Scientific Report 2011 Ongoing Research 2012

Transcript

Scientific Report 2011 Ongoing Research 2012
Centro Cardiologico Monzino
Scientific Report 2011
Ongoing Research 2012
Scientific Report 2011
Ongoing Research 2012
Chairman’s message
Cardiovascular diseases and cancer remain the most frequent causes
of death in the Western world and, in order to combat these diseases, our efforts have been directed toward the development of research in these important fields.
Despite of the deep economic crisis that has affected Europe, and in particular our country, the clinical and research
activities at the European Institute of Oncology and at Centro Cardiologico Monzino during 2011 and the first part of 2012
have had a positive trend, allowing to offer the patient the best standard of care.
In order to do this, a coordinated effort to optimize the use of economical and human resources has been developed
and, thanks to the tireless and passionate work of our researchers and clinicians, important advances in the care of these
life-threatening pathologies have been achieved.
In this context a special thank is given to our former CEO Carlo Ciani for the overall activities he carried out during
the last years.
Carlo BUORA
Chairman
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Contents
• CEO message
• Scientific Director’s Report
• The Research Laboratories
• Grants Office
• Department of Cardiovascular Sciences
• Centre of Pharmacological Research for the Study
and Prevention of Cardiovascular Diseases
• Directive Board
• General Management
• Outpatient Service and Marketing
• Safety, Enviroment and Quality
• Finance and Administration
• Human Resources
• Information & Communication Technology
• Ethics Committee
• Library
• Educational and Training
• Medical Director’s Report
• Medical Directorate’s Staff
• Clinical Governance at Centro Cardiologico Monzino
• Cardiovascular Tissue Bank
• Cardiovascular Telemedicine
Colophon
Title
Publisher
Number Month/Year
Editor Authors
Secretary of editing
Photographer
Typographer
Paper
CCM Scientific Report 2011. Ongoing Research 2012.
CCM Centro Cardiologico Monzino©, Milan (Italy)
3
December 2012
Elena Tremoli
Each Unit was in charge of the writing of its own text.
Nicoletta Malgaretti, Mirko Nesurini
Roberto Benzi
Italgrafica (NO), Printed in Italy
Printed on the uncoated paper On Offset whose elementary chlorine free
pulps came from susteinaible forests
CLINICAL UNITS
Emergency Unit
Acute Cardiac Care Unit
Intensive Cardiac Care Unit
Heart Failure, Clinical Cardiology and Cardiac
Rehabilitation Unit
Interventional Cardiology Units
Electrophysiology Unit
Cardiac Surgery Unit
Extracorporeal Circulation Service
Vascular Ultrasound Service
Vascular and Endovascular Surgery Unit
Anaesthesiology and Intensive Care Unit
Echocardiography Unit
Magnetic Resonance Imaging Unit
Radiology Unit
Laboratory Medicine Unit
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RESEARCH LABORATORIES
Laboratory of Biology and Biochemistry
of Atherothrombosis
100
Unit of Cell Biology and Biochemistry
of Atherothrombosis
102
Unit of Cardiovascular Proteomics
106
Unit of Biochemistry of Oxidative Stress and Endothelial
Function in Atherothrombosis
112
Unit of Experimental Thrombosis and Imaging in vivo 118
Program for the Control of Global Cardiovascular Risk 121
Unit of Atherosclerosis Prevention
122
Unit of Arterial Morphology and Function
124
Unit of Biostatistics
126
Laboratory of Immunology and Functional Genomics
128
Laboratory of Vascular Biology and Regenerative Medicine 132
Laboratory of Cardiovascular Tissue Engineering
138
Plasma and Gene Bank
144
DNA Bank
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CLINICAL RESEARCH
Relationship between heart and kidney function in acute
cardiovascular conditions
Candidate biomarkers for the clinical and prognostic
stratification of patients with acute coronary
syndromes
Cardiopulmonary physiopathology
Interventional cardiology
Electrophysiology Area: scientific hot topics
Cardiovascular imaging
Clinical Research on Atherothrombosis
Transcatheter aortic valve implant (TAVI)
Arrhythmia surgery
Vascular surgery
BASIC AND TRANSLATIONAL RESEARCH
Prospective evaluation of platelet activation markers in
patients treated with drug-eluting stents:
platelet-associated Tissue Factor predicts the risk
of future revascularization in a pilot study
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Proteomics-based development of biomarkers in
cardiovascular disease
Red blood cells as a source of nitric oxide
Heterogeneity of human macrophages in culture and in
atherosclerotic plaque
Experimental imaging of brain and heart ischemia
Interaction between food and cardiovascular drugs
Food-blood fatty acids and coronary heart disease
Role of the longevity genes p66sh/sirt1 in insulinresistance,
diabetes and cardiovascular disease
High resolution B-mode ultrasound imaging
High resolution B-mod ultrasound imaging for the
assesment of mechanism involved in atherosclerosis
progression
New methods to analyze complex ultrasound measures
and atherosclerosis progression
Cardiovascular epigenetics
microRNAs in cardiovascular diseases
Regenerative medicine in cardiovascular diseases
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Board of Directors
As of June 2012
Carlo BUORA
Carlo Ciani Mauro Melis Marco AGNELLI
Stefano MICHELINI
Mario Cesana
Chairman
Deputy Chairman
Chief Executive Officer
ex officio Lorenzo CAMMELLI
Elena Tremoli
Auditors
Maurizio BOZZATO
Guido CROCI
Pierluigi DAVIDE
PUBLICATIONS, CLINICAL RESEARCH PROJECTS, SEMINARS
Full papers 229
Clinical Research Projects 234
Ongoing grants
244
Seminars, conferences and courses 248
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Chief Executive Officer’s Message
During 2011 and the first part of
2012 Centro Cardiologico Monzino has been able to maintain and even to increase its level of excellence with a good
economical performance, despite the difficulty of this time period, as outlined by dr. Buora.
This means that the decision of our shareholders, who contributed to creating the Institute over the years, was an
excellent one.
We hope that the economical conditions will allow us in the future to carry out further investments in research and
technology aimed at improving even further the effectiveness of our treatments.
We are constantly engaged in clinical practice to maintain a high quality care for our patients, developing in-house
and external training for many family doctors and specialists who see the Institute as a center of excellence and
reference for cardiovascular diseases.
Mauro MELIS
Chief Executive Officer
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Scientific Director’s Report
Elena Tremoli, Ph.D
Scientific Director
Scientific Direction Secretariat and Grants Office:
Chiara Centenaro, Maria Lucarelli,
Nicoletta Malgaretti, Luisa Triglia
Lab Manager: Alessandra Terragni
Elena TREMOLI, Ph.D
Scientific Director
Centro Cardiologico Monzino is the only Institute
of excellence all over Europe exclusively devoted to
cardiovascular diseases. Our mission is to understand
the underlying causes of the diseases of the heart and
the vascular tree and to develop new treatments and
cures for our patients.
Within this frame the Scientific Direction provides
planning, directions, and support to the different
components of the Institute.
Indeed, “the patient” is central in all our activities and
everybody’s efforts are directed towards the strategic
objectives of the Institute in order to create the best
conditions to allow operators and medical staff to put
their knowledge to patients’ service and our Scientific
and Management Boards to guarantee that the everyday
activity reflects these basic principles.
In July 2011, after more than one decade of Scientific
Direction, prof. Paolo Biglioli retired and prof. Elena
Tremoli has taken the responsibility of the Scientific
Direction of Centro Cardiologico Monzino. Since then, a
number of activities have been carried out both in basic
and clinical research in order to strengthen the scientific
exchanges between cardiologists, heart and vascular
surgeons and basic researchers, with the aim to further
implement the translational feature of the research
and ultimately to develop a full model of translational
medicine to be applied to Centro Cardiologico Monzino.
The implementation of collaborations among different
scientific disciplines and through activities that will
provide to scientists and clinicians a fostering ground
for their scientific work, has been among the jobs
developed during this year.
In addition the Scientific Direction is working in
monitoring and analysing industrial trends in pertinent
areas in order to facilitate the development of new
research projects, which may ultimately result in
strengthening the relationship between public authorities
and enterprises both at Italian and European level.
Centro Cardiologico Monzino project leaders provide a
strategic focus in establishing the critical path needed to
move the research down the translational path, helping
to form project teams, bridging information across
disciplines, ensuring high quality data systems and
providing funding, also through the identification of
additional resources.
Many new clinical projects started during the year,
soon after the approval of the Scientific and Ethics
Committees.
We also had a consistent number of visitors from all part
of the world attending the research laboratories and the
clinical Units. Training of young scientists and physicians
is part of our mission. Many members of our staff were
actively involved in courses, seminars and conferences
both for external and internal attendance.
We developed collaborations with several European
countries, and specific efforts were devoted to
establish contacts and cultural relationships with new
cardiovascular centres.
Overall, 2011 has been a fruitful year for publications,
since the total impact factor has increased with respect
to 2010. The scientific production, in terms of papers
published on peer-reviewed journals, is all centred
in the cardiovascular field spanning from clinical and
interventional cardiology, to heart and vascular surgery,
with a focus on new imaging techniques to visualize the
heart and the vascular tree and on the development
of biomarkers for the identification of patients at high
risk to develop the first (primary prevention) or further
(secondary prevention) cardiovascular events.
Special focus has been given, as before, to education.
During this year the Department of Cardiovascular
Sciences of the University of Milano has been merged
with other Institutes to create the Department of
Clinical Sciences and Community Health. Centro
Cardiologico Monzino hosts the Cardiovascular
Section of this Department, chaired by Prof. Cesare
Fiorentini. In addition, the Postgraduate School
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The Research Laboratories
in Heart Surgery, chaired by Prof. Alamanni, the
Postgraduate School in Cardiology, chaired by prof.
Fiorentini, the first level Degree in Techniques of
Cardiocirculatory Pathophysiology and the Master
in Cardiovascular Nursing are based in the Institute.
Finally, Centro Cardiologico Monzino hosts the Centre of
Pharmacological Research for the Study and Prevention
of Cardiovascular Diseases of the University of Milano.
In conclusion, 2011 was a productive year, full of
positive news even though we had to deal with a severe
economic crisis and a rationalization of costs and
expenses in the research activities.
Nevertheless, thanks to the help of everybody, Centro
Cardiologico Monzino has been active and successful
in terms both of scientific goals achieved, and, most
importantly, of clinical results in the care of patients
affected by cardiovascular diseases.
Publications 2000 — 2011
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
29
26
42
41
50
56
68
98
63
76
111
114
Full Papers
Journal with Impact Factor
Total Impact Factor (*)
89,47
77,92
199,34
227,86
258,798
326,05
488,215
466,974
309,841
559,95
647,97
672,02
Average Impact factor
3,09
3,00
4,86
5,56
5,18
5,82
7,18
4,77
4,92
6,51
5,84
5,89
Full Papers
Journal without Impact factor
8
11
19
16
10
18
8
14
15
24
16
16
Total Full Paper
37
37
60
57
60
74
76
112
78
100
127
130
(*) For each year the IF was calculated using values published in the Journal Citation Reports of the previous year
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CCM — Scientific Report 2011 — Ongoing research 2012
During 2011 Centro Cardiologico Monzino has carried
out translational research focused on the study of
cardiovascular disease. A global approach to this field
has been used taking advantage from the new -omics,
e.g. genomic and proteomic, and other innovative
technologies to the emerging problems in cardiology
and heart and vascular surgery. This has allowed the
quick translation of emerging scientific knowledge into
new strategies for the prevention and treatment of
cardiovascular diseases.
The four major basic research programs (Biology and
Biochemistry of Atherothrombosis, Vascular Biology
and Regenerative Medicine, Immunology and Functional
Genomics, Cardiovascular Tissue Engineering) operate in
strict conjunction with the clinical and surgical Units of
Centro Cardiologico Monzino.
The program of the Laboratory of Biology and
Biochemistry of Atherothrombosis is focused on the
studies of both the vessel wall and circulating cells,
with a major focus on the study of platelet biochemistry
and function in patients with atherothrombosis and/or
coronary disease. The Laboratory of Vascular Biology
and Regenerative Medicine is exploring the potential
of the cellular cell therapy in cardiovascular disease,
with the aim to increase its potential. The Laboratory
of Immunology and Functional Genomics focuses on the
investigation of the immunological events associated
with the formation of atherosclerotic plaque and the
occurrence of thrombus in cardiovascular diseases.
Functional genomics goals of this laboratory include
studies on gene expression and control, investigation
of mutations that cause function loss or alteration, and
development of experimental and computational methods
for protein analyses. Researches involving genetically
modified animal models as well as new experimental
imaging approaches are also carried out.
The mission of the Laboratory of Cardiovascular Tissue
Engineering is to devise strategies to derive a new
generation of cardiovascular implants and devices,
by combining cardiovascular stem cell knowledge
and material/biomechanics expertise. Starting from
basic investigations on human-derived cardiovascular
progenitors such as cord blood-/peripheral blood-/
bone marrow-derived endothelial progenitor cells
(EPCs), cardiac resident stem cells (CSCs), saphenous
vein progenitors (SVPs) and the so called aortic valve
interstitial cells (VICs), the mechanisms linked to
physiologic or pathologic differentiation of these cells is
being studied by application of “high content” molecular
analysis and high throughput material screening
methods allowing a rapid search for novel bio-materials
(polymers, hydrogels and biological scaffolds) to embed
stem cells in culture. Particularly relevant appears,
in this scenario, also the investigation on strain- and
mechanical stress-associated stem cells response
Moreover, a Program for the Control of Global
Cardiovascular Risk is ongoing. Lifestyle and
pharmacological approaches are applied to patients’
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treatment in order to reduce their cardiovascular risk.
At Centro Cardiologico Monzino five major lines of
clinical research are ongoing:
• Critical cardiology
• Interventional cardiology
• Electrophysiology
• Cardiac and Vascular Surgery
• Imaging
And research is carried out in the following laboratories:
• Heart and vascular surgery
• Anaesthesiology
• Cardiovascular Imaging
• Electrophysiology
• Clinical Laboratory Medicine
Many new clinical projects started during the year,
soon after the approval of the Scientific and Ethics
Committees. We also had a consistent number of
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visitors from all part of the world attending the research
laboratories and the clinical Units. Training of young
scientists and physicians is part of our mission. Many
members of our staff were actively involved in the
Department and Postgraduate Schools of the Milano
University and were engaged in teaching and lecturing at
scientific meetings. Numerous meetings were organized
by Centro Cardiologico Monzino, both for external and
internal attendance.
We developed collaborations with several European
countries, and specific efforts were devoted to
establish contacts and cultural relationships with new
cardiovascular centres in Saudi Arabia, Libya and Arab
Emirates.
Thanks to national and international funds, we were
successful in supporting research activities. We expect
to further increase our fund raising capacity and are
very grateful to all granting agencies that recognized the
validity of our projects.
The Scientific Director and the Directors of the Research Laboratories: Maurizio Capogrossi, Elena Tremoli, Gualtiero Colombo,
Gianluca Polvani.
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Grants Office
University Department of Cardiovascular Sciences
Cesare FIORENTINI, Full Professor
Director
Within the Scientific Direction, the Grants Office acts as
a link between researchers and sponsoring agencies and
is a central source of information on major national and
international agencies, foundations, and institutions that
support research and scholarships. It assists researchers
in identifying appropriate research funding opportunities
providing support in the preparations of applications,
developing proposal narratives and budgets, completing
the application forms and interpreting the regulations
of the granting agencies, assuring compliance with the
sponsor’s policies and requirements. It negotiates the
terms and conditions of awards, of successful proposals
and provides support for the administration of research
grants, including funding allocation, monitoring research
expenditures and producing financial statements. It
manages research contracts, preparing, whenever
necessary, subcontracts or consortium agreements with
collaborating institutions and provides administrative
support for Centro Cardiologico Monzino.
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Vice Director: Francesco ALAMANNI, Full Professor
STAFF University: Piergiuseppe Agostoni, Assoociate Professor,
Marco Agrifoglio, Associate Professor, Vincenzo Arena,
Assistant Professor, Antonio Bartorelli, Associate
Professor, Viviana Cavalca, Assistant Professor,
Francesco Donatelli, Full Professor, Luisa Gregorini
Ventura, Associate Professor, Alessandro Lualdi,
Assistant Professor, Piero Montorsi, Associate Professor,
Alessandro Parolari, Assistant Professor, Marco Pocar,
Assistant Professor, Gianluca Polvani, Full Professor,
Giulio Pompilio, Assistant Professor
Administrative: Francesco Barresi, Eleonora Boccato,
Loredana Boccotti, Anna Cerini, Alessandro Gabrieli
Cardiology Fellows: Laura Antonioli, Francesca Baratto,
Stefano Bartoletti, Daniela Benedetto, Erika Bertella,
Filippo Billi, Costanza Boiti, Angelo Cabiati, Chiara
Colombo, Sarah Cortinovis, Maria Antonietta Dessanai,
Anna Garlaschè, Pamela Gatto, Sarah Ghulam Ali,
Giuliano Giusti, Erica Gondoni, Monica Sonia Loguercio,
Massimo Mapelli, Andrea Marinetti, Lucia Mauri,
Valentina Milazzo, Marco Valerio Morpurgo, Saima
Mushtaq, Paolo Olivares, Chiara Stefania Pandini,
Margherita Pirondini, Francesca Pizzamiglio, Ilaria
Previtali, Mara Rubino, Laura Salvini, Chiara Segurini,
Marco Vicenzi , Carlo Vignati
Heart Surgery Fellows: Francesco Arlati, Cristina
Borsetti, Laura Cavallotti, Alberto Clerici, Monica
Contino, Puma Dennis Ezra Cusihuaman, Andrea Daprati,
Tommaso Generali, Sara Filippini, Marco Gennari, Daniela
Manzone, Elisa Merati, Nuri Halkawt Ali Nuri, Davide
Patrini, Alberto Pilozzi Casado, Camillo Poloni, Gabriele
Tamagnini, Giulio Tessitore
Centro Cardiologico Monzino was founded by cavalier
Italo Monzino and professor Cesare Bartorelli in 1981
with the aim to develop cardiology in Italy. As the result
of cooperation between the Milano University and a
private foundation, Centro Cardiologico Monzino became
a monothematic hospital, home of many academic
activities. Since then, the Centre has been hosting
university competences in cardiovascular field, both
clinical and surgical.
University cardiologists and heart surgeons all
belong to the Department of Cardiovascular Sciences,
School of Medicine and Surgery, Milano University.
At present several University programs are ongoing:
Degree in Medicine and Surgery, Postgraduate School
in Cardiology, Postgraduate School in Heart Surgery,
First Level Degree in Techniques of Cardiocirculatory
Pathophysiology, Master in Cardiovascular Nursing,
Master in Cardiac and Ultrasound Techniques,
Advanced Training Course in Electrocardiography,
Arrhythmology and Advanced Life Support.
The staff of the Department of Cardiovascular Sciences
carries out research activity in strict collaboration with
the staff of Centro Cardiologico Monzino.
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University Centre of Pharmacological Research for the Study
and Prevention of Cardiovascular Diseases
It should be noted that, as part of the university
departmental reorganization (Law Gelmini 240/2010,
December 2010), during year 2012 the Department
of Cardiovascular Sciences will be joined to other
departments of the Milano University to set up the wider
Department of Clinical Sciences and Community Health.
As provided by the renewed Charter of the Milano
University, the Department of Cardiovascular Sciences
will constitute the Section of Cardiovascular Sciences,
which continues to be based at Centro Cardiologico
Monzino.
Elena TREMOLI, Full Professor
Director
Scientific Committee: Mariapia Abbracchio, Full Professor,
Francesco Alamanni, Full Professor, Damiano Baldassarre,
Assistant Professor, Marina Camera, Assistant Professor,
Donatella Caruso, Associate Professor, Susanna Colli,
Associate Professor, Alberto Corsini, Full Professor,
Giancarlo Folco, Full Professor, Alessandro Lualdi,
Assistant Professor, Rodolfo Paoletti, Full Professor,
Alessandro Parolari, Assistant Professor, Gianluca Polvani,
Full Professor, Giulio Pompilio, Assistant Professor,
Angelo Sala, Associate Professor, Cesare Sirtori, Full
Professor, Mauro Amato, Carlo Ciani, Gianni Giorgi, Pablo
Werba
The activity of the Centre is focused on the study and
prevention of cardiovascular diseases and, with the support
of external collaborations, aims at developing basic research
on cellular biology and biochemistry of atherosclerosis and
thrombosis in patients affected by cardiovascular diseases.
A further aim is to evaluate the efficacy of pharmacological
interventions in the cure and prevention of patients
undergoing surgical heart revascularization.
Activities
• Program for primary and secondary prevention of
cardiovascular diseases
• Research programs /projects in collaboration with
other Italian Atherosclerosis Centres
• Scientific communication /scientific activity
communication
• Masters and specializing courses
• Seminars, congresses, meetings with experts
• Grants for young researchers
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General Management
The mission of the Centro Cardiologico Monzino
Management is to favour patient care, creating the best
conditions to allow the operators to put their expertise
and knowledge to the patients’ service.
In a complex organization there are many ways to
play this role, but we believe that our managers must
necessarily own the following principles:
Stefano MICHELINI
General Manager
Directive Board
Chief Executive Officer
Mauro Melis
General Manager
Stefano Michelini
Chief Financial Officer
Mario Cesana
Management Director
Enrico Parma
Chief Human Resources Officer Daniele Piacentini
Chief Information Officer
Claudio Carlo Franzoni
Medical Director
Lorenzo Cammelli
Scientific Director
Elena Tremoli
Cardiology Program Director Cesare Fiorentini
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General Manager’s Assistant: Graziella Bassanello
Area Coordinators:
• Critical Cardiology
• Cardiovascular Surgery
• Interventional Cardiology
• Cardiovascular Imaging
• Electrophysiology
Piergiuseppe Agostoni
Francesco Alamanni
Antonio Bartorelli
Mauro Pepi
Claudio Tondo
Unit Directors:
• Cardiovascular Surgery 2
• Intensive Care
Gianluca Polvani
Erminio Sisillo
• a continuous stimulus towards innovation and
international dimension with the belief that a
research institute can only excel if it is based on
mature and advanced management tools
• a team culture to be searched for and promoted
every day in the relationships with all those who
operate inside the Institute
• the awareness that, in such no-profit organizations
as Centro Cardiologico Monzino, the Management
systems justify their existence inasmuch as they
strongly contribute to create the conditions to reach
and maintain excellence in the cardiovascular field
This is, in synthesis, the philosophy that guides the
behaviour of the areas that coordinate and support
Centro Cardiologico Monzino clinical and research
activities. Our job, our active contribution to the fight
against cardiovascular diseases, is to try and to put into
practice this “philosophy”.
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Outpatient Service and Marketing
Safety, Enviroment and Quality
Enrico Parma
Management Director
Fabio Tealdi
Chief Safety, Enviroment and Quality Officer
STAFF Management Director’s Assistant: Fannina Manzella
Outpatient Service Manager: Fabio Tealdi
Private Admittance: Raffaella Graticoli
Marketing, Events and Internet: Alfredo Pascali
Content Manager and Scientific Writer: Andreina Folli
The outpatient service manages the accessibility of
the clinical paths for outpatients ensuring appropriate
administrative, clinic and logistic admittance. Planning
and implementation of visits and diagnostics are
included as well as the invoicing process to public and
private companies.
• Booking management through Call Center, Front
lines, Internet
• Admittance front lines
• Clinical records services
• Service Planning
• Management of data flows and invoicing
The Marketing Team of the Centro Cardiologico Monzino,
in close collaboration with the IEO Marketing Team,
is involved in many organisational aspects (sales and
promotion, market development, internet, events) and
supports communication, fund raising, branding and
press so that group standards are efficiently applied to
the cardiovascular field.
The Team facilitates the organization of conferences,
seminars and events and in particular is involved in
the planning of the “World Heart Day”, usually held
on the last week of September, a successful traditional
event attracting hundreds of people interested in
cardiovascular prevention.
In addition, the Team takes care of the development and
updating of the web site (www.cardiologicomonzino.it) to
further expand the Centro Cardiologico Monzino virtual
community. An Alumni section has been created as well
as blogs, App and links to the main social networks.
The Marketing Team is in charge of the commercial
relationships with the private health insurance and
founds sector and the setting of the fee schedule.
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Quality Management is pursued in order to basically
ensure safety of the patients and quality of care with
reference to the JCI scheme, to the ISO9001 standard
and to the requirements of the national and regional
laws. The Management of Quality includes the handling
and follow up of complaints for both outpatients and
inpatients and customer satisfaction reviews.
Environmental requirements are satisfied ensuring
compliance to the relevant laws (Dlgs152/2006) and with
a management system that follows the BS14001 standard.
Safety of the workplace is ensured through a
management system that complies with the provisions of
the standard OHSAS18001 and with reference to the laws
(Dlgs81/08 and related requirements)
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Finance and Administration
Human Resources
Mario Cesana
Chief Financial Officer
Daniele Piacentini
Chief HR Officer
STAFF Financial and Accounting Office: Riccardo Quintini
Purchasing Office and Technical Service: Gianfranco Piantelli
Management Control and Budgeting: Enrico Parma (ad
interim)
Integration of the clinical, research and administrative
areas represents the basic principle underlying the
management model adopted by the Finance and
Administration Management.
The main tasks of the Financial Management are as
follows:
• Optimization of entire diagnostic, treatment and
administrative path of each patient
• Economic - financial management of the Institute
• Administrative, legal and tax requirements
• Managing the accounting, procurement and stock
Human Resources & Education: Federica Vaga
The central role of the individual and top-quality
assistance are the two principles that have always
inspired the organizational and management work done
by the HR Office in each of the above areas. These
principles have enabled the Institute to achieve its main
objective: improving the quality of life of each patient,
who must be considered not simply as a person who
needs treatment, but, above all, as a multifaceted human
being.
Main tasks of the HR Office are as follow:
• recruitment and selection
• education and training
• performance evaluations
• salary packages and incentives
• personnel management, planning and costs
• union relations
• work organization
The HR Office is in charge of the following services:
• Performance management & organization
• Pay Roll Office
• Recruitment
• Education and training
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Information & Communication Technology
Ethics Committee
Claudio Carlo Franzoni
Chief Information Officer
STAFF Business Applications: Paolo Zilioli
Research Area: Alessandro Dellavedova
Clinical Area: Luigi Grilli
Governance: Luigi Cassi
Innovation and Technologies: Alberto Mancin
The ICT Department handles the whole computing
infrastructure: maintenance and support of Office
Automation, management of network infrastructure and
telephony, design and development of new applications.
The main tasks of the ICT Department are as fellows:
• management, maintenance and updating of third
party applications, providing user support
• design and development of applications to enable
clinical and administrative process execution
• design, development and management of the
network infrastructure, servers and telephony
• design, development and management of the
software and the basic application services
• management of the structure, organization and
content of data
• management of the safety and security of data
and resources
• technical and usage support on Information
Services
• providing computer hardware and software technical support
• defining products and technology standards and
research innovative solutions
The ICT Department is in charge of the following
services:
• Development and Programming
• Networks and Systems
• Information Systems and Research
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Giuseppe FOLLI Chairman
ferruccio BERTI
Vice Chairman
Atanasio NONIS
Secretary
Giuseppe Arosio
Maria Santina Bonardi
Lorenzo Cammelli
Giorgio Dazzo
Susanna Dellepiane
Antonio Liverani
Maurizio Pellegrini
Elena Tremoli
Emanuela Valaguzza
SECRETARIAT Daniela TAMAGNI
OBSERVERS Mauro Melis
Stefano MICHELINI
A central role in the clinical research of the Centro
Cardiologico Monzino is played by its Ethics Committee.
The scientific aspects, the expected improvements in
the general health and well being and the overall riskbenefit ratio of the participants are the milestones of
the evaluation work carried out by the Ethics Committee
which covers the whole range of clinical research
projects, involving the evaluation of medicinal products,
devices, other interventional techniques as well as
observational studies.
The Committee is accredited at the Ministry of Health,
the National Drug Agency as well as the Regione
Lombardia.
In 2011 twenty new research projects were evaluated
during four plenary sessions of the Committee. More
of 50 % of the applications involved phase III trials on
medicinal products, almost 40 % investigation plans
on innovative medical devices and the remaining 10
% observational studies. A favourable opinion was
expressed in all studies, requiring changes only on the
patient information.
Twenty-four applications of substantial amendments on
ongoing studies were evaluated and a favourable opinion
was expressed in all cases.
Finally the Committee expressed a favourable opinion
for the treatment with advanced, though still under
investigations, techniques of two patients affected by
otherwise untreatable coronary disease.
CCM — Scientific Report 2011 — Ongoing research 2012
29
Library
Education and Training
Centro Cardiologico Monzino holds a number of on-line
subscriptions to specialized journals. so to guarantee
up-to-date information for medical staff and researchers.
Article research is made on-line using a link connecting
to the subscription list.
Morever, Centro Cardiologico Monzino is member
of BIBLIOSAN, the Italian library system under the
supervision of the Ministry of Health, involving all
IRCCS, the Istituto Superiore di Sanità, the Experimental
Zooprophylactic Institutes and other healthcare
institutions. It allows our staff to have access to about
5500 electronic and full-text periodicals, to RefWorks,
a bibliographic management software which uses
bibliography citations, to NILDE, the Network for
Inter-Library Document Exchange, (allowing Centro
Cardiologico Monzino to receive and send articles
all over Italy), to ISI Web of Science and the Journal
Citation Reports both providing data on citations and
Impact Factors, to BMJ Best Practice, an innovative
system for clinical decision support. The library is also
member of ACNP, the National Collective Catalogue of
Periodicals, an on-line catalog of printed journals which
links over 2300 libraries in Italy for a total of 110.000
journals
All full text publications of our medical and scientific
staff are available on Centro Cardiologico Monzino web
site which is updated day by day to give a complete
view of the research projects and to inform the
personnel of the ongoing scientific production.
The Scientific Secretariat is an important help desk for
researchers promoting the exchange of data among
different professional areas and specialisations and,
above all, between researchers of our Institute and the
scientific world.
Medical education is a lifelong, continuous and ongoing
process throughout the professional career of healthcare
providers.
The university staff of the Department of Cardiovascular
Sciences and the Centre of Pharmacological Research
for the Study and Prevention of Cardiovascular Diseases
together with the medical staff of Centro Cardiologico
Monzino is engaged in a continuing education program
aiming at maintaining health care at the highest level.
They offer a full range of speciality and subspeciality
education in the cardiovascular field.
A variety of educational strategies has been carried out:
formal courses, skills workshops, seminars, symposia
and conferences, all addressed both to internal and
external health operators.
During 2011, practical and/or theoretical courses for
medical staff, nurses and technicians were held at Centro
Cardiologico Monzino on the following topics:
• Beta blockers and heart failure
• Cardiac computed tomography
• Cardiopulmonary exercise testing
• Carotid artery stenting
• Echocardiography
• Endomyocardial biopsy for electrophysiologists
• Interventional cardiology
• Intravascular imaging
• Mapping and ablation of ventricular arrhythmias
• Mitral regurgitation
• Pericardial puncture and epicardial mapping
• Transseptal puncture for electrophysiologists
• Vascular Ecocolordoppler
Besides, several seminars and scientific meetings involving
both physicians and researchers were organized at Centro
Cardiologico Monzino (see p. 248 for a full list).
To develop the professional skills and performances
of nurses, technicians (radiology and laboratory
technicians, perfusionists) and physiotherapists working
30
CCM — Scientific Report 2011 — Ongoing research 2012
in the Institute, an Internal Education Program was
created by the Health Professional Working Group (see
p. 45). Its activity is aimed at improving health staff
skills in handling emergency situations and developing
the use of new technologies applied to health.
It includes courses on the following topics:
• Basic Life Support and Defibrillation (BLSD)
• Re-training BLSD
• Advanced management of cardiac emergency
• ECGs for nurses (Arrhithmias, Acute Coronary
Syndrome, Cardiac Pacing and Electrophysiology)
• Risk prevention by patient handling
• Peripheral venous catheterization
• Water balance in cardiac patients
• Evidence Based Practice: the future of health
professionals
• Prevention, assessment and treatment of skin lesions
• Noninvasive ventilation
Some of these courses, particularly BLSD, are required by
CCM — Scientific Report 2011 — Ongoing research 2012
31
Medical Director’s Report
Lorenzo CAMMELLI, MD
Medical Director
JCI (Joint Commission International) and IRC (International
Resuscitation Council) to ensure that all personnel of the
Institute is trained and updated every two years on the
protocols to be used in case of cardiac arrest.
In 2011 the following courses have provided a total of
2388 credit points awarded by the Ministry of Health
and involved 354 people.
World Heart Day
The World Heart Day was created to inform people that
heart disease and stroke are the world’s major causes
of death. Unhealthy diet, physical inactivity and smoking
are the leading causes of heart disease and stroke. To
reduce the incidence of cardiovascular disease risk,
prevention is fundamental.
Since 2000 Centro Cardiologico Monzino has been
sustaining the World Heart Federation, organizing, every
year, the Monzino World Heart Day. This successful
event, usually held on the last weekend of September,
deals with a specific prevention topic and calls hundreds
of people coming from Milano and surroundings.
In 2011 the World Heart Day prevention campaign
focused on workplace heart wellness. Seminars and
lectures were held on cardiovascular risks on workplace,
environmental stress and heart health, physical activity
at work and outdoor healthy eating.
A “healthy lunch” was offered to all participants.
32
CCM — Scientific Report 2011 — Ongoing research 2012
At the end of 2011, the Monzino health care clinical
staff included 111 physicians, 43 medical residents, 244
registered nurses, 14 lab technicians, 6 rehabilitation
technicians, 14 radiology technicians and 8 cardiology
technicians (pump specialists and sonographers).
In 2011 hospital admissions were 8085 (with a 2.75%
decrease compared to 2010), 6841 in cardiology and
1.244 in surgery. This clinical inpatient activity produced
50.287 hospital days (34.961 in cardiology and 15.326
in surgery) with a decrease (-1.07%) compared to the
previous year (50.831 in 2010). The average length of
stay was 6,6 days, with an increase of 8,9% (6,06 days
in 2010). The ratio between surgical admissions and total
admissions (“surgical index”) was of 15.39% (16.7% in
2010). Case mix complexity, represented by the Average
Relative Weight, was 1,65. The Emergency Unit treated
9.745 patients and the high rate of severe diseases led
to 2054 direct admission to the clinical wards (including
145 primary PCI in the catheterization lab for acute
myocardial infarction).
During 2011 Centro Cardiologico Monzino further
developed Transcatheter Aortic Valve Replacement
(TAVR) with 123 interventions (total amount from late
2007 is 330). Furthermore, we developed percutaneous
mitral valve repair, with 7 patients treated (MitraClip
procedure), and artificial heart implant (2 patient had
Jarvick machines).
The outpatient department showed a further growth with
45.582 consultations (3.6% increase compared to 2010).
CCM — Scientific Report 2011 — Ongoing research 2012
33
Acute care: mean lenght of stay
Clinical Activity 2011
20
8.085
Emergency Unit (episodes of care)
9.745
Catheterizations lab patients
3.999
PCI
2.189
Electrophysiology studies and/or ablations
1.060
Cardiac surgery procedures
798
Vascular surgery procedures
616
15
Days
Inpatients
CYP3A4 Inhibitors/Substrates
Cyclosporine, tacrolimus
Macrolides (azithromycin, clarithromycin,
erythromycin)
Azole antifungals (itraconazole, ketoconazole)
Calcium antagonists (mibefradil, diltiazem,
verapamil)
45.582
Outpatients visits
10
Others
Digoxin
Fibrates (gemfibrozil)
5
Niacin
0
Nefazodone
Protease inhibitors (amprenavir, indinavir,
nelfinavir, nitronavir, saquinavir)
Sildenafil
Warfarin
1998 1999 2000 2001
2002 2003 2004 2005
2006 2007 2008
2009 2010 2011
CCM
Acute Care: inpatients
Cardiology
Surgery
Acute care occupancy
PERIPROCEDURAL AND IN-HOSPITAL RESULTS
9000
8153
7671
7845
SUCCESS IN IMPLANTED PATIENTS:
6969 7070
6765 6890
7000
6148
PROCEDURAL
6000
5556
5000
7257
SUCCESS:
8044
8314
8085
162/163 (99,4%)
162/164 (98,8%)
INTRAPROCEDURAL MORTALITY:
2/164 (1,2%)
EMERGENCY SURGICAL CONVERSION:
1/164 (0,6%)
Occupancy %
8000
8130
100
75
4000
3000
50
2000
Inpatients
Boca
0
34
1998 1999 2000 2001
2002 2003 2004
2005 2006 2007
2008 2009 2010
2011
1000
74
64
1998 1999 2000
2001 2002 2003
2004 2005 2006
2007 2008 2009
2010 2011
CCM — Scientific Report 2011 — Ongoing research 2012
Total
Cardiology
Surgery
CCM — Scientific Report 2011 — Ongoing research 2012
35
Casualty department (emergency)
Surgical procedures (vascular)
400
10000
9331
8177
7987
8611
8701
9424
9802
9745
8836
300
7520 7510
7195
6577
Patients
5956
200
5000
100
0
0
1998 1999 2000 2001
2002 2003 2004
2005 2006 2007
2008 2009 2010
2001
2011
Surgical procedures (all)
2002
2003
2004
Vascular Surgery
2005
2006
2007
Enduvascolar total
2008
2009
2010
BOCA Surgery
2011
DX angio
Interventional Cardiology
1200
2205
939
939 1166 1305
1576 1780
1927
2076
1906
1921
1991
2047
2189
813
656
600
1928
2180
2402
2337
2209
2012 2000
1820 1790
0
2001
2002
Cardiac Surgery
36
2003
2004
2005
2006
Vascular surgery
CCM — Scientific Report 2011 — Ongoing research 2012
2007
2008
2009
Endovascular
2010
2011
Other surgery
1788
1998 1999
2000 2001
2002 2003
2004 2005
2006 2007
2008 2009
Cath lab total PCI
Dx procedures
2022
2010
1988
2011
Altro (pfo, etc)
CCM — Scientific Report 2011 — Ongoing research 2012
37
Primary PCI procedures for AMI
Pace-maker and ICD
800
150
150
141
136
100
100
92
130
141
139
132
700
133
124
600
500
100
95
400
300
50
200
43
100
0
1998 1999 2000 2001
2002 2003 2004
2005 2006 2007 2008
2009 2010
0
1998 1999 2000 2001
2002 2003 2004
2005 2006 2007
2008 2009 2010
2011
2011
Acute AMI Procedures
PM
Electrophisiology
ICD
Total
Out-Patient Department
2319
Encounters
2236
2129
1946
50000
2120
1915
43893
1790
39857
40000
1381 1367 1436
34016
1103 1103
30000
800
846
628
342
427 484
290
676
619
338 458
503
465
494
878 902
942
910
899
963
925
38
CCM — Scientific Report 2011 — Ongoing research 2012
28600 29621
1060
41004 41462
36120 35978
30623
25859
20000 21905
944
1036
1016
1166
1195
1998 1999
2000 2001
2002 2003
2004 2005
2006 2007
2008
Total
1093
45582
Dx ablation
1226
1176
16615
10000
2009
2010
2011
Other elph procedures
0
1998 1999 2000
2001 2002 2003
2004 2005 2006
2007 2008 2009
2010
2011
CCM — Scientific Report 2011 — Ongoing research 2012
39
Echocardiography
Exercise Stress Test
10.000
25.000
20.000
7.500
15.000
5.000
10.000
2.500
5.000
0
0
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2198
4090
4235
5315
5177
7006
7779
8765
8964
9266
10548
11857
12414
12756
1784
2957
3132
3251
3276
3604
4591
5128
5316
5694
5390
6300
7012
7519
6394
7011
7481
7724
8298
8020
8487
8628
8816
10565
10616
10017
10310
10875
1089
802
627
586
518
323
321
351
402
474
563
460
374
358
2006
2007
2008
Outpatient
Inpatient
Outpatient
Holter
Ecodoppler (vascular)
9.000
10.000
6.750
7.500
4.500
5.000
2.250
2.500
0
0
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
1209
2856
3285
3703
3316
4108
4524
4829
4961
6355
5337
1531
1432
1304
1157
1052
981
1027
1190
1215
1248
1097
Outpatient
40
Inpatient
CCM — Scientific Report 2011 — Ongoing research 2012
Inpatient
1998
1999
2000
2001
2002
6810
383
2019
2474
3156
1158
796
951
1216
1101
2010
2011
5841
6695
1108
1292
2009
2003
2004
2005
3036
4136
4030
4516
5113
4914
1542
1306
1611
1215
1288
1346
Outpatient
2009
2010
2011
6243
5193
6218
7255
1644
2038
2215
2558
Inpatient
CCM — Scientific Report 2011 — Ongoing research 2012
41
Medical Directorate's Staff
MEDICAL OFFICE
Medical Director: Lorenzo Cammelli, MD
Office Manager: Rosalba Bergami
Assistant Medical Director: Ludovica Tagliabue, MD,
Marta Saronio, MD
Consultants: Luigi Molendini, MD, Massimo Monturano
CASE-MIX & CLINICAL PATHWAYS MANAGEMENT OFFICE
Administrative Staff: Francesca Ricci, Carmen Bagnardi,
Manuela Pettignano, Paola Ingegneri
CLINICAL PATHWAYS OFFICE
Registered Nurse: Franca La Rosa, Caterina Loschiavo
Consultant: Filippo Cazzulani, MD
Administrative staff: Manuela Pettignano
MEDICAL RECORDS OFFICE
Office Manager: Elisabetta Fiore
DRG & Hospital Statistics: Emanuele Martella
Administrative Staff: Anna Ferragina, Luisa Passannante,
Cosimo Bruno
CLINICAL AUDIT & PATIENT EDUCATION OFFICE
Office manager: Maria Cristina Ghidini
42
CCM — Scientific Report 2011 — Ongoing research 2012
NURSING OFFICE
Head Officer: Massimo Moro
Vice-Head Officer: Oriana Ferrari
QUALITY & OCCUPATIONAL SAFETY
Quality Manager: Fabio Tealdi
Consultants: Roberta Campiglio, MD, Giovanni Morselli, MD
OUTPATIENT DEPARTMENT
Coordinator: Francesco Pesoli, MD
Head Nurse: Laura Carparelli
STERILIZATION SERVICE
Head Nurse: Ornella Garganese
Registered Nurses: Daniela Pozzi
Quality Manager: Monica Perazzani
PATIENT SAFETY & CLINICAL RISK MANAGEMENT
Risk Manager: Giansaverio Friolo
PHARMACY
Pharmacist: Susanna Dellepiane, Laura Ricci
OPERATIVE BLOCK
Coordinator: Lorenzo Cammelli, MD
BOP Manager: Michela Ravelli
CCM — Scientific Report 2011 — Ongoing research 2012
43
Clinical Governance
at Centro Cardiologico Monzino
Centro Cardiologico Monzino is strongly committed to
quality and safety in the treatment of cardiovascular
diseases. Continuous monitoring and analysis of the
provided health care are regularly performed. The
implementation of innovative treatments, improvement
of consolidated strategies and identification of relative
weakness are addressed by multidisciplinary teams that
operate with the methodology of “Strategic Process
Improvement”. Beginning in the second quarter of 2008,
Centro Cardiologico Monzino experienced a redesign
of hospital processes, obtaining improvements in the
essential clinical pathways. Innovative processes and
original methods are actively discussed and implemented
to improve clinical performance. The search for a more
efficacious model of governance is central in the Institute’s
mission, outlined by the declaration: “Centro Cardiologico
Monzino aims at reaching excellence in the field of
prevention, diagnosis and treatment of cardiovascular
diseases, through the development of clinical-scientific
research and organizational and managerial innovation, in
a context characterized by a firm attention to the quality
of the services provided to the patients”.
In the last years more than 20 projects of management
were developed in partnership with the Centro
di Ricerche e Studi in Management Sanitario (Ce.
Ri.S.Ma.S.). During 2011 significant improvements were
developed in the areas of Clinical and Outpatient
pathways and Clinical Audit and HTA and pathways,
under the supervision of dr. Lorenzo Cammelli, in the
areas called Cath Lab Management, under the direction
of dr. Giovanni Teruzzi, and in the area of Hospital
wide ECG Management System and Coronary Care
Improvement, managed by dr. Giancarlo Marenzi.
Quality and safety care are specifically carried out
performing surveillance and control of hospital
infections, pharmacological errors’ prevention,
monitoring and evaluation of outcome through clinical
audits, promotion of generic and specific treatment
44
CCM — Scientific Report 2011 — Ongoing research 2012
appropriateness, equity in access to healthcare, respect
of regulations and deontology, professional growth,
humanization and environmental comfort.
In the perspective of cross-disciplinary and cooperative
work, many committees operate permanently at Centro
Cardiologico Monzino. They are:
1. Director’s Committee
2.Hospital Infections Committee
3. Patient Safety and Clinical Risk Management Committee
4.Drugs and Medical Devices Committee
5.Health Education Committee
6.Clinical Management Resources Committee
Among the methodologies used to reach and maintain
high quality standards, the adherence to principles
of Clinical Governance, that provide for a strong
involvement and responsibility of health operators in the
clinical design processes, are particularly important.
Clinical Governance at Centro Cardiologico Monzino is
developed in all the components described in scientific
literature: performance evaluation, quality of care,
patient safety, clinical audit, patient education, evidencebased medicine and customer satisfaction.
Hospital Pharmacy
The Hospital Pharmacy is an essential service of
the Medical Office staff. It is involved in managing
supply and stocks of medicines (drugs, vaccines, and
experimental drugs), dressing materials, medical devices,
antiseptics and disinfectants.
Hospital Infection Committee
The Hospital Infection Committee works in order to
continuously improve the watch and control of hospital
infections. In a perspective of collaborative work, key
elements are the epidemiological nurse, devoted to ward
and services surveillance, and the clinical microbiologist.
Patient Satisfaction
During the last years, Centro Cardiologico Monzino
invested large amounts of time and money to facilitate
patient wellness and satisfaction. A new customer
satisfaction’s system was developed to join the effective
quality of health assistance and how patients perceive
it. The patient satisfaction surveys (demanded by
Lombardia Region to all Health Institutions, for outpatient
department services and inpatient area) are performed
every six months. Data are collected and analyzed
with an appositely developed software and results are
periodically sent to the local regulation centre (ASL)
and both to Centro Cardiologico Monzino General
Management and Quality Office.
Clinical Audit
In 1998 Centro Cardiologico Monzino started an
experimental program of review focused on the most
critical cardiovascular procedures, in order to obtain an
improvement of clinical healthcare through its critical
appraise. The Cardiovascular Surgery was the first
scrutinized department. Later, Interventional Cardiology
procedures were analyzed and in 2008 a clinical audit
was performed in the Arrhythmology area. In 2009 the
strategic project “Clinical Audit” was implemented, further
increasing the analysis of Centro Cardiologico Monzino’s
activities, expanding the concept of self evaluation and
periodical discussion of clinical performance.
Patient Education
Since 2009, a comprehensive program of patient education
has been developed by an interdisciplinary committee.
Principal output of the program is the publication
of booklets and innovative information media. In
cooperation with all the health operators, the booklets are
constantly increasing in number and updated, to improve
patients’ information about important topics related to
cardiovascular diseases and quality of life (i.e. treatments,
food intake, rehabilitation, physical activities).
Health Professional Working Group
Patient Safety and Clinical Risk Management
Patient safety program has been performed since
2005, with the creation of the Patient Safety and Risk
Management Committee and the nomination of a Risk
Manager figure. In these years, 11 Root Cause Analyses
of problematic cases have been performed. Furthermore,
regular meetings were activated to analyze the most
serious events happened. In June 2007, a collaboration
with other structures considered leaders in the field
of safety promotion was started. In the same period,
a survey to evaluate the operators’ opinions about
patient safety was performed, in order to monitor the
knowledge about the topic in our Institute.
Modus operandi
The Health Professional Working Group (nurses, health
technicians and physiotherapists) meets monthly to
discuss professional development of the personnel in
order to develop advanced skills in the cardiovascular
area and to improve the patient quality of life by
reducing the impact of cardiovascular diseases and
ensuring a higher and higher assistance quality. The
Group also plans events, courses and seminars and
works to foster the development of cardiology and
cardiovascular nursing through scientific exchanges and
personal contacts establishing standard of training for
those who work in the cardiovascular field.
Head Officer: Massimo Moro
Vice-Head Officer: Oriana Ferrari
Members: Pierangela Andreoletti, Gabriele Bucca, Felicia
Bucci, Laura Carparelli, Mario Diomete, Ivana Favini,
Luisa Fumagalli, Ornella Garganese, Maria Cristina
Ghidini, Annarita Leardi, Emanuela Pagani, Rocca Punzi,
Marialena Ranghetti, Fabiana Rossi, Paola Rumi, Laura
Schiaroli, Giuseppe Squilla.
CCM — Scientific Report 2011 — Ongoing research 2012
45
Developing Internal Education
Management and continuous assistance of people with
cardiovascular pathologies, prevention and rehabilitation
services both inside and outside the hospital context,
are specific skills of nurses, physiotherapists and
technicians. Nurses, in particular, always need to
follow up to date programs allowing them to effectively
assist people in the best way, guarantee continuity of
cares and plan and implement education services for
teaching patients and their families how to self manage
the illness. The Health Professional Working Group
participates in the education and training of the health
46
CCM — Scientific Report 2011 — Ongoing research 2012
operators. During 2011, 10 different courses were held
and administered to 354 internal operators for a total
of 2388 credit points awarded by the Ministry of Health
(see p. 31 for a detailed list).
Besides the specific courses attended by people
working in the Institute, the Group is involved
in projects and events in cooperation with other
institutions. In November 2011 a master in “Nursing
in the Cardiovascular Area” sponsored by the Milano
University, has been activated for a total of 16
participants coming from Northern Italy.
CCM — Scientific Report 2011 — Ongoing research 2012
47
Cardiovascular Tissue Bank
Gianluca Polvani, MD
Director
Co-Director and Technical/Scientific Referee:
Luca Dainese, MD
STAFF Technical/Organizative Referee: Anna Guarino
Technician: Barbara Micheli, Francesca Prandi
48
CCM — Scientific Report 2011 — Ongoing research 2012
The activity of the Cardiovascular Tissue Bank consists
in the procurement of human valves and vessels from
multiorgan or cadaveric donors and in their proceeding,
cryopreservation, validation for distribution and clinical
employment.
Tissues are usually employed in aortic ring abscess,
reoperation of valvular substitution (valve degeneration),
aortic ectasia, small aortic anulus, sinus Valsalve
aneurysm, native vessels infections, prosthetic infections
etc…
The Cardiovascular Tissue Bank at Centro Cardiologico
Monzino, together with the Treviso bank, is the oldest
cardiovascular bank in Italy.
Founded in 1992 with the name of Banca Italiana
Omoinnesti (BIO), in 2005 it became the only
cardiovascular bank recognized and authorized by the
Regione Lombardia Health System.
The bank activity, strictly connected with the CIR/CRR
activity (Centro Interregionale di Riferimento/Centro
Regionale di riferimento), follows both the European
(Direttiva 2004/23/CE) and the Italian guidelines
(19/06/2007).
The bank team is available 24h-365d. Since 2005 the
cardiovascular tissue bank has been certified both ISO
9001-2008 and CNT (Centro Nazionale Trapianti) for
activity of “reception, proceeding, preservation and
distribution of cardiovascular tissues of human origin”.
It has also been registered in the European Registry of
cardiovascular banks.
In 2011 the bank received cardiovascular tissues from
35 multiorgan donors (Heart Beating Donor, HBD) and
6 from non heart beating (NHB) and processed 27
aortic valves, 27pulmonary valves, 17 thoracic aortas,
4 abdominal aortas with iliac arteries, 58 iliac femoral
arteries, 15 safenous veins, and 1 cava vein. After
preparation and anatomical, serological, microbiological
evaluation, 82% of all tissues was validated and ready
for distribution.
During 2011, 124 tissues were distributed all over Italy.
The Cardiovascular Tissue Bank is involved also in
basic as well as clinical research studies. Projects
concerning engineered heart valves and tissues are
ongoing. Moreover, a study aimed at improving
functional/mechanical/biochemical heart valve and vessel
properties is being developed. Other studies about
new decontamination solutions and conditions (fresh
preservation) are being developed for tissue procured
from organ and living donors.
CCM — Scientific Report 2011 — Ongoing research 2012
49
Cardiovascular Telemedicine
Gianluca Polvani, MD
Director
Staff: Alfreda Calligaris, MD, Amedeo Mereni, MD
Head Nurse: Felicia Bucci
Nurses: Francesca Bolzoni, Marina Re
Secretary: Magda Ferrari
The Unit is dedicated to the continuous surveillance of
patients with cardiovascular diseases.
This activity is carried out by a dedicated call-centre
with 24h medical-nursing reference.
The Unit activities are listed below:
• Home-rehabilitation program after cardiac surgery
procedures which ensures accurate controls especially
in the first post-operative month and aims at the full
reintegration of the patient in the normal daily life.
This home-rehabilitation program is internationally
recognized as one of the most innovative and safe
program for patients with cardiac disease after
cardiac surgery. Cardiologists, cardiac surgeons,
physiotherapists and nurses are involved. In the call
centre, medical staff with different expertise take
advantage of the use of innovative devices for care
and control of patients at home, providing the same
safety level as the hospital
• Home-rehabilitation program for heart failure patients
which allows a tight control of the clinical status of
the patients and provides a continuous interchange
between the physician and the family doctor.
This innovative management reduces the inadequate
accesses to the Emergency Unit and allows a
continuous update of the therapy.
• Continuous electrocardiographic follow-up for
arrhythmic patients with atrial and ventricular trouble
(e.g. atrial fibrillation)
• Home monitoring for patients waiting for cardiac
surgery procedures;
• Remote consulting with other institutions (for instance
Europe Assistance Service) and Italian hospitals all
over the world (IPOCM project).
50
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CCM — Scientific Report 2011 — Ongoing research 2012
51
Clinical Units
52
CCM — Scientific Report 2011 — Ongoing research 2012
Critical Cardiology Area Piergiuseppe AGOSTONI, MDArea Coordinator
Emergency Unit 2011
Emergency Unit
Alessandro SALVIONI, MD
Director
STAFF Deputy Directors: Fabrizio Celeste, MD, Elisabetta Doria, MD
Assistants: Roberta Chiodelli, MD, Gabriella Famoso, MD,
Alessandra Magini, MD, Marco Matturri, MD,
Chiara Meloni, MD, Francesca Susini, MD.
