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ABSTRACT: To investigate which transmembrane choline transporters and intracellular choline kinases play a prominent role at gene expression level in the rise of the total choline (tCho) peak at proton MR spectra in a rodent rhabdomyosarcoma model.
Twenty-two rats bearing grafted bilateral syngenic rhabdomyosarcoma were examined on a clinical 3 T MR system. Total choline concentration was measured from proton MR spectra using cubic centimeter volumes of interest (VOIs) located contiguously along the greater axis of the tumour. After euthanasia, cubic centimetre tissue specimens corresponding to Proton magnetic resonance spectroscopy (H-MRS) VOIs were frozen in liquid nitrogen. Out of 89 H-MRS voxels, only 39 with a corresponding tissue specimen suitable for biochemical processing were included in the analysis. RNA was extracted from all the 39 samples and reverse-transcribed into cDNA. Choline kinase α and β gene expression as well as genes of the transmembrane transporters OCT1, OCT2, OCT3, CTL1, CTL3, CTL4 and CHT1 were studied using reverse transcriptase polymerase chain reaction. The expression level of each gene (ΔCt), was normalized referred to that of the RPL19 gene. The Spearman rank correlation coefficient was used to analyse variables.
There was no overexpression of genes coding for kinases; however, significant correlation was observed between kinase α sub-type and the tCho peak (P=.002; r=0.51). OCT1 was the most overexpressed transporter gene. Less overexpressed CTL1 gene was significantly correlated with the tCho peak (P=.02; r=0.38).
Choline transporters seem to play a predominant role in the increase in total choline concentration at the gene expression level in our model.
Magnetic Resonance Imaging 04/2012; 30(7):1010-6. · 1.99 Impact Factor
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ABSTRACT: Activation of Ras and its downstream signaling pathways, likely contribute to the development of hepatocarcinoma. We have previously shown that intraperitoneal injections of the Ras inhibitor S-trans, trans-farnesylthiosalicyclic acid (FTS) blocks Ras activation and prevents heptocarcinoma development in rats receiving weekly injections of the carcinogene diethylnitrosamine (DEN) for 16 wk. Using this in vivo model, we evaluated the relationship between the tumor preventive effect of Ras inhibition and activation of downstream signaling pathways, cell proliferation, cell cycle events, and angiogenesis. Western blotting, quantitative PCR, immunohistochemistry, and transcription factor activity assays were used. DEN-induced activation of NFkB and Stat3 was abrogated by FTS treatment. FTS treatment showed no effect on DEN-induced elevation of TNFα, interleukin 6 and TLR4, known activators of these transcription factors. FTS significantly reduced phosphorylation of the MAPkinase p38 and of the p70S6 kinase, a surrogate marker for mTor activation, without affecting ERK and AKT phosphorylation. These events were associated with reduced c-myc and cyclin D expression as well as reduced cell proliferation in transformed, GSTp-positive hepatocytes. Moreover, FTS treatment shifted cell proliferation from transformed hepatocytes to apparently normal, GSTp negative hepatocytes. FTS treatment did not down-regulate expression of angiogenesis markers HIFα, VEGF, VEGF receptor1, and placenta growth factor. FTS treatment inhibits important signaling pathways involved in cellular proliferation leading to strongly reduced proliferation of transformed hepatocytes without affecting normal hepatocytes. This re-adjustment of the proliferation balance likely contributes to the tumor preventive of FTS in the context of Ras inhibition in hepatocarcinogenesis. © 2011 Wiley Periodicals, Inc.
Molecular Carcinogenesis 08/2011; 51(10):816-25. · 3.16 Impact Factor
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ABSTRACT: Activation of myofibroblasts (MF) and extracellular matrix (ECM) deposition predispose the expansion and differentiation of liver progenitor cells (LPC) during chronic liver injury. Because Kupffer cells (KC) are active modulators of tissue response and fibrosis, we analyzed their role in a model of LPC proliferation. A choline-deficient diet, supplemented by ethionine (CDE) was administrated to C57Bl/6J mice that were depleted of KC by repeated injections of clodronate (CLO) and compared to PBS-injected mice. On CDE, massive KC activation was observed in the PBS group, but this was blunted in CLO-treated mice. The depletion of KC did not influence LPC proliferation but reduced their invasive behavior. Instead of being found far into the parenchyma, as was found in the PBS group (mean distance from portal vein: 209 μm), LPC of CLO mice remained closer to the portal area (138 μm), forming aggregates and phenotypically resembling cells of biliary lineage. Notably, removal of KC was also associated with a significant decrease in amount of MF and ECM and in the expression of profibrotic factors. Thus, besides ECM and MF, KC are also a significant component of the microenvironmental changes preceding LPC expansion. Depletion of KC may limit the LPC parenchymal invasion through a deficiency in chemoattracting factors, reduced activation of MF, and/or a paucity of the ECM framework necessary for cell motility.
