Anna Planagumà

The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPARγ activation

The importance of inflammation in initiating the sequence of events that lead to liver fibrosis is increasingly recognized. In this study, we tested the effects of SC‐236, a selective cyclooxygenase (COX)‐2 inhibitor, in rats with carbon tetrachloride (CCl4)‐induced liver fibrosis. Livers from CCl4‐treated rats showed increased COX‐2 expression and 15‐deoxy‐prostaglandin (PG)J2 (15d‐PGJ2) formation, as well as decreased peroxisome proliferator‐activated receptor (PPAR)γ expression. In these animals, SC‐236 reduced liver fibrosis as revealed by histological analysis and by a reduction in hepatic hydroxyproline levels, metalloproteinase‐2 activity, and α‐smooth muscle actin expression. Interestingly, SC‐236 normalized 15d‐PGJ2 levels and restored PPARγ expression in the liver of CCl4‐treated rats. In isolated hepatic stellate cells (HSCs)—the major player in liver fibrogenesis—and Kupffer cells—the cell type primarily responsible for increased hepatic COX‐2—SC‐236 exhibited remarkable pro‐apoptotic and growth inhibitory properties. Of interest, SC‐236 decreased HSC viability to a similar extent than the PPARγ ligand rosiglitazone. Moreover, SC‐236 significantly induced PPARγ expression in HSCs and acted as a potent PPARγ agonist in a luciferase‐reporter trans‐activation assay. These data indicate that, by mechanisms involving non‐parenchymal cell apoptosis and PPARγ activation, the selective COX‐2 inhibitor SC‐236 might have therapeutic potential for prevention of liver fibrosis.

Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S-hydroxy-DHA

Docosahexaenoic acid (DHA) is a ω‐3 essential fatty acid that reduces the incidence and severity of a number of diseases. Recently, a novel series of DHA‐derived lipid mediators with potent protective actions has been identified. In this study we demonstrate that dietary amplification of these DHA‐derived products protects the liver from necroinflammatory injury. In vitro, supplementation of hepatocytes with DHA significantly reduced hydrogen peroxide‐induced DNA damage, evaluated by the “comet assay,” and oxidative stress, determined by measurement of malondialdehyde levels. In vivo, dietary supplementation of mice with DHA ameliorated carbon tetrachloride‐induced necroinflammatory damage. In addition, hepatic cyclooxygenase‐2 expression and PGE2 levels were significantly reduced in mice fed DHA‐enriched diets. In these animals, increased hepatic formation of DHA‐derived lipid mediators (i.e., 17S‐hydroxy‐DHA (17S‐HDHA) and protectin D1) was detected by HPLC‐gas chromatography/mass spectrometry analysis. Consistent with these findings, synthetic 17‐HDHA abrogated genotoxic and oxidative damage in hepatocytes and decreased TNF‐α release and 5‐lipoxygenase expression in macrophages. In a transactivation assay, 17‐ HDHA acted in a concentration‐dependent manner as a PPARγ agonist. Taken together, these findings identify a potential role for DHA‐derived products, specifically 17S‐HDHA and protectin D1, in mediating the protective effects of dietary DHA in necroinflammatory liver injury.—González‐Périz, A., Planagumà, A., Gronert, K., Miquel, R., López‐Parra, M., Titos, E., Horrillo, R., Ferré, N., Deulofeu, R., Arroyo, V., Rodés, J., Clària, J. Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S‐hydroxy‐DHA. FASEB J. 20, E1844–E1855 (2006)

Aspirin (ASA) regulates 5-lipoxygenase activity and peroxisome proliferator-activated receptor α-mediated CINC-1 release in rat liver cells: novel actions of lipoxin A4 (LXA4) and ASA-triggered 15-epi-LXA4

