Yvon Calmus

Pivotal role of superoxide anion and beneficial effect of antioxidant molecules in murine steatohepatitis

Nonalcoholic fatty liver disease, frequently associated with obesity, can lead to nonalcoholic steatohepatitis (NASH) and cirrhosis. The pathophysiology of NASH is poorly understood, and no effective treatment is available. In view of a potential deleterious role for reactive oxygen species (ROS), we investigated the origin of ROS overproduction in NASH. Mitochondrial production of ROS and its alterations in the presence of antioxidant molecules were studied in livers from ob/ob mice that bear a mutation of the leptin gene and develop experimental NASH. N‐acetyl‐cysteine and the superoxide dismutase (SOD) mimics ambroxol, manganese [III] tetrakis (5,10,15,20 benzoic acid) (MnTBAP), and copper [II] diisopropyl salicylate (CuDIPS) were used to target different checkpoints of the oxidative cascade to determine the pathways involved in ROS production. Liver mitochondria from ob/ob mice generated more O2°− than those of lean littermates (P < .01). Ex vivo, all three SOD mimics decreased O2°− generation (P < .001) and totally inhibited lipid peroxidation (P < .001) versus untreated ob/ob mice. Those modifications were associated with in vivo improvements: MnTBAP and CuDIPS reduced weight (P < .02) and limited the extension of histological liver steatosis by 30% and 52%, respectively, versus untreated ob/ob mice. In conclusion, these data demonstrate deleterious effects of superoxide anions in NASH and point at the potential interest of nonpeptidyl mimics of SOD in the treatment of NASH in humans. (HEPATOLOGY 2004;39:1277–1285.)

Interleukin-4 induces the activation and collagen production of cultured human intrahepatic fibroblasts via the STAT-6 pathway

Interleukin-4 (IL-4) is overexpressed in liver grafts in a context of severe recurrent hepatitis C, during which the development of fibrosis is dramatically accelerated. In this study, we examined the effects of IL-4 on the activation and collagen production of cultured human intrahepatic (myo)fibroblasts (hIHFs), and investigated the underlying mechanisms. The myofibroblastic nature of cells was evaluated morphologically using activation markers (smooth muscle α-actin, vimentin and prolyl 4-hydroxylase). Quiescent hIHFs were obtained by cell incubation in serum-free medium or cell culture on Matrigel. We first analyzed IL-4 receptor expression, STAT-6 activation by IL-4, and STAT-6 inhibition by an anti-IL-4 antibody or by STAT-6 small-interfering RNA (siRNA) transfection. We then focused on collagen production, using quantitative real-time PCR to analyze the effect of IL-4 on the mRNA expression of collagens I, III and IV, and on collagen levels in supernatants of hIHFs, using the Sircol collagen assay. hIHFs cultured in plastic wells appeared to be morphologically activated. The expression of activation markers was reduced by serum deprivation or culture on Matrigel, and restored by IL-4 incubation. The IL-4 receptor was expressed by hIHFs, and STAT-6 was activated following incubation with IL-4. Both anti-IL-4 antibody and STAT-6 siRNA transfection inhibited this activation. The treatment of hIHFs with IL-4 increased the mRNA expression of collagens I, III and IV (P<0.05) and elevated collagen levels in supernatants (P=0.01 vs untreated cells). Therefore, IL-4 exerts profibrotic effects by activating hIHFs and inducing collagen production and secretion. This effect requires IL4-R binding and STAT-6 activation. IL-4 may thus be involved in accelerated course of fibrogenesis during recurrent hepatitis C.

Mangafodipir prevents liver injury induced by acetaminophen in the mouse.