Fellows: Denise Brusoni, MD
Head Nurse: Emanuela Pagani
Nurses: Claudio Boriani, Andrea Bosis, Lorenzo Brunaccini,
Ana Maria Codrenau, Margherita Corsini, Irina Alexandrina
Isfan, Giuseppe Nesci, Marco Perella, Detelina Ivana
Rankova, Raoul Sullcapuma Rios, Mauro Turba, Franco
Vailati, Giovanni Zamboni
Activities 2011. Centro Cardiologico Monzino
Emergency Unit is probably the only example of emergency
unit exclusively devoted to cardiac care.
The Unit is equipped with nine beds provided with
continuous ECG monitoring and main non invasive vital
indicators (non invasive blood-pressure; oxygen saturation).
Four positions are dedicated to short term observation and
four to long term observation; the ninth bed is dedicated to
cardio-pulmonary intensive therapy and is equipped with a
DC defibrillator and a mechanical ventilator for invasive and
non-invasive ventilation. The Unit is also equipped with last
generation portable ultrasound devices.
In the last few years the four long term observation
positions have been dedicated mainly to the management of
chest pain so to constitute a “Chest Pain Unit”.
Patients presenting with chest discomfort not fulfilling
criteria of acute coronary syndrome, are submitted to 12-18
hour observation period during which serial ECGs and
measurements of markers of cardiac damage in blood are
performed. In case of non diagnostic ECG and/or biomarkers,
exercise testing or echo-stress (exercise stress if invaluable
ECG or pharmacologic stress if unable to exercise) is carried
out in order to avoid inappropriate admissions or discharges.
Electric cardioversion of atrial tachyarrhytmias (more than
200 during the last year) are usually performed both for
54
CCM — Scientific Report 2011 — Ongoing research 2012
Patients
% of total
Patients checked
Admissions
Discharges
Transfers to other hospitals
9745
1924
7696
125
19,7%
79,0%
1,3%
Red code
Yellow code
Green code
White code
55
814
8462
414
0,6%
8,3%
86,9%
4,2%
patients admitted to the Emergency Unit and for patients
hospitalized in the Acute Cardiac Care Unit. In addition, the
Emergency Unit supports the telecardiology service to get
ECGs of patients with heart failure, coronary artery diseases
or cardiac arrhythmias.
Every year about 10.000 patients suffering from
cardiovascular symptoms are evaluated at this Emergency
Unit. During the years a continuous increase in the access in
the EU occurred and remarkably doubled in the last decade.
The accuracy of these admissions is very high, with 95%
of the patients presenting with cardiovascular symptoms.
The most common diseases are acute coronary syndromes,
heart failure, brady and tachyarrhytmias. During 2011, among
all patients presenting to the Emergency Unit, 1924 patients
(19,7%) were hospitalized for acute cardiac disease. In the
same year, patients with “white code” (inappropriate access
to the Emergency Unit for very low level of urgency) were
just 4,2%. Only 125 patients (1,3%) were moved to a different
hospital because of non cardiovascular disease requiring
non-cardiological specialistic evaluation. During 2011 mean
waiting time (from nursing triage to medical examination) for
the patients classified “green- code” (patients with delayed
urgency) was 49 minutes.
In 2011, 1924 patients were hospitalized from the Emergency
Unit; they accounted for about 24% of all admissions.
Main fields of research:
In 2011 the staff of the Emergency Unit and of the Acute
Cardiac Care Unit evaluated the specificity and sensitivity
of a new biochemical marker (copeptine) for the faster
rule out of patients admitted in the EU with chest pain.
The data were collected, together with other 4 Emergency
Departments in Milano, and were included in the data base
Rossini Trial.
The Emergency Unit and the Acute Cardiac Care units were
also involved in several international multicenter clinical
trials on acute coronary artery disease (phase III and
phase IV studies), on lipid metabolism and on diabetes in
the setting of cardiovascular disease and in observational
epidemiological study on atrial fibrillation:
TRILOGY ACS study comparing prasugrel vs clopidogrel in
patients affected by acute coronary syndrome with unstable
angina/non-ST elevation myocardial infarction (UA/NSTEMI)
treated with medical therapy.
TRACER study evaluating the effectiveness and safety of a
new inhibitor of platelet thrombin receptor (PARs) in reducing
ischemic events in patients with acute coronary syndrome.
CEPT Inhibitor (Roche) RO4607381: a phase III double blind
randomized controlled study evaluating the effects of
RO4607381 on cardiovascular risk in stable CHD patients
with a documented acute coronary syndrome
ALECARDIO study evaluating the efficacy of aleglitazar
(receptor agonist of PPAR-alfa and PPAR-gamma) in reducing
cardiovascular events in patients with type 2 diabetes
mellitus and recent acute coronary syndrome.
SAVOR: a multicenter phase IV trial to evaluate the effects
of saxagliptin (drug of the family of incretin) on the
incidence of cardiovascular death, myocardial infarction or
ischemic stroke in patients with type 2 diabetes.
TECOS: a randomized placebo controlled trial to evaluate
cardiovascular outcomes after treatment with sitagliptin
in patients with type 2 diabetes mellitus and inadequate
glycemic control on mono- or dual combination oral
antihyperglicemic therapy.
EXSCEL: a randomized, placebo controlled clinical trial
to evaluate cardiovascular outcomes after treatment with
exenatide once weekly in patients with type 2 diabetes
mellitus.
RE-LY AF REGISTRY: risk factors, treatments and outcomes
of emergency department patients with atrial fibrillation in
multiple regions of the world.
CCM — Scientific Report 2011 — Ongoing research 2012
55
Critical Cardiology Area Acute Cardiac Care Unit
Acute Cardiac Care Unit 2011
Admissions
Transfers to other units
Transfers from other units
Discharges
Mortality rate
Days of stay
Alessandro SALVIONI, MD
Director
STAFF Deputy Directors: Fabrizio Celeste, MD, Elisabetta Doria, MD
Assistants: Roberta Chiodelli, MD, Gabriella Famoso, MD,
Alessandra Magini, MD, Marco Matturri, MD,
Chiara Meloni, MD, Francesca Susini, MD
Fellows: Denise Brusoni, MD
Residents: Laura Salvini, MD, Carlo Vignati, MD
Head Nurse: Mario Diomete
Nurses: Katerine Barreda, Salvatore Baudanza,
Filomena Briscese, Barbara Carioni, Bruna Contini,
Sara Di Berardino, Jack Djoufack, Pina Granata, Ira Griffini,
Adela Marcu, Veronica Meza, Mihai Mocanu, Rosa Pachas,
Laura Pea, Anna Pekul, Marisa Placido, Katia Resta,
Flavio Rossi
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CCM — Scientific Report 2011 — Ongoing research 2012
979
139
40
878
1.43%
8401
Activities 2011. The activities of the Acute
Cardiac Care Unit (ACCU), along with the Intensive
Cardiac Care Unit, are dedicated to the diagnosis and
treatment of patients admitted to the Emergency Unit for
acute heart diseases.
Cardiologists of the ACCU are also involved in the
activity of the Emergency Unit establishing a strict
cooperation with the other Units in order to reach the
highest level of assistance to patients. Every year about
1000 patients are admitted to the ACCU. During 2011, this
department received 51% of the patients admitted to the
Emergency Unit (12% of all hospitalizations at Centro
Cardiologico Monzino).
In most cases (89%) the whole hospitalization until
discharge was performed in the ACCU, whereas the
remaining patients were moved to other Units for clinical
tests and treatments.
Acute coronary syndromes, congestive heart failure,
severe arrhythmias (brady- and tachyarrythmias)
and valvular heart diseases were the main conditions
treated in the ACCU. Once admitted to the ACCU,
many patients are subsequently moved to other Units
according to their pathology (coronary artery bypass graft, valvular surgery including percutaneous
techniques, vascular surgery). More than 100 procedures
of electric cardioversion of acute and chronic atrial
tachyarrhythmias were also performed during 2011.
A high proportion of elderly patients admitted to the
ACCU for acute cardiovascular diseases suffers also
from severe medical diseases such as diabetes, chronic
obstructive pulmonary disease and chronic renal failure.
During 2011 ACCU was involved in 7 international phase
III and IV trials aimed at assessing the efficacy and
safety of new cardiovascular drugs in patients with
coronary acute syndromes (2 trials), in patients with
dislipydemia or type 2 diabetes and coronary disease
(5 trials) and in 1 international survey study of patients
affected by permanent atrial fibrillation.
Publications
Mega JL, Braunwald E, Mohanavelu S, Burton P, Poulter
R, Misselwitz F, Hricak V, Barnathan ES, Bordes P,
Witkowski A, Markov V, Oppenheimer L, Gibson CM;
ATLAS ACS-TIMI 46 study group. Rivaroxaban versus
placebo in patients with acute coronary syndromes
(ATLAS ACS-TIMI 46): a randomised, double-blind, phase
II trial. Lancet. 2009;374(9683):29-38
FUTURA/OASIS-8 Trial Group, Steg PG, Jolly SS, Mehta
SR, Afzal R, Xavier D, Rupprecht HJ, López-Sendón
JL, Budaj A, Diaz R, Avezum A, Widimsky P, Rao SV,
Chrolavicius S, Meeks B, Joyner C, Pogue J, Yusuf S.
Low-dose vs standard-dose unfractionated heparin for
percutaneous coronary intervention in acute coronary
syndromes treated with fondaparinux: the FUTURA/
OASIS-8 randomized trial. JAMA. 2010;304(12):1339-49
CURRENT-OASIS 7 Investigators, Mehta SR, Bassand JP,
Chrolavicius S, Diaz R, Eikelboom JW, Fox KA, Granger
CB, Jolly S, Joyner CD, Rupprecht HJ, Widimsky P, Afzal
R, Pogue J, Yusuf S. Dose comparisons of clopidogrel
and aspirin in acute coronary syndromes. N Engl J Med.
2010;363(10):930-42
Rocket AF Investigators. Rivaroxaban versus warfarin in
nonvalvular atrial fibrillation.
N Engl J Med 2011;365;10
P. Tricoci et al, TRACER Investigators. Thrombin-receptor
antagonist vorapaxar in acute coronary syndromes.
N Engl J Med 2012;366:20-33.
CCM — Scientific Report 2011 — Ongoing research 2012
57
Critical Cardiology Area Intensive Cardiac Care Unit
Giancarlo MARENZI, MD
Director
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CCM — Scientific Report 2011 — Ongoing research 2012
Activities 2011. The Intensive Cardiac
Care Unit (ICCU) is equipped with 13 beds provided
with ventilators (5 beds) and complete invasive
(hemodynamic and volumetric) and non-invasive
monitoring. The activity of the ICCU is mainly devoted to
the treatment of patients with acute coronary syndromes
(acute myocardial infarction, unstable angina) and their
complications, with major acute cardiovascular events
(cardiac arrest, acute pulmonary embolism, pericardial
effusion, aortic dissection, life-threatening arrhythmias,
conduction disturbances, acute heart failure, cardiogenic
shock etc.), and, generally, with high-risk conditions.
Patients with non-cardiovascular emergencies, such as
acute respiratory insufficiency and acute kidney injury
requiring respiratory and renal assistance, respectively,
are also admitted. In 2011 the ICCU accepted about 900
patients. The ICCU staff is trained to perform noninvasive (electrocardiogram, echocardiogram, vascular
echo-doppler) and invasive (cardiac catheterization,
pericardiocentesis, central venous and arterial
catheterization) procedures, and to support all activities
in catheterization laboratories and in emergency rooms
in case of cardiac arrest or need for cardiopulmonary
resuscitation or emergency interventions.
Both clinical and scientific activities of the Unit can be
summarized as follows:
1) prevention and treatment of renal complications
associated with cardiovascular diseases: acute
kidney injury is frequently observed in patients
admitted to this Unit because of acute cardiovascular
events and hemodynamic instability, and its
development is associated with relevant clinical and
prognostic implications, and with increased hospital
stay and costs of care. Acute kidney injury associated
with acute coronary syndromes, heart failure, and
contrast media toxicity is usually treated or prevented
with strategies based on the application of renal
replacement therapies (ultrafiltration, hemofiltration
acute coronary syndromes ad other cardiovascular
diseases are under investigation, in collaboration
with our research and clinical laboratories. In
particular, novel markers of myocardial necrosis and
hemodynamic instability (high-sensitivity troponins,
copeptin), myocardial reperfusion injury (cytochrome
c), reduced nitric oxide synthesis (dimethylarginines),
increased mortality risk (BNP, vitamin D deficiency,
acute hyperglycemia), and impaired platelet activity
are systematically evaluated in patients admitted
to our Unit, in order to improve patients’ risk
stratification and to elucidate potential mechanisms
underlying acute cardiac diseases.
and hemodiafiltration). In 2011 about 150 renal
replacement treatments were performed.
2)pericardial effusion treatment: pericardial effusion
and cardiac tamponade are common complications
of cardiac surgery, electrophysiological procedures,
percutaneous coronary interventions and cardiac
diseases. We routinely perform pericardiocentesis
under echocardiographic and radioscopic guidance
as an emergency life-saving procedure. Subsequent
intrapericardial treatments (antiblastic, sclerosing or
anti-inflammatory therapy) are administered. In 2011
we performed about 40 pericardiocentesis
3)evaluation of novel biomarkers: novel biomarkers of
Incidence of contrast-induced nephropathy (CIN) in STEMI patients with and without acute hyperglycemia, treated with primary angioplasty. eGFR = estimated glomerular filtration rate.
Acute hyperglycemia
No Acute hyperglycemia
P=0.01
50
P<0.001
45%
38%
40
P=0.13
CIN incidence, %
STAFF Senior Deputy Director: Gianfranco Lauri, MD
Deputy Directors: Emilio Assanelli, MD, Marco Grazi, MD,
Jeness Campodonico, MD
Senior Assistants: Ivana Marana, MD
Assistants: Monica De Metrio, MD, Marco Moltrasio, MD
Fellows: Angelo Cabiati, MD, Mara Rubino, MD
Residents: Valentina Milazzo, MD
Head Nurse: Ivana Favini
Nurses: Constantin Calugaru, Luisa Castellani Bencich,
Roberto Cerino, Massimiliano Croce, Marisa Dolera,
Tatiana Dragancea, Franca Falchi, Mercedes Maria Franco
Rocha, Francesca Gaggi, Alice Giolo, Andrea Gusmaroli,
Pierpaolo Iozzia, Larisa Ivanova, Pavla Kostalova, Petra
Kozlova, Federica Moneta, Davide Morandi, Mariangela
Alessandra Pace, Miguel Angel Pandia Palomino, Marco
Riboni, Sabino Sangermano, Domenico Santoro, Veronica
Barbara Sisti, Oriana Squilla, Elisabetta Volontè
Secretaries: Annamaria Bellavia, Salvina Comignolo
P<0.001
30
29%
27%
26%
P=0.01
20
16%
10
12%
16%
11%
7%
0
n=148 n=632
n=74 n=35
n=74 n=597
n=58 n=160
n=90 n=472
All patients
Diabetes
No diabetes
eGFR ≤ 60
eGFR > 60
mellitus
mellitus
ml/min/1.73m2
ml/min/1.73m2
CCM — Scientific Report 2011 — Ongoing research 2012
59
The Unit is also involved in several multicenter
international trials evaluating new antithrombotic
therapies for the treatment of acute coronary syndromes
and for their secondary prevention. Finally, important
issues of investigative interest are represented by
the evaluation, diagnosis and treatment of pericardial
effusions, in collaboration with Istituto Europeo di
Oncologia, Milano (Cardiology Unit, Dr. Carlo Cipolla)
and San Raffaele Hospital, Milano (Hematoncology and
Bone Marrow Transplantation Unit, Dr. Fabio Ciceri).
Publications
Sisillo E, Marenzi G. N-Acetylcysteine for the prevention
of acute kidney injury after cardiac surgery. J Clin
Pharmacol 2011; 51(11):1603-10.
Marenzi G, De Metrio M, Bartorelli A. Author’s reply to
acute hyperglycemia: Is really a new risk marker for
contrast-induced nephropathy in patients with acute
myocardial infarction without diabetes and normal renal
function? Am Heart J 2011;162:e9.
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CCM — Scientific Report 2011 — Ongoing research 2012
Alexander JH, Lopes RD, James S, et al. for the
APPRAISE-2 Investigators (G. Marenzi). Apixaban with
antiplatelet therapy after acute coronary syndrome. New
Engl J Med 2011;365:699-708.
Oldgren J, Budaj A, Granger CB, Khder Y, Roberts
J, Siegbahn A, Jan G.P. Tijssen JGP, Van de Werf F,
Wallentin L, for the RE-DEEM investigators (G. Marenzi).
Dabigatran vs. placebo in patients with acute coronary
syndromes on dual antiplatelet therapy: a randomized,
double-blind, phase II trial. Eur Heart J 2011;32:2781-2789
Cristell N, Cianflone D, Durante A, Ammirati E, Vanuzzo
D, Banfi M, Calori G, Latib A, Crea F, Marenzi G, De
Metrio M, Moretti L, Li H, Uren NG, Hu D, Maseri A on
behalf of the FAMI Study Investigators. High-sensitivity
C-reactive protein is within normal levels at the very
onset of first ST-segment elevation acute myocardial
infarction in 41% of cases. A multiethnic case-control
study. J Am Coll Cardiol 2011;58:2654-2661
CCM — Scientific Report 2011 — Ongoing research 2012
61
Critical Cardiology Area Heart Failure, Clinical Cardiology
and Cardiac Rehabilitation Unit
Piergiuseppe AGOSTONI, MD
Director
STAFF Deputy Directors: Marina Alimento, MD,
Anna Apostolo, MD, Gaia Cattadori, MD, Mauro Contini, MD,
Manuela Muratori, MD, Pietro Palermo, MD,
Francesco Pesoli, MD
Assistants: Daniele Andreini, MD, Giovanni Berna, MD
Fellows: Erika Bertella, MD, Stefania Farina, MD,
Stefania Paolillo, MD, Elisa Stefanini, MD
Residents: Laura Antonioli, MD, Sarah Cortinovis, MD,
Erica Gondoni, MD, Giuliano Giusti, MD, Saima Mushtaq, MD,
Carlo Vignati, MD
Nurses: Angela Galantucci, Elena Nazzari, Maria Luisa
Scapin
Secretaries: Eleonora Boccato, Michela Palmieri
Data Manager: Elisabetta Salvioni
Activities 2011. By using advanced diagnostic
tools and innovative therapeutic strategies, the Unit aims
at assessing and treating heart failure, one of the most
common pathologies in the western countries. The Unit
provides cardiovascular rehabilitation and takes care of
patients affected by heart diseases needing a “global”
evaluation and drug treatment. The Unit is equipped with
36 beds and is in charge of the Cardiac Rehabilitation
Gym, the Cardiopulmonary Research Laboratory, the
Centre and Laboratory of Sleep Disorders, the Systemic
and Pulmonary Hypertension Centre, the outpatient
departments of Heart Failure, the clinical Cardiology and
Cardiac Rehabilitation, the telemedicine.
Piergiuseppe Agostoni is Associate Professor in
Cardiology at the Milano University and Associate
Professor in Critical Care and Respiratory Medicine at
the University of Washington, Seattle, WA. At present,
he is president of CPX International (formerly ISEIRE,
International Society for Exercise Intolerance Research
and Education), the Australia based Society
(http://www.cpxinternational.com/) dedicated to teaching
Cardiopulmonary Exercise Testing (CPET).
Under the endorsement of the University of California and
later of CPX International/ISEIRE, each year a 3-day CPET
course is organized in different European institutions
(including Centro Cardiologico Monzino) and in the last
two years also in South America. Similar courses are
organized in the United States, Japan and Australia.
Since 1995 a 2-day CPET course, and more recently
weeklong stages, have been organized by the Monzino
cardiopulmonary researchers for Italian specialists
with a bulk of around 1000 attendees. Moreover,
Piergiuseppe Agostoni has an active role in international
cooperation and specifically, through the support of the
Corti Foundation (htpp://www.fondazionecorti.it/), with
cardiology teaching and cardiac patients care at St Mary
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CCM — Scientific Report 2011 — Ongoing research 2012
Lacor Hospital in Gulu (Uganda) where he spends, starting
from 1978, one month per year almost every year.
In the Cardiopulmonary Research Laboratory of
the Centro Cardiologico Monzino, patient care and
research merge in a single work so that excellence in
treatment and research are connected to each other. The
Cardiopulmonary Research Laboratory is the result of
a total integration of the Centro Cardiologico Monzino
staff and the Department of Cardiovascular Sciences
staff so that it is now impossible to separate the two
original components. The strength of the laboratory
is the team work so that cardiologists, nurses and
researchers work together to achieve their objectives.
Several junior researchers before, during and after
their Cardiology Fellowship Program as well as some
foreign researchers participate to the activities of the
Laboratory. Several grants have been offered to and
by the Cardiopulmonary Research Laboratory to allow
external researcher to have a training or to spend a
sabbatical period of time in our laboratory.
The major research topics include:
a) Cardiopulmonary interactions in heart failure patients.
In the last 20 years, research performed in our Unit has
allowed significant advances with important implications
CCM — Scientific Report 2011 — Ongoing research 2012
63
in the clinical field. Indeed, we showed that tidal volume
changing during exercise predicts the exercise capacity
of heart failure patients and any treatment able to
improve tidal volume, e.g. ultrafiltration, increases
exercise performance.
b) Beta-blockers influence on exercise performance in
heart failure patients.
Research performed in our Unit showed that beta-blockers,
which are able to improve clinical condition and prognosis
of heart failure patients, do not improve maximal exercise
performance but increase submaximal exercise performance.
Indeed, we showed that beta-blockers, and specifically
carvedilol, reduce exercise induced hyperventilation as
shown by a lower VE/VCO2 slope and a higher PaCO2
during exercise. This effect is specific of non selective
beta-blockers and is not shared by selective beta1-blockers.
On the other side of the coin, we showed that when
hyperventilation is needed, such as at high altitude, exercise
performance is reduced in carvedilol treated subjects.
c) Adaptation to high altitude.
Several research projects have been carried out on high
altitude cardiovascular adaptation in normal subjects and
heart failure patients using both a high altitude model and
“on-site” studies (Capanna Regina Margherita, 4600 m and
Mount Everest South Base Camp, 5400 m). Particularly,
we evaluated the time course and the mechanisms
responsible of lung fluid movement across the alveolarcapillary membrane when reaching high altitude and
adaptation in time.
d) Gas exchange in heart failure.
64
CCM — Scientific Report 2011 — Ongoing research 2012
This specific topic has been heavily studied in our
Unit demonstrating the presence of active mechanisms
regulating the alveolar-capillary membrane permeability
and the role of several drugs on it. Indeed, we showed
that ACE-inhibitors improve lung diffusion, whereas
aspirin and not selective beta-blockers reduce lung
diffusion and AT1 blockers and selective beta1-blockers
have no impact on lung diffusion. Moreover, we set up a
pulmonary edema model using acute saline infusion and
high altitude as such.
e) Non invasive cardiac output determination during
exercise.
For the first time, we showed that cardiac output during
exercise can be measured in heart failure patients by
an inert gas rebreathing technique. This cardiac output
measuring technique is now used to study, during
exercise, heart failure treatment efficacy such as cardiac
resynchronization therapy, mitral insufficiency correction
by mitral clip and cardiac rehabilitation. By means of
an international collaborative study with the University
of Bern, interesting data concerning the distribution
of blood flow during exercise were obtained. We also
studied the role of anemia in exercise performance
in chronic heart failure patients, evaluating, for each
patient, the specific importance of anemia to explain
functional limitation. Moreover we evaluated the effect
of Ventricular Assistance Device (Jarvik) during exercise
at different cardiac output settings. Finally we studied
normal subjects, at present 300 of the planned 500, to
measure the normal cardiac output at peak exercise
in a wide population to build up and upgrade the
available tables of normality which were based on old
research involving a limited number of subjects due
to the invasive characteristic of previously available
measurement technique.
f) The role of cardiopulmonary exercise testing in heart
failure patients.
We performed single and multicenter pathophysiological
studies. We used either our database which collects
data on about 1000 heart failure patients, including
cardiopulmonary exercise tests and follow up, or a
multicentre database, coordinated by our Unit, with more
than 3500 enrolled patients. We are building a score for
estimating survival in HF patients based on metabolic
exercise, cardiac and kidney parameters (MECKI score).
In a sub-study involving the patients with advanced
HF, we are evaluating the improvement of survival in
the modern era arising doubts about the actual heart
transplant criteria. We also evaluated by CPET HF patient
underwent to CRT showing that CRT efficacy depends on
proper positioning of the LV lead over the posterolateral
wall and, in the setting of unfavorable CS branches of
anatomy, CRT by a surgical mini-thoracotomic approach
is preferable to trans-venous lead implantation.
g) The role of cardiopulmonary exercise testing in the
clinical management of patients affected by pulmonary
CCM — Scientific Report 2011 — Ongoing research 2012
65
arterial hypertension.
Patients affected by pulmonary arterial hypertension
(PAH) show a reduced exercise tolerance with early
occurrence of dyspnea and fatigue. The origin of
functional capacity limitation is multifactorial and
several mechanisms have been proposed, including
right heart failure, which leads to a limited increase in
cardiac output during exercise, and hyperventilation
with a reduced perfusion of properly ventilated alveoli.
In addition, abnormalities in arterial blood gases are
observed, with the occurrence of hypoxemia and
hypocapnia, related to an abnormal ventilation/perfusion
match, gas diffusion abnormalities, low mixed venous
oxygen saturation and to the development of intra- and
extra-pulmonary right-to-left shunts. At present, the
6-minute walking test is the most used method to assess
exercise tolerance in PAH; it is also useful to monitor
the response to therapy and provides prognostic
information. However, the assessment of functional
capacity by cardiopulmonary exercise test (CPET)
seems to be more complete, because CPET allows for
discrimination between the metabolic, cardiovascular and
pulmonary components of exercise limitation. Moreover,
CPET estimates the severity of disease and assesses
patient prognosis and response to therapy. In PAH, a
typical CPET-response is observed, characterized by
a severe reduction in peak VO2, work rate, O2 pulse
and anaerobic threshold and by a marked increase in
VE/VCO2 slope and in the dead space to tidal volume
ratio. However, the use of CPET should be limited to
experienced centers. This review will focus on resting
lung function and exercise tolerance tests, showing
that CPET can provide the physiological explanation of
functional limitation in PAH.
h) The role of plasma SPB and RAGE as markers of lung
injury.
We studied plasma SPB and RAGE during mechanical
ventilation in patients undergoing major vascular surgery
and in the plasma during cardiopulmonary bypass. We
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CCM — Scientific Report 2011 — Ongoing research 2012
identified a different kinetics between SPB and RAGE
during lung acute damage. Indeed SPB seems to be more
strictly related to the lung than RAGE. This allow us to
suggest a role of SPB as a lung damage marker.
Other research trials on exercise physiology in heart
failure, heart failure treatment, heart failure rehabilitation
and evaluation of heart failure prognosis, have been
done or are ongoing.
Within the Unit, the Cardiac Rehabilitation (Rehab) is a
program designed to help improving the patient overall
health. The program promotes life-style changes and
medical management to:
• prevent the occurrence and/or progression of
cardiovascular diseases
• reduce the risk of a heart attack or stroke
• reduce the need for future surgery
• enhance the quality of life by reducing symptoms
An exercise physiologist or a qualified nurse helps
patients to attain their personal goals.
Cardiac Rehab programs is addressed to patients with:
• coronary artery bypass graft (CABG) surgery
• heart attack (myocardial infarction)
• heart failure
Soon after the discharge from the hospital, the patient
can enter the outpatient Cardiac Rehab program. All
sessions are medically supervised and directed by
nationally certified rehabilitation specialists and nurses.
State-of-the-art monitoring equipment and a variety of
exercise equipments are available.
G, Andreini D, Tondo C, Agostoni PG. Long-term
effectiveness of cardiac resynchronization therapy in
heart failure patients with unfavorable cardiac veins
anatomy comparison of surgical versus hemodynamic
procedure. J Am Coll Cardiol. 2011;58(5):483-490
Karsten M, Contini M, Cefalù C, Cattadori G, Palermo P,
Apostolo A, Bussotti M, Magrì D, Salvioni E, Farina S,
Sciomer S, Catai AM, Agostoni P. Effects of carvedilol
on oxygen uptake and heart rate kinetics in patients
with chronic heart failure at simulated altitude. Eur J
Cardiovasc Prev Rehabil. 2011 (Epub ahead of print)
Agostoni P, Swenson ER, Bussotti M, Revera M, Meriggi
P, Faini A, Lombardi C, Bilo G, Giuliano A, Bonacina D,
Modesti PA, Mancia G, Parati G; HIGHCARE Investigators.
High-altitude exposure of three weeks duration increases
lung diffusing capacity in humans. J Appl Physiol.
2011;110(6):1564-1571
Agostoni P, Banfi C, Brioschi M, Magrì D, Sciomer S,
Berna G, Brambillasca C, Marenzi G, Sisillo E. Surfactant
protein B and RAGE increases in the plasma during
cardiopulmonary bypass: a pilot study. Eur Respir J.
2011;37(4):841-847
Publications
Cattadori G, Schmid JP, Brugger N, Gondoni E, Palermo
P, Agostoni P. Hemodynamic effects of exercise training
in heart failure. J Card Fail. 2011;17(11):916-922
Giraldi F, Cattadori G, Roberto M, Carbucicchio C,
Pepi M, Ballerini G, Alamanni F, Della Bella P, Pontone
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Interventional Cardiology Area
Antonio BARTORELLI, MD
Area Coordinator
Interventional Cardiology Units
Antonio BARTORELLI, MD
Director of Unit 1
Piero MONTORSI, MD
Director of Unit 2
Alessandro LUALDI, MD
Director of Unit 3
Franco FABBIOCCHI, MD
Director of Unit 4
STAFF Senior Deputy Directors: Luca Grancini, MD,
Paolo Ravagnani, MD
Deputy Directors: Stefano Galli, MD, Daniela Trabattoni, MD
Assistants: Giuseppe Calligaris, MD, Stefano De Martini, MD,
Giovanni Teruzzi, MD
Residents: Maria Antonietta Dessanai, MD, Pamela Gatto, MD,
Marco Valerio Morpurgo, MD, Paolo Olivares, MD,
Chiara Stefania Pandini, MD, Francesca Pizzamiglio, MD
Fellows: Cristina Ferrari, MD, Anna Garlaschè, MD,
Massimo Mapelli, MD, Ilaria Previtali, MD
Head Nurse: Gabriele Bucca
Nurses: Mariangela Alberti, Gabriella Battaini,
Francesca Bonfiglio, Daniele Buono, Ivan Consoli,
Fernanda Giancola, Rossella Paloschi, Gabriella Panetta,
Gaetano Seletti, Enrico Speranza
Chief Technician: Giuseppe Squilla
Technicians: Vania Battaglini, Chiara De Pasquale,
Vincenzo Lo Mascolo, Flavio Mu, Stefania Pergolizzi
Secretaries: Mariagrazia Cortesi, Fernando Di Marino
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Activities 2011. Interventional cardiology is a
modern branch of medicine with a very high technology
content, which deals with the non-surgical management of
cardiovascular diseases. The role of invasive cardiology
has rapidly grown from a predominantly diagnostic
activity to a full range of therapeutic interventions
as alternatives to traditional surgical and medical
treatments. The cardiac catheterization laboratories of
Centro Cardiologico Monzino are equipped with the most
advanced and innovative tools to offer a full range of
state-of-the-art diagnostic and therapeutic procedures.
Together with traditional angiography, additional
diagnostic modalities such as intravascular ultrasound and
virtual histology, optical coherence tomography, flowwire and quantitative angiography allow a more precise
and accurate evaluation of lesion to be treated and postprocedural results.
In 2010, Centro Cardiologico Monzino continued to be
at the forefront of elective coronary artery disease
treatment using constantly updated coronary angioplasty
techniques and state-of-the-art drug-eluting stents.
Patients with acute coronary syndromes were also treated
using coronary angioplasty and stenting and the latest
technology for protecting the coronary microcirculation,
particularly in acute myocardial infarction. This patient
subset can receive prompt treatment by a skilled team of
interventional cardiologists available on a 24/24-hour 7/7day basis. Moreover, cardiac support devices (intra-aortic
balloon pump, percutaneous cardiopulmonary support and
Impella device) are available and were used to support
patients with reduced left ventricular function. We also
offered angioplasty and stenting in the arteries of the leg
and the kidneys, the carotid arteries, and the abdominal
vessels, such as the aorta and the iliacs. Interventional
cardiologists also used their extensive expertise in the
successfully treatment of structural heart disease using
innovative therapeutic modalities such as alcohol septal
ablation for hypertrophic obstructive cardiomyopathy,
atrial septal defect and patent foramen ovale closure,
and left atrial appendage obliteration for prevention of
cardiogenic embolization in atrial fibrillation patients.
In 2008, in collaboration with the Cardiac Surgery
Unit, we started a minimally invasive procedure for the
treatment of high-risk symptomatic patients with severe
aortic stenosis using the Edwards SAPIEN transcatheter
heart valve. Among preventive strategies in high-risk
patients, a particular attention has been devoted to
contrast nephropathy using very effective prophylactic
treatments such as continuous veno-venous hemofiltration
(CVVH) and novel investigational devices. Other critical
collaborations continued for prevention, diagnosis and
treatment of patients with cardiovascular disorders and
for research purposes with the Cardiovascular Imaging
Area (MDCT, 3D-echo, TEE), the Intensive Cardiac Care
Unit, the Laboratory of Biology and Biochemistry of
Atherothrombosis and the Laboratory of Gene Therapy.
In addition to their clinical activity, many members of
our Units have university and institutional teaching
CCM — Scientific Report 2011 — Ongoing research 2012
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Electrophysiology Area
Claudio TONDO, MD, Ph.D Area Coordinator
Electrophysiology Unit
Claudio TONDO, MD, Ph.D
Director
Corrado CARBUCICCHIO, MD, Ph.D
Director of Ventricular Intensive Unit
responsibilities involving mainly student and fellow
training, and are engaged in Continuing Medical Education
(CME) programs.
Publications
Silber S, Windecker S, Vranckx P, Serruys PW;
RESOLUTE All Comers investigators. Unrestricted
randomised use of two new generation drug-eluting
coronary stents: 2-year patient-related versus stentrelated outcomes from the RESOLUTE All Comers trial.
Lancet. 2011; 377(9773):1241-7
Serruys PW, Silber S, Garg S, van Geuns RJ, Richardt
G, Buszman PE, Kelbaek H, van Boven AJ, Hofma SH,
Linke A, Klauss V, Wijns W, Macaya C, Garot P, DiMario
C, Manoharan G, Kornowski R, Ischinger T, Bartorelli
A, Ronden J, Bressers M, Gobbens P, Negoita M, van
Leeuwen F, Windecker S. Comparison of zotarolimuseluting and everolimus-eluting coronary stents. N Engl J
Med. 2010; 363(2):136-46
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Marenzi G, Assanelli E, Campodonico J, De Metrio M, Lauri
G, Marana I, Moltrasio M, Rubino M, Veglia F, Montorsi P,
Bartorelli AL. Acute kidney injury in ST-segment elevation
acute myocardial infarction complicated by cardiogenic
shock at admission. Crit Care Med. 2010; 38(2):438-44.
Pontone G, Andreini D, Bartorelli AL, Cortinovis S,
Mushtaq S, Bertella E, Annoni A, Formenti A, Nobili E,
Trabattoni D, Montorsi P, Ballerini G, Agostoni P, Pepi M.
Diagnostic accuracy of coronary computed tomography
angiography: a comparison between prospective and
retrospective electrocardiogram triggering. J Am Coll
Cardiol. 2009; 54(4):346-55.
Marenzi G, Assanelli E, Campodonico J, Lauri G, Marana
I, De Metrio M, Moltrasio M, Grazi M, Rubino M, Veglia
F, Fabbiocchi F, Bartorelli AL. Contrast volume during
primary percutaneous coronary intervention and
subsequent contrast-induced nephropathy and mortality.
Ann Intern Med. 2009; 150(3):170-7.
STAFF Senior Deputy Director: Stefania Riva, MD
Deputy Directors: Antonio Dello Russo, MD, Ph.D,
Gaetano Fassini, MD
Senior Assistant: Michela Casella, MD, Ph.D
Assistants: Massimo Moltrasio, MD,
Fabrizio Tundo, MD, Ph.D
Fellows: Benedetta Majocchi, MD, Martina Zucchetti, MD,
Vittoria Marino, MD
Nurses: Luana Barbieri, Rosario Cervellione, Pasquale De
Iuliis, Roberta Fasana, Mario La Notte, Claudia Perlotti,
Maria Lena Ranghetti, Romina Ranzato, Alberto Somenzi,
Michela Vendramin
Secretaries: Viviana Biagioli, Tiziana Peroncini
Activities 2011. The clinical activity of the
Electrophysiology Area deals with electrophysiological
procedures and cardiac pacing.
Electrophysiological procedure. Almost every type
of arrhythmia is managed in the Electrophysiology
Area: supraventricular paroxysmal tachycardia, WolffParkinson-White syndrome, atrial tachycardia, atrial
flutter, atrial fibrillation, ventricular tachycardia and
ventricular premature beats. Treatment consists mostly
in radiofrequency catheter ablation of the arrhythmia
guided by conventional mapping or, more often, after 3D
reconstruction and substrate mapping. The efforts of the
Electrophysiology Area mostly converge on ablation of
atrial fibrillation and ventricular tachycardia.
Atrial fibrillation is the most common significant cardiac
arrhythmia, responsible for approximately one-third of
all hospital admissions of patients suffering from cardiac
rhythm disturbances. It is responsible for an increased
risk of stroke, heart failure and all-cause mortality. The
vast improvement over the last decade in the long term
efficacy of catheter ablation, compared to pharmacological
treatment, supports the use of ablation in the early
management of patients suffering from atrial fibrillation. In
the Electrophysiology Area catheter ablation is currently
performed for paroxysmal and persistent atrial fibrillation
(up to 300 per year). The procedure is usually guided by
3D electroanatomical mapping systems (Carto 3/NavX)
integrated by a previously acquired CT or MRI image
of the left atrium and/or intracardiac echocardiography
(CartoSound technology).
In the setting of atrial fibrillation, the Electrophyisology
Area is also equipped to perform transcatheter closure
of the left atrial appendage in order to reduce the
cardioembolic risk in those patients with persistent/
permanent atrial fibrillation who are not candidates for
warfarin therapy.
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71
Ventricular Tachycardia. Until recently, antiarrhythmic
drugs were the preferred treatment for most patients
with recurrent ventricular tachycardia, whatever the
underlying heart disease. However, the risk of sudden
cardiac death and recurrences remains high despite drug
therapy; alternative treatment options include surgical
ablation, implantation of a cardioverter-defibrillator
(ICD), and catheter ablation. Although ICDs prolong
survival, they do not prevent ventricular tachycardia
recurrences, thus being a merely palliative treatment.
Catheter ablation, on the contrary, is a potentially
curative approach, preventing ventricular tachycardia
recurrences in more than 70% of patients.
In the Electrophysiology Area catheter ablation of
ventricular tachycardia is often performed in the setting
of dilated cardiomyopathy, ischaemic cardiopathy,
arrhythmogenic right ventricular cardiomyopathy, or
in the context of an apparently normal heart, as it is
often the case with right and left ventricular outflow
tract tachycardia. Ablation is usually guided by 3D
electroanatomical mapping systems, including noncontact systems.
Moreover, the Electrophysiology Area includes a new
and unique service focused on critical ventricular
arrhythmias, called Ventricular Intensive Care (VIC).
It strives for a “global” treatment of patients with
ventricular arrhythmias responsible for critical
impairment of clinical conditions and/or for multiple ICD
interventions. The VIC unit aims at improving quality
of life and optimizing antiarrhythmic therapies by
integrating acute and long-term care provided by the
same team of operators.
The Electrophyisology Area is also equipped to perform
endomyocardial biopsies (EMB) to improve diagnostic
accuracy and prognostic correlations in the setting
of cardiomyopathy, arrhythmogenic right ventricular
dysplasia, myocarditis, cardiac tumors, arrhythmia, etc.
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The right internal jugular vein and the femoral vein are
the most common percutaneous access sites for right
ventricular EMB, which is usually performed safely
under fluoroscopic and intracardiac echo guidance.
Histological samples are also examined for genetic
mutations and for amplification of viral genomes. In
addition to its clinical uses, EMB may be a precious
instrument to better understand the cellular and
molecular pathophysiology of cardiovascular disease.
Cardiac pacing. The Electrophysiology Area performs
implantation and replacement of permanent pacemakers,
including both single-chamber and (more often)
dual-chamber devices. Each year approximately 200
implantable cardioverter-defibrillators (ICD) and/or
biventricular pacemakers (for cardiac resynchronization
therapy: CRT) are implanted, for the prevention of
sudden cardiac death and for improvement of heart
failure respectively, often in patients with severe left
ventricular dysfunction and/or malignant ventricular
arrhythmias. Over the last years, the technique of
catheter lead extraction with the use of Locking Stylets,
Telescoping Synthetic Sheaths, Electrosurgical Dissection
and Laser Extraction has been implemented to answer
the increased demand generated by lead damage or
device infection.
Publications
Santangeli P, Dello Russo A, Pieroni M, Casella M,
Di Biase L, Burkhardt JD, Sanchez J, Lakkireddy
D, Carbucicchio C, Zucchetti M, Pelargonio G,
Themistoclakis S, Camporeale A, Rossillo A, Beheiry
S, Hongo R, Bellocci F, Tondo C, Natale A. Fragmented
and delayed electrograms within fibrofatty scar predict
arrhythmic events in arrhythmogenic right ventricular
cardiomyopathy: Results from a prospective risk
stratification study. Heart Rhythm. 2012 (Epub ahead of
print)
Santangeli P, Pieroni M, Dello Russo A, Casella M,
Pelargonio G, Di Biase L, Macchione A, Burkhardt JD,
Bellocci F, Santarelli P, Tondo C, Natale A. Correlation
between Signal-Averaged Electrocardiogram and the
Histologic Evaluation of the Myocardial Substrate
in Right Ventricular Outflow Tract Arrhythmias. Circ
Arrhythm Electrophysiol. 2012 (Epub ahead of print)
Dello Russo A, Casella M, Pieroni M, Pelargonio G,
Bartoletti S, Santangeli P, Zucchetti M, Innocenti E, Di
Biase L, Carbucicchio C, Bellocci F, Fiorentini C, Natale
A, Tondo C. Drug-Refractory Ventricular Tachycardias
Following Myocarditis: Endocardial and Epicardial
Radiofrequency Catheter Ablation. Circ Arrhythm
Electrophysiol. 2012 (Epub ahead of print)
Casella M, Dello Russo A, Pelargonio G, Bongiorni MG,
Del Greco M, Piacenti M, Andreassi MG, Santangeli P,
Bartoletti S, Moltrasio M, Fassini G, Marini M, Di Cori
A, Di Biase L, Fiorentini C, Zecchi P, Natale A, Picano E,
Tondo C. Rationale and design of the NO-PARTY trial:
near-zero fluoroscopic exposure during catheter ablation
of supraventricular arrhythmias in young patients.
Cardiol Young. 2012;3:1-8.
Dukkipati SR, Neuzil P, Kautzner J, Petru J, Wichterle
D, Skoda J, Cihak R, Peichl P, Dello Russo A, Pelargonio
G, Tondo C, Natale A, Reddy VY. The durability of
pulmonary vein isolation using the visually guided laser
balloon catheter: multicenter results of pulmonary vein
remapping studies. Heart Rhythm. 2012 (Epub ahead of
print)
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73
Cardiovascular Surgery Area
Francesco ALAMANNI, MD Area Coordinator
Cardiac Surgery Unit 1 Cardiac Surgery Unit 2
Francesco ALAMANNI, MD
Director
Gianluca POLVANI, MD
Director
STAFF Deputy Directors: Moreno Naliato, MD, Alessandro
Parolari, MD, Ph.D, Giulio Pompilio, MD, Ph.D, Maurizio
Roberto, MD, Ph.D, Marco Zanobini, MD, Ph.D
Senior Assistants: Francesco Grillo, MD
Senior Deputy Directors: Marco Agrifoglio, MD
Assistant: Samer Kassem, MD
Fellow: Eleonora Penza, MD
Residents: Francesco Arlati, MD, Laura Cavallotti, MD, Andrea
Daprati, MD, Sara Filippini, MD, Tommaso Generali, MD,
Marco Gennari, MD, Daniela Manzone, MD, Elisa Merati, MD,
Gabriele Tamagnini, MD, Giulio Tessitore, MD
Head Nurses: Annarita Leardi, Rocca Punzi
Referent Nurses: Anna Sudati, Sara Fronterrè
Nurses: Brunella Biava, Evson Capuno, Andrea
Dimastrogiovanni, Francesca Fugazza, Sonia Huaman, Alin
Pavel Huci, Jirina Kopecna, Ana Liliana Lasteros, Giuseppe
Leontino, Monica Marino, Federica Marson, Saveria Mennuti,
Tiziana Nella, Lucia Palladino, Marco Racioppo, Tommaso
Sambito, Sara Sesto, Petra Skarcova, Richard Sucapuca,
Alexandra Vicari, Ermes Visigalli,
Operating Room Head Nurse: Maria Luisa Fumagalli
Operating Room Nurses: Barbara Brizio, Maria Teresa Gallo,
Rejab Jihed, Anna Ligorio, Gabriela Makrovska, Emanuela
Mancino, Cristiano Moro, Brigitta Nobrega, Daniela Pedrini,
Stefania Pellati, Ilaria Poppa, Simona Ravani, Chiara Rosselli,
Sabrina Salerno, Jana Smerekovska, Cristina Spadotto
Secretaries: Daniela Corti, Nadia Vaccari
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Cardiac Surgery Unit 1
Cardiac Surgery Unit 2
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75
Cardiovascular Surgery Area
Activities 2011. Clinical activities involved all
fields of adult cardiac surgery; in 2011 in-hospital mortality
was 2,96% (coronary surgery 0%, valve surgery 2.4%,
combined valve and coronary surgery 7,1%).
In 2011 we performed 777 surgical interventions with
a prevalence of valvular repair-replacement; there
was a slight decrease in coronary surgery due to the
increasing number of percutaneous treatment of this
disease.
Valve surgery of the adult patient very often involves
aortic and/or mitral valve, with or without associated
coronary revascularization procedures.
Mitral valve surgery. We performed 139 procedures
with an increase of very complex repairs; also, we have
implemented our techniques expanding the concept of
“respect vs resect”; this means that we have attempted
to perform more physiologic repair procedures, trying
to avoid extensive removals of mitral valve native
tissue in order to preserve (“respect”) as much as
possible the native anatomy of the mitral valve though
warranting an optimal repair procedure. During this
year a program for “Transcatheter Mitral Valve Repair”
(MitraClip), in addition to the aortic valve transcatheter
treatment program, has been developed and a growing
number of high risk surgical patients has been treated
focusing on clinical results and physiopathology of
MitraClip effects on heart valves. This has been done
with the cooperation of the Interventional Cardiology
Units. During this year we have increased the numbers
of mitral valve repairs performed by using a minimally
invasive surgical approach.
In addition, in cooperation with the Echocardiography
Unit we have continued pre- and intraoperative studies
of anatomy and mechanisms of regurgitation (with
3D-transthoracic realtime echocardiography we are able
to plan the repair technique).
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Since mitral valve disease is frequently associated with
tricuspid valve involvement, and since an untreated
tricuspid valve disease at the time of mitral valve
surgery may adversely affect the follow-up results,
during the last year we became more aggressive in
detecting and treating this disease, according to recent
clinical evidence. We were also very active in studying
mitral valve disease, focusing on the biological pathways
activated in the course of the disease and possibly
involved in the etiology of mitral valve regurgitation.
In addition, together with the Echocardiography Unit
and the Bioengineering Department of Politecnico
of Milano, we studied mathematical and simulation
models of mitral valve disease and other common
techniques of repair. More recently, always together
with the Echocardiography Unit and the Bioengineering
Department of Politecnico of Milano and Bologna, we
have focused our attention on the impact of the mitral
valve on the function of the aortic valve.
Concerning aortic valve surgery, during 2011 we performed
240 aortic valve replacements, with an increasing percentage
of procedures done by using a minimally invasive approach;
123 of these replacements were performed with transcatheter
techniques; in 20 patients we also performed aortic valve
repair with anatomic reconstruction.
With the advent of transcatheter techniques, aortic
valve replacement can now be considered in patients
who were previously off-limits for conventional
open-chest surgery. This was possible thanks to the
cooperation with the Anaesthesiology, Vascular Surgery,
Radiology, Angiography and Echocardiography Units for
preoperative assessment and intraoperative procedures.
Thus we have further expanded our experience in
transcatheter aortic valve replacements, with about 328
cases performed up to now with this technique.
The biology of aortic valve degeneration is also a hot
topic of our research: we have studied the possible
protective effects of statin pre-treatment on the
outcomes and on the disease progression of patients
with aortic valve stenosis, showing that, when the
degree of stenosis is advanced, statins may not be an
effective preventive measure.
We have started a program of sutureless aortic valve
implantation which is clinically positioned in a midway
between TAVI and conventional AVR. Using these valves
it is possible to replace aortic valves by minimally
invasive access and to reduce ECC time in complex
procedures thus reducing trauma in the aged risk
patients not candidate to TAVI .
Coronary bypass surgery represented slightly less than
30% of our activity. Despite the decreasing clinical
conditions and the increasing age of patients, our inhospital mortality (usually considered the indicator of
care quality in a cardiac surgery program) was very
low. During the last year we have been very active in
multi-arterial grafting, especially in younger patients, we
have continued clinical assessment of off-pump coronary
bypass procedures and started exploring the potential
of hybrid (surgical revascularization associated with
coronary stenting) revascularization techniques. We have
also recruited patients for studies on cardiovascular
prevention and the role of conventional and
unconventional risk factors. In addition, in cooperation
CCM — Scientific Report 2011 — Ongoing research 2012
77
with the Radiology Unit, we are concluding a follow-up
study involving 330 patients, previously submitted to
isolated coronary bypass surgery, in order to document
patency of coronary bypasses using a 64-rows CT scan
18 months after surgery and to investigate the possible
predictors of bypass occlusion.
The surgery of cardiac arrhythmias had an important
implementation during the last year; thanks to the
cooperation with the Electrophysiology Unit we started
to treat surgically (via mini-incisions) patients affected
by atrial fibrillation and unresponsive to conventional
therapies and have recently updated the clinical protocol
extending the treatment of concomitant AF in patient
in whom a surgical approach to mitral valve is not
planned (i.e. aortic valve and coronary revascularization)
and the entire procedure is performed with the closed
left atrium. We also started the isolated treatment
of ventricular tachycardia due to ischemic disease,
dilatative cardiomyopathy or ventricular tumour
unresponsive to EPS treatments. For the first time ever,
we treated 19 patients with VT.
Our data collection and results are continuously
monitored by a public clinical Audit accredited by the
most important European agency in this field, ECTSIA
(European Cardiovascular & Thoracic Surgery Institute of
Accreditation).
Research areas:
• Genomics of coronary artery, valve and aortic
disease.
• Perioperative proteomic profiles of patients
undergoing coronary bypass, valve and aortic
procedures.
• Proteomics of non-rheumatic calcific aortic
stenosis, mitral valve prolapses, and aortic
aneurysms.
• Coagulation, inflammation and oxidative stress
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CCM — Scientific Report 2011 — Ongoing research 2012
activation during and after coronary bypass, valve
and aortic procedures.
• Predictors of patency of coronary bypass grafts
and of late outcome in patients undergoing
coronary bypass surgery.
• Biology of non-rheumatic, calcific aortic stenosis
and mitral valve prolapse.
• Evaluation of algorithms predicting the occurrence
of perioperative acute kidney injury after adult
cardiac surgery
• Application of advanced statistical models to
cardiac surgery.
• Systematic review of the role of statins in the
prevention of major complications and disease
progression in non-rheumatic, calcific aortic
stenosis.
• Minimally invasive cardiac surgery for valve
repair.
• Alternative therapies for cardiac arrhythmias
(atrial fibrillation, ventricular tachycardia) not
responsive to conventional therapies: role of
surgery.
• Role of intraoperative electro-anatomic mapping
(CARTO) during surgical ablations for refractory
ventricular tachycardia.
• Mathematical and simulation models of mitral
valve disease (assessed by 3D-echocardiography)
and of the most common techniques of repair.
• Regenerative medicine for ischemic heart disease.
• Transcatheter aortic valve implantation in calcific
aortic stenosis: predictors of outcome and choice
of techniques.
• Clinical follow up of patients submitted to
surgical treatment of concomitant or lone AF via
implantable loop recorders or EPS
• MitraClip in high risk patients: evaluation of
cardiac performance before and after implantation,
effects of the device on mitral flow and dynamics.
• Sutureless aortic prosthesis: definition of
anatomical pre-implant variables, impact of the
device on mitral-aortic coupling
• Impact of mitral valve reconstruction on the aortic
valve, left and right ventricle.
Publications
Parolari A, Pesce LL, Pacini D, Mazzanti V, Salis S,
Sciacovelli C, Rossi F, Alamanni F. Monzino Research
Group on Cardiac Surgery Outcomes. Risk factors for
perioperative acute kidney injury after adult cardiac
surgery: role of perioperative management. Ann Thorac
Surg. 2012;93(2):584-591
Cheema FH, Polvani G, Argenziano M, Pesce M.
Combining stem cells and tissue engineering in
cardiovascular repair -- a step forward to derivation of
novel implants with enhanced function and self-renewal
characteristics. Recent Pat Cardiovasc Drug Discov.
2012;7(1):10-20
Pompilio G, Filippini S, Agrifoglio M, Merati E, Lauri
G, Salis S, Alamanni F, Parolari A. Determinants
of pericardial drainage for cardiac tamponade
following cardiac surgery. Eur J Cardiothorac Surg.
2011;39(5):e107-113
Parolari A, Tremoli E, Cavallotti L, Trezzi M, Kassem S,
Loardi C, Veglia F, Ferrari G, Pacini D, Alamanni F. Do
statins improve outcomes and delay the progression
of non-rheumatic calcific aortic stenosis? Heart
2011;97(7):523-529
Loardi C, Alamanni F, Trezzi M, Kassem S, Cavallotti L,
Tremoli E, Pacini D, Parolari A. Biology of mitral valve
prolapse: the harvest is big, but the workers are few. Int
J Cardiol. 2011;151(2):129-135
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79
Cardiovascular Surgery Area
Cardiovascular Surgery Area Extracorporeal Circulation Service
Vascular Ultrasound Service
STAFF Coordinator: Marco Agrifoglio, MD, Ph.D
Assistants: Luca Dainese, MD, Francesco Grillo, MD,
Moreno Naliato, MD, Maurizio Roberto, MD, Claudio
Saccu, MD, Stefano Zoli, MD
Certified Clinical Perfusionists (CCP): Marcello Fonga,
Davide Pedroletti, Gianluca Riva, Vanessa Villani
STAFF Chief Perfusionist: Fabiana Rossi
Certified Clinical Perfusionists (CCP): Antonella Bertera,
Angelica Capozzoli, Rita Fabrizi, Marcello Fonga, Davide
Pedroletti, Gianluca Riva
The Extracorporeal Circulation (ECC) Service was
established in 1982 with the start of cardiac surgery.