American Journal Of Pathology 08/2011; 179(4):1839-50. · 4.89 Impact Factor
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ABSTRACT: Conflicting observations exist concerning the role of nuclear factor kappa B (NFjB) in alcoholic liver disease (ALD) in animal models. To date no studies have examined this aspect in human liver tissue. We here assessed cytokines and toll-like receptors (TLRs) expressions in conjunction with NFkappaB activation in non-active end-stage human ALD compared with normal livers and hepatitis C virus (HCV) related end-stage disease.
mRNA and protein expression were examined by quantitative PCR and Western blotting, DNA-binding by electrophoretic mobility shift assays and NFkappaB sub-cellular localization by immunofluorescent staining of livers.
NFkappaB mRNA and protein expression as well as strong DNA-binding were preserved in ALD but significantly down-regulated in HCV compared with normal livers. P50 immunofluorescence was found in hepatocytes and bile ducts in ALD and normal livers, whereas a shift was observed in p65 staining from non-parenchymal cells in normal livers to hepatocytes in ALD. NFkappaB responsive genes mRNA levels IkBalpha and interleukin 6 were significantly higher in ALD compared with HCV. Tumour necrosis factor alpha (TNFalpha), TLRs 3 and 7 mRNA were up-regulated in ALD and HCV compared with normal liver with TNFalpha and TLR7 being the highest in HCV. Strong induction of interferon beta was found in HCV but not in ALD or normal liver tissue.
Persistent NFkappaB activation together with high pro-inflammatory cytokine expression and upregulation of TLR3 and TLR7 is associated with end-stage ALD in humans and could contribute to disease progression even in absence of alcohol intake.
European Journal of Clinical Investigation 07/2010; 40(7):575-84. · 3.02 Impact Factor
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ABSTRACT: Dysregulation of epidermal growth factor and insulin-like growth factor signaling play important roles in human hepatocellular carcinoma (HCC), leading to frequent activation of their downstream targets, the ras/raf/extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) pathways. Salirasib is an S-prenyl-cysteine analog that has been shown to block ras and/or mTOR activation in several non hepatic tumor cell lines. We investigated in vitro the effect of salirasib on cell growth as well as its mechanism of action in human hepatoma cell lines (HepG2, Huh7, and Hep3B) and its in vivo effect in a subcutaneous xenograft model with HepG2 cells.
Salirasib induced a time and dose dependent growth inhibition in hepatocarcinoma cells through inhibition of proliferation and partially through induction of apoptosis. A 50 percent reduction in cell growth was obtained in all three cell lines at a dose of 150 μM when they were cultured with serum. By contrast, salirasib was more potent at reducing cell growth after stimulation with EGF or IGF2 under serum-free conditions, with an IC50 ranging from 60 μM to 85 μM. The drug-induced anti-proliferative effect was associated with downregulation of cyclin A and to a lesser extent of cyclin D1, and upregulation of p21 and p27. Apoptosis induction was related to a global pro-apoptotic balance with caspase 3 activation, cytochrome c release, death receptor upregulation, and a reduced mRNA expression of the apoptosis inhibitors cFLIP and survivin. These effects were associated with ras downregulation and mTOR inhibition, without reduction of ERK and Akt activation. In vivo, salirasib reduced tumour growth from day 5 onwards. After 12 days of treatment, mean tumor weight was diminished by 56 percent in the treated animals.
Our results show for the first time that salirasib inhibits the growth of human hepatoma cell lines through inhibition of proliferation and induction of apoptosis, which is associated with ras and mTOR inhibition. The therapeutic potential of salirasib in human HCC was further confirmed in a subcutaneous xenograft model.