BACKGROUND/AIMS Acute liver failure (ALF), characterized by massive hepatocyte necrosis, is often caused by drug poisoning, particularly with acetaminophen (APAP). Hepatocyte necrosis is consecutive to glutathione depletion by NAPQI, a metabolite of APAP, and to mitochondrial damages caused by reactive oxygen species (ROS) overproduction. Considering the structure of Mangafodipir, a contrast agent currently used in magnetic resonance imaging of the liver, we hypothesized that this molecule could exert an antioxidant activity and be possibly used as a treatment of APAP-induced ALF. METHODS/RESULTS Mangafodipir is endowed with superoxide dismutase, catalase, and glutathione reductase activities. It can inhibit ROS production by hepatocytes in culture, and protect those cells from oxidative stresses induced by exposure to xanthine oxidase, H(2)O(2), or UV light. Moreover, preventive or curative administration of Mangafodipir to mice with APAP-induced ALF significantly increases survival rates, and abrogates aspartate aminotransferase elevation and histological damage. CONCLUSIONS Those data point out the potential interest of Mangafodipir in the treatment of toxic ALF in humans.

Endothelin-1 induces liver vasoconstriction through both ETA and ETB receptors

BACKGROUND/AIMS We investigated which endothelin receptors mediate the vasoconstrictive effects of endothelin-1 on liver circulation. METHODS An isolated perfused rat liver model in recirculation was used. RESULTS The perfusion of 10(-10) M endothelin-1 had no significant influence on the liver flow, whereas 10(-9) M endothelin-1 induced significant vasoconstriction, with flow dropping from 3.20 +/- 0.34 to 1.48 +/- 0.28 ml. min-1.g-1 liver tissue (p < 0.01 vs controls). The liver flow was interrupted following the perfusion of 10(-8) M endothelin-1. Sarafatoxin C and BQ 3020, two agonists of ETB receptor, had vasoconstrictive effects in this model. Sarafatoxin C decreased the liver flow in a dose-dependent manner, from 3.32 +/- 0.21 to 2.18 +/- 0.20, 1.60 +/- 0.09, and 1.01 +/- 0.06 ml.min-1. g-1, respectively, with 10(-9) M, 10(-8) M, and 10(-7) M. While BQ 123, an antagonist of ETA receptor, or BQ 788, an antagonist of ETB receptor, partially reversed the effect of 10(-9) M endothelin-1, the simultaneous administration of BQ 123 and BQ 788 completely reversed these effects. CONCLUSIONS These results indicate that the vasoconstrictive effects of endothelin-1 on the liver circulation are mediated through both ETA and ETB receptors.

Interleukin-4 induces human hepatocyte apoptosis through a Fas-independent pathway

IL‐4 is overexpressed in liver grafts during severe recurrent hepatitis C and rejection. Hepatocyte apoptosis is involved in both these phenomena. We therefore examined the proapoptotic effect of IL‐4 on HepG2 cells and human hepatocytes in vitro, together with the underlying mechanisms. We first measured IL‐4 receptor expression, STAT6 activation by IL‐4, and STAT6 inhibition by an anti‐IL‐4 antibody or by STAT6 siRNA transfection. We then focused on the pathways involved in IL‐4‐mediated apoptosis and the role of STAT6 activation in apoptosis initiation. The IL‐4 receptor was expressed on both cell types, and STAT6 was activated by IL‐4. Both anti‐IL‐4 and STAT‐6 siRNA inhibited this activation. IL‐4 induced apoptosis of both HepG2 cells (P= 0.008 vs. untreated control) and human hepatocytes (P<0.001 vs. untreated control). IL‐4 reduced the mitochondrial membrane potential, activated Bid and Bax, and augmented caspase 3, 8, and 9 activity. STAT6 blockade inhibited IL‐4‐induced apoptosis. Expression of Fas and Fas ligand was unaffected when HepG2 cells and hepatocytes were cultured with IL‐4, and Fas/FasL pathway blockade failed to inhibit IL‐4‐induced apoptosis. These results show that IL‐4 induces apoptosis of human hepatocytes through IL‐4 receptor binding, STAT6 activation, decreased mitochondrial membrane potential, and increased caspase activation, independently of the Fas pathway. IL‐4 might thus contribute to the progression of severe liver graft damage.—Aoudjehane, L., Podevin, P., Scatton, O., Jaffray, P., Dusanter‐Fourt, I., Feldmann, G., Massault, P. P., Grira, L., Bringuier, A., Dousset, B., Chouzenoux, S., Soubrane, O., Calmus, Y., Conti, F. Interleukin‐4 induces human hepatocyte apoptosis through a Fas‐independent pathway. FASEB J. 21, 1433–1444 (2007)