The mechanism of action of aspirin (ASA) is related to cyclooxygenase (COX) inhibition, but additional actions cannot be excluded for their antiinflammatory properties and antithrombotic activity. In the current investigation, we examined the effects of ASA on COX and 5‐lipoxygenase (5‐LO) pathways and its impact on peroxisome proliferator‐activated receptor α (PPARα) and cytokine‐induced neutrophil chemoattractant‐1 (CINC‐1) levels in rat liver cells. In Kupffer cells, the liver resident macrophages, ASA switched eicosanoid biosynthesis from prostaglandin E2 (PGE2) to leukotriene B4 (LTB4) and 15‐epi‐lipoxin A4 (15‐epi‐LXA4) formation. In hepatocytes, ASA significantly inhibited PPARα protein expression and CINC‐1 secretion, effects that were also observed in hepatocytes exposed to the selective PPARα agonist Wy‐14643. In contrast, treatment of hepatocytes with PGE2 in association with LTB4 had no significant effect on PPARα but stimulated CINC‐1 release. Interestingly, the endogenous antiinflammatory eicosanoids LXA4 and ASA‐triggered 15‐epi‐LXA4, in addition to inhibiting macrophage 5‐LO activity to a similar extent as PGE2, significantly reduced PPARα and CINC‐1 levels in hepatocytes. Taken together and because arachidonic acid‐derived products, PPARα levels, and CINC‐1 secretion are involved in the extent and duration of an inflammatory response, these findings provide additional molecular mechanisms for the pharmacological properties of ASA.

Inhibition of 5-lipoxygenase induces cell growth arrest and apoptosis in rat Kupffer cells: implications for liver fibrosis

The existence of an increased number of Kupffer cells is recognized as critical in the initiation of the inflammatory cascade leading to liver fibrosis. Because 5‐lipoxygenase (5‐LO) is a key regulator of cell growth and survival, in the current investigation we assessed whether inhibition of the 5‐LO pathway would reduce the excessive number of Kupffer cells and attenuate inflammation and fibrosis in experimental liver disease. Kupffer cells were the only liver cell type endowed with a metabolically active 5‐LO pathway (i.e., expressed mRNAs for 5‐LO, 5‐LO‐activating protein [FLAP], and leukotriene [LT] C4 synthase and generated LTB4 and cysteinyl‐LTs). Both the selective 5‐LO inhibitor AA861 and the FLAP inhibitor BAY‐X‐1005 markedly reduced the number of Kupffer cells in culture. The antiproliferative properties of AA861 and BAY‐X‐1005 were associated with the occurrence of condensed nuclei, fragmented DNA, and changes in DNA content and cell cycle frequency distribution consistent with an apoptotic process. In vivo, in carbon tetrachloride‐treated rats, BAY‐X‐1005 had a significant antifibrotic effect and reduced liver damage and the hepatic content of hydroxyproline. Together, these findings indicate a novel mechanism by which inactivation of the 5‐LO pathway could disrupt the sequence of events leading to liver inflammation and fibrosis.

Increased susceptibility to exacerbated liver injury in hypercholesterolemic ApoE-deficient mice: potential involvement of oxysterols

The contribution of metabolic factors to the severity of liver disease is not completely understood. In this study, apolipoprotein E-deficient (ApoE-/-) mice were evaluated to define potential effects of hypercholesterolemia on the severity of carbon tetrachloride (CCl4)-induced liver injury. Under baseline conditions, hypercholesterolemic ApoE-/- mice showed increased hepatic oxidative stress (SOD activity/4-hydroxy-2-nonenal immunostaining) and higher hepatic TGF-beta1, MCP-1, and TIMP-1 expression than wild-type control mice. After CCl4 challenge, ApoE-/- mice exhibited exacerbated steatosis (Oil Red O staining), necroinflammation (hematoxylin-eosin staining), macrophage infiltration (F4/80 immunohistochemistry), and fibrosis (Sirius red staining and alpha-smooth muscle actin immunohistochemistry) and more severe liver injury [alanine aminotransferase (ALT) and aspartate aminotransferase] than wild-type controls. Direct correlations were identified between serum cholesterol and hepatic steatosis, fibrosis, and ALT levels. These changes did not reflect the usual progression of the disease in ApoE-/- mice, since exacerbated liver injury was not present in untreated age-paired ApoE-/- mice. Moreover, hepatic cytochrome P-450 expression was unchanged in ApoE-/- mice. To explore potential mechanisms, cell types relevant to liver pathophysiology were exposed to selected cholesterol-oxidized products. Incubation of hepatocytes with a mixture of oxysterols representative of those detected by GC-MS in livers from ApoE-/- mice resulted in a concentration-dependent increase in total lipoperoxides and SOD activity. In hepatic stellate cells, oxysterols increased IL-8 secretion through a NF-kappaB-independent mechanism and upregulated TIMP-1 expression. In macrophages, oxysterols increased TGF-beta1 secretion and MCP-1 expression in a concentration-dependent manner. Oxysterols did not compromise cell viability. Taken together, these findings demonstrate that hypercholesterolemic mice are sensitized to liver injury and that cholesterol-derived products (i.e., oxysterols) are able to induce proinflammatory and profibrogenic mechanisms in liver cells.