With the help of specific devices, the ECC service
supports the vital functions of patients undergoing
cardiac surgery or interventional cardiology procedures
and it is involved in all situations where patients
urgently need a cardiovascular support.
Since 1982, about 18.328 ECC procedures have been
performed.
Nowadays four operating rooms are available with a
team of six technicians.
The indication for use of ECC involves both cardiac
and vascular surgery. Today ECC is also used as a
support during cardiovascular interventional cardiology
procedures and as assistance in patients with
hemodynamic compromise.
In 2011, 628 extracorporeal circulation procedures were
performed, 4 cardiopulmonary supports, and over 200
stand-by or complex vascular interventional procedures
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CCM — Scientific Report 2011 — Ongoing research 2012
and cardiosurgery.
A teaching course for perfusionists has been available
since 1988 and in 2001 it became a first level degree for
cardiovascular pathophysiology.
The Perfusion Education Program at Centro
Cardiologico Monzino was accredited by EBCP
(European Board of Cardiovascular Perfusion) in 2004.
The ECC service is certified ISO 9001:2008
The Vascular Ultrasound Service was created at Centro
Cardiologico Monzino in 1991 and it provides a noninvasive assessment of suspected vascular pathological
conditions. The vascular scan evaluations are carried out
both by cardiac and vascular surgeons with the most
sophisticated ultrasound equipments available. In 2011,
9813 procedures were performed (8434 in 2010) and a
number of technicians, highly trained and experienced in
vascular imaging techniques, was included in the staff.
Ultrasound interrogations allow to predict a wide range
of diseases and conditions.
Research activity
• preoperative non-invasive assessment of radial
artery for coronary artery bypass grafting
• preoperative non-invasive assessment of carotid
artery for coronary artery bypass grafting
• preoperative non-invasive assessment of
superficial arterial beds
Educational activity.
The students attending the Cardiac Surgery Postgraduate
School of the University of Milano are first trained
and subsequently employed in the service. To improve
education of both technologists and physicians, a
postgraduate course in Vascular Ultrasound is organized
every year at Centro Cardiologico Monzino
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Cardiovascular Surgery Area
Vascular and Endovascular Surgery Unit
Rita SPIRITO, MD
Director
Vascular and Endovascular Surgery Unit 2011
Surgical procedures
Endovascular procedures STAFF Deputy Directors: Luca Dainese, MD, Melissa Fusari, MD,
Piero Trabattoni, MD
Assistants: Claudio Saccu, MD, Stefano Zoli, MD
Cardiovascular Ambulatory: Amedeo Mereni, MD
Head Nurse: AnnaRita Leardi
Co-Head Nurse: Anna Sudati
Secretaries: Nadia Vaccari, Daniela Corti
Activities 2011.
The Unit provides diagnosis and
treatment for a wide variety of circulatory diseases such as
aneurysms of the thoracic aorta and/or the abdominal aortic
and its branches, extracranial cerebrovascular disease,
carotid artery disease, peripheral vascular surgery of the
upper and lower extremities including arterial occlusive
diseases and vein disorders (varicose veins or chronic
venous insufficiency). The Unit performs surgical and/
or minimally invasive endovascular techniques including:
balloon angioplasty and/or stenting, aortic and peripheral
vascular endovascular stent/graft placement and other
adjuncts for vascular reconstruction. During 2011, 514
patients with vascular pathologies were treated.
A new multifunctional operating room combining
endovascular, cardiac catheterization, cardiac, surgical
and radiological capabilities was opened in October 2008,
allowing a team composed by vascular, cardiac surgeons
and anaesthetists to perform transcatheter aortic valve
implants (transfemoral or transapical approach). Nowadays
the room allows, with the maximum flexibility, the treatment
of patients with the most complex cardiac and vascular
conditions. More than 8000 ultrasound tests were performed
during the last year with the most advanced diagnostic
equipments. Moreover, the use of a Multislice Computed
Tomography Angiography allows investigation of all aspects
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of vascular beds and body, particularly heart coronary
arteries. The vascular patient is often affected by multiple
disease processes and the optimal care is provided in a true
interdisciplinary environment. Since its birth in 1982, Centro
Cardiologico Monzino has always been focused on the
cooperation between surgeons and cardiologists. Our accurate
preoperative cardiac screening prior to vascular surgery
allows us to reduce the risk to develop cardiac complications,
lowering to 1% the risk of acute myocardial infarction in major
vascular interventions. Patients suffering from cardiovascular
pathologies usually undergo electrocardiogram (ECG) and
transthoracic Doppler echocardiography. Should these tests
be positive, a Dobutamine echo stress test is performed
by a cardiologist. We routinely perform echo-stress test in
patients older than 70 years because coronary artery disease
(CAD) risk is high and echo-stress test has good accuracy
in the identification of patients with significant angiographic
CAD. Patients with a previous history of coronary artery
disease, symptoms related to CAD or electrocardiography/
echocardiography/stress test abnormalities are evaluated with
coronary angiography. Patients without echocardiography/
stress test abnormalities are considered eligible for surgery.
Once patients at risk of cardiac disease have been identified,
the appropriate medical or surgical therapy has to be started
before vascular surgery. During the last year, in addition to
the clinical activity, our Unit has been involved in different
research activities. Important studies have focused on the
composition of atherosclerotic thrombus in abdominal
artery aneurysm as well as on the alteration of gluco and
proteoglycans in the artery wall. Moreover, the analysis
of oxidative stress role in patients undergoing carotid
artery stenting vs carotid artery tromboendoarterectomy is
ongoing; the Unit has also developed a study concerning
the absorption of mesoglycans in vein wall extracellular
matrix after oral administration. In the years to come the
Vascular and Endovascular Surgery Unit will be focused on
the development of a minimally invasive vascular pathologies
endoscopic treatment project in order to reduce patient
surgical trauma.
308
206
Particular attention is dedicated to the vascular and diabetic
wound management. Vascular ulcers are often chronic and
recurrent thus causing a considerable amount of morbidity
among patients with peripheral vascular diseases, including
work incapacity.
The vascular foot lesions are treated with endovascular
and/or surgical procedures. Moreover tissue substitutes –
like engineered tissue – are used for peripheral lesions.
exposure during thoracic endovascular aneurysm repair and
subsequent follow-up. Eur J Cardiothorac Surg. 2012 (Epub
ahead of print)
Publications
Dainese L, Guarino A, Burba I, Esposito G, Pompilio G,
Polvani G, Rossini A. Heart valve engineering: decellularized
aortic homograft seeded with human cardiac stromal cells. J
Heart Valve Dis. 2012;21(1):125-34
Agrifoglio M, Zoli S, Cappai A, Trabattoni P, Spirito R,
Biglioli P. Endovascular Treatment of Abdominal Aortic
Aneurysm After Previous Left Pneumonectomy: A Sound
Choice. Ann Vasc Surg. 2011;25(4):556e7-10
Zoli S, Trabattoni P, Dainese L, Annoni A, Saccu C,
Fumagalli M, Spirito R, Biglioli P. Cumulative radiation
Camera M, Brambilla M, Facchinetti L, Canzano P, Spirito R,
Rossetti L, Saccu C, Di Minno MN, Tremoli E. Tissue factor
and atherosclerosis: not only vessel wall-derived TF, but
also platelet-associated TF. Thromb Res. 2012;129(3):279-84
Dainese L, Biglioli P. Human or animal homograft: could
they have a future as a biological scaffold for engineered
heart valves? J Cardiovasc Surg (Torino). 2010;51(3):449-56
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Cardiovascular Surgery Area
Anaesthesiology and Intensive Care Unit
Anaesthesiology and Intensive Care Unit 2011
Erminio SISILLO, MD
Director
Co-director: Luca Salvi, MD
STAFF Deputy Directors: Claudio Brambillasca, MD,
Sebastiana Gregu, MD, Glauco Juliano, MD,
Valeria Mazzanti, MD, Guido Merli, MD, Stefano Salis, MD
Assistants: Cristina Beverini, MD, Paola Moliterni, MD,
Nora Piazzoni, MD, Paola Suriano, MD
Head Nurse: Pierangela Andreoletti
Nurses: Sonia Giulia Bianchi, Francesca Caggia, Herrera
Nancy Acenelia Camacho, Domenico Cavagnuolo, Hassan
Charoub, Paolo Crippa, Vito Cucchiara, Domenica
D’Ambrosio, Aurelio Della Corte, Cristiane Dias, Lucia
Di Palo, Giulio Orlando Fabbrizi, Sandra Faini, Silvana
Falchi, Claudio Fedele, Veronica Giurgila, Pavel Alin
Huci, Dagmar Lukovska, Laura Mantoan, Valeriu Ciprian
Palamariu, Marques Carla Simone Palmeira, Alessandra
Pappalettera, Luana Lucia Pellegrino, Valencia Maximo
Rodriguez, Garcia Pilar Guadalupe Valverde
Secretary: Rosalba Lamanna
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Cardiac and vascular operations Intensive Care Unit
Off-site anaesthetic procedures
Consultations N° Procedures
1200
850
510
1400
Activities 2011. The anaesthesiology team is
involved, on daily-basis, in case assessment together
with cardiologists and surgeons. Our Unit has developed
different techniques of anaesthesia according to different
kinds of surgery. In particular, anaesthetists take
care of patient health conditions in the postoperative
Intensive Care Unit, including 11 well-equipped beds
with advanced monitoring systems, in order to avoid
any postoperative dangerous complications (acute renal
injury, respiratory distress syndrome, cardiogenic shock)
that may contribute to precipitate the heart workload.
During the last years the Unit developed loco-regional
anaesthesia techniques to be used in different settings,
mainly involving spinal cord stimulation for refractory
angina and pain related to peripheral vascular disorders.
In association with general anaesthesia, some coronary
artery bypass grafts were performed with high thoracic
epidural anaesthesia in order to reduce the intra and
postoperative cardiac stress, reduce pain and hospital
length of stay. Moreover loco-regional anaesthesia is
mainly used in vascular surgery to perform carotid
endoarterectomy; since the patient is awake, it allows
a continuous assessment of consciousness level
during surgery. Furthermore the Unit has been one
of the first in Italy to routinely apply transesophageal
echocardiography in all cardiac and main vascular
operations. All the staff members are able to evaluate
cardiac anatomy and function and to properly assist the
surgeons in the achievement of the best results. Last but
not least, due to more and more sophisticated invasive
cardiology techniques, a skilled anaesthetist has become
mandatory in the catheterization laboratory, to guarantee
an adequate hemodynamic condition and sedation.
By the end of 2010, we provided computer-supported
multimodal bed side monitoring for all patients. The
surveillance software can facilitate therapeutic errors
prevention, improve therapy management and help
achieving a higher standard of care.
Publications
Sisillo E, Marenzi G. N-acetylcysteine for the prevention
of acute kidney injury after cardiac surgery. J Clin
Pharmacol. 2011;51(11):1603-10
Tamborini G, Muratori M, Maltagliati A, Galli CA, Naliato M,
Zanobini M, Alamanni F, Salvi L, Sisillo E, Fiorentini C, Pepi
M. Pre-operative transthoracic real-time three-dimensional
echocardiography in patients undergoing mitral valve
repair: accuracy in cases with simple vs. complex prolapse
lesions. Eur J. Echocardiogr. 2010;11(9):778-85
Bartorelli AL, Andreini D, Sisillo E, Tamborini G, Fusari
M, Biglioli P. Left main coronary artery occlusion after
percutaneous aortic valve implantation. Ann Thorac Surg.
2010;89(3):953-5
Salvi L, Beverini C, Maniglia P, Piazzoni N. Le
Catecolamine in cardiochirurgia sono un bene o un male?
Min Anest. 2009;75 Suppl 1:475-476
Sisillo E, Ceriani R, Bortone F, Juliano G, Salvi L, Veglia F,
Fiorentini C, Marenzi G. N-acetylcysteine for prevention
of acute renal failure in patients with chronic renal
insufficiency undergoing cardiac surgery: a prospective,
randomized, clinical trial. Crit Care Med. 2008;36(1):338-40
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Cardiovascular Imaging Area
The Cardiovascular Imaging Area is a diagnostic
department including three main areas:
Echocardiographic Laboratories, Cardiovascular
Radiological Imaging (Computed Tomography) and
Cardiac Magnetic Resonance. It is an academic
department with about 80 employees including
cardiologists, radiologists and bioengineers dedicated
to clinical, educational and scientific activities. Centro
Cardiologico Monzino has an agreement with the
Faculty of Medicine and the Cardiology and Radiology
Postgaduate Schools of the University of Milano, and a
convention with the Bioengineering Department of the
Politecnico of Milano closely cooperating with us.
The department has the most updated imaging
techniques in the three areas: 21 ultrasound units (6
portable and 3 in the operating rooms) including three
3D echomachines, six transesophageal probes (one
real time 3D probe), echo-stress, echo-contrast and
DTI modalities, intracardiac echocardiography. In the
Radiology Unit a 64 Cardiac CT is mainly dedicated to
cardiac imaging activities (using the most advanced
technologies to reduce radiation dose exposure as
prospective ECG gating and new post-processing
software). Cardiac Magnetic Resonance (“MR450
Discovery” with the magnetic field of 1.5 Tesla and
8-channels gradient coils) is performed by a dedicated
unit equipped with all main cardiovascular tools.
Portable echocardiographic units allow diagnostic
procedures in the Emergency Unit, Intensive
Cardiac Care Unit, catheter interventional and
electrophysiological laboratories, as well as
emergency echocardiograms in all the wards of
the hospital. Main diagnostic procedures include:
standard echocardiograms, echo-stress, echocontrast, transesophageal echocardiography, 3D
echocardiography, intraoperative echocardiography,
intracardiac echocardiography, cardiac computed
tomography of the coronary arteries, cardiac chambers
and peripheral vessels. Clinical activities are not only
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Mauro PEPI, MD Area Coordinator
related to all cardiovascular pathologies in the workup of different main diagnostic-clinical protocols
(approximately 130 main diagnostic work up protocols
in congenital, acquired chronic or acute cardiovascular
diseases), but also to several monitoring procedures
(percutaneous procedures in the catheterization and EPS
laboratories, intraoperative monitoring of percutaneous/
transapical aortic valve implantation, percutaneous
pericardiocentesis). An integrated imaging approach
(standard echo and echo-stress and/or cardiac CT,
echo and cardiac CT, intracardiac echo and CT, CMR)
is further improving the diagnostic work-up in new
procedures. Several clinical and research protocols,
closely related to new advanced techniques, have
been activated trying to better demonstrate the role
of 3D imaging in several pathologies (valve disease,
congenital heart diseases) and the importance of dose
radiation reduction in cardiac CT. As concerns this
latest advancement in cardiac CT, new software allow
a marked reduction in the radiation doses maintaining
feasibility, definition and diagnostic accuracy of the
technique. Cardiac Magnetic Resonance program further
reinforces the concept of an integrated approach to
cardiovascular pathologies, particularly in the field of
cardiomyopathies, cardiac ischemia, valvulopathies,
congenital heart diseases. CMR offers the unique
advantage to evaluating all cardiac chambers with high
spatial and temporal resolution in identifying (at rest and
during pharmacologic stress) myocardial characteristics
(thickness, viability, scars, perfusion). Moreover, precise
measurements of right and left ventricular volumes and
cardiac output facilitate quantitative evaluation of cardiac
diseases.
There is an excellent collaboration with all the
other units within the frame of disease-specific
multidisciplinary teams as it can be seen from the
distribution of the diagnostic equipments, staff training
and presence in all departments. The complexity of
the link between clinical activities of the staff inside
the core laboratories and inside the different wards
(including emergency rooms, operating theatre,
catheterization laboratories) reflects the evolving role
of both the cardiologist who (except the interventional
cardiologist) is deeply involved in imaging techniques
and the radiologist dedicated to cardiovascular diseases.
This is why in our units cardiologists and radiologists
fully collaborate in several activities and a new type of
cardiologist also trained (or specialized) in radiology has
been introduced.
More recently translational research projects have
been implemented thanks to several collaborations with
engineers.
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Cardiovascular Imaging Area
Echocardiography Unit
Echocardiography Unit 2011
N° Procedures
Total echocardiographic exams 23631
In-hospital exams
13351
Outpatients exams
10280
Transesophageal echocardiographic exams
1220
Stress echocardiographic exams
1383
3D-exams
620
Mauro Pepi, MD
Director
FULL-TIME STAFF Senior Deputy Directors: Paolo Barbier, MD,
Claudia Galli, MD, Anna Maltagliati, MD,
Gloria Tamborini, MD
Assistant: Paola Gripari, MD
Fellows: Claudia Cefalù, MD, Sarah Ghulam Ali, MD
Bioengineers: Laura Fusini, Francesco Maffessanti,
Federico Veronesi
Head Nurse: Laura Carparelli
Nurses: Dos Reis Satildes, Raffaella Fasso, Michela
Ferrantino, Laura Fumagalli, Maria Gagliardo, Maria
Salvatrice Giuttari, Rosanna Guadagno, Romina Locatelli,
Maria Labori, Giuseppina Manca, Concetta Maiorana,
Ennia Manoni, Inmaculada Marin Blanco, Donatella
Miodini, Sandra Robledo, Alessandra Sartori, Enza
Stefanizzi, Sabrina Zonelli
Secretaries: AnnaMaria Bellavia, Salvina Comignolo
PART-TIME STAFF Deputy Directors: Marina Alimento, MD, Fabrizio Celeste, MD,
Elisabetta Doria, MD, Gianfranco Lauri, MD,
Manuela Muratori, MD, Gianluca Pontone, MD
Assistants: Daniele Andreini, MD, Giovanni Berna, MD,
Jenesse Campodonico, MD, Gaia Cattadori, MD,
Alessandra Magini, MD, Marco Matturri, MD,
Francesca Susini, MD
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Activities 2011. Main diagnostic procedures
include: standard echocardiograms, echo-stress,
echo-contrast, transesophageal echocardiography, 3D
echocardiography, intraoperative echocardiography,
intracardiac echocardiography. Clinical activities are
not only related to all cardiovascular pathologies in the
work-up of different main diagnostic-clinical protocols
(approximately 130 main diagnostic work up protocols
in congenital, acquired chronic or acute cardiovascular
diseases), but also to several monitoring procedures
(percutaneous procedures in the catheterization and
EPS laboratories and intraoperative monitoring of
percutaneous/transapical aortic valve implantation,
percutaneous pericardiocentesis).
Emergency echo by portable ultrasound machines
is frequently performed in all wards of the institute
(approximatively 1000 / year).
Main research activities
Transthoracic 3D echocardiography
Transesophageal 3D echocardiography
Mitral valve prolapse and mitral valve repair
Advancements in mitral valve annulus 3D reconstruction
Advancements in aortic-mitral coupling through 3D
reconstruction
Left ventricular 3D shape in normal and pathological hearts
Right ventricular systolic function
Prosthetic valve dysfunction
New insights into left atrial function
New insights into transcatheter aortic valve implantation
through combined 3D echo and CT approaches
(prediction of post-TAVI aortic regurgitation, evaluation
of aortic annulus, imaging of the coronary ostia, postTAVI LV systolic function)
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Cardiovascular Imaging Area
Magnetic Resonance Imaging Unit
Gianluca PONTONE, MD
Director
STAFF Senior Assistant: Daniele Andreini, MD
Assistant: Paola Gripari, MD
Fellow: Erika Bertella, MD
Residents: Sarah Cortinovis, MD, Saima Mushtaq, MD
Chief Technician: Giuseppe Squilla
Deputy Chief Technician: Claudia Daniela Foti
Technicians: Alessandro Agozzino, Lorenzo Bonfanti,
Giorgio Missana, Marisa Mu
Head Nurse: Laura Carparelli
Nurses: Edoarda Baraggia, Carmen Cinieri,
Michela Ferrantino, Raffaela Putignano, Raffaella Sasso,
Catia Trudu
Secretaries: Emanuela Acanfora, Naty Mariconti,
Massimo Vanni
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Activities 2011.
In October 2010 the scanner
MR450 Discovery with a magnetic field of 1.5 Tesla
and 8-channels gradient coils was installed in our
hospital. This platform is one of the few developed and
used in the world mainly dedicated to cardiovascular
applications and providing a high image quality in
these clinical settings. The magnetic resonance (MRI)
is one of the most recent and innovative diagnostic
methods in the field of cardiac diseases. In this
context, the main applications are accurate and reliable
assessments of ventricular volumes, ejection fraction
and ventricular myocardial mass, using techniques
already in use in echocardiography, but with a greater
definition of the endocardial and epicardial contours.
Thanks to its excellent reproducibility, MRI can now be
considered the gold standard for studies of myocardial
perfusion and contractile reserve and for studies of
myocardial viability by identifying areas of scarring of
the myocardial wall, which today represent a milestone
for indication of revascularization procedures and
for prognostic stratification of patients with cardiac
disease. MRI can be used in many clinical settings such
as coronary artery disease, dilated cardiomyopathy,
myocarditis, hypertrophic cardiomyopathy,
arrhythmogenic right ventricular disease, congenital
heart disease, valvular disease, pericardial disease
and study of cardiac masses. In addition, MRI is a
valid alternative to MDCT in the evaluation of vascular
disease without the use of ionizing radiation and
therefore with a higher safety for repeatability in
patients requiring long and regular follow-up.
The clinical activity includes 8 cardiovascular MRI
each day, 50% scheduled for outpatients. The exams
are performed by physicians with experience in
cardiovascular imaging including echocardiography and
cardiovascular multidetector computed tomography.
Last year, 856 cardiovascular MRI and 194 stress
cardiac MRI were performed. The three main indications
were 1) coronary artery disease assessment (20%) 2)
left atrium evaluation in patients undergoing pulmonary
veins ablation for atrial fibrillation (13%) 3) ventricular
arrhythmias (11%).
The main research activities include applications in the
field of aortic annulus evaluation in patients undergoing
transaortic valve implantation, MRI-guided transcatheter
atrial fibrillation and dilated cardiomyopathy. About
the latter point, our Unit, in cooperation with the
Electrophysiology Unit, has been chosen as core-lab
for the international multicenter trial EMOSSID on
the use of cardiac MRI as gatekeeper for intracardiac
defibrillator device. Finally two international multicenter
trials have been started: CMR-BLOOD in cooperation
with the University of Los Angeles and Myocardial
Salvage Trial with the CNR of Pisa.
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91
Cardiovascular Imaging Area
Radiology Unit
Radiology Unit 2011
Giovanni BALLERINI, MD
Director
STAFF Senior Deputy Director: Enrica Maria Nobili, MD,
Gianluca Pontone, MD
Senior Assistant: Daniele Andreini, MD
Assistants: Andrea Annoni, MD, Alberto Formenti, MD
Fellows: Erika Bertella, MD
Residents: Francesca Besana, MD, Sarah Cortinovis, MD,
Saima Mushtaq, MD
Chief Technician: Giuseppe Squilla
Technicians: Alessandro Agozzino, Lorenzo Bonfanti,
Patrizia Bravi, Roberto Casalino, Elisa Consiglio,
Claudia Daniela Foti, Giorgio Missana, Marisa Mu,
Antonietta Porru
Nurses: Edoarda Baraggia, Carmen Cinieri,
Raffaela Putignano, Catia Trudu
Secretaries: Naty Mariconti, Massimo Vanni
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N° Procedures
Cardiac computed tomographies 1800
Aortic and vascular computed tomographies 3600
Radiological evaluations
21000
Ultrasound examinations 2500
Activities 2011. Since 2004 the high level of
collaboration between physicians and the administration
management, has allowed the achievement of a high
quality level in cardiovascular radiological diagnostic
methods, particularly in cardiac Multi Detector Computed
Tomography (MDCT).
In the Radiology Unit, a 64-slice cardiac MDCT has been
mainly dedicated to cardiovascular imaging activities,
using both the most advanced technologies to reduce
radiation dose exposure while maintaining feasibility,
definition and diagnostic accuracy, and the most
innovative post-processing software.
In January 2010, the new CT-Scan Discovery 750HD was
installed. In 2011 and early 2012 new software programs
were developed allowing both an improvement of the
time and space quality of images and a further reduction
of the dose delivered to the patient in vascular exams.
Together with the MDCT development, the traditional
radiology has been modified thanks to the
transformation of all diagnostics in computerized RX
and to the consolidation of the PACS (Picture Archive
and Communication System) which allows a realtime
availability of the exams with an efficient interfacing
with other diagnostic and care Units of the Institute.
The ultrasound colordoppler imaging system allows
diagnostic examinations in aortic and splanchnic vascular
pathologies also thanks to the recent progress of studies
with echo-contrast agents.
Research is fully integrated with the daily clinical
activity. In 2011 the main activity of MDCT was focused
not only on coronary MDCT in patients with low-tointermediate pre-test risk and in patients affected by
dilated cardiomyopathy, but also on the evaluation of
revascularized patients (coronary artery bypass e coronary
stents) and on different topics related to prognostic
evaluation in patients who executed cardiac MDCT.
Several studies were carried out to assess the feasibility
and precision of MDCT exams performed by using new
low dose protocols. Moreover about prognostic role of
CT coronary angiography in patients with suspected
coronary artery disease followed for 5 years, was
concluded.
We have developed a diagnostic MDCT pre-procedural
method to be applied in transcatheter aortic valve
substitutions carried out with a transfemoral or cardiac
transapical approach.
We are continuing the MDCT evaluation of sovra-aortic
vessels and intracranial circulation in patients to be
submitted to recanalization procedures and the preprocedure grading and follow-up in patients with aortic
pathology and endoprosthesis implantation.
Due to the fact that ultrasound investigation is non
invasive, specific attention has been dedicated to this
technique especially with regard to the renal arteries
in patients with suspected nephrovascular arterial
hypertension.
The Unit is engaged in educational programs, organizing
scientific meetings and theoretical and practical courses
on cardiac MDCT and vascular Doppler ultrasonography.
CCM — Scientific Report 2011 — Ongoing research 2012
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Cardiovascular Imaging Area
Publications.
Pontone G, Andreini D, Bartorelli A, Cortinovis S,
Mushtaq S, Bertella E, Annoni A, Formenti A, Nobili E,
Trabattoni D, Montorsi P, Ballerini G, Agostoni P and
Pepi M. Diagnostic accuracy of coronary computed
tomography angiography. A comparison between
prospective and retrospective electrocardiogram
triggering. J Am Coll Cardiol 2009; 54: 346-355
Tamborini G, Ajmone Marsan, N, Gripari P, Maffessanti
F, Brusoni D, Muratori M, Caiani E, Fiorentini C, Pepi
M. Reference values for right ventricular volumes
and ejection fraction with real-time three-dimensional
echocardiography: evaluation in a large series of normal
subjects. J Am Soc Echocardiogr 2010; 23: 109-115
Pepi M, Evangelista A, Nihoyannopoulos P, Flachskampf F,
Athanassopoulos G, Colonna P, Habib G, Ringelstein E, Sicari
R, Zamorano J on behalf of the European Association of
Echocardiography. Recommendations for echocardiography
use in the diagnosis and maanagement of cardiac sources of
embolism. Eur J Echocard 2010; 11: 461-476
Maffessanti F, Caiani EG, Tamborini G, Muratori M,
Sugeng L, Weinert L, Alamanni F, Zanobini M, Mor-Avi
V, Lang RM, Pepi M. Serial Changes in Left Ventricular
Shape Following Early Mitral Valve Repair.. Am J Cardiol
2010; 106(6):836-42
Ajmone Marsan N, Maffessanti F, Tamborini G, Gripari
P, Caiani E, Fusini L, Muratori M, Zanobini M, Alamanni
F, Pepi M. Left atrial reverse remodeling and function
imporvement after mitral valve repair in degenerative
mitral regurgitation: A real-time 4-dimensional
echocardiographyy study. Am Heart J 2011; 161: 314-321
Ewe SH, Muratori M, Delgado V, Pepi M, Tamborini G,
Fusini L, Klautz RJ, Gripari P, Bax JJ, Fusari M, Schalij MJ,
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Ajmone Marsan N. Hemodynamic and Clinical Impact of
Prosthesis-Patient Mismatch After Transcatheter Aortic
Valve Implantation. J Am Coll Cardiol. 2011;58(18):1910-1918
Pontone G, Andreini D, Bartorelli AL, Bertella E, Mushtaq
S, Cortinovis S, Chiappa L, Annoni A, Formenti A,
Trabattoni D, Montorsi P, Ballerini G, Fiorentini C,
Pepi M. Comparison between low-dose multidetector
computed coronary angiography and myocardial
perfusion imaging test in patients with intermediate pretest likelihood of coronary artery disease. Int J Cardiol
2011; 147(3):454-457
Andreini D, Pontone G, Bartorelli AL, Mushtaq S,
Trabattoni D, Bertella E, Cortinovis S, Annoni A, Formenti
A, Ballerini G, Agostoni P, Fiorentini C, Pepi M. High
diagnostic accuracy of prospective ECG-gating 64-slice
computed tomography coronary angiography for the
detection of in-stent restenosis : In-stent restenosis
assessment by low-dose MDCT. Eur Radiol 2011;
21(7):1430-1438
Pontone G, Andreini D, Bartorelli AL, Annoni A, Mushtaq
S, Bertella E, Formenti A, Cortinovis S, Alamanni F,
Fusari M, Bona V, Tamborini G, Muratori M, Ballerini G,
Fiorentini C, Biglioli P, Pepi M. Feasibility and accuracy
of a comprehensive multidetetctor computed tomography
acquisition for patients referred for balloon-expandable
transcatheter aortic valve implantation. Am Heart J
2011;161:1106-1113
Pontone G, Andreini D, Bartorelli L, Bertella E, Mushtaq
S, Annoni A, Formenti A, Chiappa L, Cortinovis S,
Baggiano A, Conte E, Bovis F, Veglia F, Foti C, Ballerini
G, Fiorentini C, Pepi M. Radiation dose and diagnostic
accuracy of multi detector computed tomography for the
detection of significant coronary artery disease: a metaanalysis. Int J Cardiol 2011; [Epub ahead of print]
Montorsi P, Caputi L, Galli S, Ciceri E, Ballerini G,
Agrifoglio M, Ravagnani P, Trabattoni D, Pontone G,
Fabbiocchi F, MD, Loaldi A, Parati E, Andreini D, Veglia
F, Bartorelli AL. Microembolization during carotid artery
stenting in patients with high-risk, lipid-rich plaque. J
Am Coll Cardiol 2011;58:1656–1663
Pontone G, Andreini D, Bartorelli AL, Bertella E,
Mushtaq S, Foti C, Formenti A, Chiappa L, Annoni A,
cortinovis S, Baggiano A, Conte E, Bovis F, Veglia F,
Ballerini G, Agostoni PG, Fiorentini C, Pepi M. Feasibility
and diagnostic accuracy of a low radiation exposure
protocol for prospective ECG-triggering coronary MDCT
angiography. Clin Radiol 2011; 67(3):207-215
Fusini L, Tamborini G, Gripari P, Maffessanti F, Mazzanti
V, Muratori M, Salvi L, Sisillo E, Caiani EG, Alamanni F,
Fiorentini C, Pepi M. Feasibility of Intraoperative ThreeDimensional Transesophageal Echocardiography in the
Evaluation of Right Ventricular Volumes and Function
in Patients Undergoing Cardiac Surgery. J Am Soc
Echocardiogr. 2011;24(8):868-877
Sagit Ben Zekry, Robert M Saad, Mehmet Özkan, Maie S Al
Shahid, Mauro Pepi, Manuela Muratori, Jiaqiong Xu, Stephen
H Little, William A. Zoghbi. Flow Acceleration Time and Ratio
of Acceleration Time to Ejection Time for Prosthetic Aortic
Valve Function. J Am Coll Cardiol Img, 2011; 4:1161-1170
Lang RM, Badano LP, Tsang W, Adams DH, Agricola E,
Buck T, Faletra FF, Franke A, Hung J, Pérez de Isla L, Kamp
O, Kasprzak JD, Lancellotti P, Marwick TH, McCulloch ML,
Monaghan MJ, Nihoyannopoulos P, Pandian NG, Pellikka
PA, Pepi M, Roberson DA, Shernan SK, Shirali GS, Sugeng
L, Ten Cate FJ, Vannan MA, Zamorano JL, Zoghbi WA.
EAE/ASE Reccomendations for image acquisition and
display using three-dimensional echocardiography. J Am
Soc Echocardiogr. 2012;25(1):3-46
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Laboratory Medicine Unit
Annalisa CAVALLERO, MD, Ph.D
Director
STAFF Deputy Director: Monica Raggi, DSc
Assistants: Tiziana D’Errico, MD, Giovanni Introcaso, DSc,
Daniela Riggio, DSc, Maria Rossi, MD, Roberta Temporiti, DSc
(Alessandra Ratto, MD)
Chief Technician: Paola Rumi
Technicians: Chiara Ardeleani, Laura Cadau,
Carmela Calvara, Cristina Frassi, Vanessa Guerra,
Chiara Miggiano, Massimo Moreo, Giorgia Piras,
Carmen Riva, Lorenza Rondelli, Simona Sabella
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Activities 2011. The Laboratory Medicine Unit
encompasses the fields of biochemistry, coagulation,
haematology, infectious disease serology, cancer
markers, drug monitoring and microbiology. It serves
both in and outpatients, and is also carrying out
diagnostic laboratory tests for other hospitals or medical
centres. Clinical chemistry and immunochemistry
sections are integrated and equipped with recent and
updated analyzers providing the best analytical and
clinical performance.
The coagulation section is certified for oral anticoagulant
monitoring in patients who undergo a long-term
therapy; the haematology section provides a first step
screening on patients admitted to the hospital needing
to undergo cardiovascular surgery. The microbiology
section, besides carrying out tests on in and outpatients,
performs culture tests for the Istituto Neurologico
Besta and particularly for the ICU and neurosurgery
departments. It also performs the microbiological tests
required for the activity of the Cardiovascular Tissue
Bank located at Centro Cardiologico Monzino by
assessing tissue sterility before and after disinfection
and by performing environmental controls to satisfy the
requirements for tissue sterile preparations.
The total number of tests carried out during 2011 was
about 1.100.000. Moreover, the Unit organizes the
supply of blood products by means of a dedicated team.
The laboratory staff is composed of a team working
very well together that meets and contacts daily the
physicians of the hospital departments, resulting in a
thorough vision of the patient health.
The daily internal quality controls, and the involvement in
external quality assessment programmes, either organized
by Regione Lombardia or by private companies, guarantee
the reliability of the test results. An updated online
computerized system connects the lab with the wards, so
that the results of the tests are rapidly available for the
physician, immediately after validation.
There is also an epidemiology programme that supplies
the Infection Control Committee with the results of the
microbiological research of the Institute. Moreover, the
medical staff of the laboratory, in association with FCSA
(Federazione Centri per la diagnosi della trombosi e la
Sorveglianza delle terapie Antitrombotiche), is in charge
of the supervision of the oral anticoagulant therapy to
outpatients.
Besides traditional diagnostic laboratory tests, the Unit
is involved in a research project on the evaluation of
new cardiac biomarkers in suspected acute coronary
syndrome (ACS) in collaboration with the Coronary
Care Unit. Furthermore, the laboratory collaborates with
other groups of Centro Cardiologico Monzino and other
hospitals in Milano on several research projects and
clinical trials.
Publications
Cattadori G, Wasserman K, Meloni C, Mustaq S, Contini
M, Apostolo A, Andreini D, Magrì D, Sciomer S, Veglia F,
Berna G, Introcaso G, Palermo P, Fiorentini C, Agostoni
P. Alveolar membrane conductance decreases as BNP
increases during exercise in heart failure. Rationale
for BNP in the evaluation of dyspnea. J Card Fail.
2009;15(2):136-44
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Introcaso G, Raggi M, D’Errico T, Cavallero A. Shortterm increases of plasma cardiac troponin I are better
evaluated by comparison with the reference change
value. Biochemia Medica 2010;20(3):327-33
Marenzi G, Giorgio M, Trinei M, Moltrasio M, Ravagnani
P, Cardinale D, Ciceri F, Cavallero A, Veglia F, Fiorentini
C, Cipolla CM, Bartorelli AL, Pelicci P. Circulating
cytochrome c as potential biomarker of impaired
reperfusion in ST-segment elevation acute myocardial
infarction. P. Am J Cardiol. 2010;106(10)
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Marenzi G, De Metrio M, Rubino M, Lauri G, Cavallero
A, Assanelli E, Grazi M, Moltrasio M, Marana I,
Campodonico J, Discacciati A, Veglia F, Bartorelli AL.
Acute hyperglycemia and contrast-induced nephropathy
in primary percutaneous coronary intervention. Am Heart
J. 2010;160(6):1170-7
Research Laboratories
Gatti S, Rama P, Matuska S, Berrilli F, Cavallero A, Carletti
S, Bruno A, Maserati R, and D Di Cave J. Isolation and
genotyping of Acanthamoeba isolates from corneal
infections in Italy. Med. Microbiol. 2010;5: 1324-30
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Laboratory of Biology and Biochemistry
of Atherothrombosis (4 Units)
Elena TREMOLI, Ph.D
Director
Alessandro PAROLARI, MD, Ph.D
Deputy Director
Head of the Clinical Research Unit
1. Marina Camera, Ph.D
Head of the Unit of Cell Biology and Biochemistry of Atherothrombosis
2. Cristina BANFI, Ph.D
Head of the Unit of Cardiovascular Proteomics
3. Viviana Cavalca, Ph.D
Head of the Unit of Biochemistry of Oxidative Stress and Endothelial Function in
Atherothrombosis
Activities 2011. The main goals of the
laboratory are:
•to understand how cardiovascular system
responds to physiological and pathological changes
•to identify new biomarkers of cardiovascular diseases
•to develop new therapeutic approaches
Within the laboratory there are 4 units of research,
which strictly cooperate with clinical researchers to
develop both basic and translational research.
4. Luigi Sironi, Ph.D
Head of the Unit of Experimental Thrombosis and Imaging in vivo
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1. Unit of Cell Biology and Biochemistry
of Atherothrombosis
Marina CAMERA, Ph.D
Head of the Unit
STAFF Daniela Boselli, PhD
Marta Brambilla, PhD
Laura Facchinetti, PhD student
Laura Rossetti, PhD student
Paola Canzano, MSc
Franco Moro, Technician
Activities 2011. The research activities
performed by the Unit of Cell Biology and Biochemistry of
Atherothrombosis are focused on the biochemical, cellular
and molecular mechanisms involved in the development
and progression of the atherothrombotic disease and on
their pharmacological modulation. To this aim, the Unit
uses 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 performed
on patients with cardiovascular disease. Furthermore,
a genomic and proteomic approach is also exploited in
order 1) to study the mechanisms of regulation and the
pharmacological modulation of key proteins involved in
thrombotic processes; 2) to identify genes and proteins
with altered expression in cells, tissue or biological
fluids of patients with cardiovascular diseases in order
to discover new pathogenetic mechanisms as well as
biomarkers able to predict the presence of disease at
an early stage, the evolution of the disease, or the risk
of clinical events; 3) to correlate the interindividual
variation in response to drugs with polymorphisms in
genes encoding target proteins (receptors, enzymes, ion
channels) of drugs (pharmacogenetics) in order to tailor
a personalized therapeutic intervention; 4) to identify
the role of polymorphisms and/or mutations in 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: 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
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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.
New pathogenetic mechanisms in acute coronary
syndrome: the role of platelet-associated Tissue Factor.
Tissue Factor is a 47 kD 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 of some antiplatelet drugs.
Recently we provided the evidence that TF expression is
significantly higher in resting platelets of patients with ACS
than in patients with SA. This results in a higher capacity
of platelet to generate thrombin, which in turn contributes
to the prothrombotic phenotype of ACS patients.
Chronic Kidney Disease in Acute Myocardial Infarction:
platelet function assessment and platelet transcriptomic
and proteomic analysis. Patients with chronic kidney
disease (CKD) have a higher incidence of coronary
artery disease (CAD). CKD causes modifications in
platelet reactivity leading to a higher rate of thrombotic
complications compared to CAD patients without CKD.
The underlying mechanisms have not been clarified
yet. Flow cytometric analysis of platelet and leukocyte
activation showed a platelet- and platelet-monocyte
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aggregates associated TF expression significantly lower
in CAD patients with CKD than in those without CKD,
both under basal conditions and after stimulation with
ADP. The reduction of TF observed might be attributable
to a continued consumption or release of TF-loaded
microparticles in patients with CKD. The finding that after
in vitro ADP stimulation only the expression of TF is
significantly lower in patients with CKD would support this
hypothesis. CAD/CKD platelets showed distinct transcript
expression profiles compared to CAD. In addition, the
expression level of several genes seemed to be influenced
by the glomerular filtration rate. One molecular function,
in particular, appeared to be affected by renal failure both
in the SA and in the NSTE-ACS platelets: the regulation
of actin cytoskeleton exerted by the integrin signaling
pathway. Finally, also the proteomic study revealed that
patients with CKD have a different plasma proteome
pattern in respect to patients without CKD both in the
stable angina and NSTEMI patients groups.
Percutaneous coronary and carotid intervention and
platelet activation. Drug-eluting stents (DES) are
the treatment of choice in percutaneous coronary
intervention (PCI). Concerns have been raised, however,
about a possible association between DES and an
increased late stent thrombosis rate. Dual antiplatelet
therapy with aspirin and clopidogrel is the standard of
care following DES implantation. Recently we showed
that, despite the dual antiplatelet therapy, platelet
activation in DES-treated patients is higher than in
medically treated SA patients and further increases one
month after thienopyridine discontinuation. Although the
classical markers of platelet activation tended to those
found in SA patients at 6 months after thienopyridine
withdrawal, TF-positive platelets remained significantly
higher than those of SA patients suggesting a direct
implication for late DES thrombosis.
Carotid Artery Stenting (CAS) is an evolving method to
treat carotid stenosis. Platelet activation in CAS occurs
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as a result of vessel wall damage and subendothelium
exposure leading to local thrombosis and distal
embolization (periprocedural risk of temporary or
permanent ischemic neurological deficits). The dual
antiplatelet regimen (aspirin+thienopyridine) has a
significant impact on reducing adverse neurological
outcomes. Our studies evidenced a significant higher
levels, compared to controls, of TF-positive platelets
circulating in peripheral blood of CAS patients. Of note,
dual antiplatelet therapy inhibits the expression of all
ADP-induced platelet activation markers, but TF, thus
suggesting that this prothrombotic platelet phenotype
may have implications for CAS complications.
Interindividual variation in the response to aspirin.
Several studies have reported that aspirin sometimes fails
to completely inhibit platelet function in patients with
cardiovascular disease. The phenomenon, called “aspirin
resistance”, may be due to several reasons, including
alternative “upstream” pathways of platelet activation,
as well as acquired or genetic factors. Moreover, the
assessment of aspirin resistance is not simple because
it is highly assay-dependent, and a general agreement
on standardised, reproducible and specific tests to
detect this phenomenon is still lacking. We performed
two studies on this issue. The first one is a functional
study in patients after coronary artery bypass graft
(CABG), who are at high risk of adverse cardiovascular
events, despite aspirin treatment; the second one was a
pharmacogenetic study carried out in coronary disease
patients. Our results indicate that, early after CABG, the
incidence of aspirin resistance is lower in patients who
received 325 mg aspirin compared to those who received
100mg ASA. Moreover, evidence is provided that different
methods yield different results in the detection of aspirin
resistance, rendering them not interchangeable. As far
as the pharmacogenetic study is concerned, we assessed
the influence of the GPIIb Ile843Ser SNP on the antiplatelet effects of ASA in 889 coronary disease patients
(CHD) and found that residual platelet aggregation and
TXB2 production in ASA-treated CHD patients differ
between GPIIb Ile843Ser genotypes. Carriers of the Ile/Ile
genotype may require higher doses of aspirin than Ser/
Ser patients to optimize anti-platelet therapy.
Characterization of human atherosclerotic carotid plaque
by histological techniques and multidetector computed
angiography. Plaque morphology is an important predictor
for the risk of stroke. Thus, it should be clinically relevant
to be able to predict the risk of stroke based on plaque
composition. Digital subtraction angiography (DSA) is
the gold standard for the assessment of the degree of
stenosis but it is unable to make any predictions about
plaque composition. We carried out a study to determine
the efficacy of multidetector computed tomography
(MDCT) to characterize carotid atherosclerotic plaque
morphology and composition as assessed by absolute
quantitative analysis of histologic images using a
dedicated software. We found a good correlation between
histology and MDCT (K=0.76) indicating that MDCT
angiography allows non-invasive assessment of the
carotid plaque composition.
TF SNPs and IMT. To Tissue Factor (TF), key initiator of
coagulation, roles have been ascribed not only in thrombosis
but also in atherosclerosis. TF gene promoter haplotypes
modulate TF expression, thereby potentially affecting
atherosclerosis. We have recently observed that the
haplotype-tagging TF A-603G polymorphism is associated
with carotid intima-media thickness, independently of
TF levels in plasma, providing clinical evidence of an
involvement of TF in atherosclerosis, in addition to its wellknown roles in haemostasis and thrombosis.
Inflammation in CAD and in bypass complications.
Inflammation plays a major role in the pathogenesis of
atherosclerosis and particularly in the development of the
vulnerable plaque. We reported that patients with a history
of onset of CHD as an ACS display a significantly more
intense serum amyloid A response to influenza vaccination,
a non-vascular inflammatory stimulus, than patients
with a history of onset of CHD as exercise-inducible
myocardial ischemia, suggesting a pro-inflammatory status
in patients prone to ACS but not in those with inducible
myocardial ischemia. An inflammatory response is also
induced by cardiopulmonary bypass in patients undergoing
cardiac surgery. Our studies in this field indicate that,
after coronary bypass surgery, there is a protracted
postoperative activation of inflammation persisting several
days after surgery; this postoperative activation is not
affected by the surgical strategy (on-pump or off-pump).
Publications
Camera M, Brambilla M, Boselli D, Facchinetti L, Canzano P,
Rossetti L, Toschi V, Tremoli E. Functionally active platelets
do express tissue factor. Blood 2012; 119:4339-4341
Camera M, Brambilla M, Facchinetti L, Canzano P, Spirito R,
Rossetti L, Saccu C, Di Minno MN, Tremoli E. Tissue Factor
and Atherosclerosis: Not only vessel wall-derived TF, but
also platelet-associated TF. Thromb Res. 2012;129(3):279-84
Capra V, Bäck M, Barbieri SS, Camera M, Tremoli E,
Rovati GE. Eicosanoids and Their Drugs in Cardiovascular
Diseases: Focus on Atherosclerosis and Stroke. Medicinal
Research Reviews 2012 [Epub ahead of print]
Di Minno MN, Guida A, Camera M, Colli S, Di Minno GD,
Tremoli E. Overcoming limitations of current antiplatelet
drugs: A concerted effort for more profitable strategies
of intervention. Ann Med. 2011;43(7):531-44
Colombo G, Gertow K, Marenzi G, Brambilla M, De Metrio
M, Tremoli E, Camera M. Gene expression profiling reveals
multiple differences in platelets from patients with stable
angina or non-ST elevation acute coronary syndrome.
Thromb Res. 2011; 128(2): 161-168
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2. Unit of Cardiovascular Proteomics
Cristina BANFI, Ph.D
Head of the Unit
STAFF Maura Brioschi, Ph.D
Simona Cosentino, Ph.D
Sabrina Lento, MSc
Stefania Ghilardi, Technician
Activities 2011. Proteomics, the study of an
organism’s complete complement of proteins, 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. Proteomicsbased studies are focused on the interactions of multiple
proteins and their role as part of a biological system
rather than the structure and function of one single
component. Proteins are indeed directly involved in
virtually all cellular activities and, as such, influence
cell phenotype and hence the tissues and organs. This
phenotype is the result of a dynamic, ongoing process
of protein expression and modification and varies both
under normal physiological conditions (e.g., cell-cycle
stage, differentiation, function and age) or in response to
pathophysiological stresses. Also environmental changes
have the potential to alter protein structure and function,
and may ultimately determine disease progression
and outcome. Proteomic investigations in the field of
cardiovascular disease involve the characterisation of
these modifications in order to identify novel therapeutic
targets and strategies to prevent the development of
cardiovascular diseases.
The goal of the Unit of Proteomics is the development
of 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 Laboratory 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
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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 can provide a powerful means
of identifying patho/physiological pathways in
cardiovascular diseases as it allows the simultaneous
detection of different circulating proteins and their posttranslational 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). In CABG we described an
increase of α1-antichymotrypsin and cathepsin G levels
which can reflect the generation of a proinflammatory
and of a prothrombotic state remarking the significant
involvement of neutrophil activation. Overall these data
suggest potential new therapeutic targets which may
help improving the results of this kind of surgery that,
in recent years, has been offered to sickest patients who
were earlier not considered to be good candidates or
even unsuitable for surgery. Although the involvement of
these pathways in the systemic stress response seems
to be quantitatively and quantitatively important, its
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role, the role of possible drug modulation and whether
it is beneficial or not, needs further investigation in
larger patient series. In addition, further studies are
also needed to analyse the possible relevance and
application of the different proteins as biomarkers of
clinical prognosis. In CAD the proteomic study revealed
that patients with chronic kidney disease (CKD) have
a different plasma proteome pattern in respect to
patients without CKD both in the stable angina and
NSTEMI patients groups. Therefore, with the application
of proteomic techniques, biomarkers can be identified
without previous knowledge of their involvement in
the pathogenesis of ACS, and its comorbidities, such
as CKD. However, the field is in the early stages of
evolution, and large-scale clinical studies are required to
validate the usefulness of newly identified biomarkers in
diagnosis, risk stratification, treatment and follow-up of
cardiovascular diseases.
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. Furthermore, by understanding the
multifaceted mechanisms involved in platelet interactions
with vascular surfaces and aggregation, new approaches
can be tailored to selectively inhibit the pathways most
relevant to the pathological aspects of atherothrombosis.
In this context, we observed that the platelet proteome is
altered in patients with stable or acute coronary syndrome
possibly as a consequence of the ongoing atherosclerotic
process. The identified protein changes, not previously
connected with coronary artery disease, were an increase
in the energy metabolism enzymes, and alterations in the
proteins associated with cytoskeleton-based processes,
both of which indicate platelet activation.
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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. Lipoprotein particles are stabilized in
the bloodstream by association with highly evolved
proteins called apolipoproteins, which through lipidbinding regions containing both hydrophilic and
hydrophobic surfaces facilitate binding between protein
and phospholipids. These stabilizing proteins also
mediate particle metabolism by binding to specific
receptors on cell surfaces, as well as acting as cofactors
for enzymes. For example, apoB-100 binds to arterial
wall proteoglycans, promoting subendothelial retention
of atherogenic LDL in early atherosclerosis. Besides
known LDL-associated proteins, 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.
Cellular microparticles (MP) are fragments shed from
plasma membrane blebs of virtually all cell types when
submitted to a number of stress conditions, including
apoptosis. MP release is an integral part of the
membrane-remodeling process in which the asymmetric
distribution of constitutive phospholipids between the
two leaflets is lost. MP have recently been shown to
reflect in vitro cell stimulation, and testify to cellular
activation and/or tissue degeneration occurring in vivo
under a variety of pathophysiologic circumstances.
Recent studies report the presence of endothelialderived MP in peripheral blood from patients with
lupus anticoagulant, TTP, acute coronary syndromes,
and even in blood of healthy individuals. Furthermore,
we have recently demonstrated that cardiomyocytes
release thrombogenic microparticles which in the setting
of cardiac diseases might contribute to an increased
thrombogenicity in the heart. Besides their characteristic
markers, MP have been indeed identified as true vectors
in the transcellular exchange of biologic information.
In addition to their direct effect on the promotion
and the amplification of the coagulation cascade,
MP participate in a variety of intercellular adhesion
processes and induce cellular responses. Functions of
MP should be reflected by their protein composition,
yet a comprehensive characterization of their
antigenic composition is still lacking. Thus proteomic
investigations are currently carried out to allow formal
identification of endothelial cells- and cardiomyocytesderived microparticles proteins, offering the possibility
to better understand their physiological role.
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 clinical
benefits of 3-hydroxy-3-methyl-3-glutaryl coenzyme A
(HMG-CoA) reductase inhibitors (statins) are strongly
related to their low density lipoprotein-cholesterol
lowering properties. However, because mevalonic
acid, the product of HMG-CoA reductase reaction,
is the precursor not only of cholesterol but also of
nonsteroidal isoprenoid compounds, the inhibition of
HMG-CoA reductase may result in pleiotropic effects.
Indeed, a variety of experimental data indicates that
statins can interfere with major events involved in the
formation of atherosclerotic lesions, independently
from their hypocholesterolemic properties, although
these effects have not been fully elucidated. We are
currently applying two methods for the identification
and quantification of proteins differentially regulated
by statins: the classical “gel based method” employing
2-DE, which offers the advantage to distinguish between
proteins isoforms as well as different post-translationally
modified forms of the same proteins, and a recently
developed “gel-free MS-based method” for “label-free”
quantitation, which provided absolute quantitative
profiling of proteins. The results coming from the
application of both approaches, validated by biochemical
assays and gene expression analysis, will allow us to
fully characterize the secretome of endothelial cells and
to identify the drug-regulated proteins, and will have
the potential to become a major focus of drug discovery
programs throughout the industry.
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. Systematic phenotyping by RNAi will provide
new perspectives on gene function in the context of the
genome on a gene-by-gene level and will allow to dissect
many important cellular processes with an unprecedented
spatial and temporal resolution. We are currently
exploring the inhibition effect of the Tissue Factor (TF)
expression by its specific siRNA in cardiomyocytes.
The relevance of this issue arises from the observation
that TF, switching the “on-off” of extrinsic coagulation
cascade as the initiating factor, has been revealed
presenting characteristics of signal receptor. TF-mediated
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signaling was demonstrated to be intimately correlated
with its nonthrombotic functions in tumor, angiogenesis
and inflammatory. In the cardiovascular setting, TF is
abundantly expressed by cardiomyocytes in the human
heart where it is present in the transverse part of the
intercalated disk and co-localizes with the cytoskeletal
proteins. In patients with dilated cardiomyopathy,
hypertension and ventricular hypertrophy, the TF
level was found to be reduced in structurally altered
ventricular myocardium. The reduction in TF expression
and change in localization may influence cell-to-cell
contact stability and contractility, thereby contributing
to cardiac dysfunction in DCM. Nevertheless, it has not
been completely elucidated how TF plays its role in
maintaining the structural integrity and contractility of
the myocardial muscle.
