Untargeted metabolomics to go beyond the canonical effect of acetylsalicylic acid

15openInternationalItalian coauthor/editorGiven to its ability to irreversibly acetylate the platelet cyclooxygenase-1 enzyme, acetylsalicylic acid (ASA) is successfully employed for the prevention of cardiovascular disease. Recently, an antitumoral effect of ASA in colorectal cancer has been increasingly documented. However, the molecular and metabolic mechanisms by which ASA exerts such effect is largely unknown. Using a new, untargeted liquid chromatography–mass spectrometry approach, we have analyzed urine samples from seven healthy participants that each ingested 100 mg of ASA once daily for 1 week. Of the 2007 features detected, 25 metabolites differing after ASA ingestion (nominal p 1) were identified, and pathway analysis revealed low levels of glutamine and of metabolites involved in histidine and purine metabolisms. Likewise, consistent with an altered fatty acid β-oxidation process, a decrease in several short- and medium-chain acyl-carnitines was observed. An abnormal β-oxidation and a lower than normal glutamine availability suggests reduced synthesis of acetyl-Co-A, as they are events linked to one another and experimentally related to ASA antiproliferative effects. While giving an example of how untargeted metabolomics allows us to explore new clinical applications of drugs, the present data provide a direction to be pursued to test the therapeutic effects of ASA—e.g., the antitumoral effect—beyond cardiovascular protectionopenDi Minno, Alessandro; Porro, Benedetta; Turnu, Linda; Manega, Chiara Maria; Eligini, Sonia; Barbieri, Simone; Chiesa, Mattia; Poggio, Paolo; Squellerio, Isabella; Anesi, Andrea; Fiorelli, Susanna; Caruso, Donatella; Veglia, Fabrizio; Cavalca, Viviana; Tremoli, ElenaDi Minno, A.; Porro, B.; Turnu, L.; Manega, C.M.; Eligini, S.; Barbieri, S.; Chiesa, M.; Poggio, P.; Squellerio, I.; Anesi, A.; Fiorelli, S.; Caruso, D.; Veglia, F.; Cavalca, V.; Tremoli, E

Treatment with PCSK9 inhibitors in patients with familial hypercholesterolemia lowers plasma levels of platelet-activating factor and its precursors: a combined metabolomic and lipidomic approach

13openInternationalItalian coauthor/editorIntroduction: Familial hypercholesterolemia (FH) is characterized by extremely high levels of circulating low-density lipoprotein cholesterol (LDL-C) and is caused by mutations of genes involved in LDL-C metabolism, including LDL receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/Kexin type 9 (PCSK9). Accordingly, PCSK9 inhibitors (PCSK9i) are effective in LDL-C reduction. However, no data are available on the pleiotropic effect of PCSK9i. To this end, we performed an untargeted metabolomics approach to gather a global view on changes in metabolic pathways in patients receiving treatment with PCSK9i. Methods: Twenty-five FH patients starting treatment with PCSK-9i were evaluated by an untargeted metabolomics approach at baseline (before PCSK9i treatment) and after 12 weeks of treatment. Results: All the 25 FH subjects enrolled were on maximal tolerated lipid-lowering therapy prior to study entry. After a 12 week treatment with PCSK9i, we observed an expected significant reduction in LDL-cholesterol levels (from 201.0 ± 69.5 mg/dL to 103.0 ± 58.0 mg/dL, p < 0.001). The LDL-C target was achieved in 36% of patients. After peak validation and correction, after 12 weeks of PCSK9i treatment as compared to baseline, we observed increments in creatine (p-value = 0.041), indole (p-value = 0.045), and indoleacrylic acid (p-value= 0.045) concentrations. Conversely, significant decreases in choline (p-value = 0.045) and phosphatidylcholine (p-value < 0.01) together with a reduction in platelet activating factor (p-value = 0.041) were observed. Conclusions: Taking advantage of untargeted metabolomics, we first provided evidence of concomitant reductions in inflammation and platelet activation metabolites in FH patients receiving a 12 week treatment with PCSK9iopenDi Minno, Alessandro; Orsini, Roberta Clara; Chiesa, Mattia; Cavalca, Viviana; Calcaterra, Ilenia; Tripaldella, Maria; Anesi, Andrea; Fiorelli, Susanna; Eligini, Sonia; Colombo, Gualtiero I; Tremoli, Elena; Porro, Benedetta; Di Minno, Matteo Nicola DarioDi Minno, A.; Orsini, R.C.; Chiesa, M.; Cavalca, V.; Calcaterra, I.; Tripaldella, M.; Anesi, A.; Fiorelli, S.; Eligini, S.; Colombo, G.I.; Tremoli, E.; Porro, B.; Di Minno, M.N.D

