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Background: Atherosclerosis is a chronic inflammatory disease in which oxidative stress and macrophages play fundamental roles. Macrophages are heterogeneous cells in term of morphology and function and the prevalence of a specific morpho-phenotype may influence the progression or the regression of the plaque. Human coronary plaque macrophages are not easily obtainable and monocyte-derived macrophages (MDMs) are widely accepted as a good surrogate. Our previous study has reported that healthy subjects show two dominant MDM morphotypes, spindle and round, present in the same percentage. Aim: The aims of the study were to assess the biochemical and functional profile of MDMs obtained from CAD patients and to investigate the relationship between these characteristics and plaque features detected in vivo. Material and methods: ninety CAD patients and twenty-five healthy volunteers were enrolled. MDMs were obtained by culturing monocytes for 7 days in medium supplemented with 10% autologous serum. Transglutaminase2 (TG2), tissue factor (TF), nuclear factor erythroid 2?related factor 2 (Nrf2) and heme-oxygenase (HO-1) were determined by western blotting and immunofluorescence. The uptake of apoptotic Jurkat T cells was detected by flow cytometry and thrombin formation was assessed by thrombinoscope. Fatty acids composition and oxidative stress status, determined as the ratio between the reduced and oxidized forms of glutathione (GSH and GSSG, respectively) were analyzed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). Plaque features were detected in vivo by optical coherence tomography (OCT). Results: MDMs obtained from CAD patients were characterized by the predominance of round cells. These cells exhibited a lower efferocytic capacity and higher capacity to generate thrombin in respect to those of healthy subjects, reflecting the low expression of TG2 and the enhance of TF levels. Moreover, MDMs of CAD patients showed a higher oxidative stress status, evidenced by the lower GSH/GSSG ratio, in respect to those of healthy subjects. Finally, we found a positive correlation between oxidative stress status and membrane fluidity (MFI) as evidenced by the ratio of C18:1 (oleic acid)/C18:2 (linoleic acid), as well as MFI and capacity to engulf apoptotic cells. Regarding OCT analysis, patients with a higher round MDMs prevalence exhibited more frequently a lipid rich plaque, a TCFA (thin cap fibroatheroma), a greater intra-plaque macrophage content, and a ruptured plaque, characteristics of vulnerable plaque. Furthermore, patients with high TF levels more frequently presented a intra-plaque macrophages accumulation, a ruptured plaque and a presence of thrombus. In addition, vulnerable plaque characteristics were associated with high HO-1 levels. Conclusions: MDMs of CAD patients present a pro-inflammatory and high thrombogenic profile in respect to those of healthy volunteers. The high oxidative stress present in these cells influences the membrane fluidity that in turn influences the efferocytic capability. This specific cellular profile detected in in vitro obtained MDMs is associated with the detection of high-risk and rupture-prone coronary plaques in vivo, at OCT investigation. Different MDM phenotypes might be novel diagnostic and therapeutic target able to counteract the progression of atherosclerosis.

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.

Given 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&ndash, 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 &lt, 0.05 and variable importance in projection (VIP) score &gt, 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 &beta, oxidation process, a decrease in several short- and medium-chain acyl-carnitines was observed. An abnormal &beta, 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&mdash, e.g., the antitumoral effect&mdash, beyond cardiovascular protection.

Reactive oxygen species (ROS) induce nuclear factor erythroid 2&ndash, related factor 2 (Nrf2) activation as an adaptive defense mechanism, determining the synthesis of antioxidant molecules, including heme-oxygenase-1 (HO-1). HO-1 protects cells against oxidative injury, degrading free heme and inhibiting ROS production. HO-1 is highly expressed in macrophages during plaque growth. Macrophages are morpho-functionally heterogeneous, and the prevalence of a specific phenotype may influence the plaque fate. This heterogeneity has also been observed in monocyte-derived macrophages (MDMs), a model of macrophages infiltrating tissue. The study aims to assess oxidative stress status and Nrf2/HO-1 axis in MDM morphotypes obtained from healthy subjects and coronary artery disease (CAD) patients, in relation to coronary plaque features evaluated in vivo by optical coherence tomography (OCT). We found that MDMs of healthy subjects exhibited a lower oxidative stress status, lower Nrf2 and HO-1 levels as compared to CAD patients. High HO-1 levels in MDMs were associated with the presence of a higher macrophage content, a thinner fibrous cap, and a ruptured plaque with thrombus formation, detected by OCT analysis. These findings suggest the presence of a relationship between in vivo plaque characteristics and in vitro MDM profile, and may help to identify patients with rupture-prone coronary plaque.

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.

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