1. health and fitness
2. disease

ICIN
Netherlands
Heart Institute
Annual Report 2010
ICIN
Netherlands
Heart Institute
Directors
W.H. van Gilst, PhD
E.E. van der Wall, M.D. PhD
Scientific Council
E.E. van der Wall MD, PhD (Leiden) co-chairman
W.H. van Gilst, PhD (Groningen) co-chairman
J.M.T. de Bakker, PhD
R.M.F. Berger, MD, PhD
M.J. de Boer, MD, PhD
H. Boersma, PhD
H.J.G.M. Crijns, MD, PhD
M. Daemen, PhD
P.R.M. van Dijkman, MD, PhD
P.A. Doevendans, MD, PhD
D.J.G.M. Duncker PhD
I.C. van Gelder, MD, PhD
W.J. van der Giessen, MD, PhD
S. Heymans, MD, PhD
J.W. Jukema, MD, PhD
B.J.M. Mulder, MD, PhD
C.L. Mummery, PhD
G. Pasterkamp, PhD
W.J. Paulus, MD, PhD
J.J. Piek, MD, PhD
Y.M. Pinto, MD, PhD
A.J. Rabelink, MD, PhD
A.C. van Rossum, MD, PhD
A.F.W. van der Steen, PhD
D.J. van Veldhuisen, MD, PhD
M.A. Vos, PhD
A.A.M. Wilde, MD, PhD
A.C. van Rossum, MD, PhD
F. Zijlstra, MD, PhD
Ernst van der Wall
International Scientific Commiittee
Prof.dr. W.J. Paulus (chairman), VUMC, Amsterdam
Prof.dr. M. Böhm, Universitätsklinikum Saarland
Prof.dr. G. Breithardt, Universitätsklinikum Münster
Prof.dr. Th.F. Lüscher, Universität Zürich
Prof.dr. M. Komajda, Hôpital Pitié-Salpétrière, Parijs
Dr. M.A. Konstam MD, Tufts-New England Medical Center, Boston
Prof. dr. A. Shah, Kings College, London
Prof. dr. O. Smiseth, Rikshospitalet, Oslo
ICIN - Netherlands Heart Institute
PO Box 19258
3501 DG Utrecht
The Netherlands
030-2333600
info@icin.knaw.nl
www.icin.nl
ICIN - Netherlands Heart Institute is an institute
of the Royal Netherlands Academy of Arts and Sciences.
Wiek van Gilst
ICIN
Netherlands
Heart Institute
Table of Contents
Preface: From turbulent years to a bright new future
Durrer Center for cardiogenetic research
4
8
Research line - Atherosclerosis
11
GENDER, GENetic DEterminants of Restenosis
12
The HEBE I Trial
14
HEBE III: Effects of EPO after an acute myocardial infarction
18
Circulating Cells
19
BIOMarCS Study
20
Inhibition of RIP1-mediated necrosis
22
Reperfusion injury after myocardial infarction
23
Research line - Arrhythmias
25
Mechanisms and Treatment of Tachyarrhythmias
26
RACE 3 Routine vs aggressive rhythm control
30
Genes underlying sudden cardiac death
31
Gene therapy for sick sinus syndrome
33
Arrest 11: Determinants of sudden cardiac death
34
Activated platelets during acute mycardial infarction
35
Prevention of atrial fibrillation
36
Research line - Congenital heart disease
39
Congenital Heart Disease in Adults
40
Pregnancy in congenital heart disease - ZAHARA II
48
PROSTAVA Study
49
Hypertrophic cardiomyopathy
50
Right ventricular dysfunction and tetralogy of Fallot
52
Arrhythmogenic Right Ventricular Dysplasia
54
Pulmonary hypertension and Congenital heart disease
56
Research line - Heart failure
59
Dutch Programma on Tissue Engineering
60
ICIN-NHS Cell therapy program, translational studies
61
The PRIMA Study
63
CTMM - Triumph
64
SYNOP2L in sarcomere formation and stabilization
65
Research line - Imaging
67
Myocardial viability with MRI
68
MARCC Study
70
Therapeutic application of ultrasound
71
Directing stem cells using targeted microbubbles
72
Implications of CTA Findings
74
Modification of Risk with CT Coronary Angiography
75
Cardiovascular screening of asymptomatic patients
77
2-dimensional speckle tracking echocardiography
78
Diagnostic value of advanced echo imaging
80
Ultrasound imaging and therapy
81
MRI screening for rupture-prone vulnerable plaques
84
Imaging of Atherosclerotic Plaque and Neovascularization
85
Comparison of plaque observation in CTA and VH IVUS
86
Vulnerability assessment of atherosclerotic plaques
88
Biomechanics of vulnerable plaque rupture
90
Annexes91
Annex 1: ICIN Indicators
93
Annex 2: Doctoral Theses
94
Annex 3: Publications
96
3
Towards a new ICIN
W.H. van Gilst & E.E. van der Wall, directors of ICIN
The year 2010 was again a very interesting and challenging
year for ICIN/KNAW.
At the beginning of the year, we initiated broad discussions on
the way to redefine the organisational and scientific structure
of ICIN. To that purpose, we made a visiting tour along all
the 8 University Medical Centers and we discussed with all
our principle investigators about their views on the future of
ICIN. These views have led, after multiple dialogues with
our Scientific Council and the board of KNAW, to a strategic
document that was finalized at the end of 2010. The mission
of ICIN was redefined as follows: 1) to function as a national
scientific voice of academic cardiology in the Netherlands,
and 2) to coordinate and promote translational and clinical
research in the Netherlands and
beyond together with strong
Netherlands
European
and
International
Heart Institute
partners. As a consequence,
ICIN formally extended its name
with Netherlands Heart Institute (NHI) implying a revised logo.
ICIN
Strategy 2011-2015
Main objectives of ICIN-NHI for the next five years are
1) to function as a national clinical trial umbrella organisation
for the University Medical Centers,
2) to broaden the relationship with our strategic partners: the
Netherlands Society of Cardiology (NVVC) and the Working
Group on Cardiovascular Research in the Netherlands
(WCN),
3) to strengthen the cooperation with the Netherlands Heart
Foundation (NHS), the KNAW institutions Hubrecht and the
Netherlands Interdisciplinary Demographic Institute (NIDI),
the Netherlands Organization for Health Research and
Development (ZonMW), and with the Rembrandt Institute of
Cardiovascular Science (RICS),
4) to redefine the role of the ICIN-professors and the Scientific
Council,
5) to develop new educational activities in cooperation with the
Cardiovascular Institute for Continuous Medical Education
(CVOI),
6)to develop a strategic relationship with the Netherlands
Federation of University Medical Centers (NFU), and
7) to build a political and scientific network in Europe (Brussels).
ICIN Professors
Two new ICIN-professors were appointed in 2010: prof. dr.
Stephan N. Heymans (MUMC+, Maastricht) in July 2010, and
Prof. dr. Dominique de Kleijn (UMCU, Utrecht) in October 2010.
Prof. Heymans will focus on idiopathic cardiomyopathies, and
the scientific interest of Prof. de Kleijn deals with cardiovascular
immunology (proteomics etc).
The ICIN-professors, Prof. dr. Willem van der Giessen
(ErasmusMC, Rotterdam) and Prof. dr. Wouter Jukema (LUMC,
Leiden) were officially allowed by the KNAW to renew their first
5-year appointment with a second 5-year term.
Fellowships
In 2010, one ICIN fellowship was awarded to Drs. Marc Engels
(LUMC, Leiden) who spends his fellowship in Boston, USA
(Cardiovascular Research Center, Massachusetts General
Hospital, Harvard University). His scientific research deals with
reprogramming of ventricular fibroblasts into cardiomyocytes.
String of Pearls
The ICIN/CONCOR project (coordinator Prof.dr.Barbara
Mulder, AMC, Amsterdam) had been selected at the end
of 2009 to become the 9th pearl of the Pearl String Initiative
(PSI). On February 4th, 2010, this memorable event was
celebrated at the ‘Beurs van Amsterdam’ and launched with a
so-called ‘Beursgong’ in the presence of Prof. dr. Theo Mulder
(Director Research KNAW) and Prof. dr. Eduard C. Klasen as
representative of the NFU/PSI.
In 2010 negotiations were started to propose a 10th ICIN pearl to
PSI in 2011 entitled Acute Ischemic Heart Failure (coordinated
by Prof. dr. Eric Boersma, Erasmus MC). We hope that ICIN will
be provided with a next pearl in 2011. These initiatives are also
part of our strategy to strengthen and expand the position of the
Durrer Center.
Durrer Center
The Durrer Center for Cardiogenetic Research (supported
by ICIN, KNAW and NHS) was further expanded through the
activities of ICIN-professor prof. dr. Wouter Jukema (LUMC)
as director and Dr. Peter Lansberg (AMC) as coordinator. For
scientific and strategic reasons, the management team of the
Durrer Center had been extended with Prof. dr. G. Pasterkamp
(UMCU) and Dr. P. Van Tintelen (UMCG). Attempts are being
made to include as many cardiovascular biobanks as possible.
The Durrer Center acts therefore increasingly as a platform
to unite cardio-genetic information and to create scientific
programs.
At the Spring meeting of the Netherlands Society of Cardiology
(NVVC) in Papendal, April 9th, 2010, the ICIN/NVVC Einthoven
dissertation prizes were awarded to dr. Daniel Pijnappels
(LUMC, first prize), dr. Kevin Damman (UMCG, second prize),
4
ICIN
and dr. Bob Meyboom (ErasmusMC, third prize). Summaries of
their PhD theses can be found in edition 5 (May issue, 2010) of
the Netherlands Heart Journal.
Daniel Pijnappels en Marcel Daniels
Visiting professor
In the spring of 2010 we applied at the KNAW to install a
visiting professorship for Prof.dr Günter Breithardt (Muenster,
Germany). Prof. Breithardt is past-president of the European
Society of Cardiology (ESC), world-known expert in
electrophysiology, and member of our International Scientific
Committee. The application was granted by the KNAW in the
fall of 2010. The visiting professorship will start in May 2011
and will consist of educational activities such as giving master
classes and teaching courses to PhD students and research
fellows. In addition, prof. Breithardt will assist our post-docs and
project leaders in discussing new research projects and writing
grant proposals. We also hope to benefit from the European
network of prof. Breithardt to facilitate entries in Brussels
(European Commission).
For that purpose, we visited Brussels on 4-5 October, together
with our International Scientific Committee. We had been
invited by Nether-EU, the Netherlands House of Education and
ICIN Conference in Brussels
5
Netherlands
Heart Institute
Research in Brussels. It was discussed how Nether-EU and
ICIN could together promote cardiovascular research within
the European Union. Preferably this will be done in cooperation
with the NFU and its “Healthy Aging” program. In 2011
sequential visits will be made to Brussels to formalize European
relationships.
Singapore
In the beginning of November 2010, we (ICIN-directors, ICINprofessors Dominique de Kleijn and Ton van der Steen, and
coordinator Durrer Center Peter Lansberg) visited several
research and medical institutions in Singapore to further
expand a strategic partnership with Singapore in the field of
clinical cardiovascular research and to strengthen the current
cooperation in basic research. For several years, our ICINprofessor Prof. Dominique de Kleijn has already established
close contacts with various institutions in Singapore (technical,
basic-experimental, medical). To have this meeting orderly
arranged, we had in advance a preparatory meeting in May 2010
with the Dutch Ambassador (mr. J. Janssen) from Singapore.
It was explicitly proposed that a structural collaboration would
open many avenues for performing innovative basic and clinical
research. In this respect, the Durrer Center could play a major
role in the investigation of potential cardiogenetic differences
between Singapore and the Netherlands. We hope the KNAW
will support us to fortify and extend the existing relationship. Our
rewarding visit in Singapore ended with an outside dinner at the
house of the Dutch Ambassador in Singapore.
Strategic collaboration
In December 2010, ICIN started talks with the NFU to establish
a formal cooperation with the NFU. Points of discussion are
agreements on intellectual property, the way to deal with
funding handled by ICIN, and on ownership of publications and
promotions (PhD theses). The basis for a formal agreement in
writing has already been made and will further be formalized in
2011. Such an agreement will be beneficial to our researchers,
the academic hospitals and ICIN itself.
Throughout the year, we intensified discussions with ZonMW
in order to explore the potential for a more close collaboration.
These fruitful discussions will be continued in 2011.
Key scientific results
The RACE II trial was presented as late breaking clinical trial
at the meeting of the American College of Cardiology (March
2010, Atlanta, USA) by ICIN-professor Prof. dr. Isabelle van
Gelder (UMCG, Groningen). The main findings of RACE II were
published in the New England Journal of Medicine: Van Gelder
et al. Lenient versus strict rate control in patients with atrial
fibrillation. N Engl J Med. 2010 Apr 15;362:1363-73.
The RACE 3 trial on Routine versus aggressive upstream rhythm
control, which started off in November 2009, showed a gradual
increase in inclusion of patients over time. Indirectly based on the
findings of RACE studies, it was thought appropriate to explore
the possibilities to further investigate the field of atrial fibrillation
nationwide. Discussions were started with our strategic partners
NVVC and WCN to set out a common strategy to intensify the
awareness of atrial fibrillation and to evaluate the possibilities
for performing joint research projects.
The Netherlands Heart Journal continued to publish ICINoriginated articles every two months. Former ICIN-professor
Prof. dr. Jacques de Bakker (AMC, Amsterdam) is the Section
Editor of the ICIN articles.
The HEBE I trial on Intracoronary infusion of mononuclear cells
from bone marrow or peripheral blood compared with standard
therapy in patients after acute myocardial infarction treated by
primary percutaneous coronary intervention was published in
the European Heart Journal (Hirsch A et al Eur Heart J. 2010
Dec 10. [Epub ahead of print]).
Scientific council
Throughout the year, we had our monthly assemblies of the
Scientific Council consisting of the Heads of the Department
of Cardiology, the ICIN-professors and 10 experts in different
cardiovascular areas varying from basic to clinical in terms
of depth, and from vascular to epidemiological in terms of
broadness. In this way, the Scientific Council of the ICIN
represents a wide spectrum of cardiovascular diseases. In
November 2010, we said farewell to Prof. dr. Maarten Simoons
who attended the Scientific Council for more than 7 years. Also
Prof. dr. Joep Smeets, representing the UMCN (St. Radboud
Hospital, Nijmegen) left the Scientific Council. We thank
both individuals for their valuable contributions to ICIN. Prof.
Simoons was succeeded by Prof.dr. Felix Zijlstra, who became
head of the Department of Cardiology of the Rotterdam Thorax
Centre in November 2010. The position of Prof. Smeets was
taken over by Prof. dr. Menko Jan de Boer, who became head
of the Department of Cardiology in Nijmegen in October 2010.
The HEBE III trial on the Effects of erythropoietin after an
acute myocardial infarction was presented in August 2010 at
one of the Hot-Line Sessions at the Congress of the ESC, in
Stockholm. This multicenter ICIN-study was also published
in the European Heart Journal (Voors et al. Eur Heart J. 2010
Nov;31:2593-2600).
The KP6 Immunath project (Principle Investigator Prof. dr.
Gerard Pasterkamp) was officially closed down in 2010 in
London with a colloquium involving all the Immunath partners.
This successful project has shown to be very proliferative in
terms of international publications. It also shows the capability
of ICIN to handle large-scale projects. In September 2011 one
of the Immunath researchers, Fatih Arslan, will present his
doctoral thesis and at that occasion ICIN-NHI will organize a
meeting to discuss follow up to the Immunath project and apply
for EU funding again.
The CONCOR database was
able to include its 13.000th
patient in 2010. The ZAHARA II
study on pregnancy of women
with congenital heart disease
(coordinator dr. Els Pieper,
UMCG, Groningen) reached
in 2010 its final inclusion
after 2.5 years, consisting of
213 pregnant women with
congenital heart disease and
70 healthy pregnant women.
We are anxiously awaiting
the results of the ZAHARA II
study. The GENCOR database
showed
a
considerable
increase to almost 2000
included patients in 2010.
The organisation
In June 2010, ICIN had its annual outing for its personnel. The
ICIN Bureau had organized various ‘divertimento’s in the city of
Utrecht such as climbing of the Dom tower, museum tours, and
a boat trip through the canals. We were fortunate to have sunny
weather. The day was successfully closed with a walking buffet
along the Oude Gracht.
Apart from these registries and large multicenter trials, many
ICIN projects were publication-wise very successful, resulting
in over 400 international peer-reviewed articles. In 2010, there
were 20 ICIN-based PhD theses, testifying the considerable
annual scientific output of ICIN. The full list is included in this
report, in annexes 2 and 3.
6
ICIN
Tuesday December 28th was a tragic day for ICIN
as one of its former directors, Prof. dr. Frits Meijler,
deceased at the age of 85 years. Prof. Meijler
had been ICIN director between 1983 and 1993.
During his 10-year directorate, he established
a formal alliance with the KNAW and laid the
basis for many research projects. He himself was fascinated
by the atrio-ventricular (AV) node, not only in humans, but
from mouse to whale. Prof. Meijler finished his last scientific
article in November 2010, which was published in the 3rd
edition (March issue, 2011) of the Netherlands Heart Journal
(Archetype, adaptation and the mammalian heart, Neth Heart
J 2011;19:142-148). Until his very last day, Prof. Meijler, being
an active member of the KNAW, remained deeply involved with
ICIN. We shall commemorate him as a passionate man with
unconditional dedication to ICIN.
7
Netherlands
Heart Institute
To summarize, we can proudly look back at a successful year for
ICIN. We employ over 100 researchers, almost 80 projects are
running successfully, our scientific output is excellent, and our
financial position is healthy. We thank the KNAW for providing
financial means and administrative support. The International
Scientific Committee is gratefully acknowledged for setting out
new strategies, our Scientific Council for guiding and assisting
us in taking the appropriate decisions, the Foundation Friends
of the Cardiology (Chairman Cees Maas) for strategic support,
and the ICIN Bureau (Jan Weijers, Rachida Tallahi, Marjan
de Jonge, Corry Schouten-Schonewille, Judith Thijzen, Eelco
Soeteman, and Patrick de Bruijn) for their continuous support
and important contributions to the success of ICIN/NHI.
Durrer Center for cardiogenetic research
Principal investigator: J.W. Jukema
Mission Statement: United we share; united we care!
The Durrer Center for Cardiogenetic Research is a National
multidisciplinary collaboration that aims to provide an
infrastructure for comparing and merging results from individual
cardiovascular genetic studies. By combining these different
cohorts and making them centrally accessible it will create
the possibility to deliver more effective health care strategies
aimed at cardiovascular disease prevention, and at tailoring
medicines and other treatment regimens to individuals, families
and communities.
www.durrercenter.nl
Progress in 2010
The main aim of this National project is to provide the
cardiovascular genetic research community with resources
and know-how. The Durrer Center has the infrastructure to
facilitate storage of collected materials as well as tools for data
management and bio statistical analysis.
In 2010 the Durrer Center has expanded the cryo storage
services for (multi-center) research projects. These services
include:
• Sample storage (DNA, plasma, serum and on request tissue
samples or other types of bio-materials)
• -80°C or -196°C Storage facilities
• Set up logistic services for collecting and distributing
samples (including transport between centers)
• Initiated a web based project management system for
(multi-centre) trials in collaboration with ID Quest. This ICT
solution allows for local data access, progress reports and
data entry at participating centers.
• In collaboration with the department of Clinical Epidemiology,
Bio Statistics and Bio Informatics from the Academic
Medical Center in Amsterdam (prof. Dr. A.H. Zwinderman)
Bio-informatics support can be provided.
Website
Information on the collected materials is accessible via a public
website. The Durrer website provides an overview of collected
materials and relevant clinical information. This data is available
for on-going research projects as well as completed studies.
Durrer Center office and lab
In the 2010 the physical infrastructure of the Durrer Center was
completed with the support of the board of directors of the AMC
in Amsterdam. Office and laboratory space as well as initial cryo
storage for -80°C and -196°C samples were created within the
AMC facilities.
Legal documents
Legal and governance documents were drawn with the aid of
mr. J. van Dijk – Yellow Research bv.
The following documents are accessible through our web portal.