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, by using merging proteomics
and cellular biology approaches. Echocardiographically,
MVP is defined as a single or bileaflet prolapse, at
least 2 mm beyond the long-axis annular plane, with
or without leaflet thickening. The prevalence of MVP
is estimated at 2–3%, and is equally distributed
between men and women. MVP is expected to occur
in approximately 7.2 million individuals in the US, and
over 144 million worldwide. Currently, no diagnostic or
therapeutic approaches are available to identity patients
at high risk of developing MVP and mitral regurgitation
and/or to alter the progress of the disease. MV repair
is the most accepted type of surgical intervention for
severe MV regurgitation. The longevity of the repair, in
experienced hands, is remarkable. The biomechanical
forces present prior to the operation disappear
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postoperatively, and the valve dynamics are reset. The
removal of these mechanical forces upon the valve
leaflets in conjunction with a remodeling annuloplasty,
allows for a significantly improved leaflet cooptation
and valve performance. We could speculate that the
removal of the previously present stress forces onto the
valve leaflets is a fundamental factor in the resetting
the normal valvular cellular physiology and directly
responsible for the long-term durability of this type of
operation. Therefore, merging proteomic approach to
identify molecular determinants of MVIC cell biology
with clinical studies will help to better understand
when healthy quiescent MVICs get activated and start
remodeling ECM leading to MVP development.
Organelle proteomic. Focusing on specific organelle
proteomes is 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. In the
organelle proteomic approach, the identified proteins come
from distinct subcellular locations, which are often coupled
with the biological processes in which they participate,
thus providing the functional significance of proteomic
data. To this regard, a subproteome analysis of caveolin-1
enriched membrane microdomains of human hearts
allowed us to identify multiple proteins whose expression
is altered in heart failure, thus opening new perspectives to
determine which role they may play in this disease.
Redox proteomics. The field of redox proteomics,
although relatively new and rapidly changing, has the
potential to revolutionize how we diagnose diseases,
assess risks, determine prognoses, and target therapeutic
strategies for people with cardiovascular diseases.
Proteins are indeed the major targets for reactive oxygen
species because of their abundance in biological systems
and because they are primarily responsible for most
functional processes within cells. Oxidative changes
of protein structure are irreparable and can have a
wide range of downstream functional consequences,
such as inhibition of enzymatic and binding activities,
increased susceptibility to aggregation and proteolysis,
increased or decreased uptake by cells, and altered
immunogenicity. That oxidation of proteins plays an
essential role in the pathogenesis of an important number
of degenerative diseases is now recognized. We applied a
proteomic analysis focused at investigating the oxidative
stress-induced modification of proteins in plasma of heart
failure patients revealed the presence of specific targets
whose biological activity is altered, alpha-1-antitrypsin
and fibrinogen. Oxidation of alpha-1-antitrypsin
resulted in loss of its protease inhibitor activity, thus
leading to endothelial cell death. On the other hand,
fibrinogen, when oxidized, became otherwise cytotoxic
and induced apoptosis in endothelial cells. Overall our
findings suggest that oxidised biomolecules are not only
surrogate markers of oxidative stress, but may also
play a role in the development of heart failure. More
recently, we showed that oxidative stress-induced protein
modifications are increased in the myocardium of patients
with heart failure, and the proteomic approach made it
possible to ascertain that two proteins mainly underwent
carbonylation, M-type creatine kinase (M-CK), whose
activity is impaired, and, to a lesser extent, α-cardiac
actin. Exposure of cardiomyocytes to angiotensin II and
norepinephrine led to mitochondrial ROS generation and
M-CK carbonylation with loss of its enzymatic activity.
Our findings indicate that protein carbonylation is
increased in the myocardium during HF and that these
oxidative changes may help to explain the decreased CK
activity and consequent defects in energy metabolism
observed in heart failure.
Publications
Banfi C, Brioschi M, Barcella S, Wait R, Begum S,
Galli S, Rizzi A, Tremoli E. Proteomic analysis of
human low-density lipoprotein reveals the presence of
prenylcysteine lyase, a hydrogen peroxide-generating
enzyme. Proteomics 2009; 9(5):1344-52
Banfi C, Parolari A, Brioschi M, Barcella S, Loardi
C, Centenaro C, Alamanni F, Mussoni L, Tremoli E.
Proteomic Analysis of Plasma from Patients Undergoing
Coronary Artery Bypass Grafting Reveals a Protease/
Antiprotease Imbalance in Favor of the Serpin alpha1Antichymotrypsin. J Proteome Res. 2010;9(5):2347-57
Banfi C, Brioschi M, Marenzi G, De Metrio M, Camera
M, Mussoni L, Tremoli E. Proteome of platelets in
patients with coronary artery disease. Exp Hematol.
2010;38(5):341-50
Banfi C, Brioschi M, Lento S, Pirillo A, Galli S, Cosentino
S, Tremoli E, Mussoni L. Statins prevent tissue factor
induction by protease-activated receptors 1 and 2 in
human umbilical vein endothelial cells in vitro. J Thromb
Haemost. 2011;9(8):1608-19
Brioschi M, Polvani G, Fratto P, Parolari A, Agostoni
P, Tremoli E, Banfi C. Redox Proteomics Identification
of Oxidatively Modified Myocardial Proteins in Human
Heart Failure: Implications for Protein Function. PLoS
One. 2012;7(5):e35841
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3. Unit of Biochemistry of Oxidative Stress
and Endothelial Function in Atherothrombosis
Viviana CAVALCA, Ph.D
Head of the Unit
STAFF Sonia Eligini, Ph.D
Isabella Squellerio, Ph.D
Mauro Crisci, MSc
Benedetta Porro, MSc
Paola Songia, MSc
Loredana Boccotti, Technician
Activities 2011. Oxidative stress and
endothelial dysfunction are considered important topics
in the onset and progression of atherosclerotic and
cardiovascular diseases. Oxidative stress is the result of
the imbalance between the generation of free-radicals
and the antioxidant defence system and has been
related to physiological conditions, such as aging and
physical activity, and to many chronic and degenerative
pathologies. An important oxidant role has been ascribed
to overproduction of free radicals, in particular reactive
oxygen species (ROS). ROS are normal and beneficial
products of oxidative cellular metabolism. These
biochemically active free-radical derivatives of molecular
oxygen act as normal signalling molecules in the
vasculature; however, due to their reactivity, free radical
can induce oxidative damage to proteins, lipids and DNA
causing oxidative injury to vascular cells and promoting
endothelial dysfunction. As a consequence, a diminished
nitric oxide (NO) synthesis, an altered vasodilatation and
an increased platelet aggregation could be observed.
Besides endothelial cells, that are considered the major
source of NO, circulating cells (erythrocytes, platelets
and monocytes) are also able to synthesize NO. The
contribution of red blood cells (RBC) to NO biosynthesis
has been only recently highlighted.
Our group has a long-standing interest in metabolic
pathways involved in oxidative stress and NO synthesis.
Thus the research activities of the Unit are focused on
the study of oxidative stress in relation to endothelial
dysfunction and activation of circulating blood cells, as
platelets, leukocytes and red blood cells.
In our Unit, chromatographic methods (HPLC, LC-MS/
MS) with high sensitivity and specificity have been
set up in order to determine oxidative modifications of
proteins, lipids and nucleic acids in plasma and urine.
A pattern of antioxidant factors (vitamins, glutathione,
antioxidant enzymes etc.) and the total antioxidant
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capacity can be measured. With regards to NO synthesis
pathway, few studies have addressed the concomitant
evaluation of the multiple aspects of NO pathway, also
in consideration of the numerous compounds (substrate,
cofactors and inhibitors) involved in its biosynthesis and
metabolism. The introduction of innovative approaches
for the comprehensive and quantitative analysis of wide
arrays of metabolites has opened new perspectives for
the understanding of these mechanisms in vivo. This
emerging technology, called metabolomics, describes the
biochemical pathways in terms of metabolites with low
molecular weight present in cells, tissues, organs and
biological fluids. The components of the metabolome can
be detected as the end products of gene expression or
protein activity (enzymes), thus defining the biochemical
phenotype of a biological system as a whole. Our recent
efforts have been focused to set up an LC-MS/MS
method and detect Arginine/NO targeted metabolome in
plasma and RBC. Currently, we measure the substrate,
arginine, the inhibitors, symmetric- and asymmetric
methylarginines (ADMA, SDMA) and the enzymatic
cofactor, tetrahydrobiopterin. The metabolic products of
arginine (ornitine and citrulline ) are also measured.
During 2011 we have carried out several studies in
patients with different pathological conditions focusing
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our attention on the balance between pro and antioxidant
variables and their relationship with the production of
NO. In addition we focused our attention on the red blood
cells as important mediators of NO bioavailability. We set
up a method to investigate the arginine metabolome in
RBC to better highlight the role of these cells in Arg/NO
metabolic pathway in healthy and pathological conditions.
Oxidative stress and Arg/NO metabolic pathway in
patients undergoing cardiac surgery. We have investigated
oxidative stress and Arg/NO pathway in patients candidate
to the three most common cardiac surgical procedures
performed in adults in western countries: coronary
bypass surgery (CABG), aortic valve replacement for
calcific nonrheumatic aortic stenosis (AVS) and mitral
valve repair for degenerative mitral insufficiency (MVS).
Adult patients undergoing cardiac surgery have decreased
levels of antioxidants inducing an increase of oxidative
stress compared to control subjects and, in addition,
they show a trend towards impaired Arg/NO pathway
in respect to controls. Patients affected by mitral valve
regurgitation have more pronounced perturbations of
these pathways. In a principal component analysis, we
obtained, combining all these factors, a bi-dimensional
plot location of the four groups. As you can see in Fig 1,
MVS patients are located at a greater distance from the
controls than CABG and aortic patients, suggesting that
patients affected by mitral valve regurgitation show more
pronounced perturbations in the considered pathways.
Location of the four groups (means and SEM) in the plain defined by the two first principal components derived from markers of oxidative
balance and NO pathway
AVS is a degenerative and progressive condition which
shares some characteristics with atherosclerosis. The
association between oxidative stress and aortic fibrosis
was documented in animal studies; however, to date, few
data are available in humans. We measured markers of
oxidative and antioxidant defences and we showed an
oxidative damage due to lipid peroxidation but not to
protein oxidation in patients with aortic valve stenosis.
Plasma dimethylarginines and chronic kidney disease in
long-term outcome of non-ST elevation acute coronary
syndromes. The mechanisms linking chronic kidney disease
(CKD) to adverse outcomes in patients with acute coronary
syndromes (ACS), are not fully understood. Among potential
key players, reduced nitric oxide (NO) synthesis due to its
endogenous inhibitors, asymmetric dimethylarginine (ADMA)
and symmetric dimethylarginine (SDMA) might be involved.
In this study, we measured the concentrations of metabolites
implicated in the NO biosynthetic pathway (arginine, ADMA,
SDMA in plasma) in relation with renal function and longterm outcome in patients with ACS. Plasma arginine and
ADMA were similar in patients with or without ACS whereas
SDMA was higher in CKD patients. In ACS patients, SDMA
increases and is associated with poor long-term outcome
(the hazard ratio for adverse cardiac events was 2.95,
whereas that of CKD was 1.33) . An impaired NO synthesis,
as suggested by high SDMA levels, may account for the
adverse prognosis of ACS patients with CKD.
The Kaplam-Meier curves according to median SDMA value.
100%
SDMA<=0.46µm
SDMA>0.46µm
90%
CABG
Controls
Aortic
Mitral
% event free patients
Second principal component
80%
70%
60%
50%
40%
30%
20%
10%
0%
0
First principal component
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10
20
30
40
50
60
70
80
Months
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Metabolomic evaluation of NO production in human
erythrocytes: an LC-MS/MS method to assess arginine
and its metabolites. Experimental studies suggest that red
blood cells are involved in nitric oxide (NO) synthesis and
delivery. Erythrocytes act as producers, scavengers and
vehicles of NO affecting several physiological processes. NO
bioavailability is linked not only to arginine and its metabolic
products, ornithine and citrulline, but also to methylarginines
which are inhibitors of NO synthesis. Existing methods
do not allow a systematic evaluation of the metabolic
and biosynthetic pathway of NO in RBC. A metabolomic
approach, considering a larger number of compounds
involved in NO metabolic pathway, might be helpful to
understand the role of red blood cells in physiological and
pathological conditions.
We set up an HPLC-electrospray ionization-tandem mass
spectrometry method to simultaneously detect and quantify
arginine targeted metabolome, i.e. arginine, symmetric and
asymmetric dimethylarginine, monomethylarginine, ornithine,
citrulline. Our validated LC-MS/MS method might be useful
to clarify the role of RBCs in the synthesis of NO and will
be helpful for studies assessing the involvement of these
cells in the regulation of blood flow under physiologic
or pathological conditions. Moreover, a simple sample
processing without the derivatization step suggests that
this method might represent a valuable tool for diagnostic
evaluation of arginine metabolome in red blood cells, making
it applicable in clinical chemistry.
in response to growth factors, cytokines and chemokines,
they differentiate into macrophages by growing in size
and increasing their lysosomal compartment, hydrolytic
enzymes, mitochondria and energy metabolism. The process
of differentiation results in a heterogeneous population of
macrophages: they exhibit marked phenotypic differences
and functional diversity as a result of a differentiation
program that is subjected to environmental imprinting.
Primary tissue macrophages cannot be readily expanded
ex vivo and monocytic cell lines exposed to various agents
that promote differentiation, are commonly used. However
cell lines may not adequately address the important issue
of monocyte heterogeneity and the presence of lymphocyte
playing a pivotal role in macrophage differentiation and
maturation. In addition, different treatments may impact
on gene transcription. Although the use of cell lines is
advantageous in term of availability, data cannot be easily
Human macrophages obtained by spontaneous differentiation in
the presence of autologous serum.
extrapolated to differentiated tissue macrophages.
In our laboratory we developed a model of human
macrophages generated by spontaneous differentiation of
adherent monocytes in the presence of autologous serum
for 7 days. Mature macrophages show an increase in
cytoplasmic volume and organelles compared to monocytes.
Macrophages exhibit marked heterogeneity and the
analysis of the morphology shows distinct subpopulations
that may play distinct roles. Two different subsets are
mainly identified: spindle/elongated and round cells.
The characterization of the two subpopulations in terms
of morphology, cytoskeletal architecture, lipid content,
phagocytosis/efferocytosis and antigen expression may
be important to understand the implication of macrophage
heterogeneity in atherosclerosis and in other diseases.
Publications
Cavalca V, Colli S, Veglia F, Eligini S, Zingaro L, Squellerio
I, Rondello N, Cighetti G, Tremoli E, Sisillo E. The
anesthetic propofol enhances plasma tocopherol levels
in patients undergoing cardiac surgery. Anesthesiology
2008;108(6):988-997
Cavalca V, Veglia F, Squellerio I, Marenzi G, Minardi F, De
Metrio M, Cighetti G, Boccotti L, Ravagnani P, Tremoli E.
Glutathione, vitamin E and oxidative stress in coronary
artery disease: relevance of age and gender. Eur J Clin
Invest 2009;39(4):267-272
Veglia F, Cavalca V, Tremoli E. OXY-SCORE: a global index
to improve evaluation of oxidative stress by combining
pro- and antioxidant markers. Methods in Molecular Biology
2010;594:197-213
Squellerio I, Tremoli E, Cavalca V. Quantification of arginine
and its metabolites in human erythrocytes using liquid
chromatography-tandem mass spectrometry. Anal Biochem
2011;412:108-110
Di Minno MN, Pezzullo S, Palmieri V, Coppola A, D’Angelo
A, Sampietro F, Cavalca V, Tremoli E, Di Minno G. Genotypeindependent in vivo oxidative stress following a methionine
loading test: maximal platelet activation in subjects with
early-onset thrombosis. Thromb Res 2011;128(4):e43-48
A cell culture system to study macrophage differentiation
and heterogeneity. Monocytes and macrophages originate
from multipotent progenitor cells in bone marrow and play
a pivotal role in host defense to pathogens, wound healing,
angiogenesis, and various types of chronic inflammation
such as atherosclerosis. Under normal conditions,
monocytes migrate randomly to various tissues where they
differentiate into macrophages. The external signals that
control differentiation of peripheral blood monocytes are
incompletely defined, however once they reach a tissue
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4. Unit of Experimental Thrombosis and Imaging
in vivo
Luigi Sironi, Ph.D
Head of the Unit
STAFF Silvia Barbieri, Ph.D
Francesca Colazzo, Ph.D
Eva Tarantino, MSc
Activities 2011. The Unit is located in the
Department of Pharmacological and Biomolecular
Sciences of the University of Milano and is dedicated
to the study of vascular, cerebro- and cardiovascular diseases. These studies are carried out with
a multidisciplinary approach, taking advantage of the
strict collaboration among biologists, physicists and
engineers. The Unit has at its disposal a remarkable
combination of different imaging modalities together
with a high variety of different animal models, including
knock-outs, knock-ins and transgenic mice. All the
animals are kept and treated according to the European
rules on ethical conduct in the care and use of animals.
Research program focuses on pharmacological
control of brain ischemia, inflammatory diseases,
neurodegenerative processes, and renal failure.
Recently, the field of interest has been extended to
vascular thrombosis, heart failure and myocardial
ischemia and their therapeutical treatment. Moreover,
the pathogenesis of thrombotic disorders, particularly
regarding the role of plasmatic, vascular and cellular
mediators of thrombogenesis, is also investigated as well
as the molecular and cellular mechanisms involved in the
thrombus generation and progression.
The Unit owns an experimental scanner for magnetic
resonance imaging (MRI) analysis, a Bruker Avance II
operating at 4.7 T equipped with a super wide bore and a
micro-imaging device that provides gradients of 14 gauss/
cm. With two specific birdcage coils (6 cm and 3.8 cm
diameter) and a gas anaesthesia system, this equipment
is well suited to carry out studies in mice and rats and
allows to acquire “state of the art” diffusion tensor
images with fast imaging methods as well as to carry out
protocols for the detection of iron-oxide and gadolinium
contrast agents. This equipment has been recently
upgraded with a retrospective gating (IntraGate) for MRIanalysis of moving organs -in particular cardiac MRI.
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A VisualSonics Vevo2100 imaging platform for
echographic in vivo imaging and a Millar’s MPVS Ultra
system for the evaluation of cardiac parameters in
beating hearts are also available.
In addition, the Unit is equipped for biochemical,
histological and molecular analyses of ex-vivo tissues.
Main topics:
1. Use and development of MRI techniques in a
multidisciplinary approach in order to visualize
and follow a wide range of in vivo pathologies
(epilepsy, brain and heart ischemia, neuropathy,
spinal cord injury, renal diseases, heart failure).
Studies are also carried out in order to follow by
MRI tissue damage evolution.
2.MRI visualization of white matter organization in
animals with neurodegenerative diseases taking
advantage of fractional anisotropy (FA) and fiber
tracking (FT) MRI techniques.
3.Evaluation, by MRI scanning, of engineered
magnetic nanoparticles (MNPs) as contrast agents
for the in vivo visualization of inflammatory cells.
4.Echocardiographic studies of left atrium (LA)
in adult mice and evaluation of the relationship
between atrium and ventricle function in acute
cardiovascular conditions and in response to drug
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Program for the Control of Global Cardiovascular
Risk (3 Units)
Elena TREMOLI, Ph.D
Director
Alessandro PAROLARI, MD, Ph.D
Deputy Director
Head of the Clinical Research Unit
treatments, using also combined MRI and Echo
approaches.
5.Understanding the cellular and molecular
mechanisms involved in thrombus formation
caused by different risk factors (e.g. cigarette
smoke, hyperlipemia etc. ) using genetically
modified animals.
6.Understanding the molecular and cellular
mechanisms involved in vessel wall protection
using genetically modified animals.
7.Identification and validation of new potential
biomarkers of thrombosis in normal and
genetically modified mice.
Publications
Gelosa P, Banfi C, Gianella A, Brioschi M, Pignieri A,
Nobili E, Castiglioni L, Cimino M, Tremoli E, Sironi L.
PPAR-alpha agonism prevents the oxidative stress and
inflammatory processes involved in brain and renal
damage in stroke-prone rats. J Pharmacol Exp Ther.
2010; 335:324-331
Gelosa P, Sevin G, Pignieri A, Budelli S, Castiglioni
L, Blanc-Guillemaud V, Lerond L, Tremoli E, Sironi L.
Terutroban, a thromboxane/prostaglandin endoperoxide
receptor antagonist, prevents hypertensive vascular
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hypertrophy and fibrosis. Am J Physiol Heart Circ
Physiol. 2011;300:H762-H768
Franzosi M, Guerrini U, Castiglioni L, Sironi L, Nobili
E, Tremoli E, Caiani EG. Feasibility of quantitative
analysis of regional left ventricular function in the
post-infarct mouse by magnetic resonance imaging with
retrospective gating. Comput Biol Med. 2011;41:829-37
Barbieri SS, Ruggiero L, Tremoli E, Weksler BB.
Suppressing PTEN activity by tobacco smoke plus
interleukin-1beta modulates dissociation of VE-cadherin/
beta-catenin complexes in endothelium. Arterioscler
Thromb Vasc Biol. 2008;28:732-738
Barbieri SS, Zacchi E, Amadio P, Gianellini S, Mussoni
L, Weksler BB, Tremoli E. Cytokines present in smokers’
serum interact with smoke component to enhance
endothelial dysfunction. Cardiovasc Res. 2011; 90:475-483
Barbieri SS, Amadio P, Gianellini S, Zacchi E, Weksler
BB, Tremoli E. Tobacco smoke regulates the expression
and activity of microsomal-prostaglandin E synthase-1:
role of prostacyclin and NADPH-oxidase. FASEB J. 2011
[Epub ahead of print]
1. José Pablo WERBA, MD
Head of the Unit of Atherosclerosis Prevention
2. Damiano BALDASSARRE, Ph.D
Head of the Unit of Arterial Morphology
and Function
3. Fabrizio VEGLIA, Ph.D
Head of the Unit of Biostatistics
Cardiovascular diseases represent the number one
killer in developed countries. Scientific evidences
demonstrate the efficacy and cost/effectiveness
of specific vascular screening procedures, lifestyle changes and drug treatments in reducing
cardiovascular morbidity and mortality in patients with
overt cardiovascular disease (secondary prevention)
or at high relative or absolute risk (targeted primary
prevention). The identification and global control of
conventional and emerging risk factors is a mainstay
in decreasing the incidence and rate of recurrence of
cardiovascular events. These activities are carried
out by integrating patient care and focused clinical
research.
The cultural principle shared by all the members of the
team is: “make rational use of consolidated evidencebased early diagnostic tools and effective therapies,
and foster translational research aimed at improving
individual risk assessment and global risk reduction”.
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1. Unit of Atherosclerosis Prevention
José Pablo WERBA, MD
Head of the Unit
STAFF Costanza Boiti, MD
Monica Giroli, Biologist Nutritionist, Ph.D
Federica Laguzzi, MSc
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The Unit of Atherosclerosis Prevention provides
long-term clinical care and is run by specialists in
internal medicine, nutritionists and research fellows. A
specialized approach to prevention has demonstrated its
efficacy in improving not only the control of risk factor
but also patient prognosis in different centers both in
the United States and in Europe. So far, about 2900
patients were included in the program, most of them
in secondary or high risk primary prevention, the later
primarily focused on first degree relatives of probands
with overt disease. In tight link with patient care, the
Unit carries out original research initiatives aimed
to uncover the many yet-unidentified environmental
or genetic determinants of: 1) cardiovascular risk, 2)
earliness of vascular event presentation, 3) plaques
distribution within the arterial tree, 4) type of clinical
presentation and 5) event recurrence. In addition, the
Unit collaborates in studies aimed to investigate the
pathophysiology of heart-harmful diseases (e.g. obesity,
diabetes, liver steatosis, very high or very low HDL
cholesterol levels) as well as mechanisms of efficacy/
resistance, pleiotropic and side effects of consolidated
and novel drugs for the control of cardiovascular
risk. Dietary factors also play a fundamental role in
cardiovascular disease and its prevention. Our research
activities in this field focus on the search of an optimal
diet and potential nutrient-drug interactions. Biological
samples are systematically collected from patients to
create a plasma and DNA bank. The availability of a
clinical database and a biobank is extremely useful for
observational studies of associations between genotype
and clinical phenotype and for selection of patients to
be included in experimental studies of pharmacogenetics
and pharmacogenomic aimed to test the efficacy
and tolerability of drugs in different fields such as
thrombosis, diabetes, hyperlipidemia, hypertension, and
others. Information and biological samples collected are
safely stored to test new hypothesis that could emerge
along with upcoming scientific knowledge.
Publications
Baldassarre D, Castelnuovo S, Frigerio B, Amato
M, Werba JP, De Jong A, Ravani AL, Tremoli E,
Sirtori CR. Effects of timing and extent of smoking,
type of cigarettes, and concomitant risk factors on
the association between smoking and subclinical
atherosclerosis. Stroke. 2009;40(6):1991-8
Baldassarre D, Werba JP, Castelnuovo S, Frigerio B,
Amato M, Ravani A, Veglia F, Sirtori CR and Tremoli E.
The metabolic syndrome predicts carotid intima-media
thickness no better than the sum of individual risk factors
in a lipid clinic population. Atherosclerosis. 2010;210:214-9
Simiele F, Recchiuti A, Mattoscio D, De Luca A, Cianci
E, Franchi S, Gatta V, Parolari A, Werba JP, Camera
M, Favaloro B, Romano M.Transcriptional regulation
of the human FPR2/ALX gene: evidence of a heritable
genetic variant that impairs promoter activity. FASEB J.
2012;26(3):1323-33
Misaka S, Kawabe K, Onoue S, Werba JP, Giroli M,
Kimura J, Watanabe H, Yamada S. Development of rapid
and simultaneous quantitative method for green tea
catechins on the bioanalytical study using UPLC/ESI-MS.
Biomed Chromatogr. 2012 [Epub ahead of print]
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2. Unit of Arterial Morphology and Function
Damiano BALDASSARRE, Ph.D
Head of the Unit
STAFF Mauro Amato, Ph.D
Beatrice Frigerio, Ph.D student
Alessio Ravani, MSc
Daniela Sansaro, MSc
The Unit of Arterial Morphology and Function is focused
on the non-invasive assessment of early atherosclerosis.
Most studies are performed using B-mode ultrasound,
which provides information on the presence and extent of
atherosclerotic plaques, arterial wall thickness (intima-media
thickness or IMT) and vessel lumen diameter of superficial
arteries. These variables are used as “surrogate” markers of
atherosclerosis. The experience of the group, which was the
first to describe carotid IMT (C-IMT) in 1986, is internationally
recognized. The relevance ascribed to C-IMT by the scientific
community is evidenced by the ever-growing number of
studies which use this ultrasonic variable. Besides, changes
in C-IMT are the only marker of preclinical atherosclerosis
accepted by the American Heart Association, in place of
hard clinical endpoints, in pharmacological studies for the
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approval of new drugs. In 2008 the Unit was involved in
an International committee whose mission was to develop
Guidelines to transfer the scientific C-IMT knowledge into
clinical practice. This objective was driven also by findings
of our group, showing that C-IMT may improve the power
of vascular risk factors to predict new vascular events in the
large number of patients who, according to available risk
algorithms, fall into an intermediate risk category. In this
regard, the Unit is strongly committed in developing new
strategies for the better estimation of overall cardiovascular
risk based on the concept of “integrated biomarkers”. By
measuring acute changes induced by flow in the diameter
of the brachial artery (flow-mediated vasodilatation or
FMD), B-Mode ultrasound may also provide information
about endothelial function. Patients exposed to risk factors
show endothelial dysfunction even in the absence of overt
atherosclerotic disease. Indeed, endothelial dysfunction is a
very early index and predictor of atherosclerotic disease.
Main research topics of the Unit are:
• Identification of new atherosclerosis risk factors on the
basis of their effect on C-IMT and endothelial function.
• Improvement of new tools for the study of atherosclerosis and for improving predictability of vascular
events.
• Assessment of the association between C-IMT progression, conventional and emerging risk factors and
incidence of cardiovascular events.
• Assessment of the effects of pharmacological treatments on morphological and functional properties of
superficial arteries.
In addition, the Unit coordinates and acts as core laboratory
in national and international multicentre studies on the
epidemiology of atherosclerosis.
Publications
Baldassarre D, Castelnuovo S, Frigerio B, Amato M,
Werba JP, De Jong A, Ravani AL, Tremoli E, Sirtori
CR. Effects of timing and extent of smoking, type of
cigarettes and concomitant risk factors on the association
between smoking and subclinical atherosclerosis. Stroke.
2009;40(6):1991-8
intima-media thickness in a European population: the
IMPROVE study. Eur Heart J. 2010;31(5):614-22
Calabresi L, Baldassarre D, Castelnuovo S, Conca P, Bocchi
L, Candini C, Frigerio B, Amato M, Arca M, Boscutti G,
Cattin L, Gesualdo L, Sampietro T, Vaudo G, Veglia F,
Calandra S, Franceschini G. Functional LCAT is not required
for efficient atheroprotection in humans. Circulation.
2009;120(7):628-35
Baldassarre D, Werba JP, Castelnuovo S, Frigerio B, Amato
M, Ravani A, Veglia F, Sirtori CR, Tremoli E. The metabolic
syndrome predicts carotid intima-media thickness no better
than the sum of individual risk factors. Atherosclerosis.
2010;210(1):214-9
Baldassarre D, Nyyssönen K, Rauramaa R, de Faire U,
Hamsten A, Smit AJ, Mannarino E, Humphries SE, Giral
P, Grossi E, Veglia F, Paoletti R, Tremoli E, on behalf of
the IMPROVE study group. Cross-sectional analysis of
baseline data to identify the major determinants of carotid
Interleukin-6 Receptor Mendelian Randomisation Analysis
(IL6R MR) Consortium, A.D. Hingorani, J.P.Casas. The
interleukin-6 receptor as a target for prevention of coronary
heart disease: a mendelian randomisation analysis. Lancet
2012;379(9822):1214-24
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3. Unit of Biostatistics
Fabrizio VEGLIA, Ph.D
Head of the Unit
STAFF Francesca Bovis, MSc
Calogero C. Tedesco, MSc
The Unit of Biostatistics provides advice, support and
implementation in protocol design, quality control of
databases, data coding, input, and statistical analysis of
clinical studies performed by the clinical teams of Centro
Cardiologico Monzino. It collaborates in the design, and
is in charge of data analyses, of epidemiological, clinical
and experimental studies performed by the research Units.
It acts as reference data management and data analyses
centre in international epidemiological studies. In addition,
the Unit performs autonomous research activities on a wide
range of topics, focusing on epidemiology of cardiovascular
diseases and statistical methodology as follows:
• Epidemiology of subclinical atherosclerosis: links
with social class, geography and diet
• Development of innovative methods for the
analysis of complex ultrasound measures
• Development of summary scores based on clusters
of soluble markers of inflammation, oxidative
balance and endothelial function
• New models for the analysis of familial
aggregation of atherosclerosis
• Development of theoretical models of
atherosclerosis progression
• Development of new methods to assess carotid
IMT progression
• Improvement of statistical approaches aimed to
optimize algorithms for risk prediction.
Publications
Montorsi P, Caputi L, Galli S, Ciceri E, Ballerini G,
Agrifoglio M, Ravagnani P, Trabattoni D, Pontone G,
Fabbiocchi F, Loaldi A, Parati E, Andreini D, Veglia F,
Bartorelli AL. Microembolization during carotid artery
stenting in patients with high-risk, lipid-rich plaque.
A randomized trial of proximal versus distal cerebral
protection. J Am Coll Cardiol. 2011;58(16):1656-63
Pontone G, Andreini D, Bartorelli AL, Bertella E, Mushtaq
S, Annoni A, Formenti A, Chiappa L, Cortinovis S,
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Baggiano A, Conte E, Bovis F, Veglia F, Foti C, Ballerini
G, Fiorentini C, Pepi M. Radiation dose and diagnostic
accuracy of multidetector computed tomography for the
detection of significant coronary artery stenoses A metaanalysis. Int J Cardiol. 2011 [Epub ahead of print]
Di Carlo A, Lamassa M, Wellwood I, Bovis F, Baldereschi
M, Nencini P, Poggesi A, Cramaro A, Pescini F, Lucente
G, Wolfe C A, Inzitari D,European Registers Stroke EROS
Project. Stroke unit care in clinical practice: an observational
study in the Florence center of the European Registers of
Stroke (EROS) . Eur J Neurol 2011; 18(5):686-94
Calabresi L, Baldassarre D, Simonelli S, Gomaraschi M,
Amato M, Castelnuovo S, Frigerio B, Ravani A, Sansaro
D, Kauhanen J, Rauramaa R, de Faire U, Hamsten A, Smit
AJ, Mannarino E, Humphries SE, Giral P, Veglia F, Sirtori
CR, Franceschini G, Tremoli E. Plasma lecithin:cholesterol
acyltransferase and carotid intima-media thickness in
European individuals at high cardiovascular risk. J Lipid
Res. 2011;52(8):1569-74
Parolari A, Tremoli E, Cavallotti L, Trezzi M, Kassem S,
Loardi C, Veglia F, Ferrari G, Pacini D, Alamanni F. Do statins
improve outcomes and delay the progression of nonrheumatic calcific aortic stenosis? Heart. 2011; 97(7):523-9.
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127
Laboratory of Immunology
and Functional Genomics
Gualtiero COLOMBO, MD
Director
STAFF Federica Saporiti, Ph.D
Paolo Kunderfranco, Ph.D
Giuliano Stirparo, Ph.D student
Luca Piacentini, MSc
Elisa Bono, Technician
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The Laboratory of Immunology and Functional Genomics
was established in September 2010. The Lab work
is focused on the following research fields: (1) antiinflammatory pathways in the pathophysiology of
atherothrombosis; (2) immune-mediated mechanisms
that contribute to the formation of atherosclerotic
plaques and the occurrence of cardiovascular events; (3)
genomic approaches (such as transcriptome or targeted
genome resequencing projects) to uncover potentially
useful biomarkers and to disentangle gene functions
and interactions in atherosclerosis and cardiovascular
diseases (CVDs); (4) genetics of CVDs.
Functional genomics includes assessment of functionrelated aspects of the genome itself, such as analysis
of mutations that cause function loss or alteration,
as well as measurements of molecular activities
and investigations on dynamic aspects, such as
gene transcription and translation and their control.
The latter comprise a number of “-omics” such as
transcriptomics (gene expression, microRNA and non
coding RNA profiling), proteomics (protein expression),
phosphoproteomics and metabolomics. Together these
measurement modalities quantify the various biological
processes and power the understanding of gene and
protein functions and interactions.
The ultimate goal of the Laboratory is to identify
molecular biomarkers and/or clarify pathogenic
mechanisms that would help design therapeutic
strategies to prevent and/or reverse atherosclerosis
progression and/or cardiovascular events.
To achieve these objectives, the group components have
a solid technical experience in molecular and cell culture
techniques, as well as developed skills in analysis of
complex biological systems. The group focuses on the
analysis and mining of microarray data (genotyping,
gene expression, and microRNA profiling), proteomic
profiles (antibody arrays), and deep sequencing data
(transcriptome, epigenetic modifications, and targeted
resequencing). Using modern bioinformatics tools,
network-based analysis are performed relying upon
databases that integrate clinical, imaging, molecular,
and genomic data, in a systematic examination of the
pathophysiological bases of atherothrombosis and
related pathological conditions. The Lab work is also in
support for genomics studies in collaboration with other
research groups of the CCM.
Immunology and atherosclerosis: relevance in CVDs
Atherosclerosis is a complex chronic inflammatory
disease that affects large- and medium-size arteries
and induces alterations of the phenotypes of vascular
cells. All phases of atherosclerosis are regulated by
inflammatory mechanisms that provide overlapping
networks of pathways involved in the regulation of
immune cell functions, activation of endothelium, and
alteration of metabolic parameters. Increasing evidence
suggests that components of the immune system may
alter lipid metabolism and thus affect atherosclerosis.
Central in this field is the characterization of aberrant
responses of macrophages, B and T lymphocytes,
platelets, and vascular smooth muscle cells.
Most common forms of CVDs are believed to be
multifactorial and to result from interaction of many
genes, each with a relatively small effect, working
alone or in combination with modifier genes and/
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129
or environmental factors. The identification and the
characterization of these genes and their modifiers
would enhance prediction of cardiovascular risk.
Knowledge about when, where, and to what extent
a gene is expressed is critical for understanding the
activity and biological roles of the protein product of
the gene in relation to CVDs. Understanding the role of
these genes on inflammation and the immune system is
of great importance. All these data will help to identify
potential therapeutic targets for the prevention and
treatment of atherosclerosis and other CVDs.
Emerging technologies for functional genomics
Functional genomics apply genomic information by
investigating large sets of molecules (DNA, RNA,
proteins) in numerous samples of different origins
(extracts, cells, tissues) using a widening range of
tools, including advanced array technologies and nextgeneration sequencing. The Lab has been equipped with
a set of devices for multiple purposes.
DNA arrays are used for genotyping and expression
analysis. Genotyping technologies enable whole genome
association studies for complex disease gene mapping.
Several million common SNPs are known for the human
genome, potentially allowing inheritance to be traced
for factors that predispose to common conditions.
Transcriptome analysis, monitoring the activity of a
genome by measuring mRNA expression levels, provides
important biological insights. Recent clinical studies of
CVDs demonstrate that expression analysis of large
gene sets can identify molecular profiles correlated
with disease states, which may then be developed as
diagnostic tools.
Antibody arrays are used for protein expression studies
and as discovery tools in autoimmunity. Whole proteome
chips allow high throughput array-based characterization
of functional protein interactions.
Gene functions can be revealed by loss of function
assays. Gene knockdown by modulation of mRNA
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turnover using RNA interference (RNAi) has become the
favorite method for inhibiting expression of targeted
genes. RNAi has been combined with cell microarray
systems for screening, allowing analysis of the impact of
thousands of genes on many cell biological functions in
a single experiment.
Finally, a new generation of sequencing technologies
has provided unprecedented opportunities for high
throughput functional genomic research. To date,
these technologies have been applied in a variety of
contexts, including whole genome sequencing, targeted
resequencing, detection of transcription factor binding
sites by chromatin immunoprecipitation sequencing,
methylation analysis, gene expression profiling, and
non-coding RNA discovery and profiling. A typical
application is searching genomes and/or transcriptomes
for alterations that could explain individual phenotypic
variation, particularly disease. Another key area that
takes advantage from this technique is epigenetics,
which combines genetics and environment to address
genome plasticity and the control of gene expression by,
for instance, differential DNA methylation and histone
modification.
Bioinformatics & systems biology
In the present era of post-genome research, the
various high throughput -omics approaches allow to
think in terms of construction of conceptual models
of the living cell. This global and inter-disciplinary
approach, designated “systems biology”, requires the
organization and integration of complex experimental
knowledge and its expression in models with sufficient
predictive capacity. Systems biology aims to analyze
the relationships among the elements in a system in
response to genetic or environmental perturbations,
where a biological system may encompass molecules,
cells, organs, or individuals.
Systems biology acts as a natural link between
experimental functional genomics activities and
computational biology and bioinformatics. Relevant
computational steps are the organization of complex
genomics information, modeling of the biological
system, prediction of properties of uncharacterized
systems components, and the generation of hypotheses
concerning the reaction of the system to external
perturbation. The computational aspects of systems
biology require a combination of classical developments
in bioinformatics, followed by the application of
mathematical modeling tools. This unique combination
of expertise is inherently multidisciplinary and requires
input from biomedical experimental research, high
throughput technologies, life science image processing,
bioinformatics, and mathematics. This is the biggest
challenge to face for the Lab for the next future.
Colombo G, Sordi A, Lonati C, Carlin A, Turcatti
F, Leonardi P, Gatti S, Catania A. Treatment with
a-Melanocyte Stimulating Hormone preserves calciumhandling proteins in rat heart allografts. Brain Behav
Immun 2008; 22:817-23
Functional genomics & biomedicine
The technological advances in genotyping, expression
profiling, coupled with systems biology, offer exciting
and promising advances in cardiovascular medicine,
both in understanding the basis of CVDs and in
improved diagnosis and therapy. The combination of
-omics information will allow early and more accurate
diagnosis of disease and/or prediction of disease
onset and progression. In addition, the redefinition of
disease subtypes through functional genomics is likely
to provide insight of differential response to therapy:
understanding individual responses to drugs will have
implications for their use and development.
Colombo G, Gertow K, Marenzi G, Brambilla M, De
Metrio M, Tremoli E, Camera M. Gene expression
profiling reveals multiple differences in platelets from
patients with Stable Angina or non-ST elevation Acute
Coronary Syndrome. Thromb Res 2011; 128:161-8
Achilli F, Malafronte C, Lenatti L, Gentile F, Dadone V,
Gibelli G, Maggiolini S, Squadroni L, Di Leo C, Burba I,
Pesce M, Mircoli L, Capogrossi MC, Di Lelio A, Camisasca
P, Morabito A, Colombo G, Pompilio G. Granulocyte
Colony-Stimulating Factor attenuates left ventricular
remodelling after acute anterior STEMI: results of the
single-blind, randomized, placebo-controlled multicenter
stem cell mobilization in Acute Myocardial Infarction
(STEM-AMI) trial. Eur J Heart Fail 2010; 12:1111-21
Burba I, Colombo GI, Staszewsky LI, De Simone M,
Devanna P, Nanni S, Avitabile D, Molla F, Cosentino S,
Russo I, De Angelis N, Soldo A, Biondi A, Gambini E,
Gaetano C, Farsetti A, Pompilio G, Latini R, Capogrossi
MC, Pesce M. Histone deacetylase inhibition enhances
self renewal and cardioprotection by human cord bloodderived CD34+ cells. PLoS One 2011; 6:e22158
Publications
Cortelezzi A, Colombo G, Pellegrini C, Silvestris I,
Moronetti Mazzeo L, Bosari S, Lambertenghi Deliliers
G, Fracchiolla NS. Bone marrow glycophorin-positive
erythroid cells of myelodysplastic patients responding
to high-dose rHuEPO therapy have a different gene
expression pattern from those of non-responders. Am J
Hematol 2008; 83:531-9
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Laboratory of Vascular Biology
and Regenerative Medicine
Maurizio CAPOGROSSI, MD
Director
Giulio POMPILIO, MD, Ph.D
Deputy Director
Head of the Clinical Research Unit
STAFF Daniele Avitabile, Ph.D
Ilaria Burba, Ph.D
Chiara Cencioni, Ph.D
Yuri D’Alessandra, Ph.D
Elisa Gambini, Ph.D
Giuseppina Milano, Ph.D
Patrizia Nigro, Ph.D
Ombretta Pozzoli, Ph.D
Angela Raucci, Ph.D
Alessandra Rossini, Ph.D
Francesco Spallotta, Ph.D
Lorena Verduci, Ph.D
Valerio Azzimato, Ph.D student
Marco De Simone, Ph.D student
Viviana Meraviglia, Ph.D student
Matteo Vecellio, Ph.D student
Serena Balasso, MSc
Beatrice Bassetti, MSc
Maria Cristina Carena, MSc
Alessandro Scopece, MSc
Paolo Devanna, BSc
Consultant: Carlo Gaetano, MD
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The Laboratory of Vascular Biology and Regenerative
Medicine and the Clinical Research Unit of
Cardiovascular Regeneration work in synergy to
develop strategies for the treatment of cardiac and
peripheral vascular diseases. Basic molecular studies and
experiments in animal models of cardiac and hindlimb
ischemia are complementary to the translation of cell
therapy interventions to patients.
microRNAs in cardiovascular diseases
microRNAs in coronary artery syndromes. microRNAs
(miRNAs) are small non-coding RNAs that regulate
translation of mRNAs into proteins. Dysregulation in
the expression of cardiac miRNAs has been associated
with myocardial infarction and other forms of heart
disease. Recently, miRNAs have been identified in
the bloodstream and we have found marked changes
in the plasma levels of some circulating miRNAs in
patients and experimental animals with acute myocardial
infarction. These observations raise the possibility that
some miRNAs may be sensitive biomarkers of cardiac
damage and that they may also have biological effects
on some cell functions. Ongoing studies are aimed at
establishing circulating miRNAs sensitivity and specificity
as biomarkers of coronary artery syndromes and of MI
in patients with borderline troponin elevation.
microRNAs in oxidative stress. A variety of clinically
relevant conditions including tissue damage in response
to ischemia/reperfusion, diabetes, anti-cancer therapy
with anthracyclines and aging are associated with
enhanced oxidative stress. We have found that miR-200
family is markedly up-regulated upon cell exposure to
oxidative stress and plays a pivotal role in cell cycle
inhibition, senescence and death.
Cardiovascular epigenetics
Epigenetics and cell reprogramming. Epigenetic
mechanisms modulate lineage-decisions in uncommitted
cells; primitive stem cells have hyperacetylated histones
that become progressively deacetylated during their
commitment. Our laboratory focuses on the epigenetic
mechanisms that induce mesenchymal cell to acquire
a phenotype closer to that of myocardial and vascular
precursors and on drugs that act on chromatin structure,
including histone deacetylase inhibitor and acetylases
inhibitors/activators, and may confer enhanced plasticity
and therapeutic potential to cells for cardiovascular
transplantation. Specifically, we are currently
testing different combination of epigenetically active
molecules aimed at generating functionally competent
cardiovascular precursor from adult cardiac cells
Epigenetics of metabolic memory. Diabetes is a condition
in which all organs are exposed to high glucose for a
prolonged period of time and a generalized functional
impairment may occur as a consequence of the
hyperglycemia. Recent observations revealed that, even
after normalization of glycemia by insulin treatment
of other pharmacological interventions, the functional
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133
impairment often remains unchanged. Epigenetic cues
are called in as the mechanistic reason of this alteration
although their nature is still poorly characterized.
In our laboratory we recently observed that cardiac
and bone marrow-derived mesenchymal stem cells
may have different growth ratio in relationship to the
presence of diabetes in the donor patient. Specifically,
mesenchymal cells derived from diabetic patients do
not replicate as efficiently as those obtained from nondiabetic individuals. Biological and molecular analyses
revealed that histones and DNA methylation may play
an important role in determining this impairment.
Experiments are now in progress to investigate the
origin of this alteration with special attention paid to the
potential role of epigenetic enzymes including histone
(HMTS) and DNA (DNMTS) methylases, Lysine acetylases
(HATS) and deacetylases (HDACS) protein families.
Cardiovascular regeneration
Zebrafish as a model system for regenerative medicine.
Zebrafish (Danio rerio) is capable of complete cardiac
regeneration after partial ventricular resection and is
emerging as an excellent model for in vivo studies in
regenerative medicine. Our studies using this model
focus on the molecular mechanisms underlying the
regenerative cardiac response induced by hypoxia/
reoxygenation injury and on studies of stem cell
plasticity following their transplant in zebrafish.
HMGB1 and cardiac regeneration. High-mobility group
box 1 protein (HMGB1) is a chromatin protein that is
released by inflammatory and necrotic cells. Extracellular
HMGB1 signals tissue damage and modulates stem cell
function. We demonstrated that local delivery of HMGB1
in the mouse heart, acutely after infarction, induces a
regenerative response by activating resident cardiac stem
cells in conjunction with Notch/Wnt11 signalling pathways
activation. Further, we found that HMGB1 improves
left ventricular remodelling following myocardial
infarction and markedly enhances miR-206 expression
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in the heart; this effect was associated with diminished
collagen content in the left ventricle, increased matrix
metalloprotease (MMP) – 2 and -9 activity, and miR-206mediated down-regulation of TIMP-3.
Epicardial cells. During development, the epicardium is a
source of multipotent cells which give rise to coronary
vessels and cardiomyocytes. We have shown that the
adult epicardium contains a stem cell population which
can give origin to myocardial and vascular cells; these
cells are activated following acute myocardial infarction
and contribute to the regenerative response triggered
by acute ischemia and necrosis. Further, we have shown
that epicardial cells fuse with skeletal myotubes with
high efficiency and differentiate toward the skeletal
muscle phenotype.
Human cardiac cells for heart regeneration. The heart
contains resident stem cells that maintain homeostasis
by replacing lost myocytes. In our laboratory we
perform studies both with human cardiac c-kit+ cells
(CPCs) and Cardiac Stromal Cells (CStCs). Human
cardiac c-kit+ cells. We are correlating the clinical profile
of patients undergoing cardiac surgery with biological
features of CPCs obtained from right atrium surgical
specimens. Our preliminary results show that a number
of variables associated with patient’s demographic,
cardiologic status and medications have a different
impact on resident CPCs number and function. These
results may provide new insights for the understanding
of cardiac homeostasis. Human cardiac stromal cells. We
have identified a population of human cardiac stromal
cells with mesenchyma-like features (CStCs) which we
have compared to bone marrow stromal cells (BMMSC)
from the same patient. Although CMSC and BMMSC
share common mesenchymal markers, CstCs are more
competent than BMMSC in differentiating toward the
myocardial and vascular lineages, both in vitro and in
vivo. In addition, when injected into the heart in a rat
model of post-myocardial infarction heart failure, CMSC
differentiated into cardiomyocytes and improved cardiac
function more efficiently than BMMSC. Therefore,
CStCs appear as a potentially useful cell type for tissue
engineering approach. In this light, we are currently
evaluating the ability of CStCs to efficiently repopulate
decellularized aortic valve homografts potentially
useful for clinical applications. In addition, we are
presently comparing CStC ability to differentiate into
cardiovascular cell types with that of cells isolated from
a developmentally younger tissue, i.e. chorionic villus
stroma-derived cells.
Cardio-oncology. Cardiac toxicity is one of the
most devastating sequelae of cancer therapy with
anthracyclines and trastuzumab; it limits the possibility
of further therapy, worsens patients’ quality of life
and increases mortality. It is conceivable that CPCs
and CStCs may be obtained from endomyocardial
biopsy samples prior to initiating anticancer drugs
administration; subsequently, autologous cells may be
used to treat those patients who develop cardiac toxicity
and heart failure. Therefore we have developed a rodent
model of doxorubicin and trastuzumab cardiac toxicity
in which to examine the potential therapeutic action
of cardiac cell transplantation. Further, in this animal
model we are also examining miR-200 family role to the
response to anticancer drugs.
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135
Cell enhancement strategies. Peripheral blood- or bone
marrow-derived endothelial progenitor cells (EPCs)
induce neo-vascularization in ischemic tissues. However,
EPCs isolated from elderly patients and from individuals
with cardiovascular risk factors exhibit an impaired
therapeutic potential. Therefore, we characterized two
different pre-conditioning strategies aimed at enhancing
EPCs repair ability. We analyzed the effect of Valproic
acid (VPA), an epigenetic drug with histone deacetylase
inhibitory action. VPA treatment of CD34+ cells
isolated from human umbilical cord blood preserves an
immature subset of cells in culture and increases their
self-renewal ability. Furthermore, VPA-treated human
CD34+ cells improved ischemic myocardium protection
in an immunodeficient mouse model of myocardial
infarction. In addition, we examined the effect of
bone marrow-derived ckit+ cell (BM-derived EPC)
preconditioning with a brief exposure to low pH (acidic
pre-conditioning; APC). APC increased CXCR4 expression
and phosphorylation levels via a nitric oxide-dependent
mechanism. Further, APC enhanced SDF-1-directed
migration and differentiation into endothelial cells.
Lastly, APC-treated ckit+ cells exhibited an improved
angiogenic and regenerative potential following their
transplantation in a mouse model of hindlimb ischemia.
Clinical Research Unit of Cardiovascular Regeneration
is involved in clinical studies of cell therapy for
cardiovascular ischemic disorders, including myocardial
infarction complicated by ventricular dysfunction,
refractory ischemic cardiomyopathy, limb ischemia.
On the base of a previous multicenter trial (STEMAMI) on ST-elevated myocardial infarction, the
Unit is presently planning to coordinate a national,
multicentre, randomized, placebo-controlled, Phase III
trial to demonstrate that granulocyte colony-stimulating
factor (G-CSF) therapy, in addition to state-of-the-art
treatment, may significantly improve clinical outcome
in 1530 patients with reduced left ventricular ejection
fraction (≤45%) after successful reperfusion for
large anterior acute myocardial infarction (STEMAMI
OUTCOME). Furthermore, the Unit has consolidated
the experience in treating patients with refractory
myocardial ischemia using autologous, bone marrowderived CD133+ cells isolated under GMP conditions and
injected directly into the myocardium, finalizing a first
series of 10 patients with no-option refractory angina.
Finally, the Unit has a specific interest in critical limb
ischemia, on the base of previous Phase II and Phase III
trials (TALISMAN and TAMARIS) of gene therapy with
the growth factor FGF1.
GMP standardization of CD133+ cells isolation methods
for cardiovascular repair. The US Food and Drug
Administration (FDA) and the European Medicines
Agency (EMEA) have established regulatory frameworks
to ensure high safety standards and biological quality of
cell-based medicinal products (CBMPs) that must comply
with good manufacturing practice (GMP) specifications.
We have performed bone marrow-derived CD133+ cells
isolation and release under GMP compliant procedures;
these cells were used for autologous cell transplantation
in patients with refractory cardiac ischemia.
Publications
Spallotta F, Rosati J, Straino S, Nanni S, Grasselli A,
Ambrosino V, Rotili D, Valente S, Farsetti A, Mai A,
Capogrossi MC, Gaetano C, Illi Barbara. Nitric oxide
determines mesodermic differentiation of mouse
embryonic stem cells by activating class IIa histone
deacetylases: potential therapeutic implications in a
mouse model of hindlimb ischemia. Stem Cells. 2010.
28(3):431-42
Regenerative therapy and its clinical application. The
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Achilli F, Martelli F, Maggiolini S, Marenzi G, Pompilio
G, Maurizio MC. Circulating MicroRNAs are new and
sensitive biomarkers of myocardial infarction. Eur
Heart J. 2010. 31(22):2765-2773
Achilli F, Malafronte C, Lenatti L, Gentile F, Dadone V,
Gibelli G, Maggiolini S, Squadroni L, Di Leo C, Burba I,
Pesce M, Mircoli L, Capogrossi MC, Di Lelio A, Camisasca
P, Morabito A, Colombo G, Pompilio G. Granulocyte
colony-stimulating factor attenuates left ventricular
remodelling after acute anterior stemi.Results of the
single-blind, randomized, placebo-controlled multicenter
stem cell mobilization in acute myocardial infarction
(STEM-AMI) trial. Eur J Heart Fail. 2010. 12(10):1111-1121
Colussi C, Rosati J, Straino, Spallotta F, Berni R, Stilli
D, Rossi S, Musso E, Macchi E, Mai A, Sbardella G,
Castellano S, Chimenti C, Frustaci A, Nebbioso A, Altucci
L, Capogrossi MC, Gaetano C. Nε-lysine Acetylation
Determines Dissociation from GAP-junctions and
Lateralization of Connexin 43 in Normal and Dystrophic
Heart. Proc Natl Acad Sci. 2011. 108(7):2795-2800
Cencioni C, Melchionna R, Straino S, Romani M,
Cappuzzello C, Annese V, Wu JC, Pompilio G, Santoni
A, Gaetano C, Napolitano M, Capogrossi MC. Ex vivo
acidic preconditioning enhances bone marrow ckit+ cell
therapeutic potential via increased CXCR4 expression.
