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The authors hypothesized that the cytokine storm described in COVID-19 patients may lead to consistent cell-based tissue factor (TF)-mediated activation of coagulation, procoagulant microvesicles (MVs) release, and massive platelet activation. COVID-19 patients have higher levels of TF+ platelets, TF+ granulocytes, and TF+ MVs than healthy subjects and coronary artery disease patients. Plasma MV-associated thrombin generation is present in prophylactic anticoagulated patients. A sustained platelet activation in terms of P-selectin expression and platelet–leukocyte aggregate formation, and altered nitric oxide/prostacyclin synthesis are also observed. COVID-19 plasma, added to the blood of healthy subjects, induces platelet activation similar to that observed in vivo. This effect was blunted by pre-incubation with tocilizumab, aspirin, or a P2Y12 inhibitor.

The association between migraine and patent foramen ovale (PFO) has been documented. We aimed to investigate platelet activation, prothrombotic phenotype, and oxidative stress status of migraineurs with PFO on 100 mg/day aspirin, before and 6 months after PFO closure. Data show that, before PFO closure, expression of the classical platelet activation markers is comparable in patients and aspirin-treated healthy subjects. Conversely, MHA-PFO patients display an increased prothrombotic phenotype (higher tissue factor

Introduction: 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 &lt, 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 &lt, 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 PCSK9i.

The prevalence of a macrophage phenotype in atherosclerotic plaque may drive its progression and/or instability. Macrophages from coronary plaques are not available, and monocyte-derived macrophages (MDMs) are usually considered as a surrogate. We compared the MDM profile obtained from coronary artery disease (CAD) patients and healthy subjects, and we evaluated the association between CAD MDM profile and in vivo coronary plaque characteristics assessed by optical coherence tomography (OCT). At morphological analysis, MDMs of CAD patients had a higher prevalence of round than spindle cells, whereas in healthy subjects the prevalence of the two morphotypes was similar. Compared to healthy subjects, MDMs of CAD patients had reduced efferocytosis, lower transglutaminase-2, CD206 and CD163 receptor levels, and higher tissue factor (TF) levels. At OCT, patients with a higher prevalence of round MDMs showed more frequently a lipid-rich plaque, a thin-cap fibroatheroma, a greater intra-plaque macrophage accumulation, and a ruptured plaque. The MDM efferocytosis correlated with minimal lumen area, and TF levels in MDMs correlated with the presence of ruptured plaque. MDMs obtained from CAD patients are characterized by a morpho-phenotypic heterogeneity with a prevalence of round cells, showing pro-inflammatory and pro-thrombotic properties. The MDM profile allows identifying CAD patients at high risk.

Circulating levels of Brain Derived Neurotrophic Factor (BDNF) are lower in coronary heart disease (CHD) than in healthy subjects and are associated with coronary events and mortality. However, the mechanism(s) underling this association is not fully understood. We hypothesize that BDNF may influence fibrin fiber structure and clot stability, favoring clot lysis and thrombus resolution. We showed that recombinant BDNF (rh-BDNF) influenced with clot formation in a concentration-dependent manner in both purified fibrinogen and plasma from healthy subjects. In particular, rh-BDNF reduced the density of fibrin fibers, the maximum clot firmness (MCF) and the maximum clot turbidity, and affected the lysis of clot. In addition, both thrombin and reptilase clotting time were prolonged by rh-BDNF, despite the amount of thrombin formed was greater. Intriguingly, CHD patients had lower levels of BDNF, greater fibrin fibers density, higher MCF than control subjects, and a negative correlation between BDNF and MCF was found. Of note, rh-BDNF markedly modified fibrin clot profile restoring physiological clot morphology in CHD plasma. In conclusion, we provide evidence that low levels of BDNF correlate with the formation of bigger thrombi (in vitro) and that this effect is mediated, at least partially, by the alteration of fibrin fibers formation.