•
Governance kader Durrer center
• Kaderreglement
• Reglement gebruikersraad
• Geheimhoudingsverklaring gebruikersraad
• Master Agreement for Storage and Use of Human Samples
and Clinical Data (research (partly) financed by ICIN/NHS)
• Aanvraag beschikbaarstellen onderzoek
• Patienten informatie + toetstemmingsformulier incl
toelichting
In 2010 the GENCOR registration was incorporated in the
DurrerCenter Website and can now be accessed through our
webportal.
Renee de Ruiter started in June 2010 as the first Durrer Fellow.
At the UMCG she will responsible create an inventory of stored
materials and exiting collections at the cardiology department
and participate in research focusing on replicating SNPs in
GENDER and COACH cohorts
Partnerships
• CTMM
• ID-Quest
• Yellow Research BV
• Ontwikkelingsmaatschappij Flevoland
Funding
• Nederlandse Hartstichting
• ICIN
• KNAW
• AMC
• CTMM
Staff
Director: Prof. dr. W.J. Jukema
Coordinator:
Dr. P.J. Lansberg
Management team; Prof. dr. G. Pasterkamp and
Dr J.P. van Tintelen
DNA bank/Research: Dr. M. Mannens
Dr. A. Postma (post doc)
Ir. M. Kersenberg (lab technician)
Ing. C.Lurks (lab technician)
Data management
Prof. dr. A.H. Zwinderman
Drs. E. van Iperen (PhD student)
Data & sample collection Dr. W. Hermans van Ast (post doc)
Durrer Fellow
drs. R. de Ruiter
Durrer Center bio bank projects
New collections
• Recogida Muestera
• HELIUS
• D!Haan study
• EUPACT study
• COMPARE Study
• PROSPER
• AGE IV
Existing collections
• Circulation Cells – CTMM
• FEMCOR
8
ICIN
•
•
•
•
•
•
•
•
•
•
•
•
DICAMIN
APRAISE
EASEGO
EPIC NORFOLK subcohort
GENDER
GIRAFh
GOES
RADAR
REGRESS
PRIMA
HEBE
GENCOR
Research projects in where Durrer Center provided logistic or
lab facilitating support:
• CTMM – Circulating Cells: Logistic support for collecting,
storing and distributing samples
• GENDER study : Genome Wide Association Study and
additional SNP replicating studies have been completed
• EASEGO/RADAR: Analysis of potential bio-markers for
CHD risk prediction
• EPIC NORFOLK: Replication of PCAF analysis in GENDER
and PROSPER
• DICAM: replicating possible new mutations in a large cohort
of well characterized Dutch DCM patients (DC: analysis of
candidate genes and responsible for data collection)
• LAMIN A/C: Risk Stratification for Life-threatening Ventricular
Arrhythmias in an international cohort of Lamin A/C mutation
carriers (DC: collecting clinical information from patients).
9
Netherlands
Heart Institute
Implementing ISO 1589 CCKL Certification:
The Durrrer Center has the ambition to acquire CCKL ISO 1589
certification in 2012. The elaborate documentation process was
started in April 2010. This is done in close collaboration with
the department of DNA-diagnostics in the Academic Medical
Center of which the Durrer Center is an autonomous part . The
first assessment by means of an internal audit was successfully
executed on September 27th, 2010.
Erik van Iperen
Alex Postma
Wanda Hermans-Van Ast
Peter Lansberg
Carina Lurks
Merredith Kerssenberg
ICIN
Netherlands
Heart Institute
Research line - Atherosclerosis
GENDER, GENetic DEterminants of Restenosis
Principal investigator: J.W. Jukema
Introduction
Percutaneous Coronary Intervention (PCI) is a widely used
technique for treating patients with angina or acute coronary
events, but restenosis, the re-narrowing of the treated lesion,
is still the main drawback of PCI. Restenosis is a complex
biological processes resulting from the response of the immune
system to the damage induce (by PCI) within the vascular
wall and it is still not well understood why it occurs in some
individuals and not in others.
Restenosis is a major health problem, because it occurs in
5-25% of all treated persons, depending on which technique
was used and the extension of the disease. No pharmacologic
strategy or new device has proven effective in preventing this
phenomenon. Only coronary stenting and drug-eluting stents
(DES) have reduced the incidence of restenosis, but certainly
did not abolish it.
Identifying patients at increased risk for restenosis may improve
stratification of patients to individually tailored treatment. Thus
far, however, it has proven difficult to stratify patients with
regard to risk for coronary restenosis based only upon clinical
or procedural risk factors, since risk factors identified so far
in relation to restenosis have not been consistently reported.
There is evidence that genetic factors explain part of the
excessive risk for restenosis independently of conventional
clinical variables. So far we have already identified several
genes that might predispose to restenosis by means of pilot
candidate gene approaches in our large well phenotypic
characterized GENDER population. These include for instance,
genes involve in inflammation and cell proliferation processes.
However, a more comprehensive study consisting of searching
genetic variants (which are associated with restenosis) spread
all over the complete genome was still lacking in the GENDER
study. Therefore, a genome wide association scan (GWAS) was
performed using Illumina Human 610-Quad Beadchips. These
beadchips contain 620,901 Single Nucleotide Polymorphisms
(SNPs) and Copy Number Variants (CNV). A larger independent
and also well phenotypic characterized cohort from Munich
(Germany) was used to replicate our findings.
In principle, a disease with a genetic aetiology could be caused
by a variety of different possible DNA alterations. Some of
these alterations will be SNPs, however, some others could be
structural variants like CNVs. CNVs are insertions, deletions,
and duplications of genomic regions ranging from a kilobase
to multiple megabasepairs in length and recently have been
reported to be associated with susceptibility to common
multifactorial diseases such as psoriasis, schizophrenia,
lupus as well as congenital cardiovascular disorders. Despite
the wide use of SNP markers in identifying susceptibility loci
to certain diseases, estimates suggests that in the majority of
the common diseases only a small proportion of genetic risk
has been identified. To address this issue, a complementary
approach investigating not only SNPs but also CNVs is now
underway in the GENDER study.
The GENDER project
The GENetic DEterminants of Restenosis project was designed
to study the association between various gene polymorphisms
and clinical restenosis. It is a multicenter prospective followup study with both clinical and angiographic restenosis as
an endpoint. In total 3,104 consecutive patients were treated
successfully by PCI for an acute coronary even in four referral
centers for interventional cardiology in the Netherlands:
Academic Medical Center in Amsterdam, University Medical
center Groningen, Leiden University Medical Center and
Academic Hospital Maastricht. Only, patients treated for acute
ST elevation myocardial infarction (MI) were excluded. After
having obtained written informed consent, blood was sampled
for DNA isolation and future analysis. Clinical and procedural
data were gathered prospectively. Clinical restenosis was
established during a 9-month follow-up for death, myocardial
infarction and target vessel revascularization. A repeat
angiographic study was performed in a subpopulation after 6
months. An independent endpoint committee evaluated all
potential endpoints.
Participating centres
Participating centres in the GENDER project are the Leiden
University Medical Centre in Leiden, the Academic Medical
Centre in Amsterdam, the University Hospital Maastricht and
the University Medical Centre in Groningen. The Gaubius
Laboratory of TNO-PG in Leiden collected the blood samples
from the participating centres and isolated DNA. DNA-analysis
for the candidate gene approach was performed in the University
Hospital of Maastricht, in the Sylvius Laboratory Leiden and in
The Erasmus University of Rotterdam.
The GWAS was performed at the Leiden Genome Technology
Centre (LGTC) in collaboration with Molecular Epidemiology
and Human Genetic departments at the Leiden University
Medical Centre.
Present status
In 1998 the GENDER project was designed. Approval of the
Medical Ethics Committees of the participating centres was
obtained. Inclusion of patients started in March 1999. In June
2001 the last patient was included in the project. DNA has
been extracted for all patients and several candidate gene
approaches has been performed so far.
In 2008 the GWAS was started. Initially, 321 cases and 620
controls were selected to carry out the experiment. Cases
and controls were matched by age, gender and other clinical
factors such as diabetes and current smoking that have
been previously associated to restenosis. We conducted the
genome-wide association analysis using the Illumina Human
610-Quad Beadchips. These beadchips contain 620,901 Single
Nucleotide Polimorphism (SNP) and Copy Number Variants
(CNV) probes. The median spacing between markers is 2.7 kb,
therefore offering a dense coverage of the human genome both
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ICIN
of SNPs and CNVs and ensuring a good power for detecting
these genetic variants associated to the phenotype.
Present results
We performed the genotyping using the Illumina Beadstation
and the Infinium II assay following manufacturer’s instructions.
After genotyping, samples and genetic markers were subjected
to a stringent quality control protocol. In total, 571 controls,
295 cases and 556099 SNP’s markers passed all criteria. After
the quality control, application of SNP association test using
different model of inheritance point out to interesting genes that
might be related to the development of restenosis after PCI.
Subsequently and to reduce the chance of false positive results,
we replicate our top findings on an independent and larger
restenosis population (>3000 patients) from Munich. Using the
Sequenom iPLEX system we successfully genotyped 90 SNPs.
These results are reported in a scientific article which has been
already submitted and it is now under peer review.
Moreover, we have developed a MLPA (Multiplex Ligation
Dependent Probe Amplification) assay in order to replicate in
the German cohort the CNV regions that appear to be more
frequent in cases than in controls in the GENDER study. The
results of this experiments are now been analyzed.
In addition, several labs have been interested in the GWAS
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Heart Institute
performed in the GENDER project and during the year 2010
we have started several collaborations and the results of some
of these collaborations have been already published (see list of
publications).
Principal investigators
Prof. Dr. J.W. Jukema (project leader), Cardiology, LUMC/ICIN
Prof. Dr. P.E.Slagboom, Molecular Epidemiology, LUMC
Prof. Dr. P.de Knijff, Human Genetics, LUMC
Prof. Dr. A.H. Zwinderman, Clinical epidemiology, AMC
Scientific committee
Dr. R.J. de Winter, Cardiology, AMC
Prof. Dr. P.A.F.M. Doevendans, Cardiology, UMCU
Dr. R.A. Tio, Cardiology, AZG
Prof. Dr. A. van der Laarse, Biochemistry, LUMC
Prof. Dr. R.R. Frants, Anthropogenetica, LUMC
Mw. Dr. M.P.M. de Maat, Biochemistry, EUR
Prof. Dr. J. Waltenberger, Cardiology, AZM
Prof. Dr. E.E. van der Wall, Cardiology, LUMC
Dr. B.T. Heijmans, Molecular Epidiomology, LUMC
Study coordinator
Dr. M.L.Sampietro, Human Genetics, LUMC/ICIN
Drs. J.J.W. Verschuren, Cardiology, LUMC
The HEBE I Trial
Principal investigators: J.J. Piek and F. Zijlstra
Project
Bone marrow cell therapy after acute myocardial infarction: the
HEBE I trial
Introduction
During the last decennia, the introduction of advanced
revascularization strategies and medical therapies has led
to a marked decreased mortality and morbidity following
acute myocardial infarction (AMI). Despite optimal therapy,
approximately 30 percent of the patients suffer from postinfarct heart failure, due to poor infarct healing. Bone marrow
mononuclear cells (BMMC) have been suggested to enhance
tissue repair following AMI. Although BMMC therapy improves
left ventricular (LV) recovery in experimental models of AMI,
results from the large-scale randomized trials investigating
BMMC therapy in patients have shown contradictory results. To
address this issue, the Hebe trial was initiated and published in
2010.
The Hebe I Trial
The Hebe trial is a large, multicentre, randomized trial, designed
to evaluate the effect of intracoronary infusion of autologous
BMMCs and intracoronary infusion of autologous peripheral
blood mononuclear cells (PBMC) after primary percutaneous
coronary intervention (PCI). Between 2005 and 2008, a total
of 200 patients with ST-elevation myocardial infarction (STEMI)
successfully treated by primary PCI, were included in the trial.
All patients underwent cardiovascular magnetic resonance
(CMR) imaging, followed by randomization to treatment with
BMMCs (n=69), PBMCs (n=66), or no cell therapy (n=65).
After 4 months, CMR was repeated. The primary end point of
the Hebe trial was the change in regional myocardial function
in dysfunctional segments at 4 months relative to baseline,
based on segmental analysis as measured by CMR (figure 1).
Secondary endpoints included changes in absolute segmental
wall thickening in dysfunctional segments, changes in global LV
ejection fraction, volumes, mass, and infarct size, and changes
in regional myocardial function stratified by transmural extent of
infarction (figure 2).
A
A
B
from end-systolic wall thickness.
Figure 2 Calculation of infarct mass. The short-axis late
gadolinium enhanced (LGE) image (a) shows gadolinium
enhancement in the anterior/anteroseptal region, with a central
gap indicating an absence of gadolinium perfusion, consistent
with microvascular obstruction (MVO). (b) Infarct size (red
area) was determined on the LGE images by detecting the
amount of myocardium with a signal intensity 6 SD above the
intensity of remote myocardium (blue area).
Results of the Hebe I Trial
In summary, baseline CMR parameters were comparable in the
three groups. There were no significant differences between the
two treatment groups and the control group with regard to the
primary endpoint, the percentage of dysfunctional segments at
baseline with improved segmental wall thickening at 4 months,
and the secondary endpoints of change in left ventricular (LV)
ejection fraction LV volumes, LV mass, and infarct size (fig 3).
There were also no significant differences in the changes in
absolute segmental wall thickening in dysfunctional segments,
and changes in LV volumes, mass, and infarct size between the
two treatment groups and the control group. Since the Hebe
trial was conducted in 8 different centres multiple stem cell
laboratories were involved in stem cell isolation and analysis
to minimize storage times for bone marrow, peripheral blood
and isolated stem cells. The different stem cell laboratories
used for cell isolation did not show any relation with the primary
endpoint or the change in the LV ejection fraction in the BMMC
and PBMC group.
B
Figure 1 Calculation of segmental wall thickness, volumes and
function in the end-diastolic (a) and end-systolic (b) phase.
For analysis of regional myocardial function, each short-axis
slice was divided in 12 equi-angular segments to calculate wall
thickening (mm) of each segment by subtracting end-diastolic
Figure 3 Estimation of the effect of intracoronary injection of
BMMCs or PBMCs on LV ejection fraction (a and b). (a) Lines
indicate change observed in individual patients and squares
represent the mean with standard deviation. (b) Mean change
between baseline and follow-up at 4 months presented with
the standard error.
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ICIN
Microvascular obstruction (MVO) after AMI is known to be
associated with a worse prognosis for functional recovery and
an increased incidence of MACE. MVO was present in 59% of
the patients, with an even distribution amongst the treatment
groups. No interaction was found between the presence of
microvascular obstruction and treatment.
As expected, segments with increasing transmural extent of
infarction showed a larger decrease in end-diastolic and endsystolic wall thickness. Furthermore, the likelihood of complete
recovery of dysfunctional segments was the lowest in segments
with >75% hyperenhancement. However, these results did not
differ between the three treatment groups. Therefore, there was
no indication of enhanced recovery in any of the infarct zones
due to the cell infusion.
During follow-up, 1 patient had died, 5 patients had a recurrent
AMI and 10 patients had a target lesion revascularization. We
assessed these data for the different treatment arms. One
patient assigned to the peripheral blood group died of ventricular
fibrillation at 18 days after randomization (13 days after cell
delivery). Autopsy revealed thrombus in the infarct-related
artery. The patient was discharged with aspirin and clopidogrel,
and platelet inhibition was not discontinued. Recurrent AMI
occurred in 4 patients of the PBMC group and in 1 patient of
the control group. In the PBMC group, the recurrent AMI was
related to the cell delivery procedure in 3 patients and occurred
spontaneously in one patient. Target lesion revascularization
was performed in 4 patients of the BMMC group, 6 patients of
the PBMC group and 4 patients of the control group. Ventricular
fibrillation occurred in another patient in the peripheral blood
group 1 day after randomization (within a few hours after cell
infusion) and in one patient in the control group 3 days after
randomization. Both patients survived without sequelae
after resuscitation and received an implantable cardioverterdefibrillator. The number of adverse events in the peripheral
blood group seems higher although this was non-significant.
Figure 4 Change of wall thickening in dysfunctional segments
according to the transmural extent of infarction and treatment
between baseline and follow-up.
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Netherlands
Heart Institute
The Hebe trial in perspective
The Hebe trial is the largest study so far that used a highly
accurate and quantitative imaging technique for assessment of
regional systolic function in a multi-center setting. In comparison
to other clinical studies investigating cell therapy in AMI patients,
paired CMR images were obtained in most of the patients
(95%). Only patients with a relatively large infarct size were
included in the Hebe trial. This resulted in a population with a
markedly depressed LV ejection fraction (mean 42.6%), despite
a relatively short time between symptom onset and primary
PCI with contemporary post infarct treatment. With regard to
the primary endpoint, no interaction was found between several
subgroups in post hoc analyses.
Between the different stem cell trials, cell isolation protocols
and numbers of injected cells have differed substantially.
As earlier trials already showed, the isolation protocol and
incubation period are important and can have a major impact
on the number of isolated cells and the functional activity of
these cells. However, it has been previously demonstrated that
the cell-processing protocol applied in the Hebe trial results
in a cell fraction of which the quantity and quality are at least
similar to the positive REPAIR-AMI trial. Moreover, in the Hebe
trial cell infusion was performed at the day of cell harvesting,
thus avoiding overnight storage; a procedure that may have
a negative impact on functional activity of isolated cells.
Considering these data, the lack of beneficial effect in the trial
cannot be explained by the cell isolation protocol.
There are other possible factors that could account for the lack
of benefit of cell therapy in the Hebe trial. First, patients with
relatively short total ischemic time were included (median time
of 3.3 h). Second, studies have shown that only a small amount
of the injected cells home to the myocardium after intracoronary
cell injection, suggesting that future studies should focus on
optimizing cell delivery. Third, although no effect was found on a
functional parameter (i.e. improvement in segmental and overall
myocardial function), stem cells may have more subtle effect on
the myocardial microcirculation. Restoration of the myocardial
perfusion and myocardial flow is needed before functional
improvement can develop. Therefore, assessment of the
myocardial flow and perfusion will be performed to provide more
insight on the influence of stem cells on the microvasculature
in AMI.
Finally, follow-up CMR was performed at a relatively short period
of 4 months after cell therapy, thus possibly missing long-term
effects on LV function and LV remodeling. The two-year follow
up of the Hebe trial may show different results of intracoronary
stem cell therapy.
In conclusion, no effect of intracoronary delivery of mononuclear
cells from BM or peripheral blood on regional and global systolic
myocardial function was found at 4-month follow-up in patients
with a first AMI treated with primary PCI.
- Hirsch A, Nijveldt R, van der Vleuten PA, Tijssen JG, van
der Giessen WJ, Tio RA, Waltenberger J, Ten Berg JM,
Doevendans PA, Aengevaeren WR, Zwaginga JJ, Biemond
BJ, van Rossum AC, Piek JJ, Zijlstra F. Intracoronary infusion
of mononuclear cells from bone marrow or peripheral blood
compared with standard therapy in patients after acute
myocardial infarction treated by primary percutaneous
coronary intervention: results of the randomized controlled
HEBE trial. Eur Heart J 2010 [epub ahead of print].
PhD thesis
In 2010 Alexander Hirsch (Hebe trial coordinator), completed
his PhD research with honours at the Department of Cardiology
of the Academic Medical Center of the University of Amsterdam,
Amsterdam, The Netherlands. On the 29th of January 2010,
he successfully defended his thesis: ‘Clinical and functional
outcomes after revascularization strategies in acute coronary
syndromes’. Professors in Cardiology Jan J. Piek MD PhD, and
Robert J. de Winter MD PhD were his promotors, with professor
Jan G.P. Tijssen MD PhD as his copromotor.
Future plans
2 year and 5 year follow up of cardiac function and major
adverse cardiac events (MACE)
When assessing the major endpoints of the Hebe trial, which
consist of the percentage of dysfunctional segments with
improved segmental wall thickening at 4 months, change in LV
ejection fraction, end-systolic and end-diastolic volumes, LV
mass and infarct size, the Hebe trial showed that intracoronary
infusion of BMMCs or PBMCs did not have any demonstrable
effect on the aforementioned parameters at 4 months follow
up. It’s hypothesized that the anti-inflammatory and angiogenic
properties of BMMCs and PBMCs promote the preservation of
the LV function on a longer term than 4 months. To assess the
long-term effect of BMMC and PBMC therapy on the myocardial
function, patients underwent CMR examination at 2 years
follow-up. Currently, CMR data are analyzed by an external
core laboratory for changes in segmental wall thickening, LV
ejection fraction, infarct size, volumes and LV mass.
