E. Ceni

Oxidative stress stimulates proliferation and invasiveness of hepatic stellate cells via a MMP2-mediated mechanism

Experimental evidence indicates that reactive oxygen species (ROS) are involved in the development of hepatic fibrosis; they induce hepatic stellate cells (HSC) proliferation and collagen synthesis. To address the role of matrix metalloproteinase (MMP)‐2 in promoting HSC proliferation during hepatic injury, we investigated whether oxidative stress modulates the growth and invasiveness of HSC by influencing MMP‐2 activation. Cell invasiveness and proliferation, which were studied using Boyden chambers and by counting cells under a microscope, were evaluated after treatment with a superoxide‐producing system, xanthine plus xanthine oxidase (X/XO), in the presence or absence of antioxidants and MMP inhibitors. Expression and activation of MMP‐2 were evaluated via gel zymography, immunoassay, and ribonuclease protection assay. The addition of X/XO induced proliferation and invasiveness of human HSC in a dose‐dependent manner. The addition of antioxidants as well as MMP‐2–specific inhibitors impaired these phenomena. X/XO treatment increased MMP‐2 expression and secretion appreciably and significantly induced members of its activation complex, specifically membrane‐type 1 MMP and tissue inhibitor metalloproteinase 2. To study the intracellular signaling pathways involved in X/XO‐induced MMP‐2 expression, we evaluated the effects of different kinase inhibitors. The inhibition of extracellular signal‐regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol 3‐kinase (PI3K) abrogated X/XO‐elicited MMP‐2 upregulation and completely prevented X/XO‐induced growth and invasiveness of HSC. In conclusion, our findings suggest that MMP‐2 is required for the mitogenic and proinvasive effects of ROS on HSC and demonstrate that ERK1/2 and PI3K are the main signals involved in ROS‐mediated MMP‐2 expression. (HEPATOLOGY 2005;41:1074–1084.)

Effect of pirfenidone on rat hepatic stellate cell proliferation and collagen production

BACKGROUND/AIMS Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC). METHODS HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70(S6K) activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and alpha1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively. RESULTS Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 microM, with a maximal effect at 1000 microM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70(S6K) activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na(+)/H(+) exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 microM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 microM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor(beta1) by 54% and 92%, respectively, as well as TGF(beta1)-induced alpha1(I) procollagen mRNA expression. RESULTS Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases.

Pathogenesis of alcoholic liver disease: Role of oxidative metabolism

Alcohol consumption is a predominant etiological factor in the pathogenesis of chronic liver diseases, resulting in fatty liver, alcoholic hepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma (HCC). Although the pathogenesis of alcoholic liver disease (ALD) involves complex and still unclear biological processes, the oxidative metabolites of ethanol such as acetaldehyde and reactive oxygen species (ROS) play a preeminent role in the clinical and pathological spectrum of ALD. Ethanol oxidative metabolism influences intracellular signaling pathways and deranges the transcriptional control of several genes, leading to fat accumulation, fibrogenesis and activation of innate and adaptive immunity. Acetaldehyde is known to be toxic to the liver and alters lipid homeostasis, decreasing peroxisome proliferator-activated receptors and increasing sterol regulatory element binding protein activity via an AMP-activated protein kinase (AMPK)-dependent mechanism. AMPK activation by ROS modulates autophagy, which has an important role in removing lipid droplets. Acetaldehyde and aldehydes generated from lipid peroxidation induce collagen synthesis by their ability to form protein adducts that activate transforming-growth-factor-β-dependent and independent profibrogenic pathways in activated hepatic stellate cells (HSCs). Furthermore, activation of innate and adaptive immunity in response to ethanol metabolism plays a key role in the development and progression of ALD. Acetaldehyde alters the intestinal barrier and promote lipopolysaccharide (LPS) translocation by disrupting tight and adherent junctions in human colonic mucosa. Acetaldehyde and LPS induce Kupffer cells to release ROS and proinflammatory cytokines and chemokines that contribute to neutrophils infiltration. In addition, alcohol consumption inhibits natural killer cells that are cytotoxic to HSCs and thus have an important antifibrotic function in the liver. Ethanol metabolism may also interfere with cell-mediated adaptive immunity by impairing proteasome function in macrophages and dendritic cells, and consequently alters allogenic antigen presentation. Finally, acetaldehyde and ROS have a role in alcohol-related carcinogenesis because they can form DNA adducts that are prone to mutagenesis, and they interfere with methylation, synthesis and repair of DNA, thereby increasing HCC susceptibility.

