Incubation Of Hepatic Stellate Cells Research Articles

Antifibrogenic and apoptotic effects of Ocoxin in cultured rat hepatic stellate cells.

Ocoxin is a nutritional supplement that has been shown to exert antioxidant and immunomodulatory responses in patients with chronic hepatitis C. The present work aimed to determine the effects of Ocoxin on activated hepatic stellate cells (HSC), the cell type mainly responsible for collagen deposition in the fibrotic liver. Ocoxin was found to reduce the survival of a cell line of immortalized non-tumoral rat HSC in a dose-response fashion and to diminish collagen type I levels. This latter effect was observed even at doses not affecting cell survival, pointing to an antifibrogenic action for the supplement. The decrease in viability exerted by Ocoxin on HSC correlated with an increase in histone-associated fragments in the cytoplasm and with increased activity of caspase-3, indicating the induction of apoptosis. To determine the molecular mechanisms mediating Ocoxin-induced apoptosis, the activation of members of the MAPK family was analyzed. Incubation of HSC with Ocoxin caused a transient and dramatic enhancement on ERK, JNK, and p38 MAPK phosphorylation levels. Using specific inhibitors for these enzymes, p38 MAPK was identified as a key mediator of the apoptotic effect of Ocoxin on HSC.

Human umbilical cord perivascular cells-derived extracellular vesicles mediate the transfer of IGF-I to the liver and ameliorate hepatic fibrogenesis in mice.

Extracellular vesicles (EVs) can mediate mesenchymal stromal cells (MSCs) paracrine effects. We aimed to evaluate the therapeutic potential of human umbilical cord perivascular cells (HUCPVCs) engineered to produce Insulin Growth Factor like-I (IGF-I) in experimental liver fibrosis and the role of EVs in this effect. HUCPVCs were engineered to produce human IGF-I (AdhIGF-I) or green fluorescence protein (AdGFP) using adenoviruses, and EVs were isolated from their conditioned medium (CM). In vitro effects of CM and EVs on hepatic stellate cells and hepatic macrophages were studied. Cells or EVs-based treatments were evaluated in thioacetamide-induced liver fibrosis in mice. The application of AdhIGF-I-HUCPVCs resulted in a further amelioration of liver fibrosis when compared to AdGFP-HUCPVCs and saline. Similarly, treatment with AdhIGF-I-HUCPVCs-derived EVs resulted in a reduction of collagen deposition and gene expression of the fibrogenic related molecules TGF-β1, α-SMA, and COL1A2. In vitro incubation of hepatic stellate cells with EVs-AdhIGF-I-HUCPVCs significantly reduced activation of fibrogenic cells. In addition, EVs-AdhIGF-I-HUCPVCs trigger hepatic macrophages to switch their phenotype towards anti-inflammatory phagocytes, which might be involved in the antifibrotic effect. Consistently, high levels of IGF-I were observed within EVs-AdhIGF-I-HUCPVCs but not in controls EVs. Our results showed that hIGF-I carrying EVs could mediate the paracrine mechanism by which AdhIGF-I-HUCPVCs reduce liver fibrosis.

Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.

Tenascin-C: A Crucial Proinflammatory Mediator in Macrosteatotic Liver Ischemia and Reperfusion Injury.

