Upregulation of Epac-1 in Hepatic Stellate Cells by Prostaglandin E-2 in Liver Fibrosis Is Associated with Reduced Fibrogenesiss

Exchange protein activated by cAMP (Epac-1) is an important signaling mechanism for cAMP-mediated effects, yet factors that change Epac-1 levels are unknown. Such factors are relevant because it has been postulated that Epac-1 directly affects fibrogenesis. Prostaglandin E-2 (PGE(2)) is a well-known cAMP activator, and we therefore studied the effects of this cyclo-oxygenase product on Epac-1 expression and on fibrogenesis within the liver. Liver fibrosis was induced by 8 weeks carbon tetrachloride (CCL4) administration to mice. In the last 2 weeks, mice received vehicle, PGE(2), the cyclo-oxygenase-2 inhibitor niflumic acid (NFA), or PGE(2) coupled to cell-specific carriers to hepatocytes, Kupffer cells, or hepatic stellate cells (HSC). Results showed antifibrotic effects of PGE(2) and profibrotic effects of NFA in CCL4 mice. Western blot analysis revealed reduced Epac-1 protein expression in fibrotic livers of mice and humans compared with healthy livers. PGE(2) administration to fibrotic mice completely restored intrahepatic Epac-1 levels and also led to reduced Rho kinase activity, a downstream target of Epac-1. Cell-specific delivery of PGE(2) to either hepatocytes, Kupffer cells, or HSC identified the latter cell as the key player in the observed effects on Epac-1 and Rho kinase. No significant alterations in protein kinase A expressions were found. In primary isolated HSC, PGE(2) elicited Rap1 translocation reflecting Epac-1 activation, and Epac-1 agonists attenuated platelet-derived growth factor-induced proliferation and migration of these cells. These studies demonstrate that PGE(2) enhances Epac-1 activity in HSC, which is associated with significant changes in (myo)fibroblast activities in vitro and in vivo. Therefore, Epac-1 is a potential target for antifibrotic drugs.</p

Monoacylglycerol Lipase Inhibition Protects From Liver Injury in Mouse Models of Sclerosing Cholangitis

Background and Aims Monoacylglycerol lipase (MGL) is the last enzymatic step in triglyceride degradation, hydrolyzing monoglycerides into glycerol and fatty acids (FAs) and converting 2-arachidonoylglycerol into arachidonic acid, thus providing ligands for nuclear receptors as key regulators of hepatic bile acid (BA)/lipid metabolism and inflammation. We aimed to explore the role of MGL in the development of cholestatic liver and bile duct injury in mouse models of sclerosing cholangitis, a disease so far lacking effective pharmacological therapy. Approach and Results To this aim we analyzed the effects of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding to induce sclerosing cholangitis in wild-type (WT) and knockout (MGL(-/-)) mice and tested pharmacological inhibition with JZL184 in the multidrug resistance protein 2 knockout (Mdr2(-/-)) mouse model of sclerosing cholangitis. Cholestatic liver injury and fibrosis were assessed by serum biochemistry, liver histology, gene expression, and western blot characterization of BA and FA synthesis/transport. Moreover, intestinal FAs and fecal microbiome were analyzed. Transfection and silencing were performed in Caco2 cells. MGL(-/-) mice were protected from DDC-induced biliary fibrosis and inflammation with reduced serum liver enzymes and increased FA/BA metabolism and beta-oxidation. Notably, pharmacological (JZL184) inhibition of MGL ameliorated cholestatic injury in DDC-fed WT mice and protected Mdr2(-/-) mice from spontaneous liver injury, with improved liver enzymes, inflammation, and biliary fibrosis. In vitro experiments confirmed that silencing of MGL decreases prostaglandin E-2 accumulation in the intestine and up-regulates peroxisome proliferator-activated receptors alpha and gamma activity, thus reducing inflammation. Conclusions Collectively, our study unravels MGL as a metabolic target, demonstrating that MGL inhibition may be considered as potential therapy for sclerosing cholangitis

