The research activities performed by the Unit of Cell and Molecular Biology in Cardiovascular Diseases are focused on the biochemical, cellular and molecular mechanisms involved in the development and progression of atherothrombotic disease and on their pharmacological modulation. Thus, approaches ranging from cell biology to molecular biology, with a highly integrated strategy between basic (using both in vitro cultures of human and animal cells, animal models) and clinical research are in selected groups of patients with cardiovascular disease. A genomic and proteomic approach is also exploited in order:
- to study the mechanisms of regulation and the pharmacological modulation of key proteins involved in thrombotic processes;
- to identify genes and proteins with altered expression in cells, tissues or biological fluids of patients with cardiovascular diseases in order to discover new pathogenetic mechanisms as well as biomarkers able to predict the disease at an early stage, its follow up, and to estimate the risk of clinical event occurrene;
- to correlate the inter individual variations in the response to drugs with polymorphisms in genes encoding target proteins (receptors, enzymes, ion channels) or drugs (pharmacogenetics) in order to tailor a personalized therapeutic strategy;
- to identify the role of polymorphisms and/or mutations of proteins playing key roles within the atherothrombotic process (pharmacogenomics). All these research activities are performed in close collaboration with clinical units within the Centro Cardiologico Monzino.
New pathogenetic mechanisms in acute coronary syndrome: the role of platelet-associated Tissue Factor
Tissue Factor is a 47kD glycoprotein which triggers the blood coagulation cascade. Its expression within the atherosclerotic plaque directly correlates with the plaque thrombogenicity. We previously described the presence of TF in human platelets and we showed that this glycoprotein is expressed under the control of both platelet agonists and some antiplatelet drugs.
More recently we provided the evidence that TF expression is significantly higher in resting platelets of patients with ACS compared to patients with SA. This results in a greater capacity of platelets to generate thrombin, which in turn contributes to the prothrombotic phenotype of ACS patients.
New pathogenetic mechanisms in acute coronary syndrome: platelet transcriptomic and proteomic analysis
Although platelets do not have a nucleus, they contain more than 2000 megakaryocyte-derived mRNAs, which may influence pathophysiological functions. Comparing the platelet transcriptome of SA and NSTE-ACS patients we identified 45 differentially expressed genes involved in signal transduction, macromolecular complex assembly, and response to stress that may modulate platelet reactivity in CAD.
The analysis of the platelet proteome in SA and NSTE-ACS patients identified differential expression in proteins, not previously connected with CAD, such as energy metabolism enzymes, and proteins associated with cytoskeleton-based processes, both of which indicate platelet activation
Predictive value of circulating microparticle signature in coronary bypass graft patency
Cell-derived microparticles (MPs) are emerging as novel players in cardiovascular disease (CVD). They are involved in intercellular communication being vectors of biological messages, including nucleic acids (mRNA and microRNA [miRNA]), that participate in the pathophysiology and development of disease.
Circulating MPs have been considered as biomarkers of vascular injury and inflammation in several CVD including atherothrombosis and myocardial infarction where elevated levels of MPs have been correlated with the severity of pathology. Recently, also miRNAs have been shown to play a key role in CVD opening the possibility to use them as diagnostic surrogate markers. Graft patency and completeness of revascularization are major determinants of long-term outcome after coronary artery bypass grafting (CABG). Occlusion rates can be as high as 28% (per graft) or 45% (per patient) 12- 18 months thereafter. Endothelial damage, inflammation, and intimal hyperplasia are among the main candidates responsible for the early, and possibly late, graft failure.
The issue of identifying predictors of graft patency after CABG has been addressed by several studies, which mainly focused on the presence of conventional risk factors, genetic markers, features of coronary targets, or technical aspects. Fewer studies have focused on biological markers, such as perioperative inflammatory factors, but none has assessed the potential involvement of MPs and endogenous miRNAs. To this end we are elucidating whether a specific “signature” of circulating MPs is associated with postoperative CV adverse events, e.g., bypass graft occlusion, in patients undergoing elective surgical myocardial revascularization.
Selected ongoing research projects
PCSK9 as a positive modulator of platelet activation
The pharmacological inhibition of proprotein convertase subtilisin/kexin 9 (PCSK9), in combination with statins, represents the most effective therapy for the treatment of familial hypercholesterolemia and associated cardiovascular diseases. However, the PCSK9 physiological function, beyond the regulation of the low-density lipoprotein receptor (LDL-R), still needs to be fully characterized.
Platelet activation plays a key role not only in the thrombotic complication of atherosclerosis, but it is also deeply involved in the onset and progression of the disease. Recently, an association between PCSK9 levels and platelet count in coronary artery disease (CAD) patients and residual platelet reactivity in acute coronary syndrome (ACS) patients have been reported. From these basis, in the present study we investigated the effect of PCSK9 on platelet activation and thrombosis in in vitro and in vivo experimental models.
Tthis study adds circulating platelets to the list of targets of PCSK9 thus opening for this protein the possibility to act directly on mechanism(s) not as yet suspected. The availability of drugs able to suppress the contribution of PCSK9 not only to LDL-cholesterol metabolism, but also on specific components of arterial thrombus formation may be of great help to disentangle the role of this protein in thrombosis.
- Camera M, Rossetti L, Barbieri SS, Zanotti I, Canciani B, Trabattoni D, Ruscica M, Tremoli E, Ferri N. PCSK9 as a positive modulator of platelet activation. JACC in press.
- Parolari A, Cavallotti L, Andreini D, Myasoedova V, Banfi C, Camera M, Poggio P, Barili F, Pontone G, Mussoni L, Centenaro C, Alamanni F, Tremoli E; Coronary Bypass Grafting: Factors Related to Late Events and Graft Patency (CAGE) study investigators. D-dimer is associated with arterial and venous coronary artery bypass graft occlusion. J Thorac Cardiovasc Surg. 2018;155(1):200-207.
- Brambilla M, Rossetti L, Zara C, Canzano P, Giesen PLA, Tremoli E, Camera M. Do methodological differences account for the current controversy on tissue factor expression in platelets? Platelets. 2017 23:1-9.
- Brambilla M, Facchinetti L, Canzano P, et al. Human megakaryocytes express Tissue Factor and deliver it to a subset of shedded platelets where it contributes to thrombin generation. Thromb Haemost. 2015;114(3):579-592.
- Camera M, Toschi V, Brambilla M, et al. The role of Tissue Factor in atherothrombosis and coronary artery disease: insights into platelet tissue factor. Seminars in Thrombosis and Hemostasis 2015;41(7):737-746.
Marta Brambilla, Ph.D
Paola Canzano, Ph.D
Alessia Becchetti, MSc
Maria Conti, student