Inhibition of 5-lipoxygenase-activating protein abrogates experimental liver injury: role of Kupffer cells

Activation of Kupffer cells is a prominent feature of necro‐inflammatory liver injury. We have recently demonstrated that 5‐lipoxygenase (5‐LO) and its accessory protein, 5‐LO‐activating protein (FLAP), are essential for the survival of Kupffer cells in culture, as their inhibition drives these liver resident macrophages to programmed cell death. In the current study, we explored whether the potent FLAP inhibitor, Bay‐X‐1005, reduces the number of Kupffer cells in vivo and whether this pharmacological intervention protects the liver from carbon tetrachloride (CCl4)‐induced damage. Rats treated with CCl4 showed an increased number of Kupffer cells, an effect that was abrogated by the administration of Bay‐X‐1005 (100 mg/Kg body weight, per oral, daily). Consistent with a role for Kupffer cells in necro‐inflammatory liver injury, partial depletion of Kupffer cells following FLAP inhibition was associated with a remarkable hepatoprotective action. Indeed, Bay‐X‐1005 significantly reduced the intense hepatocyte degeneration and large bridging necrosis induced by CCl4 treatment. Moreover, Bay‐X‐1005 induced a reduction in the gelatinolytic activity of matrix metalloproteinase‐2 (MMP‐2) and a decrease in mRNA expression of tissue inhibitor of MMP‐2. The FLAP inhibitor reduced leukotriene (LT)B4 and cysteinyl LT levels and down‐regulated 5‐LO and FLAP protein expression in the liver. It is interesting that a significant increase in the hepatic formation of lipoxin A4, an endogenous, anti‐inflammatory lipid mediator involved in the resolution of inflammation, was observed after the administration of Bay‐X‐1005. These findings support the concept that modulation of the 5‐LO pathway by FLAP inhibition may be useful in the prevention of hepatotoxin‐induced necro‐inflammatory injury.

Cyclooxygenase-1 derived prostaglandins are involved in the maintenance of renal function in rats with cirrhosis and ascites.

The maintenance of renal function in decompensated cirrhosis is highly dependent on prostaglandins (PGs). Since PG synthesis is mediated by cyclooxygenase‐1 and −2 (COX‐1 and COX‐2), the present study was designed to examine which COX isoform is involved in this phenomenon. Renal COX‐1 and COX‐2 protein expression and distribution were analysed by Western blot and immunohistochemistry in nine rats with carbon tetrachloride‐induced cirrhosis and ascites and 10 control animals. The effects of placebo and selective COX‐1 (SC‐560) and COX‐2 (celecoxib) inhibitors on urine flow (V), urinary excretion of sodium (UNaV) and PGE2 (UPGE2V), glomerular filtration rate (GFR), renal plasma flow (RPF), the diuretic and natriuretic responses to furosemide and renal water metabolism were assessed in 88 rats with cirrhosis and ascites. COX‐1 protein levels were found to be unchanged in kidneys from cirrhotic rats. In contrast, these animals showed enhanced renal COX‐2 protein expression which was focally increased in the corticomedullary region. Although UPGE2V was equally reduced by SC‐560 and celecoxib, only SC‐560 produced a significant decrease in UNaV, GFR and RPF and a pronounced impairment in the diuretic and natriuretic responses to furosemide in rats with cirrhosis and ascites. Neither SC‐560 nor celecoxib affected renal water metabolism in cirrhotic rats. These results indicate that despite abundant renal COX‐2 protein expression, the maintenance of renal function in cirrhotic rats is mainly dependent on COX‐1‐derived prostaglandins.

