Isabelle Leclercq

Limited therapeutic efficacy of pioglitazone on progression of hepatic fibrosis in rats.

Aim: Peroxisome proliferator activated receptor γ (PPARγ) agonists have been shown to prevent hepatic fibrosis in rodents. We evaluated the therapeutic antifibrotic potential of the PPARγ agonist pioglitazone on established hepatic fibrosis. Methods: Repeated injections of carbon tetrachloride (CCl4), a choline deficient diet, or bile duct ligation (BDL) were used to induce hepatic fibrosis in rats. Pioglitazone treatment was introduced at various time points. Therapeutic efficacy was assessed by comparison of the severity of hepatic fibrosis in pioglitazone treated versus untreated fibrotic controls. Results: When introduced after two weeks of CCl4, pioglitazone reduced hepatic fibrosis, OH proline content, hepatic mRNA expression of collagen type I, and profibrotic genes, as well as the number of activated α smooth muscle actin positive hepatic stellate cells, compared with rats receiving CCl4 only, with no significant change in necroinflammation. When pioglitazone treatment was initiated after five weeks of CCl4, no antifibrotic effect was observed. Similarly, pioglitazone was associated with a reduced severity of fibrosis induced by a choline deficient diet when introduced early, while delayed treatment with pioglitazone remained ineffective. In contrast, pioglitazone failed to interrupt progression of fibrosis due to BDL, irrespective of the timing of its administration. Conclusion: In rats, the therapeutic antifibrotic efficacy of pioglitazone is limited and dependent on the type of injury, duration of disease, and/or the severity of fibrosis at the time of initiation of treatment.

Leptin is essential for the hepatic fibrogenic response to chronic liver injury.

BACKGROUND/AIMS Obesity is associated with hyperleptinemia and is also a risk factor for fibrosis and severity of fibrosis in several chronic liver diseases. The correlation between increased leptin, obesity and hepatic fibrosis prompted us to hypothesise that leptin has profibrogenic effects on the liver. METHODS We analysed the role of leptin in liver fibrosis in leptin-deficient mice fed a diet which generates steatohepatitis, and in chronic carbon tetrachloride-induced hepatic injury. RESULTS Leptin-deficient mice failed to develop fibrosis during steatohepatitis or in response to chronic toxic liver injury, and failed to up-regulate collagen-I while developing similar hepatic injury as their genetic controls. Restitution of physiological levels of circulating leptin by injection of exogenous leptin, but not correction of the obese phenotype by dietary manipulation, restored liver fibrosis in leptin-deficient mice during chronic liver injury. These results confirmed the absolute requirement of leptin for hepatic fibrosis. We showed that leptin deficiency did not alter hepatic TNF regulation but that leptin is necessary for induction of bioactive transforming growth factor beta 1 (TGFbeta1) protein in the context of chronic liver injury. CONCLUSIONS These data establish that leptin is an essential mediator of hepatic fibrosis in response to chronic liver injury, whether metabolic or toxic in aetiology.

Administration of the potent PPARalpha agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice.

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.)

Expression of miR-33 from an SREBP2 Intron Inhibits Cholesterol Export and Fatty Acid Oxidation

The regulation of synthesis, degradation, and distribution of lipids is crucial for homeostasis of organisms and cells. The sterol regulatory element-binding protein (SREBP) transcription factor family is post-translationally activated in situations of reduced lipid abundance and activates numerous genes involved in cholesterol, fatty acid, and phospholipid synthesis. In this study, we provide evidence that the primary transcript of SREBP2 contains an intronic miRNA (miR-33) that reduces cellular cholesterol export via inhibition of translation of the cholesterol export pump ABCA1. Notably, miR-33 also inhibits translation of several transcripts encoding proteins involved in fatty acid β-oxidation including CPT1A, HADHB, and CROT, thereby reducing fatty acid degradation. The genetic locus encoding SREBP2 and miR-33 therefore contains a protein that increases lipid synthesis and a miRNA that prevents export and degradation of newly synthesized lipids. These results add an additional layer of complexity to our understanding of lipid homeostasis and might open possibilities for future therapeutic intervention.

Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.

