Activated Rat Hepatic Stellate Cells Research Articles

Effect of somatostatin-14 on extracellular matrix expression by activated rat hepatic stellate cells

Effect of somatostatin-14 on extracellular matrix expression by activated rat hepatic stellate cells

Calcium signaling machinery in quiescent and activated rat hepatic stellate cells

Calcium signaling machinery in quiescent and activated rat hepatic stellate cells

Identification of phospholipase D gene products in activated rat hepatic stellate cells — stimulation by PDGF and nucleotides

Identification of phospholipase D gene products in activated rat hepatic stellate cells — stimulation by PDGF and nucleotides

Proteome analysis of rat hepatic stellate cells

Proteome analysis was performed on cellular and secreted proteins of normal (quiescent) and activated rat hepatic stellate cells. The stellate cells were activated either in vitro by cultivating quiescent stellate cells for 9 days or in vivo by injecting rats with carbon tetrachloride for 8 weeks. A total of 43 proteins/polypeptides were identified, which altered their expression levels when the cells were activated in vivo and/orin vitro . Twenty-seven of them showed similar changesin vivo and in vitro, including up-regulated proteins such as calcyclin, calgizzarin, and galectin-1 as well as down-regulated proteins such as liver carboxylesterase 10 and serine protease inhibitor 3. Sixteen of them showed different expression levels between in vivo and in vitro activated stellate cells. These results were reproducibly obtained in 3 independent experiments. The up-regulation of calcyclin, calgizzarin, and galectin-1, as well as the down-regulation of liver carboxylesterase 10 were directly confirmed in fibrotic liver tissues. Northern blots confirmed up-regulation of the messenger RNAs (mRNAs) of calcyclin, calgizzarin, and galectin-1 in activated stellate cells, indicating that these changes were controlled at the mRNA level. In addition a list compiling over 150 stellate cell proteins is presented. The data presented here thus provide a significant new protein-level insight into the activation of hepatic stellate cells, a key event in liver fibrogenesis. (Hepatology 2000;32:268-277.)

CCAAT/Enhancer Binding Protein β Mediates the Activation of the Murine α1(I) Collagen Promoter by Acetaldehyde

Acetaldehyde was previously shown to activate the α1(I) and α2(I) collagen promoters and to increase collagen production in activated stellate cells. Also, CCAAT/enhancer binding protein β (C/EBPβ) binds and activates the mouse α1(I) collagen promoter. This study investigates the role of C/EBPβ in mediating the activation of the α1(I) collagen promoter by acetaldehyde. Nuclear extracts isolated from cultured activated rat hepatic stellate cells formed four protein–DNA complexes on electrophoretic mobility shift assay with an oligonucleotide including the C/EBP binding site between −365 and −335 in the α1(I) collagen promoter. The four complexes were identified to represent C/EBPβ binding to the oligonucleotide by supershift with C/EBPβ antibody. The principal C/EBP isoform found in the nuclear extracts from stellate cells was C/EBPβ, with very low amounts of C/EBPα detected. Acetaldehyde (200 μM) increased C/EBPβ protein in stellate nuclear extracts, increased its binding to the promoter, and activated the α1(I) collagen promoter in transfected stellate cells. Mutation of the C/EBPβ binding site markedly decreased nuclear protein binding. A transfected promoter, mutated at the C/EBP binding site, had decreased basal activity, was not activated by acetaldehyde, and was not activated when cotransfected with a C/EBPβ expression vector. This study shows that C/EBPβ is the predominant C/EBP isoform found in activated stellate cells and that increased C/EBPβ protein and C/EBPβ binding to a proximal C/EBP binding site in the promoter mediates the activating effect of acetaldehyde.

Effect of pirfenidone, a novel antifibrotic agent, on PDGF-induced intracellular signaling in rat hepatic stellate cells