Background Takotsubo (TTS) syndrome is an acute cardiac condition characterized by transient and reversible left ventricle dysfunction that mainly affects postmenopausal women. Catecholamine burst is the most accredited mechanism underpinning TTS onset and leading to endothelial dysfunction and platelet activation. Even if the use of low dose acetylsalycilic acid (ASA) in this clinical setting is based on both clinical presentation and unfavorable long-term prognosis, its efficacy has been recently challenged. Aim This study was designed to assess endothelial function, residual thromboxane formation and platelet aggregation in TTS women on low-dose ASA treatment at long-term follow-up. Methods Twenty-eight females with previously diagnosis of TTS syndrome were enrolled. Data were compared to those obtained from 23 coronary artery disease (CAD) women with a history of acute myocardial infarction, and 26 control subjects with no TTS or clinically evident CAD. Psychological and clinical profile were assessed in all study groups at the enrollment. Main metabolites involved in L-arginine/nitric oxide pathway, urinary prostacyclin, serum and urine thromboxane metabolites were measured by LC MS/MS methods. Thrombomodulin levels were quantified using an ELISA kit, and platelet aggregation, carried out on platelet rich-plasma, was induced by ADP or by epinephrine (EPI), norepinephrine (NORE) and TRAP-6, alone or in association with ADP and evaluated by Born’s method. Results In TTS women an endothelial derangement, characterized by reduced citrulline production and increased thrombomodulin concentration, with no perturbation in prostacyclin levels, was evidenced. In addition, despite ASA treatment, TTS displayed a higher residual thromboxane formation, in parallel with an enhanced platelet response to compared to CAD. Conclusions Our study highlighted the presence of endothelial perturbation in TTS patients even at long-term from the index event. The residual thromboxane production and platelet aggregation still leave open the question about the use of low dose ASA in this clinical setting.

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.

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.

In the last years, a substantial contribution of red blood cells (RBCs) in cardiovascular homeostasis has been evidenced, as these cells are able to regulate cardiovascular function by the export of adenosine triphosphate and nitric oxide as well as to maintain redox balance through a well-developed antioxidant system. Recently a link between high-risk plaque (HRP) features and myocardial ischemia, in the absence of severe lumen stenosis, has been evidenced. Nonobstructive coronary artery disease (nonob CAD) has been associated in fact with a greater 1-year risk of myocardial infarction and all-cause mortality compared with no apparent CAD. This new evidence increases interest in searching new triggers to identify these high-risk patients, in the absence/or on top of traditional hazard markers. In this study, we investigated the existence of any association between RBC morphodynamics and HRP features in individuals with different grades of coronary stenosis detected by coronary computed tomography angiography (CCTA). Ninety-one consecutive individuals who underwent CCTA [33 no CAD; 26 nonobstructive (nonob), and 32 obstructive (ob) CAD] were enrolled. RBC morphodynamic features, i.e., RBC aggregability and deformability, were analyzed by means of Laser Assisted Optical Rotation Cell Analyzer (LoRRca MaxSis). The putative global RBC morphodynamic (RMD) score and the related risk chart, associating the extent of HRP (e.g., the non-calcified plaque volume) with both the RMD score and the max % stenosis were computed. In nonob CAD group only positive correlations between RBC rigidity, osmotic fragility or aggregability and HRP features (plaque necrotic core, fibro-fatty and fibro-fatty plus necrotic core plaque volumes) were highlighted. Interestingly, in this patient cohort three of these RBC morphodynamic features result to be independent predictors of the presence of non-calcified plaque volume in this patients group. The risk chart created shows that only in nonob CAD plaque vulnerability increases according to the score quartile. Findings of this work, by evidencing the association between erythrocyte morphodynamic characteristics assessed by LoRRca and plaque instability in a high-risk cohort of nonob CAD, suggest the use of these blood cell features in the identification of high-risk patients, in the absence of severe coronary stenosis.

