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.
Two main and integrated approaches are carried out:
- analytical investigations of clinical biochemistry and ex-vivo studies by using cellular models. In particular, the activation of circulating cells (erythrocytes, monocytes, etc.);
- the role of the oxidative stress on cell function are investigated.
A long-standing interest on oxidative stress and on its relationship with endothelial function and cellular activation in atherothrombosis characterizes the research program of the Unit and the current strategy is devoted to the identification of biomarkers and/or metabolic pathways involved in these pathological conditions. In particular, we investigate possible links between oxidative stress, endothelial dysfunction and platelet aggregation and, to this end, we have developed chromatographic methods (HPLC, LC-MS/MS) with high sensitivity and specificity which measure both oxidative modifications of lipids and proteins and the levels of antioxidant factors such as vitamins, glutathione, antioxidant enzymes, etc.
Metabolomics offers new opportunities to elucidate the pathogenic and molecular mechanisms of the disease and can be considered a new tool for the discovery of novel biomarkers and therapeutic targets. This technique allows to analyze a specific metabolic pathway through the measurement of low molecular weight metabolites in cells, tissues, organs or biological fluids, thus identifying the biochemical phenotype. 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 (e.g.erythrocytes and monocytes). In particular, we have analyzed endothelial dysfunction (by measuring of metabolites involved in L-Arginine / NO metabolic pathway in plasma and red blood cells), aspirin metabolism (by evaluating its effects and its catabolic enzymes activities) and the metabolism of arachidonic acid (by measuring eicosanoids produced by platelets and endothelium).
In this contest, the Unit has focused its attention on the following research lines:
- Analysis of oxidative stress status and the L-Arginine/NO metabolic pathway in vivo in different cardiovascular disorders (acute coronary disease, takotsubo syndrome, etc.) and in vitro on circulating and cultured cells
- Study of the red blood cell as new player in cardiovascular homoeostasis: focus on its morphology and activity on cardiovascular disease
- Study of vascular homeostasis through the evaluation of eicosanoids enzymatically produced by arachidonic acid and its perturbation by antiplatelet treatment. In particular two studies have been performed to evidence: 1) aspirin unusual metabolic effects by the untargeted metabolomics approach; 2) aspirin responsiveness in pathological setting characterized by accelerated platelet tournover rate, such as cardiovascular surgical intervention
- Definition of biological signature of monocytes and macrophages obtained from coronary artery disease patients in relation to plaque morphology and activity detected in vivo by optical coherence tomography in order to detect the acute or chronic disease and the presence of vulnerable plaques even before plaque rupture precipitates thrombotic event.
Urinary 8-OHdG as a reliable oxidative stress biomarker in patients with heart failure
Reactive oxygen species (ROS) play a role in both normal physiology and in the pathogenesis of several diseases, also including HF. In physiological conditions, ROS are scavenged by the antioxidant system, but when their concentration is too high to be balanced, an oxidative damage to proteins, lipids and DNA cannot be avoided. 8-hydroxy-2-deoxyguanosine (8-OHdG), which is generated following the repair of ROS-mediated damage to DNA, is one of the most widely recognized biomarkers of oxidative stress.
In a recent meta-analysis, we have documented:
- enhanced 8-OHdG levels in HF patients as compared to healthy controls;
- a direct association between higher than normal 8-OHdG levels and the worsening of the New York Heart Association (NYHA) class of HF. No studies are available in which 8-OHdG levels have been related to the cause of HF (dilated cardiomyopathy; ischemic heart disease, hypertensive cardiomyopathy etc).
The present ongoing study has aimed to measure urinary 8-OHdG levels in HF patients in relation to NYHA class and cause of HF. Our preliminary results confirm our previous data, highlighting that 8-OHdG levels were higher in HF patients and the difference was even more glaring when NYHA III class patients (p=0.0002) were compared to CTR.
The study is moving on the evaluation of the DNA damage in different HF aetiology (e.g. dilated cardiomyopathy, ischaemic cardiomyopathy) to evaluate whether this metabolite can help to understand if the severity rather than the mechanism leading to HF have a pivotal role in determining oxidative DNA damage in order to develop a treatment strategy to avoid potential genetic mutations in HF.
Takotsubo cardiomyopathy: focus on psychological and coagulative profile and oxidative stress assessment
Takotsubo cardiomyopathy (TC) is a syndrome mainly diagnosed in post-menopausal women characterized by transient left ventricular dysfunction and elettrocardiographic abnormalities in the absence of significant coronary artery disease. His pathophysiology is very complex and it is still unclear.
