Human Stellate Cells Research Articles

Effect of Nitric Oxide on Type I Collagen Expression in Cultured Stellate Cells

This study determined the role of nitric oxide (NO) on type I collagen expression in hepatic stellate cells. Inducible NO synthase (iNOS) mRNA and iNOS protein were detected in LX-2 cells, a human stellate cell line. INF, which suppresses collagen expression, increased iNOS mRNA and iNOS protein, while acetaldehyde and TGF , which enhance collagen expression, decreased iNOS mRNA, but had no effect on iNOS protein. The NO donor DETANONOate diminished the stimulatory effect of TGB on 1(I) collagen mRNA and abrogated the enhancing effect of TGF on type I collagen protein. In conclusion, iNOS was detected in stellate cells under basal culture conditions. While in chronic models of hepatic fibrosis there is decreased hepatic fibrosis in iNOS -/mice, this study shows that the immediate action of nitric oxide is to inhibit the enhancement by TGF of collagen expression by stellate cells.

GAS6 is produced by human stellate cells and its plasma levels reflect the severity of cirrhosis

Background and aim. In rats, growth arrest-specific 6 (Gas6), a secreted ligand of tyrosine kinase receptors, promotes oval cell accumulation and hepatic stellate cell survival during liver healing. We aimed to verify origin and meaning of Gas6 expression and release in the human setting.

Direct interaction of Cucurbitacin E isolated from Alsomitra macrocarpa to actin filament

A methanol extract of Alsomitra macrocarpa leaves and branches induced a marked alteration of cell morphology in a human stellate cell line (LX-2). Similar morphologic alterations were observed in several other cell lines. Active compound was purified from the extract and determined to be cucurbitacin E (Cuc E). It has been known that Cuc E causes marked disruption of the actin cytoskeleton, supporting our observation, but how Cuc E altered the actin cytoskeleton has not been elucidated. By using the standard fluorescence assay using copolymerization and depolymerization of native and pyrene labelled actin, this study revealed that Cuc E interacted directly with actin consequently stabilizing the polymerized actin. When NIH-3T3 cells exogenously expressing YFP-labeled actin were treated with Cuc E, firstly the aggregation of globular actin and secondly the aggregation of actin including disrupted fibrous actin in the cells was observed.

Phagocytosis of apoptotic bodies by hepatic stellate cells induces NADPH oxidase and is associated with liver fibrosis in vivo

Hepatic stellate cell activation is a main feature of liver fibrogenesis. We have previously shown that phagocytosis of apoptotic bodies by stellate cells induces procollagen alpha1 (I) and transforming growth factor beta (TGF-beta) expression in vitro. Here we have further investigated the downstream effects of phagocytosis by studying NADPH oxidase activation and its link to procollagen alpha1 (I) and TGF-beta1 expression in an immortalized human stellate cell line and in several models of liver fibrosis. Phagocytosis of apoptotic bodies in LX-1 cells significantly increased superoxide production both in the extracellular and intracellular milieus. By confocal microscopy of LX-1 cells, increased intracellular reactive oxygen species (ROS) were detected in the cells with intracellular apoptotic bodies, and immunohistochemistry documented translocation of the NADPH oxidase p47phox subunit to the membrane. NADPH oxidase activation resulted in upregulation of procollagen alpha1 (I); in contrast, TGF-beta1 expression was independent of NADPH oxidase activation. This was also confirmed by using siRNA to inhibit TGF-beta1 production. In addition, with EM studies we showed that phagocytosis of apoptotic bodies by stellate cells occurs in vivo. In conclusion, these data provide a mechanistic link between phagocytosis of apoptotic bodies, production of oxidative radicals, and the activation of hepatic stellate cells.

Fibroblast Activation Protein Increases Apoptosis, Cell Adhesion, and Migration by the LX-2 Human Stellate Cell Line *