In chronic liver injury, liver progenitor cells (LPCs) proliferate in the periportal area, migrate inside the lobule, and undergo further differentiation. This process is associated with extracellular matrix (ECM) remodeling. We analyzed LPC expansion and matrix accumulation in a choline‐deficient, ethionine‐supplemented (CDE) model of LPC proliferation. After day 3, CDE induced collagen deposits in the periportal area. Expansion of LPCs as assessed by increased number of cytokeratin 19 (CK19)‐positive cells was first observed at day 7, while ECM accumulated 10 times more than in controls. Thereafter, LPCs and ECM increased in parallel. Furthermore, ECM not only accumulates prior to the increase in number of LPCs, but is also found in front of LPCs along the porto‐venous gradient of lobular invasion. Double immunostaining revealed that LPCs are embedded in ECM at all times. Moreover, LPCs infiltrating the liver parenchyma are chaperoned by α‐smooth muscle actin (α‐SMA)–positive cells. Gene expression analyses confirmed these observations. The expression of CK19, α‐fetoprotein, E‐cadherin, and CD49f messenger RNA (mRNA), largely overexpressed by LPCs, significantly increased between day 7 and day 10. By contrast, at day 3 there was a rapid burst in the expression of components of the ECM, collagen I and laminin, as well as in α‐SMA and connective tissue growth factor expression. Conclusion: Our data demonstrate that, in a CDE model, ECM deposition and activation of matrix‐producing cells occurred as an initial phase, prior to LPC expansion, and in front of LPCs along the porto‐veinous gradient of lobular invasion. Those observations may reveal a fundamental role for the established hepatic microenvironment or niche during the process of activation and differentiation of liver progenitor cells. (HEPATOLOGY 2009.)

COX‐2 induction in mice with experimental nutritional steatohepatitis: Role as pro‐inflammatory mediator

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 α knockout (PPAR‐α−/−) 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‐α−/− mice. Induction of COX‐2 was completely prevented by dietary supplementation with the potent PPAR‐α agonist Wy‐14,643 in WT but not PPAR‐α−/− mice. COX‐2 upregulation was preceded by activation of nuclear factor κB (NF‐κB) and coincided with increased levels of tumor necrosis factor α (TNF‐α), 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‐α−/− mice. In conclusion, induction of COX‐2 occurs in association with NF‐κB activation and upregulation of TNF‐α, IL‐6, and ICAM‐1 in MCD diet–induced steatohepatitis. PPAR‐α 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 2006;43:826–836.)

Assessment of diffusion-weighted MR imaging in liver fibrosis.

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.

Kupffer Cells Mediate Leptin-Induced Liver Fibrosis

BACKGROUND & AIMS 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). METHODS 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. RESULTS 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. CONCLUSIONS Leptin mediates HSC activation and liver fibrosis through indirect effects on KC; these effects are partly mediated by TGF-beta1.

Oxidative stress, KLF6 and transforming growth factor-β up-regulation differentiate non-alcoholic steatohepatitis progressing to fibrosis from uncomplicated steatosis in rats

BACKGROUND/AIMS Pathogenesis of non-alcoholic steatohepatitis (NASH) remains poorly understood. Cytochrome P450 2E1 (CYP 2E1), cytokines, oxidative stress and activation of hepatic stellate cells seem to play a role in this process. The aim was to determine the potential implication of these factors in the progression from uncomplicated steatosis to steatohepatitis with progressive fibrosis. METHODS Animals were fed a standard diet, a 5% orotic acid-diet (OA) developing hepatic steatosis, or the methionine-choline deficient (MCD) diet inducing steatohepatitis for 2 and 6 weeks. Lipid peroxidation, CYP 2E1 expression and activity, expression of UCP-2, interleukin (IL)-6, transforming growth factor (TGF)beta1, KLF6 mRNAs, and activation of hepatic stellate cells were examined by gas chromatography, high-performance liquid chromatography, Western blotting, quantitative polymerase chain reaction and immunohistochemistry. RESULTS Lipid peroxidation increased in the MCD model whereas only minor changes occurred in the OA model. KLF6 and TGFbeta1 mRNAs were selectively up-regulated in MCD animals. Stellate cell activation, inflammation and collagen deposition only occurred in the MCD group. CYP 2E1 expression and activity increased in the OA group while both decreased in MCD animals. UCP-2 and IL-6 mRNA increased in both groups. CONCLUSIONS In the context of steatosis, lipid peroxidation is associated with inflammation and stellate cell activation with concomitant increase in TGFbeta1 production, possibly through up-regulation of KLF6.

Early Detection of Steatohepatitis in Fatty Rat Liver by Using MR Elastography.

PURPOSE To assess the potential value of magnetic resonance (MR) elastographic imaging to help detect nonalcoholic steatohepatitis in the fatty rat liver. MATERIALS AND METHODS 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. RESULTS 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). CONCLUSION 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.