To assess the prevalence of MACE in long term follow up,
patients are invited for history taking and physical examination
5 years after enrolment for assessment of functional status and
the prevalence of events. The 5 year follow-up was initiated in
2010 and is estimated to finish in 2013.
Microvascular resistance and intracoronary flow measurement
Although the results of the Hebe trial showed that intracoronary
infusion of BMMCs has no beneficial effect on the recovery
of the global and regional systolic function, BMMC therapy
may have influenced neovascularization in the infarcted area,
resulting in improved recovery of the microcirculation. To assess
this hypothesis, intracoronary Doppler flow measurements were
performed at 3 to 8 days after primary PCI, and at 4 months
follow-up. Coronary flow velocity reserve and hyperaemic
microvascular resistance index (mmHg • s/cm) were calculated
to assess recovery of the microcirculation. The results of this
substudy are awaiting acceptance for publication.
Myocardial perfusion as measured by CMR first-pass perfusion
imaging
Although intracoronary infusion of BMMCs or PBMCs did not
improve LV function as assessed by segmental wall thickening
and overall LVEF, the angiogenic properties of the mononuclear
cells might augment the restoration of the myocardial perfusion
in either the infarct core zone or the surrounding border zone.
To assess this hypothesis, first-pass perfusion imaging with
CMR was performed 2 to 7 days after primary PCI, before
intracoronary infusion, with a repeat scan at 4 months followup. Different perfusion parameters were measured in the core
and rim of the infarct zone, and were compared with perfusion
in the normal myocardium, and between the three treatment
arms. The results of this sub study are expected to be ready for
publication in 2011.
Monocytes and myocardial injury and functional outcome
following AMI
Following AMI, leukocytes play a central role in protecting viable
myocardium from ischemic damage and promoting repair of
the infarcted tissue. However, an enhanced pro-inflammatory
immune response, characterized by increased neutrophil
and monocyte counts and reduced lymphocyte counts during
the acute phase of ischemia, has been associated with poor
clinical outcome. Recent clinical studies have suggested that
monocytes aggravate myocardial injury after AMI. Therefore,
we explored the relation between the monocytic response and
myocardial injury and functional outcome in patients randomized
to the PBMC group in the Hebe trial. Using flow cytometry, the
levels of classical and nonclassical monocytes were analyzed
from peripheral blood between day 3 and 8 after primary
PCI. Additionally, expression of several monocytic surface
molecules, that are known to play important roles in monocyte
activation and inflammation, was measured, as well as the
formation of monocyte-platelet complexes. Patients underwent
cardiovascular magnetic resonance imaging at baseline and
4 months follow-up. The results of this sub study are awaiting
acceptance for publication.
Study coordination
• A. Hirsch, MD, PhD / A.M. van der Laan, MD/ R. Delewi, MD
• R. Nijveldt, MD, PhD / L.F.H.J. Robbers, MD
• P.A. van der Vleuten, MD, PhD
Executive committee
• J.J. Piek, MD, PhD - Principal Investigator
• F. Zijlstra, MD, PhD - Principal Investigator
• B.J. Biemond, MD, PhD
• A.C. van Rossum, MD, PhD
• J.G.P. Tijssen, PhD
• P.A. Doevendans, MD, PhD
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Participating centres and stem cell core laboratory
• Academic Medical Centre, Amsterdam - J.J. Piek, MD, PhD
• VU University Medical Centre, Amsterdam - K. Marques,
MD
• University Medical Centre Groningen, Groningen - R.A. Tio,
MD, PhD
• University Medical Centre Utrecht, Utrecht - P.A.
Doevendans, MD, PhD
• University Medical Centre St. Radboud, Nijmegen - W.R.
Aengevaeren MD, PhD
• Erasmus Medical Centre, Rotterdam - W.J. van der Giessen,
MD, PhD
• University Hospital Maastricht, Maastricht - J. Waltenberger,
MD, PhD
• St. Antonius Hospital, Nieuwegein - J.M. ten Berg, MD, PhD
• Sanquin Research at CLB, Amsterdam - J.J. Zwaginga, MD,
PhD
17
Netherlands
Heart Institute
Industrial partners
• Biotronik
• Boston Scientific
• Guerbet
• Abbott
• Medtronic
• Novartis
• Pfizer
• Sanofi-Aventis
Acknowledgements
This study was initiated by Interuniversity Cardiology Institute
of The Netherlands with support of the Netherlands Heart
Foundation (grant 2005T101) and generous endorsement of
several industrial partners. We would like to thank all Hebe
investigators.
HEBE III: Effects of EPO after an acute myocardial infarction
Principal investigator: A.A. Voors
Aim and method of the project
The HEBE III is a multicenter, prospective, randomised openlabel trial with blinded evaluation of the primary endpoint.
The primary objective is to study the effect on left ventricular
ejection fraction (LVEF) of a single bolus of Epoetin Alfa (EPO),
administered directly after a primary percutaneous coronary
intervention (PCI) for a first AMI. A total of 529 patients with
a successful coronary angioplasty for a first ST-elevation
myocardial infarction were included. Secondary endpoints of
this study were defined a priori and included the assessment of
myocardial infarct size, the incidence of cardiovascular events
within six weeks after PCI and safety endpoints.
Progress in 2010
In June 2009, the 529th patient was included and the final visit
of the final patient was performed in July 2009. To keep up with
the fast rate of inclusion in the final year, many monitoring visits
to all participating centres took place. The final monitoring visit
took place in September and the official date of the end of the
trial was set at October 1st 2009. During these final monitoring
visits the monitor made sure that all the data entered in the
electronic database was checked and the monitor gathered all
the electrocardiograms collected by the sites for trial purposes.
Finally, all serous adverse events were categorised based
on the MedDRA system and for the medication a code was
provided according to the international ATC classification.
In late 2009 and early 2010 the data was analysed.
The HEBE III trial was presented in August 2010 at one of the
Hot-Line Sessions at the Congress of the ESC, in Stockholm.
This multicenter ICIN-study was also published in the European
Heart Journal (Voors et al. Eur Heart J. 2010 Nov;31:25932600).
Partnerships
University Medical Center Groningen
University Medical Center Leiden
Isala Clinics Zwolle
Medical Center Alkmaar
Amphia Hospital Breda
Academic Medical Center Amsterdam
St. Antonius Hospital Nieuwegein
Funding
This study is supported by grants from the Interuniversity
Cardiology Institute of the Netherlands (ICIN), the Netherlands
Heart Foundation and a research grant was received from Ortho
Biotech, a division of Janssen-Cilag B.V., The Netherlands.
Principal Investigators
Dr. A.A. Voors; UMCG, Groningen
Prof. Dr. F. Zijlstra; UMCG, Groningen
Prof. Dr. DJ van Veldhuisen; UMCG, Groningen
Dr. J.W. Jukema; LUMC, Leiden
Dr. A.W.J. van ’t Hof; Isala Clinics, Zwolle
Dr. J.O.J. Peels; Medical Center Alkmaar
Dr. J. Vos; Amphia Hospital, Breda
Dr. J.P.S. Henriques; AMC, Amsterdam
Dr. J.M. ten Berg; St. Antonius Hospital, Nieuwegein
Sub-Investigators
A.M.S. Belonje; UMCG, Groningen
Project management and Monitoring
Trial Coordinating Center, Groningen
18
ICIN
Netherlands
Heart Institute
Circulating Cells
Principal investigator: G. Pasterkamp
The aim of the CIRCULATING CELLS consortium is the
discovery and validation of new biomarkers derived from
circulating blood cells that allow prediction and facilitate
subsequent prevention of cardiovascular events in patients
with coronary artery disease. The program has been divided
into 3 central work packages (Organization and MTA, Clinical
platform and proteomics/bioinformatics) and 8 work packages
forming a technical platform. In the technical platform academic
and private partners work in close collaboration to improve
or develop technologies in the field of cell-based biomarker
detection. There are many interactions between the separate
WPs.
The ICIN collaborates in WP1 in the discovery phase of new
targets as well as in the operational part of the program. The
Durrer centre executes the storage and handling of the blood
and cell samples that were obtained in the four academic
centres.
In the first two years the major challenge was to standardize
and implement cell isolation protocols and cell-based analyses.
In addition, patient inclusion should have been finalized in this
time frame. Secondly the WPs in the technical platform should
reveal the first results showing the feasibility to use cell derived
biomarkers in the development of diagnostic or prognostic
technologies.
The consortium reached these major milestones in the first
phase of the program. The most relevant achievements are
summarized here.
- The objective to include 500 patients has been reached.
Moreover, to be able to analyze complete datasets of
sufficient number of patients we continued inclusion and
already obtained samples of over 600 patients. Follow up is
progressing and the number of endpoints matches with the
expectations.
- FACS equipment has been installed and 17 different cellbased FACS panels were obtained from 500 patients.
Quality checks of protein, RNA and miRNA have been
executed for the 4 different centers.
- The first proteomics runs have been executed in a crosssectional analysis showing that quality of the samples is
good and resulting in the first proteins of interest that differ
between patients suffering from CAD and patients without
significant lesions.
- Chips are being ordered to execute mRNA and miRNA
analyses of cell populations of 500 patients. Results are
expected in the next few months.
19
BIOMarCS Study
Principal investigator: H. Boersma
Project
BIOMarker study to identify the Acute risk of a Coronary
Syndrome (BIOMArCS)
Aim
Clinicians currently lack the possibility to identify increased
vulnerability for the occurrence of an (repeat) acute coronary
syndrome within a precise, short timeframe at the individual
patient level. Single measurements of novel biomarkers
during admission for acute coronary syndromes (ACS), have
proven that these markers are effective long-term individual
risk predictors. Coronary artery disease is a dynamic process,
however, in which increased coronary inflammation could lead to
ACS within a short timeframe. Serial biomarker measurements
may provide the possibility to assess such rapid deterioration.
Hypotheses
Against this background, the following hypotheses were
postulated:
1. Vascular inflammation becomes activated several days to
weeks before an acute coronary syndrome;
2. Plasma levels of biomarkers that specifically reflect distorted
lipid metabolism, vascular inflammation, endothelial
dysfunction, decreased endothelial regenerative capacity
and increased thrombogenicity, are up- or down-regulated
several days to weeks before an ACS;
3. Hence, serial biomarker measurements might be used to
identify divergent biomarker patterns indicating “vulnerable
periods” in the life-time of patients with CAD, during which
these patients are at an increased short-term risk of
developing an ACS (Figure), but still prior to the actual onset
of anginal complaints.
Methods
Within the context of the overall research program, the following
specific projects are being conducted
Project I - BIOMArCS pilot study
The BIOMArCS pilot study aims at describing the evolution of
biomarker patterns in 100 patients during the first 8 weeks after
admission for ACS. Insight in these normalisation patterns is
relevant to better understand divergent biomarker patterns prior
to a repeat ACS event during follow-up.
Project II - BIOMArCS main study
The aim of the nationwide, multi-centre, prospective,
observational BIOMArCS study is to identify the vulnerable
period that preludes an ACS during 1-year follow-up in a
cohort of 700 patients previously admitted with an acute
coronary syndrome. Patients who are admitted for an ACS are
followed-up during 1 year after admission. Blood samples are
collected during admission, at the day of hospital discharge and
subsequently every two weeks during the first six months after
discharge, followed by monthly blood sample collection during
the latter six months of the total of 1 year follow-up. Thus, a
total of (maximal) 21 samples per patient will be obtained. It
is expected that the primary endpoint
of cardiovascular mortality or repeat
non-fatal ACS will occur in 10%
of patients (70 cases). Biomarker
patterns in the period preceding the
endpoint event will then be compared with biomarker patterns in
210 event-free matching controls (3 for each case).
Project III - BIOMArCS-II glucose study
Elevated admission plasma glucose (APG) is common
and predicts worse clinical outcomes in patients with acute
coronary syndromes (ACS). Still, gluco metabolic care is still
underdeveloped. The single-center (MC Alkmaar) BIOMArCSII glucose study is developed to a) study the effectiveness
of intensive glucose management on clinical and biomarker
outcomes, and b) to study the relation between biomarker
washout-patterns during the first 8 weeks. A total of 300 patients
with 7.8 ≤ APG < 16 mmol*l-1 will be enrolled, who will then
be randomised to intensive glucose regulation with intravenous
insulin or expectative glucose management.
Status per January 1, 2011
The reporting year 2010 marked the third full year of the
implementation of the BIOMArCS programme. Both the ICIN
and the Dutch Network for Cardiovascular Research (WCN)
confirmed their endorsement and organisational support to the
study. By the end of 2010 a total of 18 hospitals, among which
6 ICIN centers, are actively enrolling patients in (one of the)
specified studies.
Project I - BIOMArCS pilot study
A total of 56 patients are included in the enrolling hospitals MC
Alkmaar, Erasmus MC and MC Heerlen.
Project II - BIOMArCS main study
Nineteen (19) hospitals, among which 6 ICIN centers, are
participating in the BIOMArCS main study. The UMCs of Leiden
and Nijmegen do not participate yet, but, recently, the UMC
Nijmegen has agreed to participate. On January 1, 2011, a
total of 402 patients have been included. The Erasmus MC is
responsible for 139 (35%) patients. The average ‘enrollment
speed’ in 2010 was 18.6 patients per month (slightly less than
1 patient/month/hospital). Since the incidence of the primary
endpoint is behind expectations, it has been decided to enlarge
the sample size to a total of 1000 patients. The completion of
patient enrollment is expected by the end of 2012. The principle
investigator (EB) and the trial coordinators (RO, MA, CJ) are in
close contact with all participating hospitals, and regular sitevisits are undertaken. See appendix II for more detailed data.
Project III - BIOMArCS-II glucose study
A total of 204 patients are included in the enrolling hospital MC
Alkmaar. The completion of patient enrollment is expected in
March 2012.
Principal investigator
20
ICIN
E. Boersma (Erasmus MC, Rotterdam)
Trial coordination
R.M. Oemrawsingh, K.M. Akkerhuis, C.G. Jansen (Erasmus
MC, Rotterdam)
Executive committee members
K.M. Akkerhuis (Erasmus MC, Rotterdam), F. Asselbergs
(UMCU, Utrecht), W.H. van Gilst (ICIN, Utrecht), T. Lenderink,
(Atrium MC, Heerlen), A.J. Oude Ophuis (WCN representative),
F. Zijlstra (Erasmus MC, Rotterdam), V.A. Umans (MCA,
Alkmaar), A. Moens (AZM, Maastricht), R.J. de Winter (AMC,
Amsterdam)
Investigators
K.M. Akkerhuis (Erasmus MC, Rotterdam), Y. Appelman (VUMC,
Amsterdam), F. Asselbergs (UMCU, Utrecht), R. Dijkgraaf (St
Jansdal, Harderwijk), N.J. Holwerda (St. Elisabeth Zkh, Tilburg),
21
Netherlands
Heart Institute
B. Ilmer (Havenziekenhuis, Rotterdam), T. Lenderink (Atrium
MC, Heerlen), A.H. Liem (Oosterschelde Zkh, Goes), A. Maas
(Gelre Zkh, Zutphen), A.J. Oude Ophuis (Canisius-Wilhelmina
Zkh, Nijmegen), A. Wardeh (MCH Westeinde, The Hague), E.
Ronner (Reinier de Graaf, Delft), C.E Schotborgh (Haga Zkh,
Den Haag), P.C. Smits (Maasstad Zkh, Rotterdam), S.H.K. The
(Bethesda Zkh, Hoogeveen), V.A. Umans (MCA, Alkmaar), A.
Moens (AZM, Maastricht), R.J. de Winter (AMC, Amsterdam),
J.C.C. van der Horst (UMCG, Groningen)
Sponsors
• Netherlands Heart Foundation (2007B012)
• Interuniversity Cardiology Institute Netherlands (project
number 07101)
• ErasmusMC
• Working group on Cardiovascular research Netherlands
(WCN)
• Foreest Institute, MC Alkmaar
ICIN
Netherlands
Heart Institute
Reperfusion injury after myocardial infarction
Principal investigator: S.A.J. Chamuleau
Aim and method of the project
Cell based cardiac repair has shown to modestly improve
cardiac function and improve quality of life patients with
ischemic heart disease. However, new insights in the working
mechanisms of cell therapy revealed massive cell loss (≥90%)
following cell transplantation. The aim of this project is to
elaborate on new methods to optimize cell therapy. In order
to enhance cell survival and retention, we are working on a
paracrine approach to create stem cells that are more resilient
to the harsh environment of ischemic myocardium.
Progress in 2010
In 2010, we worked on necrostatin-1 and its inhibition of cell
death caused by reperfusion injury after myocardial infarction.
We are working an in vivo study to determine the effect of
necrostatin-1 on infarct size and improvement of cardiac
function in small rodents.
Secondly, we worked on increasing the slow release of different
growth factors by making use of a new supramolecular polymer
(UPy), in collaboration with the Technical University of Eindhoven.
We completed in vitro studies on a cocktail of different growth
factors and started a study in large animals. At present the study
is expected to be finished in June 2011. Finally, the optimal cell
delivery technique is investigated in a randomized fashion in a
porcine model of ischemic heart disease by using specific cell
labeling, MRI and nuclear imaging.
Figure 1. CMPCs were pre-incubated with 30 µM Nec-1,
inhibitor of RIP1, followed by 50 µM H2O2 stimulation. Live,
apoptotic and necrotic cells were detected by AnnV/7-AAD
staining using flow cytometric analysis.
Staff
Necrostatin-1
Reperfusion of ischemic myocardium paradoxically aggravates
the injury process via formation of radical oxygen species,
release of various pro-inflammatory cytokines and attraction of
inflammatory cells to the damaged tissue. Necrostatin-1 (Nec1) is recently reported to effectively inhibit necrosis and reduce
infarct size following myocardial infarction. We have completed
a series of in vitro experiments to investigate the effect of Nec-1
on human cardiac myoprogenitor cells (CMPCs) (figure 1).
Partnerships
Studies with the use of ureido-pyrimidinone (UPy) polymers
are conducted in collaboration with Technical University of
Eindhoven (P.Y.W. Dankers)
Funding
This project was supported by the Netherlands Heart Foundation
and ICIN
23
From left to right: S.A.J. Chamuleau (ICIN, UMCU), P.A.
Doevendans (ICIN, UMCU), S. Koudstaal (ICIN), M.
Oerlemans (UMCU), D. Feyen (UMCU), R. Gaetani (UMCU)
and T. van der Spoel (UMCU). (J. Sluijter, K. Vrijsen, and
Sridevi Jaksani not on picture)
ICIN
Netherlands
Heart Institute
Research line - Arrhythmias
Mechanisms and Treatment of Tachyarrhythmias
Principal investigator: J.M.T. de Bakker
Non-invasive Electrocardiographic Imaging to
Assess Cardiac Electrophysiological Integrity
Background
Sudden cardiac death (SCD) due to tachyarrhythmias accounts
for 50% of the total number of cardiac deaths. Moreover, SCD
often occurs without knowledge of cardiac disease. The global
electrical condition of the heart can be viewed by the 12 lead
surface electrocardiogram (ECG). This ECG provides, however,
insufficient information on the actual sequence of activation
and repolarization to assess the electrical stability of the heart.
Selection of patients prone to arrhythmias is difficult. Up to now,
invasive techniques are required to assess the electrical stability
of the heart. Invasive techniques are, however, time consuming,
costly and may lead to complications.
The development of a technique that enables estimation of the
electrical status of the heart in a non-invasive way may decrease
the number of SCDs and has the potential to reduce the burden
on patients. Moreover, in patients prone to arrhythmias, noninvasive assessment of the pro-arrhythmic effect of cardiac and
non-cardiac medication would be of great value.
Key objective of the study
The key objective of this study is to establish new technology
that allows the measurement of the electrical activity and status
of the heart muscle non-invasively using a limited set of surface
electrodes and sophisticated software.
Project outline
For patient-tailored estimation of the electrical activity and
status of the heart muscle from surface ECGs, the following
steps will be combined:
1.Determination of the heart-torso geometry, which will be
done by MRI.
2. Recording of 64 surface electrocardiograms.
3. Development of software to derive electrical activity at the
outside (epicardium) and inside (endocardium) of the heart
from the body surface ECGs. For this “inverse solution” we
will further and extend the software originally developed by
van Oosterom, Huiskamp and Oostendorp in Nijmegen,
which is based on the equivalent double layer (EDL)
source model. Importantly, this method allows estimation
of maps of both the endocardial and the epicardial activity.