Although some data evidenced that TC is often associated with an acute, emotionally intense episode, several cases have no identifiable triggering events and other pathophysiological mechanisms have been associated to TC development. There is growing evidence that exaggerated sympathetic stimulation is central to the pathogenesis of this syndrome. Precisely how catecholamines mediate myocardial stunning in Takotsubo Syndrome remains incompletely understood; but possible mechanisms include epicardial spasm, microvascular dysfunction, direct adrenergic-receptor–mediated myocyte injury, and systemic vascular effects that alter ventricular-arterial coupling. Moreover, high levels of cathecolamines can be considered a potential source of oxidative stress.
Investigators have raised the estrogen hypothesis as a possible explanation for the susceptibility of post-menopausal women to TC. Several studies have reported that therapy with estrogen supplementation may prevent the development of TC, suggesting that the lack of estrogen might predispose women to the pathogenesis of TC. In this ongoing study, women with a previous TC event are investigated both in their psychological and biochemical profiles in order to highlight a relationship between emotional/mental stress and procoagulant and oxidative status and more generally to clarify the complex mechanisms underlying the onset of this syndrome.
Untargeted urine metabolomics to go beyond aspirin canonical effects
Although the clinical efficacy of Aspirine has been well documented, a substantial variability in drug response exists, resulting in some patients in a persistent platelet reactivity and atherothrombotic events, despite the drug treatment.
Metabolomics, an emerging area of research based on highly sensitive analytical methods, allows the measurement of thousands of small molecules in biological samples. This tool is perfectly suited to describe the phenotypic changes associated with pharmacologic treatments, by enabling detailed mapping of pathways involved in Aspirin response. Therefore, to obtain a more comprehensive evaluation of pathways involved in Aspirin treatment, we use an untargeted metabolomics approach to delineated the urine metabolic profile of seven healthy subjects before (T0) and 7 days (T7) after Aspirin low-dose (100 mg/die) treatment. Among 2007 detected metabolites, 64 significantly differed after Aspirin assumption. In order to explore potential metabolic pathways affected by the treatment, the identified metabolites were investigated and classified according to their connections across the biochemical pathways in which they are involved.
Pathway analysis revealed low levels of several metabolites involved in histidine, alanine, aspartate and glutamate and purine metabolisms, after Aspirin treatment. Interestingly, we observed low levels of histidine: even if the biological significance of this observation is not well defined, we can speculate that the reduction can leads not only to the decrease of its catabolites but also to a parallel decrease in the levels of histamine, commonly considered an inflammatory mediator. In this way, the impairment of histidine metabolism might participate to the anti-inflammatory activity of Aspirin.
In addition, several acylcarnitines, were affected by Aspirin treatment: in particular, butyryl-L-carnitine, tiglylcarnitine, isovalerylcarnitine and heptanoylcarnitine were decreased. The modification of short- and medium-chain acylcarnitines levels reflects an increase in fatty acid β-oxidation process. These data suggested that Aspirin, by boosting the cellular energy metabolism, may exert beneficial effects in the metabolic, mitochondrial and cardiovascular conditions in which energy depletion is present
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.
- Turnu L, Porro B, Alfieri V, Di Minno A, Russo E, Barbieri S, Bonomi A, Dello Russo A, Tondo C, D'Alessandra Y, Cavalca V, Tremoli E, Colombo GI, Casella M. Does fluoroscopy induce DNA oxidative damage in patients undergoing catheter ablation? Antioxid Redox Signal. 2017 Sep 22. [Epub ahead of print]
- Cavalca V, Rocca B, Veglia F, Petrucci G, Porro B, Myasoedova V, De Cristofaro R, Turnu L, Bonomi A, Songia P, Cavallotti L, Zanobini M, Camera M, Alamanni F, Parolari A, Patrono C, Tremoli E. On-pump Cardiac Surgery Enhances Platelet Renewal and Impairs Aspirin Pharmacodynamics: Effects of Improved Dosing Regimens. Clin Pharmacol Ther. 2017;102(5):849-858.
- 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.
- 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.
- 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.
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
Sonia Eligini, Ph.D
Benedetta Porro, Ph.D
Alessandro Di Minno, Ph.D student
Susanna Fiorelli, Ph.D student
Chiara Manega, Ph.D student
Linda Turnu, Ph.D student