Actions Of Hepatic Stellate Cells Research Articles

Role of NADPH Oxidases in Liver Fibrosis

Hepatic fibrosis is the common pathophysiologic process resulting from chronic liver injury, characterized by the accumulation of an excessive extracellular matrix. Multiple lines of evidence indicate that oxidative stress plays a pivotal role in the pathogenesis of liver fibrosis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. In addition to phagocytic NOX2, there are six nonphagocytic NOX proteins. In the liver, NOX is functionally expressed both in the phagocytic form and in the nonphagocytic form. NOX-derived ROS contributes to various kinds of liver disease caused by alcohol, hepatitis C virus, and toxic bile acids. Recent evidence indicates that both phagocytic NOX2 and nonphagocytic NOX isoforms, including NOX1 and NOX4, mediate distinct profibrogenic actions in hepatic stellate cells, the main fibrogenic cell type in the liver. The critical role of NOX in hepatic fibrogenesis provides a rationale to assess pharmacological NOX inhibitors that treat hepatic fibrosis in patients with chronic liver disease. Although there is compelling evidence indicating a crucial role for NOX-mediated ROS generation in hepatic fibrogenesis, little is known about the expression, subcellular localization, regulation, and redox signaling of NOX isoforms in specific cell types in the liver. Moreover, the exact mechanism of NOX-mediated fibrogenic signaling is still largely unknown. A better understanding through further research about NOX-mediated fibrogenic signaling may enable the development of novel anti-fibrotic therapy using NOX inhibition strategy. Antio

Hepatitis C virus core protein induces fibrogenic actions of hepatic stellate cells via toll-like receptor 2

Hepatic stellate cells (HSCs) represent the main fibrogenic cell type accumulating extracellular matrix in the liver. Recent data suggest that hepatitis C virus (HCV) core protein may directly activate HSCs. Therefore, we examined the influence of recombinant HCV core protein on human HSCs. Primary human HSCs and the human HSC line LX-2 were stimulated with recombinant HCV proteins core and envelope 2 protein. Expression of procollagen type I α-1, α-smooth muscle actin, cysteine- and glycine-rich protein 2, glial fibrillary acidic protein, tissue growth factor β1, matrix metalloproteinases 2 (MMP2) and 13, tissue inhibitor of metalloproteinases 1 and 2 was investigated by real-time PCR. Intracellular signaling pathways of ERK1/2, p38 and, jun-amino-terminal kinase (JNK) were analyzed by western blot analysis. Recombinant HCV core protein induced upregulation of procollagen type I α-1, α-smooth muscle actin, MMP 2 and 13, tissue inhibitor of metalloproteinases 1 and 2, tissue growth factor β1, cysteine- and glycine-rich protein 2, and glial fibrillary acidic protein mRNA expression, whereas HCV envelope 2 protein did not exert any significant effect. Blocking of toll-like receptor 2 (TLR2) with a neutralizing antibody prevented mRNA upregulation by HCV core protein confirming that the TLR2 pathway was involved. Furthermore, western blot analysis revealed HCV-induced phosphorylation of the TLR2-dependent signaling molecules ERK1/2, p38 and JNK mitogen-activated kinases. Our in vitro results demonstrate a direct effect of HCV core protein on activation of HSCs toward a profibrogenic state, which is mediated via the TLR2 pathway. Manipulating the TLR2 pathway may thus provide a new approach for antifibrotic therapies in HCV infection.

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.

Suppressive Effect of Orthovanadate on Hepatic Stellate Cell Activation and Liver Fibrosis in Rats

Orthovanadate (OV), an inhibitor of protein tyrosine phosphatases, affects various biological processes in a cell-type-specific manner. In this study, we investigated the effect of OV on hepatic stellate cells (HSCs). When primary rat HSCs were cultured in the presence of 10% serum, they spontaneously lost characteristic stellate morphology, proliferated, and were transformed into an activated state with the formation of abundant stress fibers and increased expression of both alpha-smooth muscle actin and collagen type I mRNA. OV treatment inhibited proliferation and activation of HSCs and partially reversed the phenotype of activated HSCs. Among the signaling molecules investigated, phosphorylation of the Src protein at tyrosine 416 was the most striking in OV-treated HSCs. Treatment of cells with Src family inhibitors partially abrogated the effects of OV. Furthermore, transfection of v-Src into activated HSCs induced a stellate morphology similar to that in the quiescent state. We then examined whether OV could effectively suppress HSC activation in vivo after liver injury induced by either carbon tetrachloride or dimethylnitrosamine. OV significantly reduced the appearance of alpha-smooth muscle actin-positive cells and decreased collagen deposition, concomitant with an improvement in liver function. Our study showed for the first time that OV was able to suppress the activation of HSCs, possibly through the modulation of Src activity, and attenuated fibrosis after chronic liver injury.