Other inverse techniques are only able to assess (pseudo)
epicardial activity.
Applicability and expected results
The combination of the proposed experiments will provide a
non-invasive means (inverse technique) to obtain detailed
information on:
1.The origin of tachyarrhythmias to guide treatment by
catheter ablation. The technique is able to distinguish
between epicardial and endocardial locations, information
that is crucial for this treatment modality;
2.Local areas with impaired conduction and/or abnormal
repolarization. Both parameters are related to arrhythmia
vulnerability;
3. Optimal electrode positions for resynchronisation therapy
(CRT) in patients with heart failure. CRT optimizes the
activation sequence of the cardiac impulse by electrical
stimulation at multiple sites;
4.The effect of cardiac and non-cardiac medication on
conduction and electrical stability of the heart;
5. Activation patterns that are related to fractionated surface
ECGs. These ECGs are associated with arrhythmias, but
their origin is unknown;
6. Conduction velocity and action potential duration restitution;
these parameters are a measure of the electrical stability of
the heart.
Status
Two postdocs and two AIO’s have been appointed; The project
will start in January 2011
Investigators
AC Linnenbank (ICIN), C Boden (ICIN), TF Oostendorp (UMC
St Radboud), PM van Dam (UMC St Radboud), A van oosterom
(UMC St Radboud), PFHM van Dessel, (UvA), R Coronel (UvA),
M Potse (CARIM), JMT de Bakker, (ICIN).
Funding
This study is granted by STW (10959).
Increased collagen deposition in aged mice
haploinsufficient for connexin 43
Background
Arrhythmogenic ventricular remodeling is associated by both
reduced gap junction expression and increased collagen
deposition. We hypothesized that reduced Cx43 expression
is responsible for enhanced fibrosis in the remodeled heart,
resulting in an arrhythmogenic substrate. Therefore, we
investigated the effect of normal or reduced Cx43 expression
on the formation of fibrosis in a physiological (aging) and
pathophysiological (Transverse Aortic Constriction, TAC)
mouse model.
Methods and Results
Cx43fl/fl (2 alleles for Cx43, Cx43WT) and Cx43CreER(T)/
fl (one allele for Cx43, Cx43HET) mice were aged to 1821 months, or at the age of 3 months either TAC- or Shamoperated and sacrificed after 16 weeks. Epicardial activation
mapping of right (RV) and left (LV) ventricle was performed on
Langendorff perfused hearts. Sustained ventricular arrhythmias
were induced in 0/11 aged Cx43WT and 10/15 Cx43HET
mice (p<0.01). Cx43 expression was reduced by half in aged
Cx43HET compared to aged Cx43WT mice, whereas collagen
deposition was significantly increased from 1.1±0.2 to 7.4±1.3%.
Aged Cx43HET mice with arrhythmias had significantly higher
levels of fibrosis and conduction heterogeneity than aged
Cx43HET mice without arrhythmias. TAC-operation significantly
increased fibrosis in control compared to Sham (0.4±0.06 vs
26
ICIN
4.0±1.2%, respectively), but this increase was significantly
higher in Cx43HET mice (10.8±1.4%).
Fibroblast proliferation and activity: Both aged and TACoperated Cx43HET hearts showed more pronounced fibrosis
compared to aged and TAC-operated Cx43WT hearts.
Enhanced fibrosis is due to enhanced collagen deposition,
which may be caused by either enhanced fibroblast proliferation,
enhanced fibroblast activity, or both. Therefore, we analyzed
fibroblast proliferation and activity of TAC- and Sham-operated
Cx43WT and Cx43HET hearts. Discoidin domain receptor 2
(DDR2) expression, specifically present at the cell membrane of
fibroblasts in cardiac tissue, was evaluated by Western blotting.
Figure 1A shows that DDR2 expression was equal between
Cx43WT and Cx43HET hearts, and that TAC-operation did
not change DDR2 expression, indicating that the number of
fibroblasts remained unchanged. Subsequently, activity of
fibroblasts was determined by expression of the procollagen
peptide P1NP. Figure 1B shows low P1NP expression in Shamoperated Cx43WT and Cx43HET hearts, respectively. TACoperation did not statistically alter P1NP expression in Cx43WT
hearts, although the expression tended to be somewhat more
intense. However, P1NP expression was significantly increased
in Cx43HET hearts after TAC-surgery when compared to Shamoperated hearts. Comparable results were obtained for P3NP,
another procollagen peptide. Finally, we performed a qPCR on
COL1A2, the gene encoding for the alpha-2 chain of collagen
type 1. Figure 1C shows that COL1A2 mRNA levels were
comparable between Sham-operated Cx43WT and Cx43HET
hearts. However, TAC-operation significantly increased COL1A2
mRNA levels in Cx43HET hearts.
Figure 1: Fibroblast proliferation and activity. Panel A:
Western blot data with comparable DDR2 (marker for fibroblast
proliferation) expression in Sham- and TAC-operated Cx43WT
and Cx43HET hearts. Panel B: Immunofluorescent pictures
showing low P1NP (marker fro fibroblast activity) expression
in Sham-operated Cx43WT and Cx43HET hearts (upper
pictures), which was slightly increased after TAC-operation
in Cx43WT hearts, but much more pronounced and only
significantly in TAC-operated Cx43HET hearts. Bars represent
50 μm. Panel C: COL1A2 mRNA expression (marker fro
fibroblast activity) that was significantly increased in TACoperated Cx43HET hearts compared to Sham23 operated
Cx43WT and Cx43HET hearts. Values are relative increases
compared to expression in Sham-operated Cx43WT hearts.
27
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Heart Institute
Conclusions
Our data showed that both physiological (aging) and
pathophysiological (TAC) stress leads to more excessive
fibrosis in mice with a 50% reduced expression of Cx43
(Cx43Cre-ER(T)/fl mice) compared to controls (Cx43fl/fl mice),
and that this was related to an increased activity of fibroblasts
rather than an increased proliferation of these cells. Together
with a heterogeneous reduced expression of Cx43, this resulted
in dispersed conduction and pro-arrhythmia.
Status
A revised manuscript with emphasis on the relation between
fibrosis and Cx43 expression has been submitted.
Investigators
JA Janse (UMCU), AAB van Veen (UMCU), R van den Nagel,
MA. Vos (UMCU), JMT. de Bakker (ICIN, AMC, UMCU), HVM.
van Rijen (UMCU).
Funding
This study is granted by NHS 2005B170 (to HVM van Rijen)
Curing ion channel mutations through selective
silencing of aberrant gene expression and upregulation of wild-type channel function
Background
Sudden cardiac death due to cardiac arrhythmias is a major
cause of death in patients with sodium channelopathies like
Brugada syndrome (BS), Long QT (LQT) and Conduction
Disease (CD). The only treatment up to now for these patients is
the ICD implantation, which has, however, several shortcomings.
As an alternative approach to treat these patients we propose
to silence expression of mutated genes and up-regulate
expression of the wild type gene. Gene silencing can be obtained
by RNA interference (RNAi), which is a cellular pathway for
posttranscriptional gene silencing. We recently investigated
the electrophysiological characteristics of a transgenic model
of the murine SCN5a mutation 1798insD, which is equivalent
to the SCN5a-1795insD mutation in man. The mutation shows
the characteristics of both BS (reduced peak current) and LQT
(increased late sodium current) and is therefore ideally suited to
test our hypothesis that silencing expression of mutated genes
and up-regulation of the expression of wild type genes can
restore normal function of the sodium channel.
Results
The functional voltage-gated sodium channels expression were
investigated in vitro in human embryonic kidney (HEK)-293T
cell lines, a HEK-293 cell line transformed by the adenovirus
E1A gene product resulting in higher expression levels. For this
purpose cells were transfected with murine wild type SCN5a
gene (pCGI-mH1) and the 1798insD mutation (pCGI-mH11798insD) respectively. Optimal conditions for transfection
and current recording were established (Lipofectamin2000,
replacing extracellular Na+ with NMDG+ to increase the
availability of sodium channels, fluoride in the internal pipette
solution to improve patch-clamp sealing).
Sodium currents were measured in transiently transfected
HEK-293T cells with pCGI-mH1 (mouse wild type SCN5a)
and β1 subunit (without siRNA) using conventional whole cell
voltage clamp technique. On average, peak current density
was increased 2.5 fold at 37°C (3978 pA/pF +/- 1674 at -44.5
mV +/-12.9) compared to room temperature (1583 pA/pF +/615 at -29.3 mV +/-3.9), P=0.06. The transiently transfected
HEK-293T cells with pCGI-mH1-1798insD showed NO sodium
current. Unexpectedly, the cells transiently transfected with both
wildtype mH1 as well as mH1-1798insD constructs to express
both alleles (heterozygously), resulted in comparable current
densities as the cells transfected with only wildtype mH1.
A persistent inward current is, however, seen in some of the
measurements. From this, we concluded that this cell-system
is not a liable system to investigate the effect of specific siRNA
knock-down of mutant sodium channel.
SiRNA constructs designed so far comprised one siRNA
construct that was able to inhibit wild type SCN5a specifically
and one siRNA construct that inhibits mutant SCN5a but slightly
inhibits the wild type gene as well. Therefore, new constructs
were made and are being tested for specific inhibition of the
mouse wild type and 1798insD. An adenoviral construct of mH1
(MYC-tagged, IRES-GFP) for overexpression of the WT allele
has been generated using the Stratagene AdEasy system.
Virus production is being done at the moment and will be ready
at the beginning of 2011.
Continuation
This study is ongoing and involves the following steps:
1) Test putative siRNAs designed against the wild-type and
mutated SCN5A coding sequence for their ability to
selectively attenuate gene expression.
2) The most efficient siRNA construct(s) for the mutant and
wild-type SCN5A gene are being used to develop lentiviral
constructs.
3) The virus with the siRNA against the mutant gene will be
tested in neonatal cardiomyocytes derived from mutant
1798insD+/- mice. Peak sodium current
and sodium current biophysical characteristics will be measured.
The magnitude of the persistent sodium current is further
assessed through application of tetrodotoxine (TTX).
4) Conduction parameters will be determined in monolayers
and patterned grown cell cultures of neonatal mutant mouse
myocytes transfected with the viral construct for siRNA
against the mutant gene. Action potential characteristics will
be determined by optical mapping using voltage sensitive
dyes.
5) The same measurements as described in 3 and 4 will be
carried out after up-regulation of wild-type SCN5a expression
through transfection of a viral construct containing wild-type
SCN5a in mutant 1798insD+/- mice and SCN5a +/- mice.
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Investigators
T. Koopman (ICIN), SCM van Amersfoorth (AMC), MAG van
der Heyden (UMCU), M Rook (UMCU), CR Bezzina (UvA), CA
Remme (ICIN), JMT de Bakker (ICIN, AMC, UMCU).
Funding
This study is supported by the Netherlands Heart Foundation
(NHS 2008B044).
Direct detection of postinfarction myocardial
fibrosis with MRI; (CTMM/COHFAR)
Background
In myocardial infarction, the assessment of myocardial injury
after ischemia is an important predictive parameter for clinical
outcome. The standard non-invasive technique currently used
for assessment of myocardial viability is delayed contrast
enhancement with gadolinium, which allows for the detection
of both necrosis and excessive collagen deposition (fibrosis) of
the myocardium.
As gadolinium uptake in the interstitial space is non-specific,
delayed enhancement cannot discriminate between different
forms of tissue damage within the heart. Therefore, to date
there is a growing demand to a non-invasive technique that can
be used for the direct detection of fibrosis and its concomitant
left ventricular (LV) remodeling in humans. The aim of this study
is to investigate the potential of ultrashort TE MRI to generate
endogenous contrast from cardiac fibrosis after infarction.
Methods and results
Myocardial infarction was created in 11 Lewis rats by ligating
the left anterior descending artery. As a control, 6 rats
underwent sham-surgery. Six weeks after surgery, rats were
anaesthetized and the heart was excised for ex vivo analysis.
The heart was placed in a plastic tube filled with fomblin, to
provide magnetic susceptibility matching between tissue and
the surroundings. Imaging was performed with a 7 Tesla MRI
scanner (Philips Healthcare, Cleveland, USA), using a home
built quadrature transmit and receive coil with a circular shaped
element of 5 cm in diameter and a stripline element of 6 cm
length. Balanced fast field echo images (TR/TE 8.3/4.2 ms,
flip angle 5 deg) were acquired for anatomical reference with
histology. 3D gradient echo (UTE GRE) images with radial
sampling (TR/TE 14/0.15 ms, flip angle 20 deg) were acquired
to detect short T2 components. The 3D GRE was repeated with
a TE of 6 ms. All images were acquired with FOV 30 x 30 x
30 mm3, and acquired isotropic resolution of 0.35 mm. The 3D
GRE images with TE=6 ms were subtracted from the UTE GRE
images with TE=0.15 ms to suppress tissues with long T2*. On
the subtracted images, intensity of the healthy myocardium was
29
Netherlands
Heart Institute
measured and the mean + 2x SD was considered as UTE target
signal. The UTE target signal area/total myocardial area ratio
was determined on three levels of each heart.
After imaging, hearts were fixed in formalin and embedded in
paraffin. Hearts were cut in slices of 4µm thickness and stained
with 0.1% Picrosirius red to show presence of collagen in the
myocardium. The area of collagen in the myocardium and total
area of the myocardium were determined by using ImageJ
software and the collagen-rich area/total myocardial area ratio
was determined.
In subtracted images of infarcted hearts, UTE signal in
the infarcted area clearly differs from the signal in healthy
myocardium of the same heart, whereas in subtracted images
of sham hearts hardly any difference in signal is detected. The
MRI ratio and the histological ratio of collagen-rich area/total
myocardial area are positively correlated r2=0.83; initial results,
based on three hearts).
Conclusions
We showed that ultrashort TE MRI technology can be used for
the direct detection of post-infarction collagen formation. This
technique to detect fibrosis in a non-invasive way might be of
great value in stratification of patients.
Continuation
A manuscript is being prepared. UTE will be tested for interstitial
fibrosis and for human hearts
Investigators
S. de Jong (CTMM), JJ Zwanenburg (UMCU), F Visser (UMCU),
R van der Nagel (UMCU), HVM van Rijen (UMCU), MA Vos
(UMCU), JMT de Bakker (ICIN, AMC, UMCU), PR Luijten
(UMCU).
Figure 2: Transversal sections of three levels of an infarcted
heart. Panel A: histological data, red=collagen; yellow=
myocardium. Panel B: subtracted MRI images (TE 0.15 ms
– TE 6ms) of corresponding sections. White areas indicate
collagen
RACE 3 Routine vs aggressive rhythm control
Principal investigator: I.C. van Gelder
Project
RACE 3: Routine versus aggressive upstream rhythm control
for prevention of early atrial fibrillation in heart failure
Progression in 2010 in general
In 2010 approximately half of the presently participating
centers were initiated. In total 62 patients are included in the 16
collaborating centers. At present 15 patients have completed
study follow up. The inclusion period lasts until July 2012.
Publication
Editorial on upstream therapy and the relevance of the RACE 3
study. Neth Heart J. 2010;18:522–523. A design article will be
submitted in 2011.
Study design
A randomized, multi-center open label study, comparing two
treatment strategies. 16 centers in The Netherlands participate.
In order to increase the inclusion rate also one British center
(Prof. Greg. Lip) will soon be participating, other British centers
will be recruited. Patients are randomized to upstream rhythm
control or conventional rhythm control.
Study objectives
To investigate whether in patients with early atrial fibrillation and
mild to moderate early heart failure an aggressive upstream
rhythm control approach, including administration of aldosterone
receptor antagonists (ARAs), renin-angiotensin-aldosterone
system (RAAS)-inhibitors, and statins, dietary restrictions,
counseling and cardiac rehabilitation programs, increases
persistence of sinus rhythm compared with conventional
rhythm control.
Steering Committee
Prof. Dr. I.C. Van Gelder (University Medical Center Groningen)
Dr. M. Alings (Amphia Hospital, Breda)
Prof. Dr. H.J.G.M. Crijns (University Hospital Maastricht)
Prof. Dr. G.Y.H. Lip (City Hospital, Birmingham, UK)
Dr. R.Tukkie (Kennemer Gasthuis, Haarlem)
Dr. J. Brügemann (University Medical Center Groningen)
Prof. Dr. J.R.L.M. Smeets (University Medical Center Nijmegen)
Dr. F.F. Willems (Ziekenhuis Rijnstate, Arnhem/Velp)
Prof. Dr. J.L. Hillege (Trial Coordination Center, UMCG)
Prof. Dr. J.G.Tijssen (Academical Medical Center Amsterdam)
Dr. R.G. Tieleman (Martini Hospital Groningen)
Prof. Dr. D.J. Van Veldhuisen (UMCG)
Study Coordination
Drs. M.D. Smit (University Medical Center Groningen)
Drs. R.A. Vermond (University Medical Center Groningen)
Drs. M. Mol (Trial Coordination Center, University Medical
Center Groningen)
Sponsorships:
Netherlands Heart Foundation (NHS B 2008 035), ICIN,
Working group on Cardiovascular Research, the Netherlands,
Sanofi Aventis, Boehringer Ingelheim, Medtronic, Biotronik, St
Jude Medical, Boston Scientific, AstraZenenca
30
ICIN
Netherlands
Heart Institute
Genes underlying sudden cardiac death
Principal investigator: C.R. Bezzina
Project
Genes underlying cardiac electrical activity and susceptibility to
sudden cardiac death
Aim and method of the project
Research within these projects aims to identify genes
underlying cardiac electrical activity and susceptibility to sudden
cardiac death. We undertake genetic studies in families with
monogenic arrhythmia syndromes associated with increased
risk of sudden cardiac death wherein we aim to identify primary
mutations as well as other genetic variation that conspires
with the primary genetic defect in determining the final clinical
presentation. Identification of genetic variants modulating
cardiac electrical activity is also carried out in mice of different
genetic backgrounds. We also focus on genetic mechanisms
underlying the susceptibility to ventricular fibrillation during
acute myocardial infarction, which accounts for a large
proportion of (sudden) deaths on the population.
Progress in 2010
A complex double deletion in LMNA underlies progressive
cardiac conduction disease, atrial arrhythmias and sudden
death
Cardiac conduction disease is a clinically and genetically
heterogeneous disorder characterized by defects in electrical
impulse generation and conduction, and associated with sudden
cardiac death. We have studied a four-generation family with
autosomal dominant progressive cardiac conduction disease,
including atrioventricular conduction block, sinus bradycardia,
atrial arrhythmias and sudden death. Genome-wide linkage
analysis mapped the disease locus to chromosome 1p22-q21,
harbouring the LMNA gene which encodes the nuclear-envelop
protein lamin A/C. Testing of the LMNA gene coding region by
PCR did not uncover any mutation. On the other hand, multiplex
ligation-dependent probe amplification (MLPA) analysis of the
LMNA gene, revealed a novel gene rearrangement involving
a 24-bp inversion flanked by a 3.8-kb deletion upstream and
a 7.8-kb deletion downstream. The presence of short inverted
sequence homologies at the breakpoints junctions suggests a
mutational event involving serial replication slippage in trans
during DNA replication. Our findings indicate that gross genomic
LMNA lesions occur and emphasizes that if conventional PCRbased direct sequencing approaches are negative in suggestive
pedigrees, mutation detection techniques capable of detecting
gross genomic lesions involving deletions and insertions should
be considered. This work was carried out in collaboration with
Prof. L.J. Jordaens (Erasmus MC).
Insight into novel mechanisms modulating susceptibility
to sudden cardiac death in the setting of an acute
myocardial infarction
Sudden cardiac death from ventricular fibrillation (VF) during
myocardial infarction (MI) is a leading cause of total and
cardiovascular mortality. We recently conducted a genome-wide
association study in patients with (cases) and without (controls)
VF during a first MI. The most significant association to VF was
31
found at chromosome 21q21, upstream of the CXADR gene
which encodes the coxsackie and adenovirus receptor (CAR).
CAR is expressed in the intercalated disc of cardiomyocytes
and CAR knock-out mice display decreased expression of
connexin-45 and connexin-43. In order to provide insight into
the involvement of CAR, we are presently investigating it’s role
in ventricular conduction and arrhythmia inducibility at baseline,
in conjunction with reduced intracellular coupling, and under
conditions of myocardial ischemia. This work is being done in
mice haploinsufficient for CAR (CAR+/-).