Nitric Oxide Synthetic Pathway in Patients with Microvascular Angina and Its Relations with Oxidative Stress

A decreased nitric oxide (NO) bioavailability and an increased oxidative stress play a pivotal role in different cardiovascular pathologies. As red blood cells (RBCs) participate in NO formation in the bloodstream, the aim of this study was to outline the metabolic profile of L-arginine (Arg)/NO pathway and of oxidative stress status in RBCs and in plasma of patients with microvascular angina (MVA), investigating similarities and differences with respect to coronary artery disease (CAD) patients or healthy controls (Ctrl). Analytes involved in Arg/NO pathway and the ratio of oxidized and reduced forms of glutathione were measured by LC-MS/MS. The arginase and the NO synthase (NOS) expression were evaluated by immunofluorescence staining. RBCs from MVA patients show increased levels of NO synthesis inhibitors, parallel to that found in plasma, and a reduction of NO synthase expression. When summary scores were computed, both patient groups were associated with a positive oxidative score and a negative NO score, with the CAD group located in a more extreme position with respect to Ctrl. This finding points out to an impairment of the capacity of RBCs to produce NO in a pathological condition characterized mostly by alterations at the microvascular bed with no significant coronary stenosis

Visita all'Osservatorio della Biodiversità marina e terrestre della Regione Sicilia - ORBS

Con il taglio inaugurale del nastro il 16 dicembre 2015, prende vita la struttura museale permanente dell'Osservatorio della Biodiversità marina e terrestre della Regione Sicilia che porta lo stesso nome del Progetto di Ricerca "ORBS – Sistema di comunicazione, informazione e diffusione dell'Osservatorio Regionale della Sicilia", intitolata il 21 dicembre 2018 al Dott. Sandro Fiorelli. Ad oggi, la struttura, è operativa presso la Sede Secondaria dell'Istituto per lo studio degli impatti Antropici e Sostenibilità in ambiente marino del Consiglio Nazionale delle Ricerche (IAS – CNR) di Capo Granitola. Il progetto ORBS, finanziato da Regione Siciliana - Assessorato alla Cooperazione, Commercio, Artigianato e Pesca - Dipartimento Pesca, con periodo di attività 2013 - 2015, si è concluso proprio con la realizzazione della struttura museale; l'Osservatorio è stato istituito dall'Assessorato del Territorio e dell'Ambiente della Regione Siciliana nell'ambito di un accordo quadro con ARPA, ISPRA e CNR. Grazie al progetto ORBS, docenti e allievi dell'Accademia di Belle Arti di Palermo e il personale CNR – IAS (ex IAMC) S. S. di Capo Granitola, hanno collaborato sinergicamente permettendo di realizzare delle azioni didattiche e creative di valore scientifico espresse con straordinaria forza e bellezza. Ricercatori e professori si sono confrontati al fine di combinare le proprie competenze riuscendo nel progetto ambizioso di coinvolgere e fondere i diversi ambiti scientifici sensibilizzando gli artisti ai temi della Biodiversità. Le opere prodotte, corredate da schede scientifiche, hanno oltre al valore artistico un aggiunto valore didattico. L'apertura della sezione espositiva dedicata alla diffusione e alla comunicazione della biodiversità rappresenta da un lato l'importante tappa conclusiva del progetto, dall'altro l'inizio di un percorso mirato alla diffusione della biodiversità verso il mondo giovanile, le scuole e per tutto il territorio. Questa strepitosa collaborazione "CNR – Accademia di Belle Arti di Palermo" conferma l'importanza e l'opportunità di unire arte e scienza per esaltare la percezione della ricerca scientifica da parte della comunità. La divulgazione della scienza è un'attività complessa e sicuramente necessita di competenze e attitudini multidisciplinari oltreché di motivazione ed entusiasmo. La comunicazione delle tematiche scientifiche, di per sé ardua nella traduzione al grande pubblico, grazie alla forza esplicativa dell'arte, diviene opportunità di riflessione, osservazione, confronto per le comunità di visitatori. Il coordinamento delle visite delle scuole di ogni ordine e grado, Enti Pubblici, Comunità Scientifica, Cariche Istituzionali, Delegazioni di Politici Italiani e Stranieri, Associazioni Culturali, Associazioni No-Profit di Volontariato, Associazioni di Promozione Sociale, Organizzazioni di Volontariato, Onlus, pubblico in generale, presso ORBS, è affidato al qualificato personale (tecnici, tecnologi e ricercatori) dell'IAS – CNR S. S. di Capo Granitola, che gestisce in prima persona i visitatori nel percorso didattico e promuove il valore della divulgazione scientifica perseguendo la terza missione degli Enti di Ricerca, attraverso l'applicazione diretta, la valorizzazione e l'impiego della conoscenza