A shortage of organ donors has led to an increased use of steatotic livers in transplantation; however, marginal livers have higher susceptibility to ischemia and reperfusion injury (IRI). Tenascin-C (Tnc) is an extracellular matrix protein (ECM) involved in various aspects of immunity, including liver injury. Here, we dissect the functional significance of high levels of Tnc in steatotic hepatic IRI. Methods: Tnc deficient (Tnc -/-) knockout (KO) mice (C57BL/6N-TgH) and matched wild-type (WT) control littermates were fed with a high-fat diet (60% total-kilocalories); mice with about 50% macrovesicular steatosis (MaS) were submitted to 60% of partial warm ischemia, followed by reperfusion. Results: While Tnc is not normally expressed in adult tissues, it was readily detected in naïve steatotic livers and further upregulated in MaS-WT livers post-IRI. Compared to MaS-WT mice, MaS-Tnc-/-mice showed significantly reduced liver damage; Tnc ablation resulted in decreased AST levels (IU/L) at 6h (4,920±1,974 vs. 7,698±1,124 p<0.05) and 24h (990±801 vs. 6,665±3,485, p<0.05) post-IRI. Moreover, histological damage was selectively sheltered in Tnc-KO livers, which showed reduced periportal congestion and necrosis compared to MaS-WT controls. The recruitment of Ly6G (6h: 50.9±13.8 vs. 106.0±39.0; 24h:74.1±28.3 vs. 215.4±24.5; p<0.05) and Mac-1 (6h:48.5±25.7 vs. 82.9±17.4; 24h:84.7±30.7 vs. 132.8±33.8;p<0.05) leukocytes was significantly decreased in MaS Tnc KO livers. MaS-Tnc KO livers were also characterized by decreased expression of pro-inflammatory TNF-α (p<0.05) and IL-1β (p<0.05), compared to MaS-WT controls. Next, we performed a series of in vitro studies, which identified hepatic stellate cells (HSCs) as primary sources of Tnc in our settings and provided evidence of a fat-dependent mechanism for Tnc upregulation by these cells. Indeed, the incubation of HSCs in conditioned medium from steatotic hepatocytes resulted in the upregulation of Tnc expression (˜4-fold-increase), compared with HSCs cultured in medium from non-steatotic hepatocytes. Conclusion: Our results evidence a critical role for Tnc expression in the pathogenesis of steatotic hepatic IRI. They also support the view that studies aimed at further understanding how ECM molecules, such as Tnc, participate in steatotic liver injury will likely contribute to the successful utilization of marginal steatotic livers in transplantation.

Butein inhibits ethanol-induced activation of liver stellate cells through TGF-β, NFκB, p38, and JNK signaling pathways and inhibition of oxidative stress

BackgroundButein has been reported to prevent and partly reverse liver fibrosis in vivo; however, the mechanisms of its action are poorly understood. We, therefore, aimed to determine the antifibrotic potential of butein.MethodsWe assessed the influence of the incubation of hepatic stellate cells (HSCs) and hepatoma cells (HepG2) with butein on sensitivity to ethanol- or acetaldehyde-induced toxicity; the production of reactive oxygen species (ROS); the expression of markers of HSC activation, including smooth muscle α-actin (α-SMA) and procollagen I; and the production of transforming growth factor-β1 (TGF-β1), metalloproteinases-2 and -13 (MMP-2and MMP-13), and tissue inhibitors of metalloproteinases (TIMPs). The influence of butein on intracellular signals in HSCs; i.e., nuclear factor-κB (NFκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol was estimated.ResultsButein protected HSCs and HepG2 cells against ethanol toxicity by the inhibition of ethanol- or acetaldehyde-induced production of ROS when cells were incubated separately or in co-cultures; butein also inhibited HSC activation measured as the production of α-SMA and procollagen I. As well, butein downregulated ethanol- or acetaldehyde-induced HSC migration and the production of TGF-β, TIMP-1, and TIMP-2; decreased the activity of MMP-2; and increased the activity of MMP-13. In ethanol-induced HSCs, butein inhibited the activation of the p38 MAPK and JNK transduction pathways as well as significantly inhibiting the phosphorylation of NF κB inhibitor (IκB) and Smad3.ConclusionsThe results indicated that butein inhibited ethanol- and acetaldehyde-induced activation of HSCs at different levels, acting as an antioxidant and inhibitor of ethanol-induced MAPK, TGF-β, and NFκB/IκB transduction signaling; this result makes butein a promising agent for antifibrotic therapies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00535-012-0619-7) contains supplementary material, which is available to authorized users.

