BUSCADOR RECOLECTA

[ES] La lesión por isquemia-reperfusión (I/R) hepática (IRI) es una causa importante de mortalidad y morbilidad en la resección hepática y el trasplante de hígado. Durante la hipoxia, el hígado permanece sin oxígeno, cambiando su metabolismo y parando la síntesis de ATP. Paradójicamente, el restablecimiento del flujo de oxígeno causa más daño, activando el sistema inmunitario que generará una gran cantidad de especies reactivas de oxígeno (ROS) causando daño celular y tisular. La ciclooxigenasa-2 (COX-2) es una enzima clave en la biosíntesis de prostaglandinas y su importancia en la IRI es controvertida. La PGE2, prostaglandina E2, es el principal producto de la COX-2, y participa en la mediación de procesos patológicos como la inflamación, la fiebre y el dolor. El uso de AINEs, inhibidores específicos de la COX, apunta a un efecto beneficioso en la resolución del proceso inflamatorio, pero cada vez más estudios apoyan el papel antiinflamatorio de la COX-2. De hecho, estudios previos han demostrado que la sobreexpresión de COX-2 en hepatocitos protege a los ratones de la apoptosis y el estrés celular, además de reducir la respuesta inflamatoria en diferentes modelos de enfermedad hepática.
En esta tesis doctoral, se utilizó un ratón transgénico que sobreexpresa COX-2 en los hepatocitos (h-COX-2 Tg) para dilucidar el papel y la implicación de la COX-2 en la IRI. Los animales de tipo silvestre (Wt) y h-COX-2 Tg fueron sometidos a 90 min de isquemia, seguidos de 4 o 24 h de reperfusión. Comparando los animales h-COX-2 Tg con sus hermanos Wt, el daño celular y tisular se atenúa tras la IRI. Entre las distintas vías modificadas, la cascada inflamatoria está menos activada, con menor liberación de citoquinas pro-inflamatorias, menor reclutamiento hepático, e infiltración de neutrófilos. Las vías de necrosis y apoptosis también se atenúan así como se reduce del estrés del retículo endoplásmico, y aumenta la autofagia. La respuesta antioxidante se potencia en el contexto de la sobreexpresión de COX-2 y la producción total de ROS es menor, lo que contribuye a un menor daño tisular. Cuando los animales Wt se someten un pre-condicionamiento (PC), la COX-2 endógena se induce a niveles más altos que sin PC, mostrando menos daño, una inflamación atenuada, y una respuesta antioxidante mejorada. Además, se muestra que el papel de la COX-2 en esta protección es específico, ya que su inhibición con DFU revierte los efectos observados e iguala el daño causado a los animales Wt. Las mitocondrias son actores centrales en la fisiopatología de la IRI. En este sentido, la función mitocondrial está preservada en los hígados que sobreexpresan COX-2, con un potencial de membrana mitocondrial conservado y una tasa respiratoria preservada. Estos efectos pueden explicarse por una estabilización de las crestas mitocondriales, invaginaciones de la membrana mitocondrial interna (IMM) que se mantienen mediante interacciones de varias isoformas de la proteína OPA1. Su procesamiento está mediado por proteasas, como OMA1. En ratones h-COX-2 Tg hay un menor procesamiento de OPA1, que se correlaciona con una actividad atenuada de OMA1. Por otro lado, se realizó un estudio retrospectivo en pacientes que habían sido sometidos a un trasplante hepático. Se analizaron los niveles de PGE2 y se correlacionaron con las funciones hepáticas tras el trasplante. Este análisis muestra que la presencia de PGE2 en el plasma de los pacientes receptores se correlaciona con un mejor pronóstico, mientras que unos niveles más bajos de PGE2 se asocian con una disfunción precoz del injerto.
Todos estos resultados presentan a la COX-2 como un nuevo actor en la protección del hígado tras I/R, mostrando un papel antiinflamatorio y antioxidante, así como reduciendo el daño mitocondrial, el estrés celular y la muerte celular. Además, se demuestra cómo las prostaglandinas derivadas de la COX-2 en condiciones fisiológicas pueden desempeñar un papel protector en casos de trasplante hepático., [CA] La lesió per isquèmia-reperfusió (I/R) hepàtica (IRI) és una causa important de mortalitat i morbiditat en la resecció hepàtica i el trasplantament de fetge. Durant la hipòxia, el fetge roman sense oxigen, canviant el seu metabolisme i aturant la síntesi d'ATP. Paradoxalment, el restabliment del flux d'oxigen causa més danys, activant el sistema immunitari que genera una gran quantitat d'espècies reactives d'oxigen (ROS) causant dany cel·lular i tissular. La ciclooxigenasa-2 (COX-2) és un enzim clau en la biosíntesi de prostaglandines i la seva importància a l'IRI és controvertida. La PGE2, prostaglandina E2, és el principal producte de la COX-2, i participa en la mediació de processos patològics com la inflamació i la febre. Mentre que l'ús d'AINEs, inhibidors específics de la COX-2, apunta a un efecte beneficiós en la resolució del procés inflamatori, cada cop més estudis donen suport a un paper antiinflamatori de la COX-2. De fet, estudis previs han demostrat que la sobreexpressió de COX-2 en hepatòcits protegeix els ratolins de l'apoptosi i l'estrès cel·lular, a més de reduir la resposta inflamatòria, en diferents models de malaltia hepàtica.
En aquesta tesi, s'ha utilitzat un ratolí transgènic que sobreexpressa la COX-2 en els hepatòcits (h-COX-2 Tg) per dilucidar el paper i la implicació de la COX-2 a l'IRI. Els animals de tipus silvestre (Wt) i h-COX-2 Tg van ser sotmesos a 90 min d'isquèmia, seguits de 4 o 24 h de reperfusió. Comparant els animals h-COX-2 Tg amb els seus germans Wt, el dany cel·lular i tissular s'atenua després de l'IRI. Entre les diferents vies modificades, la cascada inflamatòria està menys activada, s'alliberen menys citocines proinflamatòries , hi ha un menor reclutament hepàtic i menor infiltració de neutròfils. Les vies de necrosi i apoptosi també s'atenuen, així com es redueix l'estrès del reticle endoplasmàtic, i l'autofàgia augmenta. La resposta antioxidant es potencia i la producció total de ROS també és menor, fet que contribueix a un menor dany tissular. Quan els animals Wt se sotmeten a un precondicionament (PC), la COX-2 endògena s'indueix a nivells més alts que sense PC, i aquests fetges mostren menys dany, una inflamació atenuada i una resposta antioxidant millorada. A més, es mostra que el paper de la COX-2 en aquesta protecció és específic, ja que la seva inhibició amb DFU, reverteix els efectes observats i