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3.0 Unported License.-- et al., The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD., We acknowledge the following grant support: SAF2012-33283 (MINECO, Spain), Comunidad de Madrid S2010/BMD-2423, EFSD and Amylin Paul Langerhans Grant and Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII, Barcelona, Spain) to AMV.; SAF2010-16037, SAF2013-43713-R (MINECO) and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas
y Digestivas (CIBEREHD, ISCIII) to PMS. RD12/0042/0019 (ISCIII) and S2010/BMD-2478 (Comunidad de Madrid) to LB, PI 13/01299 and Fundación Mutua Madrileña 2012 to C G-M and AIRC IG-2012 to GMF., Peer Reviewed

Cyclooxygenase (COX) catalyzes the first step in prostanoid biosynthesis and exists as two isoforms. COX-1 is a constitutive enzyme involved in physiological processes, whereas COX-2 is induced by a variety of stimuli. MicroRNAs (miRNAs) are noncoding RNAs that function as key posttranscriptional regulators of gene expression. Although it is known that COX-2 expression is regulated by miRNAs, there are no data regarding COX-2 involvement in miRNA regulation. Considering our previous results showing that COX-2 expression in hepatocytes protects against insulin resistance, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs implicated in insulin signaling in liver cells. Our results provide evidence of the molecular basis for a novel function of COX-2 in miRNA processing. COX-2 represses miRNA 23b (miR-23b), miR-146b, and miR-183 expression in liver cells by increasing the level of DEAD-box helicase p68 (DDX5) through phosphatidylinositol 3-kinase (PI3K)/p300 signaling and by modulating the enzymatic function of the Drosha (RNase type III) complex through its physical association with DDX5. The decrease of miR-183 expression promotes protection against insulin resistance by increasing insulin receptor substrate 1 (IRS1) levels. These results indicate that the modulation of miRNA processing by COX-2 is a key event in insulin signaling in liver and has potential clinical implications for the management of various hepatic dysfunctions., This work was supported by the Ministerio de Economía y Competitividad, Spain (SAF2010-16037 and SAF2013-43713-R to P.M.-S., BFU2011-24760 to L.B., and SAF2012-397329 and CIBERehd to M.C.), Comunidad de Madrid, Spain (S2010/BMD-2378 to L.B. and P.M.-S.), and Instituto de Salud Carlos III, Spain (RD12/0042/0019 and CIBERehd to L.B. and P.M.-S. and PI13/01299 to C.G.-M). N.A. is supported by Red de Investigación Cardiovascular (RD12/0042/0019). D.E.F. is supported by Financing Program for short stays abroad CONICET-Argentina. L.C.-S. is supported by a postdoctoral fellowship from CONACYT, Mexico., Peer Reviewed

Accumulation evidence links obesity-induced inflammation as an important contributor to the development of insulin resistance, which plays a key role in the pathophysiology of obesity-related diseases such as type 2 diabetes and nonalcoholic fatty liver disease. Cyclooxygenase (COX)-1 and -2 catalyze the first step in prostanoid biosynthesis. Because adult hepatocytes fail to induce COX-2 expression regardless of the proinflammatory stimuli used, we have evaluated whether this lack of expression under mild proinflammatory conditions might constitute a permissive condition for the onset of insulin resistance. Our results show that constitutive expression of human COX-2 (hCOX-2) in hepatocytes protects against adiposity, inflammation, and, hence, insulin resistance induced by a high-fat diet, as demonstrated by decreased hepatic steatosis, adiposity, plasmatic and hepatic triglycerides and free fatty acids, increased adiponectin-to-leptin ratio, and decreased levels of proinflammatory cytokines, together with an enhancement of insulin sensitivity and glucose tolerance. Furthermore, hCOX-2 transgenic mice exhibited increased whole-body energy expenditure due in part by induction of thermogenesis and fatty acid oxidation. The analysis of hepatic insulin signaling revealed an increase in insulin receptor-mediated Akt phosphorylation in hCOX-2 transgenic mice. In conclusion, our results point to COX-2 as a potential therapeutic target against obesity-associated metabolic dysfunction., This work was supported by Financing Program for Short Stays Abroad (Consejo Nacional de Investigaciones Científicas y Técnicas-Argentina) to D.E.F., by a Post-Doctoral fellowship from Consejo Nacional de Ciencia y Tecnología, México, SAF2012-39732 (Ministerio de Economía y Competitividad [MINECO], Spain) to L.C.-S., by CIBERehd (Instituto de Salud Carlos III [ISCIII], Spain) to M.C., by BFU2011-24760 (MINECO, Spain) to L.B., by S2010/BMD-2378 (Comunidad de Madrid) to L.B. and P.M.-S., by RD12/0042/0019 (ISCIII, Spain) and CIBERehd (ISCIII, Spain) to L.B. and P.M.-S., by SAF2010-16037, SAF2013-43713-R (MINECO, Spain) to P.M.-S., and by SAF2012-33283 (MINECO, Spain), S2010/BMD-2423 (Comunidad de Madrid), European Foundation for the Study of Diabetes and Amylin Paul Langerhans Grant, and CIBERDEM (ISCIII, Spain) to Á.M.V., Peer Reviewed

