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

N-Acetylcysteine Inhibits Platelet Function through the Regeneration of the Non-Oxidative Form of Albumin

N-acetylcysteine (NAC) is able to break down protein disulfides, generating free thiols. This mechanism occurs on mixed disulfides of albumin (HSA) to form mercaptoalbumin (HMA), the main antioxidant species in the plasma. Circulating HSA exists in two main forms: the reduced form (HMA), and the oxidized forms, whose predominant modification is cystenylation (HSA-Cys). Increased levels of oxidized HSA have been detected in several diseases associated with oxidative stress. This study showed that NAC inhibits platelet aggregation by restoring HMA. In addition, the regeneration of HMA by NAC inhibits platelet functions such as intracellular calcium mobilization, reactive oxygen species generation, arachidonic acid metabolites synthesis, and adhesion to the collagen matrix. In our conditions, the exposure of platelets to NAC did not increase GSH levels. However, the inhibition of platelet aggregation was also detected following treatment of platelet-rich plasma with GSH, which, similarly to NAC, reduced HSA-Cys levels. Furthermore, this study showed that cysteine, another compound able to restore HMA by reducing the HSA-Cys content, inhibited platelet aggregation to a similar extent as NAC. The results obtained in this study suggest a new mechanism by which NAC can modulate platelet activation and suggest its possible use as an antiplatelet drug in conditions associated with oxidative stress

Macrophage Phenotyping in Atherosclerosis by Proteomics

Macrophages are heterogeneous and plastic cells, able to adapt their phenotype and functions to changes in the microenvironment. They are involved in several homeostatic processes and also in many human diseases, including atherosclerosis, where they participate in all the stages of the disease. For these reasons, macrophages have been studied extensively using different approaches, including proteomics. Proteomics, indeed, may be a powerful tool to better understand the behavior of these cells, and a careful analysis of the proteome of different macrophage phenotypes can help to better characterize the role of these phenotypes in atherosclerosis and provide a broad view of proteins that might potentially affect the course of the disease. In this review, we discuss the different proteomic techniques that have been used to delineate the proteomic profile of macrophage phenotypes and summarize some results that can help to elucidate the roles of macrophages and develop new strategies to counteract the progression of atherosclerosis and/or promote regression

Cyclooxygenase-2 Glycosylation Is Affected by Peroxynitrite in Endothelial Cells: Impact on Enzyme Activity and Degradation

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (&gt;8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity

An Optimized MRM-Based Workflow of the <span style="font-variant: small-caps">l</span>-Arginine/Nitric Oxide Pathway Metabolites Revealed Disease- and Sex-Related Differences in the Cardiovascular Field

Clinical data indicate that low circulating l-homoarginine (HArg) concentrations are associated with cardiovascular (CV) disease, CV mortality, and all-cause mortality. A high number of LC-based analytical methods for the quantification of HArg, in combination with the l-arginine (Arg)-related pathway metabolites, have been reported. However, these methods usually consider a limited panel of analytes. Thus, in order to achieve a comprehensive picture of the Arg metabolism, we described an improved targeted metabolomic approach based on a multiple reaction monitoring (MRM) mass spectrometry method for the simultaneous quantification of the Arg/nitric oxide (NO) pathway metabolites. This methodology was then employed to quantify the plasma concentrations of these analytes in a cohort of individuals with different grades/types of coronary artery disease (CAD) in order to increase knowledge about the role of HArg and its associated metabolites in the CV field. Our results showed that the MRM method here implemented is suitable for the simultaneous assessment of a wide panel of amino acids involved in the Arg/NO metabolic pathway in plasma samples from patients with CV disease. Further, our findings highlighted an impairment of the Arg/NO metabolic pathway, and suggest a sex-dependent regulation of this metabolic route

Oxidative Stress and Arginine/Nitric Oxide Pathway in Red Blood Cells Derived from Patients with Prediabetes

The effects of the oral glucose tolerance test (OGTT) on red blood cells (RBCs) have not been thoroughly investigated, although it is known that the ingestion of 75 g of glucose during OGTT results in a systemic state of inflammation and oxidative stress. Therefore, we evaluated the effect of OGTT on oxidative stress and L-arginine/Nitric Oxide (L-Arg/NO) metabolic pathway in RBCs obtained from patients with prediabetes. Blood samples were collected from all participants before (T0) and at 10 (T1), 20 (T2), 30 (T3), 60 (T4), 90 (T5), 120 (T6), 150 (T7), and 180 (T8) minutes after glucose loading. Results showed a significant increase in oxidative stress status characterized by a rise in the GSSG/GSH ratio at T4 and T6 that increased in parallel with a reduction of NO production in RBCs. In addition, in this time frame, increased exposure of phosphatidylserine on RBCs membrane was observed. These metabolic modifications were rescued at T8, together with an increase in activated RBC NO synthase expression. These findings provide a possible explanation of the phenomena occurring after glucose loading and suggest that, even in the early stages of diabetes, it may be important to avoid acute variations in glycemia in order to prevent diabetic complications