A novel in vitro model of alcohol-induced hepatic fibrogenesis

Chronic alcohol consumption is one of the main etiological factors for liver disease worldwide, however, only a fraction of drinkers develops significant hepatic inflammation, and even less progresses to significant hepatic fibrosis and cirrhosis. Still, there are no experimental models that lead to relevant hepatic fibrosis, and there is a high need to unravel the mechanisms and factors promoting hepatic injury in alcoholic liver disease (ALD).The aim of this study was to establish an in vitro model to study the hepatocyte mediated effects of alcohol on hepatic stellate cells (HSCs), the central mediators of liver fibrogenesis. Methods and Results: HepG2 hepatoma cells and primary human hepatocytes (PHH) were incubated with different concentrations of alcohol (0.1–1%) for 48h, which did not affect cell viability or mitochondrial activity but led to dose-dependent hepatocellular lipid accumulation as assessed by analysis of triglyceride content and free fatty acid levels, and as visualized by oil red o staining. Subsequently, human HSC were incubated with conditioned media (CM) from alcohol stimulated (Alc) or control hepatocytes. Further controls included control medium with and without alcohol (at concentrations determined in the CM from Alc-treated cells). Noteworthy, incubation of HSC with CM from Alc-treated hepatocytes significantly induced proliferation as well as pro-fibrogenic (TGF-beta) and NFkapaB-mediated pro-inflammatory gene (IL-8, RANTES) expression compared to the control groups. Conclusion: Our in vitro data indicate release of soluble factors as a potential mechanism for development and progression of fibrogenesis in alcoholic liver disease, the identification of which may lead to novel prognostic markers. Thus, this study provides an attractive in vitro model to study molecular mechanisms of alcohol-induced fibrogenesis, and to identify and test novel targets for anti-fibrotic therapies in alcoholic liver disease.

Gc-globulin (vitamin D binding protein) is synthesized and secreted by hepatocytes and internalized by hepatic stellate cells through Ca 2+-dependent interaction with the megalin/gp330 receptor

Background Gc-globulin or vitamin D binding protein is a highly expressed, multifunctional and polymorphic serum protein, which also serves as the major transporter for vitamin D metabolites in the circulation. The present study was performed to analyze the interaction between gc-globulin of hepatocytes and hepatic stellate cells, the most important fat-/retinol-storing cell type in the liver, which spontaneously transdifferentiates to myofibroblasts in culture. Methods Hepatic stellate cells and hepatocytes were isolated by the pronase/collagenase reperfusion method, hepatocytes by collagenase reperfusion of the organ. Gc-globulin expression was monitored by immunocytochemistry, immunoblotting, RT-PCR, metabolic labelling with [ 35S]-methionine, and its intracellular binding to α-smooth-muscle actin was investigated by co-immunoprecipitation. Cytoskeletal stainings of gc-globulin and α-smooth-muscle actin in hepatic stellate cells and the identification of the receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2 were performed with confocal immunocytochemistry, immunoblotting and/or FACS-analysis. Results Hepatocytes synthesize and secrete gc-globulin as shown by RT-PCR and [ 35S]-methionine labelling, which could be suppressed by cycloheximide. Also, a strong signal for gc-globulin was detected in the immunoblot of native hepatic stellate cell lysates. However, no mRNA for gc-globulin was found in this cell type, which suggests no active synthesis by hepatic stellate cells. Hepatic stellate cells were tested positively for the presence of known gc-globulin interacting receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2. Inhibition of the megalin/gp330 receptor by a competitive, neutralizing antibody resulted in decreased intracellular availability of gc-globulin in hepatic stellate cells. The latter effect was enhanced by additional incubation of hepatic stellate cells with EDTA for complexing Ca 2+, suggesting a Ca 2+-dependent internalization of gc-globulin into hepatic stellate cells via the megalin/gp300 receptor. This was supported by confocal microscopy which showed a co-localization of gc-globulin with the multifunctional megalin/gp330 receptor on this cell type. Inside hepatic stellate cells, a linkage between gc-globulin and α-smooth muscle actin filaments of hepatic stellate cells was detected by immunocytochemistry. Intracellular binding of gc-globulin to α-smooth-muscle actin filaments was confirmed by co-immunoprecipitation. Conclusion We give evidence to the expression of the megalin/gp330 receptor on hepatic stellate cells and that this receptor is involved in the Ca 2+-dependent internalization of gc-globulin into hepatic stellate cells, a protein synthesized and secreted into the extracellular space and circulation by hepatocytes. Inside hepatic stellate cells, it co-localizes with and binds to α-smooth muscle actin filaments. Under consideration of the available literature, these findings propose a participation of gc-globulin in hepatic vitamin D metabolism as well as in hepatic stellate cell stability and apoptosis as important mechanisms of liver regeneration.