Macrophages are present in a large variety of locations, playing distinct functions that are determined by its developmental origin and by the nature of the activators of the microenvironment. Macrophage activation can be classified as pro-inflammatory (M1 polarization) or anti-inflammatory-pro-resolution-deactivation (M2), these profiles coexisting in the course of the immune response and playing a relevant functional role in the onset of inflammation (Figure 1). Several groups have analysed the metabolic aspects associated with macrophage activation to answer the question about what changes in the regulation of energy metabolism and biosynthesis of anabolic precursors accompany the different types of polarization and to what extent they are necessary for the expression of the activation phenotypes. The interest of these studies is to regulate macrophage function by altering their metabolic activity in a 'therapeutic way'., This work was supported by the Ministerio de Economía y Competitividad [grant numbers SAF2013-43713, SAF2014-52492 and IPT2012-1331-60000]; the Comunidad de Madrid [grant number S2010/BMD-2378]; and the Red Cardiovascular [grant numbers RD12/0042/0019]., Peer Reviewed

The resolution of inflammation is an active process driven by specialized pro-resolving lipid mediators, such as 15-epi-LXA4 and resolvin D1 (RvD1), that promote tissue regeneration. Macrophages regulate the innate immune response being key players during the resolution phase to avoid chronic inflammatory pathologies. Their half-life is tightly regulated to accomplish its phagocytic function, allowing the complete cleaning of the affected area. The balance between apoptosis and autophagy appears to be essential to control the survival of these immune cells within the inflammatory context. In the present work, we demonstrate that 15-epi-LXA4 and RvD1 at nanomolar concentrations promote autophagy in murine and human macrophages. Both compounds induced the MAP1LC3-I to MAP1LC3-II processing and the degradation of SQSTM1 as well as the formation of MAP1LC3(+) autophagosomes, a typical signature of autophagy. Furthermore, 15-epi-LXA4 and RvD1 treatment favored the fusion of the autophagosomes with lysosomes, allowing the final processing of the autophagic vesicles. This autophagic response involves the activation of MAPK1 and NFE2L2 pathways, but by an MTOR-independent mechanism. Moreover, these pro-resolving lipids improved the phagocytic activity of macrophages via NFE2L2. Therefore, 15-epi-LXA4 and RvD1 improved both survival and functionality of macrophages, which likely supports the recovery of tissue homeostasis and avoiding chronic inflammatory diseases., This work was supported by grants BFU2011/24760, SAF2013-43271-R, SAF2013-43713-R, SAF2014-52492 and IPT-2012-1331-060000 from MINECO; RD12/0042/0019 from Instituto de Salud Carlos III (ISCIII), Spain, and S2010/BMD2378 from Comunidad de Madrid., Peer reviewed