Injury and repair in chronic liver disease involve cell adhesion, migration, apoptosis, proliferation, and a wound healing response. In liver, fibroblast activation protein (FAP) has both collagenase and dipeptidyl peptidase IV (DPIV) activities and is expressed only by activated hepatic stellate cells (HSC) and myofibroblasts, which produce and degrade extracellular matrix (ECM). FAP was colocalized with collagen fibers, fibronectin, and collagen type I in human liver. FAP function was examined in vitro by expressing green fluorescent protein FAP fusion protein in cell lines cultured on collagen-I, fibronectin, and Matrigel. Glutamates at 203 and 204 as well as serine624 of FAP were essential for peptidase activity. Human embryonic kidney 293T cells overexpressing FAP showed reduced adhesion and migration. FAP overexpression in the human HSC line LX-2 caused increased cell adhesion and migration on ECM proteins as well as invasion across transwells in the absence or presence of transforming growth factor beta-1. FAP overexpression enhanced staurosporine streptomyces-stimulated apoptosis in both cell lines. Interestingly, the enzyme activity of FAP was not required for these functions. Overexpressing FAP increased the expression of matrix metalloproteinase-2 and CD44 and reduced integrin-beta1 expression in 293T cells, suggesting potential pathways of FAP-mediated impairment of cell adhesion and migration in this epithelial cell line. In conclusion, these findings further support a pro-fibrogenic role for FAP by indicating that, in addition to its enzymatic functions, FAP has important nonenzymatic functions that in chronic liver injury may facilitate tissue remodeling through FAP-mediated enhancement of HSC cell adhesion, migration, and apoptosis.

Hepatocyte apoptosis enhances fibrogenic activity of human stellate cells following engulfment of apoptotic bodies - a novel link between two key features of chronic liver disease: Natalie J Torok, Pavel Taimr, Mayo Clinic, Rochester, MN; Scott L Friedman, Mount Sinai Medical Center, New York, NY; Gregory J Gores, Mayo Clinic, Rochester, MN

Hepatocyte apoptosis enhances fibrogenic activity of human stellate cells following engulfment of apoptotic bodies - a novel link between two key features of chronic liver disease: Natalie J Torok, Pavel Taimr, Mayo Clinic, Rochester, MN; Scott L Friedman, Mount Sinai Medical Center, New York, NY; Gregory J Gores, Mayo Clinic, Rochester, MN

Cell-cell junctions between mammalian (human and rat) hepatic stellate cells.

To investigate intercellular junctions between mammalian hepatic stellate cells, we examined cultured human and rat hepatic stellate cells at the ultrastructural and molecular levels. Intercellular junctions between cultured human stellate cells, which developed irrespective of the type of culture substratum, were detected by transmission electron microscopy. On the basis of their characteristic ultrastructure, these junctions were identified in cultured human hepatic stellate cells as adherens junctions but not as tight junctions, desmosomes, or gap junctions. N-cadherin, alpha-catenin and beta-catenin, and p120ctn were detected by Western blotting in rat stellate cells as molecular components of the intercellular adhesive structures. Immunofluorescence for pan-cadherin, alpha-catenin, and beta-catenin were also detected in cultured human stellate cells. Moreover, pan-cadherin and beta-catenin were co-localized at the contact regions between the cultured human stellate cells. These data suggest that the junctional adhesion between the stellate cells can be formed both in vivo and in vitro. Thus, hepatic stellate cells may participate in the structural organization of the cells in liver lobules through the formation of intercellular adherens junctions. This is the first description of the presence of cell-cell junctions between hepatic stellate cells in mammals at the fine structural and molecular levels.

Engagement of alphavbeta3 integrin regulates proliferation and apoptosis of hepatic stellate cells.

Hepatic stellate cells are the major source of the extracellular matrix that accumulates in fibrotic liver. During progressive liver fibrosis, hepatic stellate cells proliferate, but during resolution of fibrosis there is extensive stellate cell apoptosis that coincides with degradation of the liver scar. We have examined the possibility that the fate of stellate cells is influenced by the extracellular matrix through the intermediary of alpha(v)beta(3) integrin. alpha(v)beta(3) integrin was expressed by activated, myofibroblastic rat and human stellate cells in culture. Antagonism of this integrin using neutralizing antibodies, echistatin, or small inhibitory RNA to silence alpha(v) subunit expression inhibited stellate cell proliferation and their expression of proliferating cell nuclear antigen and activated forms of p44 and p42 MAPK. These alpha(v)beta(3) antagonists also increased apoptosis of cultured stellate cells, and this was associated with an increase in the BAX/BCL-2 protein ratio, induction of nuclear DNA fragmentation, and activation of intracellular caspase-3. Expression of tissue inhibitor of metalloproteinases-1 by activated stellate cells was reduced by the alpha(v)beta(3) antagonists, while matrix metalloproteinase-9 synthesis was enhanced. Stellate cells incubated with active recombinant matrix metalloproteinase-9 showed enhanced apoptosis, while cells treated with a synthetic inhibitor of this protease showed increased survival. Our studies suggest that alpha(v)beta(3) integrin regulates the fate of hepatic stellate cells. Degradation of alpha(v)beta(3) ligands surrounding activated stellate cells during resolution of liver fibrosis might decrease alpha(v)beta(3) integrin ligation, suppressing stellate cell proliferation and inducing a fibrolytic, matrix metalloproteinase-secreting phenotype that may prime stellate cells for apoptosis.