Effect of fetuin, a TGFβ antagonist and pentoxifylline, a cytokine antagonist on hepatic stellate cell function and fibrotic parameters in fibrosis

We have previously shown that monocyte conditioned medium (MCM) from patients with liver fibrosis stimulated proliferation of hepatic stellate cells (HSCs), the major cell involved in hepatic fibrosis. To investigate the potential role of fetuin and pentoxifylline in fibrosis we used MCM samples obtained from patients with biopsy proven hepatic fibrosis related to Hepatitis C (HCV). Our results indicate that the MCM obtained from patients with HCV-related liver fibrosis significantly stimulated collagen synthesis in HSCs as assessed by tritiated proline incorporation into a collagenase sensitive trichloroacetic acid (TCA) precipitate. Collagen synthesis was also stimulated in HSCs using transforming growth factor beta (TGFbeta) and this effect was neutralized using TGFbeta antibody. Incubation of HSCs with fetuin (but not TGFbeta antibody) significantly inhibited collagen synthesis in HSCs that were stimulated by HCV MCM samples. Patient MCM samples would also stimulate proliferation of HSCs as assessed by tritiated thymidine uptake but this effect was not attenuated by fetuin. Likewise the significant stimulatory effect of platelet derived growth factor (PDGF) on HSC proliferation and collagen synthesis was not inhibited by fetuin but could be significantly reduced by 70% and 40% respectively, when treated with pentoxifylline. We also investigated the ability of samples obtained from patients with hepatic fibrosis to inhibit HSC apoptosis, as determined by okadaic acid-induced 4-hydroxynonenal immunocytochemistry in HSCs. We have previously reported that okadaic acid induces apoptosis in HSCs as assessed by Hoescht and TUNEL. Okadaic acid treatment produced a positive 4-hydroxynonenal (4-HNE) immunoreactivity in HSCs and treatment with HCV patient MCM or TGFbeta decreased the 4-HNE positive immunoreactivity in HSCs treated with okadaic acid. Our results suggest that fetuin may be beneficial in hepatic fibrosis and suggest that combination of fetuin and pentoxifylline may target the two key events in hepatic fibrosis by modifying the effects of TGFbeta and PDGF, the two major growth factors in fibrosis.

Effects of radix salviae miltiorrhizae on endothelin-1-induced calcium changes in hepatic stellate cells

AIM: To investigate the effect of radix salviae miltiorrhizae (RSM) on the changes of [Ca]i induced by endothelin-1 (ET-1) in hepatic stellate cells (HSCs). METHODS: After preparation of RSM cream, the effects of RSM on ET-1-induced changes of HSCs [Ca]i were observed by laser scanning confocal microscopy. RESULTS: In normal buffer (including Ca, buffer A), the fluorescence intensity was enhanced accordingly with the increase of ET-1 concentration. The cumulative-response curve showed EC50 was 1.1×10 mol/L. After incubation of HSCs with ET-1 in buffer A and buffer B (absence of extracellular calcium, EGTA), the duration of calcium peak had significant difference (165.2 ± 10.1 s vs 91.0 ± 7.2 s, P 0.05). In the cells pre-incubated with RSM, KCl-induced elevation of [Ca]i was decreased, and the calcium peak value (78.0% ± 6.1% → 26.3% ± 1.2%, P < 0.01) and duration of calcium wave (70.8 ± 10.4 s → 15.9 ± 5.1 s, P < 0.01) were decreased significantly. CONCLUSION: RSM inhibits ET-1-induced depletion of intracellular calcium, which has no correlations with the influx of extracellular calcium. RSM can also inhibit KCl-induced influx of calcium, indicating its characteristic of blocking voltage-operated Ca channel.