Molecular mechanisms on sepsis progression are linked to the imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity. Previous studies demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, has immunomodulatory effects, increasing survival in C57BL/6 mice in a model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). The mechanism by which BZL inhibits inflammatory response in sepsis is poorly understood. Also, our group recently reported that BZL is able to activate the nuclear factor erytroide-derived 2-Like 2 (NRF2) in vitro. The aim of the present work was to delineate the beneficial role of BZL during sepsis, analyzing its effects on the cellular redox status and the possible link to the innate immunity receptor TLR4. Specifically, we analyzed the effect of BZL on Nrf2 regulation and TLR4 expression in liver of mice 24 hours post-CLP. BZL was able to induce NRF2 nuclear protein localization in CLP mice. Also, we found that protein kinase C (PKC) is involved in the NRF2 nuclear accumulation and induction of its target genes. In addition, BZL prompted a reduction in hepatic CLP-induced TLR4 protein membrane localization, evidencing its immunomodulatory effects. Together, our results demonstrate that BZL induces hepatic NRF2 activation with the concomitant increase in the antioxidant defenses, and the attenuation of inflammatory response, in part, by inhibiting TLR4 expression in a murine model of sepsis., This work was supported by Research Grants PICT 2007-1730 from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and by Secretaría de Estado de Ciencia, Tecnología e Innovación del Gobierno de la Provincia de Santa Fe, Argentina (112/10) to M.T.R.; PICT 0360 from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) to V.A.C.; PICT 2012-2383 from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) to D.E.F. and SAF2012-33283 (MINECO, Spain), S2010/BMD-2423 (Comunidad de Madrid, CAM), SAF2013-43713-R (MINECO, Spain) to O.M. and P.M.S., Peer Reviewed

Cyclooxygenase-2 (COX-2) is involved in different liver diseases but little is known about the significance of COX-2 in the development and progression of non-alcoholic steatohepatitis (NASH). This study was designed to elucidate the role of COX-2 expression in hepatocytes in the pathogenesis of steatohepatitis and hepatic fibrosis. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either fed methionine-and-choline deficient (MCD) diet to establish an experimental non-alcoholic steatohepatitis (NASH) model or injected with carbon tetrachloride (CCl) to induce liver fibrosis. In our animal model, hCOX-2-Tg mice fed MCD diet showed lower grades of steatosis, ballooning and inflammation than Wt mice, in part by reduced recruitment and infiltration of hepatic macrophages, with a corresponding decrease in serum levels of pro-inflammatory cytokines. Furthermore, hCOX-2-Tg mice showed a significant attenuation of the MCD diet-induced increase in oxidative stress and hepatic apoptosis observed in Wt mice. Even more, hCOX-2-Tg mice treated with CCl had significantly lower stages of fibrosis and less hepatic content of collagen, hydroxyproline and pro-fibrogenic markers than Wt controls. Collectively, our data indicates that constitutive hepatocyte COX-2 expression ameliorates NASH and liver fibrosis development in mice by reducing inflammation, oxidative stress and apoptosis and by modulating activation of hepatic stellate cells, respectively, suggesting a possible protective role for COX-2 induction in NASH/NAFLD progression., This work was supported by Financing Program for short stays abroad for Assistant Researchers (CONICET-Argentina) (2618/13) and ANPCyT-PICT2383-2012 to D.F.; SAF2012-39732 (MINECO, Spain) and CIBERehd (ISCIII, Spain) (CB06/04/1069) to M.C.; SAF2014-52492 (MINECO, Spain) to L.B.; S2010/BMD-2378 (Comunidad de Madrid, CAM) to L.B. and P.M.S.; RD12/0042/0019 (ISCIII, Spain) and CIBERehd (ISCIII, Spain) to L.B. and P.M.S.; SAF-2015-65267-R (MINECO/FEDER), S2010/BMD-2423 (Comunidad de Madrid, CAM), EFSD and Amylin Paul Langerhans Grant and CIBERdem (ISCIII, Spain) to A.M.V.; PI13/01299 (ISCIII, Spain) to C.G-M.; SAF2013-43713-R (MINECO, Spain) to P.M.S., Peer Reviewed

Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, the balance between which decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β. We have previously shown that caveolin-1 (CAV1) is required for activation of the metalloprotease tumour necrosis factor (TNF)-α-converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17), and hence transactivation of the EGFR pathway. The specific mechanism by which TACE/ADAM17 is activated has not yet been determined. Here we show that TGF-β induces phosphorylation of sarcoma kinase (Src) in hepatocytes, a process that is impaired in Cav1(-/-) hepatocytes, coincident with a decrease in phosphorylated Src in detergent-resistant membrane fractions. TGF-β-induced activation of TACE/ADAM17 and EGFR phosphorylation were blocked using the Src inhibitor PP2. Cav1(+/+) hepatocytes showed early production of reactive oxygen species (ROS) induced by TGF-β, which was not seen in Cav1(-/-) cells. Production of ROS was inhibited by both the NADPH oxidase 1 (NOX1) inhibitor STK301831 and NOX1 knock-down, which also impaired TACE/ADAM17 activation and thus EGFR phosphorylation. Finally, neither STK301831 nor NOX1 silencing impaired Src phosphorylation, but PP2 blocked early ROS production, showing that Src is involved in NOX1 activation. As expected, inhibition of Src or NOX1 increased TGF-β-induced cell death in Cav1(+/+) cells. In conclusion, CAV1 is required for TGF-β-mediated activation of TACE/ADAM17 through a mechanism that involves phosphorylation of Src and NOX1-mediated ROS production., This work was supported by grants from the SpanishMinistry of Economy and Competitiveness (co-fundedby FEDER funds and the European Regional Development Fund: A Way To Build Europe) (BFU2012-35538 and ISCIII-RTICC: RD12-0036-0029 to I.F.,SAF2013-43713R to P.M. -S. and BFU2012-33932 toG.E.), the Agencia de Gestio d’Ajuts Universitaris i de Recerca, Generalitat de Catalunya (2014SGR0334 toI.F.), and the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007–2013) under REA grant agreement number PITN-GA-2012-316549 (IT LIVER) toI.F. J.M. -C. was the recipient of a pre-doctoral fellowship from the Ministry of Education, Culture and Sport, Spain (AP2010-3036). The I.F. research group is supported by the European Cooperation in Science and Technology (COST Action BM1203/EUROS)., Peer Reviewed

The biosynthesis of prostaglandins and thromboxanes has been a focus of interest in the management of many liver diseases. Cyclooxygenases are the enzymes involved in the first step of the biosynthesis of these lipid mediators and selective inhibitors for these isoenzymes as well as pharmacological analogues of prostaglandins have been developed and are currently applied therapeutically. Here we discuss the implications of these enzymes in the onset of metabolic and lipid disorders in the liver and their potential role in the progression of the diseases towards fibrosis and hepatocellular carcinogenesis., Supported by MINECO, Spain, No. SAF2014-52492R; COOPB20213 (CSIC), No. SAF2013-43713R and No. SAF2016-75004R; ISCIII, Spain, CIBERcv and CIBERehd; We thank FEDER for financial support., Peer reviewed

Título del pdf-manuscrito: PGE2 attenuates liver fibrosis in mice by downregulating miR-23a-
5p and inducing apoptosis of hepatic stellate cells., MicroRNAs (miRNAs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver diseases, whose end stage is hepatic fibrosis, a major global health burden. Pharmacological strategies for prevention or treatment of hepatic fibrosis are still limited, what makes it necessary to establish a better understanding of the molecular mechanisms underlying its pathogenesis. In this context, we have recently shown that cyclooxygenase-2 (COX-2) expression in hepatocytes restricts activation of hepatic stellate cells (HSCs), a pivotal event in the initiation and progression of hepatic fibrosis. Here, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs on a mouse model of CCl4 and bile duct ligation (BDL)-induced liver fibrosis. Our results provide evidence that COX-2 represses miR-23a-5p and miR-28-5p expression in HSC. The decrease of miR-23a-5p and miR-28-5p expression promotes protection against fibrosis by decreasing the levels of pro-fibrogenic markers α-SMA and COL1A1 and increasing apoptosis of HSC. Moreover, we demonstrate that serum levels of miR-28-5p are decreased in patients with chronic liver disease. These results suggest a protective effect exerted by COX-2-derived prostanoids in the process of hepatofibrogenesis., This work was supported by Financing Program for short stays abroad for Assistant Researchers (CONICET-Argentina) and i-COOP2016-20213 to D.F.; SAF2014-52492 (MINECO, Spain) to L.B.; CIBERcv and CIBERehd (ISCIII, Spain) to L.B., P.M.S. and M.C.; PI13/01299 (ISCIII, Spain) to C.G-M.; SAF2013-43713-R, SAF2016-75004-R and SAF2015-70270-REDT (MINECO, Spain) to L.B., P.M.S. and M.C. We thank FEDER for financial support., Peer reviewed