Eur Heart J. 2011. [Epub ahead of print]
D’Alessandra Y, Devanna P, Limana F, Straino S,
Di Carlo A, Brambilla PG, Rubino M, Carena MC,
Spazzafumo L, De Simone M, Micheli B, Biglioli P,
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Laboratory of Cardiovascular Tissue Engineering
Gianluca POLVANI, MD
Director
Maurizio PESCE, Ph.D
Deputy Director
STAFF Maria Cristina Vinci, Ph.D
Rosaria Santoro, Ph.D
Francesca Prandi, Ph.D student
The mission of the Laboratory of Cardiovascular Tissue
Engineering is to devise strategies to derive a new
generation of cardiovascular implants and devices,
by combining cardiovascular stem cell knowledge
and material/biomechanic expertise. Starting from
basic investigations on human-derived cardiovascular
progenitors such as cord blood-/peripheral blood-/
bone marrow-derived endothelial progenitor cells
(EPCs), cardiac resident stem cells (CSCs), saphenous
vein progenitors (SVPs) and the so called aortic
valve interstitial cells (VICs), the mechanism linked to
physiological or pathological differentiation of these cells
is being studied by applying “high content” molecular
analysis and high throughput material screening methods
which allow a rapid search for novel bio-materials
(polymers, hydrogels and biological scaffolds) to
embed stem cells in culture. In this scenario, also the
investigation on strain- and mechanical stress-associated
stem cells response appears particularly relevant.
Histone code of human endothelial progenitor cell
function and disease: from memory to risk. (Fig. 1)
Bone marrow-derived endothelial progenitor cells (EPCs)
play a role in physiological endothelial cell turnover
through innate endothelialization capacity, and protect
arteries from atherosclerosis and damage-associated
stenosis. The amount and the activity of circulating EPCs
are dramatically decreased with age, in patients with
diabetes, and in other risk conditions associated with
cardiovascular diseases (CVD). Therefore, the use of these
cells for engineering blood vessels appears limited. While
attempts have been made to restore innate EPCs functions
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through various pharmacological interventions, the
molecular basis of these defects remain elusive. Recent
data have highlighted the existence of an epigenetic
memory, which can be established as a consequence
of metabolic dysfunction and risk conditions. At the
molecular level, these defects are “written” in the genome
as changes in the DNA and chromatin modifications
underpinning an altered gene expression.
In the attempt to mimick risk conditions for potential
phenotypic and functional transformation of human EPCs,
we have set up an assay to expose these progenitors to
oxidized LDL, a form of lipoprotein abundantly present in
the atherosclerotic plaque and known to activate innate
immunity associated to atherosclerosis progression. We
have found that these cells are converted in functionally
active antigen presenting cells, able to activate
T-lymphocytes proliferation in mixed leukocyte reactions
(MLR), and secrete a variety of pro-inflammatory
cytokines. Molecular profiling of these cells and their
epigenetic makeup in response to Ox-LDL exposure, are
currently being analyzed.
This research is performed by our Laboratory in
collaboration with the College of Physicians and
Surgeons, Columbia University, New York, USA, and
the Laboratory of Functional Genomics at Centro
Cardiologico Monzino.
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139
Fig. 1
Fig. 2
Circulating pro-angiogenic cells (CFU-ECs)
Human SVPs - Passage 1
oxidized-LDL
SV CONDITIONING BIOREACTOR
Antigen presenting cells
(APCs)
Pressure transducer
Pc
Patm
Valve
SV housing
Reservoir
Pump
SV
Human SVPs - Passage 4
Lymphocyte (➞) activation
A mechano-biology approach to understand the role of saphenous
vein-resident vascular progenitors (SVPs) in the establishment and
progression of vein bypass graft disease. (Fig. 2)
Implantation of saphenous vein (SV) segments is routinely
used to generate aorto-coronary bypass grafts (CABG)
to revascularize the ischemic myocardium. It is estimated
that between 10 and 15 years after implantation, 50%
of vein bypasses undergo functional failure (vein graft
disease, VGD), due to accumulation of smooth muscle
cells (SMCs) in the intima. While molecular and signaling
pathways linked to smooth muscle cell activation in VGD
have been widely investigated, the role of other veinintrinsic and vein-extrinsic cells is less clear.
This issue is relevant in the light of the recent results
obtained in animal models, showing that cells recruited
from circulation (i.e. inflammatory cells) reside in the
graft intima, where they may activate vessel-resident
cells or directly differentiate into SMCs, or that vesselresident progenitors are triggered to differentiate
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CCM — Scientific Report 2011 — Ongoing research 2012
into SMCs by signaling associated to vascular injury/
mechanical stress. Vascular progenitors endowed with
clonogenic potential and differentiation ability have
been recently found into human SVs. These cells, named
saphenous vein progenitors (SVPs), can be grown ex vivo
from expansion of primary CD34+CD31- cells obtained
from enzymatic digestion of SV segments. To assess
the role of biomechanics in possible SVP-mediated
phenotype changes, we have performed stimulations with
a commercially available bioreactor (FlexCell System),
using a custom made bioreactor platform tailored to
perform vessel conditioning in an organ culture condition.
Use of these tools showed a crucial role of biomechanics
in SVPs differentiation and growth control, as well as in
changes of vein structure and remodeling.
This research is performed by our Laboratory in
collaboration with the Dept of Biomedical Engineering of the
Politecnico of Milan, and the Laboratory of Regenerative
Medicine of the Bristol Heart Institute, Bristol, UK.
Analysis of the interaction between stem cells and biopolymers using high-throughput screening techniques:
towards engineering of the artificial cardiovascular stem
cell niche. (Fig. 3)
Recent integration of the high content imaging platforms
with the “micro-patterning” (e.g microcontact- or inkjetprinting) techniques has allowed emergence of a new bioengineering branch tailored to study (stem) cell behavior
under condition of confinement into geometrically defined
or chemically definite composed micro-environments. In
addition, reproduction of physiological or pathological
mechanical forces in micrometer scale by use of the
so called “lab-on-chip” fabrication philosophy, allows
stem cells interrogation for multiple basic responses
(differentiation, proliferation, markers modulation,
modifications in the epigenetic landscape) to complex
and combinatorial environmental conditions, in a single
experimental setting.
In the present project, the interactions between cardiac-
derived c-kit+ cells and of the so called valve interstitial
cells (VICs) onto polymer arrays is currently being
analyzed making use of this recent approach. This
will lead to identify the best polymers to grow cardiac
and valve derived progenitors with “niche”-compliant
conditions. The final result will be the formulation
of “smart” materials to instruct auto-assembly of
myocardial tissue and aortic valve artificial leaflets with
native-like properties.
This research, is carried by our laboratory in
collaboration with the Laboratory of High Throughput
Chemistry of the University of Edinburgh, UK.
Decellularization of the human pericardium: towards the
generation of a biocompatible scaffold for aortic valve
tissue engineering. (Fig. 4)
Engineering of aortic valve (AoV) leaflets with
characteristics of native tissue is an important step
toward derivation of AoV implants with maximal
CCM — Scientific Report 2011 — Ongoing research 2012
141
Fig. 3
Fig. 4
INKJET-PRINTING/CHIP ARCHITECTURE AND DESIGN
13x16 polymers grid
Fresh
Spot shape, pitch and dimensions
MICROCONTACT PRINTING (μCP)/CELL CULTURE
biological compliance and durability. This might be
advantageous compared with the use of animal-derived
bio-prosthetic valves, donor-derived homografts,
or mechanical valves. This possibility is particularly
attractive for pediatric AoV surgery. In fact, pediatric
patients need to be transplanted with valve tissues able
to grow in concert with the heart, thereby generating
durable grafts that do not need further substitution
during the life span.
Pericardium from different animal species has been
introduced to generate the so called bio-prosthetic
valves. The major limitation of these devices is
that aldehyde fixation, commonly used to prepare
pericardium of animal origin, causes calcification and
mechanical failure over time due to the absence of an
active cellular machinery necessary for remodeling
and renewing extracellular matrix components and the
presence of cellular remnants, which act as a calcium
“nucleation” centers. We have setup a method to
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CCM — Scientific Report 2011 — Ongoing research 2012
Decellularized
decellularize the human pericardium using hypotonic
buffers preserving mechanical resistance of the tissue in
strain-stress tests, also after cryo-preservation. Further,
decellularized pericardium was implanted subcutaneously
for up to two months into immunocompetent mice, to
analyze rejection and calcification. The results showed
that decellularization using the adopted method, with or
without cryopreservation, granted an optimal tolerance
of the human tissue.
This research is performed by our laboratory in
collaboration with the Dept of Biomedical Engineering of
the Politecnico of Milan, the Dept of Pharmacology of
the University of Milan and the Cardiovascular Tissue
Bank at Centro Cardiologico Monzino.
40x
40x
40x
40x
20x
20x
Publications
Burba I, Colombo GI, Staszewsky LI, De Simone M,
Devanna , Nanni S, Avitabile D, Molla F, Cosentino S,
Russo I, De Angelis N, Soldo AR, Biondi A, Gambini E,
Gaetano C, Farsetti A, Pompilio G, Latini R, Capogrossi,
MC, Pesce M. Histone Deacetylase inhibition enhances
self renewal and cardioprotection by human cord bloodderived CD34+ cells. PLoS One 2011;6(7): e22158
Cheema FH, Polvani GL, Argenziano M, Pesce M.
Combining stem cells and tissue engineering in
cardiovascular repair – a step forward to derivation of
novel implants with enhanced function and self renewal
characteristics. Recent Pat. Cardiovasc. Drug Discov.
2012;7(1): 10-20
perspectives from pericardium? IBiomaterials and Stem
Cells in regenerative Medicine (Murugan Ramalingam,
Seeram Ramakrishna and Serena West Eds),2012, CRC-Press
Pesce M, Pompilio G, Polvani GL, Capogrossi MC. When
Stemness meets Engineering: towards “Niche” Control
of Stem Cell Functions for Enhanced Cardiovascular
Regeneration. In (Gustav Steinhoff Ed.), Regenerative
medicine (Second Edition). Springer 2012
Vinci MC, Polvani GL, Pesce M. Epigenetic programming
and risk: the birthplace of cardiovascular disease? Stem
Cell Rev Rep, 2012
Vinci MC, Prandi F, Micheli B, Tessitore G, Guarino A,
Dainese L, Polvani GL, Pesce M. Natural membranes
as scaffolds for biocompatible aortic valve leaflets:
CCM — Scientific Report 2011 — Ongoing research 2012
143
Plasma and Gene Bank
The bank collects plasma and genes from patients
undergoing aortocoronary bypass grafts (CEC or no
CEC) in order to develop research on cardiovascular risk
factors
Clinical Research
DNA Bank
Since 2002, a genomic DNA bank collecting DNA
from patients affected by coronary diseases and/
or other atherosclerotic pathologies as well as from
healthy patients, has been established at Centro
Cardiologico Monzino in order to investigate markers
(polymorphisms) useful both in the identification of
cardiovascular risk factors and in the characterization of
acute vascular events in high risk subpopulations. The
DNA bank is connected to a database which collects the
patient clinical information allowing the identification of
subjects with specific characteristics (age, gender, family
history, etc…).
In the last years the bank has turned out to be a
precious help in carrying out studies concerning
the development and progression of atherosclerotic
pathology in patients with candidate gene
polymorphisms
144
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145
Relationships between heart and kidney function
in acute cardiovascular conditions
146
CCM — Scientific Report 2011 — Ongoing research 2012
disease and acute kidney injury have been carried out
at Centro Cardiologico Monzino, in collaboration with
the Interventional Cardiology and the Post-Operative
Intensive Care Units. The goal of these studies is to
characterize, in terms of acute kidney injury risk and
associated morbidity and mortality, subsets of patients
undergoing interventional coronary procedures or
cardiac surgery. The clinical scenarios considered in this
area of research were ST-elevation acute myocardial
infarction (STEMI) and cardiogenic shock treated with
primary percutaneous coronary intervention, severe
renal insufficiency (stage IV and V of the classification
proposed by the National Kidney Foundation)
undergoing elective cardiovascular procedures, and
chronic kidney disease patients undergoing cardiac
surgery. All these patients have been identified as
at very high risk for acute kidney injury and poor
outcomes, because of the presence of multiple comorbidities and of additional factors, other than
contrast exposure (hemodynamic instability, bleeding,
cardiopulmonary support, nephrotoxic drugs, etc.),
that may negatively impact on kidney function. The
combination of these factors increases the development
of cardiac disease, contributes to progression of renal
dysfunction, and ultimately perpetuates mortality risk.
New prophylactic strategies for renal protection
have been prospectively evaluated in these highrisk populations. They are based on pharmacological
(N-acetylcysteine), mechanical (hemofiltration), or
combined (furosemide-induced diuresis with matched
hydration) approaches. The general concept emerging
from these studies is that no universal strategy
for renal protection exists. Indeed, several lines of
preventive therapy are possibly required, and the best
therapeutical option should be selected on the bases
of the single patient’s risk profile and the considered
clinical setting. As the poor prognosis associated with
renal insufficiency may partially, or totally, thwart the
benefit obtained by percutaneous or surgical coronary
No AKI
AKI Stage 1
AKI Stage 2-3
57%
60
50
In-hospital mortality (%)
A large body of experimental and clinical evidences,
as well as an increasing awareness, indicates that
both chronic kidney disease and acute kidney injury
are relevant issues in cardiovascular medicine from an
epidemiological, clinical, and prognostic point of view.
Diagnosis and treatment of patients with cardiovascular
disease rely heavily on cardiovascular imaging and on
interventional coronary procedures and cardiac surgery.
Contrast-mediated imaging studies and interventions
are a relevant part of modern medical practice. An
increasing number of patients, estimated at 30 million
per year in the United States, receive contrast agents
during diagnostic or interventional procedures.
Iodinated contrast media have a strategic place in
this expanding field and, although they are essential
for contrast enhancement and diagnostic precision,
they are also toxic for the kidney. Indeed, one of the
most troublesome complications of contrast agent
administration is kidney toxicity, and contrast-induced
nephropathy represents one of the leading causes of
renal impairment and the third cause of in-hospital
acquired renal failure. Development of contrast-induced
nephropathy is linked to a higher risk of cardiovascular
complications, prolonged hospitalization, and increased
in-hospital and long-term mortality. Acute kidney injury
is also a common complication after cardiac surgery,
associated with significant morbidity and mortality,
and with a prolonged intensive care unit and hospital
stay. Temporal trends in cardiac surgery show that
patients with co-morbidities and complex surgical
procedures are increasing. Thus, acute kidney injury
after cardiac surgery is an important health problem
that is expected to increase in incidence. Therefore, the
effective prevention of renal deterioration, particularly
in patients with preexisting renal insufficiency, should
improve morbidity and mortality following percutaneous
coronary procedures and cardiac surgery, and decrease
hospital stay and costs.
In recent years, several studies on chronic kidney
43%
40
30
22%
20
0
12%
9.5%
10
6%
1%
N=2801
1.3%
N=262
N=147
Overall population
0%
N=1467
N=155
STEMI
N=181
N=1339
N=104
N=64
NSTEMI/UA
In-hospital mortality in patients with acute coronary sindromes, developing, or not (no AKI), mild (Stage 1) and severe ( Stage 2-3) acute
kidney injury (AKI). STEMI = ST-elevation acute myocardial infarction; NSTEMI = non-ST-elevation acute myocardial infarction.
revascularization, exploration of promising new
strategies continues, with the hope that pharmacological
or mechanical kidney protection might improve
outcomes in these patients.
Finally, alternative non pharmacological approaches for
the therapy of acute and chronic heart failure have been
investigated. In collaboration with the Heart Failure Unit,
large experience has been acquired in the treatment of
patients with refractory congestive heart failure and
severe fluid overload by means of different kind of
renal replacement therapies, in particular ultrafiltration
and hemofiltration.
CCM — Scientific Report 2011 — Ongoing research 2012
147
Candidate biomarkers for the clinical
and prognostic stratification of patients
with acute coronary syndromes
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Inflammatory prothrombotic biomarkers. Several
research activities are ongoing, regarding the
Course after hospital presentation for CK-MB and serum
cytochrome c in STEMI patients treated with primary angioplasty.
Data are presented as median and interquartile range.
250
CK-MB (ng/mL)
Citochrome c. In collaboration with the Experimental
Oncology Department of Istituto Europeo di Oncologia
(IFOM-IEO), the Intensive Cardiac Care Unit is evaluating
serum levels of cytochrome c, a mitochondrial protein,
as a potential marker of apoptosis associated with
myocardial reperfusion injury in STEMI patients.
Primary percutaneous coronary intervention is the most
effective strategy for reducing infarct size and improving
clinical outcomes in patients presenting with a STEMI.
The process of myocardial reperfusion, however, can
induce injury to the myocardium caused by the abrupt
restoration of coronary blood flow after an ischemic
episode, thereby reducing the beneficial effects of
myocardial reperfusion. Indeed, reperfusion-induced
myocardial injury has been experimentally shown to
induce cardiac cells death and to increase infarct size,
accounting for up to 50% of its final size. Several
studies have generated interest in the reperfusion phase
of STEMI, as a possible target of cardioprotection.
Until recently, however, the efficacy shown by most
cardioprotective agents in animal models, has been
difficult to confirm in clinical trials. One possible reason
is the lack of available biomarkers of reperfusion injury
that allow us to detect in clinical practice the presence
of reperfusion-induced myocardial damage, to measure
its extent, and to evaluate the effects of cardioprotective
interventions. Myocardial reperfusion injury is mainly
due to apoptotic cell death that occurs in response to
oxidative stress, ATP depletion and other stimuli triggered
by restoration of blood flow after coronary occlusion.
Mitochondrial cytochrome c release is a key event
in the activation of caspase-3, a downstream pivotal
step of early stages of apoptosis. Thus, cytochrome c
concentration in plasma of patients with STEMI represents
a possible new index of apoptotic cell death associated
with coronary reperfusion, and its measurement could
provide important clinical, prognostic, and therapeutic
information in this setting. Recently published data
(Marenzi G. et al. Am J Cardiol 2010) seem to confirm this
hypothesis; further investigations on a larger population
of patients with acute coronary syndromes are ongoing
to establish the possible complementary prognostic value
of this marker, when considered together with classical
indexes of myocardial necrosis.
200
characterization of genomic, proteomic, thrombotic, and
inflammatory profiles of patients with acute coronary
syndromes (for details see the research activities
of the Laboratory of Biology and Biochemistry of
Atherothrombosis). In these patients the mechanisms
underlying the association between high risk clinical
features and enhanced mortality have not been
fully elucidated but are possibly associated with the
presence of an abnormal pathological background,
including platelets activation and coagulation processes,
inflammatory response, and cellular characteristics
and functions. We investigate the biological aspects
that distinguish low from high risk patients with
acute coronary syndromes, in terms of thrombotic,
inflammatory, and cellular characteristics, oxidative
stress and nitric oxide pathway. The acquisition of a
new knowledge about the complex substrate underlying
high risk patients suggests new therapeutic targets and
strategies aimed at significantly reducing the increased
mortality rate of these patients.
Vitamin D deficiency. Recent studies have highlighted
the role of vitamin D deficiency in a variety of basic
biological and physiological processes throughout the
body, including the cardiovascular system. Accumulating
150
100
evidences suggest that vitamin D levels in plasma are
lower in the presence of cardiovascular risk factors such
as diabetes, hypercholesterolemia, hypertension, and
chronic kidney disease, and that vitamin D deficiency is
associated with an increased cardiovascular risk. Vitamin
D deficiency might also play a role in the pathogenesis
of acute coronary syndromes (ACS). However, no
information is available on the levels of vitamin D in
blood of patients hospitalized with ACS, as well as their
relationship with disease severity and short-term clinical
outcome. Recognition of the mechanisms linking vitamin
D deficiency to increased mortality risk in ACS patients
is a critical challenge which might justify a strategy
based on “acute vitamin D supplementation” as a novel
effective therapy for high-risk ACS patients. A new
research project is ongoing, promoted by the Intensive
Cardiac Care Unit, in collaboration with the Nephrology
and Dialysis Unit, Multimedica IRCCS, the Laboratory
of Biology and Biochemistry of Atherothrombosis
and the Clinical Chemistry Unit. The project is aimed
at investigating the role of vitamin D deficiency in
the setting of ACS, its relationship with in-hospital
outcomes, and its possible influences on platelet function
and thrombus formation.
50
0
0
6
12 18
24 30
36 48 72
96
0
6
12 18
24 30
36 48 72
96
hours
2,0
Cytochrome c (ng/mL)
Research activity is focusing on the study of
new possible biomarkers to be used for clinical
characterization and prognostic stratification of patients
with acute coronary syndromes.
1,5
1,0
0,5
0,0
hours
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149
Cardiopulmonary pathophysiology
Lung functional abnormalities. It is now well known
that lung function abnormalities are part of the chronic
heart failure syndrome, as both lung mechanics and gas
exchange are impaired. Impairment of lung mechanics is
Pulmonary function in patients grouped in 4 functional classes
according to peak VO2
120
100
80
VO2 Max <12 ml/Kg/min
VO2 Max 12-16 ml/Kg/min
VO2 Max 16-20 ml/Kg/min
VO2 Max >20 ml/Kg/min
° == pp << 0.01
* 0.01
°
°
°
°
°
°
*
°
°
60
40
20
0
150
FVC (%)
FEV1 (%)
FEV1/FVC
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MVV
present at rest in patients with severe HF. However, lung
mechanics abnormalities are evident, even in patients with
moderate HF, during physical activity. Indeed, at rest,
pulmonary function is normal in patients with moderate
HF whereas a restrictive lung disease is frequently
observed in patients with severe HF. We have grouped in
four classes HF patients according to exercise capacity
and identified a restrictive lung disease in patients with
peak VO2 < 12 ml/min/kg and a strong tendency toward
a restrictive lung disease in HF subjects with an exercise
capacity ranging between 12 and 16 ml/min/kg
Cardiac enlargement and lung fluid increase are among
the major causes of the restrictive lung disease. Indeed,
we showed an inverse relationship between cardiac
volume and pulmonary function.
Lung function abnormalities during exercise. In an
international multicenter collaborative study it has been
shown that, at a given VO2, the higher the ventilation
the greater the HF severity. The increase of ventilation
is due to an increase in respiratory rate with the tidal
volume being lower in patients with severe heart failure.
In figure 1a, VO2 is plotted vs VE. Data show that for the
same VO2, patients in a lower functional class have a
higher ventilation than normal subjects or patients with
less severe heart failure. In figure 1b, VE is increased
due to an increase of respiratory rate with a lower tidal
volume in more compromised patients.
We have also shown that heart failure patients,
at variance with normal subjects, have a reduced
residual functional capacity (FRC) at the beginning
of exercise but, when they reach an early expiratory
flow limitation, they increase FRC in order to increase
ventilation. The increase in FRC and both the reduced
expiratory time and the presence of uneven lung
compliance, produce disomogenity of end-expiratory
alveolar pressure which leads to ventilation/perfusion
inhomogeneity.
Finally, we published a paper about the evolution of the
Fig. 1a
concept of ventilatory limitation during exercise combining
the pneumologist and cardiologist point of view.
Lung diffusion abnormalities in chronic HF patients. In
humans, alveolar gas exchange is measured in terms
of total lung diffusion for carbon monoxide (DLCO) and
specific membrane diffusion capacity (Dm). We have
suggested that DLCO abnormalities have a prognostic
capacity in HF, are a target for therapy and also a limiting
factor for exercise performance. Indeed, heart failure
patients with low DLCO have a reduced exercise capacity.
For a given VO2 the oxygen pressure gradient across the
alveolar capillary membrane has to increase more than in
subjects with normal DLCO. Consequently, when exercise
is performed below sea level, as in the Dead Sea, or at a
high altitude, HF subjects with reduced DLCO improve or
reduce their exercise performance more than HF patients
with normal DLCO. DLCO and DM abnormalities have a
relevant prognostic capacity in HF albeit it is yet unknown
whether or not the improvement of DLCO obtained with
treatment is associated with an improvement of prognosis.
Fig. 1b
We have done several high altitude experiments using
a simulated altitude model which has been developed
at Centro Cardiologico Monzino/Department of
Cardiovascular Science, Milano University, as well as
participating to a Highcare International Research Mount
Everest Expedition (http://www.highcare2008.eu/).
There is a linear correlation between DLCO and peak VO2
Peak VO2
Over the last twenty years the Cardiopulmonary Research
Laboratory at Centro Cardiologico Monzino has played a
major role in research advancement in the understanding
of cardiopulmonary interaction in heart failure (HF).
This has been made possible by the strong collaborative
network with the Emergency Unit for BNP (natriuretic
peptide) and pulmonary edema studies, with the Intensive
Cardiac Care Unit for ultrafiltration studies, with the
Interventional Cardiology Unit for coronary angiography,
with the Electrophysiology Unit for biventricular pace
makers, with the Anaesthesiology and Intensive Care Unit
for cardiopulmonary interaction in the operating room,
with the Echocardiography and Radiology Units for echo
and TC studies and with the Laboratory of Biology and
Biochemistry of Atherothrombosis for the identification
of biomarkers of disease, e.g. metalloproteasis, surfactant
derived proteins etc.
CCM — Scientific Report 2011 — Ongoing research 2012
151
Moreover, we performed several studies on gas
exchange abnormalities in HF and have identified the
first biomarker of alveolar capillary membrane damage.
Indeed, we reported an increased circulating plasma
level of surfactant protein B (SPB) in HF patients whose
value is higher when HF is severe. SPB level correlates
with lung diffusion, both total DLCO and membrane
specific Dm, and with peak VO2 and VE/ VCO2 slope.
Gas exchange abnormalities are associated with
anatomical changes in the alveolar-capillary membrane,
which include reduction in the number of the alveolarcapillary units, interstitial fibrosis, local thrombosis and
an increase in cellularity. All this has been extensively
studied in our laboratory elaborating two heart failure
models (rapid saline infusion and hypoxia exposure,
both with reducing FiVO2 and with high altitude
exposure) and demonstrating the active role on DLCO
in HF of several therapeutic interventions: increase
in DLCO by ACE-inhibitors, anti-aldosteronic drugs, a
during exercise, is associated to a reduction in DLCO
which is greater when the HF severity is serious.
We have now a more clear understanding of gas diffusion
in heart failure patients thanks mainly to our twenty
five years studies at the Cardiopulmonary Research
Laboratory of Centro Cardiologico Monzino. Several
research studies are still ongoing or under development.
Artero-venous difference vs cardiac output for different peak
VO2, measured with rebreathing and with Fick
20
18
16
VO2
3
14
2,5
12
2
10
neutral effect by ultrafiltration and AT1 blockers, and a
negative role by B1.B2 receptors blockers (carvedilol).
We have also shown that the capillary blood volume at
rest is reduced in patients with severe exercise limitation
and that reduction of lung fluids by ultrafiltration is not
associated to DLCO improvement. Differently a transitory
increase in pulmonary vascular pressure, as it happens
CO determination during exercise. We are now capable to
measure simultaneously during exercise cardiac output,
VO2 and the artero-venous O2 differences, in other words
to split the Fick equation into its three components.
Clinically this allows to understand and separate from
each other, in HF patients, the role of anaemia, muscle
deconditioning, and cardiac output reduction guiding
us in a data based decision on CRT, drug and surgical
treatment. New insights are now available on exerciseinduced blood-flow redistribution before and after
training.
1,5
8
1
Rebreathing
6
0,5
Flick
4
2
0
0
2
4
6
8
10
12
14
16
18
C(a-v)O2 estimated/measured (ml/100ml)
DLCO and its components, Dm (membrane diffusion capacity), Vc (capillary volume) and Va (alveolar volume), in different
functional class.
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153
Interventional Cardiology
The Interventional Cardiology Unit is involved in several
lines of clinical research focusing on the percutaneous
treatment of coronary artery disease, peripheral vascular
disease and structural heart disease. The aim of our
scientific work is to improve treatment outcome by
means of new transcatheter techniques and innovative
interventional devices and to explore innovative
diagnostic modalities in the area of coronary and
peripheral atherosclerotic disease.
Coronary artery disease
The Unit is in the vanguard in the area new coronary
stent evaluation, investigating the efficacy of second
generation drug-eluting stents (DES) and assessing the
clinical outcome of DES use for off-label indications in
percutaneous coronary intervention (PCI).
New coronary stents. Currently available stents were
designed for straight lesions in which they have shown
to provide superb acute and long-term results. One lesion
subset that continues to challenge the interventional
cardiologist is bifurcation lesions. A number of different
strategies have been employed with standard stents to
address bifurcation lesions each of which has significant
limitations including decreased procedural success
and increased restenosis and thrombosis rates. These
limitations prompted the development of stents designed
specifically to treat bifurcation lesions. We are currently
evaluating the Tryton Side-Branch Stent™, a cobalt
chromium balloon expandable stent designed specifically
for bifurcation lesion treatment. The aim is to assess the
safety and feasibility of the Tryton Side-Branch Stent™ in
this lesion subset and to evaluate in detail intravascular
ultrasound findings immediately after stent deployment
and at six-month follow-up in order to correlate them
with clinical outcome.
Second-generation DES. Few data substantiate long-term
safety and efficacy of second-generation DES in the real-
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CCM — Scientific Report 2011 — Ongoing research 2012
world. We are enrolling real-world patients and coronary
lesions in our registry to assess acute, mid-term (6
months) and long-term (2 year) outcome of two second
generation DES, the EndeavorTM-zotarolimus-eluting stent
and the Xience VTM-everolimus eluting stent. Moreover,
we are involved in a multicenter randomized trial (Spirit
women randomized trial) comparing a first generation
DES, the Cypher sirolimus eluting stent, with a second
generation DES, the Xience VTM-everolimus-eluting stent,
in women.
Bioabsorbable Vascular Scaffold (BVS). We started
for the first time in Italy a clinical study for the
assessment of the safety and performance of the BVS,
a novel polymeric bioabsorbable stent in patients with
obstructive coronary artery disease characterized by a
maximum of two de novo native coronary artery lesions
each located in different epicardial vessels. The BVS is
comprised of 4 main components: an anti-proliferative
drug (everolimus), expected to be 80% eluted by 28
days, a polymer drug reservoir, expected to be fully
absorbed in 9 months and a polymer stent, expected to
be fully absorbed in 18 to 20 months. The BVS, despite
being a polymer that is naturally absorbed and fully
metabolized, performs like a metallic DES in terms of
deliverability, conformability and scaffolding. The key
advantages are that it leaves behind, after bioabsorption,
a healed natural vessel without any permanent implant
and is fully compatible with MDCT imaging.
Off-label use of DES. We are participating in the GISESICI registry, an ongoing retrospective observational
multicenter registry promoted by the Italian Society of
Invasive Cardiology, in which 19 high-volume centers
enrolled 1,453 consecutive patients who underwent
percutaneous coronary intervention on unprotected left
main coronary artery. The aim is to compare long-term
clinical outcome following DES or bare-metal stents
(BMS) implantation on lesions located at the ostium, the
shaft or the bifurcation of the left main in a large realworld population. In this registry, the risk of ischemic
events in relation to the duration and time of suspension
of dual antiplatelet therapy is also assessed as well as
the impact of acute coronary syndrome, gender, left
ventricular function and diabetes on mid- and long-term
outcome.
Another field of interest is the implantation of longer
and multiple overlapping DES in diffusely diseased
vessels, as well as total vessel reconstruction. These
interventions are increasing in daily clinical practice in
relation to the significant decrease of restenosis rate
after DES implantation even in very complex coronary
lesions. Recently, however, concerns have been raised
regarding the safety of DES use in off-label indications
such as very long lesions, especially with regards to
the risk of late and very late stent thrombosis, often
coinciding with the cessation of one or both antiplatelet
agents. Therefore, we are conducting a study that is
aimed at the evaluation of all patients who underwent
long lesions stenting with a first-generation sirolimuseluting DES (Cypher) since its introduction in our
institution (April 2002). Data were collected from a
prospectively created database in which the specific
target stents length, longer stented length (>60 mm)
subgroup and clinical follow-up were decided prior
to data interpretation. Analysis of these data will
provide critical information regarding the safety and
effectiveness of multiple and overlapping implantations
of this specific DES in patients with long and diffuse
coronary artery disease.
Acute myocardial infarction. Several randomized trial
have demonstrated that primary angioplasty is the
more effective reperfusion modality in acute myocardial
infarction resulting in fewer deaths, reinfarctions and
strokes than fibrinolysis does. One limitation of primary
PCI is the “no-reflow phenomenon”, a dire event defined
as the inability to perfuse a portion of myocardium after
re-establishment of patency of a previously occluded
epicardial coronary artery. No-reflow is associated with
myocardial injury, lack of myocardial function recovery
and increased mortality. We are focusing our research
activity on the prevention and management of the
no-reflow phenomenon in acute myocardial infarction
patients undergoing primary PCI. Use of innovative distal
protection and thrombus aspiration devices, in order to
prevent distal embolization, and specific pharmacological
treatment approaches for preservation of the coronary
microcirculation function are currently evaluated in this
clinical setting.
Stress cardiomyopathy (Tako-Tsubo syndrome). This is a
relatively rare but dramatic acute cardiac condition that
mimics myocardial infarction and is mostly observed in
women. We are exploring the pathogenesis and triggers
of the Tako-Tsubo syndrome, as well as the therapeutic
strategies and long-term outcome.
Unstable coronary plaque. Propensity of subcritical
coronary artery plaques to induce acute coronary
events is investigated by intravascular virtual histology
technique (VH-IVUS). This technique allows the
differentiation between stable fibrotic plaques, which
do not require invasive treatment, from plaques filled
with lipid components that may be prone to abrupt
rupture and able to trigger an acute coronary syndrome.
Detection and categorization of subcritical unstable
coronary plaques using VH-IVUS in order to correlate
them with clinical outcome and their passivation by
stenting represent specific field of research.
Peripheral interventions
Carotid stenting. This field of research is based on an
interventional experience in almost 600 patients who
were treated with carotid stent implantation using
different types of stents and brain protection devices.
The clinical results compare very favorably with those
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reported in the literature and the standard of care
requirements of the scientific societies for the treatment
of carotid artery disease. Areas of clinical research in
this field include:
a)The comparison of different types of brain
protection devices (distal vs. proximal protection)
and carotid stenting (CAS) technique (predilation
vs. direct stenting), and evaluation of the clinical
value of trans-cranial Doppler monitoring during
CAS in high-risk patients.
b)The investigation of CT-angiography role as a
pre-CAS diagnostic assessment tool to identify
patients at high anatomical risk for cerebral
complication or CAS failure
c)The comparison between CT-angiography and
Doppler US in the assessment of stenosis severity
and composition
d)The study in detail of intracranial circulation
variant in relation to patient tolerance of a
proximal protection device (MoMa).
Comparative evaluation of invasive vs. non-invasive
coronary artery imaging techniques.
With the introduction of multidetector computed
tomography (MDCT), coronary computed tomography
(CT) angiography has emerged as a new tool for
diagnosing coronary artery disease and patient followup. We are involved in several studies in collaboration
with the Area of Cardiovascular Imaging aimed at
assessing the diagnostic performance, as compared
to invasive coronary angiography, of 64-slice MDCT
for the detection of coronary stenosis in patients with
low probability of coronary artery disease such as
patients with dilated cardiomyopathy. Other studies
focus on the feasibility and diagnostic accuracy of
64-slice MDCT for the evaluation of in-stent restenosis
comparing the finding of this non-invasive imaging
modality to the results of invasive coronary angiography
and intravascular ultrasound in patients who were
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previously treated with coronary stent implantation.
Moreover, we are assessing the prevalence of coronary
artery anomalies in patients who undergo MDCT and
correlating the MDCT anatomical findings to the patient
clinical symptoms. The aim is to assess in which cases
MDCT has an additional clinical value compared to
invasive coronary angiography in the diagnosis and
characterization of these congenital pathologies.
Structural heart disease
Atrial septal defect and patent formane ovale. Since
2000, our Unit has been involved in the percutaneous
treatment of atrial septal defect (ASD) and patent
foramen ovale (PFO) patients. A causal relationship
between PFO and migraine has been recently
hypothesized. Indeed, increased frequency of PFO in
migraineurs was first reported in 1998 in a case-control
study and PFO has a prevalence of 15% to 25% in the
general population and up to 60% in patients with
migraine with aura. In consideration of the reports
of several retrospective uncontrolled studies that
have indicated improvement of migraine with aura
after percutaneous PFO closure, we are prospectively
assessing, using a validated migraine severity score,
the impact of PFO closure on migraine attacks over
time in consecutive patients who were treated with the
Amplatzer PFO occluder in our institute since May 2000.
Mitral valve regurgitation. Mitral regurgitation affects
millions of patients worldwide, yet only about 20% of
the 600,000 patients diagnosed in the United States
and Europe each year undergo open heart surgery,
the traditional treatment for mitral regurgitation. We
are conducting a clinical study with a new device
(MitraClip system) for non-surgical repair of mitral valve
regurgitation. This breakthrough mitral valve repair
technology clips the leaflets of the mitral valve together
to reduce regurgitation offering a minimally invasive
alternative to open heart surgery – not unlike the
opportunity that stents provided more than two decades
ago for the treatment of coronary artery disease. In 2011,
we are continuing a clinical study to assess the safety,
feasibility and efficacy of this new interventional treatment
approach in patients with severe mitral regurgitation
considered high risk for mitral valve surgery.
therapy at 1 and 6 months after thienopyridine
suspension. As a comparison, we are analyzing platelet
activation in 20 medically-treated stable angina patients.
Therapeutic renal denervation
We initiated a study to evaluate the safety and blood
pressure-lowering efficacy of selective renal denervation
using a novel percutaneous, catheter-based treatment
in patients with difficult to-control and resistant
hypertension. In this approach, renal sympathetic nerve
ablation is achieved percutaneously via the lumen of
the main renal artery using a catheter connected to a
radiofrequency (RF) generator. After gaining access
via the femoral artery and confirmation of anatomic
eligibility with renal angiography, the treatment catheter
(Symplicity, Ardian, Inc) is introduced into each renal
artery, and discrete RF ablations lasting ≤2 minutes
each are applied to achieve ≤6 ablations separated both
longitudinally and rotationally within each renal artery.
Catheter tip temperature and impedance are constantly
monitored during ablation, and RF energy delivery is
regulated according to a predetermined algorithm.
Antiplatelet therapy and platelet function inhibition
after drug-eluting stent implantation
Discontinuation of antiplatelet therapy, in particular
thienopyridine withdrawal, has emerged as one of
the most important predictors of stent thrombosis.
In this regard, a rebound effect has been suggested
to be responsible for the adverse events reported
within the initial 90 days after stopping clopidogrel
in patients previously treated with DES implantation.
In collaboration with the Unit of Cell Biology and
Biochemistry of Atherothrombosis, we are evaluating
patients treated with DES who underwent evaluation
of platelet activation markers during dual antiplatelet
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Electrophysiology Area: scientific hot topics
Our recent research efforts are mainly focused on:
a) catheter ablation of atrial fibrillation;
b) catheter ablation of life-threatening ventricular
arrhythmias;
c) substrate mapping and diagnostic endomyocardial
biopsy in different subsets of cardiomyopathy;
d) new techniques to achieve clinically significant
reduction of the exposure to ionizing radiation in
supraventricular tachycardia ablation;
e) identification of the best strategies for left ventricular
stimulation in patients implanted with biventricular
pacemaker and/or cardioverter defibrillator.
f) evaluation of the multielectrode pulmonary vein
isolation system for the treatment of paroxysmal
atrial fibrillation (REVOLUTION Study)
g) Advantages of a novel surround-flow irrigated
catheter for ventricular tachycardia ablation
(THERMOCOOL SF Study)
Atrial fibrillation (AF) is the most common significant
cardiac arrhythmia, responsible for approximately onethird of all hospital admissions of patients suffering
from cardiac rhythm disturbances. It is responsible for
an increased risk of stroke, heart failure and all-cause
mortality. Over the last decade the vast improvement in
the long term efficacy of catheter ablation, compared to
pharmacological treatment, supports the use of ablation
in the early management of patients suffering from atrial
fibrillation. The success rate of AF ablation varies between
65% and 85% in patients with AF, depending on type
of AF (paroxysmal or persistent or permanent), patient
selection, technique used, and methods of follow up.
Our research activities are aimed at identifying 1) the
biochemical and structural predictors of AF, and 2) the
procedural techniques associated with improved success
rates of catheter ablation procedures, particularly in
patients with persistent AF.
Regarding biochemical predictors of AF, the recent rapid
evolution of microRNA (miRNA) research has shown that
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this class of small non-coding RNAs plays important
roles in diverse fundamental biological processes
including cell growth, differentiation, aging and death. In
particular some of these miRNAs have been shown to be
involved in AF and are considered to have the potential
to regulate AF based on their target genes. Thus aim of
our studies is the identification of tissue or circulating
miRNAs in young patients suffering from AF in the
absence of any apparent predisposing cause.
Another research thread is the study of atrial fibrosis
identified by cardiac MRI and low potentials on
electroanatomical atrial substrate mapping. To this
regard an analysis will be performed to determine
whether the presence or absence of atrial fibrosis
influences the long-term efficacy of catheter ablation.
In the first of our ongoing studies on procedural
techniques, we are testing a “step-wise” AF ablation
strategy to improve the effectiveness of the procedure
in patients with persistent AF. Patients undergo catheter
ablation guided by 3D electroanatomical mapping
systems (CARTO 3), with the aim of electrically
isolating pulmonary veins, and performing left atrial
substrate modification by means of ablation of complex
fragmented atrial electrograms and linear ablation
on the left atrial roof, left mitral isthmus, right cavotricuspid isthmus. The follow-up of these patients is
strictly based on data recorded from a loop recorder
implanted immediately after ablation. These patients
also undergo a functional cardiac evaluation, by means
of cardiopulmonary exercise testing and non-invasive
measurement of cardiac output at baseline and during
exercise. The aim is to evaluate whether persistent AF
compromises cardiac function even in cases with no
overt reduction in left ventricular ejection fraction.
Another topic of our research activity is the evaluation
of new mapping and ablation catheters to electrically
isolate the pulmonary veins in patients with paroxysmal
AF. The catheters under study in our laboratory
use different sources of ablation energy, including
radiofrequency, extreme cold (cryoablation) and laser
energy.
We are also studying genetic determinants predisposing
to intra-atrial thrombus formation in patients with
persistent AF despite regular oral anticoagulant therapy
(i.e. INR between 2 and 3). Previous studies conducted
in our echocardiography laboratory demonstrated
that 6-7% of patients undergoing a transoesophageal
echocardiogram before electrical cardioversion have
intra-atrial thrombus despite a correct value of INR.
Blood samples are also obtained from patients who
undergo transoesophageal echocardiography before
electrical cardioversion to evaluate possible genetic
determinants predisposing to thrombus formation.
Ventricular arrhythmias. Radiofrequency catheter
ablation is now considered a curative approach for
ventricular tachycardia (VT), either idiopathic or in the
setting of ischaemic or non-ischaemic cardiomyopathy.
In our Electrophysiology laboratory, the success rate of
VT ablation approximates 70%. However, management
of recurrent VT often remains difficult, as in some
patients neither antiarrhythmic drugs nor conventional
endocardial radiofrequency ablation can prevent all
recurrences. Ongoing research of the Electrophysiology
Area is focused on the possibility to improve the
success rate of VT ablation by means of different 3D
mapping systems, often integrated by intracardiac
echocardiography of the ventricular chambers.
Figure 1. Primary and secondary cure rates (freedom from atrial fibrillation recurrence) in 1404 patients undergoing pulmonary vein antrum
isolation guided by a circular mapping catheter and intracardiac echocardiography at 4 different Institutions with 12 different operators.
P<0.001
%
100
90
80
70
60
50
40
30
20
10
0
P=0.006
92,2
77,6
P=0.28
86,4
83,3
75,8
60,6
Paroxysmal
Persistent
Primary cure rate
Permanent
Secondary cure rate
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One of our studies on VT ablation aims to evaluate the
impact on success rate of catheter ablation of a software
for automated pace-mapping (PaSo) in patients with
frequent ventricular premature beats. Coupled with
standard electroanatomic CARTO mapping, the PaSo
software can perform automated template matching,
which helps to localize the optimal ablation site of
ventricular premature beats arising from either the left
or right ventricle.
Another ongoing trial is aimed at evaluating the
usefulness of intracardiac echocardiography of the
left ventricle integrated with 3D electroanatomic
mapping (CartoSound). Intracardiac echocardiography
is possible because lower-frequency transducers
have been miniaturized and mounted onto catheters
which can be inserted transvenously into the heart.
These lower-frequency transducers are capable of
enhanced tissue penetration, permitting high-resolution
2D “whole-heart” imaging. Intracardiac ultrasound
may facilitate electrophysiological procedures and VT
ablation by guiding trans-septal catheterization, enabling
visualization of endocardial anatomy and more precise
targeting of arrhythmogenic substrates, ensuring
optimal ablation electrode/tissue contact and promptly
diagnosing procedural complications.
A third ongoing multicentric study (EMOSIDD) is aimed
at evaluatong the morphologic and electrophysiological
substrate markers of increased arrhythmic risk in
patients with dilated cardiomyopathy undergoing ICD
implantation for the primary prevention of sudden
cardiac death. We also aim to identify whether there
is any electrophysiological substrate modification at
the time of the first arrhythmic event in these patients.
We will prospectively correlate electroanatomic
mapping and cardiac magnetic resonance findings
with arrhythmic events, in order to identify substrate
markers of increased arrhythmic risk in patients with
dilated cardiomyopathy, who are therefore more
likely to benefit from a ICD implantation. In addition,
electroanatomic mapping will be repeated at the time of
the first arrhythmic event and compared with that one
at baseline, in order to evaluate any electrophysiological
substrate changes.
Reduction of exposure to ionizing radiation.
Radiofrequency catheter ablation is the mainstay of
therapy for supraventricular tachyarrhythmias (SVT).
Conventional ablation techniques require the use of
fluoroscopy to navigate and position catheters within the
heart, thus exposing patients to ionizing radiation with an
attributable life-time risk of cancer. We recently reported
the feasibility and safety of non-fluoroscopic ablation of
a wide range of SVTs using the EnSite NavXTM mapping
system. The NO-PARTY is a multicentre, randomized
controlled trial designed to test the hypothesis that
radiofrequency catheter ablation of SVTs guided by the
non-fluoroscopic EnSite NavXTM mapping system results
in a clinically significant reduction of the exposure to
ionizing radiation compared with conventional ablation
techniques. The study will randomize 210 patients
undergoing ablation of SVT to either a conventional
technique or one guided by the non-fluoroscopic EnSite
NavXTM mapping system. All SVTs except for AF and non
isthmus-dependent atrial flutter are considered suitable
for randomization. Other elements of care, including drug
therapy, are to follow best evidence-based practice. The
primary end-point is the reduction of the total radiation
dose to the patient, as assessed by dose-area product
(DAP). Others pre-specified end-points include reduction
of the radiation dose to the operator and reduction
Fig. 3 Panel (a) shows a three-view reconstruction of both right- and left-sided chambers in a 16-year-old with a left posterior accessory
pathway, obtained without the help of fluoroscopy. The LAO projection shows ablation pulses (white) from inside the coronary sinus. Panel
(b) shows two modified views to better illustrate ablation pulses (white) at the mitral annulus. AP antero-posterior, LAO left anterior oblique
Panel A: Bipolar voltage map of the left ventricle in left anterior oblique view.
Panel B: Intracardiac echo fan intersecting the low-voltage area, showing vacuolization (asterisks) and an endocardial hyperechogenic area
(empty arrow) at the basis of the anterior papillary muscle. White arrows indicate a sacculated pericardial effusion.
Panel C: T2-weighted MRI short-axis (top) and long-axis (bottom) views, showing focal hyperintensity at the base of the antero-lateral
papillary muscle.
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of the total procedural fluoroscopy time. Moreover, a
cost-effectiveness analysis will be performed, taking into
account the additional costs associated with an ablation
approach guided by the non-fluoroscopic EnSite NavXTM
mapping system, and the life-time estimable benefits for
the patient associated with reduced exposure to ionizing
radiation.
Cardiac resynchronization therapy (CRT). In the setting
of cardiac pacing, our research activity is mainly focused
on left ventricular pacing in CRT. The advent of CRT
brought great advancement to the management of HF,
with significant results over morbidity and mortality.
However, a reasonable percentage of patients does not
benefit from this therapy. Therefore, complementary
methods such as ECG, tissue Doppler echocardiogram
and electroanatomic mapping try to identify parameters
capable of distinguishing the best candidates for CRT
and to reduce the incidence of non-response. However,
the best method to locate the optimal RV and LV
pacing sites in individual patients remains unclear. A
potential reason for non-response to CRT may be the
presence of extensive scar tissue in the region of the
tip of the LV pacing lead. Pacing the LV in non-viable
or scarred myocardium may result in less effective
or even ineffective LV pacing and, as a consequence,
failure of LV resynchronization and no response to CRT.
Accordingly, in our study (SHARE-M) we try to establish
an immediately available and helpful evaluation of the
acute response to CRT in relation to the myocardium
substrate, scar tissue, LV dyssynchrony, fixed and
functional conduction blocks, based on the novel
Dynamic Substrate Mapping algorithm which permits in
vivo reconstruction of cardiac anatomy and assessment
of the activation sequence with high spatial resolution.
Pulmonary vein isolation system in the treatment
of paroxysmal atrial fibrillation (REVOLUTION
Study). Radiofrequency (RF) catheter ablation has
provided excellent results for treating many types of
supraventricular arrhythmias. The ACC/AHA/ESC AF
Management Guidelines now consider catheter ablation
to prevent recurrent AF a reasonable alternative to
pharmacological therapy (i.e. a second treatment option)
in all categories of the treatment algorithm to maintain
sinus rhythm. In fact, catheter ablation techniques
targeting the isolation of the PVs are now commonly
used in the electrophysiology laboratory. The 2007
115±5cm
USABLE LENGTH
IRRIGATION
PORT
64±3cm
HANDPIECE
CONNECTOR
CONNECTOR
SHAFT
LOOP LUMEN WITH
IRRIGATED RING ELECTRODES
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Novel surround-flow irrigated catheter for ventricular
tachycardia ablation. Sustained ventricular tachycardia
(VT) is an important cause of mortality and morbidity
in patients with ischemic dilated cardiomyopathy
(DCM). Most patients have several different ventricular
tachycardia indicating a complex arrhythmia substrate.
Implantable cardioverter-defibrillators often can
terminate most malignant ventricular episodes with
success, reducing sudden death but ICD shocks reduce
quality of life and ventricular tachycardia predict
increased risk of death and heart failure despite effective
ICD functioning. Beyond this ICD cannot change the
underlying arrhythmic substrate. Catheter ablation
has been shown to be useful to reduce ventricular
tachycardia recurrences refractory to antiarrhythmic
therapy. Patients submitted for ventricular tachycardia
ablation with a previous myocardial infarction usually
show cardiac failure with low ejection fraction. In these
patients fluid overloading due to the use of saline
irrigation during ablation with an irrigated catheter could
be critical during an ablation procedure with a large
115±5cm
USABLE LENGTH
64±3cm
ROTATING
KNOB
HANDPIECE
SHAFT
DEFLECTABLE LUMEN
DEFLECTABLE LUMEN
162
Electrode Irrigated PV Isolation System when used for
the treatment of drug refractory symptomatic PAF.
CURVE DEFLETION
THUMB KNOB
CURVE DEFLETION
THUMB KNOB
ROTATING
KNOB
IRRIGATION
PORT
HRS/EHRA/ ECAS consensus statement on catheter and
surgical ablation states that electrical isolation of the
PVs from the left atrium is “the cornerstone for most AF
ablation procedures.” It is widely considered the best
method to treat AF in the paroxysmal AF population.
Episodes of atrial fibrillation are initiated by ectopic
sites which derive in more than 90% of cases from
the pulmonary veins. One or several pulmonary veins
may be implicated and often in a single vein there is a
multiple initiation site. The foci originate in the muscular
cardiac bundles that line the pulmonary veins. These
muscular extensions occupy varying proportions of the
vein perimeter, ranging from one quadrant to the whole
circumference. Using electrophysiology catheters their
position and extent can be precisely evaluated from
the electric potentials that they generate. By isolating
the pulmonary veins with radiofrequency, most cases
of paroxysmal AF can be corrected (episodes lasting
<1 week that resolve spontaneously) and about 20% of
persistent AF cases (>1 week or requiring electric shocks
long standing persistent AF is excluded). The purpose
of this study is to assess the safety and effectiveness
of the nMARQ™Circular and nMARQ™Crescent Mapping
& Ablation catheters and the workflow of the Multi-
A Circular Mapping &
Ablation Catheter
A Crescent Mapping &
Ablation Catheter
LOOP LUMEN WITH
IRRIGATED RING ELECTRODES
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Cardiovascular Imaging
number of RF applications and long procedure time.
The new ThermoCool SF® irrigated ablation catheter
TCSF (Biosense Webster, Inc., Diamond Bar, CA.) with a
bi-directional technology has been designed to provide
optimal flow characteristics with a porous tip that allows
highly efficient cooling and requires therefore half the
flow rate to deliver the same RF power.
The catheter design with uniform cooling of the catheter
tip without hotsposts, claims to produce consistent
lesions independent of tip orientation and could be more
effective requiring a shorter application time.
Use of the TCSF catheter is associated with a statistically
significant reduction in fluid infusion volumes during
prolonged VT ablation procedures, which may benefit
patients with LV dysfunction. A trend towards reduced
mean RF application time per area unit might suggest
improved efficacy with the TCSF catheter.
The research activity is deeply integrated with the daily
clinical work including echocardiographic and computed
tomographic examinations.
Regarding recent research projects, our main efforts are
focused on:
a)new methods in transthoracic and transesophageal
3D-echocardiography demonstrating advantages
of 3D vs 2D-echocardiography in the evaluation of
right ventricular function, mitral valve prolapse,
intraoperative monitoring of cardiac surgery, left
atrial function (Fig. 1-2-3).
