A. Gentilini

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.

PI-3-kinase and MAPK regulate mesangial cell proliferation and migration in response to PDGF

Proliferation and migration are important biological responses of mesangial cells to injury. Platelet-derived growth factor (PDGF) is a prime candidate to mediate these responses in glomerular disease. PDGF and its receptor (PDGFR) are upregulated in the mesangium during glomerular injury. We have recently shown that PDGF activates phosphatidylinositol 3-kinase (PI-3-kinase) in cultured mesangial cells. The role of this enzyme and other more distal signaling pathways in regulating migration and proliferation of mesangial cells has not yet been addressed. In this study, we used two inhibitors of PI-3-kinase, wortmannin (WMN) and LY-294002, to investigate the role of this enzyme in these processes. Pretreatment of mesangial cells with WMN and LY-294002 dose-dependently inhibited PDGF-induced PI-3-kinase activity assayed in antiphosphotyrosine immunoprecipitates. WMN pretreatment also inhibited the PI-3-kinase activity associated with anti-PDGFRβ immunoprecipitates prepared from mesangial cells treated with PDGF. Pretreatment of the cells with different concentrations of WMN resulted in a dose-dependent inhibition of PDGF-induced DNA synthesis. Both WMN and LY-294002 inhibited PDGF-stimulated migration of mesangial cells in a dose-dependent manner. It has recently been shown that PI-3-kinase physically interacts with Ras protein. Because Ras is an upstream regulator of the kinase cascade leading to the activation of mitogen-activated protein kinase (MAPK), we determined whether activation of PI-3-kinase is necessary for activation of MAPK. Pretreatment of mesangial cells with WMN and LY-294002 significantly inhibited PDGF-induced MAPK activity as measured by immune complex kinase assay of MAPK immunoprecipitates. Furthermore, PD-098059, an inhibitor of MAPK-activating kinase inhibited PDGF-induced MAPK activity and resulted in significant reduction of mesangial cell migration in response to PDGF. These data indicate that MAPK is a downstream target of PI-3-kinase and that both these enzymes are involved in regulating proliferation and migration of mesangial cells.

Phosphatidylinositol-3 kinase and extracellular signal-regulated kinase mediate the chemotactic and mitogenic effects of insulin-like growth factor-I in human hepatic stellate cells

BACKGROUND/AIM Several studies have shown that proliferation of hepatic stellate cells is stimulated by insulin-like growth factor-I. The aim of this study was to investigate the effect of insulin-like growth factor-I on human hepatic stellate cells chemotaxis and the intracellular pathways involved in both mitogenic and chemotactic effects. METHODS/RESULTS Insulin-like growth factor-I, at the concentration of 100 ng/ml, was able to induce a 2- to 3-fold increase in human hepatic stellate cells migration in a modified Boyden chamber system. This effect was associated with a marked activation of phosphatidylinositol 3-kinase by insulin-like growth factor-I, as evaluated by measurement of phosphatidylinositol 3-kinase activity in phosphotyrosine immunoprecipitates In order to establish a functional link between these observations, we then performed experiments employing two selective phosphatidylinositol 3-kinase inhibitors, namely wortmannin and LY294002. These compounds blocked activation of phosphatidylinositol 3-kinase and inhibited insulin-like growth factor-I-induced hepatic stellate cells migration. Since phosphatidylinositol 3-kinase activation has been shown to be necessary for platelet-derived growth factor-induced mitogenesis in hepatic stellate cells, we verified the effects of phosphatidylinositol 3-kinase inhibition on insulin-like growth factor-I-induced DNA synthesis. Incubation with either wortmannin or LY294002, dose-dependently reduced the mitogenic potential of insulin-like growth factor-I. Since phosphatidylinositol 3-kinase is involved, at least in part, in the activation of the Ras/extracellular signal-regulated kinase pathway in hepatic stellate cells, the role of extracellular signal-regulated kinase activation in mediating the biological effects of insulin-like growth factor-I was explored. Insulin-like growth factor-I induced mitogenesis and chemotaxis were markedly reduced by pre-incubation of hepatic stellate cells with PD-98059, a selective inhibitor of MEK. CONCLUSIONS Activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase is required for both insulin-like growth factor-I-dependent hepatic stellate cells proliferation and chemotaxis. Insulin-like growth factor-I, together with other soluble mediators, may contribute to the hepatic wound-healing response by modulating hepatic stellate cells migration and proliferation.