Intercellular Adhesive Structures Between Stellate Cells – An Analysis in Cultured Human Hepatic Stellate Cells

To investigate whether or not hepatic stellate cells can form intercellular junctions with each other, we cultured human stellate cells (LI90) on different kinds of substrata. Intercellular junctions were detected between these cultured stellate cells by transmission electron microscopy (TEM). The molecular components of the intercellular adhesive structures were identified by immunofluorescence microscopy. Immunofluorescence for cadherin and catenins was detected at the adhesion sites between the cultured stellate cells. Thus, the intercellular junctions were indicated to be adherens junctions at the molecular level. The junctions developed in the cultured stellate cells irrespective of the type of substratum. These data suggest that the junctional formation between the stellate cells occurs in vivo as well as in vitro.

Apoptotic Body Engulfment by a Human Stellate Cell Line Is Profibrogenic

Hepatocyte apoptosis and stellate cell activation are both features of chronic liver diseases, but a relationship between these events has not been explored. In macrophages, engulfment of apoptotic bodies induces expression of transforming growth factor-β (TGF-β), a profibrogenic cytokine. We examined whether a similar response occurs in stellate cells. Fluorescently labeled hepatocyte apoptotic bodies were added to cultures of primary and immortalized human stellate cells. Stellate cells, but not hepatocytes, readily engulfed apoptotic bodies in a time-dependent manner as assessed by confocal microscopy. The activation of primary and immortalized human stellate cells after incubation with apoptotic bodies, as well as their fibrogenic activity, was indicated by an increase in α-smooth muscle actin (primary cells), TGF-β1, and collagen α1(I) mRNA (primary and immortalized cells). The profibrogenic response was dependent upon apoptotic body engulfment, because nocodazole, a microtubule-inhibiting agent, blocked both the engulfment and the increase of TGF-β1 and collagen α1(I) mRNA. As described in primary rodent stellate cells, up-regulation of collagen α1(I) mRNA was inhibited by a PI-3K inhibitor (LY294002) and a p38 mitogen-activated protein kinase inhibitor (SB203580) in LX-1 cells. In conclusion, these data support a model in which engulfment of hepatocyte apoptotic bodies by stellate cells leads to a fibrogenic response by eliciting a kinase-signaling pathway.

Activated stellate cells express the TRAIL receptor-2/death receptor-5 and undergo TRAIL-mediated apoptosis

Apoptosis has emerged as an important mechanism to reduce numbers of activated stellate cells during the resolution phase of hepatic fibrosis. These observations suggest that activated stellate cells may be more susceptible to apoptotic stimuli than their quiescent counterparts. Because other activated cell types are more sensitive than their quiescent phenotypes to apoptosis by tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), we examined the expression of TRAIL death receptors (DRs) and susceptibility to TRAIL cytotoxicity in stellate cells undergoing progressive activation. A spontaneously immortalized human stellate cell line, LX-2, was analyzed during 14 days of progressive activation following plating, during which time α–smooth muscle actin (α-SMA) and a β-crystallin (markers of stellate cell activation) messenger RNA (mRNA) increased 7-fold and 5-fold, respectively. During this same interval, TRAIL-R1/DR4 and TRAIL-R2/DR5 mRNA expression increased 18-fold and 17.6-fold, although TRAIL-R2/DR5 expression was quantitatively 103-fold greater than TRAIL-R1/DR4; parallel changes occurred in TRAIL/DR5 protein expression and cellular susceptibility to TRAIL-mediated apoptosis. Similar findings were observed in primary murine stellate cells undergoing activation on a plastic surface. In conclusion, stellate cells show activation-dependent TRAIL-R2/DR5 expression and TRAIL-mediated apoptosis. Because TRAIL-R2/DR5 is not expressed by hepatocytes, TRAIL/DR5 agonists may be useful in reducing fibrosis by inducing stellate cell apoptosis. (H EPATOLOGY 2003;37:87-95.)