Quantitative trait loci for electrocardiographic parameters
and arrhythmia in the mouse and rat.
Cardiac arrhythmias associated with sudden death are
influenced by multiple biological pathways and are modulated
by numerous genetic and environmental factors. Elevated
heart rate and prolonged ECG indices of conduction and
repolarization have been associated with risk of sudden death.
Insight into the genetic underpinnings of these parameters thus
provides an important means to the dissection of the genetic
components modulating risk of sudden cardiac death.
In one study we have mapped quantitative trait loci (QTL)
modulating heart rate, ECG indices of conduction and
repolarization, and susceptibility to arrhythmia, in a conduction
disease-sensitized F(2) mouse population. Heart rate,
P-duration, PR-, QRS- and QT-interval were measured at
baseline (n=502) and after flecainide administration (n=370)
in mutant F(2) progeny (F(2)-MUT) resulting from the FVB/NJScn5a1798(insD/+) X 129P2-Scn5a1798(insD/+) mouse cross.
Episodes of sinus arrhythmia and ventricular tachyarrhythmia
occurring post-flecainide were treated as binary traits. F(2)MUT mice were genotyped using a genome-wide 768 single
nucleotide polymorphism (SNP) panel. Interval mapping
uncovered multiple QTL for ECG parameters and arrhythmia. A
sex-interacting scan identified QTL displaying sex-dependency,
and a two-dimensional QTL scan unmasked locus-locus
(epistasis) interactions influencing ECG traits. A number of
QTL coincided at specific chromosomal locations, suggesting
pleiotropic effects at these loci. Through transcript profiling
in myocardium from the parental mouse strains we identified
genes co-localizing at the identified QTL that constitute highly
relevant candidates for the observed effects.
To further complement the studies in the Scn5a1798(insD/+)
mice, we have started an additional project in outbred mice
of known ancestry, which enable highly efficient fine-mapping
of genetic effects (QTLs). In collaboration with Dr. P. Potter at
MRC Harwell (Oxford, UK) and Prof. J. Flint and Dr. J. Nicod
at the Welcome Trust (UK), we are currently involved in a
large-scale phenotyping project of outbred mice, where we
aim to generate ECG and echocardiography data in these
mice. Combining large-scale phenotyping with highly detailed
genotyping in these outbred mice, this unique project will allow
fine-mapping and identification of genetic variants and QTLs for
cardiac electrophysiology, structure and function.
In a third project, being carried out in collaboration with Dr.
Michal Pravenec (Prague), we aim to identify genes and
chromosomal loci modulating sudden arrhythmic death risk
in heart failure (HF). For this, ECG data for conduction and
repolarization (as markers for susceptibility to ventricular
arrhythmias) are generated across 30 recombinant inbred (RI)
rat strains derived from the spontaneously hypertensive rat and
the Brown Norway rat (HXB and BXH RI strains) before and
after spontaneous onset of left ventricular hypertrophy. Through
classical physiological quantitative trait locus (pQTL) analysis
as well as combined expression quantitative trait loci (eQTL)
and quantitative trait transcript (QTT) analyses of the cardiac
transcriptome, genes regulating cardiac electrophysiology are
identified.
The detection of QTL influencing ECG indices and arrhythmia
is an essential step towards identifying genetic networks for
sudden, arrhythmic, cardiac death. Crucially, combining results
obtained from both rat and mouse studies will significantly
strengthen the outcome of these projects. The data generated in
these experiments will provide a unique data set of phenotypic
and genetic markers which will also be available for future
genomic studies.
Funding
This research is funded by the Fondation Leducq (CVD05, ICIN
project 0610), the Netherlands Heart Foundation (2005T024/
ICIN project 06401, 2007B202/ICIN project 06402) and ICIN
(06102).
Staff
Dr. Connie R. Bezzina, Prof. A.A. Wilde (Principal Investigators)
Dr. C.A. Remme (Senior Research Scientist, ICIN)
Dr. J.G.Barc (Post-doc, NHS)
Drs Brendon P. Scicluna (PhD student, NHS)
Drs I.C. Kolder (PhD student, NHS)
Dr. R. Pazoki (PhD student, NHS)
Ms. M. Westerveld (research analyst, NHS)
Ms. R. Wolswinkel (research analyst, ICIN)
Ms. N. Bruinsma (research nurse, NHS)
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Netherlands
Heart Institute
Gene therapy for sick sinus syndrome
Principal investigator: H.L. Tan
Aim and method of the project
Electronic pacemakers have an excellent safety profile;
however, their function is sub-optimal. An important drawback of
electronic pacemakers is the lack of autonomic modulation of the
heart rate. This problem could be circumvented by developing
a biological pacemaker in which working myocardium is
transformed into myocardium with a nodal phenotype capable
of controlling heart rate.
Progress in 2010
In 2010, we primarily conducted large animal experiments
in collaboration with the laboratory of Dr. Michael Rosen at
Columbia University. In this year, we tested various approaches
to optimize HCN2 based biological pacemakers; one of such an
approach is illustrated here.
Introducing the Ca2+-stimulated adenylyl cyclase AC1 into
HCN2 based biological pacemakers enhances their function
Proof-of-concept has been provided regarding biological
pacemakers based on HCN2, but basal and maximal rates are
less than ideal. We previously reported that co-expression of
HCN2 and the Ca2+-stimulated adenylyl cyclase 1 (AC1) in
cultured ventricular myocytes results in elevation of intracellular
cAMP and faster beating rate than HCN2 plus GFP or HCN2
plus AC6. In the present study, we investigated whether this
also occurred in vivo and whether it was entirely explained by
the effect of cAMP on expressed HCN2.
The adenoviral constructs of HCN2 and AC1 or GFP were
implanted into the left bundle branch of AV-blocked dogs to test
in vivo biological pacemaker function as described previously.
To establish HCN2 dependent and AC subtype specific effects
on pacemaker function, we co-expressed HCN2/RE with GFP,
AC1 and AC6 in neonatal rat ventricular myocytes.
In vivo, we found that co-expression of HCN2 plus AC1
compared to HCN2 plus GFP results in faster basal rates which
could be increased still further by epinephrine (155% increase
at 1 μg/kg/min) and faster maximal rates (fig. 1). Hence, the
prior demonstration of in vitro efficacy was replicated in vivo.
In cell culture, replacing HCN2 with HCN2/RE - to eliminate
cAMP binding to the expressed channel - did not prevent AC1
from enhancing rate compared to AC6 or GFP controls (HCN2/
RE+AC1 210±22 bpm, P<.05 vs HCN2/RE+GFP, 160±15 and
HCN2/RE+AC6, 125±22 bpm). The increased rate in the AC1
group occurred with no effect on HCN2/RE current indicating
that the outcome at least in part reflects actions on endogenous
targets of cAMP.
We therefore concluded that co-expressing AC1 with HCN2
enhances both HCN2 and other -most likely Ca2+ based
- pacemaker mechanisms, while maintaining intact betaadrenergic responsiveness. Hence, AC1 co-expression may
provide a useful strategy to enhance HCN2 based biological
pacemakers.
33
Figure 1. Basal beating rates, maximal beating rates,
dependence on electronic back-up pacing and demonstration
of gene transfer. A, Basal pace-mapped beating rates, as
recorded upon daily check-up on day 5-7, were significantly
faster in HCN2/AC1 injected animals as compared to HCN2/
GFP (P<0.05). Maximal pace-mapped beating rates, as
recorded using 24hr Holter, were also faster in HCN2/AC1 as
compared to HCN2/GFP (P<0.05). As a result of these faster
beating rates, the dependence on electronic back-up pacing
was reduced in HCN2/AC1 injected animals as compared to
HCN2/GFP (P<0.05). B, Immunohistochemistry in tissue slabs
from a HCN2/AC1 injected animal. In the upper panel, the
injected region is positive for HCN2 (green) and AC1 (red).
Nuclei are counterstained using DAPI. In the lower panel, noninjected region is negative for HCN2 and SkM1. Scale bare
represents 100 µm.
Partnerships
Large animal studies are conducted in collaboration with
Columbia University, New York NY, USA (Dr. M.R. Rosen) as
part of the ICIN fellowship (awarded to GJJB).
Studies employing the use of cardiac myocyte progenitor cells
are conducted in collaboration with Utrecht University (Dr. J.P.
Sluijter) and Leiden University (Dr. M.J. Goumans).
Funding
This project is supported by the Netherlands Heart Foundation,
the NFCVE and ICIN (fellowship).
Staff
GJJ Boink (ICIN), HL Tan (AMC), A.D. den Haan (AMC), AO
Verkerk (AMC), Diane Bakker (AMC), SCM van Amersfoorth
(ICIN), AAB van Veen (UMCU), J Seppen (UvA), JMT de
Bakker (ICIN).
Arrest 11: Determinants of sudden cardiac death
Principal investigator: H.L. Tan
Project
ARREST11: a prospective population-based study into clinical,
genetic and pharmacological determinants of sudden cardiac
arrest
Aim
To establish clinical, genetic and pharmacological determinants
of sudden cardiac arresr (SCA)
Method
Prospective inclusion of all patients with out-of-hospital SCA with
ECG-documented ventricular tachycardia/fibrillation (VT/VF)
(cases) in a contiguous region of The Netherlands (population
2.5 million) Collection of resuscitation details (including ECG),
past and present medical history, medication use prior to SCA,
and DNA samples. Medical history, medication use, and genetic
variations of cases are compared to controls (case-control
design) by estimating odds ratios using conditional logistic
regression, with/without adjustment for prognostic factors of VT/
VF (e.g., ischemia, heart failure).
Progress in 2010
In the third year of this project, we have expanded the data
collection infrastructure to cover the province North Holland
completely, and continue to expand the region of data inclusion
to Utrecht province. At present, we have identified 3695 patients
with VT/VF, of whom we are collecting medical information and
medication use. Of 1700 of these patients, we have collected
DNA samples. We have established collaborations with various
other investigators, for collaborative studies (CAREFUL, IPCI,
Oregon-SUDS, SEIN), replication studies (AGNES), and to draw
suitable control patients (AMC-HAG-net). This has resulted in
publication 1 (with AGNES study, listed below) and a paper in
press in J Am Coll Cardiol (with CAREFUL study). In this study,
we have comprehensive investigated the incidence, causes,
and neurologic outcome of SCA in children. We found that SCA
accounts for a significant proportion of pediatric mortality, and
cardiac causes are the most prevalent causes of SCA. The vast
majority of SCA survivors have a neurologically-intact outcome
(Figure 1).
Figure 1. Causes of SCA in children (<21 years). Left group of
bars represent natural causes, right group non-natural causes.
All causes are shown as the percentage of the total number
of out-of-hospital cardiac arrest cases. Numbers above bars
indicate numbers of patients.
Partnerships
1.Prof. Dr. A. de Boer, Dept. of Pharmacoepidemiology &
Pharmacotherapy, Utrecht University, Utrecht
2. Prof. Dr. M.C.J.M. Sturkenboom, Depts. of Epidemiology
& Biostatistics and Medical Informatics, Erasmus Medical
Center, Rotterdam.
3.Prof. Dr. A.A.M. Wilde, Dept. Of Cardiology, Academic
Medical Center, Amsterdam
4. Prof. Dr. I.M. van Langen, Dept. of Genetics, University of
Groningen, Groningen
Funding
This study is supported by The Netherlands Organization for
Scientific Research (NWO Mozaiek 017.003.084 to A. Bardai,
and ZonMW Vici 918.86.616 to H.L. Tan) and the Dutch
Medicines Evaluation Board (MEB/CBG).
Staff
A. Bardai, MD (ICIN)
P. Homma, BSc (ICIN)
M.T. Blom, MA (AMC)
H.L. Tan, MD, PhD (AMC)
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Netherlands
Heart Institute
Activated platelets during acute mycardial infarction
Principal investigator: H.L. Tan
Project
The role of activated platelets in the occurrence of ventricular
fibrillation during acute myocardial infarction.
Aim and method of the project
Sudden cardiac death is mainly caused by ventricular fibrillation
(VF) in the setting of acute ischemia resulting from coronary
thrombi. Animal experiments have shown that platelet activation
may increase susceptibility of ischemic myocardium to VF, but
the mechanism is unknown. In the present study, we evaluated
the effects of activated blood platelet products (ABPPs) on
electrophysiological properties and intracellular Ca2+ (Ca2+i)
homeostasis.
Platelets were collected from healthy volunteers. After
activation, their secreted ABPPs were added to superfusion
solutions. Rabbit ventricular myocytes were freshly isolated, and
membrane potentials and Ca2+i were recorded using patchclamp methodology and indo-1 fluorescence measurements,
respectively.
Progress in 2010
ABPPs prolonged action potential duration and induced early
and delayed afterdepolarizations. ABPPs increased L-type
Ca2+ current (ICa,L) density, but left densities of sodium current,
inward rectifier K+ current, transient outward K+ current, and
rapid component of the delayed rectifier K+ current unchanged.
ABPPs did not affect kinetics or (in)activation properties of
membrane currents.
In the second year of this project, we have completed the
data from intracellular Ca2+ measurements. Figure 1A shows
typical Ca2+i traces of a myocyte stimulated at 0.2-Hz in control
conditions (black line) and in the presence of ABPPs (red line).
Figure 1B summarizes the average diastolic and systolic Ca2+i
concentrations, Ca2+i transient amplitudes, systolic Ca2+i
rise, and time constant of the Ca2+i transient decay in control
conditions and in the presence of ABPPs. ABPPs significantly
increased the systolic Ca2+i concentration, the Ca2+i transient
amplitude, and the systolic Ca2+i rise, while the Ca2+i transient
decay significantly decreased. Figure 1C demonstrate increased
sarcoplasmic reticulum Ca2+ content (SR Ca2+). The increase
in systolic Ca2+i occurred within 3 min of superfusion with
ABPPs, and dissipated after 4 min washout. Trypsin-treated
ABPPs still increased APD90 significantly. However, while
untreated ABPP induced AP prolongation of 12%, trypsin-treated
ABPPs prolonged APs only by 4% (n=7), suggesting that AP
prolongation is partially mediated by peptides. Likewise, trypsin
treatment blunted the effect of ABPPs on Ca2+i transients
(Fig. 2). ABPPs did not affect the Na+- Ca2+ exchange current
(INCX) in Ca2+-buffered conditions, but the increase in Ca2+i
transient amplitude suggests an increase in functional INCX.
Publication of the data is expected in 2011.
Figure 2
Partnerships
Dr. R. Nieuwland, Laboratory of Experimental Clinical Chemistry,
Academic Medical Center, Amsterdam
Funding
Netherlands Heart Foundation (NHS2007B020)
Figure 1
35
Staff
Olga Zakhrabova,MD, PhD (ICIN)
Arie O Verkerk, PhD (AMC)
Jonas de Jong, MD (AMC)
Rienk Nieuwland, PhD (AMC)
Hanno L Tan, MD, PhD (AMC)
Prevention of atrial fibrillation
Principal investigator: I.C. van Gelder
Project
Prevention of Atrial Fibrillation: Markers for Stretch and Pacing
Induced Structural Remodeling and the Preventive Efficacy of
Upstream Therapy
Background
Atrial fibrillation (AF) is the most common cardiac arrhythmia,
having a prevalence of 1% in young patients up to 9% in patients
at the age of 75 years. Risk factors for AF include hypertension,
heart failure and mitral valve disease, which all are complicated
by hemodynamic overload of the ventricles and the atria.
Pressure or volume overload of the atria causes elongation of the
cardiomyocytes, i.e. increased stretch. AF promoting changes,
atrial remodeling, occur in atria from patients with heart failure,
hypertension and mitral valve disease, before the first episode
of AF, as such creating a substrate for AF. Stretch is thought
to be an important mediator in causing the structural changes
seen before onset of atrial fibrillation, caused by underlying
diseases. Models to more specifically investigate the effects of
atrial stretch are cell stretching in vitro on flexible membranes
and ex vivo using isolated hearts with increased pressure or
volume. In vivo models include transverse aorta constriction
and models of mitral valve disease and hypertension. We aim
to establish a cell culture model in which early remodeling in
pressure overload of the atria is mimicked. This represents the
atrial situation after start of hypertension or heart failure, i.e. the
underlying disease, but before start of AF. In addition, we aim to
investigate the effects in vivo pressure overload via transverse
aortic constriction on atrial remodeling.
In vitro - stretch
Cells were isolated, cultured and set under cyclical stretch on
elastic membranes using the Flexercell-4000 system. We started
with HL-1 atrial cardiomyocytes, the only atrial cardiomycoyte
cell line available. Now we use primary neonatal rat atrial and
ventricular cardiomyocytes (NRAM and NRVM). Cells are
analyzed for induction of the fetal gene program, induction of
stress makers and breakdown of contractile elements as seen
in myolysis.
Figure 1. Primary neonatal rat atrial (1A) and ventricular
(1B) myocytes and fibroblasts were stained for a-actinin with
specific antibodies (green) and total actin was stained with
Texas red-phalloidin. Cardiac myocytes stain both red and
green, whereas fibroblasts stain red for actin exclusively.
Nuclei are stained blue with DAPI.
Aim: Our goal is to develop a cell culture model mimicking early
atrial remodeling in pressure overload of the atria, representing
the atrial situation after start of hypertension or heart failure.
Methods: Neonatal rat atrial cardiomyocytes (NRAM) were
cultured and set under cyclical stretch on elastic membranes.
Cells were analyzed after 3-24hr of stretch. mRNA levels of alpha
and beta-myosin heavy chain (MHC) and skeletal alpha-actin
(ACTA) were used as markers of dedifferentiation. Breakdown
of troponin T was used as a measure of myolysis. To investigate
the involvement of calcineurin we measured myocytes enriched
calcineurin interacting protein (MCIP1) mRNA expression and
used cyclosporine A to inhibit calcineurin.
Results: Stretching with 1Hz and 15% elongation for 24hr
showed 1.8-fold increased beta/alpha-MHC ratio. In addition,
skeletal alpha-actin expression was 1.5 fold increased after 6hr
of stretch. Stretching with 1Hz and 15% elongation or 3Hz and
10% elongation did not reduce troponin T protein expression.
Thus, these stretch regimens show no evidence for myolysis,
but dedifferentiaton was suggested. ANP mRNA levels are 1.5fold increased and ANP is released in the medium. BNP and
GDF-15 mRNA levels are 2-fold increased after 3hr and 1.4-fold
after 24hr, respectively. Involvement of calcineurin is suggested
by 2.2-fold increased mRNA expression of MCIP1. Furthermore
stretch induced effects on mRNA level are attenuated with
cyclosporine A. In addition, we investigated the effects of stretch
on ventricular cardiomyocytes (NRVM), the effects are more
pronounced in ventricles.
Conclusions: Stretch of neonatal rat atrial cardiomyocytes
results in cellular changes which may resemble those seen
before and in the early course of AF such as dedifferentiation
and changes in expression of natriuretic peptides. Calcineurin
seems to be involved in the changes caused by stretch.
Figure 2: Comparison of mRNA expression of atrial (ANP)
and brain-type (BNP) natriuretic peptides in primary neonatal
rat atrial (NRAM) and ventricular (NRVM) myocytes. Cells
were also treated with 20 μM phenylephrine (PE) to induce
hypertrophy which leads to increases in both ANP and BNP in
NRVM only to levels comparable with NRAM at baseline.
36
ICIN
In vitro – pacing
Aim: In addition, we investigated the effects of pacing, as a
model of AF, on NRAM and NRVM.
Methods: Neonatal rat atrial and ventricular cardiomyocytes
were paced with 1Hz and 3Hz for 24hr. Cells were analyzed for
changes in mRNA levels of ANP, BNP and GDF15 and markers
of dedifferentiation: skeletal alpha-actin and beta/alpha-myosin
heavy chain. Expression of myocytes enriched calcineurin
interacting protein was measured as a marker of calcineurin
activity.