Molecular Cancer 01/2010; 9:256. · 3.99 Impact Factor
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ABSTRACT: Inhibition or blockade of HSCs (hepatic stellate cells), the main matrix-producing cells involved in the wound-healing response, represents an attractive strategy for the treatment of liver fibrosis. In vitro studies have shown that activation of AMPK (AMP-activated protein kinase), a key player in the regulation of cellular energy homoeostasis, inhibits proliferation of myofibroblasts derived from HSCs. If AMPK is a true regulator of fibrogenesis then defective AMPK activity would enhance fibrogenesis and hepatic fibrosis. To test this, in the present work, in vitro studies were performed on mouse primary HSCs treated or not with the AMPK activator AICAR (5-amino-4-imidazolecarboxamide ribonucleotide) or isolated from mice lacking the AMPKalpha1 catalytic subunit (AMPKalpha1-/-) or their littermates (AMPKalpha1+/+). Liver fibrosis was induced in vivo in AMPKalpha1-/- and AMPKalpha1+/+ mice by repeated injections of CCl(4) (carbon tetrachloride). During culture activation of HSCs, AMPK protein and activity significantly increased and regulatory AMPKgamma3 mRNA was specifically up-regulated. Stimulation of AMPK activity by AICAR inhibited HSC proliferation, as expected, as well as collagen alpha1(I) expression. Importantly, AMPKalpha1 deletion inhibited proliferation of HSCs, but not fibrogenesis, in vivo. Moreover, AMPKalpha1 deletion was not associated with enhanced CCl(4)-induced fibrosis in vivo. In conclusion, our present findings demonstrate that HSC transdifferentiation is associated with increased AMPK activity that could relate to the stabilization of AMPK complex by the gamma3 subunits. Activation of AMPK in HSCs inhibits in vitro fibrogenesis. By contrast, low AMPK activity does not prevent HSC activation in vitro nor in in vivo fibrosis.
Clinical Science 10/2009; 118(6):411-20. · 4.61 Impact Factor
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ABSTRACT: The mechanistic significance of oxidative stress to fibrogenesis in the methionine and choline-deficient (MCD) diet-induced model of steatohepatitis was evaluated by antioxidant intervention, using either vitamin E or L-2-oxothiazolidine-4-carboxylate (OTC), a cysteine precursor that promotes glutathione synthesis. Significant depletion of hepatic reduced glutathione (GSH) and elevation of thiobarbituric acid reactive substances (TBARS) occurred from week 3 in association with hepatic injury in mice fed the MCD diet. Hepatic stellate cell (HSC) activation and increased collagen alpha1(I) mRNA expression, together with morphologic fibrosis were evident from week 5. Vitamin E repleted GSH, reduced TBARS, steatosis, inflammation, HSC activation and collagen alpha1(I) mRNA expression, and ameliorated fibrosis. Vitamin E did not effect the expression of either profibrogenic cytokines (transforming growth factor-beta 1, connective tissue growth factor) or matrix remodeling enzymes (tissue inhibitor of metalloproteinase-1 and -2, matrix metalloproteinase-2 and -13). Despite repletion of hepatic GSH in OTC-supplemented mice, the initial benefit in the reduction of hepatic TBARS and inhibition of collagen alpha1(I) mRNA expression at week 5, failed to protect these mice from hepatic injury or fibrosis at later time points. Oxidative stress or products of lipid peroxidation mediate HSC activation and collagen gene expression directly in the MCD model of steatohepatitis. Vitamin E but not glutathione augmentation can interrupt this pathogenic process.
International Journal of Molecular Medicine 09/2009; 24(2):171-80. · 1.98 Impact Factor
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ABSTRACT: To assess the potential value of magnetic resonance (MR) elastographic imaging to help detect nonalcoholic steatohepatitis in the fatty rat liver.
This study was approved by the regional ethics committee. Fifty-four rats were imaged after being fed either a standard diet, a choline-deficient diet for up to 8 weeks to induce steatohepatitis, or a 2-week orotic acid diet to induce steatosis; or were imaged 48 hours after carbon tetrachloride injection to model acute liver injury. MR elastography was performed at 7.0 T to assess viscoelastic liver parameters. Steatosis and fibrosis were quantified with morphometric and biochemical analysis. Myofibroblast activation was assessed with morphometric analysis of alpha-smooth muscle actin. Expression of transforming growth factor beta1 and procollagens 1 and 3 as markers of fibrogenesis was evaluated with real-time reverse transcription polymerase chain reaction. Inflammation was scored at histologic analysis.
