Proteomics, the study of the complete complement of proteins encoded by an organism, is an emerging field that has the potential to uncover new therapeutic targets for the treatment and prevention of cardiovascular diseases, as well as new diagnostic biomarkers for early disease detection. Proteomics-based studies are focused on the interactions of multiple proteins and on their role as part of a biological system rather than the structure and function of one single component.
By properly applying proteomics, at least three different types of biomarkers can be potentially developed for cardiovascular medicine: mechanistic markers, clinical disease markers, and therapeutic markers.
The Unit of Proteomics aims at improving diagnosis and treatment of cardiovascular diseases with an approach based on two principal activities: identification of candidate disease markers from well-defined human clinical cohorts, animal studies or in vitro cell systems, and validation of protein patterns in clinical data sets.
So far, there are only a few examples of the systematic approach to define such protein fingerprints, but these appear very promising. The use of protein mass spectra has been proposed as a powerful diagnostic tool for ovarian cancer. This, then, is the opportunity for clinical proteomics to define patterns of proteins that provide clinically useful information about susceptibility to disease, diagnosis, prognosis, and guided therapy.
Consistently with this objective, the Unit of Proteomics aims at improving diagnosis and treatment of cardiovascular diseases with an approach based on two principal activities: identification of candidate disease markers from well-defined human clinical cohorts, animal studies or in vitro cell systems, and validation of protein patterns in clinical data sets.
Cardiovascular proteomics has therefore important direct 'bedside' applications, even if we are still in the early stages. However, a fruitful interaction with the clinical units will enable us to test the relevance of new molecular markers for diagnostic, prognostic and therapeutic improvements, aiming at a rapid translation of basic findings to the clinical setting.
Selected ongoing research projects
The goal of the Unit of Proteomics is to develop new, generally applicable technologies for proteomic analysis for generating new discoveries in specific areas of cardiovascular research using a synergistic combination of contemporary two-dimensional gel electrophoresis, a well established mass spectrometry facility, and state-of-the-art methods of biochemistry and gene expression/regulation analysis.
Several proteomic approaches have been recently applied by the Unit of Proteomics to the study of: a) plasma; b) circulating cells; c) circulating mediators (lipoprotein, microparticles); d) secreted proteins (secretome); e) cell cultured systems; f) tissues; and g) organelles in the search for new mechanistic or diagnostic biomarkers for cardiovascular diseases.
- Plasma proteome
The incorporation of proteomic analysis of plasma into functional biochemical and biological approaches provides a powerful mean of identifying patho/physiological pathways in cardiovascular diseases as it allows the simultaneous detection of different circulating proteins and their post-translational modifications that cannot be identified by conventional measurements. We applied this approach to the investigation of the complex network of molecular mechanisms involved in the deleterious effects of coronary artery bypass graft (CABG) surgery and in coronary artery disease (CAD).
- Circulating cells
Circulating cells represent an important target of proteomics because they can bear information reflecting directly an inflammatory or pro-coagulant state related to the pathology. We focused on the analysis of platelets whose participation in the genesis of chronic atherosclerotic lesions and the formation of thrombi that acutely occlude arteries, causing serious disease, is now well established.
- Circulating mediators: lipoprotein and microparticles
The mechanisms by which human low density lipoprotein LDL manifests its atherogenic properties have been, indeed, the topic of intense investigation during the past decades but, still, few data have been reported on proteins contained in human LDL and their possible functional roles. Our proteomic analysis revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol-binding protein, and paraoxonase-1. Of interest, PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. The integration of the proteomic data with biochemical and gene expression analysis allowed us to assess that PCL1 is generated along with nascent lipoprotein and that it can itself generate an oxidant, thus suggesting that PCL1 may play a significant role in atherogenesis.
- The secretome
The secretome recently emerged as a new term to describe the global study of proteins that are secreted by a cell at any given time or under certain conditions, constitutes an important class of proteins that control and regulate a multitude of biological and physiological processes, thus making it a clinically relevant source for biomarkers and therapeutic target discoveries. In this respect, the application of a global proteomic approach to determine the effect of statins on the proteins released, “secretome”, by endothelial cells, could help to understand novel mechanisms by which statins promote some of their beneficial effects.
- The unexplored human mitral valve prolapse proteome
This translational research program is aimed to investigate the cellular and molecular regulators of tissue remodeling during the development of human Myxomatous Mitral Valve Prolapse (MVP), the most common indication for mitral valve surgery due to severe mitral regurgitation merging proteomics and cellular biology approaches. Echocardiographically,
- Organelle proteomic
Focusing on specific organelle proteomes affords an attractive alternative to reduce the tremendous complexity of the cellular/tissue proteomes and represents an attainable goal for better spatial and functional correlations of the identified proteins.
- The Italian MT-HPP Initiative
The mitochondrial Human Proteome Project (mt-HPP) is a Human Proteome Organization (HUPO) initiative led by the Italian Proteomics Association (ItPA), focused on human mitochondrial proteins. Due to the intrinsic nature of the mitochondrial proteome, this action is part of both the chromosome-centric Human Proteome Project (c-HPP) and the Biology/Disease Human Proteome Project (B/D-HPP). The main goal of this effort is to obtain robust information about the integrative role of proteins acting at the mitochondrial level, considering both those encoded by the mitochondrial DNA (mt-DNA) and by the nuclear DNA. The project is conducted with the support of HUPO and under supervision of external advisors (Peipei Ping, University of California Los Angeles; Mohano Babu, University of Regina, Canada).