Effect of cyanidin 3- O-β-glucopyranoside on hepatic stellate cell proliferation and collagen synthesis induced by oxidative stress

Background. Reactive oxygen species play a role in the pathogenesis of hepatic fibrosis, mainly through the activation of hepatic stellate cells. Cyanidin-3- O-β-glucopyranoside is a natural antioxidant compound distributed in several fruits and vegetables. Aim. To evaluate the effect of cyanidin-3- O-β-glucopyranoside on hepatic stellate cells proliferation and collagen synthesis induced by a pro-oxidant agent. Methods/results. Oxidative stress was induced by incubation of hepatic stellate cells with a ferric nitrilotriacetate complex (100 μmol/L). Incubation with ferric nitrilotriacetate induced an increased intracellular hydroperoxide formation, which was completely inhibited by cyanidin-3- O-β-glucopyranoside at a concentration of 50 μmol/L. Similarly, cyanidin-3- O-β-glucopyranoside was able to inhibit ferric nitrilotriacetate-induced hepatic stellate cells proliferation, evaluated by an ELISA method, with a maximal effect at 50 μmol/L. Incubation of hepatic stellate cells with cyanidin-3- O-β-glucopyranoside inhibited ferric nitrilotriacetate-induced extracellular signal-regulated kinase 1/2 activation, evaluated by western blot, whereas it did not affect p70S6 kinase and AKT expression. Finally, cyanidin-3- O-β-glucopyranoside reduced ferric nitrilotriacetate-induced Na +/H + exchange activation, evaluated by a spectrofluorimetric method, and collagen type I synthesis, evaluated by northern blot. Conclusion. Cyanidin-3- O-β-glucopyranoside is able to modulate hepatic stellate cells proliferation and type I collagen synthesis induced by a pro-oxidant agent, thus suggesting a potential role for this antioxidant compound in the prevention of fibrosis in chronic liver diseases.

Effects and regulation of osteopontin in rat hepatic stellate cells

Using a cDNA microarray, we identified osteopontin (OPN) as one of the genes upregulated in cultured activated hepatic stellate cells (HSCs). Northern and western blot analyses showed that OPN was increasingly expressed during the progressive activation of cultured rat HSCs, and a significant increase in OPN was observed in carbon tetrachloride-induced rat liver fibrosis. In biliary atresia, OPN protein was predominantly expressed in Kupffer cells and HSCs in the necrotic areas. Incubation of HSCs with recombinant OPN-induced significant proliferative and migratory effects, and induced matrix metalloproteinase 2 production and activation. Moreover, OPN increased type I collagen production and type II transforming growth factor-β receptor mRNA and protein. In conclusion, this study shows that OPN is expressed in activated HSCs and suggests that the upregulation of OPN might be a central pathway of HSC activation.

Somatostatin suppresses endothelin-1–induced rat hepatic stellate cell contraction via somatostatin receptor subtype 1

Background & Aims: Hepatic stellate cells (HSCs) are considered therapeutic targets to decrease portal hypertension. To elucidate some of the hemodynamic effects of somatostatin (SST) on portal pressure, the presence and function of SST receptors (SSTRs) on HSCs were investigated. Methods: SSTR messenger RNA expression, and SSTR presence was investigated using reverse-transcription polymerase chain reaction, real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The function of SSTRs was studied by examining the effects of SST and specific SSTR agonists on endothelin-1(ET-1)–induced HSC contraction. Results: Specific amplicons for SSTR subtypes 1, 2, and 3 were demonstrated in rat liver and in activated HSCs. The presence of SSTR subtypes 1, 2, and 3 was confirmed by Western blotting. With immunohistochemistry, a strong staining of HSCs was obtained for SSTR subtypes 1, 2, and 3 in CCl4-treated rats, but not in normal rat liver. Incubation of HSCs on collagen gels with buffer, 10−8 mol/L SST, and 2 × 10−8 mol/L ET-1 resulted in collagen surface area decreases of 5.5% ± 3.3%, 6.8% ± 4.4%, and 49.8% ± 8.3%, respectively. Relative contraction of gels preincubated with 10−8 mol/L SST followed by 2 × 10−8 mol/L ET-1 or vice versa as compared with maximal contraction (100%) with 2 × 10−8 mol/L ET-1 were 72.6% ± 17.9% and 76.2% ± 12.6%, respectively (P < 0.05). SSTR agonist 1, but not SSTR agonist 2 or 3, was able to counteract the contractile effect of ET-1. Conclusions: Activated rat HSCs bear SSTR subtypes 1, 2, and 3. SST causes significant partial inhibition of ET-1–induced contraction of activated HSCs, mainly by stimulation of SSTR subtype 1.GASTROENTEROLOGY 2001;121:915-930