EFFECT OF PIRFENIDONE, A NOVEL ANTIFIBROTIC AGENT, ON PDGF-INDUCED INTRACELLULAR SIGNALING IN RAT HEPATIC STELLATE CELLS A. Di Sario, E. Bendia, G. Svegliati Baroni, S. Saccomanno 1, R. Ridolfi, U. Ugili t, L. Trozzi, A.M. Jezequel I, A. Benedetti Gastroenterology, University of Ancona, Ancona, Italy. 1Exp. Pathology, University of Ancona, Ancona, Italy. Pirfenidone (5 methyl-l-phonyl-2-(1H)-pyridone) is a potentially useful novel broad spectrum anfifibrotic agent which has been shown to inhibit fibroblast proliferation in response to nmneaetm growth factors, and to produce antitibrotic effects in a variety of nninml models. Aim of this study was to evaluate the effect o f pirfenidone on PDGF-mediated intracellular events in activated rat hepatic stellate cells (HSC). Incubation of HSC with PDGF-BB induced a six-fold increase in cell proliferation, evaluated by measuring bmmodeoxyur id i~ (BrdU) incorporation (12.4--t:I.2 vs 2.15±1.2 % BrdU positive cells in controls, P<0.001). The contemporaff incubation with pirfenidone, significantly inhibited PDGF-indueed HSC proliferation starting at a concentration of 1 pM (7.0~1.6; P<0.01), with a maximal effect at 1000 ~ (1.9-a:0.4; P<0.001). Measmement of LDH release in the culture medium, Uypan blue exclusion test and electron microscopy showed no cytotoxic effect of pirfenidone at coneenlrations that inhibited HSC proliferation (1, 10, 100, 500 and 1000 ldVl). The inhibition of P D G F i n d m ~ HSC proliferation was not associated with variations in PDGF-receptor autophosphorylation. Moreover, incubation with pirfenidoue did not inhibit PDGF-induced ERKI aml ERK2 activation, as well as pTOS6k (a downstream component of the PI-3 kinn.qe pathway) phosphorylation. On the contrary, piffenidone was able to inhibit PDGF-indueed activation of the Na+/H + exchange, which plays an important role in regadafing PDGF-induced HSC proliferation, with a maximal effect at I000 pM. These results suggest that l~fenidone could be a new candidate for antifibmtic thexa W in chronic liver diseases. Molecular and cell biology (gene expression, signalling, fibrosis)

Activated rat hepatic stellate cells bear functionally active somatostatin receptors

Activated rat hepatic stellate cells bear functionally active somatostatin receptors

Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells.

The tissue repair response to hypoxic stimuli during wound healing includes enhanced production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Hepatic stellate cells are oxygen-sensing cells, capable of producing VEGF. We hypothesized that hypoxia-stimulated signaling in activated stellate cells mediate VEGF secretion during liver injury. The specific aim was to evaluate the effect of hypoxia on the gene expression of VEGF in HSC-T6 cells, an immortalized rat hepatic stellate cell line, and in rat primary cultures of stellate cells. Hypoxic induction of VEGF mRNA was dose- and time-dependent. The hypoxic stimulation of VEGF messenger RNA (mRNA) correlated with the secretion of VEGF protein in conditioned media by hypoxic T6 cells. S-Nitroso-N-acetyl-D, L-penicillamine (SNAP), a nitric oxide (NO) donor, and desferrioxamine (DFx) and cobalt chloride, mimics of cellular hypoxia, similarly stimulated VEGF mRNA expression and secretion. Four previously described splice variants of the VEGF mRNA (VEGF-120, 144, 164, 188) were detected in both normoxic- or hypoxic-activated stellate cells. There was differential expression of the VEGF receptors, Flt-1 and Flk-1, in hypoxic T6 cells. Hypoxic conditions selectively stimulated Flt-1 mRNA expression, whereas Flk-1 mRNA remained unchanged. Hypoxic induction of VEGF was also demonstrated in primary stellate cell cultures and after in vivo injury. Hypoxia stimulates cell signaling in stellate cells, culminating in the rapid induction of VEGF and Flt-1 mRNA expression and VEGF secretion. The hypoxic induction of VEGF is mimicked by NO and may be of mechanistic importance in the pathogenesis of hepatic wound healing and hepatocarcinogenesis.

Effects of retinoic acid on proliferation, phenotype and expression of cyclin-dependent kinase inhibitors in TGF-beta1-stimulated rat hepatic stellate cells.