iguala el dany causat als animals Wt. Els mitocondris són actors centrals en la fisiopatologia de l'IRI. En aquest sentit, la funció mitocondrial és preservada als fetges que sobreexpressen COX-2, com es pot demostrar per un potencial de membrana mitocondrial conservat i una taxa respiratòria preservada. Aquests efectes es poden explicar per una estabilització de les crestes mitocondrials, invaginacions de la membrana mitocondrial interna (IMM) que es mantenen mitjançant interaccions de diverses isoformes d'OPA1, una proteïna de la IMM. El seu processament està mediat per proteasas, com OMA1. En ratolins h-COX-2 Tg hi ha un menor processament d'OPA1, que es correlaciona amb una activitat atenuada d'OMA1, mostrant una estabilització de les crestes. D'altra banda, es va fer un estudi retrospectiu amb pacients que havien estat sotmesos a un trasplantament hepàtic. Es van analitzar els nivells de PGE2 i es van correlacionar amb les funcions hepàtiques després del trasplantament. Aquesta anàlisi mostra que la presència de PGE2 en el plasma dels pacients receptors es correlaciona amb un millor pronòstic, mentre que uns nivells més baixos de PGE2 s'associen amb una disfunció precoç de l'empelt.
Tots aquests resultats presenten a la COX-2 com un nou actor en la protecció del fetge després d'I/R, mostrant un paper antiinflamatori i antioxidant, així com reduint la lesió mitocondrial, l'estrès cel·lular i la mort cel·lular. A més, es demostra com les prostaglandines derivades de la COX-2 en condicions fisiològiques poden exercir un paper protector en casos de trasplantament hepàtic., [EN] Hepatic ischemia-reperfusion (I/R) injury (IRI) is a major cause of mortality and morbidity in liver resection and liver transplantation. During the hypoxia, the liver remains without oxygen supply, shifting its metabolism and stopping the ATP synthesis. Paradoxically, the restoration of oxygen flow causes the most damage with an activation of the immune system that will generate a burst of reactive species of oxygen (ROS) that will cause cell and tissue damage. Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandin biosynthesis and its importance in IRI is controversial. PGE2, prostaglandin E2, is the main product of COX-2, and is mainly involved in mediating pathological processes such as inflammation, fever and pain. While the use of NSAIDs, specific COX inhibitors, points to a beneficial effect in the resolution of the inflammatory process, several studies support the idea of an anti-inflammatory role of COX-2. In fact, previous studies have shown that COX-2 overexpression in hepatocytes protects mice from apoptosis and cellular stress, as well as reducing the inflammatory response, in different liver disease models.
In this PhD thesis, a hepatocyte-specific COX-2 transgenic mouse (h-COX-2 Tg) was used to elucidate the role and involvement of COX-2 in IRI. Wild type (Wt) and h-COX-2 Tg animals were subjected to 90 min of ischemia, followed by 4 or 24 h of reperfusion. Comparing h-COX-2 Tg animals with their Wt littermates, cellular and tissue damage resulting from IRI is attenuated. Among these pathways, the inflammatory cascade is less activated, with less pro-inflammatory cytokine release, less hepatic recruitment and neutrophil infiltration. Necrosis and apoptosis pathways are also attenuated such as reduced endoplasmic reticulum stress, and increased autophagy. The antioxidant response appears to be enhanced in the context of COX-2 overexpression and total ROS production is also lower, contributing to less tissue damage. When Wt animals are subjected to preconditioning (PC), endogenous COX-2 is induced at higher levels than without PC, and these livers show less damage, attenuated inflammation, and an enhanced antioxidant response. Furthermore, the role of COX-2 in this observed protection has been shown to be specific, as its inhibition with DFU, reverses the observed effects, and matched the damage caused to Wt animals. Mitochondria are central players in the pathophysiology of IRI. In this regard, mitochondrial function is preserved in COX-2-overexpressing livers, as can be demonstrated by a conserved mitochondrial membrane potential and a preserved respiratory rate. These results can be explained by a stabilisation of mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) that maintained through interactions of various isoforms of OPA1. Its processing is mediated by proteases, such as OMA1, which acts under certain stimuli. In h-COX-2 Tg mice, there is a reduced OPA1 processing that correlates with attenuated OMA1 activity, showing a stabilisation of cristae in the context of COX-2 overexpression after I/R. On the other hand, a retrospective study was conducted in patients who had undergone liver transplantation. In this part of the study, PGE2 levels were analysed and correlated with liver functions after transplantation. This analysis shows that the presence of PGE2 in the plasma of recipients correlates with a better prognosis, while lower PGE2 levels are associated with early graft dysfunction.
All these results present COX-2 as a new player in liver protection after I/R, showing an anti-inflammatory and antioxidant role, as well as reducing mitochondrial damage, cell stress and cell death. Furthermore, it is shown how COX-2-derived prostaglandins under physiological conditions can play a protective role in cases of liver transplant., This work has been carried out with the financial support of the Spanish Ministry of
Science and Innovation (SAF2016-75004R and PID2019-108977RB-100), the
CIBERehd (Centro de Investigaciones Biomédicas En Red de Enfermedades
Hepáticas y Digestivas) and the COST Action (CA15203 - Mitochondrial mapping:
Evolution - Age - Gender - Lifestyle - Environment (MITOEAGLE)).
Marina Fuertes Agudo benefited from a pre-doctoral FPI contract (BES-2017-
081928) associated with the SAF2016-75004R project. She spent 3 months in the
laboratory of Dr. Pau Sancho Bru at the Institut d’Investigacions Biomèdiques August
Pi I Sunyer (IDIBAPS, Barcelona, Spain) funded by a short stay grant awarded by the
CIBERehd and 3 months in the laboratory of Dr. Anne Dubart Kupperschmitt and Dr.
Jean Charles Duclos Vallée at the Institut Nationale de la Santé et la Recherche
Médicale (INSERM, Villejuif, France) funded by a short stay grant awarded by the
European Molecular Biology Organisation (EMBO, SEG_9771).