Fig. 1
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Fig. 2
Fig. 3
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Fig. 4
b)feasibility and accuracy of non-invasive evaluation of
coronary arteries, stents and by pass grafting through
cardiac computed tomography (Fig.4-5)
c)new technical advances in radiation dose reduction
in cardiac computed tomography maintaining a high
accuracy in native and stented coronary arteries
evaluation.
d)cardiac CT and prognosis
e)new bioengineering approaches to cardiac
imaging computation, mainly in the field of
3D-echocardiography (Fig. 6-7-8)
f)collaboration with other Units within the framework of
a disease-specific multidisciplinary team including new
imaging data in the evaluation of patients undergoing
electrophysiological approaches, cardiac failure
therapies, PCI procedures, cardiac surgery (including
percutaneous/transapical aortic valve implantation,
mitral clip, left atrial appendage closure).
Collaboration with other Units and integrated advanced
imaging techniques are well exemplified in a clinical
protocol concerning mitral valve diagnostic approaches
and mitral valve surgery. In this field transthoracic
3D-echocardiography is performed before surgery to
better define anatomy of the valve (recent studies from
our group showed a 95% accuracy). During surgery
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Fig. 7
Fig. 8
Fig. 5
Fig. 6
in the pre- and postoperative phases, transesophageal
3D-echocardiography further defines anatomic details
and facilitates the understanding of optimal surgical
planning and surgical results. In each of these steps
and during the follow-up (1, 3, 6 and 12 months) 2D and
3D examinations are performed. Data on left ventricular
function and 3D shape, right ventricular 3D function,
mitral valve annulus (both native and postoperative
prosthetic annulus) function and shape are analyzed
throughout the study. New data have been published
on the remodelling of the left atrium after mitral
valve repair, therefore having a very complete and
comprehensive definition of changes of heart chambers
after a “correct early timing” of surgery (left ventricular
remodelling and shape, right ventricular dimensions and
function, left atrial remodelling and function). Moreover
all these 3D data are not only analyzed with available
software, but also with new software and methods
of analysis set up thanks to novel bioengineering
approaches.
Another field of interest is a novel approach to the aortic
valve annulus analysis and coronary arteries in patients
undergoing transfemoral aortic valve implantation.
In these cases, 3D TEE allows a precise assessment
of several parameters including prediction of postimplantation aortic regurgitation and the evaluation of
the distance between the left main coronary ostium and
the aortic valve.
This integrated clinical and advanced imaging protocol
is applied to the several cardiovascular fields previously
mentioned.
A recent field of scientific interest is the new realtime
3D-echocardiographic imaging. In particular, our
attention is focused on the quantification of novel 3D
left ventricular shape indices, able to overcome the
limitations relevant to prior 2D indices, to better follow
ventricular remodelling after surgery and to study
the coupling between left ventricular function and
morphology.
Another field of interest is the evaluation of the dynamic
motion of mitral and aortic valves. By the development
of specific 3D tracking algorithms, it is possible to
follow the movement and deformation of the valvular
annuli in the 3D space, and quantify them by innovative
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Fig. 9
parameters. In this way, the immediate (by TEE) and
mid-term (by TTE) follow-up effect of mitral valve repair
with annuloplasty on the mitral and aortic valves, as well
as on their coupling, can be quantified. Moreover, the
unique information derived from this analysis, together
with papillary muscle tips positions, are utilized to
generate a realistic computational model of the mitral
valve structure, customized to the patient specific
characteristics, which could be useful both in the surgical
planning and in the follow-up evaluation phases. Recently
a new approach has been evaluated concerning the
simultaneous analysis of aortic-mitral coupling in patients
undergoing mitral valve repair. These research projects
are carried out in the mainframe of the “Surgaid” project
(www.surgaid.org ), in collaboration with the Politecnico
of Milano and the University of Bologna.
In the area of non invasive evaluation of native and
stented coronary arteries or by pass grafting, studies
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Fig. 10
have been focused on the diagnostic accuracy of 64
Slice Multi Detector Computed Tomography (MDCT)
and on low radiation doses (trying to reduce with new
protocols the high radiation exposure). ProspectiveECG-triggering MDCT was recently introduced at Centro
Cardiologico Monzino and we showed that this protocol
allows an accurate detection of coronary stenosis with
a low radiation dose. Moreover, diagnostic accuracy
is high not only in native coronary arteries but also in
stented lesions.
Another area of collaboration among different Units at
Centro Cardiologico Monzino concerns MDCT advances
and heart failure (including new integrated techniques
for electrophysiological procedures). In details, studies
are focused on the very high accuracy of MDCT in the
diagnosis of idiopatic vs ischemic cardiomyopathy (thus
avoiding invasive coronarography), on the evaluation
of the venous coronary circulation (essential in the
Fig. 11
pre-procedural planning of biventricular pacing), the
CARTO techniques (integrated approaches of MDCT and
electrophysiological mapping facilitating navigation of
catheters inside cardiac cavities). Moreover new insights
into the evaluation of the aortic annulus have been
published mainly related to the importance of a precise
assessment of annulus size and geometry in patients
undergoing transfemoral aortic valve implantation.
In the field of the long term prognosis, MDCT has been
studied in patients with intermediate risk of coronary
artery disease and patients affected by diabetes showing
the importance of the atherosclerotic burden on long
term prognosis.
method of acquisition and reconstruction of images of
the tricuspid annulus and right ventricle. Interestingly
tricuspid annulus is relatively easily evaluated through
this novel MRI method and an accurate dynamic
reconstruction has been shown to be feasible. (Fig. 11).
Recently MRI (Fig 9-10) has been integrated in our area
and, even though the Unit start-up allowed us only
a 2 years experience (with more than 1000 cases per
year), we have already started the evaluation of new
fields of interest such as the tricuspid valve with a new
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Clinical Research on Atherothrombosis
The research activity of the Atherothrombosis Clinical
Research Unit is deeply integrated with the clinical
activities of the Cardiac and Vascular Surgery Units and
focuses on the translational cardiovascular features of
the main diseases affecting the adult:
1. coronary artery disease
2.valve disease
3.diseases of the aorta
In order to improve our capabilities to study valve and
aortic diseases, we have started a clinical and research
project to create a Clinical and Research Center, called
MALVA (Centro di alta specialità per la prevenzione,
diagnosi e cura delle MALattie Valvolari ed Aortiche).
The mission of this Center is to bring together a
knowledgeable and experienced multidisciplinary team
of cardiologists, cardiac/vascular surgeons and other
cardiology, imaging and biomarkers experts to provide:
• a thorough evaluation of patients using state-ofthe art diagnostic tests
• on-going comprehensive care of patients affected
by valve and aorta diseases
• genetic screening for families of patients with
genetic disorders, such as Marfan Syndrome
• ongoing research and education to provide
patients with high quality and innovative therapies
Finally, this unit is also focused on the study of the
predictors of the main complications occurring during
and after adult cardiac surgical procedures.
Coronary artery disease. Coronary artery disease is one
of the most diffuse diseases in the western countries,
and bypass surgery is the most reliable and durable
therapy for the severe forms of this disease. Even if
coronary bypass surgery enables the best results in the
long term, patients, particularly the younger ones, are
not ensured they will be free from problems related to
coronary disease during their lifetime. The clinical benefit
of myocardial revascularization is related to the lack
of adverse cardiovascular events and to graft patency
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of great saphenous vein, which is nowadays the main
autologous vessel used for grafting coronaries different
from anterior interventricular artery. It is well known that
bypasses tend to occlude over time as well as native
coronaries tend to progress their disease. Unfortunately,
phenomena leading to early and late complications and to
graft occlusion are not fully clarified and it is not possible
to explain interindividual and temporary variability of
progressive stenosis rate only on the basis of classical
atherosclerosis risk factors. We know that, after coronary
bypass surgery, a protracted prothrombotic and a proinflammatory status ensues, and, even if a great effort
to understand the physiology and pathophysiology of
the response to CPB has occurred over the last 50 years
and several targeted strategies using different drugs
modifying CPB components or even abolishing CPB itself
have been tested in order to attenuate the systemic
inflammatory and prothrombotic response occurring
after CABG, none of these was able to blunt effectively
the whole body activation occurring after this kind of
surgery. Also, the role of this whole-body response, as
330 patients were assessed for eligibility
24 declined to partecipate before d/c from
the hospital after provifing informed consent
306 patients eligible for subsequent follow-up
9 not found or not reached with phone call
297 patients contacted for phone interview at follow-up
7 dead
59 declined to accept
50 not eligible
2 CT performed elsewhere
179 Patients underwent CT scan
well as the role of different antithrombotic strategies, is
still not well defined.
In 2011 we concluded the 18 months follow-up of the
BAPPY study; in this study we collected blood samples
from 330 consecutive patients before and after coronary
bypass surgery and followed them up clinically
performing a 64-rows CT scan at 18 ± 3 months after the
surgical intervention in order to document the patency
of coronary bypasses and to identify possible predictors
of bypass occlusion. Concerning CT-scan assessment of
patency, among the 330 enrolled patients, 179 underwent
assessment of graft patency, as shown in the flowchart
on the page before.
patency per patient (that means how many patients have
perfectly patent grafts) is 135/179 (75.4%).
The patency rates of the different types of grafts are the
following:
LIMA n=12/75
RIMA n=6/20
Radialn= 1/5
SVG n=27/303
(6.8%)
(30%)
(20%)
(8.9%)
The main features of the study population concerning
coronary disease and coronary bypass grafts that were
performed during surgery, are reported in theTable 1.
Patients had on average 2.7 coronaries affected by
coronary atherosclerosis and received an average of
2.7-2.8 grafts per patients, mainly saphenopus vein graft
and left internal mammary artery.
The results of 64-rows CT scan on 179 patients show an
overall patency of 366/503 graft (72.7%), whereas the
Table 1
VARIABLE
ENROLLED
(pts, n=330)
FOLLOW-UP
(pts, n=297)
CT SCAN
(pts, n = 179)
Diseased coronary vessels, (n) ± SD
2.75 ± 0.52
2.74 ± 0.53
2.77 ± 0.52
Distal anastomoses, (n) ± SD
2.73 ± 0.75
2.73 ± 0.77
2.81 ± 0.74
Saphenous vein (SVG), n (%)
531 (59.0%)
481 (59.2%)
303 (60.2%)
Left internal mammary artery (LIMA), n (%)
319 (35.4%)
285 (30.1%)
175 (34.8%)
Right internal mammary artery (RIMA), n (%)
35 (3.9%)
33 (4.1%)
20 (4.0%)
Radia artery (RA), n (%)
15 (1.7%)
12 (1.5%)
5 (1.0%)
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The next steps will be the prosecution of the follow-up
to 5 years, and the study of the biomarkers, mainly
inflammatory and haemostatic, possibly associated with
poor clinical outcomes and reduced graft patency at
follow-up, in order to identify new predictors, molecular
mechanisms and targets of unfavourable clinical
outcomes.
Valve disease. Nonrheumatic calcific aortic stenosis and
mitral valve insufficiency are respectively the first and
the second cause of valve disease requiring surgery,
whereas tricuspid valve disease is less frequent and is
usually associated to advanced mitral valve disease.
Calcific aortic valve stenosis is the most common heart
valve disease in the western world, especially in elderly
people; on average, every year in Europe and in the
United States around 50.000 aortic valve replacements
occur due to this pathology. The prevalence of clinically
significant aortic stenosis increases progressively with
age, being 2% in people over 65, and more than 4%
in octogenarians. It is expected to reach the widest
diffusion by the year 2030, when the “baby boomers”
of the sixties will reach the age of 65 or more. The
behavior of aortic valve sclerosis, a milder form of
aortic valve disease characterized by calcification and
stiffening of the aortic valve without a transvalvular
gradient, parallels the one of calcific stenosis, the
prevalence being around 20-30% in patients aged over
65 years, and reaching 45-60% in octogenarians. Now it
is clear that calcific aortic stenosis is not a passive, agerelated disease. Although the progression of this disease
is a multi-factorial process resembling atherosclerosis,
there are some important differences in the final results.
In atherosclerosis the final result is plaque development
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and plaque instability whereas calcific aortic disease
ends up with a severe calcification of the aortic valve.
This suggests that calcific aortic stenosis is not a
single disease process but, more likely, a common
macroscopic anatomic equivalent of a series of partly
related processes (inflammation, matrix remodeling,
myofibrobasts metabolism, angiogenesis, oxidative
stress, and haemostatsis perturbations) that ultimately
leads to severe calcification of the valve. Mitral valve
prolapse (MVP), an abnormal systolic protrusion of
mitral valve leaflets into the left atrium, represents
a common cause of severe mitral regurgitation (MR)
and it often requires surgical correction, especially in
people living in industrialized countries. Available data
suggest that it has a prevalence of 2% to 8% among
the adult population; thus, MVP is expected to occur
somehow in the life course in approximately 7,2 million
individuals in the United States and in over 144 million
worldwide. Besides, a marked effect on its spread and
severity is played by increasing age, indeed important
mitral anatomical changes are found at post-mortem
in approximately 5-7% of elderly people, with males
showing about twice the risk of females of developing
severe regurgitation when ageing. Regarding younger
people, some studies show that both genders are
equally affected, whereas others document a female
preponderance. In most cases this disease is a primary
condition characterized by a progressive myxomatous
degeneration of the mitral valve leaflets and of chordae
tendinae; it represents a slowly developing process
which usually shows a benign course, as less than
10% of all prolapsing mitral valves progress to severe
regurgitation requiring surgery during their lifetime;
however, given the wide diffusion of MVP, this translates
into very high costs for health organizations in the adult
population, even if only a small portion of patients will
eventually need surgical treatment. Although clinical
features and pathophysiology of MVP have been
known from several decades, limited data are available
regarding biological mechanisms or biochemical
perturbations possibly implicated in its progression.
The multitude of the mechanisms potentially involved
in aortic and mitral valve disease progression, together
with the available clinical evidence, strongly suggests
that there are several unknown mechanisms possibly
contributing to the progression of this disease and we
have been actively investigating them with several lines
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of research. Through a combined genomic, proteomic
and transcriptomic approach we are trying to identify
new genes and proteins involved in this process,
studying both diseased valves and plasma of patients
undergoing aortic valve replacement and mitral valve
repair in order to assess what are the derangements
in haemostatic, inflammatory, and oxidative stress
pathways before surgery and what are the changes
that surgery may correct or cause in these patients.
It is very interesting to note that very limited studies
are available concerning the role of oxidative stress in
these two most frequent valve diseases in the western
countries. For this reason we have recently completed
an observational study investigating whether oxidative
stress, antioxidants and nitric oxide pathways differ
in control subjects and in adult patients candidate to
the three most common cardiac surgical procedures
performed in the adults of western countries: coronary
bypass surgery (CABG), aortic valve replacement for
calcific nonrheumatic aortic stenosis (Aortic), or mitral
valve repair for degenerative mitral insufficiency (Mitral).
We have enrolled 165 consecutive patients undergoing
surgery from January to May 2011 (CABG, n=63; Aortic,
n=51; Mitral, n=51, respectively). In addition, thirtythree healthy subjects with cardiovascular risk factors
similar to surgery patients were also studied (Controls).
Candidates to cardiac surgery had increased levels of
oxidative stress and reduced levels of antioxidants with
respect to Controls; also, there was a trend in nitric
oxide pathways impairment in surgical candidates.
Concerning differences among surgical procedures,
Mitral patients had impaired glutathione and nitric oxide
pathways with respect to CABG and Aortic patients.
Overall, the patterns of oxidative stress and NO pathway
of Mitral Patients were more dissimilar from Controls
than those of CABG and Aortic patients. Moreover,
an additional study investigated whether oxidative
stress, antioxidants and nitric oxide pathways differ
between control subjects and candidates to isolated
aortic valve replacement with or without coronary
atheroscle-rosis has shown that candidates to isolated
aortic valve replacement had increased oxidative stress,
reduced antioxidant levels and more pronounced nitric
oxide pathway impairment with respect to control
healthy subjects with similar cardiovascular risk
factors. Interestingly, there were no differences, within
aortic stenosis patients, between patients with normal
coronaries and patients with coronary atherosclerosis.
These data are the proof of concept that valve diseases
are sensibly associated with marked levels of oxidative
stress; the next step will be to determine whether
oxidative stress improves after surgery and whether and
how other molecular mechanisms, namely inflammation
and haemostasis, are also involved in these diseases.
Concerning tricuspid valve disease, we are carrying
out a study to ascertain whether the different repair
techniques now used to correct functional tricuspid
insufficiency are equivalent or not in term of early and
late results. The main point of this study is to answer
a simple question: we know that over time there are
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many different techniques for repairing a functionally
insufficient tricuspid valve. These techniques can be
arbitrarily categorized into two main groups as shown in
the figure:
Tricuspid valve
repair
Anular
stabilization
Complete
Partial
Rigid ring
Pericardial
ring
Flexible ring
Bex/incomplete
rings
NO anular
stabilization
Suture
plasties
Bicuspidization
(Kay)
There are techniques that warrant annular stabilization,
whereas others do not. We are actually running a metaanalysis of the studies currently available in literature
to see whether there are differences between these two
main repairing philosophies.
Aortic disease. Thoracic aortic aneurysms (TAAs) are
important causes of mortality and morbidity in western
countries. Overall, they represent the 15th leading cause
of death in USA, and have an estimated incidence of 1
case per 10.000 persons/year, whereas the incidences
for aortic dissection and aortic ruptures are both around
3 per 100,000 persons/year. The two major etiologies
of TADs are, in order of importance, degenerative/
atherosclerotic and genetic; interestingly, more than 20%
of patients affected by TAA has a first-degree relative
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with the same problem; less frequently, TAAs recognize
an inflammatory/infective problem or trauma as a
possible cause. The main problem a physician encounter
when dealing with TAAs is that the diagnosis is usually
made by an imaging study done for unrelated problems;
otherwise, TAAs are usually clinically silent until one of
the dramatic complications (usually recognized as acute
aortic syndromes, dissection, rupture, hematoma, and
penetrating aortic ulcer), ensues threatening non only
patient’s life in the immediate but also often prejudicing
survival and quality of life at follow-up.
For this reason prevention and prediction have become
a very active area of research in this field; on one side,
it is important to monitor and predict TAA progression
in size, on the other it is essential not only to predict
but also to diagnose the occurrence of complications
in a timely way. Although biological testing for
disease prediction has been already discussed several
times on media, the role of biomarkers in TAAs is still
under definition either for routine patient screening,
or for periodic follow-up, or for a prompt diagnosis
in emergency conditions. Actually we are reviewing
the rapidly accumulating knowledge and the new
trends on the role of biomarkers in the diseases of
thoracic aorta, focusing on established and emerging,
arbitrarily defined, biomarkers in the fields of genetics,
inflammation and haemostasis, matrix remodelling,
substances/cell components/ released upon cell damage,
in order to identify new possible biomarkers that will
be studied in patients affected both by TAAs and acute
aortic syndromes.
Moreover, our unit is also active in the study of
the outcomes of surgical therapies for acute aortic
syndromes, especially in genetically triggered aortic
aneurysms. It is known that Marfan syndrome (MFS)
is the most frequent inherited disorder of connective
tissue, and is strongly associated to aortic dilatation,
dissection and rupture; in these patients Type B
dissection occurs frequently. At present It is not known
whether stent grafting, which is now frequently used
in type B aortic dissection and descending thoracic
aneurysms in non-Marfan patients, is a valuable
option in Marfans, and reports from the literature are
somehow sparse and sporadic. For this reason we are
performing, together with the Department of Cardiac
Surgery of Bologna University and the Department
of Cardiovascular Surgery, Thoracic Aortic Surgery
Program of UPENN, a systematic review of studies
reporting the early and late results of endovascular
stent grafting in MFS patients with type B dissection in
the attempt to quantify possible benefits or potential
drawbacks of this approach in these patients.
adult cardiac surgery, it leads to dialysis in 1% to
5%, and increases perioperative morbidity, mortality
and costs. Previous papers have studied risk factors
associated with the occurrence of AKI mainly focusing
on factors measurable before surgery, occasionally
adding a handful of perioperative factors, mainly related
to transfusion requirements, bleeding or low-output
syndrome. However, the occurrence of AKI after cardiac
surgery is often not immediate and other factors
pertaining to intraoperative, cardiopulmonary bypass
(CPB) and postoperative management of the patients
could be relevant. Moreover, some factors might be
measurable some time before AKI occurrence and, more
importantly, could suggest appropriate strategies to
prevent or limit AKI. We have recently performed a
study in order to investigate: 1) what are risk factors
for AKI, expanding to other perioperative variables
observable before AKI; 2) whether the expansion to
new variables —related to intraoperative, CPB and
postoperative management of the patients — improves
our ability to predict AKI. To do that, we have studied
3219 patients operated from January 2006 to December
2009. Patient preoperative characteristics, as well as
intraoperative, CPB and postoperative management
variables were evaluated for association with AKI
with different statistical models, first including only
preoperative variables followed by the sequential
addition of intraoperative, CPB and postoperative
management variables, and ROC analysis was used
to evaluate and compare models’ discriminatory
power. AKI occurred in 288/3219 patients (8.8%).
Multivariable analysis identified 15 predictors of
AKI; four of them were preoperative (age, diabetes,
smoking and serum creatinine), four intraoperative
(inotropes, erythrocytes transfusion, crossclamp time
and need of a new pump run), two CPB-related (urine
output and furosemide administration during CPB)
and five postoperative (erythrocytes transfusion,
administration of vasoconstrictors, inotropes, diuretics
and antiarrhythmics). Model-discrimination performance
improved from an area under the curve of 0.830 (95% CI
0.807-0.854) for the model including only preoperative
variables to an area under the curve of 0.904 (95%
CI 0.886-0.921) for the model including all variables
(p<0.001). Our study documented that several factors
influence AKI development after cardiac surgery and
perioperative patients management significantly affects
AKI occurrence. Predictive models can be sensibly
improved by the addition of these variables, and
this will be the basis for substantial changes in our
clinical practice, following, as always, the postulates of
translational surgical cardiovascular research.
Predictors of the main complications occurring during
and after adult cardiac surgical procedures. Perioperative
acute kidney injury (AKI) complicates 1% to 30% of
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177
Transcatheter aortic valve implant (TAVI)
Prolonged life expectancy has resulted in an aging
population and, consequently, in an increased number
of patients with degenerative calcific aortic stenosis.
Surgical aortic valve replacement (AVR) is the treatment
of choice for a vast majority of patients and it is one of
the most effective, well-known and 50 years old surgical
procedure, offering symptomatic relief and improving
long-term survival. Nevertheless, the surgical approach
is associated with substantial operative mortality rates in
high-risk patients. Consequently, surgery is not offered
to almost one-third of patients with severe aortic stenosis
owing to a combination of reasons such as advanced age,
impaired left ventricular function, re-do procedure, or
multiple comorbidities. Moreover, as longevity within the
general population is increasing, the proportion of aortic
stenosis patients with contraindications for surgery is also
expected to increase. Given the limited therapeutic options
in this subset of patients, there has been interest in the
development of alternative, minimally invasive techniques.
TAVI has less than 10 years since first in man implantation
and less than 5 years in a real post-commercial setting.
TAVI has rapidly become a valuable alternative to
surgery and medical therapy in high–risk patients with
a transfemoral (TF) or transapical (TA) approach, in case
of narrow, kinked, diseased aortic and/or iliac arteries
(PARTNER Trial). This innovative procedure eliminates
some of the main risks associated with conventional
surgical aortic valve replacement, such as median
sternotomy and the need of cardiopulmonary bypass, thus
opening new perspectives for those patients currently
excluded from surgical practice due to advanced age and/
or the presence of concomitant diseases.
TF-TAVI is less invasive than TA implant, because it can
be carried out under local anaesthesia, with a small groin
incision or total percutaneous approach and it does not
require chest incision. With the last delivery systems, the
precision of TF-TAVI is equal to the TA-TAVI, the risk of
vascular complications is mitigated and the number of TFTAVI has increased more and more.
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Patients’ selection and access choice for TAVI is based
on a preoperative multidisciplinary team approach and
this procedure has to be always executed in an hybrid
operative room (multifunctional operating room with
fixed ceiling-mounted angiography equipment. Fig 1) by a
specialized TAVI team.
In the Cardiac Surgery Unit of Centro Cardiologico
Monzino Hospital, TAVI program started in March 2008
with Edwards Sapien device via TA or TF approach and
TAVI team is composed by a small group of cardiovascular
surgeons, interventional cardiologists, non invasive
imaging cardiologists (TT and TEE echocardiography),
anaesthesists and nurses.
In December 2011, 2 Corevalve devices have been
successfully implanted via TF approach.
From March 2008 to December 2011 289 patients
underwent TAVI procedures (TF 195, TA 94); preoperative
logistic EuroScore was 20.6 ± 11.7%, procedure success
was 98.8% with 3 cases of intraoperative death and 30day mortality was 3.1% (Fig.2).
Echocardiographic assessment revealed an excellent
hemodynamic function of the prostheses with a
physiologic mean aortic gradient and a significantly
improved effective orifice area at discharge and at one
month follow-up; patients improved of one NYHA class at
least at one month follow-up.
Transcatheter aortic valve implantation makes possible to
quickly perform heart valve replacement in the clinic and
with shorter recovery time, thus reducing dramatically the
costs to ensure proper treatment to the population.
The Unit is involved in the multicenter European SOURCE
and SOURCE XT registries of transcatheter aortic valve
implantation using the Edwards Sapien and Edward Sapien
XT valves.
Finally, the further diameter reduction of the introducer
(expandable introducer) has allowed minimally invasive
percutaneous technique in patients previously not eligible
for the transfemoral approach due to bad vascular
Fig 1 Multifunctional operating room
anatomy; the availability of the Edwards Sapien 29 mm
transapical and now transfemoral model has allowed
TAVI treatment in patients previously inoperable due to
excessive size of the native valve.
Seminars and teaching courses are currently held at
Centro Cardiologico Monzino with the aim to promote
the technique of TAVI for severe and symptomatic aortic
stenosis in high-risk patients and to develop a new figure
of cardiovascular surgeon, able to employ these new
technologies and techniques to offer the best range of
therapeutic options for aortic stenosis patients.
Due to the complexity of the procedure and the rapid
rate at which the technology is evolving, TAVI team has
to be the meeting point between cardiovascular surgeons
and cardiologists for a collaborative effort to establish a
successful program for these high risk patients.
Fig 2 A TAVI delivered by TF approach
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Arrhythmia surgery
Conventional treatment of atrial and ventricular
tachyarrhythmias is usually endovascular performed.
There is, however, a part of these arrhythmias that does
not respond to this approach; in case of ventricular
arrhythmias, this may lead to patient’s death. We have
been exploring the feasibility and developing new
minimally invasive and standard approaches to surgically
treat refractory atrial and ventricular arrhythmias in
order to offer this potentially life-saving treatment to a
wider patient population.
Arrhytmia surgery. The treatment of heart rhythm
disorders has gone through significant changes. Initially
it was mainly limited to pharmacologic therapy and the
transformation and adaptation of surgical procedures
to a minimally invasive catheter-based approach, and
subsequent hybridization of the approach, has led to a
newly recognized interest in the development of surgical
approaches in this field, also taking advantage from the
introduction of radiofrequency energy as an ablative
energy source. Presently two main fields are under
development: surgical treatment of atrial fibrillation (AF)
and of ventricular tachyarrhythmias.
Atrial fibrillation. The prevalence of AF is 2% in the
general population and approximately 10% in patients
over 60 years, being the most common form of
sustained cardiac arrhythmia. The incidence of AF,
however, may be substantially higher, due to undetected
asymptomatic AF or undersampling in patients
with paroxysmal AF. With the increasing age of the
population, there is no question that the prevalence will
continue to rise. AF is undoubtedly a costly public health
issue. Unfortunately, the actual numeric data on the
economic burden of AF are sparse compared to those on
other significant cardiac disease states such as stroke
and congestive heart failure.
Although AF is often considered an innocuous
arrhythmia, it is associated with serious morbidity and
mortality due to its three detrimental sequelae:
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1. palpitations, resulting in patient discomfort and
anxiety;
2.loss of synchronous atrioventricular (AV)
contraction, which compromises cardiac
haemodynamics, resulting in varying degrees of
ventricular dysfunction or congestive heart failure;
3.stasis of blood flow in the left atrium, which
increases the risk of thromboembolism and stroke
Atrial fibrillation consists of multiple re-entrant circuits
within the atrium; around the vena cava, pulmonary
veins, and appendages; and around areas of functional
block. Creation of multiple lines of block between these
non-conducting structures may prevent propagation
of arrhythmic circuits. The currently available surgical
ablation systems offer a wide variety of options for
surgical ablation as a concomitant or stand alone atrial
fibrillation procedure. Saline-irrigated radiofrequency
monopolar and bipolar energy is used to produce
electrically isolating lines in the atrial tissue.
Two groups of patients are eligible for this type of
treatment:
• patients undergoing other cardiac surgery (with
the prevalence of mitral valve disease);
• patients with atrial fibrillation refractory to
pharmacological therapy, electric cardioversion or
EPS treatment
Patients in the first group are always treated
concomitantly for valve or, less frequently, for coronary
disease requiring surgery. Bipolar and monopolar
probes are used and lesions set is discussed with EP
(Fig 1) according to clinical status, AF duration, left
atrium dimensions, left ventricular function. Left atrial
appendage exclusion is always performed.
Patients of the second group are under investigation to
develop a minimally invasive surgical treatment with
single or bilateral minithoracothomy in order to isolate
pulmonary veins and to exclude left atrial appendage,
allowing less discomfort and fast recovery.
For better follow-up AF recurrence is monitored with loop
Fig 1
recorder and we offer muldisciplinar ambulatory control.
Ventricular tachiarrhytmias. Ventricular tachycardia
(VT) is a life-threatening arrhythmia that is common
to all forms of heart disease and is an important
cause of sudden death. The number of patients with
cardiac arrhythmias is nowadays higher and higher
due to people increasing age along to occurrence of
hypertension, diabetes mellitus, metabolic syndrome and
ischemic heart diseases.
Primary prevention of ventricular arrhythmias is
usually done with the aid of implantable cardioverter
defibrillators (ICD), and it is well known that the use
of these devices in selected patients may improve
patient survival. In addition, for patients with frequent
occurrences of ventricular tachycardia (VT), catheter
mapping and radiofrequency ablation is available,
although technically challenging. There are several
factors limiting the role of catheter ablation of ischemic
VT. The hemodynamic instability of patients with
coronary artery disease and depressed left ventricular
function limits mapping during the tachycardia. A patient
may have multiple re-entrant circuits. The VT re-entrant
circuits involve scarred myocardium or can be epicardial
as locations that may be out of reach for RF energy to
penetrate. Finally, short-term success may be eclipsed by
development of new VTs with further myocardial injury.
This approach controls VT in most of patients but for
those with recurring VT after catheter ablation, surgery
is the only available option.
This is particularly true nowadays due to an emerging
population of patients with new clinical characteristics,
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In our experience we treat 19 patients (17M-2F, mean age 64.11 ± 9.7)
presenting advanced forms of heart disease (mainly
ischemic dilated cardiomyopathy) and malignant
ventricular arrhythmias (complex arrhythmia pattern).
We are now exploring the feasibility of the development
of new minimally invasive and standard approaches to
surgically treat refractory ventricular tachyarrhythmias
in order to offer this potentially life-saving treatment to
a wider patient population.
An innovative surgical approach is under development
for these patients, when transcatheter ablation
(with endocardial and epicardial approach) has
not been effective or when it is not indicated. In
our multifunctional operating room we perform an
electroanatomical map of the epicardial and endocardial
surface with CARTO system; this allows a precise
location of the areas that need to be treated in order
to eliminate VT. We also do pacemapping inside the
pathological area to evaluate the presence of re-entry
circuits. Then the linear ablation strategy is carried
out to cross the channels and connect scar areas with
anatomical structure, ablation of zone with late potentials.
We use cryoenergy to freeze tissues with a cryoprobe
which destroys the arrhytmogenic tissue while
maintaining structural integrity.
Death: 3 patients
•
HF + MOF (after 2 months)
•
no VT recurrence Heart failure (after 6 months)
Superimposed endo-epi voltage maps of LV showing conducting channel within lateral basal portion of LV epicardium. Arrows point to
concealed entrainment site and VT termination site
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Vascular Surgery
The scientific activity of the Vascular Surgery Unit is
strictly linked to the clinical activity. The Unit supports
a broad range of both clinical and basic research
programs in order to improve diagnosis, treatment, and
prevention of diseases involving the vascular system.
The Vascular Surgery Unit scientific activity is focused
on six major areas:
Proteomics of atherosclerotic plaques
Proteomics of atherosclerotic carotid artery plaques.
By applying proteomics to human carotid artery plaque
extracts, we have identified a panel of proteins that are
differently represented in stable or unstable plaques,
thus providing an additional mean for analyzing the
molecular process involved in plaque destabilization in
vivo. Western blotting was used to confirm different
levels of selected proteins in plaque extracts.
Evaluation of low molecular mass thiol content in carotid
atherosclerotic plaques. Recently several methods for
thiol determination in different sample types investigating
intraplaque thiols and the relationship existing between
plasma and plaque thiol levels obtained from patients
undergoing carotid endarterectomy, have been developed.
Moreover, the amounts of intraplaque thiols bound to
proteins and their correlation with total intraplaque
thiols has been analyzed. Our results have shown that
increased levels of intraplaque glutathione may induce
important effects on plaque fate by perturbing the normal
low molecular weight of thiol redox state.
Oxidative stress and carotid artery stenosis and
treatment
Evaluation of oxidative stress in patients with carotid
artery stenosis that underwent surgical carotid
endarterectomy or carotid angioplasty. The oxidative
stress parameters in carotid artery stenosis and the
relation between oxidative stress markers and carotid
artery surgical or endovascular treatments have been
evaluated.
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Oxidants and antioxidants possible role in pathogenesis
of calcific aortic valve stenosis. Previous epidemiological
studies have shown that the aortic sclerotic process
shares risk factors and pathogenetic similarities with
atherosclerosis, in particular a common event in both
conditions is the oxidative stress. Evidences indicate that
oxidized low density lipoprotein (LDL) are present both
in isolated cardiac valves and in atherosclerotic plaques.
The role of oxidative stress is not yet well defined and
clear, but it is now recognized to be a prominent feature
of many acute and chronic diseases, and even of the
normal aging process. In particular there are very few
information about the role of oxidative damage towards
lipids and proteins in heart valvular disease, because it
is difficult and not even clearly defined how to assess
the presence of oxidative stress.
Another important area of study is “antioxidant systems”
that are part of the natural defences of the human body
towards oxidative stress. We are evaluating the markers
of physiological antioxidant systems - free and total
malondialdehyde (MDA), hydroperoxides, products of
the advanced oxidation of proteins (AOPP), reduced
glutathione (GSH) and glutathione disulfide (GSSG)- and
markers of oxidative stress (the two main isophorms of
vitamine E, α and γ tocopherol). The preliminary results
have shown that oxidants and antioxidants seem to play
a key role in the pathogenesis of calcific aortic stenosis;
further studies are essential to better define their roles
and to elaborate new and innovative therapeutic and
preventive strategies.
Glycosaminoglycans and cardiovascular cellular matrix
The extracellular matrix (ECM) of heart valve consists
predominantly of fibrillar collagens, elastic fibers,
glycoproteins, proteoglycans (PGs) and complex
polysaccharides, glycosaminoglycans (GAGs). ECM
and GAGs are fundamental to maintain the mechanical
and functional properties of heart valves and represent
an active, dynamic compartment capable of providing
instructional signals to adjacent cells, determining many
cellular functions.
A wide variety of GAGs exert considerable and variable
control over the physical and mechanical properties of
ECM. As a consequence, the fine structure of GAGs
changes during certain diseases involve changes in the
relative and/or total amounts of GAGs. In particular,
modifications of the relative percentage of single
polysaccharides, e.g. the ratio of Condroitin Sulfate /
Dermatan Sulfate (CS/DS) that are uronic acids and
of 4-sulfated disaccharides to 6- and non-sulfated
disaccharides of galactosaminoglycans, and the overall
charge density may occur.
Therefore, CS/DS analysis, which identifies
structural variations due to differential expression
of the corresponding proteins responsible for their
biosynthesis, may reflect tissue or cell status.
The research carried out in collaboration with the
School of Biosciences and Biotechnologies, University of
Modena, is focused on GAG determination in normal and
pathological heart valves and vein vessels. In particular
the composition of aortic valves, both normal and
stenotic or with insufficiency, and of mitral insufficiency
in terms of GAGs is measured.
Preliminary results indicate changes in the relative
amount and distribution of GAGs in the posterior
leaflets of mitral valves in patients suffering from
mitral regurgitation. Similar data have been obtained in
aortic valve degenerative disease with a decrease in the
tension to which these tissues are subjected and with an
abnormal matrix microstructure capable of influencing
the hydration and of conditioning the mechanical
weakness of these pathological tissues.
As we know, the vein insufficiency represents the most
common cause of lower leg swelling, affecting up to
2% of the entire population, and up to 20% of elderly
people. Thus we have performed specific studies in
order to assess the GAG content of saphenous veins
before and after oral administration to patients of
specific of glycosaminoglycans. Preliminary data indicate
that the content of GAGs in pathological vein is lower
than the one of normal veins. Oral GAGs administration
helps to maintain the mechanical vein characteristics.
Tissue engineered heart valve
Heart valve substitutes, both mechanical and
bioprosthetic, currently in use are unable to grow, repair
or remodel within an individual. This represents a major
problem for children needing valve replacement because
they have to undergo multiple major surgical procedures
as they grow or get older with fast-increasing mortality
risks.
Tissue-Engineered Heart Valves (TEHVs) represent the
ideal heart valve to be replaced since they have the
potential to grow and repair within the host, to minimize
inflammatory and immunological responses and to limit
thromboembolism. In fact, viable cells included in TEHVs
can theoretically adapt to a growing and changing
environment just as a native biological structure does.
The aim of the project is to develop TEHVs from
natural and synthetic tissue sources using an acellular
biomatrix (homograft valve and polymers) as a scaffold
and seeding it with viable autologous (recipient’s) cells
through the following steps:
1. Biological/synthetic scaffold creation. These
activities concern the decellularisation method
of cryopreserved aortic valves. Valvular tissues,
taken from a donor and clinically unsuitable for
transplants, are processed according to the EATB
cryopreservation protocols to obtain a scaffold.
We are studying the possibility to develop a
biopolimeric scaffold.
2.Culture and expansion of the seeding cells.
Innovative dynamic culture systems (bioreactors)
have been developed for this purpose in the last
year. These are also fundamental for the sowing
and growing of cells on the scaffold.
3.Valves recellularisation. The bioreactors
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mechanically stimulate the scaffold and foster
a proper cellular differentiation (together with
different growth factors) and a correct tissue
remodeling, the valves are repopulated with
recipient cells.
4.Animal tests and in humans valve implants. During
the lifecycle of the project, TEHVs will be tested
with test rigs (stand test), animals (sheep) and
finally implanted in humans for a full clinical trial.
Evolution from one type of test to the other will
require full clearance of TEHVs through previous
method to limit animal sufferance and sacrifice,
and risks for humans. At present time we are
evaluating different homograft decellularization
and recellularization techniques.
Moreover, in cooperation with the Bioengineering
Department, Politecnico of Milano, we have created a
bioreactor able to mimic the human blood flow in order
to test the functioning of different valves.
thickness, myocardial infarction and stroke as well as
congestive heart failure.
The presence of vascular disease and its possible
association with 25-hydroxyvitamin D levels has been
evaluated using a a Doppler ultrasound.
Basic and Translational Research
Gene expression of molecules involved in
atherosclerotic aneurismatic aortic wall perivascular
fat and also in abdominal visceral to subcutaneous
abdominal fat process.
The aim of this project is to study the patterns
of gene expression involved in inflammation,
angiogenesis, cellular proliferation, thrombosis and
fibrinolisis, vascular calcification and remodelling in
the perivascular fat. Patient abdominal fat samples
are compared with perivascular aneurysm samples
fat obtained during surgical abdominal aneurysms
treatment.
Evaluation of vascular peripheral ultrasound disease
and vitamin D levels.
Vitamin D deficiency has often been associated
with cardiovascular disease risk factors such as
hypertension and diabetes mellitus, with markers
of subclinical atherosclerosis such as intima-media
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Our data indicate that DES-treated patients show a
prothrombotic phenotype both during DAT and after
clopidogrel suspension. Indeed, in DES-treated patients,
despite five months of DAT, the percentages of TF
positive-platelets and -MPA were markedly higher
as compared to those of MT patients and remained
significantly higher also when patients were on aspirin
only. At clopidogrel suspension, the percentage of PAC1 positive platelets, which was, during DAT, comparable
to that found in MT patients, significantly increased too,
further highlighting a platelet activated phenotype.
Values, evaluated during DAT, of platelet–associated
TF delta stimulation or F1+2 plasma levels above the
median (296% and 199 pMol/L, respectively) were
significant predictors of need for reintervention in
the four-year follow up. Therefore, in DES patients
the evaluation during DAT of parameters related to
blood coagulation such as platelet-associated TF delta
stimulation and F1+2 plasma levels may assist in the
identification of patients who are at greatest risk of
future coronary interventions.
p < 0.001
p = 0.004
p = 0.002
p = 0.0001
p = 0.0005
TF Positiv e platelets (%)
p = 0.006
p = 0.05
DAT
ADP STIMULATED
1 month 6 months Medicallytreated
ASA only
patients
DAT
1 month 6 months Medicallytreated
ASA only
patients
DES-treated patients
DES-treated patients
Figure 2. The risk score, obtained combining values of platelet-associated TF and of F1+2, was highly associated with adverse events
(p=0.0001 by log-rank test; HR 7.5, 95% C.I. 2.2-25.1, p=0.001, for one-step score increase). The area under the ROC curve is 0.82 (95%
C.I. 0.65-0.98, p<0.0001), indicating a very good prognostic value.
ROC Curve for Model
Area Under the Curve = 0,8182
Both below the median
One of the two above the medium
Sensitivity
Up to a decade ago the role of activated platelets in
atherosclerosis was believed to be confined to thrombus
formation, responsible for the clinical manifestation of
the disease. More recent studies, however, emphasize
that activated platelets express molecules contributing
also to the onset and evolution of atherosclerotic plaque
and of vascular restenosis. We recently reported the
finding that platelet reactivity to classical agonists
results in expression, on the platelet surface, of
functionally active tissue factor (TF), the key activator
of blood coagulation and protein also involved in a wide
range of biological processes including induction of
pro-inflammatory response and proliferation of vascular
smooth muscle cells. Moreover, we also provided
evidence that a significantly greater number of TFpositive platelets, with an higher thrombin generation
capacity, is present in acute coronary syndrome (ACS)
patients compared to either stable angina patients or
healthy subjects providing a further explanation of the
increased prothrombotic potential characteristic of these
patients.
No data are available on the behaviour of plateletassociated TF in patients treated with DES. Thus,
we sought to assess it together with other platelet
activation markers such as P-selectin, activated
GpIIbIIIa expression as well as the number of total and
TF-positive monocyte-platelet aggregates (MPA) by
whole-blood flow cytometry in DES-treated patients
during DAT and at one and six months after clopidogrel
suspension. In order to have reference values of platelet
activation in coronary artery disease (CAD), the same
markers were also assessed in 40 medically-treated
stable angina patients (MT). Finally we evaluated
whether elevated levels of platelet activation markers
in stent-treated patients, if any, may predict the risk of
future revascularization in a four-year follow-up.
RESTING
TF Positiv e platelets (%)
Drug-eluting stent (DES) implantation is the treatment
of choice for patients with symptomatic coronary artery
disease undergoing percutaneous coronary intervention
(PCI). By reducing the rates of late lumen loss and
restenosis, DES have expanded the indications for PCI
into more complex anatomic subsets (long lesions,
small arteries, bifurcations) in high-risk patients (renal
failure, diabetes). A low rate of in-stent restenosis (ISR)
after DES, however, still exists and since the population
treated with DES is large, its prevalence is not
negligible. DES implantation causes a massive platelet
activation and dual antiplatelet therapy (DAT) with
aspirin and clopidogrel for 6-12 months is the standard
of care for PCI-treated patients. It is worth mentioning,
on this regard, that the length of DAT is empirically set,
since no study has so far assessed platelet activation at
clopidogrel suspension.
Figure 1. The percentage of TF positive resting platelets measured by whole-blood flow cytometry in DES-treated patients during DAT was
3 fold higher than that observed in MT patients; it further increased one month after clopidogrel suspension, and even more six months
after clopidogrel suspension. After in vitro blood challenging with ADP, the percentage of TF positive platelets markedly increased (~3-4
fold delta stimulation) with respect to the unstimulated blood sample at all time points. Particularly, despite the DAT, the percentage of TF
positive platelets was comparable to that found in MT patients who are in aspirin only. At clopidogrel suspension, ADP stimulation resulted
in a number of TF-positive platelets significantly higher than that found during DAT, as expected, and that in MT patients.
Event-free survival
Prospective evaluation of platelet activation
markers in patients treated with drug-eluting
stents: platelet-associated Tissue Factor predicts
the risk of future revascularization in a pilot study.
Risk score:
Plat-TF+F1+2
Both above the median
Follow up (Months)
1 - Specificity
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Proteomics-based development of biomarkers
in cardiovascular disease
Understanding cardiovascular diseases and their
modifications requires clarification of mechanistic
changes in organ phenotype over time, the
influence of genetic variations, and the effects
of pharmacologic, surgical, and interventional
treatments. These requirements have been addressed
by genetic and biochemical approaches, but a more
meaningful understanding of gene expression can be
achieved through characterization of the products
of that expression, proteins, the essential biological
determinants of disease phenotype. The ultimate
phenotype of cells, organs, and organisms is reflected in
the instantaneous global protein profile, the proteome;
similarly, changes in human health are the result
of changes in the proteomes of individual patients
over time in response to endogenous and/or external
stimuli. The advent of novel proteomic approaches to
investigate the complexity of human illness promises
to shed new light on the pathogenesis of a broad
range of cardiovascular diseases. These inferences are
multifaceted and include the commonly recognized
role of proteomics in characterizing biomarkers and
biosignatures for the prognosis and diagnosis of
disease, the capability of these technologies of revealing
information regarding functional subproteomes of
organelles and networks in the heart and vasculature,
and the importance of proteomics in defining changes in
these functional subproteomes to guide future therapy.
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. Firstly,
changes in the subcellular phenotype (subproteome)
of the organism can lead to alterations in proteins
detectable as mechanistic markers. These changes
closely reflect what is going on in the cell and how
the signaling pathways are manipulated. Secondly,
the arrival of the disease carries changes in proteins
that are detected by proteomics, the so-called clinical
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disease markers. These markers are specific of the
individual disease state and can indicate the state of
progression, severity, and location of the syndrome.
Lastly, therapeutic markers are those that become
evident as the treatment of a chronic disease progresses
in the patient. These markers are influenced by many
factors, including individual nature of the disease,
drug treatment, patient activities, etc. Under ideal
circumstances, a combination of markers from these
different groups would be used for a more accurate
diagnosis and treatment.
Despite these promising expectations, proteomics is
inherently challenging because of the complexity of
the molecules with which it deals, proteins, the main
working parts of biological systems. Their multiple
functional roles require them to be made, modified, and
often quickly remodified under the control of numerous
multilevel systems. Clinical proteomics is further
complicated by the fact that proteins can reflect genetic
differences among patients at both the structural and
quantitative levels. Compared with nucleic acids, proteins
show much wider variations in physical properties
(which leads to greater difficulties in global separation
and detection) and in abundance (protein concentrations
vary by more than 1010 in plasma, which generates
additional technical challenges for detection of minor
components). Despite these difficulties, proteins offer
the best general picture of what is working in a cell
or tissue, and what is not. Thus, although proteomics
is currently far from comprehensive, it can point to
numerous proteins with intriguing, even if unproven,
clinical utility.
Furthermore, improvements in proteomic technologies
that allow a more detailed characterization of protein
gene products, coupled with carefully constructed clinical
databases and sophisticated analytic techniques, will
serve as an important bridge to connect our nascent
appreciation of biological complexity with our ultimate
goal of understanding and treating disease. Therefore,
a deep understanding of early protein abnormalities
associated with major diseases will result in a revolution
in preventive medicine by greatly enhancing our ability to
identify patients at risk and those with preclinical disease.
Improved patient care through the use of protein
markers is indeed a well-proven paradigm. For
instance, use of new protein markers has become
an integral part of clinical cardiology in the past ten
years. Blood measurements of C-reactive protein,
Indicative of therapeutic
mechanism
Used to adjust
clinical treatment
Therapeutic
Markers
Mechanistic
Markers
Clinical
Disease
Markers
Reflective of disease molecular origin
Suggestive of cellular therapies to modulate phenotype
troponin, and B-type natriuretic peptide are becoming
routine measures to determine risk, to diagnose, to
prognosticate, and to guide treatment of coronary heart
disease, acute coronary syndromes, and heart failure.
Up to now, the study of protein markers has been
focused on “candidate” protein approaches, whereas
“omics” technologies allow unbiased assessment of how
patterns of proteins differ in various disease states. Yet
an obvious hypothesis is that definition of patterns of
disease-related changes, including novel disease marker
proteins, would provide substantially more useful clinical
information than a single marker. Observational studies
have shown that the combination of protein markers (for
example, troponin and C-reactive protein) into panels
can provide valuable additional information in stratifying
risk in acute coronary syndromes.
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
Cardiovascular 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 can have therefore important
direct ‘bedside’ applications, even if we are still in the
early stages. However, a fruitful interaction with the
clinical departments 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.
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Red Blood Cells as a source of Nitric Oxide
The endothelial dysfunction is a pathological condition
frequently occurring in patients with coronary
artery disease. It is associated with an impaired
endothelium-dependent vasodilation, due to a decreased
bioavailability of nitric oxide (NO). This mediator plays
a crucial role in the regulation of vascular homeostasis,
vasomotor tone and platelet aggregation. Several factors
may reduce the bioavailability of NO and, among them,
the increased oxidative stress, secondary or not to an
inflammatory condition, is of particular importance.
Oxidative stress impairs the NO synthesis and it may
inactivate the produced NO by transforming it into
peroxynitrate.
Besides endothelial cells that are considered the major
source of NO, also circulating cells (erythrocytes,
platelets and monocytes) are able to synthesize NO.
The production of NO by red blood cells (RBC) has
been only recently recognized. Initially, these cells
were considered only scavengers of NO produced
by endothelial cells. In fact, NO is inactivated by a
dioxygenation reaction with oxyhemoglobin (HbO2) to
form methemoglobin (MetHb) and nitrate. Alternatively,
it may bind the heme group of deoxygenated
hemoglobin to form NO-Hb and/or the S-nitrosated
derivative of HbO2, SNO-Hb, the latter probably
involved in the regulation of blood flow. More recently,
evidence that RBC constitutively carry an active NO
synthase (NOS) has been provided. This calciumdependent enzyme, located both in RBC membrane
and cytoplasm, metabolizes L-arg, with an activity well
comparable to that of the endothelial enzyme. Based on
this knowledge, the role of RBC in the NO metabolism
has been revised. It is now accepted that RBC are not
only mere passive scavengers but also “transporters”
of bioactive NO forms (SNO-Hb and the oxidized
form nitrite) and active contributors to vascular NO.
Similarly to nitrite and SNO-Hb, NO produced by RBC
participates in the regulation of blood flow and platelet
function. In fact, stimulation of RBC-NOS with L-arg
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decreases platelet aggregation whereas its inhibition
has opposite effects. NO produced by RBC acts also
in an autocrine manner through the modulation of RBC
deformability and their passage through the capillaries
thus affecting the blood flow in the microcirculation.
Under specific conditions (e.g. fall in Hb saturation by
O2 or the passage in resistance capillary), erythrocytes
release bioactive NO from SNO-Hb and from low
molecular weight nitrosothiols. Erythrocytes are also
able to release ATP, which in turn stimulates the NO
synthesis by endothelial cells and platelets. In addition,
it has been recently shown that large amounts of ADMA
and SDMA are incorporated into erythrocyte proteins
and that RBC carry other important enzymes of L-arg
metabolism, i.e. L-arg degrading arginase and ADMA
metabolizing dimethylarginine dimethylaminohydrolase
(DDAH), and cationic amino acid transporters. Alteration
of these enzymatic systems in RBC may be responsible
for a decreased NO bioavailability either per se or in
combination with a deficiency of L-arg or a decreased
BH4 availability or an increase of reactive oxygen
species. Consequently, alterations of RBC L-arg/NO
metabolic pathway induced by specific conditions (e.g.
inflammation and oxidative stress) in pathological
processes such as atherosclerosis and cardiovascular
diseases can be hypothesised. However, to date
little is known about the conditions that impair NO
bioavailability in this cell compartment. Studies carried
out in our laboratory are aimed at defining the synthetic
and metabolic profile of NO (NO metabolome) in RBC
and plasma of healthy subjects and patients affected
by coronary artery disease, in order to understand the
effect of NO RBC-derived on vascular homeostasis.
To this end, an integrated and innovative approach
is used to define multiple metabolic changes caused
by the pathological condition. In our laboratory this
approach has been already set up: a number of
substrates/metabolites involved in NO synthetic and
metabolic pathways are simultaneously analyzed by an
LC-MS/MS method. NO synthesis in RBC will be also
assessed both in terms of NO synthase (NOS) enzyme
expression (immunohistochemistry and Western blot
method) and of aminoacid transporter expression and
activity. A study is in progress to determine arginine
targeted metabolome in RBC of patients with coronary
macro and/or microvascular dysfunction The enzyme
deputed to NO synthesis, RBC-NOS, will be detected by
immunofluorescence in patients and in controls.
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193
Heterogeneity of human macrophages in culture
and in atherosclerotic plaque.
Macrophages are immune cells which play a central
role in atherosclerosis, the major underlying cause of
cardiovascular events. They are involved in the initiation,
progression and vulnerability of the atherosclerotic
plaque. Like other immune cells, macrophages are not
static but respond to a multitude of stimuli they receive
from the microenvironments and consequently show
high plasticity and heterogeneity.
Macrophage functions can broadly be categorized by a
series of dichotomies, for example, innate or acquired
immunity, tissue destruction or repair, immigration
or emigration, cholesterol accumulation or release,
pro-inflammatory or anti-inflammatory activity. These
functions could be carried out by distinct subpopulations
of macrophages. A current simplified classification of
macrophage phenotypes is the discrimination between
pro-inflammatory (M1) and anti-inflammatory (M2)
macrophages. Whereas M1 macrophages are induced
by bacterial compounds or inflammatory cytokines and
are mainly involved in the expansion of inflammation
through the production of inflammatory mediators,
M2 have immunoregulatory and immunosuppressive
functions, and promote tissue repair and healing. A wide
range of markers has been described to characterize
different populations in vivo and in vitro. For identifying
particular macrophage subsets, cell surface proteins
are generally evaluated, but the study of one or only
few related phenotypical surface markers greatly
oversimplifies the macrophage phenotype in an in
vivo situation. In addition M1 and M2 phenotypes are
obtained upon in vitro stimulation and the paradigm of
macrophage polarization is essentially based on in vitro
results. Currently, there is still a great need for more
extensive characterization of the different macrophage
population, i.e. identifying a panel of markers and
functional properties of distinct macrophage subset.