In rats with steatohepatitis, mean elasticity (2.24 kPa +/- 0.19 [standard deviation] vs 1.82 kPa +/- 0.22) and mean viscosity (0.86 kPa +/- 0.10 vs 0.59 kPa +/- 0.12) increased significantly (P < .005) after the 2-week orotic acid diet, while steatosis, inflammation, myofibroblast activation, and increase of other fibrogenesis markers were observed. Fibrosis appeared only after 5 weeks. In rats with steatosis, viscosity increased (0.77 kPa +/- 0.11, P < .005), elasticity remained constant. In rats with acute liver injury, elasticity (2.96 kPa +/- 0.63) and viscosity (0.85 kPa +/- 0.22) increased (P < .005), while fibrogenesis and inflammation were observed without substantial fibrosis or steatosis. At multivariate analysis in all rats, liver elasticity correlated only with myofibroblast activation (P < .001, r > 0.6).
The results suggest that in nonalcoholic fatty rat liver, MR elastography may be useful in the early detection of steatohepatitis by showing increased elasticity and appearing before fibrosis development, which was linked to myofibroblast activation. Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.2523081817/-/DC1.
Radiology 08/2009; 253(1):90-7. · 5.73 Impact Factor
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ABSTRACT: Leptin has profibrogenic effects in liver, although the mechanisms of this process are unclear. We sought to elucidate the direct and indirect effects of leptin on hepatic stellate cells (HSCs).
HSCs from Sprague-Dawley rats were exposed to leptin and expression of collagen-I, tissue inhibitor of matrix metalloproteinases-1 (TIMP1), transforming growth factor beta1 (TGF-beta1), and connective tissue growth factor (CTGF/CCN2) was assessed. The effects of medium from Kupffer cells (KCs) and sinusoidal endothelial cells (SECs) following leptin were evaluated in HSCs; alpha-smooth muscle actin (alphaSMA) production and KC signaling were analyzed.
HSCs were not activated by incubation with leptin. However, HSCs cultured with medium taken from KCs that were incubated with leptin had increased expression of collagen I, TIMP1, TGF-beta1, and CTGF/CCN2, as well as alphaSMA protein levels and proliferation. These effects were leptin receptor dependent because conditioned medium from KCs isolated from leptin receptor-deficient Zucker (fa/fa) rats did not activate HSCs. In KCs incubated with leptin, messenger RNA and protein expression of TGF-beta1 and CTGF/CCN2 increased. Leptin potentiated signal transducer and activator of transcription 3, AKT, and extracellular signal-related kinase 1/2 phosphorylation in KCs and increased AP-1 and nuclear factor-kappaB DNA binding. Finally, addition of anti-TGF-beta to KC-conditioned medium inhibited HSC expression of collagen I, TIMP1, and CTGF/CCN2, whereas signal transducer and activator of transcription 3 inhibitor attenuated TGF-beta1 production by KC.
Leptin mediates HSC activation and liver fibrosis through indirect effects on KC; these effects are partly mediated by TGF-beta1.