Oral Methylthioadenosine Administration Attenuates Fibrosis and Chronic Liver Disease Progression in Mdr2−/− Mice

BACKGROUND: Inflammation and fibrogenesis are directly related to chronic liver disease progression, including hepatocellular carcinoma (HCC) development. Currently there are few therapeutic options available to inhibit liver fibrosis. We have evaluated the hepatoprotective and anti-fibrotic potential of orally-administered 5'-methylthioadenosine (MTA) in Mdr2(-/-) mice, a clinically relevant model of sclerosing cholangitis and spontaneous biliary fibrosis, followed at later stages by HCC development. METHODOLOGY: MTA was administered daily by gavage to wild type and Mdr2(-/-) mice for three weeks. MTA anti-inflammatory and anti-fibrotic effects and potential mechanisms of action were examined in the liver of Mdr2(-/-) mice with ongoing fibrogenesis and in cultured liver fibrogenic cells (myofibroblasts). PRINCIPAL FINDINGS: MTA treatment reduced hepatomegaly and liver injury. α-Smooth muscle actin immunoreactivity and collagen deposition were also significantly decreased. Inflammatory infiltrate, the expression of the cytokines IL6 and Mcp-1, pro-fibrogenic factors like TGFβ2 and tenascin-C, as well as pro-fibrogenic intracellular signalling pathways were reduced by MTA in vivo. MTA inhibited the activation and proliferation of isolated myofibroblasts and down-regulated cyclin D1 gene expression at the transcriptional level. The expression of JunD, a key transcription factor in liver fibrogenesis, was also reduced by MTA in activated myofibroblasts. CONCLUSIONS/SIGNIFICANCE: Oral MTA administration was well tolerated and proved its efficacy in reducing liver inflammation and fibrosis. MTA may have multiple molecular and cellular targets. These include the inhibition of inflammatory and pro-fibrogenic cytokines, as well as the attenuation of myofibroblast activation and proliferation. Downregulation of JunD and cyclin D1 expression in myofibroblasts may be important regarding the mechanism of action of MTA. This compound could be a good candidate to be tested for the treatment of (biliary) liver fibrosis

Cannabinoid CB2 Receptor Potentiates Obesity-Associated Inflammation, Insulin Resistance and Hepatic Steatosis

BACKGROUND: Obesity-associated inflammation is of critical importance in the development of insulin resistance and non-alcoholic fatty liver disease. Since the cannabinoid receptor CB2 regulates innate immunity, the aim of the present study was to investigate its role in obesity-induced inflammation, insulin resistance and fatty liver. METHODOLOGY: Murine obesity models included genetically leptin-deficient ob/ob mice and wild type (WT) mice fed a high fat diet (HFD), that were compared to their lean counterparts. Animals were treated with pharmacological modulators of CB2 receptors. Experiments were also performed in mice knock-out for CB2 receptors (Cnr2 -/-). PRINCIPAL FINDINGS: In both HFD-fed WT mice and ob/ob mice, Cnr2 expression underwent a marked induction in the stromal vascular fraction of epididymal adipose tissue that correlated with increased fat inflammation. Treatment with the CB2 agonist JWH-133 potentiated adipose tissue inflammation in HFD-fed WT mice. Moreover, cultured fat pads isolated from ob/ob mice displayed increased Tnf and Ccl2 expression upon exposure to JWH-133. In keeping, genetic or pharmacological inactivation of CB2 receptors decreased adipose tissue macrophage infiltration associated with obesity, and reduced inductions of Tnf and Ccl2 expressions. In the liver of obese mice, Cnr2 mRNA was only weakly induced, and CB2 receptors moderately contributed to liver inflammation. HFD-induced insulin resistance increased in response to JWH-133 and reduced in Cnr2 -/- mice. Finally, HFD-induced hepatic steatosis was enhanced in WT mice treated with JWH-133 and blunted in Cnr2 -/- mice. CONCLUSION/SIGNIFICANCE: These data unravel a previously unrecognized contribution of CB2 receptors to obesity-associated inflammation, insulin resistance and non-alcoholic fatty liver disease, and suggest that CB2 receptor antagonists may open a new therapeutic approach for the management of obesity-associated metabolic disorder