Leptin enhances the effect of transforming growth factor β in increasing type I collagen formation

Hepatic fibrosis produced by carbon tetrachloride and by Schistosoma masoni is markedly decreased in leptin deficient ob/ob mice as compared to control mice. Leptin is present in activated rat stellate cells, which are the principal collagen producing cells in the liver. The purpose of this study was to identify the leptin receptor and to determine the effects of leptin on type I collagen expression in the human stellate cell line, LX-1. Leptin protein was detected in the LX-1 cells. The leptin receptor (OB-R) was demonstrated by immunofluorescent staining and confocal microscopy. However, only the short forms (Ob-R s), but not the long forms (Ob-R l), of leptin receptor mRNA expression were detected. Leptin increased α 1(I) collagen mRNA and type I collagen production. Leptin did not increase TGFβ1 mRNA or protein in the cultured LX-1 cells. Leptin, however, increased TGFβ type II receptor mRNA and protein and augmented the effect of TGFβ1 on collagen production. In conclusion, this study shows that the effect of leptin in increasing type I collagen production in stellate cells is mediated by actions of leptin in increasing the effectiveness of TGFβ on fibrogenesis by means of an enhancement of the TGFβ type II receptor.

Characterization of human stellate cell activation-associated protein and its expression in human liver

This study cloned cDNA of human homologue (hSTAP) of rat stellate cell activation-associated protein (rSTAP). hSTAP gene is on chromosome 17q and is composed of four exons. Various types of cells including hepatic stellate cells expressed hSTAP mRNA. Recombinant hSTAP was a heme protein with the activity of peroxidase. hSTAP can be used as a marker of quiescent stellate cells in human liver.

Morphology of sites of adhesion between hepatic stellate cells (vitamin A‐storing cells) and a three‐dimensional extracellular matrix

Background Hepatic stellate cells lie in the perisinusoidal space in a three-dimensionally distributed extracellular matrix (ECM). This three-dimensional structure of the ECM regulates the proliferation, morphology, and functions of the stellate cell. To investigate how the three-dimensional structure of ECM regulates behavior of the cells, we cultured stellate cells two- or three-dimensionally and examined the morphology of the cells in both cases as well as the localization of cell-surface adhesion molecules specific for the ECM. Methods Isolated rat stellate cells and human stellate cells were cultured in Dulbecco's modified Eagle's medium. Rat stellate cells were cultured in non-coated polystyrene culture dishes, or on or in type I collagen gels. The morphology of cell-ECM adhesion was examined under transmission and scanning electron microscopes. Localization of integrin α2 and integrin β1 in human stellate cells was examined by immuno-electron microscopy. Immunostaining was performed with a mouse monoclonal anti-human integrin α2 or integrin β1 antibody and goat anti-mouse IgG coupled with 10-nm immunogold. Results Hepatic stellate cells cultured in polystyrene dishes spread well. However, the cells cultured on or in the type I collagen gel became slender. The cells extended long cellular processes onto or into the gel. The cellular processes were entangled three-dimensionally with the type I collagen fibers and directly adhered to these fibers. The cells inoculated in type I collagen gels formed a large number of adhesive structures that resembled focal adhesions. These adhesive structures were distributed not only on the lower side but also on the upper side of both the cell bodies and cellular processes. Moreover, each adhesive area formed a face but not a point. Integrin α2 and integrin β1 were detected on the surfaces of cell bodies, cellular processes, and microprojections. Conclusions The cells cultured in type I collagen gel develop a three-dimensional adhesive structure. Anat. Rec. 250:430–437, 1998. © 1998 Wiley-Liss, Inc.

Morphology of sites of adhesion between hepatic stellate cells (vitamin A-storing cells) and a three-dimensional extracellular matrix.