Gastroenterology 05/2009; 137(2):713-23. · 11.68 Impact Factor
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ABSTRACT: Ras oncoproteins are probably implicated in normal and malignant cell growth in various organs. Inhibition of Ras interferes with cell proliferation of non-hepatic cells in vitro and in vivo. A potential role for Ras in normal and malignant hepatocyte proliferation prompted us to evaluate the impact of Ras inhibition by FTS (S-farnesylthiosalicylic acid) on hepatocyte proliferation in vitro in the human hepatic tumour cell line HepG2 and in vivo after PH (partial hepatectomy) in rats. Rats were administered with FTS intraperitoneally (1, 8 and 16 h after PH) and killed 12, 24 and 48 h after PH. Cell proliferation, phosphorlyation of members of the MAPK (mitogen-activated protein kinase) pathway and levels and activity of cell cycle effectors (cyclin D, cyclin E, Cdk2 and Cdk4) were assessed in FTS-treated rats compared with controls. FTS significantly decreased overall cell count, PCNA (proliferating-cell nuclear antigen) expression and BrdU (bromodeoxyuridine) incorporation into HepG2 cells after 7 days of culture. FTS treatment significantly reduced BrdU incorporation and PCNA expression in hepatocytes after PH. Unlike control rats, cell-membrane expression of Ras was decreased in FTS-treated animals after PH, resulting in decreased Raf membrane recruitment and phosphorylation and in reduced phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2). The antiproliferative effect of FTS was linked to a decrease in expression and activity of the cyclin E/Cdk2 complex, without affecting cyclin D and Cdk4. Ras inhibition by FTS significantly decreased proliferation of HepG2 cells and normal hepatocytes after a strong and highly synchronized proliferation stimulus elicited by PH. The inhibitory effect was at least partially mediated by inhibition of Ras/Raf/MAPK signalling. It appears worthwhile to evaluate the impact of Ras inhibition on the development of hepatocarcinomas in vivo in adequate animal models.
Clinical Science 02/2008; 114(1):73-83. · 4.61 Impact Factor
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ABSTRACT: To determine the correlations between the viscoelastic parameters of the liver measured with in vivo MR elastography and quantitative analysis of liver fibrosis.
MR elastography of the liver was performed in 10 rats with hepatic fibrosis induced by intraperitoneal carbon tetrachloride (CCl(4)) injections and five normal rats. Longitudinal waves of 200 MHz were transmitted into the liver with a mechanical transducer. Wave propagation into the liver was analyzed with a phase-locked spin-echo sequence at 1.5 T. The viscoelastic parameters, obtained with the Voigt model, were correlated with automatic image analysis of the fibrotic areas and with analysis of the hydroxyproline content of the liver.
Substantial correlations were observed between the shear viscoelastic parameters and the percentage of fibrosis at automatic image analysis (r = 0.7, P = 0.005 for the elasticity, and r = 0.8, P = 0.001 for the viscosity) and moderate correlations were seen between the shear viscoelastic parameters and the hydroxyproline content (r = 0.6, P = 0.016 for the elasticity and r = 0.5, P = 0.041 for the viscosity).
The viscoelastic parameters of the liver measured with in vivo MR elastography correlate with quantitative analysis of liver fibrosis. These results suggest that MR elastography is a promising noninvasive method to quantify liver fibrosis.
Journal of Magnetic Resonance Imaging 11/2007; 26(4):956-62. · 2.70 Impact Factor
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ABSTRACT: In vitro and animal data suggest that hepatitis C virus (HCV) proteins might interfere with signal transducer and activator of transcription 3 (Stat3) signaling. It remains unknown whether Stat3 influences the apoptotic-proliferation balance and how this may relate to liver fibrosis progression in HCV-infected patients. We assessed Stat3 expression and DNA-binding as well as expression of its regulators protein inhibitor of activated Stat 3 (Pias3) and suppressor of cytokine signaling 3 (Socs3) in 65 HCV-infected livers at various stages of fibrosis progression. We then determined the level of expression of the proliferation markers cyclin D1 and proliferating cell nuclear antigen (PCNA) in conjunction with pro- and antiapoptotic markers Bax and Bcl-2 in the same liver samples. With the onset of fibrosis, Stat3 DNA-binding decreased and became almost undetectable in livers with bridging fibrosis or cirrhosis. Stat3 DNA-binding inversely correlated with Pias3 expression and Stat3-Pias3 interaction increased with the progression of fibrosis. Cyclin D1 and PCNA in hepatocytes decreased dramatically during fibrosis progression and levels highly correlated with Stat3 expression. In addition, an antiapoptotic profile due to upregulation of Bcl-2 principally in infiltrating inflammatory cells was observed with progressing fibrosis. In conclusion, fibrosis progression is characterized by a continuous decline in Stat3 DNA-binding activity related to overexpression and progressive interaction of Pias3-Stat3. The decrease in Stat3 activity correlated with reduced hepatocytes proliferation and a positive antiapoptotic balance in infiltrating inflammatory cells that are known mediators of cell damage in HCV.
Laboratory Investigation 03/2007; 87(2):173-81. · 3.64 Impact Factor
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ABSTRACT: To assess whether hepatic fibrosis is associated with a restriction in the diffusion of water that can be analyzed with diffusion-weighted MR imaging (DWI) of the liver.