- Proteomic analysis of in vitro cell systems
In the context of cellular proteome we are currently interested in the cellular phenotyping following gene silencing by RNAi. We are currently exploring the inhibition effect of the Tissue Factor (TF) expression by its specific siRNA in cardiomyocytes.
Redox proteomics – The MASSTRPLAN European Project
The field of redox proteomics, although relatively new and rapidly changing, has the potential to revolutionize how we diagnose disease, assess risks, determine prognoses, and target therapeutic strategies for people with cardiovascular diseases.
In this study we are going to analyse the oxidative modifications to a broad range of oxidative-modified biomolecules with the final aim at identifying oxidative molecular signature of pathophysiological and therapeutic interest in the management, treatment, and risk assessment of patients.
The team of researchers at CCM will take advantage of their participation, as partners, in MASSTRPLAN (MASS Spectrometry Training network for Protein Lipid adduct Analysis), an European training network (proposal number 675132) within the EU Framework Programme for Research and Innovation, Horizon 2020. The MASSTRPLAN network brings together 10 main European teams (6 academic, 2 hospitals, and 2 commercial) with strong and complementary skills in chemistry, Liquid Chromatography-Mass spectrometry (LC-MS) analysis, bioinformatics, molecular and cellular biochemistry, and diagnostic development.
CCM will benefit from the participation to the MASSTRPLAN network through a constant and fruitful interactions with the other partners; indeed, the research program of MASSTRPLAN is designed to develop methods of detecting and quantifying challenging heterogeneous oxPTMs including lipoxidation using LC-MS, novel bio-informatics tools, and complementary analytical techniques, including fluorescence microscopy and mutagens, to validate the oxPTMs in biological samples. Once established using model systems, the methodology will be translated to the analysis of biological samples, and, potentially used to develop diagnostic tests.
- Banfi C, Baetta R, Gianazza E, Tremoli E. Technological advances and proteomic applications in drug discovery and target deconvolution: identification of the pleiotropic effects of statins. Drug Discov Today. 2017;22(6):848-869.
- Banfi C, Guarino A, Brioschi M, Ghilardi S, Mastrullo V, Tremoli E, Polvani G. Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve. J Vis Exp. 2017 Jun 14;(124).
- Brioschi M, Martinez Fernandez A, Banfi C. Exploring the biochemistry of the prenylome and its role in disease through proteomics: progress and potential. Expert Rev Proteomics. 2017;14(6):515-528.
- Susilovic-Grabovac Z, Banfi C, Brusoni D, Mapelli M, Ghilardi S, Obad A, Bakovic-Kramaric D, Dujic Z, Agostoni P. Diving and pulmonary physiology: Surfactant binding protein, lung fluid and cardiopulmonary test changes in professional divers. Respir Physiol Neurobiol. 2017;243:27-31
- Amadio P, Colombo GI, Tarantino E, Gianellini S, Ieraci A, Brioschi M, Banfi C, Werba JP, Parolari A, Lee FS, Tremoli E, Barbieri SS. BDNFVal66met polymorphism: a potential bridge between depression and thrombosis. Eur Heart J. 2017;38(18):1426-1435.
- Banfi C, Baetta R, Gianazza E, Tremoli E. Technological advances and proteomic applications in drug discovery and target deconvolution: identification of the pleiotropic effects of statins. Drug Discov Today. 2017 Mar 8. pii: S1359-6446(17)30108-3. doi: 10.1016/j.drudis.2017.03.001. [Epub ahead of print]
- Baetta R, Lento S, Ghilardi S, Barbati E, Corsini A, Tremoli E, Banfi C. Atorvastatin reduces long pentraxin 3 expression in vascular cells by inhibiting protein geranylgeranylation. Vascul Pharmacol. 2015 Apr-Jun;67-69:38-47. doi: 10.1016/j.vph.2014.11.008. Epub 2015 Apr 4.
- Magrì D, Banfi C, Maruotti A, Farina S, Vignati C, Salvioni E, Morosin M, Brioschi M, Ghilardi S, Tremoli E, Agostoni P. Plasma immature form of surfactant protein type B correlates with prognosis in patients with chronic heart failure. A pilot single-center prospective study. Int J Cardiol. 2015 Dec 15;201:394-9. doi: 10.1016/j.ijcard.2015.08.105. Epub 2015 Aug 15.
- Lento S, Brioschi M, Barcella S, Nasim MT, Ghilardi S, Barbieri SS, Tremoli E, Banfi C. Proteomics of tissue factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control. J Proteomics 2015;119C:75-89.
Roberta Baetta, Ph.D
Maura Brioschi, Ph.D
Erica Gianazza, Ph.D
Marta Pontremoli, Ph.D student
Valeria Mastrullo, Ph.D student
Alma Martínez Fernández, Ph.D student
Stefania Ghilardi, Technician