Alcohol induced hepatic fibrosis: Role of acetaldehyde

Alcohol abuse is one of the major causes of liver fibrosis worldwide. Although the pathogenesis of liver fibrosis is a very complex phenomenon involving different molecular and biological mechanisms, several lines of evidence established that the first ethanol metabolite, acetaldehyde, plays a key role in the onset and maintenance of the fibrogenetic process. This review briefly summarizes the molecular mechanisms underlying acetaldehyde pro-fibrogenic effects. Liver fibrosis represents a general wound-healing response to a variety of insults. Although mortality due to alcohol abuse has been constantly decreasing in the past 20 years in Southern Europe and North America, in several Eastern-European countries and Great Britain Alcoholic Liver Disease (ALD) shows a sharply increasing trend [Bosetti, C., Levi, F., Lucchini, F., Zatonski, W.A., Negri, E., La, V.C., 2007. Worldwide mortality from cirrhosis: an update to 2002. J. Hepatol. 46, 827-839]. ALD has a complex pathogenesis, in which acetaldehyde (AcCHO), the major ethanol metabolite, plays a central role. Ethanol is mainly metabolized in the liver by two oxidative pathways. In the first one ethanol is oxidized to acetaldehyde by the cytoplasmic alcohol dehydrogenase enzyme (ADH), acetaldehyde is then oxidized to acetic acid by the mitochondrial acetaldehyde dehydrogenase (ALDH). The second pathway is inducible and involves the microsomal ethanol-oxidizing system (MEOS), in which the oxidation of ethanol to acetaldehyde and acetic acid also leads to generation of reactive oxygen species (ROS). Chronic ethanol consumption significantly inhibits mitochondrial ALDH activity while the rate of ethanol oxidation to acetaldehyde is even enhanced, resulting in a striking increase of tissue and plasma acetaldehyde levels [Lieber, C.S., 1997. Ethanol metabolism, cirrhosis and alcoholism. Clin. Chim. Acta 257, 59-84]. This review will focus on the molecular mechanisms by which acetaldehyde promote liver fibrosis.

Antidiabetic thiazolidinediones inhibit invasiveness of pancreatic cancer cells via PPARγ independent mechanisms

Background/Aims: Thiazolidinediones (TZD) are a new class of oral antidiabetic drugs that have been shown to inhibit growth of some epithelial cancer cells. Although TZD were found to be ligands for peroxisome proliferators activated receptor γ (PPARγ) the mechanism by which TZD exert their anticancer effect is currently unclear. Furthermore, the effect of TZD on local motility and metastatic potential of cancer cells is unknown. The authors analysed the effects of two TZD, rosiglitazone and pioglitazone, on invasiveness of human pancreatic carcinoma cell lines in order to evaluate the potential therapeutic use of these drugs in pancreatic adenocarcinoma. Methods: Expression of PPARγ in human pancreatic adenocarcinomas and pancreatic carcinoma cell lines was measured by reverse transcription polymerase chain reaction and confirmed by western blot analysis. PPARγ activity was evaluated by transient reporter gene assay. Invasion assay was performed in modified Boyden chambers. Gelatinolytic and fibrinolytic activity were evaluated by gel zymography. Results: TZD inhibited pancreatic cancer cells’ invasiveness, affecting gelatinolytic and fibrinolytic activity with a mechanism independent of PPARγ activation and involving MMP-2 and PAI-1 expression. Conclusion: TZD treatment in pancreatic cancer cells has potent inhibitory effects on growth and invasiveness suggesting that these drugs may have application for prevention and treatment of pancreatic cancer in humans.

