The research activities of the Unit have been focused on the study of biochemical, cellular, and metabolic mechanisms involved in the onset and progression of atherothrombosis. In particular, the activation of circulating (red blood cells, platelets) and resident (macrophages) cells and the role of the oxidative stress on cell function were investigated.
To this aim, the current strategy includes the study of pathways involved in oxidative damage, arachidonic acid metabolism and NO dependent vasodilation, in order to highlight possible links between oxidative stress, endothelial dysfunction and platelet aggregation. We have developed chromatographic methods (HPLC, LC-MS/MS) with high sensitivity and specificity, in order to determine both oxidative modifications of lipids and proteins and the levels of antioxidant factors such as vitamins, glutathione, antioxidant enzymes, etc. A relative new emerging technology, called metabolomics, has opened new perspectives for the understanding of the interactions between oxidative stress and different metabolic pathways in vivo. This technique allows to analyze a particular metabolic route through the measurement of low molecular weight metabolites involved in it and present in cells, tissues, organs and biological fluids, thus defining the biochemical phenotype of a biological system as a whole. In this context, our recent studies have been focused on the set up and validation of metabolomics methods using different biological matrices, i.e. blood, urine and circulating cells (firstly erythrocytes and monocytes), to investigate several metabolic pathways. In particular we have analyzed pathways related to endothelial dysfunction (by measurement of metabolites involved in L-Arginine / NO metabolic pathway in plasma and red blood cells), to aspirin metabolism (by the evaluation of the enzyme activities involved in its hydrolysis both in plasma and in red blood cells) and to the metabolism of arachidonic acid (by measurement of eicosanoids produced by platelets and endothelium).
As regard to resident cells, macrophages are the first inflammatory cells to invade atherosclerotic lesions, and they are the main component of atherosclerotic plaque. However, since it is very difficult to obtain these cells from human atherosclerotic plaques, macrophages, obtained from spontaneously differentiated monocytes isolated from peripheral blood (MDMs), are considered as good surrogate. On these bases, we developed a model of human macrophages generated by adherent monocytes differentiated for 7 days in the presence of autologous serum.
The Unit focused its attention on several research lines:
- the analysis of oxidative stress status and the L-Arginine/NO metabolic pathway in vivo in different cardiovascular disorders and in vitro on circulating and cultured cells;
- the study of the red blood cell as new player in cardiovascular homoeostasis: focus on its morphology and activity on cardiovascular disease;
- the study of vascular homeostasis through the evaluation of eicosanoids enzymatically produced by arachidonic acid and its perturbation by aspirin treatment. Two aspects are investigated: (1) Aspirin pharmacokinetics in heathy subjects by determination of its catabolic products and of the enzyme involved in its metabolism; (2) Aspirin responsiveness in pathology with accelerated platelet tournover rate, such as cardiovascular surgical intervention;
- the analysis of atherosclerotic plaque morphology and activity by means of optical coherence tomography (OCT) and the association of in vivo plaque characteristics with the biological signature of MDMs obtained from coronary artery disease (CAD) patients, in order to detect the acute or chronic disease and the presence of vulnerable plaques even before plaque rupture precipitates thrombotic event.
Evaluation of DNA damage induced by oxidative stress
The purpose of this study is to evaluate the DNA damage induced by trans-catheter ablation and to highlight potential intervention to protect patients from this damage.
The study is moving on the evaluation of the DNA damage in terms of strand breaks in relation to epigenetic changes. In particular, it will be assessed whether these modifications are directly proportional to the ionization dose (DAP) exposition. This study could be an essential premise to the understanding of the mechanism and the amount of cell damage caused by low dose ionizing radiation and, consequently, it could lay the groundwork for identifying potential preventive therapeutic interventions in order to minimize ionizing radiation damage.
Inter-individual variability of aspirin metabolism: focus on aspirin esterase activity
Up to now, the physiologic basis for this “aspirin resistance” phenomenon is undefined and a high number of different factors are involved in it. Considering ASA catabolism, the 50-70% of aspirin is hydrolyzed in the systemic circulation by two pathways: a spontaneous pH-dependent autolysis and an enzymatic hydrolysis catalyzed by plasma and erythrocyte esterases. This latter pathway involves different types of butyrylcholinesterase and carboxylesterase and, probably, albumin. However, the contribution of these enzymatic activities in aspirin catabolism is still a matter of debate.
Aim of this study is to characterize the activity of ASA esterase in both plasma and red blood cell (RBC) in order to define the importance of their contribution in ASA hydrolysis.
The next step of this project will be the evaluation of ASA esterase activity in physiological and pathological settings, or in pharmacological conditions characterized by transient or stable changes in aspirin pharmacodynamics. The importance of this study is based on the the knowledge that conditions such as drug interactions, diabetes, increase of platelet turnover, obesity and aging potentially increase the variability of aspirin response. Understanding the underlying aspirin metabolism-related mechanisms is crucial to design strategies able to restore a normal response, restrain variability, and tailor the therapeutic intervention to the therapeutic need.