MDR1 gene expression in hepatocellular carcinoma and the peritumoral liver of patients with and without cirrhosis

P-glycoprotein (P-gp) and MDR1 mRNA expressions were assessed in tumoral and peritumoral specimens from patients with hepatocellular carcinoma (HCC) and in cirrhotic livers without HCC, using immunohistochemistry (C494 monoclonal antibody) and reverse transcription-polymerase chain reaction (RT-PCR) analysis. P-gp overexpression was detected in 24/28 tumoral livers (85%). In the peritumoral liver, staining was strong in cirrhotic nodules, and fainter in non-cirrhotic specimens. P-gp expression was as intense in the cirrhotic specimens free of HCC as in the peritumoral tissue of HCC developing in cirrhotic patients. These results were confirmed by RT-PCR analysis.

Increased expression of interleukin-4 during liver allograft rejection

BACKGROUND/AIMS To investigate the respective roles of interleukin-2 (IL-2) and IL-4 during rejection, we evaluated the expression of IL-2, IL-2 receptor and IL-4 in human liver allografts. METHODS Immunohistochemistry and RT-PCR were performed in liver biopsies. To determine the effects of immunosuppression and cholestasis in IL-4 production, in vitro experiments were also designed. RESULTS IL-2 protein and its mRNA were absent in the liver, with minimal expression of IL-2 receptor, during rejection. In contrast, IL-4 protein and its mRNA were highly expressed during acute and chronic rejection, whereas this expression was absent in stable liver transplant recipients. In vitro, cyclosporine potently inhibited IL-2 and IL-2 receptor expression of activated mononuclear blood cells, but poorly inhibited IL-4 expression. Chenodeoxycholic acid decreased IL-2 and IL-2 receptor expression, but increased IL-4 expression. CONCLUSIONS During liver allograft rejection, IL-2 pathway is down-regulated, while IL-4 expression is increased by cholestasis and poorly inhibited by cyclosporine. These data suggest that IL-4 is involved in the mechanisms of liver allograft rejection in patients treated with cyclosporine.

Shaping macrophages function and innate immunity by bile acids: Mechanisms and implication in cholestatic liver diseases

The liver is selectively enriched in innate immune cells, macrophages (Kupffer cells), natural killer, and natural killer T cells. These cells release an array of mediators with cytotoxic, pro- and anti-inflammatory, angiogenic, fibrogenic, and mitogenic activity that function to fight infections, limit tissue injury, and promote wound healing. The diverse activity of macrophages is mediated by distinct subpopulations that develop in response to signals within their microenvironment. Understanding the mechanisms and role of the microenvironment contributing to modulation of macrophage populations is crucial for comprehension of the pathophysiology of liver injury in diverse conditions. Several studies initiated in the 1990s have shown that bile acids modulate innate and adaptive immunity. In the last decade, bile acids turned into hormones and signalling molecules involved in many metabolic and inflammatory processes. Biological properties of bile acids are thought to be mediated mainly through activation of the nuclear receptor FXR, the membrane receptor TGR5, as well as PK, ERK, MAP kinases signalling pathways. FXR and TGR5 agonists are currently under development for clinical purpose. This review analyses the mechanisms involved in the immunomodulatory effects of bile acids on the macrophage and discuss their implications in the pathophysiology of cholestasis, primary biliary cirrhosis and primary sclerosing cholangitis.

Use of lidocaine metabolism to test liver function during the long-term follow-up of liver transplant recipients

Abstract:  Background/Aims:  The aim of this study was to assess the usefulness of the monoethylglycinexylidide (MEGX) test to monitoring the long‐term function of liver allografts.

Hypodermin A, a new inhibitor of human complement for the prevention of xenogeneic hyperacute rejection

Abstract: Background: Hyperacute rejection (HAR) of discordant xenografts in the pig‐to‐human combination can be prevented using tranplants expressing transgenic molecules that inhibit human complement. Hypodermin A (HA), a serine esterase that degrades C3, was tested in the guinea‐pig‐to‐rat and in the pig‐to‐human combinations.