Background Macrophages play a key role in coronary plaque destabilization. In-vitro human monocyte-derived macrophages (MDMs) are used to study macrophages infiltrating tissue. Optical coherence tomography (OCT) provides an in-vivo insight of the coronary arteries. We compared the MDMs morpho-phenotype and culprit plaque features at OCT in acute coronary syndrome (ACS) patients according to the underlying plaque pathobiology. Methods Sixty-six patients undergoing coronary angiography and pre-angioplasty OCT of the culprit vessel were allocated to three groups according to mechanism of ACS at OCT and C-reactive protein levels (cut-off: 2 mg/Ll): 1) plaque rupture with systemic inflammation; 2) plaque rupture without systemic inflammation, 3) plaque with intact fibrous cap. A blood sample was collected to obtain MDMs, categorized as having “round” or “spindle” morphology. Results Thirty-two patients (48.5%) were assigned to Group 1, 10 (15.2%) to Group 2 and 24 (36.4%) to Group 3. The “round” MDMs were significantly more frequent in Group 1 (39.25 ± 4.98%) than in Group 2 (23.89 ± 3.10%) and Group 3 (23.02 ± 7.89%), p = 0.008. MDMs in Group 1 as compared to Groups 2 and 3 showed lower efferocytosis (8.74 ± 1.38 vs 9.74 ± 2.15 vs 11.41 ± 2.41; p = 0.012), higher tissue factor levels (369.84 ± 101.13 vs 301.89 ± 59.78 vs 231.74 ± 111.47; p = 0.001) and higher heme oxygenase-1 expression (678.78 ± 145.43 vs 419.12 ± 74.44 vs 409.78 ± 64.33; p = 0.008). Conclusions MDMs of ACS patients show morpho-phenotypic heterogeneity with prevalence of pro-thrombotic and pro-oxidative properties in case of plaque rupture and systemic inflammation. Such MDMs subpopulation may take part to the cellular pathways leading to fibrous cap rupture with the subsequent thrombus formation.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin growth factor family, well known for its role in the homeostasis of the cardiovascular system. Recently, the human BDNF Val66Met single nucleotide polymorphism has been associated with the increased propensity for arterial thrombosis related to acute myocardial infarction (AMI). Using cardiac magnetic resonance imaging and immunohistochemistry analyses, we showed that homozygous mice carrying the human BDNF Val66Met polymorphism (BDNFMet/Met) undergoing left anterior descending (LAD) coronary artery ligation display an adverse cardiac remodeling compared to wild-type (BDNFVal/Val). Interestingly, we observed a persistent presence of pro-inflammatory M1-like macrophages and a reduced accumulation of reparative-like phenotype macrophages (M2-like) in the infarcted heart of mutant mice. Further qPCR analyses showed that BDNFMet/Met peritoneal macrophages are more pro-inflammatory and have a higher migratory ability compared to BDNFVal/Val ones. Finally, macrophages differentiated from circulating monocytes isolated from BDNFMet/Met patients with coronary heart disease displayed the same pro-inflammatory characteristics of the murine ones. In conclusion, the BDNF Val66Met polymorphism predisposes to adverse cardiac remodeling after myocardial infarction in a mouse model and affects macrophage phenotype in both humans and mice. These results provide a new cellular mechanism by which this human BDNF genetic variant could influence cardiovascular disease.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin growth factor family, well known for its role in the homeostasis of the cardiovascular system. Recently, the human BDNF Val66Met single nucleotide polymorphism has been associated with the increased propensity for arterial thrombosis related to acute myocardial infarction (AMI). Using cardiac magnetic resonance imaging and immunohistochemistry analyses, we showed that homozygous mice carrying the human BDNF Val66Met polymorphism (BDNFMet/Met) undergoing left anterior descending (LAD) coronary artery ligation display an adverse cardiac remodeling compared to wild-type (BDNFVal/Val). Interestingly, we observed a persistent presence of pro-inflammatory M1-like macrophages and a reduced accumulation of reparative-like phenotype macrophages (M2-like) in the infarcted heart of mutant mice. Further qPCR analyses showed that BDNFMet/Met peritoneal macrophages are more pro-inflammatory and have a higher migratory ability compared to BDNFVal/Val ones. Finally, macrophages differentiated from circulating monocytes isolated from BDNFMet/Met patients with coronary heart disease displayed the same pro-inflammatory characteristics of the murine ones. In conclusion, the BDNF Val66Met polymorphism predisposes to adverse cardiac remodeling after myocardial infarction in a mouse model and affects macrophage phenotype in both humans and mice. These results provide a new cellular mechanism by which this human BDNF genetic variant could influence cardiovascular disease.