A New Mechanism Involving ERK Contributes to Rosiglitazone Inhibition of Tumor Necrosis Factor-α and Interferon-γ Inflammatory Effects in Human Endothelial Cells

Objective—Microvascular endothelium is one of the main targets of the inflammatory response. On specific activation, endothelial cells recruit Th1-lymphocytes at the inflammatory site. We investigated the intracellular signaling mediating tumor necrosis factor (TNF)-α and interferon (IFN)-γ inflammatory response in human microvascular endothelial cells (HMEC-1) and the interfering effects of the peroxisome-proliferator-activated-receptor (PPARγ) agonist, rosiglitazone (RGZ). Methods and Results—TNFα and IFNγ, mainly when combined, stimulate IFNγ-inducible protein of 10 kDa (IP10) and fractalkine production evaluated by ELISA and TaqMan analyses. This effect is not only mediated by activation of the NFkB and Stat1 classic pathways, but also involves a rapid increase in phosphorylation and activation of extracellular signal-regulated kinases (ERK1/2) as measured by Western blot. RGZ interferes with TNFα and IFNγ stimulation of IP10, fractalkine, and adhesion molecule through a novel rapid mechanism which involves the blocking of ERK activation. Conclusions—Our findings shed new light on the mechanisms underlying the inflammatory response of microvascular endothelium and on the possible therapeutic use of RGZ in vasculopathies involving Th1-responses.

The potential of antidiabetic thiazolidinediones for anticancer therapy

The thiazolidinediones (TZDs) are a class of synthetic compounds for treatment of insulin-resistant Type 2 diabetes mellitus. TZDs are known activators of the peroxisome proliferator-activated receptor-γ (PPAR-γ), and exert their antidiabetic action largely through this nuclear receptor family. Moreover, increasing experimental evidences of PPAR-γ-independent effects are accumulating. Apart from the established metabolic actions, TZD treatment exerts additional biological effect such as control of cell growth, differentiation, motility and programmed cell death. In this context, considerable interest has focused on TZDs as potential chemopreventive agents in oncology; however, despite encouraging observation on the potential anticancer effect of these drugs in several in vitro experimental models, controversial results have been obtained with animal models and in pilot clinical trials. This review summarises the molecular mechanisms of the antineoplastic actions of TZDs and the relevance of these findings in human pathology and therapy.

Acetaldehyde regulates the gene expression of matrix-metalloproteinase-1 and -2 in human fat-storing cells

Altered degradation of extracellular matrix has been implicated in the pathogenesis of hepatic fibrosis. We studied the effect of acetaldehyde (AcCHO) on gene expression of matrix-metalloproteinase (MMP)-1 (fibroblast type- interstitial collagenase) and MMP-2 (72 kDa gelatinase-type IV collagenase) in comparison with the AcCHO effect on collagen type I and IV synthesis in cultures of fat-storing cells (FSC) isolated from normal human livers. Cultured human FSC expressed single mRNA transcripts (2.7 and 3.2 kb) specific for MMP-1 and MMP-2, respectively. AcCHO inhibited MMP-1 mRNA levels, whereas it stimulated collagen type I mRNA and protein expression. Opposite AcCHO effects were evident on MMP-2 mRNA and collagen IV synthesis, being MMP-2 up-regulated and collagen IV down-regulated. These data suggest that regulation of MMP-1 and MMP-2 genes by AcCHO may contribute to disruption of the normal basement membrane and its replacement with fibrillar collagens in the early stages of alcoholic liver fibrosis.