© 2020 by the authors., Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML., M.G. Contreras Mostazo and I. Alshamleh were supported by the EU grant HaemMetabolome H2020-MSCA-ITN-2015-675790. H. Schwalbe, H. Serve, F. Schnütgen, S. Marin and M. Cascante acknowledge support from the European Commission (HaemMetabolome [EC-675790]). M. Cascante and S. Marin acknowledge the Spanish Ministerio de Economia y Competitividad (MINECO)-European Commission FEDER funds—“Una manera de hacer Europa” (SAF2017-89673-R), the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) Generalitat de Catalunya (2017SGR1033) and CIBERehd (CB17/04/00023) (ISCIII, Spain). M. Cascante also received support through the prize “ICREA Academia” for excellence in research, funded by ICREA foundation–Generalitat de Catalunya. F. Schnütgen was supported by the Wilhelm Sander-Foundation (2015.138.1) and the Deutsche Forschungsgemeinschaft (SCHN1166/4-1). H. Schwalbe acknowledges support for the Center for Biomolecular Magnetic Resonance (BMRZ) by state of Hesse. M. Casado and P. Martín-Sanz acknowledge support from SAF2016-75004-R (MINECO, Spain, AEI/FEDER, UE), PID2019-108977RB-I00 (MICINN, Spain,AEI/FEDER, UE), and CIBERehd (CB06/04/1069) (ISCIII, Spain). D. Fuhrmann, B. Brüne, N. Kurrle and H. Serve acknowledge Deutsche Forschungsgemeinschaft (SFB 815, TP A08, TP A10).

Póster presentado al: Liver Meeting AASLD 2021: American Association fort eh Study of Liver Diseases. (Anaheim, CA, USA and virtual), 12-15 de novembre 2021 Abstract: 470, pag. 293A, In this work, we analyzed different aspects in order to characterize the impact of COX-2 in mitochondrial function after IRI., This work was supported by SAF2016-75004-R (MINECO, Spain) and PID2019-108977RB-I00 (MICINN, Spain), CIBERehd (ISCIII, Spain). M F-A. is recipient of FPI fellowship from MINECO (BES-2017-081928), Peer reviewed

© 2022 The Authors., It has long been known that thyroid hormones have implications for multiple physiological processes and can lead to serious illness when there is an imbalance in its metabolism. The connections between thyroid hormone metabolism and the immune system have been extensively described, as they can participate in inflammation, autoimmunity, or cancer progression. In addition, changes in the normal intestinal microbiota involve the activation of the immune system while triggering different pathophysiological disorders. Recent studies have linked the microbiota and certain bacterial fragments or metabolites to the regulation of thyroid hormones and the general response in the endocrine system. Even if the biology and function of the thyroid gland has attracted more attention due to its pathophysiological importance, there are essential mechanisms and issues related to it that are related to the interplay between the intestinal microbiota and the immune system and must be further investigated. Here we summarize additional information to uncover these relationships, the knowledge of which would help establish new personalized medical strategies., The review was written by authors that work in a laboratory where projects are supported by: Ministerio de Economía, Industria y Competitividad (RTI2018-094727-B-100, SAF2017-82436-R, RYC-2015-18083, PID2019-107650RB-C22 and PID2019-108977RB-100), Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CB16/11/00222), Consorcio de Investigación en Red de la Comunidad de Madrid, S2017/BMD-3686 and Fondo Europeo de Desarrollo Regional.