Background & aims: Patients with cystathionine β-synthase deficiency (CBSD) exhibit high circulating levels of homocysteine and enhanced lipid peroxidation. We have characterized the plasma lipidome in CBSD patients and related lipid abnormalities with reactions underlying enhanced homocysteine levels. Methods and results: Using an ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry method, plasma lipids were determined with an untargeted lipidomics approach in 11 CBSD patients and 11 matched healthy subjects (CTRL). Compared to CTRL, CBSD patients had a higher medium and long-chain polyunsaturated fatty acids (PUFA) content in phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE) species (p < 0.02), and depletion of phosphatidylcholine (PC; p = 0.02) and of lysophosphatidylcholine (LPC; p = 0.003) species containing docosahexaenoic acid (DHA), suggesting impaired phosphatidylethanolamine-N-methyltransferase (PEMT) activity. PEMT converts PE into PC using methyl group by S-adenosylmethionine (SAM) thus converted in S-adenosylhomocysteine (SAH). Whole blood SAM and SAH concentrations by liquid chromatography tandem mass spectrometry were 1.4-fold (p = 0.015) and 5.3-fold (p = 0.003) higher in CBSD patients than in CTRL. A positive correlation between SAM/SAH and PC/PE ratios (r = 0.520; p = 0.019) was found. Conclusions: A novel biochemical abnormality in CBSD patients consisting in depletion of PC and LPC species containing DHA and accumulation of PUFA in PE and LPE species is revealed by this lipidomic approach. Changes in plasma SAM and SAH concentrations are associated with such phospholipid dysregulation. Given the key role of DHA in thrombosis prevention, depletion of PC species containing DHA in CBSD patients provides a new direction to understand the poor cardiovascular outcome of patients with homocystinuria.

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.

Background and aims Transglutaminase 2 (TGM2), a member of the transglutaminase family of enzymes, is a multifunctional protein involved in numerous events spanning from cell differentiation, to signal transduction, apoptosis, and wound healing. It is expressed in a variety of cells, macrophages included. Macrophage TGM2 promotes the clearance of apoptotic cells (efferocytosis) and emerging evidence suggests that defective efferocytosis contributes to the consequences of inflammation-associated diseases, including atherosclerotic lesion progression and its sequelae. Of interest, active TGM2 identified in human atherosclerotic lesions plays critical roles in plaque stability through effects on matrix cross-linking and TGFβ activity. This study explores the mechanisms by which TGM2 controls efferocytosis in human macrophages. Methods and results Herein we show that TGM2 increases progressively during monocyte differentiation towards macrophages and controls their efferocytic potential as well as morphology and viability. Two experimental approaches that took advantage of the inhibition of TGM2 activity and protein silencing give proof that TGM2 reduction significantly impairs macrophage efferocytosis. Among the mechanisms involved we highlighted a role of the receptors CD14 and SR-AI whose levels were markedly reduced by TGM2 inhibition. Conversely, CD36 receptor and α v β 3 integrin levels were not influenced. Of note, lipid accumulation and IL-10 secretion were reduced in macrophages displaying defective efferocytosis. Conclusion Overall, our data define a crucial role of TGM2 activity during macrophage differentiation via mechanisms involving CD14 and SR-AI receptors and show that TGM2 inhibition triggers a pro-inflammatory phenotype.