AIM:To study the molecular mechanisms of retinoic acid (RA)on prolix-feration and expression of cyclin-dependent kinase inhibitors (CKI), i.e.p16, p21 and p27 in cultured rat hepatic stellate cells (HSC) stimulated with transforming growth factor beta 1 (TGF-beta1).METHODS:HSC were isolated from healthy rat livers and cultured.After stimulated with 1mg/L TGF-beta1, subcultured HSC were treated with or without 1nmol/L RA. MTT assay, immunocytochemistry (ICC) for p16, p21, p27 and alpha-smooth muscle actin (alpha-SMA) protein, in situ hybridization (ISH) for retinoic acid receptor beta 2 (RAR-beta2) and p16, p21 and p27 mRNA and quantitative image analysis (partially) were performed.RESULTS:inhibited HSC proliferation (41.50%,P<0.05),decreased the protein level of alpha-SMA (55.09%, P<0.05), and induced HSC to express RAR-beta2 mRNA. In addition, RA increased the protein level of p16 (218.75%, P <0.05) and induced p21 protein expression; meanwhile, p27 was undetectable by ICC in both control and RA-treated HSC. However, RA had no influence on the mRNA levels of p16, p21 or p27 as determined by ISH.CONCLUSION:Up-regulation of p16 and p21 on post-transcriptional level may contribute, in part, to RA inhibition of TGF-beta 1-initiated rat HSC activation in vitro.

Glutathione Levels Discriminate between Oxidative Stress and Transforming Growth Factor-β Signaling in Activated Rat Hepatic Stellate Cells

Reactive oxygen species are implicated in the pathogenesis of several diseases, including Alzheimer's disease, multiple sclerosis, human immunodeficiency virus, and liver fibrosis. With respect to liver fibrosis, we have investigated differences in antioxidant enzymes expression in stellate cells (SCs) and parenchymal cells from normal and CCl(4)-treated rat livers. We observed an increase in the expression of catalase in activated SCs. Treatment with transforming growth factor-beta (TGF-beta) increased the production of H(2)O(2). Treatment with catalase decreased TGF-beta expression. Addition of H(2)O(2) resulted in increased TGF-beta production. 3-Amino-1,2,4-triazole abolished the capacity of SCs to remove H(2)O(2). A paradoxical increase in capacity was observed when the cells were pretreated with diethyl maleate. Treatment with 3-amino-1, 2,4-triazole increased TGF-beta production. A paradoxical decrease of TGF-beta production was observed with diethyl maleate. Treatment of the cells with N-acetylcysteine resulted in increased TGF-beta production. TGF-beta decreased the capacity of the SCs to remove H(2)O(2.) An increase in the capacity to remove H(2)O(2) was observed when TGF-beta was removed by neutralizing antibodies. In conclusion, our results suggest: 1) a link between cellular GSH levels and TGF-beta production and 2) that cellular GSH levels discriminate whether H(2)O(2) is the result of oxidative stress or acts as second messenger in the TGF-beta signal transduction pathway.

Role of Rho small GTP binding protein in the regulation of actin cytoskeleton in hepatic stellate cells

Backgroundl Aims: In the fibrotic response to liver injury, hepatic stellate cells are activated, leading to the myofibroblastic cell shape, with actin cytoskeletal reorganization and increased extracellular matrix production. The reorganization of actin cytoskeleton suggests that the small GTP binding protein Rho might modulate the process of this myofibroblastic change. The aim of this study was to investigate the role of Rho in the phenotypic changes of hepatic stellate cells. Methods: The phenotypic changes were investigated by the overexpression of Rho regulator, Rho GDI or dominant negative mutant of Rho in mouse hepatic stellate cell line, GRX cells. In activated rat hepatic stellate cells, the effects of microinjection of Botulinus toxin C3, which is the specific inhibitor for Rho, were analyzed. Furthermore, the effect of C3 on the type I collagen accumulation in hepatic stellate cells was investigated. Results: Overexpression of Rho GDI or the dominant negative mutant of Rho caused the shrinkage cell shape and suppressed stress fiber formation. Microinjection of toxin C3 caused a markedly distorted cell shape and the disappearance of stress fibers in rat stellate cells. In addition, C3 strongly suppressed collagen accumulation in activated stellate cells. Conclusions: These results suggest that Rho regulates the actin cytoskeletal reorganization, and may be implicated in the collagen accumulation in activated stellate cells. These findings provide evidence for the role of Rho in the myofibroblastic phenotype in hepatic stellate cells.

Transforming growth factor beta and tumor necrosis factor alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells.