L’ex stabilimento Ellesse. Riflessioni sulla tutela e valorizzazione del patrimonio industriale in Italia

Obiettivo Novecento raccoglie riflessioni sul tema della città ideale e sulle sperimentazioni compiute nel secolo scorso. Interesse particolare è la recente iscrizione della città di Ivrea tra i siti UNESCO: il riconoscimento tangibile del valore architettonico e sociale della visione riformatrice di Adriano Olivetti. Obiettivo Novecento raccoglie una selezione di contenuti originali volti all’interpretazione dell’esperienza eporediese nel contesto attuale e nell’ottica di una prospettiva futura. L’intento non è solamente la divulgazione di visioni e previsioni in ambito locale, ma delle teorie più rilevanti del secolo scorso e la verifica puntuale sullo stato dell’arte in merito al dibattito sulla città pianificata, diffusa e dispersa. Professionisti ed esperti del settore espongono e promuovono la propria vision all’interno della raccolta che alterna teorie storicizzate a possibili scenari futuri nell’intento di (re)interpretare in maniera attuale il secolo appena trascorso

12(S)-Hydroxyeicosatetraenoic acid downregulates monocyte-derived-macrophage efferocytosis: new insights in atherosclerosis

The involvement of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipooxygenase product of arachidonic acid, has been suggested in atherosclerosis. However, its effect on macrophage functions is not completely understood, so far. The uptake of apoptotic cells (efferocytosis) by macrophages is an anti-inflammatory process, impaired in advanced atherosclerotic lesions. This process induces the release of the anti-inflammatory cytokine interleukin-10 (IL-10), and it is regulated by Rho-GTPases, whose activation involves the isoprenylation, a modification inhibited by statins. We assessed 12-HETE levels in serum of coronary artery disease (CAD) patients, and explored 12(S)-HETE in vitro effect on monocyte-derived macrophage (MDM) efferocytosis. Sixty-four CAD patients and 24 healthy subjects (HS) were enrolled. Serum 12-HETE levels were measured using a tandem mass spectrometry method. MDMs, obtained from a spontaneous differentiation of adherent monocytes, were treated with 12(S)-HETE (10-50 ng/mL). Efferocytosis and RhoA activation were evaluated by flow cytometry. IL-10 was measured by ELISA. CAD patients showed increased 12-HETE serum levels compared to HS (665.2 [438.1-896.2] ng/mL and 525.1 [380.1-750.1] ng/mL, respectively, p < 0.05) and reduced levels of IL-10. MDMs expressed the 12(S)-HETE cognate receptor GPR31. CAD-derived MDMs displayed defective efferocytosis vs HS-MDMs (9.4 [7.7-11.3]% and 11.1 [9.6-14.1]% of MDMs that have engulfed apoptotic cells, respectively, p < 0.01). This reduction is marked in MDMs obtained from patients not treated with statin (9.3 [7.4-10.6]% statin-free CAD vs HS, p = 0.01; and 9.9 [8.6-11.6]% statin-treated CAD vs HS, p = 0.07). The in vitro treatment of MDMs with 12(S)-HETE (20 ng/mL) induced 20% decrease of efferocytosis (p < 0.01) and 71% increase of RhoA activated form (p < 0.05). Atorvastatin (0.1 μM) counteracted these 12(S)-HETE-mediated effects.These results show a 12(S)-HETE pro-inflammatory effect and suggest a new potential contribution of this mediator in the development of atherosclerosis

Characterization of aspirin esterase activity in health and disease: In vitro and ex vivo studies