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

This data article is referred to the research article entitled Human monocyte-derived macrophages are heterogeneous: proteomic profile of different phenotypes by Eligini et al. Eligini S., Brioschi M., Fiorelli S., Tremoli E., Banfi C., Colli S. Human monocyte-derived macrophages are heterogeneous: proteomic profile of different phenotypes. J. Proteomics 124, 2015, 112-123. Macrophages obtained in vitro from blood monocytes are largely used as surrogate model of tissue macrophages that are heterogeneous and not easy to obtain and handle. Under spontaneous differentiation in vitro, monocyte-derived macrophages (MDMs) display two dominant subsets (round and spindle) that show different transcriptional, antigenic, and functional profiles mimicking, at least in part, the heterogeneity of tissue macrophages. This article reports the nano-LC-MS(E) analysis of the proteome of round and spindle MDMs allowing a deeper comprehension of macrophage heterogeneity.

Tissue macrophages play a key role in many aspects of human physiology and pathology. These cells are heterogeneous both in term of morphology and function. As an example, heterogeneity has been reported within the atherosclerotic lesions where distinct populations exert opposite functions driving plaque progression or stability. Tissue macrophages are not easily obtained and differentiated blood-derived monocytes are largely used as surrogate model. We previously reported that human macrophages spontaneously differentiated from adherent monocytes show two dominant subsets, distinct for morphology (spindle and round) and functions. The aim of this study was to evaluate the intracellular proteome of these two macrophage subsets by means of a microproteomic workflow properly set up to simultaneously identify and quantify proteins from a minimal number of morphotypically heterogeneous cells in culture. We report two distinct proteomic profiles that distinguish round from spindle macrophages. In particular, differential abundances were observed for proteins involved in membrane traffic regulation, lipid handling, efferocytosis, and protection against stress conditions. Results reinforce and extend previous data on the functional and antigenic profile of these macrophage phenotypes strengthening the suitability of our model to focus on macrophage heterogeneity. Biological significance Tissue macrophages patrol homeostatic functions, immune surveillance, and resolution of inflammation. The spectrum of macrophage activation states is, therefore, wide and gives ground for the heterogeneity of these cells, documented in health and disease. This study provides knowledge of the distinct proteome that characterises the two dominant morphotypes (round and spindle) of human macrophages that, in our culture condition, are generated by spontaneous differentiation from blood-derived monocytes. Results extend previous data about the different antigenic, transcriptional, and functional profiles of these morphotypes and further strengthen the suitability of this in vitro model to study macrophage heterogeneity and to address the effects of environmental challenges and drugs.

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.

This paper describes a microproteomic workflow that is useful for simultaneously identifying and quantifying proteins from a minimal number of morphotypically heterogeneous cultured adherent cells. The analytical strategy makes use of laser capture microdissection, an effective means of harvesting pure cell populations, and label-free mass spectrometry. We optimised the workflow with particular reference to cell fixation which is crucial for successful laser-based microdissection and also downstream molecular studies. In addition, we defined the minimum number of cells to be isolated and analysed for satisfactory proteome coverage. To set up this workflow, we choose human monocyte-derived macrophages spontaneously differentiated in vitro. These cells, under our culture conditions, show distinct morphotypes, reminiscent of the heterogeneity observed in tissues in various homeostatic and pathological states, e.g. atherosclerosis. This optimised workflow may provide new insights into biology and pathology of heterogeneous cell in culture, particularly when other cell selection approaches are not suitable.

A decreased nitric oxide (NO) bioavailability and an increased oxidative stress play a pivotal role in different cardiovascular pathologies. Recent studies have shown that 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). We determined substrate, products (Arg, L-citrulline and L-ornithine) and inhibitors (asymmetric- and symmetric dimethylarginine and monomethylarginine) of NO synthesis in parallel to reduced and oxidized form of glutathione. MVA patients show alterations in the ability of RBCs to produce NO, based on an increase of NO synthesis inhibitors parallel to that found in plasma and a reduction of NO synthase expression. When summary scores of NO synthesis and oxidative stress were computed, both patient groups were associated with a positive score and a negative NO score. This finding points out to an impairement 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. ![Figure][1] [1]: pending:yes