DWI was performed in 10 normal rats and 15 rats with liver fibrosis. Echo-planar DWI was performed in the living rats at 1.5 T and repeated immediately after the animals were killed. Afterwards the livers were explanted, fixed in Bouin solution, and imaged with a DW spin-echo sequence at 4.7 T. Fibrosis was quantified by densitometry on Sirius red-stained histological sections.
In living rats the apparent diffusion coefficient (ADC) decreased with the severity of liver fibrosis (controls: 1535 +/- 294 mm(2)/second; CCl(4) (5 weeks) 1129 +/- 273 mm(2)/second; CCl(4) (9 weeks): 943 +/- 132 mm(2)/second; P = 0.002). An inverse correlation between ADC and liver fibrosis volume density was observed (r = -0.712, P < 0.001). In contrast, these findings were not observed in the rats after they were killed or in the fixated livers.
Decreased ADC correlated with increased liver fibrosis in living rats, but not after death. These results suggest that restricted water diffusion cannot be assessed by DWI in liver fibrosis. Other factors, such as a decrease of perfusion, may explain the decrease of the hepatic ADC measured in vivo in rats with liver fibrosis.
Journal of Magnetic Resonance Imaging 01/2007; 25(1):122-8. · 2.70 Impact Factor
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ABSTRACT: The underlying mechanisms that perpetuate liver inflammation in nonalcoholic steatohepatitis are poorly understood. We explored the hypothesis that cyclooxygenase-2 (COX-2) can exert pro-inflammatory effects in metabolic forms of fatty liver disease. Male wild-type (WT) C57BL6/N or peroxisome proliferator-activated receptor alpha knockout (PPAR-alpha-/-) mice were fed a lipogenic, methionine- and choline-deficient (MCD) diet or the same diet with supplementary methionine and choline (control). COX-2 was not expressed in livers of mice fed the control diet. In mice fed the MCD diet, hepatic expression of COX-2 messenger RNA and protein occurred from day 5, continued to rise, and was 10-fold higher than controls after 5 weeks, thereby paralleling the development of steatohepatitis. Upregulation of COX-2 was even more pronounced in PPAR-alpha-/- mice. Induction of COX-2 was completely prevented by dietary supplementation with the potent PPAR-alpha agonist Wy-14,643 in WT but not PPAR-alpha-/- mice. COX-2 upregulation was preceded by activation of nuclear factor kappaB (NF-kappaB) and coincided with increased levels of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, and intercellular adhesion molecule 1 (ICAM-1). Selective COX-2 inhibitors (celecoxib and NS-398) protected against the development of steatohepatitis in WT but not PPAR-alpha-/- mice. In conclusion, induction of COX-2 occurs in association with NF-kappaB activation and upregulation of TNF-alpha, IL-6, and ICAM-1 in MCD diet-induced steatohepatitis. PPAR-alpha suppresses both COX-2 and development of steatohepatitis, while pharmacological inhibition of COX-2 activity ameliorates the severity of experimental steatohepatitis. COX-2 may therefore be a pro-inflammatory mediator in metabolic forms of steatohepatitis.
Hepatology 05/2006; 43(4):826-36. · 11.66 Impact Factor
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ABSTRACT: To analyze the effects of NF- kappa B inhibition by antioxidant pyrrolidine dithiocarbamate (PDTC) or TNF inhibitor pentoxifylline (PTX) on liver regeneration after partial hepatectomy (PH).
Saline, PDTC or PTX were injected 1 h before PH and rats were killed at 0.5 and 24 h after PH. Several control groups were used for comparison (injection control groups).
Compared to saline injected controls, NF- kappa B activation was absent 0.5 h after PH in rats treated with PDTC or PTX. At 24 h after PH, DNA synthesis and PCNA expression were identical in treated and control rats and thus occurred irrespectively of the status of NF- kappa B activation at 0.5 h. Signal transducer and activator of transcription 3 (Stat3) activation was observed already 0.5 h after PH in saline, PDTC or PTX group and was similar to Stat3 activation in response to injection without PH.