Inhibition by pentoxifylline of extracellular signal‐regulated kinase activation by platelet‐derived growth factor in hepatic stellate cells

1 It has been proposed that pentoxifylline (PTF) acts an antifibrogenic agent by reducing the synthesis of extracellular matrix components, and this possibility has been confirmed in animal models of hepatic fibrosis. In this study the effects of PTF on the proliferation of extracellular matrix producing cells induced by platelet‐derived growth factor (PDGF) were evaluated. The study was performed on hepatic stellate cells, currently indicated as the major source of extracellular matrix in fibrotic liver. 2 PTF caused a dose‐dependent reduction of PDGF‐induced mitogenesis with an IC50 of 170 μm, identical to the EC50 for the increase in intracellular cyclic AMP levels. Preincubation with PTF did not affect either PDGF‐receptor autophosphorylation or phosphotidylinositol 3‐kinase activity, whereas it markedly reduced PDGF‐stimulated extracellular signal‐regulated kinase (ERK) activity and ERK isoform phosphorylation. PTF also reduced PDGF‐induced c‐fos mRNA expression, which is dependent on activation of the RAS/ERK pathway. In addition, the PDGF‐induced increase in cytsolic‐free calcium was almost completely prevented by pretreating the cells with PTF. 3 The results of the present study indicate that PTF, in addition to its effect on collagen deposition and degradation, may exert an antifibrogenic effect by reducing the PDGF‐induced proliferation of extracellular matrix producing cells. This effect appears to be mediated by a reduction of PDGF‐stimulated ERK activity as well as of other intracellular signalling pathways such as the PDGF‐induced elevation of cytosolic‐free calcium.

Role of the stromal-derived factor-1 (SDF-1)-CXCR4 axis in the interaction between hepatic stellate cells and cholangiocarcinoma

BACKGROUNDS & AIMS Cholangiocarcinoma (CCA) is highly fatal because of early invasion, widespread metastasis, and lack of an effective therapy. Migration, invasion, and metastasis of CCA cells are modulated by signals received from stromal cells. The SDF-1-CXCR4 axis emerges as a pivotal regulator of migration and survival of different tumor cells. The aim of the present study was to characterize the interaction between CCA cells and human hepatic stellate cells (hHSC) focusing on the role of SDF-1. METHODS The intrahepatic CCA cell line HuCCT-1 and primary hHSC were used for this study. RNA expression was examined by RTQ-PCR and protein expression by Western blotting. Immunofluorescence microscopy and immunohistochemistry were also employed. Migration of CCA cells was assessed using modified Boyden chambers. RESULTS CXCR4 was clearly expressed in CCA cells of human CCA liver specimens. SDF-1 and hHSC conditioned medium (CM) promoted HuCCT-1 cell migration, which was abrogated by pre-incubation with AMD3100, a non-peptide antagonist of the CXCR4 receptor. In addition, HuCCT-1 cells silenced for CXCR4 did not migrate in presence of SDF-1. Both P-ERK and p-AKT were implicated in HuCCT-1 migration and showed a biphasic trend under stimulation of SDF-1. Finally, SDF-1 induced apoptotic rescue of HuCCT-1 cells by binding to CXCR4. CONCLUSIONS Our study demonstrates that CCA cells migration and survival are modulated by the crosstalk between SDF-1, released by hHSC, and HuCCT-1 cells bearing CXCR4.

Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells.

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin‐like growth factor‐I (IGF‐I) and IGF binding protein (IGFBP) activity. IGF‐I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF‐I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP‐1 through IGFBP‐6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF‐I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP‐2 through IGFBP‐6 mRNAs and release detectable levels of IGFBP‐2 through IGFBP‐5. Because IGF‐I, platelet‐derived growth factor‐BB (PDGF‐BB), and transforming growth factor‐β (TGF‐β) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF‐I induced IGFBP‐3 and IGFBP‐5 proteins in a time‐dependent manner without an increase in the corresponding mRNAs. IGFBP‐4 protein levels decreased in response to IGF‐I. TGF‐β stimulated IGFBP‐3 mRNA and protein but decreased IGFBP‐5 mRNA and protein. In contrast, PDGF‐BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP‐3 (rhIGFBP‐3) was then tested for its effect on IGF‐I‐induced mitogenesis in HSCs. rhIGFBP‐3 inhibited IGF‐I‐stimulated DNA synthesis in a dose‐dependent manner, with a peak effect observed at 25 nM IGFBP‐3. Because TGF‐β is highly expressed in cirrhotic liver tissue, we determined whether IGFBP‐3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral‐induced chronic active hepatitis. In the majority of these samples, IGFBP‐3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF‐I and TGF‐β differentially regulate IGFBP‐3, IGFBP‐4, and IGFBP‐5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF‐I. J. Cell. Physiol. 174:240–250, 1998.