Resistin as an Intrahepatic Cytokine: Overexpression during Chronic Injury and Induction of Proinflammatory Actions in Hepatic Stellate Cells

Obesity and insulin resistance accelerate the progression of fibrosis during chronic liver disease. Resistin antagonizes insulin action in rodents, but its role in humans is still controversial. The aims of this study were to investigate resistin expression in human liver and to evaluate whether resistin may affect the biology of activated human hepatic stellate cells (HSCs), key modulators of hepatic fibrogenesis. Resistin gene expression was low in normal human liver but was increased in conditions of severe fibrosis. Up-regulation of resistin during chronic liver damage was confirmed by immunohistochemistry. In a group of patients with alcoholic hepatitis, resistin expression correlated with inflammation and fibrosis, suggesting a possible action on HSCs. Exposure of cultured HSCs to recombinant resistin resulted in increased expression of the proinflammatory chemokines monocyte chemoattractant protein-1 and interleukin-8, through activation of nuclear factor (NF)-kappaB. Resistin induced a rapid increase in intracellular calcium concentration, mainly through calcium release from intracellular inositol triphosphate-sensitive pools. The intracellular calcium chelator BAPTA-AM blocked resistin-induced NF-kappaB activation and monocyte chemoattractant protein-1 expression. In conclusion, this study shows a role for resistin as an intrahepatic cytokine exerting proinflammatory actions in HSCs, via a Ca2+/NF-kappaB-dependent pathway and suggests involvement of this adipokine in the pathophysiology of liver fibrosis.

Ligands of peroxisome proliferator-activated receptor γ modulate profibrogenic and proinflammatory actions in hepatic stellate cells

Background & Aims: Proliferation and migration of hepatic stellate cells (HSCs) and expression of chemokines are involved in the pathogenesis of liver inflammation and fibrogenesis. Peroxisome proliferator–activated receptor (PPAR)-γ is a receptor transcription factor that controls growth and differentiation in different tissues. We explored the effects of PPAR-γ agonists on the biological actions of cultured human HSCs. Methods: HSCs were isolated from normal human liver tissue and used in their myofibroblast-like phenotype or immediately after isolation. Activation of PPAR-γ was induced with 15-deoxy-Δ12,14-prostaglandin J2 or with troglitazone. Results: PPAR-γ agonists dose-dependently inhibited HSC proliferation and chemotaxis induced by platelet-derived growth factor. This effect was independent of changes in postreceptor signaling or expression of c-fos and c-myc and was associated with inhibition of cell cycle progression beyond the G1 phase. Activation of PPAR-γ also resulted in a complete inhibition of the expression of monocyte chemotactic protein 1 at the gene and protein levels. Comparison of quiescent and culture-activated HSCs revealed a marked decrease in PPAR-γ expression in activated cells. Conclusions: Activation of PPAR-γ modulates profibrogenic and proinflammatory actions in HSCs. Reduced PPAR-γ expression may contribute to confer an activated phenotype to HSCs.GASTROENTEROLOGY 2000;119:466-478

Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat.

Upon liver injury, hepatic stellate cells (HSC) show increased proliferation, motility, and extracellular matrix (ECM) production. The extracellular signal-regulated kinases (ERK) control different functions in a cell-specific manner. In this study, we evaluated the role of ERK activation in cultured HSC stimulated with platelet-derived growth factor (PDGF) and after induction of liver injury in vivo. HSC were isolated from normal human liver tissue, cultured on plastic, and used in their myofibroblast-like phenotype. In in vivo experiments, HSC were isolated from normal rats or at different time points after a single intragastric administration of CCl(4). Nontoxic concentrations of PD98059, a specific inhibitor of ERK activation, reduced PDGF-induced activation of ERK in a dose-dependent fashion. Suppression of ERK activation was associated with complete inhibition of HSC proliferation and with a 57% reduction in chemotaxis. In the presence of the ERK inhibitor, binding of the AP-1 complex and of STAT1 to the related regulatory elements was inhibited. The inhibition of the DNA binding activity of STAT1 was mediated by a reduction in PDGF-induced tyrosine phosphorylation. Expression of c-fos in response to PDGF was also reduced, but not suppressed, by treatment with PD98059. In HSC isolated from CCl(4)-treated rats, ERK activity increased as early as 6 hours following liver damage, and declined thereafter. The results of this study indicate that ERK activation regulates proliferation and chemotaxis of HSC, and modulates nuclear signaling. Acute liver damage in vivo leads to activation of ERK in HSC.