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)

Intracellular pathways mediating Na +/H + exchange activation by platelet-derived growth factor in rat hepatic stellate cells

Background & Aims: The Na +/H + exchanger is the main intracellular pH regulator in hepatic stellate cells (HSCs), and its activity is increased by platelet-derived growth factor (PDGF). Amiloride, an Na +/H + exchange inhibitor, reduces PDGF-induced HSC proliferation, suggesting that the Na +/H + exchanger plays a role in regulating HSC proliferative response. The aim of this study was to characterize the intracellular pathways mediating activation of the Na +/H + exchanger by PDGF in HSCs. Methods: The activity of the Na +/H + exchanger and HSC proliferation rate were evaluated under control condition and after incubation with PDGF in the absence or presence of specific inhibitors of the main intracellular pathways of signal transduction. Na +/H + exchange protein expression was evaluated by means of Western blot. Results: PDGF induced a significant increase in the activity of the Na +/H + exchanger without modifying protein expression. Inhibition of the calcium/calmodulin– and protein kinase C–dependent pathways resulted in a significant inhibition of both Na +/H + exchange activity and of PDGF-induced HSC proliferation. The involvement of the two pathways was confirmed by showing that incubation of HSCs with both phorbol-12-myristate-13-acetate, a potent protein kinase C activator, and thapsigargin, which increases intracellular calcium levels, significantly increased both the Na +/H + exchanger activity and HSC proliferation rate. Inhibition of the protein kinase A pathway did not modify either PDGF-induced Na +/H + exchange activation or PDGF-induced HSC proliferation. On the contrary, inhibition of the mitogen-activated protein kinase– and of phosphatidylinositol 3-kinase–dependent pathways significantly reduced PDGF-induced HSC proliferation without affecting the activity of the Na +/H + exchanger. Conclusions: Activation of the Na +/H + exchanger by PDGF in HSCs is mediated by calcium/calmodulin- and protein kinase C–dependent pathways. PDGF-induced HSC proliferation is mediated by Na +/H + exchange–dependent and –independent pathways. GASTROENTEROLOGY 1999;116:1155-1166

Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells

BACKGROUND & AIMS: Platelet-derived growth factor (PDGF) is the most potent mitogen for hepatic stellate cells (HSCs) in vitro. The aim of this study was to investigate the role of phosphatidylinositol 3-kinase (PI 3-K) activation in mediating the biological effects of PDGF on cultured HSCs and its involvement in vivo. METHODS: HSCs were isolated from normal human livers. PI 3-K was assayed on phosphotyrosine or PDGF- receptor immunoprecipitates by in vitro kinase assay. RESULTS: Incubation of HSCs with PDGF caused a time-dependent increase in PI 3-K activity. Immunoprecipitation of PDGF-alpha and -beta receptors showed that both subunits associate with active PI 3-K in PDGF-stimulated HSCs. Wortmannin, a specific PI 3-K inhibitor, dose-dependently blocked PI 3-K activity induced by PDGF and inhibited DNA synthesis. PDGF (homodimer)-BB also stimulated HSC chemotaxis, which was inhibited by pretreatment with wortmannin. To explore the potential role of PI 3-K in vivo, liver homogenates from rats treated with CCl4 and from control rats were immunoprecipitated with anti-PDGF-beta-receptor antibodies. Liver injury was associated with increased PDGF-beta-receptor autophosphorylation, and greater PI 3-K activity associated with the receptor itself. CONCLUSIONS: This study shows that in cultured HSCs, PI 3-K activation is necessary for both mitogenesis and chemotaxis induced by PDGF and that this pathway is up-regulated during liver injury in vivo. (Gastroenterology 1997 Apr;112(4):1297-306)