Results: In NRVM 1Hz and 3Hz pacing caused increased
expression of ANP (2.9-fold; p < 0.01 and 2.2-fold; p < 0.05),
BNP (3.5-fold; p = 0.01 and 2.3-fold; n.s.) and GDF15 (3.1fold; p < 0.01 and 4.3-fold; p < 0.001). Expression of ACTA
and beta/alpha-MHC was increased with 1Hz and 3Hz pacing
respectively. In NRAM pacing did not change expression of
ANP and beta/alpha-MHC. Levels of BNP and ACTA were 30%
and 39% decreased upon 3Hz pacing. Expression of GDF15
(2.4-fold; p < 0.01 and 3.6-fold; p < 0.001) and MCIP (1.9 and
2.0-fold; p < 0.05) was increased.
Conclusion: In conclusion, pacing causes a pathological
response in NRVM only. These differences are important to take
into account when targeting remodeling.
Future directions: We will investigate the effects of 5Hz pacing
on NRAM and NRVM.
In vivo
Aim: Our goal was to investigate the effects of pressure overload
of the left ventricle by transverse aortic constriction (TAC) and
subsequent increase in left atrial pressure on atrial remodeling.
Methods: Male mice were subjected to TAC or sham for 4 weeks
with and without the angiotensin II type 1 receptor blocker,
losartan (30 mg/kg/day), n = 6 for all groups, except TAC control,
n = 4. Atria were harvested and assayed with real time RT-PCR
for expression of ANP, BNP, skeletal alpha-actin, collagen type I
alpha 1 (Col1a1) and myocytes enriched calcineurin interacting
protein.
Results: Four weeks of TAC caused ventricular and atrial
hypertrophy as shown by increased left ventricular and atrial
weight. A reduced cardiac function was shown by reduced
ejection fraction, contractility and relaxation. In addition an
increased LVEDP was found. Increased mRNA expression of
1.4-fold of ANP, 2.6-fold of BNP and ACTA suggested atrial
stress, dedifferentiation and hypertrophy. Increased fibrosis is
suggested by increased expression of Col1a1 and increased
activity of calcineurin by increased MCIP1 expression. The
latter could be a possible mechanism governing these effects.
TAC in combination with losartan diminished the effect of TAC
on BNP, as well as on ACTA and MCIP1 mRNA expression,
but not on ANP mRNA expression. Treatment with slightly
improved hemodynamic parameters compared to TAC.
Losartan diminished the effect of TAC on BNP, as well as on
ACTA and MCIP1 atrial mRNA expression, but not on ANP
mRNA expression.
Conclusions: These data show that pressure overload of the
37
Netherlands
Heart Institute
Figure 3: Flow chart showing the series of events caused
by stretch. Hypothetical scheme of stretch induced by
hypertension, heart failure and possibly extreme endurance
exercise leading to calcium overload, activation of the renin–
angiotensin–aldosterone system (RAAS) and release of
different factors, resulting in structural remodelling and finally
in AF.
ventricles induces atrial remodeling, which may be a substrate
for atrial fibrillation. Atrial hypertrophy, fibrosis and increased
activity of calcineurin are suggested. Concomitant treatment
with losartan prevented these changes, suggesting protective
effects of losartan in atrial remodeling due to LV pressure
overload.
Future directions: 3 animals will be added to the TAC group
and histological analysis on hypertrophy and fibrosis will
be performed. In addition, atria from other models will be
investigated as well as other known pharmaceuticals.
Summary
Stretch of atrial cardiomyocytes results in cellular changes which
may resemble those seen before AF such as dedifferentiation
and changes in expression of natriuretic peptides. Interestingly,
the response of atrial and ventricular cardiomyocytes is different
upon pacing and mechanical stretch. Also in vivo pressure
overload leads to changes in the atria, which potentially make
the atria more prone to develop AF.
Researchers:
Anne-Margreet R. de Jong, graduated in 2007, master Medical
Pharmaceutical Sciences at the University of Groningen
(employed by ICIN/UMCG).
Dr. Alexander H. Maass, cardiologist
Silke Oberdorf-Maass, technical assistant
Prof. Dr. Isabelle C. Van Gelder, cardiologist, group leader
Thoraxcenter, Department of Cardiology and Experimental
Cardiology, University Medical Center Groningen, University of
Groningen and Interuniversity Cardiology Institute Netherlands,
Utrecht, The Netherlands.
ICIN
Netherlands
Heart Institute
Research line - Congenital heart disease
Congenital Heart Disease in Adults
Principal investigator: B.J.M. Mulder
Introduction
Adults with a congenital heart defect comprise a relatively
new and young population, because cardiac surgery became
available only in the 1970’s. Long term outcome and underlying
genetic causes are investigated in CONCOR, the national
registry and DNA-bank for patients with CONgential CORvita..
Since its initiation in 2001 nearly 13.000 patients have been
included. Most frequent main diagnoses are depicted in figure
1. In 2010 drs M.J.Schuuring coordinated the project as ICIN
investigator. Dr ET van der Velde had to withdraw as ICT
consultant.and has been replaced by drs. Maurice Langemeijer.
Lia Engelfriet, Irene Harms and Sylvia Mantels were travelling
along the ICIN centers and non-university hospitals as research
nurses.
To increase awareness and retrieve patients with congenital
heart disease that were lost to follow-up a nationwide media
campaign entitled: “Wanted! 8000 Heart Patients” was started in
2009. (figure 2). Until the end of 2010 applications were retrieved
from over 1000 subjects with CHD, of whom 30 patients had
to undergo an intervention within due time. Our successful
campaign will be continued for the coming years.
CONCOR ( www.concor.net) has an important spinoff with 40
scientific projects using its registry and/or DNA bank; 66 articles
have been published in international peer reviewed journals and
11 PhD students have finished their thesis using CONCOR.
In 2010, 12 studies using CONCOR were coordinated from
the AMC: 10 studies were running on long term outcome of
congenital heart disease (Quality of Life, Future and prognosis,
Eisenmenger syndrome, Marfan syndrome, Transposition of
the great arteries, Tetralogy of Fallot, Pulmonary atresia, Aortic
Coarctation, Arrhythmias in congenital heart disease and Down
Syndrome) and 2 studies on the genetic basis of congenital heart
disease (Zebrafish and genes for heart development,and Euro
Heart Repair). In 2010, 34 articles have been published and 1
thesis was completed. The core staff comprised dr.B.J.Bouma,
dr.M.Groenink and prof B.J.M.Mulder.
Drs. M.M.Winter defended his thesis entitled: ”The systemic
right ventricle”, November 26th in Amsterdam (supervisors Prof
BJM Mulder, Dr BJ Bouma ).
Angiontensin II receptor blockers
Drs. T.van der Bom continued the project on the efficacy of
angiontensin II receptor blockers in patients with a systemic
RV. These patients have a morphologic right ventricle
(RV) supporting the systemic circulation (e.g. patients with
congenitally corrected transposition of the great arteries
(ccTGA) and patients with complete transposition of the great
arteries (TGA) after an atrial switch operation). Complications
early in life are common in these patients, with dysfunction
of the systemic RV being the most important contributor to
morbidity and mortality. Unfortunately, little is known on the
pathophysiology of commonly seen complications and evidence
based treatment regimens are lacking. In patients with left
ventricular dysfunction, Angiotensin II receptor blockers (ARBs)
and Angiotensin Converting Enzyme (ACE) inhibitors reduce
both morbidity and mortality. In theory, it is highly likely that
patients with a systemic RV would benefit equally from these
medications.
Figure 1: Most frequent main diagnoses of 12.800 adult patients with a congenital heart defect in CONCOR.
40
ICIN
Progress
Starting December 2010, exclusion investigations are being
conducted in all participating centers. At present 16 patients
have been excluded after end-of-study investigations. The
study design of the abovementioned study has been published
in the American Heart Journal. The last patient visit is expected
in the summer of 2011, after which the blinding will be lifted and
the results will be analysed.
In a separate though related retrospective study, the ECGs
of patients with a systemic right ventricle were analyzed. As
arrhythmia is the most frequent complication in patients with a
systemic right ventricle after ventricular failure, we are interested
whether certain characteristics in the regular 12 lead ECG will
identify those patients that are at risk. Results are pending.
Moreover, drs. T. van der Bom has published two reviews. One
concerns the epidemiology of congenital heart disease and was
published in Nature Reviews Cardiology. Another concerns risk
reducing measures in adult congenital heart patients.
Partnerships
The multicentre trial “Valsartan in patients with a systemic right
ventricle” is performed in collaboration with the AMC, LUMC,
UMCG, UMCN, Erasmus MC, VUMC and UMCU.
Funding
This project is funded by an unrestricted educational grant from
Novartis.
Figure 2: MR image of a 26-year old male with atrial correction
of transposition of the great arteries, showing the aorta anterior
to the pulmonary artery and arising from the right ventricle. AO
= aorta; AV = aortic valve; LV = left ventricle; PA = pulmonary
valve; PV = pulmonary valve; RV = right ventricle
41
Netherlands
Heart Institute
Effect of high dose statins on the IMT
Drs P Luijendijk continued his project on “the effect of high dose
statins on the intima-media thickness (IMT), a reproducible
endpoint for atherosclerosis, in adult post-coarctectomy
patients”.
Aortic coarctation is a congenital cardiovascular malformation
comprising a circumscript narrowing of the aortic lumen usually
located distally of the left subclavian artery, at the site where
the ductus arteriosus enters the aorta. Coarctation of the aorta
(CoA) is a common malformation, accounting for 6 to 8 percent
of all congenital heart defects. In the Netherlands, more than 700
adult patients are registered in the national registry for patients
with CONgenital CORvitia (CONCOR).Studies have shown
decreased survival rates in patients after surgical correction
of CoA, post coarctectomy patients. The most common cause
of this premature death is coronary artery disease, followed
by sudden death, heart failure, cerebrovascular accident and
ruptured aortic aneurysm.
Progress:
P. Luijendijk continued the inclusion for this multi- centre trial
and reached the intended number of 150 patients necessary
for this study. Furthermore up to 80 adult post-coarctectomy
patients that were screened during an observational study in
2001 have been included in a longitudinal follow up study.
Awaiting the results of this prospective multicentre trial,
P.Luijendijk analysed retrospective data of 80 adult post
coarctectomy patients who underwent serial Magnetic
Resonance Imaging between 2001 and 2008, to evaluate the
presence and progression of enlargement of the thoracic aorta.
This study, for the first time quantitatively, demonstrates an
increased progression rate of ascending aortic dilatation in adult
post-coarctectomy patients with 2.2 mm/5 years. The increased
progression rate of ascending aortic dilatation in CoA patients
was associated with a history of VSD and the presence of an
increased left ventricular mass index. It is known that aortic
coarctation (CoA) occurs as a “simple” isolated disorder, and
in a more “complex” form, combined with associated congenital
cardiac abnormalities. The progression rate of ascending aortic
dilatation is increased in complex CoA patients, as compared
to simple CoA patients. These findings point towards a more
comprehensive genetic subset of patients with an increased risk
for progressive ascending aortic dilatation.
Atherosclerosis progression was studied in 80 adult postcoarctectomy patients who were previously studied in an
observational study in our institution in 2001. Intima-media
thickness (IMT) is nowadays considered a validated and
reproducible endpoint for atherosclerosis. In a previous report
from our institution it was shown that the IMT was significantly
increased both in normotensive and in hypertensive postcoarctectomy patients as compared to healthy controls.
The current study demonstrated that IMT progression was
increased in CoA, which suggests that CoA patients suffer from
accelerated atherosclerosis. These findings give more insight in
the pathofysiology of the process of atherosclerosic disease in
these patients. (Presented at ACC Atlanta and ESC Stockholm
2010)
Another study concerned the predictive value of exercise induced
hypertension for the development of chronic hypertension in
CoA patients. These findings are of major clinical importance
as chronic hypertension is a major concern in adult postcoarctectomy patients (CoA). These findings demonstrated
that maximal exercise systolic blood pressure is a predictor for
chronic hypertension in CoA patients. These findings indicate
that the presence of exercise induced hypertension is of clinical
significance, and might point towards early hypertension
treatment in these patients, who already have a high burden of
cardiovascular disease.
Partnerships
The multicentre trial “High dose statins in post-coarctectomy
patients is performed in collaboration with AMC, LUMC, UMCN,
UMCU, VUMC, UMCG.
Funding
Pfizer B.V. is funding this study with an unrestricted educational
grant.
Long-term prognosis of adult patients with CHD
Drs. C Zomer continued her project research on the long-term
prognosis of adult patients with CHD, with the CONCOR registry
as a backbone of her studies.
An increasing number of patients with congenital heart disease
(CHD) reach adulthood due to improved developments in
paediatric cardiology, cardiac surgery and thorough monitoring.
However, little is known about long-term complications and longterm prognosis, since this is a relatively ‘new’ patient group.
Progress
Drs. C. Zomer initiated a study to examine the impact of a
congenital heart defect on social life and lifestyle in adults.
Nearly 2000 patients received an invitation to participate in a
web-based questionnaire, and eventually 1496 patients filled
out the questionnaire (response=76%). Reference data were
available for all outcomes. Main findings were a higher risk
of low educational attainment, unemployment, low income
and having no relationship for patients with a congenital heart
defect compared to the reference group. The risk was strongly
correlated with the severity of defect. Adults with CHD did seem
to have a healthier lifestyle than the reference group. (Presented
at the NVVC, ESC and AHA 2010).
Together with drs T van der Bom she wrote a review on the
changing epidemiology of congenital heart disease together with
a colleague .This review gives an overview of the etiology, birth
prevalence, current prevalence, mortality, and complications
of congenital heart disease. (Published jan 2011 in Nat. Rev.
Cardiol.).
Finally, C. Zomer spent the last few months of 2010 studying
heart failure in adults with CHD. Survival of patients with CHD
has increased dramatically. However, this goes together with
the development of long-term complications, including heart
failure (HF). Our aim was to assess outcome of CHD patients
who were hospitalised for HF, and to predict which patients are
most at risk for being hospitalised for HF. The mortality data
used in this study were obtained from CONCOR and by linking
the National Mortality Registry to the CONCOR registry. The
hospitalisation data were obtained by linkage to the Dutch
Medical Registration. We found that 221 out of 10,816 adult
patients (2.0%) were admitted for HF during a mean follow up
period of 21 years per patient. The cumulative observed risk
of re-admission for HF was 23% after one year. Corrected for
gender and defect, patients admitted for HF had 5 times higher
risk of death compared to the overall CONCOR population.
Patients with highest risk of fatal outcome had single ventricles,
pulmonary atresia with ventricle septum defect, and transposition
of the great arteries. (Will be presented at ACC in April 2011).
Figure 3: Magnetic resonance image of a dilated ascending
aorta( arrow) in a patient with previous correction of aortic
coarctation.
Predictors for HF-admission were the congenital heart defect,
multiple defects in one patient, palliative surgery in childhood
and pacemaker implantation in childhood. (Presented at NVVC
2010)
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Alongside her research, C. Zomer started a Master’s programme
Epidemiology in September 2009. In 2010 she continued this
program and it is expected she will graduate in summer of 2011.
Partnerships
This PhD project is mediated by collaboration with AMC and the
Julius Center (UMCU).
Netherlands
Heart Institute
The role of Bosentan in Fontan patients
Drs. M.J. Schuuring continued the project “the role of bosentan
in Fontan patients: improvement of exercise capacity?” started
in 2009 by drs. J.C. Vis. The main goal of this prospective
multi-centre study was to determine whether bosentan, an
endothelin-1 receptor antagonist, improves maximum exercise
capacity in Fontan patients by reducing the pulmonary vascular
resistance. This multicentre trial was performed in collaboration
with AMC, LUMC, UMCN, UMCU and UMCG.
Furthermore, Mark Schuuring continued the registry of adult
patients with pulmonary arterial hypertension started by dr.
M.G. Duffels. Pulmonary arterial hypertension (PAH) associated
with congenital heart disease due to systemic-to-pulmonary
shunting is associated with a high risk of morbidity and mortality.
Seventy patients with CHD and PAH were treated with bosentan
and evaluated with laboratory tests, 6-minute walk test, quality
of life questionnaires, and Doppler echocardiography. In 2010,
a prolonged beneficial effect of bosentan treatment for these
patients was demontsrated.
Figure 4. Survival curves of adults with CHD who were
admitted ( green line) and who were not admitted for heart
failure( blue line)
Impaired cardiac reserve
Drs. S Romeih continued her study on “Impaired cardiac reserve
in asymptomatic patients with residual moderate pulmonary
stenosis after pulmonary valvulotomy: evidence for diastolic
dysfunction”.Residual pulmonary stenosis (PS) is still a common
finding after pulmonary valvulotomy. The clinical outcome in
patients with residual moderate PS has been underdocumented.
The response to physical and pharmacological stress was
compared between moderate PS patients with and without
previous pulmonary valvulotomy. All patients underwent a
cardiopulmonary exercise test, dobutamine stress MRI, and
delayed contrast enhanced MRI to detect myocardial fibrosis.
RV stroke volume increased only in patients without previous
intervention.VO2max and O2-pulse during physical stress were
strongly related to RV-SV with dobutamine. Exercise capacity
was limited in patients with residual moderate PS after previous
pulmonary valvulotomy. This is caused by inability to increase
stroke volume during exercise, probably by disturbed RV filling
properties.
This study is a collaboration between pediatric ( Prof N.A.Blom)
and adult cardiology in LUMC (dr H.W.Vliegen) Leiden and
AMC Amsterdam.
Drs S Romeih finished her MRI studies early 2010 and will finish
her thesis in 2011.
43
A new multicenter retrospective study has been started named
“congenital heart disease patients with segmental pulmonary
arterial hypertension benefit from bosentan treatment”. This
multicentre trial is performed in collaboration with Royal
Brompton Hospital (London), Royal Prince Alfred Hospital
(Sydney) and Childrens Hospital (Boston). Some patients
develop a heterogeneous pulmonary pressure distribution
(segmental PAH). Aim of this study is to investigate efficacy of
bosentan in these patients.
Figure 5: (A) Schematic drawing of major aortic-pulmonary
collateral arteries (MAPCA) stenosis; (B) Magnetic resonance
angiography showing inferior MAPCA stenosis.
In a retrospective study it was demonstrated that right ventricular
function declined post-operatively in all CHD patients undergoing
cardiac surgery .Therefore, a new prospective study has been
initiated to investigate the effect of peri-operative bosentan in
these patients. In this prospective randomized open label with
blinded end-points study right ventricular function will be studied
in patients with and without peri-operative bosentan.
Partnerships
The study is a collaboration between UMCG, LUMC, UMCN
and AMC.
Funding
Actelion B.V. is funding this study with an unrestricted
educational grant.
Figure 6: Inappropriate shock after atrial fibrillation in a
57-years old man with reoaired VSD and ICD for secondary
prevention of SCD.
Arrhythmias in adults with CHD
Drs Z. Koyak started her ICIN project on arrhythmias in adults
with CHD in November 2009. Despite the tremendous progress
in cardiac surgery, diminished long term survival remains a
major concern in adults with congenital heart disease (CHD).
In this population sudden cardiac death (SCD) is the most
common cause of late mortality. To date, no single risk factor has
convincingly been identified to predict ventricular arrhythmias or
sudden death in this population. Internal Cardiac Defibrillator
(ICD) therapy has been proven to be effective in primary
and secondary prevention of SCD in adults with ischemic or
dilated heart disease and an impaired left ventricular function.
Although the number of patients with CHD that receive an ICD
is steadily increasing risk stratification and the indication for ICD
implantation for primary prevention are poorly defined in this
group. Moreover, the effects of ICD therapy during long term
follow-up are still unknown.
The aim of her first study on arrhythmias is to identify risk
factors for SCD in adults with CHD. This is a retrospective, multi
center case controlled study. The aim of her second study is to
investigate the long term outcome of ICD therapy in prevention
of SCD in adults with CHD.
Progress
Z. Koyak included 171 sudden cardiac death cases and 200
control patients in her study on predictors for SCD and the
inclusion is still ongoing. The first results of this study have been
presented at the ESC in 2009. The final results are expected in
May 2011.
Meanwhile she finished her second study on arrhythmias. The
goal of this study was to investigate the long term outcome of
ICD therapy in adults with CHD. There are 136 patients included
in this study. The results of this study will be presented at the
ACC in April 2011.
Partnerships
The multicentre study “Predictors of Sudden Cardiac Death
in Adults with Congenital Heart Disease’’ is performed in
collaboration with AMC, UMCG, University Hospitals Leuven
(Belgium) and Toronto General Hospital (Canada).
The multicentre study ‘’Long Term Results of Implantable
Cardioverter Defibrillators in Adults with Congenital Heart
Disease’’ is performed in collaboration with AMC, UMCG and
LUMC.