© 2021 The Author(s)., Nucleotide-binding oligomerization domain 1 (NOD1), a pattern recognition receptor (PRR) that detects bacterial peptidoglycan fragments and other danger signals, has been linked to inflammatory pathologies. NOD1, which is expressed by immune and non-immune cells, is activated after recognizing microbe-associated molecular patterns (MAMPs). This recognition triggers host defense responses and both immune memory and tolerance can also be achieved during these processes. Since the gut microbiota is currently considered a master regulator of human physiology central in health and disease and the intestine metabolizes a wide range of nutrients, drugs and hormones, it is a fact that dysbiosis can alter tissues and organs homeostasis. These systemic alterations occur in response to gastrointestinal immune adaptations that are not yet fully understood. Even if previous evidence confirms the connection between the microbiota, the immune system and metabolic disorders, much remains to be discovered about the contribution of NOD1 to low-grade inflammatory pathologies such as obesity, diabetes and cardiovascular diseases. This review compiles the most recent findings in this area, while providing a dynamic and practical framework with future approaches for research and clinical applications on targeting NOD1. This knowledge can help to rate the consequences of the disease and to stratify the patients for therapeutic interventions., The review was written by authors that work in a laboratory where projects are supported by: Ministerio de Economía, Industria y Competitividad/Agencia Estatal de Investigación 10.13039/501100011033 (RTI2018-094727-B100, SAF2017-82436-R, BIO2016-77639-P, PID2019-108977-RBI00 and PID2020-113238-RBI00), Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CB16/11/00222), Consorcio de Investigación en Red de la Comunidad de Madrid, S2017/BMD-3686 and Fondo Europeo de Desarrollo Regional.

Trabajo presentado en las XV Jornadas CIBER Enfermedades Hepáticas y Digestivas: Jornades científiques del CIBERehd, celebradas en modalidad virtual y de forma presencial en Barcelona (España) del 08 al 09 de noviembre de 2021., Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostanoids. Prostaglandins are involved in multiple homeostatic processes, as well as playing an important role in the onset of inflammation. COX-2 is an isoform that is expressed and induced by different stimuli in various tissues and cell types; however, in liver, COX-2 expression is restricted to those situations where proliferation and dedifferentiation occur (1). Our previous results have shown that COX-2 expression in hepatocytes protects against hyperglycemia-induced liver damage, peripheral insulin resistance and adiposity in mice fed a high-fat diet (2), and also protects against experimental non-alcoholic steatohepatitis and fibrosis (3)., This work was supported by SAF2016-75004-R and PID2019-108977RB-I00 (MINECO, Spain) and CIBERehd (ISCIII, Spain). M F-A. and M L-T are recipient of FPI fellowship from MINECO (BES-2017-081928 and PRE2020-094885, respectively). We thank FEDER for financial support.

10 páginas, 2 figuras, 2 tablas. This article belongs to the Special Issue Molecular Research of Genes Involved in Metabolic Diseases.
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/biomedicines10051178/s1, Figure S1: Comparison of the c.-
405A>G position in SREBF2 gene in five Hominidae species; Table S1: Sequences of primers used in
this work., Patients with high cholesterol and glucose levels are at high risk for cardiovascular disease. The Sterol Regulatory Element Binding Protein (SREBP) system regulates genes involved in lipid, cholesterol and glucose pathways. Autosomal Dominant Hypercholesterolemias (ADHs) are a group of diseases with increased cholesterol levels. They affect 1 out of every 500 individuals. About 20-30% of patients do not present any mutation in the known genes (LDLR, APOB and PCSK9). ADHs constitute a good model to identify the genes involved in the alteration of lipid levels or possible therapeutic targets. In this paper, we studied whether a mutation in the SREBP system could be responsible for ADH and other metabolic alterations present in these patients. Forty-one ADH patients without mutations in the main responsible genes were screened by direct sequencing of SREBP system genes. A luciferase reporter assay of the found mutation and an oral glucose tolerance test in carriers and non-carriers were performed. We found a novel mutation in the SREBF2 gene that increases transcription levels and cosegregates with hypercholesterolemia, and we found increased glucose levels in one family. SREBP2 is known to be involved in cholesterol synthesis, plasma levels and glucose metabolism in humans. The found mutation may involve the SREBF2 gene in hypercholesterolemia combined with hyperglycemia., This work was funded by the CIBER of Diabetes and Associated Metabolic Diseases
(CIBERDEM, which is an initiative of the Instituto de Salud Carlos III, Madrid, Spain), research grants
PI17/0497 and PI21/00506 from FIS to F.J.C., grant AP11-091 from Generalitat Valenciana to F.J.C.
and grant PID2019-108977RB-I00 from the Spanish Ministry of Science and Innovation to M.C., Peer reviewed