These data strongly suggest that (1) NF- kappa B p65/p50 DNA binding produced in response to PH is not a signal necessary to initiate the liver regeneration, (2) Stat3 activation is a stress response unrelated to the activation of NF- kappa B. In conclusion, NF- kappa B activation is not critically required for the process of liver regeneration after PH.
World Journal of Gastroenterology 01/2006; 11(46):7345-50. · 2.47 Impact Factor
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ABSTRACT: We explored the roles of nuclear factor-kappaB (NF-kappaB) and tumor necrosis factor (TNF) alpha (TNF-alpha) as mediators of inflammation in a nutritional model of steatohepatitis.
Wild-type (wt), TNF null -/-, and TNF receptor (R)-1-/- mice were fed a methionine- and choline-deficient (MCD) diet for up to 5 weeks. Liver injury (serum alanine aminotransferase [ALT]), hepatic inflammation, triglycerides, and lipid peroxide levels were determined. Hepatic NF-kappaB activation and expression of TNF and intercellular adhesion molecule-1 (ICAM-1) were assayed.
Irrespective of genotype, MCD diet-fed mice developed hepatic lipid peroxidation and serum ALT elevation; at day 10, livers from wt, TNF-/-, and TNFR-1-/- mice showed equivalent steatohepatitis. NF-kappaB/DNA binding was enhanced in hepatic nuclear fractions from MCD diet-fed wt mice compared with dietary controls; there were corresponding increases of ICAM-1 and TNF messenger RNA (mRNA). Likewise, NF-kappaB activation and ICAM-1 expression were enhanced by MCD dietary feeding in TNF-/- and TNFR-1-/- mice compared with respective controls. To establish whether NF-kappaB is a primary mediator of inflammation in experimental steatohepatitis, we over-expressed a mutant, nondegradable IkappaB (mIkappaB), delivered by adenovirus in vivo. As expected, hepatic mIkappaB expression reduced NF-kappaB/DNA binding induced by MCD dietary feeding, with resultant abrogation of ICAM-1 and TNF synthesis. Such blockade of NF-kappaB transcriptional activation substantially protected against development of steatohepatitis, with significant reductions in liver injury and hepatic inflammation.
In the MCD dietary model of steatohepatitis, NF-kappaB is activated early and is an important proinflammatory mediator of lesion development, but steatohepatitis occurs independently of TNF synthesis and TNFR-1 activation.
Gastroenterology 11/2005; 129(5):1663-74. · 11.68 Impact Factor
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ABSTRACT: In vitro and animal data suggest that alcohol and hepatitis C virus (HCV) proteins might interfere with Stat3 signaling, a potential regulator of liver cell apoptosis and proliferation.
We assessed Stat3 expression, activity and the apoptotic-proliferation balance in end-stage HCV and alcoholic liver disease (ALD) in man. Explanted livers of HCV and ALD patients were compared to normal and primary biliary cirrhosis (PBC) livers.
Although Stat3 expression and phosphorylation was not altered in HCV and ALD cirrhosis, Stat3 DNA-binding was not detected in all ALD and most HCV samples. Deficient Stat3 DNA-binding was associated with high Pias3 expression, but not with increased Socs3 levels. Bcl-2 was up-regulated in HCV and ALD together with decreased Caspase3 activity. Compared to base-line cell proliferation in normal donor livers, HCV cirrhosis showed a marked reduction in cyclin D1 and PCNA, whereas both markers were only slightly reduced in ALD.
End-stage HCV and ALD cirrhosis is characterized by impaired Stat3 DNA-binding possibly through up-regulation of Pias3. Therefore, impaired activation of Stat3 target genes might contribute to disturbed liver regeneration and repair. The attempt in cirrhotic livers to favor anti-apoptotic over pro-apoptotic pathways is not sufficient to compensate for the low cellular proliferation rates.