Comparative Protection against Liver Inflammation and Fibrosis by a Selective Cyclooxygenase-2 Inhibitor and a Nonredox-Type 5-Lipoxygenase Inhibitor

In this study, we examined the relative contribution of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), two major proinflammatory pathways up-regulated in liver disease, to the progression of hepatic inflammation and fibrosis. Separate administration of 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-236), a selective COX-2 inhibitor, and CJ-13,610, a 5-LO inhibitor, to carbon tetrachloride-treated mice significantly reduced fibrosis as revealed by the analysis of Sirius Red-stained liver sections without affecting necroinflammation. Conversely, combined administration of SC-236 and 4-[3-[4-(2-methylimidazol-1-yl)-phenylthio]]phenyl-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide (CJ-13,610) reduced both necroinflammation and fibrosis. These findings were confirmed in 5-LO-deficient mice receiving SC-236, which also showed reduced hepatic monocyte chemoattractant protein 1 expression. Interestingly, SC-236 and CJ-13,610 significantly increased the number of nonparenchymal liver cells with apoptotic nuclei (terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive). Additional pharmacological profiling of SC-236 and CJ-13,610 was performed in macrophages, the primary hepatic inflammatory cell type. In these cells, SC-236 inhibited prostaglandin (PG) E2 formation in a concentration-dependent manner, whereas CJ-13,610 blocked leukotriene B4 biosynthesis. Of note, the simultaneous addition of SC-236 and CJ-13,610 resulted in a higher inhibitory profile on PGE2 biosynthesis than the dual COX/5-LO inhibitor licofelone. These drugs differentially regulated interleukin-6 mRNA expression in macrophages. Taken together, these findings indicate that both COX-2 and 5-LO pathways are contributing factors to hepatic inflammation and fibrosis and that these two pathways of the arachidonic acid cascade represent potential targets for therapy.

5-Lipoxygenase (5-LO) is Involved in Kupffer Cell Survival. Possible Role of 5-LO Products in the Pathogenesis of Liver Fibrosis

A wealth of evidence indicates that inflammation plays a central role in the current paradigm of liver fibrosis. Kupffer cells, which represent the largest population of resident macrophages in the body [1], are uniquely positioned as the predominant primary inflammatory effector cells to initiate the inflammatory cascade leading to tissue remodeling and fibrosis. For this reason, the presence of an increased population of Kupffer cells together with the bulk release of inflammatory mediators by these macrophages are considered to be critical events during the early stages of liver inflammation and fibrosis [2,3]. Arachidonic acid metabolites derived from 5-lipoxygenase (5-LO) are essential regulators of cell growth and survival [4]. Given that we recently demonstrated that 5-LO expression and leukotriene (LT) formation are increased in livers from rats with carbon tetrachloride (CCl4)-induced cirrhosis [5], it is our hypothesis that 5-LO products play a role in Kupffer cell survival and in the pathogenesis of liver inflammation and fibrosis. Therefore, in the current study we examined the 5-LO pathway in sinusoidal liver cells and specifically analyzed the role of 5-LO in Kupffer cell growth and survival.

Renal Effects of Selective Cyclooxygenase Inhibition in Experimental Liver Disease

Renal synthesis of vasodilator prostaglandins (PGs) plays a key role in the maintenance of renal function in decompensated cirrhosis [1,2]. In fact, acute PG inhibition with nonsteroidal anti-inflammatory drugs (NSAIDs) in patients with cirrhosis and ascites is associated with a significant impairment in renal hemodynamics, sodium excretion, free water clearance and the renal response to furosemide and spironolactone [1-3]. Thus, in clinical practice, patients with decompensated cirrhosis cannot be treated with NSAIDs on a long-term basis because of the high risk of developing renal failure and refractory ascites.