Studies carried out in our laboratory are focused on the
characterization of the distinct macrophage morphotypes
identified in our culture conditions to delineate a profile
in term of antigen expression and function. This cellular
model might be adopted as a suitable model to study
macrophage heterogeneity, a feature that has been
disclosed in settings spanning from the inflammatory
and wounding response to the atherosclerotic lesion.
Also in atherosclerosis, macrophage heterogeneity is
an accepted concept. However in this condition the M1/
M2 division is a very strong simplification of reality,
indeed in atherosclerotic plaque, the micro-environment
is very heterogeneous and macrophages do not interact
with individual cytokines but with many other factors
produced in an autocrine manner by other tissue
and inflammatory cells. However the identification of
macrophage phenotypes that promote different aspects
of atherosclerotic disease may have implications for
the development of therapeutic interventions and it has
been suggested that promoting each of these subsets
at different stages of disease may have therapeutic
benefits.
Model of induction of macrophage heterogeneity in atherosclerosis. Macrophage can polarise towards different phenotypes according
to various stimuli present in the surrounding microenvironments.
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195
Experimental Imaging of brain and heart ischemia
Imaging techniques play now a pivotal role in biomedical
sciences, both at clinical and basic level.
Their use in clinical medicine is not limited to
diagnosis, but is also addressed to the identification
and understanding of the etiology and pathophysiology
of many diseases and is essential for monitoring the
efficacy of therapeutic approaches. Moreover, due to
their non-invasiveness, high-definition and versatility,
imaging techniques provide a unique opportunity
for translating basic, in vivo, research into human
applications. The goal of our research - pursued
through an interdisciplinary application of different
imaging approaches and, in particular, magnetic
resonance imaging (MRI) and echography - is to move
scientific discoveries, in particular those in the area of
pharmacology, from the laboratory to clinical practice.
Magnetic resonance imaging (MRI) for the evaluation of
acute stroke and its response to therapy.
Stroke is the second leading cause of death worldwide
and has the most devastating consequences among
vascular diseases, causing long-term neurological
disability and incurring high societal and financial costs.
Although during the last years the understanding of
its pathophysiology has remarkably improved, stroke
still remains an unmet medical need. In our laboratory,
brain ischemia and identification of new pharmacological
targets are studied principally in two models:
1. Permanent middle cerebral artery occlusion (pMCAO)
surgically induced in rats or mice. The development
of acute cerebral lesions are assessed over time in
MRI longitudinal studies by using trace of apparent
diffusion coefficient maps [Tr(D)] and T2-weighted.
T2-map and gradient-echo images are performed to
track inflammatory cells infiltration into the ischemic
brain damage labelled in vivo by USPIO, a cellspecific MRI contrast agent taken up by microglia
and macrophages. Recently we are setting up
and validating fractional diffusion anisotropy (FA)
Figure 1: MRI Unit (spectrometer 4.7 Tesla) (a).. Coronal section of rat brain visualized by MRI, Tr(D) ( b) and T2 weighted (c)
images. Visualization, by immunofluorescence, of P2Y12-receptor expression (green) on microglia cells (red) in areas surrounding
ischemic lesion (d and f) and in healthy tissue (e) from MCAO rats.
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analysis in order to visualize white matter alterations
and reorganization after ischemic brain damage in
rodents.
2. Spontaneously hypertensive rats stroke-prone
(SHRSP). This animal model develops spontaneously
a complex form of cerebrovascular pathology
resembling the human disease. Characterization of
molecular and cellular mechanisms underlying brain
damage and assessment of pharmacological treatment
aimed at the prevention of tissue ischemia have been
extensively investigated by the MRI Unit.
Delivery, trafficking and toxicity of engineered magnetic
nanoparticles in macrophages and CNS cells in animal
models of stroke.
The goal of this research is to develop an efficient
way to harness monocytes/macrophages to selectively
transport MNPs to inflamed brain areas for the diagnosis
and follow-up of inflammatory and ischemic diseases by
MRI. Developing this diagnostic method is an ambitious
goal that implies a significant scientific and technological
effort and the integration of different scientific
disciplines: physics, chemistry, and materials science at
the nanometer scale. The potential of engineered MNPs
as contrast agents for MRI will be assayed in vitro and
validated in vivo in animal models of stroke.
Pathophysiological characterization of renal disease by
MRI and echography.
Implementation of MRI analysis to the study of kidney
focuses on: (i) characterization of the end-organ
damage mechanisms occurring spontaneously in the
kidney of hypertensive stroke-prone rats (SHRSP)
with particular emphasis on the role of iron overload
in the development of hypertensive nephropathy, (ii)
investigation of the relationship between renal and
cerebral damage, both spontaneously occurring in
SHRSP, (iii) evaluation of the effectiveness of several
renoprotective therapies evaluated by the use of specific
micro-bubbles that allow the echographic evaluation of
tissue perfusion.
Figure 2: MRI gradient-echo coronal renal sections of a health rat (arrows, a) and a proteinuric SHRSP (arrows, b) with T2*
decreased signal in the cortex, an index of iron accumulation.
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197
Interactions between food and cardiovascular
drugs
Imaging approach to ischemic heart disease: cardiac-MRI
and high-resolution echocardiography.
In our laboratory, a model of cardiac infarction occluding
permanently the left anterior descendent coronary
artery has been set up in mice. This model has been
used to elucidate functional and structural changes
associated with ischemic heart disease and to evaluate
novel pharmacological therapies. Characterization
of the temporal evolution of the damage is possible
using MRI and echocardiography. Both methods allow
to have anatomical information of the whole cardiac
cycle by cine imaging and functional information of the
organ by the evaluation of parameters such as ejection
fraction, end diastolic, end systolic and stroke volumes.
Moreover cardiac-MRI allows to identify the ischemic
area by contrast agents like gadolinium at the very
beginning of the damage. Another field of interest is the
echocardiographic and morphometric characterization of
left atrium (LA) in adult mice and the evaluation of the
relationships between atrium and ventricle function in
acute cardiovascular conditions.
Figure 3: High resolution Vevo2100 platform (a); e.cho 2D and color Doppler of mouse appendage at telediatolic and telesystolic
frame (b); mouse heart morphology Sagittal four chamber view of mouse hearth with cardiac-MRI (b), echocardiography (c).,
LA = left atrium, histology (d); LV = Left Ventricle, RV = Right Ventricle.
Statins are compounds that reduce blood levels of
LDL-cholesterol by competitively inhibiting HMG-CoA
reductase activity in liver cells. This effect promotes
the expression of lipoprotein receptors in hepatocytes,
augmenting the uptake of atherogenic lipoproteins,
thus reducing their circulating levels. Although formally
labeled as lipid-lowering drugs, statins are actually
anti-atherothrombotic compounds, due to their lipid
effects and, putatively, to many other “pleiotropic”
effects (antioxidant, antiproliferative, antinflammatory,
and many others) demonstrated in vitro and sometimes
in vivo. Numerous clinical studies have demonstrated the
efficacy of statins to reduce cardiovascular morbidity
and mortality of about 25 to 50%, in direct relationship
with the baseline cardiovascular risk of the population.
These studies prompted the massive use of statins in
clinical practice all over the developed world for the
primary and secondary prevention of atherosclerotic
cardiovascular diseases in the last decade. In fact,
statins are absolutely the most frequently prescribed
cardiovascular drugs.
Many other cardiovascular and non cardiovascular
drugs have pharmacologically relevant interactions with
a
c
Figure 1. Selected drugs that may increase risk of side effects
of statins when used concomitantly
CYP3A4 Inhibitors/Substrates
Cyclosporine, tacrolimus
Macrolides (azithromycin, clarithromycin,
erythromycin)
Azole antifungals (itraconazole, ketoconazole)
Calcium antagonists (mibefradil, diltiazem,
verapamil)
Nefazodone
Protease inhibitors (amprenavir, indinavir,
nelfinavir, nitronavir, saquinavir)
b
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CCM — Scientific Report 2011 — Ongoing research 2012
Sildenafil
Warfarin
Others
Digoxin
Fibrates (gemfibrozil)
Niacin
Figure 2. Incidence and severity of muscle side effects of
statins
statins (Figure 1), generally leading to increased statin
levels in blood and statin toxicity, mostly in liver and
skeletal muscle.
These side effects may be clinically expressed as
hepatitis to liver failure in the former, or as myalgia,
myopathy, or even life-threatening rabdomyolisis in the
later (Figure 2). Concern of severe toxicity is one of
the most frequent patients’ and physicians’ arguments
for statin omission, suboptimal dosing and early
withdrawal. Awareness of drug-drug interactions is very
important to reduce this risk. Yet, recent observations
show that not only drugs but also some specific foods
may significantly interact with statins. Paradigmatic
examples are the interactions of some fruit juices with
specific statins. For example, numerous cases of muscle
toxicity by grapefruit juice-statin interactions were
reported, and pharmacokinetic studies demonstrated that
consumption of grapefruit juice consistently increase
simvastatin lactone [AUC(0-24) 3.6-fold; CMax 3.9fold] and simvastatin acid [AUC(0-24) 3.3-fold; CMax
4.3-fold] bioavailability (i.e. Lilja et al. British Journal
CCM — Scientific Report 2011 — Ongoing research 2012
199
Figure 3. Examples of demonstrated interactions between nutrients and statins
Grapefruit juice increases serum
concentrations of atorvastatin and has no
effect on pravastatin
Atorvastatin acid
2.5-fold
Atorvastatin lactone
3.3-fold
Lilja JJ etal. Clin Pharmacol Ther 1999;66:1187
of Pharmacology 2004;58:56-60). Similar results had
been obtained with lovastatin (Clin Pharmacol Ther 1998,
63:397-402). Moreover, grapefruit juice also significantly
interacts with atorvastatin acid [AUC(0-72) 2.5-fold] as
well as with atorvastatin lactone [AUC(0-24) 3.3-fold;
CMax 2.6-fold] but not with pravastatin (Lilja et al. Clin
Pharmacol and Ther 1999;66:118-27). Pravastatin, instead,
showed a significant although modest interaction with
a specific type of orange juice [AUC 0-240 min 1.5 fold]
(Koitabashi Y et al. Life Sciences 2006;78:2852-2859).
More recently, pomegranate juice, which interacts with
several other drugs, was also implicated in a case of
rhabdomyolysis by statins (Am J Cardiol. 2006;98:7056). (Figure 3).
Herbs, which are erroneously regarded as innocuous
beverages, may also affect statin pharmacokinetics.
In fact, the herbal antidepressant St. John’s wort
(Hypericum perforatum) decreases plasma concentrations
of simvastatin (Sugimoto et al. Clin Pharmacol and
Figure 4. Putative favourable green tea properties
Figure 5. Kinetics design
Effects of regular consumption of
grapefruit juice on the pharmacokinetics
of simvastatin
Simvastatin acid
3.3-fold
Simvastatin lactone
3.6-fold
Lilja JJ etal. BritishJ Clin Pharmacol 2004;58:560
Ther 2001;70:518-524). A recent report also suggests a
possible interaction of St John’s wort with rosuvastatin
(Gordon RY et al.Am J Med, 122:2, 2009).
Green tea is an herbal product massively consumed in
Asian countries. As a consequence of the popularization
of the many healthy properties attributed to green tea
(Figure 4), the consumption of this typically oriental
infusion has extended widely to western countries not
only as herb teas but also as industrialized drinks.
In a recent study from our group, we observed that
consumption of three cups/day of green tea doubles the
bioavailability of simvastatin. Based on this observation
(Werba JP et al, Ann Intern Med. 2008;149:286-7;
Figure 5) and the available information about the
metabolic pathways of simvastatin and green tea, we
speculate that the interaction observed may be related
to a competition for mechanisms of transport and/
or metabolism between specific green tea cathechins
and simvastatin. Extended pharmacokinetic interaction
studies are being performed in collaboration with Prof.
Shizuo Yamada (University of Shizuoka, Japan) and Prof.
Hiroshi Watanabe (University of Hamamatsu, Japan).
of the subjects with this response on green tea had
lower total simvastatin bioavailability (lactone + acid)
and higher acid/lactone ratio on water. These findings
suggest that subjects with a low spontaneous drug
bioavailability, presumably due to an increased activity
of drug efflux pumps (MDR1), are more susceptible to
the effect of green tea. Therefore, in 2011 we plan to
search for MDR1 gene variants that could explain this
phenotype. The identification of gene variants that affect
MDR1 function may have wider implications in terms of
potential drug-drug and drug-nutrient interactions.
Similar studies were performed in Italy (Milan) and
in Japan (Hamamatsu) to assess the consistency of
the putative interaction in two ethnically different
populations. Healthy male young adult volunteers (n=
12 in Italy and n=12 in Japan) have been recruited for
the studies. (Fig. 5)
The results obtained so far (n=12 Italians and n=7
Japanese) show a wide inter-individual variability in the
effect of green tea on simvastatin kinetics. Indeed, we
observed similar changes to the original case report
in about one quarter of participants. Remarkably, most
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201
Food-blood fatty acids and coronary heart
disease
Coronary heart disease (CHD) is a major cause of morbidity
and mortality in Europe. There are well-established risk
factors for CHD, including smoking, high blood pressure,
raised total cholesterol, low HDL-cholesterol and type 2
diabetes. In terms of nutrition, a diet being high in saturated
fat has been shown to be associated with the incidence of
CHD, moreover, most evidence suggests that the type of fat
is more important than the amount of fat. The observation
that Greenland Eskimos (Inuit) have a low incidence of CHD
despite a high saturated fat intake, has led the scientific and
public interest to focus on the role of the various fatty acids
in the prevention and treatment of disease, particularly CHD.
Saturated and trans-fatty acids increase both n-6 and n-3
polyunsaturated fatty acids (PUFA) and decrease the risk
of CHD. In fact, several evidences indicate that moderate
doses of n-3 PUFA significantly decrease the risk of fatal
CHD. Higher doses and longer duration of intervention
may also protect from non-fatal CHD events. The exact
mechanisms through which n-3 PUFA affect CHD are not
well established but may include a decrease in fasting and
postprandial triglyceride levels, a decrease in arrhythmias,
modulation of platelet aggregation and decreased synthesis
of pro-inflammatory agents. However, most of the
knowledge about the effects of dietary fatty acids on CHD
risk is based on observational cohort studies and controlled
dietary experiments evaluating clinical effects of fatty acid
intake. Instead, there is limited information on blood levels
of fatty acids in patients with coronary heart disease in
relation to the diet of these subjects. Aim of our ongoing
study is to assess the differences in the fatty acid blood
profile between healthy subjects and patients with overt
coronary heart disease and to evaluate to what extent the
fatty acid pattern is determined by dietary habits in these
groups. This study started enrolment in 2008 and ended in
December 2010. We recruited 179 patients with CHD (defined
after coronary angiography and admitted to the hospital for
Fig. 1: Blood fatty acids comparison between CHD and control. *p=0.03; **p= 0.004; # p=0.001; ## p=0.0001
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coronary artery bypass surgery) and 150 healthy volunteers.
Blood samples were obtained by collecting a drop of whole
blood from a fingertip and analysed by gas-cromatography.
The analyses have been performed in collaboration with
Prof.Claudio Galli from the Department of Pharmacological
Sciences, University of Milan. Information on dietary habits
referred to the previous year was based on a validated
food-frequency questionnaire (FFQ) of European Prospective
Investigation into Cancer and Nutrition (EPIC). We conducted
also interviews using a structured questionnaire that
include information on socio-demographic factors, smoking,
physical activity, history of coronary heart disease in
relatives. Also blood parameters (total cholesterol, LDL, HDL,
triacylglycerols, glycemia), anthropometric variables (weight,
height and BMI), waist circumference, blood pressure and
heart rate were measured. The preliminary results of this
study (71 patients and 55 controls) showed that patients with
CHD have higher blood concentration of palmitic and stearic
acid and total saturated fatty acids compared to controls;
moreover, coronary patients showed lower blood levels
of total PUFA, linoleic acid (the major n-6 fatty acid) and
eicosapentaenoic acid (EPA, a n-3 fatty acid). In contrast, we
found no significant differences between docosahexaenoic
acid (DHA) and EPA+DHA between groups (fig. 1). DHA is
considered a n-3 fatty acid that improves cardiovascular
risk. Yet, no significant difference is observed in terms of
saturated and polyunsaturated fatty acids intake between
cases and controls. This discrepancy could be related to
recall bias: cases reporting a desirable diet instead of actual
diet, a time gap between adoption of correct diet and blood
fatty acid modification or a different desaturation activity
between groups.
We also considered the associations between nutritional
habits and blood level of fatty acids in the whole population.
We observed a direct correlation between intake of fish and
blood level of n-3 PUFA (fig. 2).
Fig. 2: Correlation between fish dietary intake and blood fatty acids in all subject. **p<0.01, ## p=0.0001
CCM — Scientific Report 2011 — Ongoing research 2012
203
Role of the longevity genes p66sh/sirt1 in insulinresistance, diabetes and cardiovascular disease.
T2DM is regarded as the advanced stage of a longlasting period of pre-diabetes, which includes impaired
fasting glucose and glucose intolerance, frequently
observed in patients with the metabolic syndrome.
T2DM is one of the most deleterious risk factors for the
cardiovascular system and its prevalence is alarmingly
increasing, in parallel with the worldwide epidemic of
obesity. Although polygenic determinants are probably
involved in the genesis of T2DM, the subject’s life-style
crucially affects the chance to develop pre-diabetes
and T2DM. Indeed, a very frequent trigger of these
disturbances is abnormal weight gain as a result of
scarce physical activity and excessive caloric intake.
Excessive fat in the body behaves as an expanded
organ which secretes numerous mediators potentially
involved not only in reducing insulin-sensitivity and
worsening pancreatic beta-cell dysfunction but also
Figure 1
in the pathogenesis of the life-threatening vascular
complications of T2DM (Figure 1).
Most of the deleterious effects of pre-diabetes and
T2DM on health is related to the occurrence of microand macro-vascular complications in the medium-longterm. These include chronic renal disease, progressive
retinopathy and polyneuropathy among the former
and the consequences of coronary, cerebrovascular
and peripheral atherosclerosis among the later.
Hyperglycaemia per se, the hallmark of pre-diabetes
and T2DM and the target of current anti-diabetic
therapies, leads to an increase of functionally altered
glycated proteins and to the formation of advanced
glycosilated endproducts (AGEs), which have been
involved in the pathogenesis of T2DM complications.
Yet, recent clinical trials demonstrate that even the
most modern drugs for glucose control, only lightly
prevent T2DM complications, especially macro-vascular,
which account for more than 70% of its mortality.
This percentage clearly contrasts with the much lower
cardiovascular mortality in non-diabetics (Figure 2).
Therefore, other mediators need to be identified as
possible therapeutic targets to reduce cardiovascular
morbidity and mortality in the ever-growing population
of diabetics.
p66Shc is an ubiquitously expressed redox enzyme that
generates mitochondrial ROS, which regulate insulin
signalling. Recent basic studies using a gene knock out
mice model (p66Shc -/-) led to ascribe p66Shc a patent
role in determining body fat accretion and weight gain
(Figure 3), susceptibility to AGE-induced renal disease
(Figure 4) and susceptibility to atherosclerosis (MartinPadura I et al. Endothelium. 2008;15:276-87), the most
typical consequences of T2DM. Hence, p66SHC and
its related protein Sirt1, might turn into relevant targets
for the prevention of obesity, T2DM and micro- and
macrovascular diabetic complications in humans.
Only one small study (Pagnin et al, J Clin Endocrinol
Metab 2005) addressed the relationship between
p66SHC and T2DM in humans, demonstrating an
increased gene expression in patients with T2DM
vs controls, which correlated with plasma levels of
8-isoprostanes, a marker of systemic oxidative stress
(Figure 5). Therefore, more information is needed
Figure 2: Causes of death in diabetics and non diabetics in
western population
Figure 3. p66Shc-/- prevention of obesity. Body weight curves of
wild type and p66Shc-/- male mice fed standard or high-fat diets.
From Berniakovich I et al. J. Biol Chem 2008
Figure 4. Serum and kidney tissue AGEs, plasma isoprostane 8-epi-PGF2α levels and renal NF κB/p65 activation in WT and p66Shc KO mice
injected with mouse serum albumin or carboxymethyl-lysine. From Menini S et al. Diabetologia 2007.
WT-MSA
KO-MSA
WT-CML
KO-CML
Serum AGEs (U/ml)
Kidney AGEs (U/mg)
Isoprostane 8-epi PGF2α (pg/ml)
NFkB/p65 activation (OD)
2.74±0.60
7.78±0.99
83.63±7.06
0.153±0.021
2.36±0.41
6.14±0.49
77.29±5.09
0.135±0.035
9.94±1.06 30.11±2.53a
120.11±8.74a
0.543±0.097a
5.50±0.55a,b
12.02±1.24a,b
86.75±7.11b
0.188±0.036b
a
Values are means±SD; n=7 per group, except for NFkB/p65 activation (n=4)
a
p<0.001 in CML-treated vs the corresponding MSA-treated mice
b
p<0.001 in KO vs the corresponding WT mice
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205
High resolution B-mode ultrasound imaging
to substantiate the involvement of the p66shc/Sirt1
pathway in human obesity, insulin-resistance and T2DM
and to investigate the possibility and clinical effects of
modulating the expression and activity of these proteins
by life-style changes or drugs.
The Unit of Atherosclerosis Prevention and the Unit
of Biochemistry of Oxidative Stress and Endothelial
Function in Atherothrombosis of the Centro Cardiologico
Monzino are involved, in collaboration with the
Department of Experimental Oncology of the Istituto
Europeo di Oncologia and the Department of Clinical
and Experimental Medicine of the University of Padova,
in a project aimed to investigate and dissect out, for
the first time in humans, the relationship between the
p66SHC/Sirt1 pathway and two tightly linked clinical
expressions in patients with prevalent abnormalities of
glucose metabolism: hyperglycemia and overweight/
obesity.
The protocols have been designed with the aim to
provide original clinical information about: 1) the
relationship between the severity of fat accretion,
insulin-resistance, expression and activity of the
p66SHC/Sirt1 pathway in peripheral blood mononuclear
cells (PBMC), subcutaneous and omental adipose tissue,
and levels of markers of oxidative stress (Associations
Study; 80 patients); 2) the effect of acute hyperglycemia
on the expression and activity of the p66shc/Sirt1
pathway in PBMC and the association of its changes
with variations of markers of oxidative stress (Glucose
Challenge Study; 20 patients); 3) the effect of glucose
control on the p66shc/Sirt1 pathway in type 1 and type
2 diabetic patients (Glucose Control Study; 40 patients);
4) the effect of a clinically significant weight-loss by
life-style measures (diet and physical activity) on the
expression and activity of the p66shc/Sirt1 pathway
in PBMC and in subcutaneous adipose tissue (Weight
Loss Study; 30 patients). Data analysis will also provide
information about the possibility to utilize P66 SHC
expression and activity in circulating PBMC as a marker
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Figure 5. Linear regression between total plasma 8-isoprostane levels and p66shc gene expression in PBMC from diabetics (r2 = 0.47;
P = 0.0284). From Pagnin et al, J Clin Endocr and Metab 2005.
of P66 SHC expression and activity in adipose tissues
and about the influence of other specific vascular risk
factors (hypercholesterolemia, hypertension, current
smoking) on the expression and activity of P66SHC in
PBMC and subcutaneous and intra-abdominal adipose
tissue.
The studies described above started enrolment of
patients in June 2010. The studies are co-sponsored
by the Centro Cardiologico Monzino, the University
of Padova, the Istituto Europeo di Oncologia and the
Ministry of Health (Ricerca Finalizzata).
High resolution B-mode ultrasound of superficial arteries
is one of emerging technologies with strong evidence for
its value in risk detection of asymptomatic individuals.
Amongst the imaging tests, this technique is the most
powerful due to the wide spectrum of underlying
morphological and functional abnormalities that can be
detected, such as: vessels dimensions and geometry,
wall thickness, luminal stenosis, occurrence and
extent of atherosclerotic plaque as well as arterial
functional properties. The assessment of abnormal
arterial relaxation in terms of decreased brachial artery
flow mediated dilation (B-FMD) or decreased arterial
compliance, as measured by this technique also allows,
in fact, the identification of endothelial dysfunction; the
primary pathophysiologic mechanism of the disease
which can be detected in the earliest stage of the disease.
composition) potentially useful for predicting the
likelihood of plaque rupture.
One of the most important strength of high resolution
ultrasound imaging lies in the fact that it allows not
only the non-invasive and direct visualization of vessels
lumen but also of vessel walls. Beside detection and
measurement of atherosclerotic plaque, this technique
allows the identification on the artery walls of two
echogenic lines separated by a relatively anechoic space.
In 1986 our group demonstrated for the first time that
these two lines were generated by the blood-intima and
media-adventitia interfaces, respectively. The distance
between these two lines was defined carotid intimamedia thickness (C-IMT).
B-mode ultrasound may also provide prognostic
information directly related to atherothrombotic
properties of vessels (calcifications and plaque
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207
After about thirty years of researches both C-IMT and
B-FMD, are now considered the most widely accepted
non-invasive markers of sub-clinical atherosclerosis.
Both these variables, in fact, have been widely used in
clinical and epidemiological studies to investigate the
effects of established and non-traditional vascular risk
factors, as well as to investigate the association with
end-organ damage in high-risk patients.
Against this background, these ultrasound variables
have a high potential to be included in a next future
among the most important diagnostic tools available
in the clinical practice not only for the early detection
of atherosclerosis but also for the early recognition of
patients at high risk of cardiovascular diseases.
The general mission of our group is to increase
the current understanding of the pathophysiology
of atherosclerotic diseases with the final goal to
implement strategies aimed at reducing the incidence
of cardio- and cerebro-vascular events and minimizing
the related morbidities.
New knowledge in this field is absolutely desirable in order
to create the scientific background needed to bring these
ultrasonic markers, which until now have been mainly
of research pertinence, in the clinical practice for both
diagnosis and prevention purposes and to monitor the
efficacy of pharmacological or dietary interventions. In this
direction we are mainly interested in evaluating the reliability
of methods used for the measurements of these ultrasonic
variables and in understanding whether these surrogate
markers: a) add independent information on risk or
prognosis, b) account for a clinically significant proportion
of the disease, c) provide good sensitivity, specificity and
predictive value in the prediction of cardiovascular events.
The strategy chosen to achieve these objectives is to use
these markers from the earlier stages of the disease, well
before the exordium of clinical manifestations.
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Studies performed to reach the Unit goals are aimed at:
1) investigating the role of conventional and emerging
atherosclerosis risk factors on the basis of their effect on
C-IMT and endothelial function, 2) improving the current
capacities to identify patients at high cardiovascular risk by
using imaging techniques.
Researches aimed at investigating the role of conventional
and emerging atherosclerosis risk factors on the basis of
their effect on C-IMT and endothelial function.
A large part of the risk to develop atherosclerosis is not
explained by traditional risk factors. For example, it is known
that conventional risk factors explain less than 30% of C-IMT
variability, which suggests that other determinants, not yet
considered, may exist. This underscores the importance of
search for novel risk factors. Based on this rationale, we
are now performing a series of projects that use C-IMT or
B-FMD as surrogate markers of atherosclerosis to identify
new vascular risk and protecting factors affecting the
atherosclerotic process as well as to attempt to elucidate
some uncertainties on the role of already identified
atherosclerosis determinants.
One of our interests in this field concerns the study of family
aggregation of atherosclerosis. The role of family history of
premature cardiovascular events (FHPCE) as an independent
risk factor for atherosclerosis and for vascular events is a
quite well established issue. However, it is not well known
whether this role comes from genetic or environmental
factors. Some authors have tried to confirm the nature of
FHPCE as a risk factor for atherosclerosis by evaluating the
amount of “offspring-carotid IMT variability” explained by
parent’s C-IMT. These studies, however, reported contrasting
results with an amount of offspring C-IMT variability
explained by parent’s C-IMT ranging from 20 to 92%. In
order to investigate whether these discrepancies might be
related to the age of generational pairs considered (parent
– offspring), we are currently evaluating the contribution of
parent’s-IMT to offspring-IMT variability in young (parentage <60) and old (parent-age>75) generational pairs.
The generic term “chronic-degenerative diseases” includes
a number of clinical entities with different impact on
quality of life and patient survival. Among these there are:
the metabolic syndrome, the fatty liver disease and the
autoimmunity; all expression of a chronic inflammatory
state. Chronic inflammation, in turn, is an important
risk factor for atherosclerosis. Greater knowledge of
the individual etiological conditions could produce a
significant reduction of the impact of these diseases. This
raised our interest in the study of interaction among these
chronic-degenerative diseases. Therefore, the Unit started
a collaborative study with the Department of Medicine,
Surgery and Odontoiatry , University of Milano, with
the ASL Milano 1- Az Osp Legnano/ Presidio Ospedaliero
di Abbiategrasso, with the Association of Hepatology
(ACE) of Reggio Calabria and with National Centre for
Epidemiology, Surveillance and Health Promotion, Istituto
Superiore di Sanita, aimed at determining the prevalence
of metabolic, inflammatory and autoimmune risk factors
for early cardiovascular damage and their association with
early cardiovascular damage.
One of the uncertainties on the pathophysiologic role
of already identified vascular risk factors concerns the
relationship between high density lipoprotein cholesterol
(HDL-C), enzymes involved in HDL metabolism and
carotid atherosclerosis. The relationship between
C-IMT and HDL-C may strongly depend on the arterial
effects of the specific/prevalent molecular mechanism
that determine low or high HDL-C levels. For example,
the role in atherosclerosis of enzymes involved in the
reverse cholesterol transport such as plasma cholesteryl
ester transfer protein (CETP) and lecithin: cholesterol
acyltransferase (LCAT) is still not completely defined. In
the attempt to give an answer to these questions, the
Unit designed a study aimed at: a) analysing in a large
population the correlation between plasma levels of
LCAT and CETP and C-IMT; b) analysing the correlation
between plasma levels of LCAT and CETP and plasma
lipid concentrations; and c) investigating whether the
relationship between plasma levels of LCAT and CETP
and carotid atherosclerosis is affected by different lipid
concentrations.
Cigarette smoking is one of the most important risk
factors for atherosclerosis. Although the marked harmful
effect of cigarette smoking on cardiovascular health
is well established, many people continue or even
start smoking. The demonstration of vascular benefits
consequent on smoking cessation would encourage
abandoning this habit, particularly in asymptomatic
subjects, who are less motivated than patients with
overt cardiovascular symptoms. In this field, we are now
concluding a study designed to evaluate whether brachial
flow mediated dilation (B-FMD) may be used as surrogate
biomarkers of subclinical atherosclerosis to develop
clinical models which may be used to deeply investigate
the vascular effects of active smoking. Specifically, we are
now performing studies designed to determine the effect
of acute (single cigarette) and chronic use of light or
heavy cigarettes on B-FMD.
Studies focused on the role on subclinical atherosclerosis
of other emerging vascular risk factors, such as
C-Reactive Protein and other inflammation markers, and
socio-economic risk factors, such as occupation, are
currently ongoing.
Research aimed at improving our current capacities to
identify patients at high cardiovascular risk by using
imaging techniques.
A large number of cardiovascular events occurs in
asymptomatic individuals, therefore prevention has to include
not only secondary prevention in patients who survive a
vascular event but also primary prevention for the early
identification and treatment of patients at significant risk. This
early recognition of high risk patients is extremely important
since coronary heart disease is often lethal at presentation.
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Cumulative event-free rate (%)
Cumulative event-free rate (%)
A number of recent clinical and experimental evidences show
that diagnostic possibilities may increase if vascular risk
factors are managed by using a “Global Risk Assessment’
approach. In fact, provided they are included in specific
mathematical algorithms, the determination of traditional
risk factors such as hyperlipidaemia, hypertension, smoking,
diabetes mellitus and physical inactivity, may actually
represent a useful first step to identify, among asymptomatic
individuals, those who could be at risk for a vascular event
from those who are not. This approach, for example, allows
the stratification of individuals into high, intermediate and
low risk categories. Unfortunately, also using the “Global
Risk Assessment” approach, our capacity to predict the
cardiac risk is quite limited and does not exceed the 6065%. The result is that patients considered at intermediate
risk often develop unexpected vascular events and this
suggests that at least subjects at intermediate risk need to
be considered as candidates for additional tests to better
refine their risk estimate and to select the most appropriate
intervention. The need to improve the risk prediction has
prompted the search for novel markers of cardiovascular
risk. Amongst these, imaging techniques have been proved
to have the highest potential to improve the estimation of
cardiovascular risk provided by traditional risk factors and
1.00
Panel a
0.98
1st quintile
2nd quintile
3rd quintile
0.96
4th quintile
0.94
0.92
0.90
Quintiles of IMT mean-max
0
6
12
18
5th quintile
24
30
36
Months
1.00
Panel b
0.98
1st quintile
0.96
2nd quintile
3rd quintile
0.94
0.92
0.90
4th quintile
5th quintile
Quintiles of ICCAD
0
6
12
18
24
30
36
Months
Kaplan-Meier event-free curves with respect to the combined end
point (any cardiovascular, cerebrovascular or peripheral vascular
event) according to quintiles of the IMTmean-max (panel a) and
ICCAD (panel b)
Cumulative event-free rate (%)
The logical workup for risk assessment in primary
prevention should initially be based on traditional risk
factors. However, exposure to one or more cardiovascular
disease (CVD) risk factors is highly prevalent also in
individuals who do not develop clinical disease. On the
other hand, cardiac events occur also in many individuals
who have no established risk factors for atherosclerosis.
The experience from epidemiological and clinical research
also suggests that no single atherosclerosis risk factor
possesses the necessary requirements for an accurate
recognition of patients at CVD risk. Thus, the diagnostic
possibilities to discriminate between individuals who are
actually at risk from those who are not by using single
vascular risk factors is quite limited.
1.00
0.98
Low IMT - Low ICCAD
High IMT - Low ICCAD
Low IMT - High ICCAD
0.96
0.94
0.92 IMT mean-max and ICCAD
above below the median
0.90
0
6
12 18
24
30
High IMT - High ICCAD
36
Months
Additive effect of IMTmean-max and ICCAD assessed by stratifying
the study population into 4 groups according to the presence of
IMTmean-max and ICCAD values above or below the median.
global risk scores and to enhance the detection of the high
risk or vulnerable patients.
With this objective, we have recently observed that
the mean-maximal value of carotid IMT (IMTmean-max)
and the carotid diameter measured in plaque free areas
(ICCAD) are both associated to successive vascular
events in this large group of European subjects at high
risk of atherosclerosis for the presence of at least three
vascular risk factors.
-0.16
Framingham Risk Factors (FRFs)
CC-IMTmean
Bif-IMTmean
ICA-IMTmean
CC-IMTmax
Bif-IMTmax
ICA-IMTmax
ICCAD
IMTmax
IMTmean
IMTmean-max
ICCAD+IMTmax
ICCAD+IMTmean
ICCAD+IMTmean-max
FRFs+CC-IMTmean
FRFs+Bif-IMTmean
FRFs+ICA-IMTmean
FRFs+CC-IMTmax
FRFs+Bif-IMTmax
FRFs+ICA-IMTmax
FRFs+ICCAD
FRFs+IMTmax
FRFs+IMTmean
FRFs+IMTmean-max
FRFs+ICCAD +IMT max
FRFs+ICCAD +IMT mean
FRFs+ICCAD +IMT mean-max
-0.04 -0.02
The additive effect of these two variables in the risk
stratification was evident also when the study population
was stratified into four groups according to the presence
of an IMTmean-max and ICCAD above or below median.
In the same study we have also shown that the
combination of IMTmean-max and ICCAD with vascular
Risk factors can be used to enhance the predictability of
cardiovascular events.
-0.00
0.02
0.04
P
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0001
<0.0001
0.048
0.042
0.43
0.99
0.89
Werse
reclassification
0.0001
0.0008
0.0001
0.002
0.0005
<0.0001
0.0002
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
Better
reclassification
P Bonferroni
0.0004
<0.0001
0.0004
<0.0001
<0.0001
0.0006
0.0006
0.003
0.001
0.006
0.0003
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
Integrated Discrimination Improvement (IDI) as compared to a model including only Framingham Risk Factors (age, gender, total
cholesterol, HDL-cholesterol, systolic blood pressure, hypertension, diabetes and current smoking).
CCM — Scientific Report 2011 — Ongoing research 2012
211
High resolution B-mode ultrasound imaging
for the assessment of mechanisms involved in
atherosclerosis progression
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CCM — Scientific Report 2011 — Ongoing research 2012
recently concluded in our Institute. The main objective
of this study, funded by the European Union, was
to evaluate the association between carotid IMTprogression within 15 months and the rate of subsequent
(from the 15th to the 36th month of follow up) vascular
events in an European population of patients at high risk
of cardiovascular disease. To reach the study objectives,
3703 patients have been followed for 36 months. Of
these patients, 1050 have been recruited in Finland (2
clinical centers both in Kuopio), 533 in Sweden, 532 in
The Netherlands, 501 in France, 1095 in Italy (2 clinical
centers: Milan and Perugia). The analyses of baseline
results of this study clearly showed that latitude is an
important determinant of C-IMT, which is not explained
by between-country differences in established vascular
risk factors (Baldassarre D. et al., Eur Heart Journal. 2010; 31: 614-622).
Researches aimed at understanding the molecular
mechanisms involved in atherosclerosis progression.
New advances in B-mode imaging technologies have
led to improved quality in detecting minute changes
in ultrasonic images, allowing the assessment of even
microscopic changes, of carotid atherosclerotic plaque
and overall of carotid intima media thickness (C-IMT).
The possibility to perform these measurements with
an extremely high accuracy and precision joined with
the possibility to identify different C-IMT progression
patterns suggests that this high ultrasonic technique
may be also used to monitor the natural evolution of
the atherosclerotic disease as well as to monitor the
effects of antiatherosclerotic interventions. Against
this background, and in view of a) its correlation with
coronary atherosclerosis and b) its capacity to predict
incident coronary events, C-IMT has been widely used
in clinical trials, as a substitute for angiography or
vascular events, to investigate the effectiveness of
pharmacological and dietary interventions.
Despite hundreds of studies published so far, a number of
important issues related to the evolution of atherosclerosis
remains to be addressed. Therefore studies specifically
focused on the assessment of mechanisms involved in
atherosclerosis progression are desirable.
The general mission of our group in this field is
to increase the current understanding on carotid
atherosclerotic progression with the final goal to
understand the mechanism involved in the natural
evolution of the disease as well as in the changes
of this evolution induced by antiatherosclerotic
interventions.
In this direction, besides the assessment of reliability
of methods used for the measurements of C-IMT
progression, the Unit is mainly interested in the
recognition of determinants of C-IMT-progression and
in understanding whether C-IMT progression a) may
add independent information on risk or prognosis, b)
provides good sensitivity, specificity and predictive
value in the prediction of cardiovascular events c)
may be actually used as a surrogate endpoint in place
of vascular events in studies aimed at evaluating the
efficacy of antiatherosclerotic intervention.
1.00
Cumulative event-free rate (%)
In an epidemiological study carried out in middle-aged
American adults (ARIC study), significant associations
were found between C-IMT progression (9 years of
follow-up) and change in LDL cholesterol, triglycerides,
onset of diabetes and onset of hypertension.
Interestingly, the associations between C-IMT progression
and changes in risk factors over the same period were
somewhat stronger than those observed with the same
vascular risk factors measured at baseline. In another
study (the Framingham study) investigators observed
that the association of carotid atherosclerosis with
time-integrated risk factor levels is stronger than with
concurrently measured risk factors, thus suggesting that
those factors, emerging as most important for C-IMT
change analysis, may represent the current determinants
of atherosclerosis progression rather than those long
persisting factors that affect such progression over
a lifetime. Taken together these findings suggest that
while a one time measure of C-IMT probably reflects
the effect of exposure to risk factors which have acted
over the whole patient’s lifetime, the C-IMT progression
better reflects the effect of risk factors which have acted
in the period shortly preceding the scan. In fact, if an
individual changes his lifestyle or his pharmacological
treatment shortly before the ultrasonic scan (or perhaps
even 1 to 2 years before), the C-IMT may be poorly
related to concurrently measured risk factors and
ultimately, to outcome. In this kind of individuals, it has
been hypothesized that C-IMT-progression might be a
better index of future CVD risk than any cross-sectional
estimation of subclinical arterial disease. Despite all these
evidences, unfortunately it is still currently unknown
if this technique could provide a specific or sensitive
clinical predictor of incident CVD for an individual
patient; thus, studies of the ability of change in C-IMT to
predict clinical events in subsets of symptomatic as well
as asymptomatic individuals are needed. A multicenter,
longitudinal, observational study (The IMPROVE Study)
specifically designed to address this topic has been
0.99
0.98
0.97
1st tertiles
0.96
2nd tertiles
0.95
0.94
0.00
3rd tertiles
Tertiles of Fastest IMT progr
(P=0.005 by Log-Rank Test)
5.00
10.00
15.00
20.00
25.00
Time (Months) to event/censoring
Kaplan-Meier event-free curves with respect to the combined
endpoint stratified for tertiles of the Fastest-IMTprogr.
In this contest we have observed that the fastest
progression detected in the whole carotid tree, regardless
the location in which it was occurred (Fastest-IMTprog) is
the unique IMT progression variable significantly associated
to successive vascular events in European subjects
More than 20 years of experience in the field have
conferred to the Unit the role of Coordinator and Core
Laboratory in national and international multicentre
studies on the epidemiology of atherosclerosis.
In order to reach the Unit goals, our studies aim at:
1) understanding the molecular and environmental
mechanisms involved in atherosclerosis progression,
2) investigating the possibility to interfere with this
progression by using life-style and drug interventions.
Research aimed at investigating the possibility to
interfere with atherosclerosis progression by using lifestyle and drug interventions.
There is a substantial residual risk of CVD in patients
optimally treated with LDL-C lowering drugs. While
LDL-C lowering remains the primary lipid therapeutic
goal, evidences suggest that raising plasma levels of
HDL-C and lowering triglycerides with either nicotinic
acid or fibrates also reduces CVD risk (Coronary
Drug Project, HHS, VA-HIT, BECAIT, etc). Although
different types of novel HDL-C therapies are currently
in development, nicotinic acid and fibrates still remain
the only HDL-C drug therapies approved/available so
far. Nicotinic acid and fibrates, however, modify HDL-C
levels by different molecular mechanisms and may
differently affect the functionality of HDL particles. The
attention to the mechanism of HDL-C increase and to
HDL particle function was recently drawn by the results
of the ILLUMINATE trial, which showed a paradoxical
increased risk of cardiovascular events in patients that
achieved a more than 70% rise in HDL-C levels with
the CETP inhibitor torcetrapib. Thus, understanding the
effect of drugs not limitedly on the HDL-C level but
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213
New methods to analyze complex ultrasound
measures and atherosclerosis progression
also on the HDL composition (or quality) and function
is emerging as a critical issue for the choice of optimal
HDL-modifying therapies. Therefore, the Unit aimed to
assess comparatively the effects of nicotinic acid and
fibrates on HDL levels, composition and functionality
in patients with low (< 40 mg/dL) and normal (between
40 and 59 mg/dL) HDL-C levels in a multicenter, open,
randomized, crossover study. In collaboration with the
Department of Pharmacological Sciences, University of
Milan, exploratory research technologies have been used
to conduct these investigations, including assessment
of drug effects on: a) Reverse Cholesterol Transport
(RCT), as measured by the capacity of serum to promote
cholesterol efflux from macrophages and hepatoma cells;
b) HDL particle number, size and composition; c) Levels
and activity of HDL modifying enzymes/transporters; d)
Anti-oxidant, anti-inflammatory and anti-thrombotic HDL
properties; e) Endothelial Function (indexed by B-FMD).
Carotid Intima-Media Thickness (c-IMT) is a well accepted
surrogate marker of subclinical atherosclerosis. IMT
measurements can be performed at different locations in
the carotid artery and with different protocols (e.g. as the
average or the maximal of a series of measures taken in
a specific carotid segment). In order to identify the best
measure, or combination of measures, we can use, as a
criterion, the optimal prediction of future cardiovascular
events. To accomplish this task, however, not only
sensitivity and specificity must be considered, but also
feasibility and costs, in view of the large number of
exams to be performed in clinical research and patients
screening. Our group is engaged in a study based on two
different cohorts, one of about 2000 subjects enrolled
in the Milano area, and one of 3700 subjects from five
Europeans countries. Both cohorts were followed-up for
at least 36 months and incident cardio- and cerebrovascular events were recorded.
Epidemiology of subclinical atherosclerosis: links with
social class, geography and diet.
A low socioeconomic status (SES) is associated with the
majority of diseases, including CVD and atherosclerosis.
The interest of our group has focused for a long time
on the analysis of Carotid Intima-Media Thickness
(c-IMT), a well accepted surrogate marker of subclinical
atherosclerosis. In this study we employed the database
including more than 3700 subjects from five different
European countries, and we analysed the relationships
between c-IMT, social class (as denoted by three job
categories: manual workers/ service workers/ white
collars) and geographical area (Northern Europe vs
Italy). As expected, c-IMT was strongly associated with
low social class, with a positive trend going from white
collars to manual workers. The novel, unexpected finding
was that this trend was observed only in northern
Europe and completely absent in Italy (Fig.1). White
collars from northern Europe had the same IMT levels
as white collars in Italy, but the spread was apparent
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when considering service and manual workers. To be
noticed that this analysis was adjusted not only for age,
gender and conventional vascular risk factors, but also
for education. Thus, some other factor must explain
why social class is associated with c-IMT in France,
Netherlands, Sweden and Finland but not in Italy. A
potential explanation may be found in the different
dietary patterns in the North and South of Europe. For
long-time the Mediterranean diet (Med-Diet), rich in
cereals, vegetables, fish, olive oil and red wine, has been
thought to protect from cardiovascular disease, whereas
the typical northern-Europe diet, based on meat, dairy
products and saturated fatty acids, has been linked to
an increased risk of atherosclerosis. In our study we
observed that in northern Europe the adoption of a
Med-Diet (as measured by an appropriate score) was,
as expected, much lower than in Italy; however, the
interesting finding was that, whereas in Italy all social
classes exhibit, on average, about the same Med-Diet
high
SOCIAL CLASS
Figure 1 Subclinical atherosclerosis and lifelong profession, in
Northern Europe and in Italy
CCM — Scientific Report 2011 — Ongoing research 2012
215
score, in the North there is an apparent negative trend
form the highest to the lowest class (Fig 2). The reason
may be that the foods typically associated to the Meddiet (e.g. pasta, olive oil, wine) are more expensive in
the North than in Italy, and thus are consumed mainly
by the most affluent classes.
These results corroborate the hypothesis that the
adoption of a Med-Diet may be a crucial factor in
preventing atherosclerosis.
Development of summary scores based on clusters of
soluble markers of inflammation, oxidative balance and
endothelial function.
Oxidative stress is usually defined as an imbalance
between radical oxygen species (ROS) production and
antioxidant defenses, which leads to oxidative damage
and dysfunction of macromolecules, cells and tissues.
Our group has recently proposed the OXY-SCORE, a
comprehensive index of oxidative stress, derived from
a computation of different variables relevant for the
oxidative balance. We have previously reported that the
Figure 2 Adoption of a Mediterranean diet and lifelong profession,
in Northern Europe and in Italy
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OXY-SCORE is more sensitive than individual oxidative
markers in detecting even slight differences in oxidative
status between age and gender groups in healthy subjects.
In 2008 we carried out a study to compare OXY-SCORE
with its individual components in a specific clinical
setting characterized by significant oxidative changes.
A panel of plasma markers was measured in patients
subjected to coronary artery bypass graft surgery with
or without utilization of cardiopulmonary bypass (CPB
or OPCAB, respectively), two clinical circumstances with
patent differences in the fluctuations of the oxidative
balance. Plasma malondialdehyde, oxidized and reduced
glutathione, individual antioxidant capacity, alpha- and
gamma-tocopherol and urinary isoprostanes were
measured before, during, and after surgery. OXY-SCORE
was computed at each time point. The performances of the
individual biomarkers and of OXY-SCORE in discriminating
the two classes of patients and in assessing major and
minor variations in the oxidative balance were then
compared. The results indicate that the ability of OXYSCORE to correctly classify the patients is excellent, with
an area under the ROC curve of 0.90 and 0.78 during and
after surgery respectively. Free MDA performed slightly
better in this task, but, in contrast with OXY-SCORE, it was
insensitive to the minor variations of the oxidative balance
in the OPCAB group. The performance of all the other
individual biomarkers was definitely worse.
In addition, we are planning to devise and validate
a similar score based on biochemical markers
associated with endothelial (dis)function and nitrogen
monoxide (NO) metabolism: L-arginine; asymmetric
dimethylarginine (ADMA); symmetric dimethylarginine
(SDMA); tetrahydrobiopterin (BH4).
New models for the analysis of familial aggregation of
atherosclerosis.
The association between the level of c-IMT in the
probands and a reported cardiovascular disease in
first degree relatives has been widely documented.
Development of theoretical models of atherosclerosis
progression.
The aim of this project is to test different life-long models
of atherosclerosis progression, as assessed by c-IMT.
Specifically, we consider two competing models, one
based on a steady, linear progression throughout the
entire life-span, and one based on an “S” shaped growth
curve, characterized by a flat stage followed by an abrupt
increase and a subsequent stasis at a ‘saturation’ level.
The study is based on two datasets: a subjects with a
wide age range (6-90), and a dataset of longitudinal
measurements (3700 subjects dataset of cross-sectional
c-IMT measures taken on about 3500) that will allow
computing actual progression rates. The results from the
two datasets will be merged with an approach analogous
to that proposed by Ware (Am J Epidemiol 1990;132) and
by Xu (Am J Epidemiol 1995;141). Another important
objective of this study is the analysis of the differences in
the time course of c-IMT progression in men and women,
in order to identify potential genetic, environmental
or physiologic (such as menopause occurrence) risk
modifiers. Figure3 shows the plot of c-IMT vs age in the
cross-sectional dataset. Figure 4 shows the corresponding
vascular mortality rates in the Italian population.
Figure 3: Cross sectional IMT data in 3500 subjects age 6-90
Figure 4: Vascular mortality rates in Italy, 2002.
This association is considered to be the result of both
genetic and shared environmental components. We are
developing statistical models to measure the differential
effects depending on the kind of affected first-degree
relatives (father, mother, brothers or sisters), in order
to understand the pattern of disease inheritance. A
database including 3711 subjects, recruited in five
European countries, will provide the data for this project.
The database includes, for each subject, a variety of
c-IMT measures and a complete collection of information
about a history of cardio-, cerebro- and peripheral
vascular disease for all first-degree relatives. We are
testing a model similar to that proposed by HouwingDuistermaat and Van Houwelingen (Houwing-Duistermaat
JJ, Van Houwelingen HC. Stat Med 1998;17:2865) for
the development of a family indicator summarizing the
information available for all relatives (CVD Family Score).
CCM — Scientific Report 2011 — Ongoing research 2012
217
Cardiovascular epigenetics
Development of new methods to assess carotid IMT
progression.
Cross sectional C-IMT measurements are considered a
well established marker of the advance of subclinical
atherosclerosis and have been successfully employed
in the prediction of future cardio/cerebro-vascular
events. However, longitudinal C-IMT measurements and,
specifically, the assessment of C-IMT progression have
yielded to conflicting results as predictors of vascular
events. A likely explanation for this inconsistency is
that C-IMT progression, as computed by the traditional
approach (i.e. as the change of segment-specific
measurements or of summary measures, such as the
overall mean or maximum IMT), is too affected by
measurement error, which dilutes all potential statistical
associations with vascular events. The aim of this project
is to identify novel C-IMT progression variables less
affected by measurement error and thus exhibiting a
stronger association with vascular events. An example
of such variables is the measure of the ‘fastest IMT
progression’, i.e. a sharp increase of IMT occurring
anywhere in the carotid tree. Interestingly, if the fastest
IMT progression is proved to be the best variable to
describe atherosclerosis evolution, this will imply that
this phenomenon tends to be focalized, at least in a
short term, in definite spots, instead of being diffused in
all carotid segments (Figure 5).
Improvement of statistical approaches aimed to optimize
algorithms for risk prediction.
Our group is engaged in a study aimed to identify the
most effective procedure to integrate new variables
(specifically c-IMT measures) in the existing algorithms
for cardiovascular risk prediction. We are focusing on
the Framingham Risk Score (FRS) and on the new ‘Cuore’
algorithm, developed by the Italian National Institute of
Health. The main goal of the study is to verify whether
intermediate-risk individuals (say with an FRS between
10 and 20), positioned in a sort of “grey area” between
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CCM — Scientific Report 2011 — Ongoing research 2012
low and high risk, can be better classified by taking into
account the c-IMT. An obvious benefit is that individuals
reclassified as at high-risk could beneficiate of potentially
life-saving treatments. The analyses are carried out on
a database of about 1000 patients with a median followup of 5 years. To test the impact of new variables in the
prediction of future events we are evaluating innovative
approaches such as the ROC curves analysis applied to
survival data (Pencina MJ. Stat Med 2004;23:2109), and
methods based on the computation of reclassification
indexes, such as NRI (Net Reclassification Index) or IDI
(Integrated Discrimination Improvement) (Pencina MJ. Stat
Med 2008;27:157–172).
Figure 5: Different models for the dynamics of IMT progression
Background
The molecular basis of epigenetics is complex; it involves
modifications of the transcriptional status of certain genes
but not the basic structure of DNA. This mechanism
enables differentiated cells in a multi-cellular organism
to express only the genes that are necessary for their
own activity whereas the others are silenced. Epigenetic
changes are preserved across cell generations and, in
addition to gene silencing, specific epigenetic processes
include imprinting and X chromosome inactivation.
There are several layers of gene expression regulation.
Among them, chromatin remodeling represents a
hot field of investigation. Chromatin is a polymer of
nucleosomes, constituted by DNA wrapped around
histones. Modifications in DNA wrapping usually
correspond to gene expression changes. Chromatin
remodeling depends on two main mechanisms, both
ruling the differentiation of higher eukaryotes.
Post-translational modification of aminoacids in histone
proteins. Aminoacids in histones are usually positively
charged. If histone charge changes, the shape of
the histone sphere may be modified. Since DNA is
not completely unwound during replication, modified
histones may be carried into each new copy of the DNA
in which they may act as templates. The unstructured
N-termini of histones, i.e. the histone tail, can be highly
modified by acetylation, methylation, ubiquitylation,
phosphorylation and sumoylation. Acetylation generally
correlates with transcriptional competence as it removes
the positive charge, causing DNA unwinding from the
nucleosome. When this occurs, complexes like SWI/SNF
and other transcriptional factors can bind to the DNA,
allowing RNA polymerase-dependent gene transcription.