[Methods]
Peptides and proteins from cardiomyocytes were trypsin-digested using the whole proteome in-gel digestion protocol, followed by 18O labeling as previously described (Bonzon-Kulichenko, E. et al Mol. Cell. Proteomics 2011, 10, M110 003335, doi:10.1074/mcp.M110.003335). The peptide pools were separated in 24 fractions ranging from pH 3-10 by IEF on a 3100 OFFGel fractionator (Agilent, Santa Clara, CA, USA) using the standard methods for peptides recommended by the manufacturer. The recovered fractions were desalted using OMIX C18 tips (Varian, Inc, Agilent, USA), and dried down before reverse phase-high performance liquid chromatography (RP-HPLC)-LIT analysis using a Surveyor LC System coupled to a linear ion trap mass spectrometer LTQ (Thermo Fisher Scientific, Waltham, MA USA). The LTQ was operated in a data-dependent ZoomScan and MS/MS switching mode as previously described (Lopez-Ferrer, D. et al. Proteomics 2006, 6 Suppl 1, S4-11, doi:10.1002/pmic.200500375). Protein identification was done using SEQUEST algorithm (Bioworks 3.2 package, Thermo Fisher Scientific). MS/MS raw files were searched against a Rat/Mouse Swissprot database supplemented with the sequence of bovine and porcine trypsin. SEQUEST results were analyzed using the probability ratio method (Martinez-Bartolome, S et al. Mol. Cell. Proteomics 2008, 7, 1135–1145, doi:10.1074/mcp.M700239-MCP200) and discovery rates (FDR) of peptide identifications were calculated as previously described ((Navarro, P. et al. J Proteome Res 2009, 8, 1792–1796, doi:10.1021/pr800362h). Peptide identification and quantification were done as previously described (Bonzon-Kulichenko, E. et al Mol. Cell. Proteomics 2011, 10, M110 003335, doi:10.1074/mcp.M110.003335; Navarro, P.; et al. J Proteome Res 2014, 13, 1234–1247, doi:10.1021/pr4006958). Statistical significance of protein abundance changes was assayed by controling the FDR, being a FDR less than 0.05 considered to be significant. Threshold-free analysis of coordinated protein responses was performed using the SBT model, as described (García-Marqués, F. et al. Mol. Cell. Proteomics 2016, 15, 1740–1760, doi:10.1074/mcp.M115.055905)., The biochemical mechanisms of cell injury and myocardial cell death after myocardial infarction remain unresolved. Cyclooxygenase 2 (COX-2), a key enzyme in prostanoid synthesis, is expressed in human ischemic myocardium and dilated cardiomyopathy, but it is absent in healthy hearts. To assess the role of COX-2 in cardiovascular physiopathology, we developed transgenic mice, thatconstitutively express functional human COX-2 in cardiomyocytes under the control of the α-myosin heavy chain promoter. These animals had no an apparent phenotype, but were protected against ischemia-reperfusion injury in isolated hearts, with an enhanced functional recovery and diminished cellular necrosis. To further explore the phenotype of this animal model, we carried out a differential proteome analysis of wild-type vs. transgenic cardiomyocytes. Here we include the results of this proteomic study with the list of identified proteins and their quantification, Ministerio de Economia y Competitividad (SAF2013-43713-R) and Generalitat Valenciana (ACOMP/2011/120), No