Figure 7: Appropriate shock after ventricular fibrillation in a 23
years old man with repaired bicuspid aortc valve and an ICD
for secondary prevention of SCD.
Predicting health care use and perceived health care
needs in patients with congenital heart disease
Drs. D. Schoormans continued her project “Predicting health
care use and perceived health care needs in patients with
congenital heart disease”. To deliver adequate and tailored
care to those who need it, we must identify characteristics of
patients with congenital heart disease that predict health care
use and perceived need of health care. Interventions targeted at
modifying influential factors (e.g., reducing anxiety) may lower
unnecessary health-care consumption and improve quality of
life. The results of this study will thus enable a more appropriate
and cost-effective allocation of resources.
Progress
In 2010 drs. D. Schoormans analyzed the psychosocial data
of 1109 patients. Two manuscripts were drafted and submitted.
The first manuscript is entitled: The perspective of patients with
congenital heart disease: does health care meet their needs?
The objective of this study is to examine patients’ health careuse and needs and patients’ perspective on the delivered
care. Results show that for most patients both the amount and
the quality of care is sufficient. Patients in need for additional
contact, rated the communication skills of cardiologists as
insufficient. Therefore, in addition to recommended training
programs described by the ACC/AHA and ESC guidelines, we
recommend the incorporation of communication training for
cardiologists.
The second manuscript is titled: Patients with a congenital heart
defect and Type D personality feel functionally more impaired,
worry more, but use less health care. This study examines
whether personality type, clinical factors and quality of life are
associated with health-care utilization. Type D personality is
characterized by high scores on both negative affect and social
inhibition. Patients with versus without a Type D personality are
labeled “Type D patients” and “non-Type D patients”. Type D
patients report a poorer functional status and more worries, but
less health-care utilization In clinical practice patients should be
screened for Type D personality, since social inhibition, present
in Type D patients, may prevent them to contact a health-care
provider in case of need.
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Additionally, in line with research on satisfaction with care
among recently operated patients data collection has been
continued and is currently being analyzed.
Partnerships
This study is a collaboration between AMC, UMCG, LUMC,
UMCU, UMC St Radboud, TweeSteden hospital in Tilburg, and
Orbis Medical Centre in Sittard-Geleen.
COMPARE study: COzaar in Marfan PAtients
Reduces aortic Enlargement
Drs. T. Radonic en drs.P.de Wittte continued their COMPARE
study: COzaar in Marfan PAtients Reduces aortic Enlargement.
Marfan syndrome (MFS) is one of the most common systemic
disorders of connective tissue with the incidence of 2-3 per
10,000 individuals. Aortic disease, leading to progressive
aneurismal dilatation and dissection is the main cause of
morbidity and mortality of Marfan patients. Studies in the mouse
model of MFS have found losartan to prevent formation of aortic
aneurysm due to its antagonism of Tissue Growth Factor beta
Main objective of this ICIN study is to investigate the effect of
losartan on the rate of aortic dilatation. The study is desiged as
a multi-centre, prospective, randomized trial with a three-year
follow-up; 330 adult patients (>18 yrs) with MFS are randomized
and assigned to receive 100 mg Cozaar or not for three years.
They continue taking their medication (usually beta blocker) in
same dosage. Aortic diameters are measured with MRI or multislice CT and echo.
Progress
At the end of 2010 232 patients were included in the study. In
all patients baseline CMR was performed with measurements
of aortic dimensions and elasticity and ventricular function.
Blood samples were obtained in all patients, and punch skin
biopsies in consenting patients. Fifty percent of patients were
randomised to losartan therapy. Three patients have developed
an aortic dissection. Three patients are lost to follow-up. Eight
patients stopped with losartan treatment due to side-effects.
Two patients in the control group got pregnant and consequently
dropped out of the study. Below we show the results obtained
from our first data.
Part 1
Because losartan is hypothesized to be beneficial in reducing
aortic root growth of Marfan syndrome (MFS) patients we
investigated the changes of whole transcriptome gene
expression ( WTGE) and correlated the baseline WTGE with
aorta dilatation rate measured over 12 years prior to treatment
start.
Punch skin biopsies were obtained in consenting participants
of the COMPARE trial before therapy (baseline), after 4 weeks
and one year of losartan therapy. In 88 samples RNA was
isolated and WTGE was measured using Human Exon 1.0 ST
Arrays (Affymetrix). WTGE changes and splicing events caused
by losartan were investigated in patients before and 4 weeks
45
Netherlands
Heart Institute
after losartan therapy. Baseline WTGE measurements were
correlated with aortic dilatation rate.
Paired analysis of gene expression changes after 4 weeks of
losartan therapy revealed 20 differentially expressed genes
(delta=0.54, q<10-7). Two genes of the TGF-beta pathway were
significantly changed after losartan therapy: CIDEA and ENG.
When correlated with the aortic dilatation rate, baseline
expression of 2 genes was significant: HLA-DRB5 and HLADRB1 (r=0.46, r=0.42; q<10-7). Validation revealed p values
of 0.0002 and 0.014 respectively again, suggesting an
inflammation process in the connective tissue modifying the
aortic dilatation rate. Histologic analysis of aortic tissue of 11
Marfan patients confirmed these findings (figure 9).
The modifying role of inflammation on the aortic dilatation rate
is a novel finding in the MFS. It may be used as a biomarker of
progressive aortic dilatation and offers new perspectives for the
treatment of a progressive aortic dilatation. Losartan seems to
decrease the TGF-beta signaling pathway in Marfan patients.
Part 2
Marfan syndrome is an autosomal dominantly inherited
connective tissue disorder caused by mutations in the fibrillin-1
gene. As fibrillin-1 is a component of the extracellular matrix of
the myocardium, ventricular function in Marfan syndrome could
be impaired. Echocardiographic studies evaluating ventricular
function in Marfan syndrome have been inconclusive. We
assessed biventricular function in Marfan syndrome by means
of cardiac MRI (CMR).
CMR was performed in 144 Marfan patients without significant
valvular dysfunction, previous valvular surgery or previous
aortic root surgery. Biventricular diastolic and systolic volumes
were measured and ejection fraction was calculated.
Left ventricular ejection fraction (LVEF) was mildly impaired
(mean 53±7%, impaired in 76% of patients), as was right
ventricular ejection fraction (RVEF) (mean 51±7%, impaired
in 46% of patients). LVEF and RVEF were strongly correlated
(r=0.7, p<0.001). No significant differences were found between
patients on beta-blocker therapy and those without. There was
no correlation between aortic elasticity as measured by flow
wave velocity and LVEF.
Biventricular ejection fraction is mildly impaired with normal end
diastolic volumes in adult
patients with Marfan syndrome. This impairment is independent
of beta-blocker usage and aortic elasticity. There is a strong
correlation between LVEF and RVEF, suggesting intrinsic
myocardial dysfunction.
Partnerships
The study is a collaboration between AMC, UMCG, LUMC,
VUMC and UMCN
Funding
This study is funded by the Netherlands Heart Foundation
(grant 2008B115)
Figure 9. Histologic analysis of aortic tissue of 11 Marfan
patients revealed elastin fiber breaks (EvG staining, arrows),
increased counts of macrophages segregating along these
fiber breaks predominately on adventitial side of aortic media.
Increased counts of CD4+ positive Th cells were found in de
aortic adventitia while the counts of cytotoxic CD8+ T cells
were not elevated. MFS= Marfan syndrome. Co=control.
probands’ families were studied, and familial CHD and/or LVNC
was present in 3 of 5 families that were available for study,
segregating with the MYH7 mutation (Figure 1). We concluded
that Ebstein’s anomaly is a congenital heart malformation that is
associated with mutations in MYH7, and that MYH7 mutations
are predominantly found in Ebstein’s anomaly associated with
LVNC. This may warrant genetic testing and family evaluation
in this subset of patients (Circ Cardiovasc Genet. 2010 Dec 2.
[Epub ahead of print]).
Another major part of the project is the study of families with
multiple affected patients with CHD (linkage, candidate gene
analysis, next generation sequencing). Several families are
under study. A locus was identified in a large family with CHD
and electrocardiographic abnormalities reminiscent of left atrial
isomerism, though the causative gene has not been identified
yet. The manuscript is now under review for publication.
A different focus of the project is the clinical aspect of genetics
of CHD. By means of a questionnaire that has been sent to
486 adult patients with CHD, we investigated information
provision as well as patients’ knowledge and concerns about
inheritance of their CHD. We concluded that a substantial
proportion of adult CHD patients lacks knowledge and desires
more information about inheritance, indicating a need for better
patient education. This implies that current guidelines and/or
their implementation do not seem to meet the needs of these
Genetics of Congenital Heart Defects
Drs. Klaartje van Engelen continued her project on “Genetics of
Congenital Heart Defects” (CHD).
In this project, we search for genes and mutations implied
in cardiac development and human CHD, using different
approaches and techniques.
Progress
As part of the HeartRepair project (a pan-European research
consortium established in 2006 as a 6th Framework Programme
Integrated Project of the European Commission, searching for
cardiac repair mechanisms after myocardial infarction), DNA of
more than 400 patients with different types of underdeveloped
hearts has been collected from the CONCOR DNA bank, as
well as from patients from our collaborating partners from the
University of Newcastle, United Kingdom, and the National
register for Congenital Heart Defects in Berlin, Germany. Over
400 genes related to cardiac development have been screened
by next-generation sequencing, mutations and variations being
found in different genes in several patients. Although the project
formally reached its end in June 2010, the mutations that have
been found are currently being investigated further.
Within the HeartRepair project, we screened the MYH7 gene
in a cohort of 141 unrelated Ebstein’s anomaly patients and we
identified a heterozygous mutation in 8 (6%). In 6 of 8 probands
LVNC was identified in addition to Ebstein’s anomaly. The
Figure 10. MRI and echocardiographic images of a 24-year
old woman with an MYH7 mutation. Ebstein anomaly and left
ventricular noncompaction are present.
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ICIN
patients and a dedicated program of counselling for adults
with CHD has to be developed to optimize knowledge and
satisfaction with information provision and to reduce or manage
concerns regarding inheritance of CHD. Our manuscript is now
under review for publication.
Partnerships
In the HeartRepair project, we collaborate with the Institute
47
Netherlands
Heart Institute
of Human Genetics and the dept. of Cardiology, Newcastle
University, Newcastle upon Tyne, United Kingdom and the
National Register for Congenital Heart Defects in Berlin,
Germany.
Funding
The Heart Repair project is funded by a grant from European
Union Sixth Framework Programme.
Pregnancy in congenital heart disease - ZAHARA II
Principal investigator: P.G. Pieper
Background
Previous research has shown that pregnant women with
congenital heart disease (CHD) are more susceptible to
cardiovascular, obstetric and offspring events. The causative
pathophysiologic mechanisms underlying maternal and offspring
complications are incompletely understood. Inadequate
uteroplacental circulation is an important denominator
in adverse obstetric events and offspring outcome in the
general population. The relation between cardiac function and
uteroplacental perfusion has however not been investigated
in women with CHD. Moreover the effects of physiological
changes on pregnancy related events are unknown.
Methods
The ZAHARA II study is a prospective multi-centre cohort
study, which investigates changes in and relations between
cardiovascular parameters and uteroplacental Doppler flow
patterns during pregnancy in women with CHD, compared with
matched healthy controls. The relation between cardiovascular
parameters and uteroplacental Doppler flow patterns and
the occurrence of cardiac, obstetric and offspring events
will be investigated. At 20 and 32 weeks of gestation clinical,
neurohumoral and echocardiographic evaluation and fetal
growth together with Doppler flow measurements in fetal en
maternal circulation are performed. Maternal evaluation is
repeated 1-year postpartum.
We would like to thank all participating centres and all colleagues
who made a great effort to achieve this inclusion and hope to
count on the same great dedication during follow up.
The first preliminary results of ZAHARA II were presented at the
ESC 2010. The main results are to be expected in the course
of 2011. An article describing the study design of the ZAHARA
II study was accepted for publication in the American Heart
Journal.
At the moment we are working on 2 manuscripts concerning the
validations of existing risk scores and risk estimation models
for both maternal and offspring risk. Several other manuscripts
concerning the main objectives of the study are expected in the
second half of 2011.
Investigators
Project leader: Mrs. Dr. P.G. Pieper, UMCG
PhD student: Drs. A. Balci, UMCG
Implications
By identifying the factors, responsible for pregnancy related
events in women with CHD, risk stratification can be refined
which may lead to better pre-pregnancy counseling and
eventually improve treatment of these women.
Results
The ZAHARA II study has reached its final inclusion in September
2010. A Total of 237 eligible patients were addressed for
participation and 233 of them signed informed consent. (figure
1) After drop out of 21 women, 212 could be included for follow
up. This is 33% more than the expected 160 patients that we
had planned to enrol in three years. The most important reasons
for drop out of patients were miscarriage before the 20th week
of gestation and serious protocol violations. Baseline data of the
patients were recorded at first prenatal visit and during follow
up and included clinical and obstetric data, echocardiographic
data, electrocardiographic data and blood and urine samples.
Most patients (95%) have already given birth and are now in the
postpartum follow up phase. Only 5% of the included patients
have not given birth yet. In addition to the included patients,
we were able to enrol 69 healthy controls. Almost 93% of the
healthy women have given birth by now.
Figure 1. Inclusion of patients in and exclusion from the
ZAHARA II study.
Figure 2. Distribution of main congenital heart disease in
ZAHARA II
48
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Netherlands
Heart Institute
PROSTAVA Study
Principal investigator: P.G. Pieper
Background
In patients with congenital heart disease, often mechanical
or biological prosthetic valves are implanted. These patients
differ from adults who have prosthetic valves implanted for
acquired valve disease because of differences in age, lifestyle
and variety of valve location, and because of the probably high
incidence of prosthesis-patient-mismatch. However, data about
the long-term complications of prosthetic valves, including the
prevalence and complications of prosthesis-patient mismatch,
in adults with congenital heart disease are scarce.
Objectives
The primary objective of this study is to investigate the relation
between characteristics of valve prosthesis (i.e. valve type,
location and size) on functional outcome and quality of life in
adult patients with congenital hert disease.
The secondary objectives of this study are:
• To investigate the prevalence and predictors of prosthesispatient-mismatch in a population with congenital heart
disease.
• To investigate the incidence of prosthesis related
complications in a population with CHD (re-operation, valve
thrombosis, bleeding complications, hemolysis, paravalvular
regurgitation, endocarditis, arrhythmias, pregnancy-related
complications, heart failure).
• To investigate the relation between prosthesis related
complications and valve type (especially for pulmonary
prosthetic valves).
Methods
We intend to include 600 patients with CHD and a prosthetic
valve, who are identified through the CONCOR database.
Primary outcomes are VO2max and quality of life. Secondary
outcomes are the prevalence of prosthesis-patient-mismatch
49
(according to pre-defined criteria) and the incidence of valve
related complications.
Patient files of congenital heart disease patients with prosthetic
valves identified from the CONCOR database will be studied
for past medical history including surgical procedures and the
occurrence of valve-related complications. The cross-sectional
evaluation encomprises history, physical examination, VO2max,
quality of life questionnaires, echocardiogram including
prosthetic valve area, MRI (ventricular volumes and mass),
laboratory evaluation (including NT-pro-BNP).
Expectations
We expect the spectrum of prosthetic valve-related complications
and the prevalence of prosthesis-patient mismatch in our CHD
population to differ from adult populations with acquired valve
disease as reported in the literature.
Implications
Our results may influence the choice of valve prosthesis, the
indication for more extensive surgery (e.g. annulus enlargement)
and the indication for re-operation in patients with prosthesispatient-mismatch.
Current state of the PROSTAVA study
Inclusion has started in UMCG and will soon commence in AMC
and UMCN. Several other centres will join the study during 2011
Duration
September 2010 - September 2013
Investigators
Project leader: Mrs. Dr. P.G. Pieper, UMCG
Researchers: Drs. Ymkje J. van Slooten, Drs. Hendrik G. Freling
Hypertrophic cardiomyopathy
Principal investigator: A.C. van Rossum
Aim and method of the project
This project focuses on the pathogenesis of wall thickening in
hypertrophic cardiomyopathy (HCM), which is a complex genetic
heart disease affecting ~1:500 individuals. In order to evaluate
the hypertrophic and fibrotic response in HCM, patients with
sarcomeric mutations and transgenic animals are subjected
to state-of-the-art serial cardiovascular magnetic resonance
(CMR) imaging investigations.
Progress in 2010
The clinical and morphological expression of HCM is highly
variable. The typical HCM patient displays asymmetrical left
ventricular (LV) hypertrophy of the interventricular septum,
frequently accompanied by left ventricular outflow tract (LVOT)
obstruction and heart failure symptoms. Current advances
in genetic testing however, yield an increasing amount of
asymptomatic mutation carriers without signs of overt disease
manifestation. The investigation of these pre-hypertrophic
patients is of particular interest (together with preclinical studies
in animals), since it may provide valuable insights into the
complex pathogenesis of hypertrophy in HCM and may even
lead to targeted preventive therapy [1].
To this end a group of HCM carriers underwent cardiovascular
magnetic resonance (CMR) imaging with tissue tagging.
Tagging allows the non-invasive quantification of torsion
and circumferential strain, which are both markers of cardiac
deformation. Torsion and the ratio of torsion-to-circumferential
strain (TECS) were significantly higher in carriers with respect
to controls (see figure 1), indicating that pre-hypertrophied HCM
hearts exhibit functional abnormalities before the occurrence
of hypertrophy [2]. Besides these functional changes,
localised morphological changes (called crypts, figure 2) at
the inferoseptum occur in high numbers of carriers [3]. We
are currently investigating the prevalence and characteristics
of crypts in a large heterogeneous group of carriers and are
comparing these findings with a group of consecutive CMR
patients without HCM mutations.
Fig. 2. CMR two-chamber cine image displaying several crypts
located at the inferior LV wall, indicated by the white arrows.
Experimental studies
In order to gain more insight into the complex hypertrophic
response in HCM, we subjected transgenic HCM (cMyBP-C)
null mice to serial CMR acquisitions and treated half of the study
population with cariporide (Na+/H+ exchange blocker). We used
the Bruker 9.4 T at the Utrecht animal facility scanner to generate
cine images to enable the off-line calculation of left ventricular
mass and volumes (see figure 3). After two months of treatment,
cariporide failed to regress the amount of pre-existent LV
hypertrophy in these mice. We are currently analysing whether
blockage of the Na+/H+ exchanger influences the formation of
fibrosis in these animals.
Fig. 3. Short-axis cine image of a cMyBPC null mouse.
Manually drawn epicardial (green) and endocardial (red)
borders allow off-line quantification of LV volumina and mass.
Fig. 1. Differences in torsion and TECS between healthy
volunteers, mutation carriers and manifest HCM patients.
A possible hypothesis that provides an overall explanation for
the hypertrophic response in HCM prescribes that inefficient
ATP-usage of mutated sarcomeres causes enhanced energy
metabolism relative to cardiac work. As a result, intracellular
ATP-dependant molecular pathways may become perturbated
and may ultimately stimulate pro-hypertrophic signalling. In
order to confirm this hypothesis with a translational approach,
we started a collaboration with the cardiology and cardiothoracic
surgery department of the St. Antonius Hospital in Nieuwegein.
50
ICIN
Netherlands
Heart Institute
Hypertrophic obstructive cardiomyopathy patients eligible
for surgery (myectomy) will undergo both positron emission
tomography (PET) and CMR investigations at the VU University
Medical Center before the intervention. After surgery, efficiency
of contraction of myocardial tissue (which is obtained during
surgery) will be determined and related to in vivo data.
Funding
The project (04805) is financed by the ICIN and a part by a
grant of the Netherlands Heart Foundation (NHS 2006B213).
The preclinical studies are financed by the Seventh Framework
Program of the European Union ‘BIG-HEART’ grant agreement
241577 obtained by Jolanda van der Velden.
Partnerships
The research regarding HCM mutation carriers is based upon
a collaboration between the VU University Medical Center
(imaging) and the Academic Medical Center (AMC) Amsterdam.
A collaboration between VU University Medical Center
Amsterdam (department of physiology) and the Animal Facility
in Utrecht (University Medical Center Utrecht (UMCU) enables
the preclinical studies.