Biochemical and morphological characterization of red blood cell in coronary artery disease
The aim of this project is to evaluate whether the reduced production of NO found in RBCs of patients with CAD is able to decrease the deformability of the erythrocyte membrane itself.
Preliminary data have shown that RBCs from CAD patients have a reduced ability to change their shape in response to an increase in shear stress. This characteristic is associated with an alteration of membrane deformability in hypertonic conditions (p<0.001 vs Ctrl) and to an increase in the content of the main cytoskeleton protein spectrin (p<0.01 vs Ctrl).
In this contest, also RBC aggregation is an important, shear dependent, determinant of blood viscosity. Regarding this point, we demonstrated that in blood samples from CAD patients, erythrocyte aggregates are smaller but more stable than that found in healthy subjects. These results may suggest an impairment not only of NO synthesis but also of RBC capacity to transfer it to the peripheral tissues confirming an active role of RBCs in cardiovascular system. These findings need to be confirmed by a large number of patients and the specific impaired mechanism have to be highlighted yet. The knowledge deriving from this study could open new field of intervention in CVD and pave the way for discovering new prognostic tools in this disease setting.
Monocytes in coronary heart disease: implications for plaque morphology and activity
Monocytes are primitive hematopoietic cells that primarily arise from the bone marrow, circulate in the peripheral blood and give rise to differentiated macrophages. Emerging evidence suggests that monocytes can initiate inflammatory responses, carry antigen to lymph nodes, patrol and clean up the vasculature, recognize pathogens, and help kill tumor cells.
Epidemiological studies either report absolute cell count of monocyte subset, or relative cell counts, but information about genomic, proteomic and metabolomic profile of monocytes isolated from coronary artery disease (CAD) patients, in relation to characteristics of atherosclerotic plaque are scanty. By means a high-resolution visualization (10 µm) of coronary plaque morphology and its microstructures with in vivo optical coherence tomography (OCT), is possible to obtain relevant information on coronary plaques including fibrous cap thickness, lipid core, and macrophages accumulation, that can be combined with phenotypic profile of monocytes, thus providing a unique signature for identification of those atherosclerotic lesions that are most likely to cause a coronary event.
The aim of our study was to delineate a transctiptomic, proteomic, metabolomic, and functional profile of the monocytes isolated from CAD patients and to investigate whether these in vitro findings reflect the in vivo morphology features and the activity of coronary plaques in acute and chronic CAD patients undergoing OCT assessment.
- Eligini S, Porro B, Lualdi A, Squellerio I, Veglia F, Chiorino E, Crisci M, Garlaschè A, Giovannardi M, Werba JP, Tremoli E, Cavalca V. Nitric oxide synthetic pathway in red blood cells is impaired in coronary artery disease. PLoS One 2013;8(8):e66945.
- Cavalca V, Rocca B, Squellerio I, Dragani A, Veglia F, Pagliaccia F, Porro B, Barbieri SS, Tremoli E, Patrono C. In vivo prostacyclin biosynthesis and effects of different aspirin regimens in patients with essential thrombocythaemia. Thromb Haemost 2014;112(1):118-127.
- Porro B, Eligini S, Veglia F, Lualdi A, Squellerio I, Fiorelli S, Giovannardi M, Chiorino E, Dalla Cia A, Crisci M, Werba JP, Tremoli E, Cavalca V. Nitric oxide synthetic pathway in patients with microvascular angina and its relations with oxidative stress. Oxid Med Cell Longev 2014;2014:726539.
- Eligini S, Brioschi M, Fiorelli S, Tremoli E, Banfi C, Colli S. Human monocyte-derived macrophages are heterogenous: proteomic profile of different phenotypes. J Proteomics 2015;124:112-123.
- Zaccardi F, Rizzi A, Petrucci G, Ciaffardini F, Tanese L, Pagliaccia F, Cavalca V, Ciminello A, Habib A, Squellerio I, Rizzo P, Tremoli E, Rocca B, Pitocco D, Patrono C. In Vivo Platelet Activation and Aspirin Responsivenes in Type 1 Diabetes. Diabetes 2016;65(2):503-509.
- Tarantino E, Amadio P, Squellerio I, Porro B, Sandrini L, Turnu L, Cavalca V, Tremoli E, Barbieri SS. Role of thromboxane-dependent platelet activation in venous thrombosis: Aspirin effects in mouse model. Pharmacol Res 2016;107:415-425.
- Di Minno A, Turnu L, Porro B, Squellerio I, Cavalca V, Tremoli E, Di Minno MN. 8-Hydroxy-2-Deoxyguanosine Levels and Cardiovascular Disease: A Systematic Review and Meta-Analysis of the Literature. Antioxid Redox Signal 2016;24(10):548-555.
Andrea Anesi, PhD
Sonia Eligini, Ph.D
Benedetta Porro, Ph.D
Susanna Fiorelli, Ph.D student
Alessandro Di Minno, Ph.D student
Chiara Manega, Ph.D student
Linda Turnu, Ph.D student
Loredana Boccotti, Technician