Hepatic stellate cells lie in the perisinusoidal space in a three-dimensionally distributed extracellular matrix (ECM). This three-dimensional structure of the ECM regulates the proliferation, morphology, and functions of the stellate cell. To investigate how the three-dimensional structure of ECM regulates behavior of the cells, we cultured stellate cells two- or three-dimensionally and examined the morphology of the cells in both cases as well as the localization of cell-surface adhesion molecules specific for the ECM. Isolated rat stellate cells and human stellate cells were cultured in Dulbecco's modified Eagle's medium. Rat stellate cells were cultured in non-coated polystyrene culture dishes, or on or in type I collagen gels. The morphology of cell-ECM adhesion was examined under transmission and scanning electron microscopes. Localization of integrin alpha2 and integrin beta1 in human stellate cells was examined by immunoelectron microscopy. Immunostaining was performed with a mouse monoclonal anti-human integrin alpha2 or integrin beta1 antibody and goat anti-mouse IgG coupled with 10-nm immunogold. Hepatic stellate cells cultured in polystyrene dishes spread well. However, the cells cultured on or in the type I collagen gel became slender. The cells extended long cellular processes onto or into the gel. The cellular processes were entangled three-dimensionally with the type I collagen fibers and directly adhered to these fibers. The cells inoculated in type I collagen gels formed a large number of adhesive structures that resembled focal adhesions. These adhesive structures were distributed not only on the lower side but also on the upper side of both the cell bodies and cellular processes. Moreover, each adhesive area formed a face but not a point. Integrin alpha2 and integrin beta1 were detected on the surfaces of cell bodies, cellular processes, and microprojections. The cells cultured in type I collagen gel develop a three-dimensional adhesive structure.

Epimorphin expressed in human liver stellate cells is upregulated by transforming growth factor-beta

Epimorphin expressed in human liver stellate cells is upregulated by transforming growth factor-beta

Effects of nitric oxide (NO)-donors on activated human hepatic stellate cells (HSC): cellular basis for the treatment of portal hypertension

EFFECTS OF NITRIC OXIDE (NO)-DONORS ON ACTIVATED HUMAN HEPATIC STELLATE CELLS (HSC): CELLULAR BASIS FOR THE TREATMENT OF PORTAL HYPERTENSION. R.M.S. DeFranco, A. Calipiuri, *P. Failli, A. Gentilini, R. Romanelli, “A. Czsini, C. Tosti-Guerra, P. Gentilini, G. Laffi, and M. Pinzani. Istituto di. Medicina Interna, *Dipartimento di Farmacologia, and “Centro di Alcologia, Universita di Firenze, Firenze, Italy.

9,13-di- cis-Retinoic acid induces the production of tPA and activation of latent TGF-β via RARα in a human liver stellate cell line, LI90

We studied the mechanism by which 9,13-di- cis-retinoic acid (9,13dcRA), a novel and endogenous stereoisomer of all- trans-RA, induces TGF-β formation in a human liver stellate cell line, LI90. 9,13dcRA induced the expression of RARα and RARβ, enhanced the production of tissue-type plasminogen activator (tPA), thereby, surface plasmin levels, and induced the activation of latent TGF-β. Similar effects were obtained with RARα-selective retinoid, but not with RARβ- or RARγ-selective retinoid, and the induction was inhibited by RARα-selective antagonist. These results suggest that 9,13dcRA up-regulates tPA expression, resulting in the formation of TGF-β by LI90 cells, at least in part, via induction and activation of RARα.

Liver stellate cells in chronic viral hepatitis: the effect of interferon therapy

Background/Aims: Liver stellate cell proliferation and differentiation into myofibroblast-like cells is related to the development of liver fibrosis. Several cytokines, including interferons, regulate liver stellate cell proliferation and phenotypic modulation. Recent studies indicate that human liver stellate cells express the α-isotype of actin, specific to smooth muscle cell differentiation. We aimed to evaluate the expression of α-smooth muscle actin-positive liver stellate cells in patients with chronic viral hepatitis and to evaluate whether and how such expression can be modified by α-interferon treatment. Methods: Using immunohistochemistry and a semi-quantitative scoring method, we evaluated α-smooth muscle actin expression in liver stellate cells before and after α-interferon therapy in a series of liver biopsies from 44 patients with chronic viral hepatitis Results: Before therapy, a large number of liver stellate cells expressing α-smooth muscle actin were present throughout all acinar zones. A significant reduction in α-smooth muscle actin expression by liver stellate cells was demonstrated in biopsies performed after suspending the interferon treatment. The drop in the number of α-smooth muscle actin-labelled cells after therapy correlated closely with the improvement in the histological index of activity. Conclusions: The results suggest a specific effect of interferon on liver stellate cells, possibly related to its anti-inflammatory action.