18 páginas, 7 figuras, Cyclooxygenase 2 (COX-2) is a key enzyme in prostanoid biosynthesis. The constitutive hepatocyte expression of COX-2 has a protective role in hepatic ischemia-reperfusion (I/R) injury (IRI), decreasing necrosis, reducing reactive oxygen species (ROS) levels, and increasing autophagy and antioxidant and anti-inflammatory response. The physiopathology of IRI directly impacts mitochondrial activity, causing ATP depletion and being the main source of ROS. Using genetically modified mice expressing human COX-2 (h-COX-2 Tg) specifically in hepatocytes, and performing I/R surgery on the liver, we demonstrate that COX-2 expression has a beneficial effect at the mitochondrial level. Mitochondria derived from h-COX-2 Tg mice livers have an increased respiratory rate associated with complex I electron-feeding pathways compared to Wild-type (Wt) littermates, without affecting complex I expression or assembly. Furthermore, Wt-derived mitochondria show a loss of mitochondrial membrane potential (ΔΨm) that correlates to increased proteolysis of fusion-related OPA1 through OMA1 protease activity. All these effects are not observed in h-COX-2 Tg mitochondria, which behave similarly to the Sham condition. These results suggest that COX-2 attenuates IRI at a mitochondrial level, preserving the proteolytic processing of OPA1, in addition to the maintenance of mitochondrial respiration., The experiments were performed in laboratories where projects are supported by: Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación 10.13039/501100011033 (SAF2016-
75004-R and PID2019-108977RB-I00), Centro de Investigación Biomédica en Red en Enfermedades
Hepáticas y Digestivas (CB06/04/1069), Centro de Investigación Biomédica en Red de Enfermedades
Cardiovasculares (CB/11/00222), Consorcio de Investigación en Red de la Comunidad de Madrid,
S2017/BMD-3686,and Fondo Europeo de Desarrollo Regional. M.F.-A. and M.L.-T. are recipient of
FPI fellowship from MINECO (BES-2017-081928 and PRE2020-094885, respectively)., Peer reviewed

Methods

20 μL of plasma samples were spiked with deuterated internal standards stock solution. Then proteins were precipitated and supernatants were dried and reconstituted in methanol:water (50:50, V/V). Besides, approximately 50 mg of each tissue were placed in 2 ml tubes containing CK14 ceramic beads (Precellys). For each 50 mg of tissue, 300 μl of methanol and the deuterated internal standards were added and tissues were homogenized in a Precellys 24 Dual system equipped with a Criolys cooler (Precellys). Samples were analyzed using an Acquity UPLC system (Waters, UK) equipped with an Acquity UPLC BEH C18 column (1.7μm, 2.1 x 100 mm; Waters). The MS analysis was performed using a Waters Xevo TQ-XS mass spectrometer (Waters) with an ESI source working in the negative-ion mode., Cyclooxygenase-2 (COX-2) is involved in different liver diseases, but little is known about the significance of COX-2 or its metabolites in cholestatic injury. This study was designed to elucidate the role of COX-2 expression during the pathogenesis of cholestasis. Thus, we investigated the mechanisms underlying the role of COX-2 and its derived prostaglandins in modulating cell survival, inflammation, oxidative stress status and the synthesis and excretion of bile acids (BA) in response to cholestatic liver injury. We used genetically modified mice constitutively expressing human COX-2 (hCOX-2-Tg) specifically in hepatocytes. Transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either subjected to a common bile duct ligation (BDL) to establish an experimental model of obstructive cholestasis. We performed an exhaustive analysis of the different types of bile acids (total, primary, secondary, conjugated, non-conjugated and hydrophilic, α-, β- and ω-muricholic acid) in plasma and in liver tissue from Wt and h-COX-2 Tg mice. Samples were analyzed at Instituto de Investigación Sanitaria La Fe (Valencia, Spain) detecting a total of 31 analytes., Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-108977RB-I00), No

19 páginas, 9 figuras, 1 tabla, 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 that constitutively express functional human COX-2 in cardiomyocytes under the control of the α-myosin heavy chain promoter. These animals had no apparent phenotype but were protected against ischemia-reperfusion injury in isolated hearts, with 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. The results revealed a tissue-specific proteomic profile dominated by mitochondrial proteins. In particular, an increased expression of respiratory chain complex IV proteins was observed. This correlated with increased catalytic activity, enhanced respiratory capacity, and increased ATP levels in the heart of COX-2 transgenic mice. These data suggest a new link between COX-2 and mitochondria, which might contribute to the protective cardiac effects of COX-2 against ischemia-reperfusion injury., This work was supported by Ministerio de Economia y Competitividad (BIO2012-37926),
Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-
108977RB-I00; PID2020-113238RB-I00), by Generalitat Valenciana (ACOMP/2011/120), by grant PRB2
(IPT13/0001—ISCIII-SGEFI/FEDER, ProteoRed), Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CB06/04/1069) and Centro de Investigación Biomédica en Red de
Enfermedades Cardiovasculares (CB/11/00222). M.S.A. was supported by a fellowship from CSIC
(Spanish National Research Council) (JAE-predoc). M.F.-A. is a recipient of the FPI fellowship from
MINECO (BES-2017-081928)., Peer reviewed