Asmase Regulates autophagy and lysosomal membrane permeabilization and its inhibition prevents early stage nonalcoholic steatohepatitis

Background & Aims: Acid sphingomyelinase (ASMase) is activated in nonalcoholic steatohepatitis (NASH). However, ASMase's contribution to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined ASMase's role in high fat diet (HFD)-induced NASH. Methods: Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase-/- mice fed HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed HFD. Results: ASMase deficiency determined resistance to HFD or methionine and choline deficient diet-mediated hepatic steatosis. ASMase-/- mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress and steatosis, indicating selectivity in the resistance of ASMase-/- mice to ER stress. Autophagic flux determined in the presence of rapamycin and/or chloroquine was lower in primary mouse hepatocytes (PMH) from ASMase-/- mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldinA caused ER stress in PMH from ASMase+/+ mice but not ASMase-/- mice. ASMase-/- PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by the oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD- induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH. Conclusions: These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH

Characterization of Blood Immune Cells in Patients With Decompensated Cirrhosis Including ACLF

Background and Aims: Patients with cirrhosis and acute-on-chronic liver failure (ACLF) have immunosuppression, indicated by an increase in circulating immune-deficient monocytes. The aim of this study was to investigate simultaneously the major blood-immune cell subsets in these patients. Material and Methods: Blood taken from 67 patients with decompensated cirrhosis (including 35 critically ill with ACLF in the intensive care unit), and 12 healthy subjects, was assigned to either measurements of clinical blood counts and microarray (genomewide) analysis of RNA expression in whole-blood; microarray (genomewide) analysis of RNA expression in blood neutrophils; or assessment of neutrophil antimicrobial functions. Results: Several features were found in patients with ACLF and not in those without ACLF. Indeed, clinical blood count measurements showed that patients with ACLF were characterized by leukocytosis, neutrophilia, and lymphopenia. Using the CIBERSORT method to deconvolute the whole-blood RNA-expression data, revealed that the hallmark of ACLF was the association of neutrophilia with increased proportions of macrophages M0-like monocytes and decreased proportions of memory lymphocytes (of B-cell, CD4 T-cell lineages), CD8 T cells and natural killer cells. Microarray analysis of neutrophil RNA expression revealed that neutrophils from patients with ACLF had a unique phenotype including induction of glycolysis and granule genes, and downregulation of cell-migration and cell-cycle genes. Moreover, neutrophils from these patients had defective production of the antimicrobial superoxide anion. Conclusions: Genomic analysis revealed that, among patients with decompensated cirrhosis, those with ACLF were characterized by dysregulation of blood immune cells, including increases in neutrophils (that had a unique phenotype) and macrophages M0-like monocytes, and depletion of several lymphocyte subsets (including memory lymphocytes). All these lymphocyte alterations, along with defective neutrophil superoxide anion production, may contribute to immunosuppression in ACLF, suggesting targets for future therapies

Monoacylglycerol lipase reprograms hepatocytes and macrophages to promote liver regeneration