In addition, the positively charged tails of histone
proteins from one nucleosome may interact with the
histone proteins on a neighbouring nucleosome, causing
them to pack closely. Lysine acetylation may interfere
with these interactions, causing the chromatin structure
to open. Lysine acetylation may also help recruit other
activating chromatin modifying enzymes and basal
transcription machinery as well. Methylation of histone
lysines has long been associated with constitutively
transcriptionally silent chromatin. However, recently it
has been shown that tri-methylation of histone H3 at
lysine 4 is strongly associated with and required for
full transcriptional activation. It should be emphasized
that the transcriptional effect of histone modifications is
position-dependent. Multiple modifications may occur at
the same time, working together to change nucleosome
behaviour.
Addition of methyl groups to the DNA. DNA
methylation, mostly at CpG sites, converts cytosine
to 5-methylcytosine. Basically, certain enzymes (such
as DNMT1) have a higher affinity for the methylated
cytosine. If this enzyme reaches a methylcytosine in one
of the DNA strands, it will methylate the other. Highly
methylated areas tend to be transcriptionally less active,
through a mechanism not fully understood. Methylated
cytosines also marks inherited parental chromosomal
areas in the zygote, a phenomenon termed genetic
imprinting. Because 5-methylcytosine is chemically
very similar to thymidine, CpG sites are frequently
mutated and become rare in the genome, except at CpG
islands where they remain unmethylated. Thus these
epigenetic changes have the potential to direct increased
frequencies of permanent genetic mutation.
Epigenetics and cardiovascular diseases
Recent genetic and biochemical analyses indicate
that epigenetic changes play an important role in the
development of cardiac hypertrophy and heart failure;
e.g. dysregulation in histone acetylation status (see
figure 1) has been linked to an impaired contraction
ability of cardiac myocytes. Although such epigenetic
changes should eventually lead to alterations in the
expression of genes associated with the affected
CCM — Scientific Report 2011 — Ongoing research 2012
219
histones, little information is available on the genes
responsible for the development of heart failure. Current
efforts at the Centro Cardiologico Monzino have focused
on deciphering the network of genes which are under
abnormal epigenetic regulation in dystrophic skeletal
muscle and heart. To this end, coupling chromatin
immunoprecipitation to high-throughput profiling
systems is being applied to endothelial, smooth muscle
cells and cardiac myocytes in normal as well as in
affected hearts. Further, members of different classes
of histone modifying enzymes as well as high weight
molecular complexes formed by chromatin modifiers
are studied. Given the pivotal role of nitric oxide in
the pathophysiology and epigenetics of cardiovascular
cells (see figure 2), the possible involvement of a
dysregulation of nitric oxide-dependent epigenetic
mechanisms is currently investigated.
The results of these studies should not only improve
our understanding of the molecular basis of important
cardiovascular alterations including hypertrophy and heart
failure but also provide essential information that will
facilitate the development of new epigenetic-based therapies.
Nitric oxide epigenetic effects in endothelial cells. In the upper panels it is shown the effect of the nitric oxide donor DETA/NO on the
subcellular localization of Histone Deacetylase 4 (HDAC4). HDAC4 shuttles from the cytoplasm to the nucleus of endothelial cells upon
DETA/NO treatment. This event corresponds to a marked deacetylation of histone H3 (lower panel; NTG = nitroglycerin).
DETA/NO
Solvent
Specific fields of interest
a)Characterization of epigenetic changes associated to
aging and to the cellular senescence processes which
affect stem cells function;
b)Understanding the biological relevance of epigenetic
processes involved in heart failure and hypertrophy;
c)Investigation of the epigenetic mechanisms underlying
the mechanisms of vasorelaxation and nitrate tolerance;
d)Identification of genetic and/or pharmacological
strategies to modulate chromatin changes associated
to diabetes and metabolic memory.
60’
120’
240’
8h
HDAC4
DAPI
Merge
10% FBS
H3AcK14
ctr
H3Ph-S10Ac-K14
TSA
ctr
TSA
30 KD
H1
14 KD
H3AcK14
NTG -
-
-
-
+
DAPI
Histone modifications are differentially regulated in hypertrophic hearts. Picture shows the effect of Trichostatin A (TSA) administration
on acetylation and phospho-acetylation of histone H3 in the hypertrophic heart left ventricle. TSA was cronically administered by subcutaneous minipumps in which an aortic banding was performed to induce cardiac hypertrophy. After 21 days mice were sacrificed and
immunofluorescence was performed to detect modified histones. Control hearts did not show any phospho-acetylated histone H3, which
were reactivated upon TSA treatment. No differences were observed in the pool of monoacetylated histones H3 betweeen control and
treated hearts.
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CCM — Scientific Report 2011 — Ongoing research 2012
221
microRNAs in cardiovascular diseases
microRNAs (miRNAs) are 21-23-nucleotide non-proteincoding RNA molecules that act as negative regulators
of gene expression, modulating the stability and/or the
translational efficiency of target messenger RNAs. To
date, more than 900 human miRNAs have been cloned
and, since each miRNA can modulate hundreds of targets,
it may prove difficult to find a biological process or
function that is not at least in certain aspects, under the
influence of miRNAs. Indeed, miRNA activity is involved
in the control of a wide range of biological functions
and processes, such as development, differentiation,
metabolism, growth, proliferation and apoptosis.
miRNAs and oxidative stress. Different tissues and
physiological conditions are each associated to a
particular pattern of miRNA expression (miRNA
signatures) and altered miRNA signatures have been
associated to specific diseases. Indeed, our laboratory
contributed to the identification of a miRNA signature
specific for endothelial cells exposed to reactive
oxygen species (ROS). Specifically, we found that the
entire miR-200 family expression level increased upon
oxidative stress in human umbilical vein endothelial
cells (HUVEC) exposed to 200 μM hydrogen peroxide
(H2O2) for 8 to 24 hours. Specifically, miRNAs profiling
showed that miR-200c and the co-transcribed miR-141
increased more than eightfold. The other clustered
miR-200 gene family members (miR-200a, miR-200b
and miR-429) were also induced, albeit to a lower
level. Furthermore, miR-200c up-regulation was not
endothelium restricted, and occurred also in other cell
lines and upon exposure to an oxidative stress-inducing
drug such as 1,3- bis(2 chloroethyl)-1nitrosourea (BCNU).
We found that miR-200c overexpression induced HUVEC
growth arrest, apoptosis and senescence. Interestingly,
all these phenomena were also induced by H2O2 and
were partially rescued by miR-200c inhibition. Moreover,
miR-200c target ZEB1 , both messenger RNA and protein,
were down-modulated by H2O2 and by miR-200c
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overexpression. We also found that ZEB1 knockdown
recapitulated miR-200c-induced responses, and that
the forced expression of a ZEB1 allele non-targeted by
miR-200c was able to prevent miR-200c phenotype (see
figure). We also elucidated the mechanism of H2O2mediated miR-200c upregulation, demonstrating the
involvement of the oncosuppressor proteins p53 and
retinoblastoma. Furthermore, we found that miR-200
family is also induced following acute hindlimb ischemia
in skeletal muscles and this induction is oxidative stress
dependent, since in p66ShcA-null mice, which exhibit
less oxidative stress than wt mice, this induction is
significantly attenuated. In conclusion, we demonstrated
that ROS induce miR-200c and other miR-200 family
members and that the ensuing down-modulation of
ZEB1 plays a key role in ROS-induced apoptosis and
senescence.
miRNAs as biomarker of coronary artery disease.
Recent evidence suggests that miRNAs are present in
the systemic circulation and may constitute excellent
disease biomarkers. Recent results from our laboratory
have identified a number of miRNAs (miR-1, -133a, -133b
and -499-5p) that increase acutely, both in patients
and rodents with myocardial infarction, and return to
baseline within a few days after coronary occlusion.
This result has led to intense investigation about the
possibility that, at least in some clinical conditions,
miRNAs may be more sensitive and specific indicators of
myocardial infarction than commonly used biomarkers,
i.e. cardiac troponins.
ZEB1 downmodulation is required for miR-200-c-mediated induction of growth arrest apoptosis and senescence.
HUVEC were co-infected with lentiviruses encoding miR-200c and ZEB1D for 2 h. Single infection either of miR-200c or ZEB1D was
performed together with ZEB1D backbone vector (vec) or miR-scramble viruses, respectively. Control cells were co-infected with
miR-scramble and vec viruses. After infection cells were allowed to recover in complete fresh medium for additional 72 h, without
puromycin selection and then plated.
(a) Representative western blot demonstrating the expression level of ZEB1 in cells co-infected with miR-200c and ZEB1D miR- 200c
forced expression decreased ZEB1, but this effect was largely inhibited in cells expressing ZEB1D. This experiment was performed three
times with similar results. Tub, α-tubulin.
(b) HUVEC co-infected with miR-200c and ZEB1D showed a higher proliferation rate compared with miR-200c overexpressing cells (n=5
at each time point; *P<0.001; #P<0.05).
(c) HUVEC were infected with ZEB1D, miR-200c, miR-200c and ZEB1D, or control viruses. After 72 h, cells were plated and after
additional 40 h apoptosis was measured by fragmentation of cellular DNA. The proapoptotic effect of miR-200c was abolished by the
expression of ZEB1D (n=3; *P<0.05).
(d) HUVEC were infected with ZEB1D, miR-200c, miR-200c and ZEB1D, or control viruses. After 72 h, cells were seeded and after 8 h
were fixed and stained for SA-b-gal. Cells co-infected with miR-200c and ZEB1D showed a decrease of the % of SA-βgal-positive cells
compared with miR-200c- infected cells (n=3; *P<0.001)
CCM — Scientific Report 2011 — Ongoing research 2012
223
Regenerative medicine in cardiovascular diseases
Introduction
Cardiovascular regenerative medicine represents a major
emerging area of investigation. Several key findings have
given momentum to cell therapy studies and contributed
to the broad interest in this field:
• It has been demonstrated that the heart is not a
terminally differentiated organ and that, even as
part of the physiologic aging process, new cardiac
cells form and replace old ones.
• It has been identified a c-kit+ resident cardiac
stem cell that can differentiate both toward
the myocardial and vascular lineages and that,
in experimental animal models, can repair the
damaged heart following myocardial infarction (MI).
• In animal models it has been shown that, beside
the c-kit+ cardiac stem cell, other cell types, both
cardiac- and bone marrow-derived, once injected
into the infarcted heart appear to differentiate
toward the myocardial and vascular lineages and
improve cardiac function.
• Ongoing clinical studies are providing evidence that
patients with ischemic heart disease and depressed
left ventricular function benefit from autologous
bone marrow-derived cell transplantation, their
symptoms improve and the function of their heart
is better than in control patients.
• There is accumulating evidence that in patients
with refractory ischemia cardiac injection of bone
marrow-derived cells improves symptoms and
objective evidence of myocardial perfusion.
• Two recent studies have suggested that autologous
cardiac cells, c-kit+ and cardiosphere-derived,
injected intracoronary in patients with ischemic
cardiomyopathy, may have a beneficial action.
Historical note. The field of regenerative medicine
started in the early 1980s when angiogenic growth
factors were discovered and growth factor proteins were
used in preclinical studies to induce new blood vessel
224
CCM — Scientific Report 2011 — Ongoing research 2012
formation and enhance blood flow to the ischemic heart
and limb. However, these proteins have a short half-life
in vivo and the economic cost associated with their
prolonged use is exceedingly high. Further, systemic
growth factor administration for extended periods of
time carries the risk of new blood vessel development
in areas where they could be detrimental, such as
the retina, brain or subclinical neoplastic foci. These
limitations prevented the widespread use of “therapeutic
angiogenesis” interventions with proteins. In the
early 1990s, gene therapy became an area of intense
investigation in regenerative medicine; it appeared to
provide a completely novel approach to transfer a gene
into the cells of a living organism and to locally enhance
the production of a potentially therapeutic protein. This
would have avoided the costs of prolonged protein
administration and the risks of systemic distribution of
the protein. Unfortunately, in clinical experimentation,
gene therapy of cardiovascular ischemic diseases met
significant failures and only some limited success.
This has led to progressive waning in the interest
in this potential therapeutic strategy, at least in the
cardiovascular arena.
Cell therapy entered the regenerative medicine field in
the mid-1990s, however it was in 2001 that a pivotal study
demonstrated that bone marrow-derived c-kit+/Lin- cells
could regenerate the infarcted left ventricle in the mouse.
In 2003 another key study demonstrated the existence of
a resident c-kit+ cardiac stem cell capable of forming both
myocardial cells and blood vessels. Despite the enormous
amount of experimental data that both proponents and
detractors of cell therapy for cardiovascular diseases have
produced, the field has become one of the most intensively
debated and investigated, with studies going from clinical
experimentation to basic molecular mechanisms of cell
transdifferentiation and reprogramming.
The road ahead of us. There are a number of challenges
that still need to be overcome in order to establish the
role of cell therapy in cardiovascular diseases.
Which cell is the best one. Cells from different sources
are being used in cardiovascular cell therapy studies. At
Centro Cardiologico Monzino we use autologous bone
marrow-derived CD133+ cells, which have been selected
under GMP conditions, to treat patients with refractory
myocardial ischemia. Patient enrolment is also open for
the use of the same cell type in the treatment of patients
with hibernating myocardium undergoing coronary artery
bypass surgery. In basic and preclinical studies we have
identified and characterized a cardiac mesenchyma-like
stromal cell which appears highly prone to differentiate
toward the myocardial and vascular lineages and may
have a significant therapeutic potential. The Centro
Cardiologico Monzino is also investigating the properties
of resident cardiac c-kit+ stem cells, for their clinical
use, and also induced pluripotent stem cells (iPS) derived
from cardiac stromal cells.
Ex vivo enhancement of a cell therapeutic potential. Cell
transplantation offers the opportunity to manipulate
cells ex vivo, prior to reinjecting them into the patient.
At Centro Cardiologico Monzino we have focused on
the use of epigenetic drugs which modulate chromatin
remodelling and gene expression, miRNA manipulation
and classical preconditioning interventions to enhance
cell plasticity, survival and, ultimately, their therapeutic
potential.
How cells should be injected. Cell transplant is usually
performed either by intracoronary or by direct
intramyocardial injection. At Centro Cardiologico
Monzino we have elected to use direct intramyocardial
injection via the epicardial route. An alternative
approach is to inject via the endocardial route in
ischemic areas which exhibit electromechanical
dissociation and that are identified by NOGA
electromechanical mapping. Systemic cell injection
and injection into the coronary sinus are also under
evaluation by other laboratories.
Bone marrow cells mobilization with G-CSF. Recent
clinical evidences suggest that mobilization of stem
cells from the bone-marrow by granulocyte colonystimulating factor (G-CSF) may improve left ventricular
function and remodelling in patients after ST-elevated
acute myocardial infarction (STEMI). The mobilization
process is not completely understood but the strategy
takes advantage to the non-invasive approach of the
treatment that seems to mimic the exposure of injured
myocardium to higher concentrations of circulating stem
and progenitor cells during the first phase after necrosis.
The next step will be to corroborate these data with
larger and longer-term randomized controlled trials.
Drug therapy. The alternative to cell transplantation
is represented by drugs that activate the patient’s
own stem cells and therefore avoid the need for cell
transplant. Few pharmacological interventions have
been shown to promote cardiac stem cell activation and
cardiac regeneration, i.e the combination of hepatocyte
growth factor and insulin-like growth factor 1, thymosin
β4 and HMGB1. Centro Cardiologico Monzino has
focused on HMGB1; in animal models this protein
modulates progenitor cell function, induces cardiac
regeneration and improves left ventricular function after
acutely MI and also in the chronically failing heart.
The other target for pharmacologic intervention is
represented by the mechanisms which induce cell cycle
arrest; it has been proposed to interfere with these
mechanisms to induce adult myocardial cell proliferation.
Which diseases should be treated. To date, most
studies worldwide have focused on left ventricular
dysfunction following MI, i.e. the most common form
of heart disease. At Centro Cardiologico Monzino we
have treated patients with refractory ischemia and
CCM — Scientific Report 2011 — Ongoing research 2012
225
we have consistently found an improvement in angina
class and objective evidence of enhanced perfusion
(see Figure below). In our preclinical studies we have
worked on animal models of acute MI and heart failure
secondary to MI. Other laboratories worldwide are
working on the use of cell therapy to treat non-ischemic
cardiomyopathy, cardiac arrhythmias and pulmonary
hypertension.
The economic hurdle. The development of any
therapeutic intervention requires enormous financial
investments and, in the case of drugs and medical
devices, the industry supports the research activities,
from basic studies to clinical trials. In regenerative
medicine the only interest for the industry is in drug
therapy and in the development of cell products for
allogenic use, as it is presently being done for bone
marrow-derived mesenchymal cells. Autologous cell
therapy falls in a different category; the financial
support comes from taxpayers money and directly
from institutions which aim at developing a “service” to
offer their patients, somewhat similar to bone marrow
transplant. Therefore, finding the financial resources
to support autologous cell therapy studies is a major
obstacle that has to be overcome in order to bring cell
therapy to clinical practice.
Publications, Clinical Research Projects,
Seminars, Conferences and Courses
Conclusions. The studies performed so far have shown
the great potential of cardiovascular regenerative
medicine. However, many issues have still to be
addressed in order to establish regenerative medicine
full potential. The ongoing worldwide effort in this field
may ultimately lead to cell therapy interventions which
best suit each patient’s need.
Improvement of myocardial perfusion at stress (see arrows) 6-months after intramyocardial injection
of bone-marrow-derived CD133+ cells in a patient with refractory untreatable angina.
226
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CCM — Scientific Report 2011 — Ongoing research 2012
227
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CCM — Scientific Report 2011 — Ongoing research 2012
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article ID
304562
0
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HUMAN APOLIPOPROTEIN A-I
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1,839
Quantitative Analysis of Mitral Valve
Apparatus in Mitral Valve Prolapse
Before and after Annuloplasty: A
Three-Dimensional Intraoperative
Transesophageal Study.
J Am Soc Echocardiogr
1133-1140
Maffessanti F, Marsan NA, Tamborini G,
Sugeng L, Caiani EG, Gripari P, Alamanni F,
Jeevanandam V, Lang RM, Pepi M.
251-254
6,802
1,626
J Interv Card
Electrophysiol
32
125-135
1,228
Multidetector computed tomography
vs multiplane transesophageal
echocardiography in detecting atrial
Thrombi in patients candidate to
radiofrequency ablation of atrial
fibrillation
152
55-60
Maltagliati A, Pontone G, Annoni A, Formenti A,
Galli CA, Tamborini G, Alimento M, Andreini D,
Tondo C, Pepi M
Int J Cardiol
41
161
314-321
5,052
J Interv Card
Electrophysiol
31
69-80
1,228
Left atrial reverse remodeling and
functional improvement after mitral
valve repair in degenerative mitral
regurgitation: A real-time 3-dimensional
echocardiography study
Am Heart J
Catheter ablation of atrial fibrillation:
randomized controlled trials and
registries, a look back and the view
forward
Marsan NA, Maffessanti F, Tamborini G, Gripari
P, Caiani E, Fusini L, Muratori M, Zanobini M,
Alamanni F, Pepi M.
Pepi M, Tamborini G, Fusini L, Maffessanti F
21
109-117
0
J Cardiovasc
Electrophysiol
22
1359-1366
3,288
Three-dimensional echocardiography
and mitral valve prolapse diagnosis: new
insights into leaflet and cardiac chamber
morphology, and annulus dynamics
J Cardiovsc Echogr
Imaging of Scar in Patients with
Ventricular Arrhythmias of Right
Ventricular Origin: Cardiac Magnetic
Resonance Versus Electroanatomic
Mapping
CCM — Scientific Report 2011 — Ongoing research 2012
30
58
109-118
273-277
483-490
1,228
1,228
14,292
CCM — Scientific Report 2011 — Ongoing research 2012
237
Pontone G, Andreini D, Bartorelli AL, Annoni A,
Mushtaq S, Bertella E, Formenti A, Cortinovis
S, Alamanni F, Fusari M, Bona V, Tamborini G,
Muratori M, Ballerini G, Fiorentini C, Biglioli
P, Pepi M
Feasibility and accuracy of a
comprehensive multidetector computed
tomography acquisition for patients
referred for balloon-expandable
transcatheter aortic valve implantation
Am Heart J
161
1106-1113
5,052
Pontone G, Andreini D, Bartorelli AL, Bertella E,
Mushtaq S, Cortinovis S, Chiappa L, Annoni A,
Formenti A, Trabattoni D, Montorsi P, Ballerini
G, Fiorentini C, Pepi M.
Comparison between low-dose
multidetector computed coronary
angiography and myocardial perfusion
imaging test in patients with intermediate
pre-test likelihood of coronary artery
disease
Int J Cardiol
147
454-457
6,802
Pontone G, Andreini D, Bertella E, Cortinovis
S, De Martini S, Pirondini M, Fabbiocchi F,
Bartorelli AL, Pepi M.
Detection of left main coronary artery
anomalous origin with low-dose
multidetector computed tomography
using prospective ECG gating.
J Cardiovasc Med
12
506-509
0,786
Pontone G, Andreini D, Galli S, Montorsi P,
Bartorelli AL, Pepi M
Detection of a large left anterior
descending coronary artery aneurysm
using low-dose coronary multidetector
computed tomography.
J Cardiovasc Med
Detection of bronchocoronary collateral
by low-dose multidetector computed
tomography.
Eur J Cardiothor Surg
Pontone G, Grancini L, Andreini D, Pepi M.
Clinical Research Projects in Progress during 2011
0,712
Title
Principal Investigator
RESEARCH LABORATORIES
40
272
2,397
Erythrocyte NO metabolic pathway in patients with microvascular angina
Cavalca
Effect of acute hyperglycemia on the synthesis pathway of nitrogen monoxide (NO) in erythrocytes
Cavalca
Effect of aspirin chronic treatment on platelet function in coronary patients with type II diabetes
Cavalca
In vivo evaluation of the effect of treatment with folic acid on the metabolic pathway of nitric oxide synthesis
Cavalca
N-acetylcysteine as
​​ a treatment in the iperomocysteinuric patient not responding to folic acid therapy
Cavalca
Transcriptome analysis in blood leukocytes of patients with coronary artery disease
Colombo
Association between the BDNF Val66Met polymorphism and coronary artery disease
Colombo
Circulating microRNAs in atrial fibrillation
D'Alessandra
Analysis of circulating microRNAs in patients with coronary artery disease
D'Alessandra
Indices of systemic and cellular activation, modulation of oxidative stress in aortic valve replacement
Nava
Prevention of atherothrombotic risk
Parolari
Assessment of the carotid intima-media thickness contribution to the cardiovascular risk. Comparison among different
European populations at high and low risk.
Tedesco
Transcriptomics of antiplatelet resistance
Tremoli
Antiplatelet effect of aspirin at low doses every 12 hours in patients undergoing coronary artery bypass graft and / or aortic
valve replacement using bioprosthesis
Tremoli
Impact of the measurement of carotid intima-media thickness on cardiovascular risk classification in patients in primary
prevention
Veglia
Evaluation of the flow-mediated dilatation (FMD) of the brachial artery using the lumen area in place of the diameter
Veglia
Calculation of the minimum amount of measurements needed to correctly estimate the progression of subclinical atherosclerosis
Veglia
Nutrient-drug interaction: green tea and simvastatin
Werba
Oral niacin flush. Predictive value of skin tests using methylnicotinate
Werba
CRITICAL CARDIOLOGY
238
CCM — Scientific Report 2011 — Ongoing research 2012
Influence of ACE genotype on lung diffusion (DLCO) at rest and after intravenous water load in patients with chronic heart
failure (CHF) receiving ACE inhibitors
Agostoni
Treatment of severe heart failure by ultrafiltration
Agostoni
A multicenter, randomized, double-blind, parallel group, active-controlled study to evaluate the efficacy and safety of both
aliskiren monotherapy and aliskiren/enalapril combination therapy compared to enalapril monotherapy, on morbidity and
mortality in patients with chronic heart failure (ALISKIREN - CSPP100F2301 - ATMOSPHERE)
Agostoni
Evaluation of the effects of 4 oral dosages of S 44121 vs placebo on cardiac function and NT-proBPN in patients with chronic
heart failure and left ventricular dysfunction not treated with a beta-blocker (CL2 4412-003)
Agostoni
Evaluation of the effects of 4 oral dosages of S 44121 vs placebo on cardiac function and NT-proBPN in patients with chronic
heart failure and left ventricular dysfunction not treated with a beta-blocker (CL2 4412-004)
Agostoni
CCM — Scientific Report 2011 — Ongoing research 2012
239
240
International registry on cardioversion of atrial fibrillation (RHYTHM-AF)
Agostoni
INTERVENTIONAL CARDIOLOGY
"Sleep and Heart" Italian Multicenter Project (PROMISES)
Agostoni
Percutaneous mitral repair using Evalve MitralClip
Bartorelli
Effects of beta 2-receptor blocking on pulmonary function in a human model of acute fluid overload
Agostoni
Agostoni
Clinical evaluation of a new procedure for percutaneous denervation of the renal sympathetic nervous system in patients with
hypertension refractory to drug treatment
Bartorelli
Evaluation of the dead space in patients with chronic heart failure
Evaluation of the effects of beta-blocker therapy on exercise tolerance in uncomplicated hypertension
Alimento
A continuation in the clinical evaluation of the BVS everolimus eluting coronary stent system in the treatment of patients with
de novo native coronary artery lesions (ABSORB EXTEND)
Bartorelli
Reference values ​​of cardiac output during exercise in healthy subjects.
Cattadori
Bartorelli
Psychological assessment and dyastolic dysfunction in hypertension (Paddy study)
Fiorentini
A randomized comparison of the NEVOTM Sirolimus-eluting coronary stent system using a bioresorbable polymer vs the
Everolimus-eluting coronary stent system (XIENCE V/ XIENCE PRIME/PROMUS) using a durable polymer in coronary artery
lesions (NEVO II)
Clinical and prognostic significance of vitamin D plasma levels in patients with acute coronary syndrome
Marenzi
Bartorelli
Prognostic significance of acute renal failure in course of acute coronary syndrome
Marenzi
A phase IV pilot study to evaluate kidney measured by neutrophil gelatinase-associated lipocalin (NGAL) as a new bio-marker
in patients with normal GFR undergoing percutaneous coronary intervention with IOPAMIDOL injection 370 or IODIXANOL
320 (IOP 116)
Prevention of contrast nephropathy in patients affected by renal failure in diuretic therapy and controlled hydration (Renal
Guard)
Marenzi
Bartorelli
Impact of different therapeutic approaches in patients with cardiorenal syndrome in course of acute congestive heart failure
Marenzi
A multicenter, double-blind, randomized trial to determine the clinical benefit and safety of vytorin versus simvastatin
monotherapy in high-risk patients with acute coronary syndrome (IMPROVE-IT)
Marenzi
A prospective, single blind, randomized controlled study to evaluate the safety and effectiveness of the tryton side branch
stent™ used in conjunction with a drug-eluting stent compared to side branch balloon angioplasty in conjunction with a
drug-eluting stent in the treatment of de novo bifurcation lesions involving the main branch and side branch within the native
coronary circulation (TRYTON)
Evaluating Xience V in Multi Vessel Disease (EXECUTIVE registry)
Bartorelli
A multicenter, randomized, double blind, placebo controlled study to evaluate the safety and efficacy of SCH530348 in
addition to standard of care in subjects with a history of atherosclerosis disease: thrombin receptor antagonist in secondary
prevention of atherothrombotic ischemic events (TRA2P TIMI 50)
Marenzi
Randomized evaluation of intracoronary nitroprusside vs adenosine after thrombus aspiration during primary PErcutaneous
coronary intervention for the prevention of No reflow in Acute Myocardial Infarction (REOPEN-AMI )
Fabbiocchi
Marenzi
Multicenter registry of the treatment of acute myocardial infarction according to the currently available best practice
(OPTIMA)
Fabbiocchi
Long-term follow-up of management models of antithrombotic therapies in patients with acute coronary syndrome (EPICOR)
Safety and efficacy evaluation of short-term treatment with A-2002 in patients with acute coronary syndromes (VISTA 16)
Marenzi
PROMUSTM ElementTM Everolimus-eluting coronary stent system European post-approval surveillance study . A prospective,
open-label, multicenter, observational study with all-comers enrollment (PE-PROVE)
Fabbiocchi
A randomized, double-blind, placebo controlled, parallel group, multinational trial, to assess the prevention of thrombotic
events with ticagrelor compared to placebo on a background of acetyl salicylic acid (ASA) therapy in patients with history
of myocardial infarction (PEGASUS-TIMI 54)
Marenzi
Cangrelor versus standard therapy to achieve optimal management of platetel inhibition (PHOENIX-TMC CAN 10-01)
Galli S
Marenzi
AnGiox in elderly subjects with myocardial infarction (with or without ST-segment elevation) undergoing percutaneous
coronary intervention (PCI) in Lombardy, Lombardy AGE-MI Registry. Coordinated by the Regional Registry Study Group
LombardIMA - Hippocrates Research
Galli S
Randomized, double-blind, triple-dummy trial to compare the efficacy of otamixaban with Unfractionated Heparin +
eptifibatide, in patients with Unstable angina/Non ST segment Elevation Myocardial infarction scheduled to undergo an early
invasive strategy (SEPIA 2 -EFC6204 TAO)
Marenzi
Myocardial contrast-echocardiography role in the assessment of acute and chronic outcome in PCI and CTO. Comparison with
MR angiography by dipyridamole stress
Grancini
A phase II, randomized, double-blind, parallel-arm, placebo-controlled, with variation of the dose study to evaluate the safety
and efficacy of apixaban in patients with recent-onset of acute coronary syndrome (APPRAISE-APIXABAN)
Short-term clinical outcome in patients with percutaneous or surgical coronary revascularization combined with valve surgery
Lualdi
Project to implement adherence to guidelines in the management of acute coronary syndromes during intra-and posthospitalization (BLITZ 4)
Marenzi
Assessment of myocardial functional recovery after percutaneous reopening of a chronic total occlusion in patients with
monovascular coronary artery disease. Role of MR angiography
Lualdi
Evaluation of early cessation or reduction of cigarette smoking through the use of electronic cigarette used as an adjunct to
standard counseling activities (SMOKING)
Marenzi
Tolerability and safety of the proximal cerebral protection during carotid stenting in patients with intracranial variants of
the Willis circle
Montorsi
Identification of proteomic / proinflammatory and immune biomarkers in patients with acute coronary syndromes at high risk
to develop clinical events: genomic and proteomic approaches
Marenzi
Evaluation of carotid stenosis by CT angiography. Comparison with intravascular carotid assessment by IVUS
Montorsi
Long-term follow-up of management models of antithrombotic therapies in patients with acute coronary syndrome (EPICOR)
Palermo
Endovascular treatment of carotid "near-occlusion"
Montorsi
Using the biomarker copeptin for the early exclusion of a serious condition in patients presenting with symptoms of "acute
chest pain."
Salvioni
A phase III, double blind, randomized, placebo controlled study to evaluate the effects of R04607381 on cardiovascular risk
in stable CHD patients with a documented recent acute coronary syndrome (EFFETTI RO 4607381)
Salvioni
A multicenter, randomized, double-blind, placebo-controlled study to evaluate the safety and efficacy of SCH 530348 in
addition to standard of care in subjects with acute coronary syndrome: thrombin receptor antagonist for clinical event
reduction in acute coronary syndrome (TRACER )
Salvioni
Study of cardiovascular events to evaluate the potential of aleglitazar in reducing cardiovascular risk in patients with Type-2
diabetes mellitus (T2D) and recent episode of acute coronary syndrome (ALECARDIO)
Salvioni
Steroids In cardiac surgery (SIRS)
Parolari
Phase III, multicenter, randomized, double-blind, placebo-controlled trial to evaluate the effect of saxagliptin on cardiovascular
death , myocardial infarction or ischemic stroke incidence in patients with type 2 diabetes (SAVOR)
Salvioni
Compassionate therapy with intramyocardial injection of autologous marrow cells in patients with refractory myocardial
ischemia
Pompilio
RE-LY registry on atrial fibrillation (AF): risk factors, treatments and outcomes of patients with atrial fibrillation admitted to
the emergency department in multiple regions of the world (RE-LY )
Salvioni
Pompilio
Randomized, placebo-controlled trial to assess cardiovascular outcomes after treatment with sitagliptin in patients with type
2 diabetes mellitus and inadequate glycemic control (TECOS)
Salvioni
A double-blind, randomized, placebo-controlled, parallel group study to evaluate the efficacy and safety of four 4 mg doses
of XRP0038/NV1FGF at intervals of 2 weeks on the events of amputation or death from any cause in patients with critical
ischemia of lower limbs and skin lesions (TAMARIS)
Salvioni
A prospective, randomized, multicenter, international drug vs placebo to assess the impact of clonidine and acetylsalicylic
acid (ASA) in patients undergoing noncardiac surgery at risk for cardiovascular events in the perioperative (POISE 2)
Salvi
Randomized, placebo-controlled trial to assess cardiovascular outcomes after treatment with exenatide once weekly in
patients with diabetes mellitus type 2 ( EXSCEL )
N-acetylcysteine in
​​ the prevention of contrast nephropathy in transcatheter aortic prosthesis implantation
Sisillo
A comparison between prasugrel and clopidogrel in acute coronary syndrome (ACS) subjects with UA/NSTEMI who are
medically managed (TRILOGY ACS Study)
Salvioni
CCM — Scientific Report 2011 — Ongoing research 2012
Correlation among clinical, angiographic and intra-hospital outcomes in myocardial infarction with ST-segment elevation
Italian AvantgarDE Dual AntiplateLet Therapy Registry (IDEAL)
Ravagnani
Trabattoni D
CARDIOVASCULAR SURGERY
SOURCE Registry: Edwards Sapien Aortic Bioprosthesis European Outcome registry
Fusari
SOURCE XT Registry: Edwards SAPIEN XT Aortic Bioprothesis Multi-Region Outcome Registry
Fusari
Omega-3 Fatty Acids for Prevention of Post-Operative Atrial Fibrillation (OPERA)
Parolari
TM
CCM — Scientific Report 2011 — Ongoing research 2012
241
Relationship between lipoprotein profile, plasma thiol level of inflammatory markers and carotid atherosclerosis. Identification
of plasma biomarkers of cardiovascular disease
Spirito
Expression of molecules involved in athero-thrombotic and aneurysmal process in infrarenal aortic perivascular adipose
tissue
Spirito
Endovascular treatment of steno-occlusions in the SFA by Zilver PTX stent
Trabattoni P
Acute type A dissection: emergency surgical treatment combined with E-XL Aortic Stent
Trabattoni P
Hybrid treatment of aortic arch aneurysms
Trabattoni P
Modification of peri-and postoperative right ventricular function in mitral valve plastic: influence of the surgical approach
and the mode of myocardial protection
Zanobini
Electrophysiology
Coronary atherosclerosis in outlier subjects: protective and individual risk factor evaluation (GISSI Outliers CAPIRE )
Andreini
Pathophysiological determinants of mitral annulus tissue Doppler systolic velocity obtained by high resolution echocardiography
Barbier
Accurate real-time 3D echocardiography in the localization of trans esophageal mitral valve prolapse: comparison between
visual method and with dedicated software
Pepi
Quantitative intraoperative evaluation of mitral valve apparatus pre-and post- annuloplasty intervention
Pepi
New techniques of morphofunctional evaluation of the tricuspid ring and valve and right ventricle by reconstruction from
MRI and 3D echocardiographic images
Pepi
Role of multidetector CT (MDCT) in evaluation of patients undergoing percutaneous aortic valve implantation (TAVI)
Pontone
Feasibility and diagnostic accuracy of ultra low dose'''' multidetector CT in the evaluation of coronary artery disease
Pontone
New anatomical parameters of the left atrium as predictors of atrial fibrillation
Pontone
Role of low-dose MDCT in the preoperative evaluation of patients with mitral valve prolapse
Pontone
Clinical study on the use of a electrophysiology low flow irrigation electrocatheter for the ablation of ventricular tachycardia
Carbucicchio
Pacemapping techniques for identification of the ablation site in patients with ventricular arrhythmias: validation and utility
of a new system of mathematical analysis (PAcing SOftware).
Carbucicchio
Comparison between 64-slice cardiac CT and stress echo in the diagnosis of significant coronary artery disease
Pontone
Endomyocardial biopsy via transeptal puncture in patients undergoing atrial fibrillation ablation. Searching for new biomarkers
using analysis of tissue microRNAs
Casella
Role of Magnetic Resonance Imaging (MRI) in assessing aortic annulus in patients undergoing percutaneous aortic valve
implantation (TAVI).
Pontone
Mapping and ablation of ventricular extrasystoles: usefulness of the automatic algorithm "Paso" implemented in the mapping
system CARTO (MAE-Paso)
Casella
3D transesophageal assessment of right ventricular size and function in the operating room
Tamborini
Near zerO fluoroscopic exPosure during catheter ablAtion of supRavenTricular arrhYthmias (NO-PARTY)
Casella
3D assessment of left atrial size and function pre-and post-plastic mitral surgery.
Tamborini
Percutaneous implantation of aortic valve prosthesis: 3D transesophageal echocardiography usefulness
Tamborini
Impact of percutaneous aortic valve replacement (TAVI) on left ventricular systolic and diastolic function : echocardiographic
long-term follow-up
Tamborini
Morphological and electrophysiological characteristics of myocardial substrate associated with increased arrhythmic risk in
patients with dilated cardiomyopathy (EMOSIDD)
Dello Russo
Usefulness of cardiac MRI in the identification of intraventricular desynchronization area in patients undergoing ventricular
resynchronization therapy
Dello Russo
Substrate in Heart failure And effectiveness of Resynchronization Therapy by EnSite Array system (SHARE-M)
Dello Russo
Evaluation of cardiac output by cardiopulmonary exercise testing with non-invasive measurement in patients with chronic
atrial fibrillation undergoing catheter ablation
Fassini
Monitoring after catheter ablation of atrial fibrillation by implantation of implantable recorder (loop recorder)
Moltrasio Massimo
Interference between the modern implantable cardiac devices and instrumentation used in the dental setting. An in vivo study
Moltrasio Massimo
Atrial fibrillation ablation registry
Moltrasio Massimo
Monitoring resynchronization in cardiac patients (MORE CARE)
Moltrasio Massimo
Genetic polymorphism and incidence of intra-atrial thrombosis in patients with permanent AF / chronic despite adequate oral
anticoagulation
Riva
Assessing the usefulness of the delayed-enhancement MRI in determining the degree of atrial fibrosis and its prognostic
correlation with the medium / long term efficacy of chronic AF transcatheter ablation
Riva
Identification of the levels of autonomy of patients undergoing implantation of mono and bicameral pacemaker
Tondo
Role of endomyocardial biopsy guided by electroanatomic mapping in the definition of the arrhythmic substrate in patients
with ventricular arrhythmias without apparent organic heart disease or uncertain diagnostic tests
Tondo
Endoscopic Guided Ablation and Voyage mapping (IRIS)
Tondo
A prospective, randomized, milticenter, interventional study designed to evaluate the safety and efficacy of percutaneous
ablation catheter TactiCath for the treatment of symptomatic paroxysmal atrial fibrillation using an irrigated catheter for
radiofrequency ablation with full recognition of the contact force (ToccaStar)
Tondo
Clinical WoRkflow Study for the EValuation Of the Multi-ELectrode PUlmonary Vein Isolation System for the Treatment of
Paroxysmal AtrIal FibrillatiON (REVOLUTION)
Tondo
Post-market clinical study of the CardioFocus endoscopic ablation system-adaptive contact (CF-EAS-AC)
Tondo-Dello Russo
CARDIOVASCULAR IMAGING
242
Additional clinical role of 64 slice cardiac MDCT in the visualization of anatomical variants and of coronary anomalies
Andreini
Feasibility and diagnostic accuracy of MDCT in the evaluation of coronary circulation in diabetic patients with suspected
coronary artery disease
Andreini
Feasibility and diagnostic accuracy of multidetector low-dose CT in the evaluation of coronary stents patency
Andreini
Prognostic role of multidetector CT in patients undergoing coronary artery bypass graft
Andreini
Comparison between nuclear magnetic resonance (NMR) and two-dimensional echocardiography in the quantification of
mitral regurgitation in mitral valve prolapse
Andreini
CCM — Scientific Report 2011 — Ongoing research 2012
CCM — Scientific Report 2011 — Ongoing research 2012
243
Ongoing Grants in 2011
Age.na.s
•Training of pharmacists: estimation of cardiovascular risk and approach to critical situations
European Commission
•IMPROVE
•Role of cyclophilin A in bone marrow vascular progenitor cell mobilization and recruitment in the angiogenic response to
ischemia - CAPI
•Nucleolar-dependent secretion of microRNA in a model of doxorubicin and trastuzumab cardiomyopathy - NUMIRDT
Fondazione Monzino
•Development of engineered cardiac valves
•Laboratory of experimental surgery: cardiovascular regenerative therapy
•New pathogenetic mechanisms of acute coronary syndromes
•Study of the pathogenic mechanisms of heart failure during emotional stress
Fondazione Veronesi
•The circulating transcriptome in coronary artery disease: a next generation sequencing approach
Ministero della Salute
Ricerca Corrente
•Absorption of the glycosaminoglycans (GAGs) into saphenous vein wall after their oral administration
•Analysis of the functional role of pentraxin 3 in endothelial cells through an integrated approach of proteomics and gene
silencing
•Analysis of the functional role of tissue factor in cardiomyocytes through an integrated approach of proteomics and gene
silencing
•Analysis of transcriptional profiles associated to brain damage in spontaneously hypertensive stroke prone (SHRSP) rats
•Aortic valve with minimally-invasive implantation
•Assessing oxidative stress in patients with carotid stenosis underwent surgical treatment and / or carotid stenting
•Assessment of the effectiveness of a medium / long term complex multistep strategy in the transcatheter ablation of
chronic AF
•Brachial artery diameter: a new marker of atherosclerosis?
•CABG, genomics, perioperative changes in hemostasis and inflammation and clinical outcomes at follow-up.
•Carotid atherosclerotic plaque: characterization by imaging, biochemical and histopathological techniques
•Caveolin-rich membrane microdomains and Tissue Factor modulation by protease receptors (PARS): role of cholesterol
biosynthesis inhibitors
•Cell therapy of heart failure and ischemic heart injury
•Characterization by MRI techniques of heart ischemia in ApoE KO mice
•Characterization of cells derived from cardiac mesenchyme: epigenetic and functional studies, stem cell reprogramming
•Characterization of human carotid plaques by imaging techniques and histopathology
244
CCM — Scientific Report 2011 — Ongoing research 2012
•Characterization of the epigenetic role of nitric oxide in aging and cellular senescence
•Clinical evaluation of safety and efficacy of a new system of hemostasis (QuikClot® Interventional) after diagnostic
percutaneous coronary or interventional procedures
•Correlation between platelet and megakaryocyte trascriptome in CAD patients
​​
•Development of a procedure for “decellularisation” and cryo-preservation of human pericardial patches in order to
engineer cardiovascular tissue
•Development of innovative diagnostic systems for atherosclerotic disease: ANN-IDA study
•Development of methods based on mass spectrometry for the absolute quantitative analysis of proteins in biological samples
•Development of murine model of dilated cardiomyopathy induced by doxorubicin and trastuzumab
•Echocardiographic and histological study of the influence of aging on left atrial function in mice
•Effect of cigarette smoke in the modulation of endothelial dysfunction
•Effect of drugs in drug-eluting stents on endothelial function and oxidative stress
•Effectiveness of nitric oxide and Histone Deacetylases inhibitors in dystrophic cardiomyopathy: identification of common
targets using a proteomic approach
•Endothelial cell secretoma
•Endovascular exclusion of aneurysms of the thoraco-abdominal aorta by fenestrated endograft: advantages of the
radiology suite in elective and emergency implantation
•Engineered tissues: cryopreserved valves cellularized by autologous cells of the recipient.
•Epigenetic modifications and impaired endothelialization of circulating endothelial progenitor cells in normal subjects and
patients at risk
•Epigenetic role of chronic electrical stimulation in cardiac cells
•Evaluation of the platelet activation state following stent placement in the carotid and / or iliac-femoral artery.
Observational study
•Evaluation of the hemostatic system in the mouse COX-2 knockout
•Evaluation of the molecular mechanisms involved in the stabilization of platelet mRNA
•Functional adaptation of the stem cell population of the saphenous vein (SVPs) as a factor predisposing to the development
of restenosis in coronary artery bypass grafting
•Genomic and proteomic expression profile of diabetic patients affected by platelet coronary syndromes
•Glycosaminoglycan and proteoglycan determinations in human aortic valves in normal and pathological conditions.
•GMP development of resident cardiac progenitors
•GPR17 purinergic receptor expression in stem cells residing in the heart
•Identification of human macrophage markers associated to the phenotype 5/1000
•Identification of the molecular targets of aspirin by a proteomic approach
•Identification of the role of the new purinergic receptor GPR17 in cardiomyocytes using a proteomic approach
•Impact of the BDNF Val66Met polymorphism on the hemostatic system: possible differences between the sexes
•Inflammation and cerebral ischemia
•Influence of dyslipidemia on left atrial size and compliance in subjects with isolated hypercholesterolemia
•Interactions between stem cells and biopolymers using’’ high throughput’’ systems, towards the engineering of artificial
stem cell niches for cardiovascular tissue bio-engineering
•Mecki score (Metabolic, Exercise, Cardiac, Kidney Index) for the identification of cardiovascular risk in patients with heart failure
•MicroRNAs and aging
•Minimally invasive aortic and mitral valve surgery
•Mitochondrial proteome of cardiac cells HL-1
•Mitral valve disease: genetic and phenotypic markers predictive of progression of the disease: systematic review of the
literature
•Modulation of endothelial prostanoids by cigarette smoke in association with IL-1 beta
•New markers of vascular imaging as predictors of vascular events in a European population of patients at high risk (new
projects based on the IMPROVE study)
•New model of left atrial function in mice
•Optimization of time / temperature in the decontamination of cardiovascular tissues with BASE.128
•Pharmacological control of cerebral ischaemia
•Plasma fatty acids and nutrition in patients with overt coronary artery disease
CCM — Scientific Report 2011 — Ongoing research 2012
245
•Platelet function in cardiac patients with chronic renal failure
•Possible role of brain-derived neurotrophic factor (BDNF) in the thrombosis
•Precursors of endothelial and smooth muscle cells in the abdominal aortic aneurysm: role of Cyclofillin A and microRNAs
modulated by aging
•Predictors of peri-operative renal injury after adult cardiac surgery: role of the pre-, intra-and postoperative variables and
of the extracorporeal circulation management
•Proteomics of atherosclerotic plaque
•Proteomics of C. elegans and effects of the silencing of the gene R53.1, the human ortholog of the gene FLAD1
•Proteomics of human heart valves
•Proteomics of plasma lipoproteins: identification of new associated molecules and clinical implications
•Proteomics of procoagulant microparticles
•Regeneration in adult zebrafish heart as a model for repair of cardiac ischemia
•Registry and Biobank of aortic valve disease
•Registry and Biobank of mitral valve disease
•Registry and Biobank of thoracic and abdominal aortic aneurysms and acute aortic syndromes
•Regulation of the alveolar capillary membrane function: is protein SP-B a useful marker?
•Regulation of the biosynthesis of Dickkopf-1 (Dkk-1) by statins in human endothelial cells
•Regulation of the pentraxin 3 biosynthesis by statins in human endothelial cells
•Relationship between platelet reactivity index and risk of adverse cardiovascular events in patients with acute coronary
syndrome treated with coronary angioplasty
•Role of cyclooxygenase-2 (COX-2) metabolites in the regulation of Tissue Factor (TF) induced by cigarette smoke
•Role of cyclooxygenase-2 in a mouse model of thrombosis
•Role of melanocortins in the restenosis and stent thrombosis prevention
•Study of the role of RAGE and HMGB1 in cardiovascular aging and development of cardiac fibrosis
•Surgical techniques to support electrophysiological procedures
•TF-dependent procoagulant contribution in leukocytes and platelets of patients with CAD
•The sum of early family aggregation for vascular disease in different districts and for risk factors as a novel risk factor for
atherosclerosis
•Thrombosis of drug-eluting stent
•Use of antithrombotics in atrial fibrillation (ATA - AF)
Ricerca Finalizzata
•Breathing disorders in heart failure: high altitude hypoxia as a model to define diagnostic tools, therapeutic strategies
•Identification of proteomic/proinflammatory and immune biomarkers in patients with acute coronary syndromes at high risk
to develop clinical events: genomic and proteomic approaches
•Insulin resistance and coronary disease: insights into inflammation, thrombosis and metabolic syndromes
•Role of longevity genes P66SH/SIR1 in insulin-resistance, diabetes and cardiovascular diseases
•Implementing brain repair in stroke via the exploiment of adult neurogenesis and gliogenesis: focus on the cross-talk
between microglial and endogenous precursor cells
•MicroRNA bioengeneering of human cardiac mesenchymal cells and heart regeneration
Progetti interni
•Cardiovascular Tissue Bank in Lombardia
•Clinical audit- Heart Surgery
•Clinical audit-Cardiology
•Patient analysis and tracking system data form
Regione Lombardia
•microRNA as new early markers of acute myocardial ischemia
•Echocardiographic imaging of the left atrium as a marker of cardiovascular events
•Setting up diagnostic and therapeutic pathways with the aid of telemedicine to identify mechanisms for the presentation
and development of atrial fibrillation. A prospective randomized multicenter study (PER.DI.FIA.TO)
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CCM — Scientific Report 2011 — Ongoing research 2012
247
Seminars, conferences and courses 2011
Seminars
April 19 - Salvatore Cuzzocrea (Messina) “Strategies to
reduce I/R injury”
April 29 - Germano Ferrari (Milano) “Best practices in
technology transfer: from intellectual property protection
to market”
May 24 - Nicholas W. Shworak (Lebanon, NH, USA)
“A novel anti-inflammatory pathway of the blood vessel
wall”
May 31 - Giuseppe Gaipa (Monza) “Single cell phosphoflow analysis in haematological malignancies: methods
and applications”
June 07 - Silvia Soddu (Roma) “HIPK2: dall’attivazione di
p53 in risposta al danno al DNA alla citochinesi”
June 14 - Marco Bianchi (Milano) “HMGB1, inflammation
and tissue regeneration”
June 30 - Lina Badimon (Barcellona) “Atherosclerotic
plaque complications: plaque angiogenesis”
July 12 - Bianca Rocca (Roma) “Variability in the
responsiveness to low dose aspirin: pharmacological and
disease-related mechanisms”
September 20 - Massimiliano Gnecchi (Pavia) “Use of
mesenchymal stem cells to repair infarcted hearts”
September 27 - Andrea Urbani (Roma) “Clinical
proteomics strategies and investigations: a perspective”
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CCM — Scientific Report 2011 — Ongoing research 2012
October 11 - Elisabetta Cerbai (Firenze) “Alterazioni
elettrofisiologiche cellulari nell’ipertrofia e insufficienza
cardiaca umana: basi molecolari e prospettive
farmacologiche”
October 25 - Huei-Sheng Vincent Chen (La Jolla)
“Modeling inherited cardiac diseases with patientspecific iPScells”
November 22 - Angelo Scuteri (Roma) “L’ importanza
del fenotipo per valorizzare i risultati della ricerca di
base. Il modello della sindrome metabolica”
November 30 - Mauro Giacca (Trieste) “How can we
mend a broken heart” (Bee Gees, 1971): searching for
genes that induce myocardial protection or repair by in
vivo gene transfer using AAV vectors””
September 24 - Cesare Fiorentini, Elena Tremoli
“Giornata mondiale per il cuore 2011”
November 16 - Piergiuseppe Agostoni “Beta-bloccanti e
scompenso:come scegliere”
November 03-04 - Elena Tremoli “Aterotrombosi: dalla ricerca
di base alla clinica” V Convegno monotematico della SIF
November 21-22 - Claudio Tondo “Biopsia
endomiocardica per elettrofisiologi”
November 16 - Piero Montorsi “ 3° Audit Stenting
carotideo”
November 21-22 - Piero Montorsi “19° Carotiday” Corso
di stenting carotideo
December 14 - Rita Spirito “Il piede vascolare: percorso
diagnostico-terapeutico”
November 21-23 - Alessandro Lualdi “Corso teorico
applicativo di Cardiologia Interventistica”
Courses
March 21-22- Piero Montorsi “17° Carotiday” Corso di
stenting carotideo
Feb-Mar-Apr-May-Jun - Marco Agrifoglio “Corso di
perfezionamento in Ecocolordoppler Vascolare”
Jan-Feb-Apr-Jun-Oct-Nov - Claudio Tondo “Tecnica
della puntura transettale e trattamento della fibrillazione
atriale mediante crioablazione”
April 01 - Stefano Galli, Piero Montorsi, Paolo Ravagnani
“Focus IVUS Advanced”
May 9-10 - Piergiuseppe Agostoni: “Il test da sforzo
cardiopolmonare (CPET): basi fisiologiche, indicazioni e
interpretazione”
May 27 - Stefano Galli, Piero Montorsi, Paolo Ravagnani
“Focus IVUS Basic”
September 13-15 - Mauro Pepi, Paolo Barbier “Corso
base di ecocardiografia”
Conferences
March 03 - Elena Tremoli “Il cuore del Monzino per le
donne”
March 30 - Paolo Biglioli, Francesco Alamanni, Cesare
Fiorentini “Live Cardiac Surgery. Treatment of heart
failure”
September 26-28 - Claudio Tondo “Corso teoricopratico sulle diverse tecniche di puntura transettale per
elettrofisiologi”
October 24 - Paolo Barbier “Rigurgito mitralico: dalla
quantificazione ecodoppler all’analisi morfologica 3D
nella routine diagnostica”
May 09 - Paolo Biglioli. Cesare Fiorentini “ Audit Day
2011”
October 24-25 - Corrado Carbucicchio, Claudio Tondo
“Mappaggio e ablazione transcatetere di aritmie
ventricolari”
June 08 - Daniela Trabattoni “Forame Ovale Pervio:
quando, come, perché intervenire”
November 7-8 - Corrado Carbucicchio, Claudio Tondo
“Puntura pericardica e mappaggio epicardico”
June 10 - Paolo Ravagnani “La cardiopatia ischemica nel
terzo millennio: è tempo per una riflessione”
November 15-17 - Mauro Pepi, Paolo Barbier “Corso
avanzato di ecocardiografia”
CCM — Scientific Report 2011 — Ongoing research 2012
249
Centro Cardiologico Monzino
Istituto di Ricovero e Cura a Carattere Scientifico
Via Parea 4 20138 Milano
T +39 02 580021
F +39 02 504667
W www.cardiologicomonzino.it
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