In the course of atherogenesis, the spleen plays an important role in the regulation of extramedullary hematopoiesis, and in the control of circulating immune cells, which contributes to plaque progression. Here, we have investigated the role of splenic nucleotide-binding oligomerization domain 1 (NOD1) in the recruitment of circulating immune cells, as well as the involvement of this immune organ in extramedullary hematopoiesis in mice fed on a high-fat high-cholesterol diet (HFD). Under HFD conditions, the absence of NOD1 enhances the mobilization of immune cells, mainly neutrophils, from the bone marrow to the blood. To determine the effect of NOD1-dependent mobilization of immune cells under pro-atherogenic conditions, Apoe−/− and Apoe−/−Nod1−/− mice fed on HFD for 4 weeks were used. Splenic NOD1 from Apoe−/− mice was activated after feeding HFD as inferred by the phosphorylation of the NOD1 downstream targets RIPK2 and TAK1. Moreover, this activation was accompanied by the release of neutrophil extracellular traps (NETs), as determined by the increase in the expression of peptidyl arginine deiminase 4, and the identification of citrullinated histone H3 in this organ. This formation of NETs was significantly reduced in Apoe−/−Nod1−/− mice. Indeed, the presence of Ly6G+ cells and the lipidic content in the spleen of mice deficient in Apoe and Nod1 was reduced when compared to the Apoe−/− counterparts, which suggests that the mobilization and activation of circulating immune cells are altered in the absence of NOD1. Furthermore, confirming previous studies, Apoe−/−Nod1−/− mice showed a reduced atherogenic disease, and diminished recruitment of neutrophils in the spleen, compared to Apoe−/− mice. However, splenic artery ligation reduced the atherogenic burden in Apoe−/− mice an effect that, unexpectedly was lost in Apoe−/−Nod1−/− mice. Together, these results suggest that neutrophil accumulation and activity in the spleen are driven in part by NOD1 activation in mice fed on HFD, contributing in this way to regulating atherogenic progression., This work has been supported by: Ministerio de Ciencia, Investigación y Universidades, Agencia Estatal de Investigación *** (PID2019-104284RB-I00, PID2020-113238RB-I00, RTI2018-098113B-I00, PID2019-108977RB-I00), Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CB16/11/00222) and Consorcio de Investigación en Red de la Comunidad de Madrid, S2017/BMD-3686, Fondo Social Europeo and Fondo Europeo de Desarrollo Regional.

The bioavailability and regulation of iron is essential for central biological functions in mammals. The role of this element in ferroptosis and the dysregulation of its metabolism contribute to diseases, ranging from anemia to infections, alterations in the immune system, inflammation and atherosclerosis. In this sense, monocytes and macrophages modulate iron metabolism and splenic function, while at the same time they can worsen the atherosclerotic process in pathological conditions. Since the nucleotide-binding oligomerization domain 1 (NOD1) has been linked to numerous disorders, including inflammatory and cardiovascular diseases, we investigated its role in iron homeostasis. The iron content was measured in various tissues of Apoe-/- and Apoe-/-Nod1-/- mice fed a high-fat diet (HFD) for 4 weeks, under normal or reduced splenic function after ligation of the splenic artery. In the absence of NOD1 the iron levels decreased in spleen, heart and liver regardless the splenic function. This iron decrease was accompanied by an increase in the recruitment of F4/80+-macrophages in the spleen through a CXCR2-dependent signaling, as deduced by the reduced recruitment after administration of a CXCR2 inhibitor. CXCR2 mediates monocyte/macrophage chemotaxis to areas of inflammation and accumulation of leukocytes in the atherosclerotic plaque. Moreover, in the absence of NOD1, inhibition of CXCR2 enhanced atheroma progression. NOD1 activation increased the levels of GPX4 and other iron and ferroptosis regulatory proteins in macrophages. Our findings highlight the preeminent role of NOD1 in iron homeostasis and ferroptosis. These results suggest promising avenues of investigation for the diagnosis and treatment of iron-related diseases directed by NOD1., The experiments were performed in a laboratory where projects are supported by: Ministerio de Economía, Industria y Competitividad/Agencia Estatal de Investigación and Next Generation EU funds 10.13039/501100011033 (BIO2016–77639-P, PID2019–108977RB-I00 and PID2020–113238RB-I00), Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CB16/11/00222), Consorcio de Investigación en Red de la Comunidad de Madrid, S2017/BMD-3686 and Fondo Europeo de Desarrollo Regional and Fondo Solcial Europeo.

Trabajo presentado en el FASEB Science Research Conference: The Liver Biology Conference: Fundamental Mechanisms and Translational Applications,, [Background]: NAFLD is the most prevalent chronic liver disease in the world and is considered the hepatic manifestation of metabolic syndrome. It begins with an accumulation of fat in the liver, which can lead to cellular damage and inflammation (NASH) and, if unaddressed, progresses to liver fibrosis, where parenchymal tissue is replaced by extracellular matrix. Cyclooxygenase (COX) is a key regulatory step in the biosynthesis of prostanoids. The COX-2 isoform is expressed and induced by different stimuli in various tissues and cell types; however, in liver, COX-2 expression is restricted to those situations in which proliferation or dedifferentiation occur (1). Our previous results revealed that COX-2 expression in hepatocytes protects against hyperglycemia-induced liver damage and against peripheral insulin resistance and adiposity in mice subjected to a high-fat diet (2). Furthermore, COX-2 expression in hepatocytes protects against experimental nonalcoholic steatohepatitis and fibrosis in mice (3).
[Aims]: Since our previous results are performed in mice, we wanted to bridge from animal models to a more translational research, close to human. Therefore, we wanted to establish an in vitro model based on 3D human cell culture to evaluate the role of COX-2, specifically the role of prostaglandins, in a human model of NAFLD/NASH.
[Methods]: To analyze the role of COX-2 in steatosis and fibrosis, we established a 3D spheroid culture composed of HepG2 and LX-2 cells. These spheroids were treated with palmitic acid and TGF¿ to induce NAFLD/NASH and fibrosis, respectively. They were then treated with PGE2 to evaluate their effect in this model.
[Results]: In summary, our results demonstrate that PGE2 treatment reduces lipid levels after fatty acid exposure. Moreover, it reduces fatty acid-induced cell damage and insulin resistance. Furthermore, PGE2 slows fibrotic progression by decreasing stellate cells activation after TGF¿ stimulation.
Conclusions: These preliminary results indicate that a therapeutic strategy for the treatment of NAFLD/NASH may be COX-2-derived prostaglandin therapy., This work was supported by SAF2016-75004-R and PID2019-108977RB-I00 (MINECO, Spain) and CIBERehd (ISCIII, Spain). M F-A. and M L-T are recipient of FPI fellowship from MINECO (BES-2017-081928 and PRE2020-094885, respectively). We thank FEDER for financial support.