Background &amp; Aims: Liver regeneration is a repair process in which metabolic reprogramming of parenchymal and inflammatory cells plays a major role. Monoacylglycerol lipase (MAGL) is an ubiquitous enzyme at the crossroad between lipid metabolism and inflammation. It converts monoacylglycerols into free fatty acids and metabolises 2-arachidonoylglycerol into arachidonic acid, being thus the major source of pro-inflammatory prostaglandins in the liver. In this study, we investigated the role of MAGL in liver regeneration. Methods: Hepatocyte proliferation was studied in vitro in hepatoma cell lines and ex vivo in precision-cut human liver slices. Liver regeneration was investigated in mice treated with a pharmacological MAGL inhibitor, MJN110, as well as in animals globally invalidated for MAGL (MAGL-/-) and specifically invalidated in hepatocytes (MAGLHep-/-) or myeloid cells (MAGLMye-/-). Two models of liver regeneration were used: acute toxic carbon tetrachloride injection and two-thirds partial hepatectomy. MAGLMye-/- liver macrophages profiling was analysed by RNA sequencing. A rescue experiment was performed by in vivo administration of interferon receptor antibody in MAGLMye-/- mice. Results: Precision-cut human liver slices from patients with chronic liver disease and human hepatocyte cell lines exposed to MJN110 showed reduced hepatocyte proliferation. Mice with global invalidation or mice treated with MJN110 showed blunted liver regeneration. Moreover, mice with specific deletion of MAGL in either hepatocytes or myeloid cells displayed delayed liver regeneration. Mechanistically, MAGLHep-/- mice showed reduced liver eicosanoid production, in particular prostaglandin E2 that negatively impacts on hepatocyte proliferation. MAGL inhibition in macrophages resulted in the induction of the type I interferon pathway. Importantly, neutralising the type I interferon pathway restored liver regeneration of MAGLMye-/- mice. Conclusions: Our data demonstrate that MAGL promotes liver regeneration by hepatocyte and macrophage reprogramming. Impact and Implications: By using human liver samples and mouse models of global or specific cell type invalidation, we show that the monoacylglycerol pathway plays an essential role in liver regeneration. We unveil the mechanisms by which MAGL expressed in both hepatocytes and macrophages impacts the liver regeneration process, via eicosanoid production by hepatocytes and the modulation of the macrophage interferon pathway profile that restrains hepatocyte proliferation.The authors thank V. Fauveau, Institut Cochin, for help in surgery experiments; Olivier Thibaudeau of the Plateau de Morphologie Facility (INSERM UMR 1152, France) and Nicolas Sorhaindo of the Plateforme de Biochimie (CRI, INSERM UMR1149) for their help in the histology and liver function tests; and K. Bailly from the cytometry platform of Cochin Institute and H. Fohrer-Ting from the Centre de Recherche des Cordeliers, Paris University, for cell sorting analyses.Scopu

Rôle clé de la COX-2 dans l'inhibiton de la prolifération des cellules étoilées du foie myofibroblastiques humaines

PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Pathophysiology of NASH: perspectives for a targeted treatment.

International audienceNon alcoholic steatohepatitis (NASH) is the more severe form of nonalcoholic fatty liver disease. In NASH, fatty liver, hepatic inflammation, hepatocyte injury and fibrogenesis are associated, and this condition may eventually lead to cirrhosis. Current treatment of NASH relies on the reduction of body weight and increase in physical activity, but there is no pharmacologic treatment approved as yet. Emerging data indicate that NASH progression results from parallel events originating from the liver as well as from the adipose tissue, the gut and the gastrointestinal tract. Thus, dysfunction of the adipose tissue through enhanced flow of free fatty acids and release of adipocytokines, and alterations in the gut microbiome generate proinflammatory signals that underlie NASH progression. Additional 'extrahepatic hits' include dietary factors and gastrointestinal hormones. Within the liver, hepatocyte apoptosis, ER stress and oxidative stress are key contributors to hepatocellular injury. In addition, lipotoxic mediators and danger signals activate Kupffer cells which initiate and perpetuate the inflammatory response by releasing inflammatory mediators that contribute to inflammatory cell recruitment and development of fibrosis. Inflammatory and fibrogenic mediators include chemokines, the cannabinoid system, the inflammasome and activation of pattern-recognition receptors. Here we review the major mechanisms leading to appearance and progression of NASH, focusing on both extrahepatic signals and local inflammatory mechanisms, in an effort to identify the most promising molecular targets for the treatment of this condition