Transforming growth factor beta (TGF-beta) as well as tumor necrosis factor alpha (TNF-alpha) gene expression are up-regulated in chronically inflamed liver. These cytokines were investigated for their influence on apoptosis and proliferation of activated hepatic stellate cells (HSCs). Spontaneous apoptosis in activated HSC was significantly down-regulated by 53% +/- 8% (P <.01) under the influence of TGF-beta and by 28% +/- 2% (P <.05) under the influence of TNF-alpha. TGF-beta and TNF-alpha significantly reduced expression of CD95L in activated HSCs, whereas CD95 expression remained unchanged. Furthermore, HSC apoptosis induced by CD95-agonistic antibodies was reduced from 96% +/- 2% to 51 +/- 7% (P <.01) by TGF-beta, and from 96% +/- 2% to 58 +/- 2% (P <.01) by TNF-alpha, suggesting that intracellular antiapoptotic mechanisms may also be activated by both cytokines. During activation, HSC cultures showed a reduced portion of cells in the G0/G1 phase and a strong increment of G2-phase cells. This increment was significantly inhibited (G1 arrest) by administration of TGF-beta and/or TNF-alpha to activated cells. In liver sections of chronically damaged rat liver (CCl4 model), using desmin and CD95L as markers for activated HSC, most of these cells did not show apoptotic signs (TUNEL-negative). Taken together, these findings indicate that TGF-beta and/or TNF-alpha both inhibit proliferation and also apoptosis in activated HSC in vitro. Both processes seem to be linked to each other, and their inhibition could represent the mechanism responsible for prolonged survival of activated HSC in chronic liver damage in vivo.

Increased 9,13-di- cis-retinoic acid in rat hepatic fibrosis: implication for a potential link between retinoid loss and TGF-β mediated fibrogenesis in vivo

Background/Aims: During hepatic fibrosis, hepatic stellate cells (HSCs) transform into myofibroblastic cells and lose their intracellular droplets of retinyl esters, the storage form of vitamin A. Recently, we have demonstrated that 9,13-di- cis-retinoic acid (RA), a geometric isomer identified as a stable and major metabolite of vitamin A in circulation, stimulates the synthesis of plasminogen activator (PA) and induces PA/plasmin-dependent latent transforming growth factor (TGF)-β activation in HSC cultures, probably via induction and activation of RA receptor (RAR) α. The aim of the present study was to address a potential link between the loss of retinyl esters to increased formation of RA(s), which might play a role in facilitating TGF-β-mediated liver fibrogenesis in vivo. Methods: We examined the effect of 9,13-di- cis-RA on transactivating activity of RARα in HeLa cells as well as its effect on PA- and TGF-β-dependent collagen synthesis in rat and human HSC cultures. We measured the changes in 9,13-di- cis-RA levels both during activation of rat HSCs in vitro and during porcine serum-induced rat hepatic fibrosis in vivo and correlated this with RAR α/β, PA, TGF-β and type I procollagen mRNA expression in the fibrotic liver. Results: 9,13-di- cis-RA transactivated RARα, and provoked PA/plasmin and TGF-β-dependent procollagen synthesis in HSCs. 9,13-di- cis-RA levels were increased both in activated HSCs in vitro and in fibrotic liver accompanying the enhanced expression of RAR α/β, PA, TGF-β and procollagen in vivo. Conclusions: These findings suggest a potential link between 9,13-di-cis RA formation and hepatic fibrosis via formation of TGF-β in vivo, and thus provide further insight into the biologic role of retinoids during hepatic fibrogenesis.

Decreased expression of smooth muscle α actin in activated rat hepatic stellate cells at the S-phase of the cell cycle in vitro

Hepatic stellate cells contain actin and myosin, and can contract in nature. With hepatocyte injury, these cells are activated to proliferate, change to a myofibroblast-like phenotype and produce extracellular matrices including collagens. This activation is accompanied by increased expression of cellular smooth muscle α actin. Generally, cells fail to exert specific functions during proliferation. The function of activated hepatic stellate cells was investigated in relation to the cell cycle. Hepatic stellate cells were isolated from normal rats and activated by culture on plastic dishes for 7 days. Western and Northern blot analyses revealed that these cells markedly expressed smooth muscle α actin and transforming growth factor-beta (TGF-β) mRNA. When recombinant human platelet derived growth factor (PDGF)-BB was added to the culture medium, such smooth muscle α actin expression was decreased in a dose-related manner, but TGF-β mRNA expression increased. DNA synthesis by these cells was also increased, but collagen synthesis was unchanged. Immunocytochemically, apparent staining of smooth muscle α actin was seen in 9% of 5-bromo-2′-deoxyuridine (BrdU)-positive cells, but in 80% of BrdU-negative cells. We conclude that hepatic stellate cells at the S-phase of the cell cycle after activation by culture express no, or less, smooth muscle α actin.

Organic osmolyte transport in quiescent and activated rat hepatic stellate cells (Ito cells).