Due to its ability to irreversibly inactivate platelet cyclooxygenase, low-dose aspirin (ASA) is the most widely used antithrombotic agent. Although, studies in specific types of patients with cardiovascular disease (CVD) have shown an incomplete inhibition of platelet's cyclooxygenase, which may increase the variability in drug response. Some aspects of ASA pharmacokinetics (PK) still need further investigation. In this study, we aimed to characterise the contribution of esterase enzymes to ASA hydrolysis in the peripheral blood and to assess their activity in 36 healthy subjects (Ctrl) and 77 CVD patients. To this aim, an in vitro assay testing esterase activity in parallel to a liquid chromatography-tandem mass spectrometry method simultaneously detecting ASA and its main metabolites salicylic (SA) and gentisic acids, have been developed. Michaelis-Menten constant (Km) calculation, ASA esterase isoforms characterisation, and ASA PK study were then achieved. The calculated Km identified plasma esterases as the enzymes with the higher affinity for the substrate compared to the RBC ones. Both rivastigmine and 4-bis-nitrophenyl phosphate inhibited plasma esterase activities, suggesting that acetylcholinesterase and carboxylesterase largely contribute to ASA hydrolysis. The feasibility of the method here developed has been explored in Ctrl and CVD patients. The effect of ASA treatment on enzyme activity was further evaluated on an age, sex and BMI matched subgroup of patients and Ctrl (n = 10 for each subgroup, on and off ASA). No overall variations were evidenced in both CVD and Ctrl groups, even when the effect of ASA treatment was tested. This result suggests the absence of any influence of disease state, drug treatments, and comorbidities on plasma esterase and the inability of ASA intake to induce esterase function. In conclusion, the method here developed allows a better characterisation of ASA esterase activity and could be helpful to define the PK-related determinants of ASA responsiveness in order to personalise regimen in specific pathological conditions

Netrin-1 in Atherosclerosis: Relationship between Human Macrophage Intracellular Levels and In Vivo Plaque Morphology

Netrin-1 is a laminin-like protein that plays a pivotal role in cell migration and, according to the site of its release, exerts both pro and anti-atherosclerotic functions. Macrophages, key cells in atherosclerosis, are heterogeneous in morphology and function and different subpopulations may support plaque progression, stabilization, and/or regression. Netrin-1 was evaluated in plasma and, together with its receptor UNC5b, in both spindle and round monocyte-derived macrophages (MDMs) morphotypes from coronary artery disease (CAD) patients and control subjects. In CAD patients, plaque features were detected in vivo by optical coherence tomography. CAD patients had lower plasma Netrin-1 levels and a higher MDMs expression of both protein and its receptor compared to controls. Specifically, a progressive increase in Netrin-1 and UNC5b was evidenced going from controls to stable angina (SA) and acute myocardial infarction (AMI) patients. Of note, spindle MDMs of AMI showed a marked increase of both Netrin-1 and its receptor compared to spindle MDMs of controls. UNC5b expression is always higher in spindle compared to round MDMs, regardless of the subgroup. Finally, CAD patients with higher intracellular Netrin-1 levels showed greater intraplaque macrophage accumulation in vivo. Our findings support the role of Netrin-1 and UNC5b in the atherosclerotic process

Lipidomics analysis of monocytes from patients with acute myocardial infarction reveals lactosylceramide as a new player in monocyte migration

Monocyte recruitment after vascular injury and their migration through the vessel wall represent crucial events in the initiation, progression, and destabilization of atherosclerotic plaque. Circulating monocytes are exposed to stimuli that alter their physiological state, and among them, lipids play a key role. Several studies investigated the mechanisms by which lipids affect monocyte functions promoting coronary atherosclerotic plaque initiation, but information on the relationship between lipid composition and function of monocyte is scant. We aimed at studying the migration of circulating monocytes isolated from patients with acute myocardial infarction (AMI) at hospital presentation and investigating its correlation with cellular lipid profile. The migration of monocytes was tested using both fetal bovine serum (FBS) and autologous serum as chemoattractant stimuli. Monocyte lipid profile was evaluated through an untargeted lipidomics approach, using a liquid chromatography/time-of-flight mass spectrometry platform. We observed that AMI patients' monocytes showed a significant increase in FBS and autologous serum-mediated migration compared to controls. Moreover, a different monocyte lipidomic profile between the two study groups was detected. In particular, AMI patients' monocytes showed an altered composition in ceramides, with an increase in lactosylceramide and in phospholipids (ie, phosphatidylethanolamine and lisophosphatidylethanolamine). Of note, a positive correlation between lactosylceramide levels and monocyte migration was observed. Furthermore, the lactosylceramide synthase inhibition significantly reduced FBS-induced monocyte migration. Our results highlight the influence of lactosylceramide on the monocyte migration capacity, pointing out a new possible mechanism of lipids in the onset of atherothrombosis and, hence, in AMI