Iron participates in myriad processes necessary to sustain life. During the past decades, great efforts have been made to understand iron regulation and function in health and disease. Indeed, iron is associated with both physiological (e.g., immune cell biology and function and hematopoiesis) and pathological (e.g., inflammatory and infectious diseases, ferroptosis and ferritinophagy) processes, yet few studies have addressed the potential functional link between iron, the aforementioned processes and extramedullary hematopoiesis, despite the obvious benefits that this could bring to clinical practice. Further investigation in this direction will shape the future development of individualized treatments for iron-linked diseases and chronic inflammatory disorders, including extramedullary hematopoiesis, metabolic syndrome, cardiovascular diseases and cancer., Work in the authors laboratory is supported by: Ministerio de Economía, Industria y Competitividad/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-108977RB-I00 and PID2020-113238RB-I00), Centro de Investigación Biomédica en Red en Enfermedades Cardiovasculares (CB16/11/00222), Consorcio de Investigación en Red de la Comunidad de Madrid, S2017/BMD-3686 and Fondo Europeo de Desarrollo Regional.

Oxidative stress and inflammation play an important role in the pathophysiological changes of liver diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that positively regulates the basal and inducible expression of a large battery of cytoprotective genes, thus playing a key role in protecting against oxidative damage. Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandin biosynthesis. Its expression has always been associated with the induction of inflammation, but we have shown that, in addition to possessing other benefits, the constitutive expression of COX-2 in hepatocytes is beneficial in reducing inflammation and oxidative stress in multiple liver diseases. In this review, we summarized the role of NRF2 as a main agent in the resolution of oxidative stress, the crucial role of NRF2 signaling pathways during the development of chronic liver diseases, and, finally we related its action to that of COX-2, where it appears to operate as its partner in providing a hepatoprotective effect., The laboratory is supported by: Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-108977RB-I00), CIPROM/2022/42 from Generalitat Valenciana, and Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CB06/04/1069). M F-A and M L-T are recipients of FPI fellowship from MINECO (BES-2017-081928 and PRE2020-094885, respectively)., Peer reviewed