Journal of Hepatology 11/2005; 43(4):687-95. · 9.26 Impact Factor
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ABSTRACT: Activation of the cyclin E/Cdk2 complex may play an important role in mid-G1/S-phase progression in proliferating mammalian cells. We evaluated the effect of targeted inhibition of Cdk2 activity by CYC202 (R-roscovitine) on hepatocytes proliferation in vivo after 70% partial hepatectomy (PH) in rats. In controls, Cdk2 activity and DNA synthesis peaked 24 h after PH. CYC202 abrogated Cdk2 activity, prevented BrdU incorporation and PCNA expression and increased mortality 24 h after PH. Cyclin E and Cdk2 protein expression and complex formation was not affected by CYC202 nor was cyclin D1, Cdk4 and c-ras mRNA expression. Two consecutive injections 8 and 20 h after PH were required to elicit the inhibitory effect of CYC202, which was lost when either the injection at 8 h or at 20 h was withheld. Cdk2 activity and cell progression resumed 48 h after PH in surviving animals suggesting that CYC202 induced a reversible inhibition of the cell cycle. Our results confirm an important role for Cdk2 in hepatocytes proliferation in the regenerating liver. We demonstrate that molecular events, including Cdk2 activation, occurring within the 8th and 24th hour after PH (G1/S-phase transition) are crucial in determining whether or not DNA synthesis and hepatocytes proliferation proceed normally after PH.
Laboratory Investigation 05/2005; 85(4):562-71. · 3.64 Impact Factor
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ABSTRACT: Administration of a methionine and choline deficient (MCD) diet to rodents causes progressive fibrosing steatohepatitis pathologically similar to human metabolic steatohepatitis. We have previously shown that the peroxisome proliferator-activated receptor-alpha (PPARalpha) agonist, Wy-14,643, prevented the development of MCD diet-induced steatohepatitis. We have now tested whether Wy-14,643 ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice were fed the MCD diet for 51 days to induce severe steatohepatitis. They were then treated with Wy-14,643 together with the MCD diet for 5 or 12 days; positive controls continued on the MCD diet for 5 or 12 days. After 5 days of Wy-14,643 treatment, alanine aminotransferase (ALT) levels were significantly decreased, steatohepatitis less severe, and hepatic lipoperoxides significantly reduced. After 12 days, hepatic triglycerides were normalized and there was near resolution of histological changes. MCD dietary feeding was associated with increased expression of vascular cell adhesion molecule (VCAM)-1, and increased numbers of activated macrophages in the liver. Treatment with Wy-14,643 reduced VCAM-1 expression and macrophage numbers. MCD diet-fed mice developed hepatic fibrosis with increased hepatic collagen alpha1(I), tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and matrix metalloproteinase (MMP)-13 mRNA levels. After treatment with Wy-14,643, expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells and near resolution of liver fibrosis. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by treatment with Wy-14,643. It is likely that activation of PPARalpha reverses fibrosis indirectly by reducing stimuli, such as lipid peroxides, and activation of cells responsible for promoting hepatic fibrosis.
Hepatology 06/2004; 39(5):1286-96. · 11.66 Impact Factor
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ABSTRACT: Administration of a methionine and choline deficient (MCD) diet to rodents causes progressive fibrosing steatohepatitis pathologically similar to human metabolic steatohepatitis. We have previously shown that the peroxisome proliferator-activated receptor-α (PPARα) agonist, Wy-14,643, prevented the development of MCD diet-induced steatohepatitis. We have now tested whether Wy-14,643 ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice were fed the MCD diet for 51 days to induce severe steatohepatitis. They were then treated with Wy-14,643 together with the MCD diet for 5 or 12 days; positive controls continued on the MCD diet for 5 or 12 days. After 5 days of Wy-14,643 treatment, alanine aminotransferase (ALT) levels were significantly decreased, steatohepatitis less severe, and hepatic lipoperoxides significantly reduced. After 12 days, hepatic triglycerides were normalized and there was near resolution of histological changes. MCD dietary feeding was associated with increased expression of vascular cell adhesion molecule (VCAM)-1, and increased numbers of activated macrophages in the liver. Treatment with Wy-14,643 reduced VCAM-1 expression and macrophage numbers. MCD diet-fed mice developed hepatic fibrosis with increased hepatic collagen α1(I), tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and matrix metalloproteinase (MMP)-13 mRNA levels. After treatment with Wy-14,643, expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells and near resolution of liver fibrosis. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by treatment with Wy-14,643. It is likely that activation of PPARα reverses fibrosis indirectly by reducing stimuli, such as lipid peroxides, and activation of cells responsible for promoting hepatic fibrosis. (HEPATOLOGY 2004;39:1286–1296.)
Hepatology 04/2004; 39(5):1286 - 1296. · 11.66 Impact Factor