Effect of pentoxifylline on the degradation of procollagen type I produced by human hepatic stellate cells in response to transforming growth factor‐β1

1 Pentoxifylline (PTF) may act as a potential antifibrogenic agent by inhibiting cell proliferation and/or collagen deposition in cell type(s) responsible for the accumulation of extracellular matrix. The aim of the present study was to investigate at which level PTF may affect synthesis and degradation of type I collagen in human hepatic stellate cells (HSCs), a key source of connective tissue in fibrotic liver. 2 Procollagen type I synthesis and release were evaluated in cells maintained in serum free/insulin free medium for 48 h and then stimulated with transforming growth factor‐β1 (TGF‐β1) for different time periods in the presence or absence of PTF. TGF‐β1 caused an upregulation of procollagen I mRNA levels with a peak increase after 3–6 h of stimulation. This effect was followed by an increase in both the cell associated and the extracellular levels of the corresponding protein, with a peak effect at 9–12 h after the addition of TGF‐β1. Co‐incubation with PTF slightly but consistently reduced basal as well as stimulated procollagen I mRNA levels, with negligible effects on the cell‐associated expression of the corresponding protein. Conversely, PTF dose‐dependently reduced procollagen type I levels detected in supernatants from unstimulated and stimulated cells. 3 Pulse‐chase experiments employing L‐[3H]‐proline revealed that PTF was able to induce significantly the degradation of procollagen, mainly in the extracellular compartment. We next analysed the effect of PTF on the major pathway involved in type I collagen degradation. PTF did not affect the expression of metalloproteinase 1 (MMP‐1) mRNA both in basal and stimulated conditions, whereas it markedly reduced the expression of tissue inhibitor of metalloproteinase 1 (TIMP‐1) mRNA. Accordingly incubation with PTF increased the levels of ‘activated MMP‐1’ in cell supernatants in both basal and stimulated conditions. 4 These results suggest that the antifibrogenic action of PTF on human HSCs is mainly mediated by extracellular collagen degradation rather than by a reduction of collagen synthesis.

Thiazolidinediones Inhibit Hepatocarcinogenesis in Hepatitis B Virus―Transgenic Mice by Peroxisome Proliferator-Activated Receptor γ―Independent Regulation of Nucleophosmin

Antidiabetic thiazolidinediones (TZD) have in vitro antiproliferative effect in epithelial cancers, including hepatocellular carcinoma (HCC). The effective anticancer properties and the underlying molecular mechanisms of these drugs in vivo remain unclear. In addition, the primary biological target of TZD, the ligand‐dependent transcription factor peroxisome proliferator‐activated receptor γ (PPARγ), is up‐regulated in HCC and seems to provide tumor‐promoting responses. The aim of our study was to evaluate whether chronic administration of TZD may affect hepatic carcinogenesis in vivo in relation to PPARγ expression and activity. The effect of TZD oral administration for 26 weeks was tested on tumor formation in PPARγ‐expressing and PPARγ‐deficient mouse models of hepatic carcinogenesis. Proteomic analysis was performed in freshly isolated hepatocytes by differential in gel electrophoresis and mass spectrometry analysis. Identified TZD targets were confirmed in cultured PPARγ‐deficient hepatocytes. TZD administration in hepatitis B virus (HBV)–transgenic mice (TgN[Alb1HBV]44Bri) reduced tumor incidence in the liver, inhibiting hepatocyte proliferation and increasing apoptosis. PPARγ deletion in hepatocytes of HBV‐transgenic mice (Tg[HBV]CreKOγ) did not modify hepatic carcinogenesis but increased the TZD antitumorigenic effect. Proteomic analysis identified nucleophosmin (NPM) as a TZD target in PPARγ‐deficient hepatocytes. TZD inhibited NPM expression at protein and messenger RNA levels and decreased NPM promoter activity. TZD inhibition of NPM was associated with the induction of p53 phosphorylation and p21 expression. Conclusion: These findings suggest that chronic administration of TZD has anticancer activity in the liver via inhibition of NPM expression and indicate that these drugs might be useful for HCC chemoprevention and treatment. HEPATOLOGY 2010