Tesis doctoral, 217 p., [EN] Hepatic ischemia-reperfusion (I/R) injury (IRI) is a major cause of mortality and morbidity in liver resection and liver transplantation. During the hypoxia, the liver remains without oxygen supply, shifting its metabolism and stopping the ATP synthesis. Paradoxically, the restoration of oxygen flow causes the most damage with an activation of the immune system that will generate a burst of reactive species of oxygen (ROS) that will cause cell and tissue damage. Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandin biosynthesis and its importance in IRI is controversial. PGE2, prostaglandin E2, is the main product of COX-2, and is mainly involved in mediating pathological processes such as inflammation, fever and pain. While the use of NSAIDs, specific COX inhibitors, points to a beneficial effect in the resolution of the inflammatory process, several studies support the idea of an anti-inflammatory role of COX-2. In fact, previous studies have shown that COX-2 overexpression in hepatocytes protects mice from apoptosis and cellular stress, as well as reducing the inflammatory response, in different liver disease models. In this PhD thesis, a hepatocyte-specific COX-2 transgenic mouse (h-COX-2 Tg) was used to elucidate the role and involvement of COX-2 in IRI. Wild type (Wt) and h-COX-2 Tg animals were subjected to 90 min of ischemia, followed by 4 or 24 h of reperfusion. Comparing h-COX-2 Tg animals with their Wt littermates, cellular and tissue damage resulting from IRI is attenuated. Among these pathways, the inflammatory cascade is less activated, with less pro-inflammatory cytokine release, less hepatic recruitment and neutrophil infiltration. Necrosis and apoptosis pathways are also attenuated such as reduced endoplasmic reticulum stress, and increased autophagy. The antioxidant response appears to be enhanced in the context of COX-2 overexpression and total ROS production is also lower, contributing to less tissue damage. When Wt animals are subjected to preconditioning (PC), endogenous COX-2 is induced at higher levels than without PC, and these livers show less damage, attenuated inflammation, and an enhanced antioxidant response. Furthermore, the role of COX-2 in this observed protection has been shown to be specific, as its inhibition with DFU, reverses the observed effects, and matched the damage caused to Wt animals. Mitochondria are central players in the pathophysiology of IRI. In this regard, mitochondrial function is preserved in COX-2-overexpressing livers, as can be demonstrated by a conserved mitochondrial membrane potential and a preserved respiratory rate. These results can be explained by a stabilisation of mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) that maintained through interactions of various isoforms of OPA1. Its processing is mediated by proteases, such as OMA1, which acts under certain stimuli. In h-COX-2 Tg mice, there is a reduced OPA1 processing that correlates with attenuated OMA1 activity, showing a stabilisation of cristae in the context of COX-2 overexpression after I/R. On the other hand, a retrospective study was conducted in patients who had undergone liver transplantation. In this part of the study, PGE2 levels were analysed and correlated with liver functions after transplantation. This analysis shows that the presence of PGE2 in the plasma of recipients correlates with a better prognosis, while lower PGE2 levels are associated with early graft dysfunction. All these results present COX-2 as a new player in liver protection after I/R, showing an anti-inflammatory and antioxidant role, as well as reducing mitochondrial damage, cell stress and cell death. Furthermore, it is shown how COX-2-derived prostaglandins under physiological conditions can play a protective role in cases of liver transplant., [ES] La lesión por isquemia-reperfusión (I/R) hepática (IRI) es una causa importante de mortalidad y morbilidad en la resección hepática y el trasplante de hígado. Durante la hipoxia, el hígado permanece sin oxígeno, cambiando su metabolismo y parando la síntesis de ATP. Paradójicamente, el restablecimiento del flujo de oxígeno causa más daño, activando el sistema inmunitario que generará una gran cantidad de especies reactivas de oxígeno (ROS) causando daño celular y tisular. La ciclooxigenasa-2 (COX-2) es una enzima clave en la biosíntesis de prostaglandinas y su importancia en la IRI es controvertida. La PGE2, prostaglandina E2, es el principal producto de la COX-2, y participa en la mediación de procesos patológicos como la inflamación, la fiebre y el dolor. El uso de AINEs, inhibidores específicos de la COX, apunta a un efecto beneficioso en la resolución del proceso inflamatorio, pero cada vez más estudios apoyan el papel antiinflamatorio de la COX-2. De hecho, estudios previos han demostrado que la sobreexpresión de COX-2 en hepatocitos protege a los ratones de la apoptosis y el estrés celular, además de reducir la respuesta inflamatoria en diferentes modelos de enfermedad hepática. En esta tesis doctoral, se utilizó un ratón transgénico que sobreexpresa COX-2 en los hepatocitos (h-COX-2 Tg) para dilucidar el papel y la implicación de la COX-2 en la IRI. Los animales de tipo silvestre (Wt) y h-COX-2 Tg fueron sometidos a 90 min de isquemia, seguidos de 4 o 24 h de reperfusión. Comparando los animales h-COX-2 Tg con sus hermanos Wt, el daño celular y tisular se atenúa tras la IRI. Entre las distintas vías modificadas, la cascada inflamatoria está menos activada, con menor liberación de citoquinas pro-inflamatorias, menor reclutamiento hepático, e infiltración de neutrófilos. Las vías de necrosis y apoptosis también se atenúan así como se reduce del estrés del retículo endoplásmico, y aumenta la autofagia. La respuesta antioxidante se potencia en el contexto de la sobreexpresión de COX-2 y la producción total de ROS es menor, lo que contribuye a un menor daño tisular. Cuando los animales Wt se someten un pre-condicionamiento (PC), la COX-2 endógena se induce a niveles más altos que sin PC, mostrando menos daño, una inflamación atenuada, y una respuesta antioxidante mejorada. Además, se muestra que el papel de la COX-2 en esta protección es específico, ya que su inhibición con DFU revierte los efectos observados e iguala el daño causado a los animales Wt. Las mitocondrias son actores centrales en la fisiopatología de la IRI. En este sentido, la función mitocondrial está preservada en los hígados que sobreexpresan COX-2, con un potencial de membrana mitocondrial conservado y una tasa respiratoria preservada. Estos efectos pueden explicarse por una estabilización de las crestas mitocondriales, invaginaciones de la membrana mitocondrial interna (IMM) que se mantienen mediante interacciones de varias isoformas de la proteína OPA1. Su procesamiento está mediado por proteasas, como OMA1. En ratones h-COX-2 Tg hay un menor procesamiento de OPA1, que se correlaciona con una actividad atenuada de OMA1. Por otro lado, se realizó un estudio retrospectivo en pacientes que habían sido sometidos a un trasplante hepático. Se analizaron los niveles de PGE2 y se correlacionaron con las funciones hepáticas tras el trasplante. Este análisis muestra que la presencia de PGE2 en el plasma de los pacientes receptores se correlaciona con un mejor pronóstico, mientras que unos niveles más bajos de PGE2 se asocian con una disfunción precoz del injerto. Todos estos resultados presentan a la COX-2 como un nuevo actor en la protección del hígado tras I/R, mostrando un papel antiinflamatorio y antioxidante, así como reduciendo el daño mitocondrial, el estrés celular y la muerte celular. Además, se demuestra cómo las prostaglandinas derivadas de la COX-2 en condiciones fisiológicas pueden desempeñar un papel protector en casos de trasplante hepático., This work has been carried out with the financial support of the Spanish Ministry of Science and Innovation (SAF2016-75004R and PID2019-108977RB-100), the CIBERehd (Centro de Investigaciones Biomédicas En Red de Enfermedades Hepáticas y Digestivas) and the COST Action (CA15203 - Mitochondrial mapping: Evolution - Age - Gender - Lifestyle - Environment (MITOEAGLE)). Marina Fuertes Agudo benefited from a pre-doctoral FPI contract (BES-2017- 081928) associated with the SAF2016-75004R project. She spent 3 months in the laboratory of Dr. Pau Sancho Bru at the Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS, Barcelona, Spain) funded by a short stay grant awarded by the CIBERehd and 3 months in the laboratory of Dr. Anne Dubart Kupperschmitt and Dr. Jean Charles Duclos Vallée at the Institut Nationale de la Santé et la Recherche Médicale (INSERM, Villejuif, France) funded by a short stay grant awarded by the European Molecular Biology Organisation (EMBO, SEG_9771)., Peer reviewed