Update on ascites and hepatorenal syndrome

Ascites is the most common complication occurring during liver cirrhosis. Even if a significant decrease in renal clearance may be observed in the first step of chronic active liver disease, renal impairment, at times complicated by the typical signs of hepatorenal syndrome, occurs only in patients with ascites, especially when tense and refractory. Experimental and clinical data seem to suggest a primary sodium and water retention in the pathogenesis of ascites, in the presence of an intrahepatic increase of hydrostatic pressure, which, by itself, physiologically occurs during digestion. Abnormal sodium and water handling leads to plasma volume expansion, followed by decreased peripheral vascular resistance and increased cardiac output. This second step is in agreement with the peripheral arterial vasodilation hypothesis, depicted by an increase in total blood volume, but with a decreased effective arterial blood volume. This discrepancy leads to the activation of the sympathetic nervous and renin-angiotensin-aldosterone systems associated with the progressive activation of the renal autacoid systems, especially, that of the arachidonic acid. During advanced cirrhosis, renal impairment becomes more sustained and renal autacoid vasodilating substances are less available, possibly due to a progressive exhaustion of these systems. At the same time ascites becomes refractory inasmuch as it is no longer responsive to diuretic treatment. Various pathogenetic mechanisms leading to refractory ascites are mentioned. Finally, several treatment approaches to overcome the reduced effectiveness of diuretic therapy are cited. Paracentesis, together with simultaneous administration of human albumin or other plasma expanders is the main common approach to treat refractory ascites and to avoid a further decrease in renal failure. Other effective tools are: administration of terlipressin together with albumin, implantation of the Le Veen shunt, surgical porto-systemic shunting or transjugular intrahepatic portosystemic stent-shunt, or orthotopic liver transplantation, according to the conditions of the individual patient.

Molecular Pathogenesis of NASH.

Nonalcoholic steatohepatitis (NASH) is the main cause of chronic liver disease in the Western world and a major health problem, owing to its close association with obesity, diabetes, and the metabolic syndrome. NASH progression results from numerous events originating within the liver, as well as from signals derived from the adipose tissue and the gastrointestinal tract. In a fraction of NASH patients, disease may progress, eventually leading to advanced fibrosis, cirrhosis and hepatocellular carcinoma. Understanding the mechanisms leading to NASH and its evolution to cirrhosis is critical to identifying effective approaches for the treatment of this condition. In this review, we focus on some of the most recent data reported on the pathogenesis of NASH and its fibrogenic progression, highlighting potential targets for treatment or identification of biomarkers of disease progression.

Evaluation of intracellular signalling pathways in response to insulin-like growth factor I in apoptotic-resistant activated human hepatic stellate cells

BackgroundHuman hepatic stellate cells have been shown to be resistant to apoptotic stimuli. This is likely dependent on the activation of anti-apoptotic pathways upon transition of these cells to myofibroblast-like cells. In particular, previous studies have demonstrated an increased expression of the anti-apoptotic protein Bcl-2 and a decreased expression of the pro-apoptotic protein Bax during the transition of the hepatic stellate cell phenotype from quiescent to myofibroblast-like cells. However, the role and expression of other key anti-apoptotic and survival pathways elicited by polypeptide growth factors involved in the chronic wound healing process remain to be elucidated. In particular, insulin growth factor-I promotes chemotactic and mitogenic effects in activated human hepatic stellate cells and these effects are mediated by the activation of PI 3-K. The role of insulin growth factor-I as a survival factor in human hepatic stellate cells needs to be substantiated. The aim of this study was to evaluate the involvement of other key anti-apoptotic pathways such as PI-3K/Akt/p-Bad in response to insulin growth factor-I.ResultsInsulin growth factor-I induced activation of Akt followed by Bad phosphorylation after 15 minutes of incubation. These effects were PI-3k dependent since selective inhibitors of this molecule, wortmannin and LY294002, inhibited both Akt and Bad phosphorylation. The effect of insulin growth factor-I on the activation of two downstream targets of Akt activation, that is, GSK3 and FHKR, both implicated in the promotion of cell survival was also investigated. Both targets became phosphorylated after 15 minutes of incubation, and these effects were also PI-3K-dependent. Despite the activation of this survival pathway insulin growth factor-I did not have a remarkable biological effect, probably because other insulin growth factor-I-independent survival pathways were already maximally activated in the process of hepatic stellate cell activation. However, after incubation of the cells with a strong apoptotic stimuli such as Fas ligand+cycloheximide, a small percentage of hepatic stellate cells underwent programmed cell death that was partially rescued by insulin growth factor-I.ConclusionIn addition to Bcl-2, several other anti-apoptotic pathways are responsible for human hepatic stellate cell resistance to apoptosis. These features are relevant for the progression and limited reversibility of liver fibrosis in humans.

Ascites and hepatorenal syndrome.

Ascites is a frequent complication of chronic liver disease with severe portal hypertension. Moreover, in the presence of tense ascites, renal dysfunction and hepatorenal syndrome may occur. Unfortunately, there is no explanation that thoroughly describes the complex relationship between the liver and kidney in either physiological or pathological conditions. Nevertheless, available evidence indicates that early sodium and water retention precedes decompensation, characterized by hyperdynamic circulation. The best approach to the treatment of these patients should be aimed at the prevention of ascites formation. An accurate sequential treatment is indicated in patients with ascites. In the case of hepatorenal syndrome, the only definitive approach is liver transplantation.