 was stimulated by LPS. The expression of Septin4 was analyzed by Western blot and quantitative real-time PCR. To observe the relationship among Toll-like receptor 4 (TLR4), TGF-β, and Septin4, proteins from the anti-TLR4 antibody blocked cells, as well as the TGF-β-induced cells, were analyzed by the method of Western blot. As the results, LPS could induce the alteration of α-smooth muscle actin and Septin4 expression in LX-2 cells. Septin4 expression was regulated by LPS stimulation through TLR4 and TGF-β pathway. These results therefore suggest that Septin4 may be involved in the process of activation of hepatic stellate cells by LPS stimulation. Further work would focus on the function of Septin4 in hepatic inflammatory injury and fibrosis.

Hepatic stellate cells (HSCs) are a major fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. With increasing interest in developing antifibrotic therapies, there is a need for cell lines that preserve the in vivo phenotype of human HSCs to elucidate pathways of human hepatic fibrosis. We established and characterised two human HSC cell lines termed LX-1 and LX-2, and compared their features with those of primary human stellate cells.

The development of techniques for isolating hepatic lipocytes (Ito, stellate or fat-storing cells) from rodents has been instrumental in defining their role in hepatic vitamin A storage and fibrogenesis. In this study, we developed a method for the purification of lipocytes and Kupffer cell from wedge sections of normal human liver and examined their properties in primary culture. Sections of donor liver (400 to 600 gm) harvested but not used for transplantation were perfused in situ with University of Wisconsin solution and used for lipocyte isolation within 48 hr. Cells were isolated by catheter perfusion of the wedge through several large vessels with L-15 salts, Pronase and collagenase, followed by Larex density gradient centrifugation. Lipocytes were plated on either uncoated plastic or a basement membrane-like gel. Lipocyte and Kupffer cell yields were 2.3 +/- 0.6 x 10(5) and 8.6 +/- 1.4 x 10(5) cells, respectively, per gram of liver (n = 5). Lipocyte purity was 91% as assessed by vitamin A autofluorescence, and Kupffer cell purity was 83% as determined by uptake of fluorescinated staphylococci. Lipocytes cultured on the plastic spread within 48 to 72 hr, displaying slightly more heterogeneous retinoid droplet size than comparable rat cells; on a basement-membrane gel, the cells remained aggregated and spherical with occasional spindlelike extensions. Lipocytes on plastic expressed procollagens I and III, collagen IV and laminin by immunocytochemistry, and types I, III and IV procollagen messenger RNAs by RNAse protection. Northern blot and polymerase chain reaction, respectively. Transmission electron microscopy of lipocytes at 7 days demonstrated a prominent rough endoplasmic reticulum and contractile filaments. Scanning electron microscopy revealed a smooth cell surface with perinuclear droplets beneath the cell membrane. With continued primary culture on plastic (more than 7 days), cells appeared "activated" (i.e., increased spreading and diminished retinoid droplets) and began proliferating as assessed by nuclear autoradiography and [3H]thymidine incorporation. Kupffer cells observed by scanning electron microscopy in early primary culture displayed prominent membrane ruffling and lamellipodia. In summary, we have established a reproducible method for the isolation and primary culture of human lipocytes and Kupffer cells.