Activation of hepatic stellate cells (HSCs) results in multiple alterations of cell function, but nothing is known about organic osmolytes in these cells. Organic osmolyte transport and transporter messenger RNA (mRNA) expression was studied in quiescent rat HSCs and after their transformation into alpha1-smooth muscle actin-positive myofibroblastlike cells. Quiescent stellate cells expressed in an osmosensitive manner the mRNA levels of the transporters for taurine (TAUT) and myoinositol (SMIT), whereas that for betaine was not detectable. However, these cells showed osmosensitive uptake not only of taurine and myoinositol but also of betaine. Osmosensitive betaine uptake was mediated by amino acid transport system A. After transformation into myofibroblasts, taurine and myoinositol uptake increased 5.5-fold and 4.5-fold, respectively, together with the respective transporter mRNA levels. Betaine uptake increased twofold because of osmosensitive induction of BGT1 expression. In both quiescent and activated HSCs, hypoosmotic cell swelling induced a rapid and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid-sensitive osmolyte efflux. In quiescent HSCs, hyperosmotic exposure increased the messenger RNA (mRNA) level of cyclooxygenase-2, which was counteracted by taurine but not by betaine or myoinositol. The study identifies taurine, myoinositol, and betaine as osmolytes in HSCs. Transformation of HSCs is accompanied by enhanced osmolyte transport activity and induction of the BGT1 transporter, which may be another activation marker of HSCs.

Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor.

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFkappaB activity in activated but not in quiescent HSCs with subsequent expression of NFkappaB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IkappaB super-repressor on the cytokine mediated activation of NFkappaB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1beta or tumor necrosis factor alpha (TNFalpha) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IkappaB super-repressor (Ad5IkappaB) or beta-galactosidase (Ad5LacZ) as a control. NFkappaB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IkappaB protein was measured by Western Blot. ICAM-1 and IL-6 expression was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IkappaB, and the Ad5IkappaB, which provides an exogenous nondegradable IkappaB, block the stimulation of NFkappaB activity by TNFalpha and IL-1beta in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFkappaB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IkappaB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFkappaB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IkappaB in activated HSCs. Inhibition of IkappaBalpha degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.

Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells.

The aim of this study was to evaluate intracellular pH (pHi) regulation in nonactivated and activated rat hepatic stellate cells (HSC). The fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was used to measure pHi in the presence and absence of HCO3-. In the absence of HCO3-, baseline pHi was significantly higher (P < 0.001) in activated than in nonactivated HSC (7.1 +/- 0.1 vs. 6.9 +/- 0.2) and decreased, in both groups, after amiloride administration and after Na+ removal. After an acid-loading maneuver, pHi recovery was significantly higher (P < 0.03) in activated than in nonactivated HSC (H+ flux = 11.0 +/- 3.8 vs. 7.7 +/- 2.9 mM/min at pHi 6.6) and was inhibited by amiloride and Na+ removal. In the presence of HCO3-, baseline pHi was higher in both groups and decreased after amiloride administration. Amiloride and Na+ removal inhibited pHi recovery after an intracellular acid load by 77 and 93%, respectively, in nonactivated and by 82 and 92%, respectively, in activated HSC, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited pHi recovery by only 27%. Acute Cl- removal increased pHi by 0.07 +/- 0.01 pH unit/min in the absence but not in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid in nonactivated and activated HSC in an Na(+)-independent manner. In activated HSC, 24 h of incubation with 25 ng/ml platelet-derived growth factor (PDGF)-BB (in 0.5% serum) did not modify baseline pHi (7.07 +/- 0.1 vs. 7.08 +/- 0.1 in HSC cultured in 0.5% serum only) but significantly (P < 0.02) increased, with respect to controls, pHi recovery after an acute acid load. Incubation with PDGF for 24 h induced a fivefold increase in HSC proliferation expressed as percentage of bromodeoxyuridine-positive cells (30.8 +/- 6.7 vs. 6.1 +/- 1.9% in controls). When amiloride (0.1 mM) was present, PDGF-induced HSC proliferation was significantly inhibited (8.1 +/- 0.4%, P < 0.001). Our results show that 1) the Na+/H+ exchanger is the main pHi regulator in rat HSC, 2) activation of HSC is associated with an increase in pHi and in the activity of the Na+/H+ exchanger, 3) PDGF increases the activity of this exchanger, and 4) amiloride is able to inhibit HSC proliferation induced by PDGF.

Receptor tyrosine kinase expression, a central component of rat hepatic stellate cell activation - characterization by homology PCR

SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.