The multicenter study titled ‘ENerGetIcs in hypertrophic
cardiomyopathy: traNslation bEtween MRI, PET and cardiac
myofilament function’ (‘ENGINE’-study) is a collaboration
between the VU University Medical Center Amsterdam and St.
Antonius Hospital Nieuwegein.
Staff
Promotor: Prof Dr. A.C. van Rossum
Co-promotors: Dr. T. Germans (CMR imaging) and Dr. J. vd
Velden (physiology).
51
Collaborations:
AMC: Prof. Dr. A.A. Wilde and Dr. I. Christiaans
UMCU: Prof. Dr. G. Pasterkamp
Animal facility (GDL): M.G.J. Nederhoff and K. vd Kolk
St. Antonius Hospital: Dr. J.M. ten Berg and Dr. A. Yilmaz.
Right ventricular dysfunction and tetralogy of Fallot
Principal investigator: W.A. Helbing
Project
Early diagnosis of right ventricular dysfunction in patients
operated for tetralogy of Fallot. A multicenter study with serial
follow-up.
of RV overload and hypertrophy.
3. Correlate circulating levels of identified markers with cardiac
and systemic parameters, and to identify (a) potential
biomarker(s) as a diagnostic and prognostic tool(s).
Background, aims and method
Residual pulmonary regurgitation (PR) plays a crucial role in
the long-term outcome of patients operated for tetralogy of
Fallot. Ultimately, longstanding PR can lead to right ventricular
(RV) dilatation, impairment of RV function, deterioration of
exercise capacity, arrhythmias, and an increased risk of sudden
cardiac death. Patients with severe PR are often treated with
a pulmonary valve replacement (PVR), but the optimal timing
to perform a PVR remains subject for debate. Serial follow-up
in patients with corrected tetralogy of Fallot is limited and the
course of deterioration of ventricular function has not yet been
clarified. Serial follow-up data may provide insight in the course
of RV size and function after surgery, which could contribute to
decision making on optimal timing of PVR.
Progress in 2010
The last patients were successfully included in 2010. In total,
87 patients after surgical repair of tetralogy of Fallot had
completed the serial follow-up study protocol, which consisted
of: taking history, performing physical examination, performing
an MRI study, performing a bicycle exercise test, blood sample
collection, ECG and 24-hour Holter registration.
Additionally, a subgroup of patients had also undergone an MRI
study during low-dose dobutamine stress (7.5 µg/kg/min.) at the
baseline and the follow-up study. The results of this study showed
us that in 5 years time, important progression of RV size occurs
in patients, who have moderate to severe PR and an RVEDV of
≥ 150 ml/m2 (for RVEDV 4 ml/m2 increase per year). This was
accompanied by a tendency towards a decrease in VO2 max,
but not in other exercise parameters. This increase in RV size
over time did not occur in patients with smaller right ventricles
(RVEDV < 150 ml/m2). The increase in RV size in patients
with an RVEDV ≥ 150 ml/m2 was not associated with relevant
changes in LV size, biventricular function or contractile reserve.
Importantly, these results did not differ from those in patients
with an RVEDV < 150 ml/m2 or in patients who had undergone
a PVR. Biventricular contractile reserve was preserved in all
patients, even in those with the largest right ventricles.
The project aims to improve early diagnosis of right ventricular
failure during long-term follow-up of patients with tetralogy
of Fallot, by integrating information from history, physical
examination, imaging techniques, and blood tests. This will
ultimately result in improved guidelines for treatment of potential
right ventricular failure, required for prevention of heart failure in
patients with tetralogy of Fallot.
In patients who have undergone surgical repair of tetralogy of
Fallot, we aim to:
1. Assess the development of RV dimensions and function in
relation to clinical outcome, in a time dependent manner.
2. Identify, characterize and quantify markers in blood samples,
using high density antibody arrays and ELISA’s, obtained
from Fallot patients at different stages of the development
In another study, a comparison was made between a cohort of
patients who had undergone balloon valvuloplasty for isolated
pulmonary valve stenosis and a group of patients with repaired
tetralogy of Fallot, matched for gender, age at treatment and age
at study. Patients underwent an MRI scan, echocardiography
and a bicycle exercise test. Results were also compared to
Figure 1:Serial follow-up results of RVEDV (ml/m2) and RVEF (%) assessed with MRI at rest and during low-dose dobutamine
stress in subgroups of patients with repaired tetralogy of Fallot.
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ICIN
a group of healthy controls, matched for gender and age at
study. We demonstrated that patients with isolated PS had a
lower exercise capacity, a larger RV and a lower RV function
than healthy controls, despite an excellent clinical condition.
However, exercise performance and biventricular function were
similar to those in patients with tetralogy of Fallot, despite more
severe PR and larger RV volumes in Fallot patients.
High-density antibody arrays were used to test 272 proteins
in 96 Fallot patients and in 70 healthy controls. Patients with
different degrees of RV volume overload and different follow-up
durations were selected for this study. The analysis resulted in
the identification of a selected number of proteins which might
potentially be useful as diagnostic and prognostic markers. This
will quantitatively be tested with ELISA’s.
Collaboration
The project involves collaboration between the departments
of Pediatric Cardiology / Cardiothoracic surgery of Erasmus
MC-Sophia and UMCU-WKZ, and between the departments of
Cardiology / Cardiothoracic surgery AMC and LUMC (Center for
Congenital Heart Disease Amsterdam Leiden).
The project also involves collaboration with the departments
of Cardiology, Radiology, Clinical Chemistry and Molecular
Biology of Erasmus MC.
Staff
- Prof. W.A. Helbing, PhD, MD
Department of Paediatrics, division of Cardiology, Erasmus
MC
- Prof. B.J.M. Mulder, PhD, MD, Department of Cardiology,
AMC
-Prof. M.G. Hazekamp, PhD, MD, Department of
Cardiothoracic Surgery, LUMC-AMC
-Prof. A.J.J.C. Bogers, PhD, MD, Department of
Cardiothoracic Surgery, Erasmus MC
- H.W. Vliegen, PhD, MD, Department of Cardiology, LUMC
- H.J. Duckers, PhD, Department of Cardiology, Molecular
Biology, Erasmus MC
- S.E. Luijnenburg, MD, Research Fellow, ICIN
Funding
This project is funded by the Netherlands Heart Foundation.
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Heart Institute
Arrhythmogenic Right Ventricular Dysplasia
Principal investigator: R.N.W. Hauer
Background
Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy
(ARVD/C) is considered as an inherited desmosomal disease.
Desmosomes contribute to cardiac mechanical intercellular
coupling. Recent studies have shown that mutations in genes
encoding desmosomal proteins appear to be associated with
the ARVD/C phenotype. These mutations may affect number
and integrity of desmosomes and may influence amount
and distribution of other intercalated disk proteins, including
gap junctions (connexin43). This may decrease intercellular
electrical coupling and thus enhance arrhythmogenicity. On
the other hand, histology in ARVD/C showed altered tissue
architecture due to surviving myocardial strands embedded
in accumulated fibro-fatty tissue. Both intercellular electrical
uncoupling and these interconnecting myocardial strands
give rise to lengthened conduction pathways, load mismatch
at pivotal points, and conduction slowing, all contributing to
activation delay and reentrant circuits.
Hypotheses
Arrhythmogenicity in early genetically determined ARVD/C
may be due to gap junction remodeling, followed at a later
stage of the disease by replacement fibrosis due to cell
death. This hypothesis is strongly supported by recent
immunohistochemical observations by Asimaki et al. showing
in ARVD/C patients the occurrence of alterations in intercalated
disk proteins in pathohistologically still unaffected tissue.
However, since sporadic ARVD/C cases are well described,
not all types of ARVD/C are necessarily genetically determined,
but alternatively sporadic occurrence may be due to incomplete
penetrance or spontaneous mutations.
Aims
1) How are desmosomal changes related to the contribution
of different arrhythmogenic substrates in ARVD/C? 2) Quantify
the genetic contribution to ARVD/C in a large cohort of patients.
3) Are different types of mutation or mutation sites related to
differences in penetrance and clinical characteristics? If so,
are these differences understandable at the molecular/cellular
level?
Methods
Originally Index-patients fulfilling predefined diagnostic ARVD/C
criteria (generally accepted Task Force criteria, defined by
consensus in 1994) were collected. Since publication of the
modified Task Force criteria in both Circulation and European
Heart Journal in 2010 these new criteria were used to redefine
fulfilment of ARVD/C diagnosis in all included patients.
After genotyping index patients, their family members are
approached, genotyped and clinically screened. All genes
encoding desmosomal proteins will be screened. Family history
will be analyzed. In each family, the mutation will be related to
predefined clinical outcome parameters. The amount of fibrosis
will be studied by delayed-enhancement MRI.
ARVD/C relevant mutant constructs will be expressed after
viral transfer to neonatal cardiac myocytes. Different mutations
will be related to amount and distribution of desmosomal
proteins, connexin43 and ion channel proteins by Western
blotting and immunofluorescence techniques. Electrophysiologic characteristics of transfected myocytes will be
determined and related to clinical outcome parameters. Amount
and distribution of intercalated disk proteins will also be studied
in ARVD/C patients and controls. This methodology is needed
to answer clinical and basic questions in Aims.
Results Clinical study
1) In order to study genotype-phenotype relationships of
ARVD/C, patients fulfilling the diagnostic Task Force criteria
defined in 2010 have been collected nationwide. All 8 University
Medical Centers cooperated in this part of the project. Together,
the number of index-patients included could be increased from
115 in 2008 to 137 in 2009, and to 169 in 2010. In all patients,
the gene encoding Plakophilin-2 (PKP2) was analyzed. More
important, in 149 of these 169 index-patients, all 5 genes
encoding desmosmal proteins (DSG2, DSC2¸ PKP2, DSP,JUP)
have been analyzed. Pathogenic mutations were identified
in 87/149 (58%) of index-patients, mainly truncating PKP2
mutations. Multiple desmosomal mutations were found in
only 2% of patients. A total number of 23 different pathogenic
mutations (primarily PKP2) were found, and in addition 8
most likely pathogenic DNA variants (predominantly missense
mutations). Pathogenicity of these missense mutations was
likely because of analysis with in silico predictive programs.
Furthermore, at the end of 2010 in total 302 family members from
93 different families of the 149 index-patients were clinically and
genetically characterized. The diagnosis “ARVD/C” was made
in 60 family members. Thus, in total from all families with DNA
analysis of 5 desmosomal genes 149+60=209 patients fulfilled
the Task Force criteria from 2010 for ARVD/C diagnosis. This
is one of the largest series of ARVD/C patients in the world.
Since 137 family members had pathogenic mutations, the total
number of mutation-carriers was 149+137= 286 individuals
(mainly truncating PKP2)! Of the 57 of 282 (20%) initially
asymptomatic relatives that showed any sign of ARVD/C 84%
carried a mutation.
Compared to relatives of index-patients without mutations,
mutation-carrying relatives had:
1) A six-fold risk of ARVD/C diagnosis.
2) Markedly increased risk of arrhythmias and
3) Earlier onset of ARVD/C signs and symptoms. In young
relatives, age <20 years, sudden death and signs of ARVD/C
occurred exclusively in PKP2 mutation-carriers. Prolonged
terminal activation duration, a marker of activation delay,
appeared to be an early ARVD/C sign. A manuscript has
been submitted and was accepted for publication in
Circulation in 2011, very recently. Another manuscript
prepared in collaboration with Mayo Clinics (Ackerman)
on background genetic noise in ARVD/C was accepted for
publication in J Am Coll Cardiol, also in 2011.
2) Cox and Hauer co-authorized in the international consensus
paper on New Task Force criteria for ARVD/C diagnosis,
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which has been published in both Circulation and Eur Heart
J. Prolonged terminal activation duration (TAD) and superior
axis of ventricular tachycardia with left bundle branch block
morphology, previously identified in our study, were accepted
to include in the new Task Force criteria. In addition, a report
has been published in Circ Arrhythm Electrophysiol on clinical
impact of the modified criteria.
Status and continuation: Inclusion of patients will be continued.
Although the target of 150 ARVD/C index-patients and their
family members has been reached and important data have
been obtained on diagnostic improvement, molecular-genetic
involvement, and in the comparison of mutation-carriers versus
non-carriers and sporadic cases, many scientific challenges
persist:
1) More patients should be included to have more relatives per
specific DNA mutation to identify specific mutation-related
phenotypic differences and differences within a family by
genetic modifiers and environmental factors.
2)Prolonged terminal activation duration (TAD) has been
identified as important parameter for activation delay.
However, prolongation of TAD will be typically due to
presence of right ventricular outflow tract involvement,
which may be not always the case. Identification of universal
delay parameters is needed and will be studied by relating
electrophysiologic data with sites of hemodynamic and
structural alteration (advanced echocardiography, MRI with
late enhancement).
3) Immunohistochemical analysis is very promising, but much
more data are needed.
4) Ultimate goal is early diagnosis and risk stratification.
In April 2011 MGPJ Cox will finalize her thesis on the clinical
part. Since September 2010 a new PhD student JA Groeneweg
is involved.
Results Experimental studies
1) A human PKP2 cDNA expression construct has been
obtained, and a series of 6 mutations covering the entire coding
region and resulting in a premature stopcodon have been made.
The mutations are based on identified mutations in the (Dutch)
ARVD/C patient population. Ectopic expression of wild type and
mutant proteins, N-terminal HA-tagged to ensure immunogenic
detection, was checked by western blot and immunofluorescence microscopy. Localization of mutant and wild type
PKP2 protein has been determined in COS7, HEK293t and
BWEM cells by immunofluorescence. On basis of localization,
mutant proteins were categorized in three groups: 1. localized
in the nucleus, 2. in protein synthesis and transport associated
vesicles, or 3. diffusely throughout the cytoplasm. Importantly,
they all displayed an abnormal subcellular localization in COS7
and HEK293t cells when compared to wild type PKP2, which is
normally localized at the plasma membrane. To allow further in
vitro analysis, adenoviruses of the PKP2 constructs are made.
The constructs will be expressed in neonatal cardiomyocytes to
study their effect on conduction.
Results from clinical cardiologic evaluation to assess diagnostic
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Heart Institute
Task Force criteria in ARVD/C patients with the corresponding
PKP2 mutations were obtained, including arrhythmias, ECG,
and structural abnormalities. The ARVD/C phenotype appeared
to be associated with a loss of plasma membrane localization
of PKP2 in COS7 and BWEM cells. Additional loss- or gain–
of functions of truncated PKP2 protein appeared not to be
correlated to the severity of the ARVD/C phenotype.
2) Post-mortem cardiac samples and right ventricular septal
biopsies from 10 ARVD/C patients and 6 controls have been
analyzed using immunohistochemistry for the presence
and localization of Connexin43, Plakophilin2, Plakoglobin,
N-Cadherin, Desmoplakin and Collagen. Four patients
had mutations in PKP2 and 1 in DSG2. In 8 of 10 ARVD/C
patients connexin43 expression was down-regulated and was
heterogeneously distributed compared to controls. In contrast,
expression and distribution of PKP2, DSP, and N-cadherin and
also JUP were remarkably unaffected when using standard
protocols. Alterations in expression profiles did not differ
between the left ventricular free wall and the right ventricular
free wall and importantly, were similar to those seen in right
ventricular septal biopsies. Recently, Asimaki et al., from
Harvard also published down-regulation of connexin43 but
unlike our first results also universal down-regulation of JUP.
This was published in New England Journal of Medicine, March
2009. Differences were studied in cooperative studies were
tissue samples were exchanged. After carefully reviewing the
staining protocol we were able to see the downregulation of
JUP in 7/10 patients.
3) A PKP2 haploinsufficient mouse has been obtained and is
currently being extensively characterized. Cohorts of 3 and 6
months old mice will be studied, as well as mice that undergo a
TAC surgery and a group subjected to voluntary running. ECGs,
echocardiograms and epicardial activation mappings of these
animals will be analyzed. Furthermore the hearts of these mice
will be used for western blot, PCR and immunohistochemistry.
Status and continuation
The effect of plakophilin 2 mutation on the composition of the
desmosome and gap junctions will be studied in neonatal
cardiomyocytes. Molecular analyses will be performed on the
PKP2 haploinsufficient mice and will be compared to wildtype.
Investigators: RNW Hauer (UMCU), MGPJ Cox (UMCU), M
Noorman (UMCU), JJ van der Smagt (UMCU), HVM van Rijen
(UMCU), MAG van der Heyden (UMCU), AAB van Veen (UMCU),
MA Vos (UMCU), D Dooijes (UMCU), JG Post (UMCU), P Loh
(UMCU), MJ Cramer (UMCU), JA Groeneweg (UMCU), JP
van Tintelen (UMCG), IC van Gelder (UMCG), ACP Wiesfeld
(UMCG), IM van Langen (UMCG), JDH Jongbloed (UMCG), PA
vd Zwaag (UMCG), JMT de Bakker (ICIN, UvA, UMCU), AAM
Wilde (UvA), C vd Werf (UvA), ZA Bhuiyan (UvA), PGA Volders
(AZM), Avd Wijngaard (AZM), MJ Schalij (LUMC), DE Atsma
(LUMC), L Jordaens (EMCR), AC Houweling (VUMC).
Financial support: This study is granted by: ICIN, HLSU
Foundation, and the Netherlands Heart Foundation (2007B139).
Pulmonary hypertension and Congenital heart disease
Principal investigator: G. Tj. Sieswerda
Project
The pressure overloaded right ventricle in pulmonary
hypertension and congenital heart disease; a deformation
imaging study’
Aim and method
The aim of the scientific part of this project is threefold:
1.Patients with pulmonary hypertension have a pressure
overloaded right ventricle, this is also the case in several
congenital heart defects. However there is a substantial
difference in time to right ventricular failure and survival
between these groups, detrimental to the patients with
pulmonary hypertension. We aim to explain this difference
by comparing the characteristics of the right ventricles of
these groups and healthy controls. Understanding the
adaptive capacity of the heart gives more insight in the
different disease courses and could give new perspective
on therapeutic options. A total of 8 groups will be compared
to each other, using different new imaging techniques
in both echocardiography and MRI, and an extensive
cardiopulmonary exercise test.
2.There is a need for prognostic markers in pulmonary
hypertension patients that are available early in the
disease and preferably noninvasive. We therefore aim
to use the new echocardiographic techniques and
elaborate cardiopulmonary exercise testing to find early
prognostic markers. A group of 150 patients with pulmonary
hypertension will be evaluated twice (once at entrance in
the study and once at 6 months) and followed for 2 years
using the primary endpoint ‘clinical worsening’.
3.Three-dimensional speckle tracking is one of these new
imaging techniques and has yet to be validated. We
will therefore investigate its clinical applicability of this
technique and validate with MRI tagging as a reference.
This will be done in a group of 36 healthy individuals as well
as in patients with congenital heart disease/right ventricular
dysfunction.
Progress in 2010
In addition to the scientific goal of this project, optimizing
patient care for pulmonary hypertension in the UMCU is also
an important goal of this project. Starting this project, no clear
referral structure for pulmonary hypertension was present in
the UMCU, also dedicated specialists (of each of the involved
divisions) were lacking.
For the cardiology two dedicated cardiologist, the GUCH
cardiologists F.J. Meijboom and G.Tj. Sieswerda, are now the
dedicated specialists when diagnostic aid is needed in patients
suspected of pulmonary hypertension. Patients can be referred
to the secretary of ‘grown-up with congenital heart disease’.
Patients with clinically suspected pulmonary hypertension can
now be evaluated at a one-stop outpatient clinic within 1-2 weeks.
Also referral for right heart catheterization (in the suspicion of
pulmonary hypertension) is now structured and planned by the
investigator (M. Driessen). With these adjustments, the time to
diagnosis has been shortened.
A second adjustment is the introduction of a dedicated
pulmonary hypertension meeting, which takes place every
4 weeks. This meeting is multidisciplinary including the
pulmonology, cardiology and rheumatology department. Dr. R.
Snijder from the St. Antonius hospital, whom is specialized in
pulmonary hypertension, also takes place in these meetings.
These meetings are used to determine further diagnostic tests
needed and appropriate therapeutic strategy. Also a summary
of each meeting with diagnosis and advice is included in the
patient charts.
All and all, a lot of progress has been made in optimizing the
patient care for pulmonary hypertension. For 2011 though,
still many points can be optimized; the responsible specialists
in each division need to be appointed and known within the
involved departments and planning of diagnostic tests (for
categorizing the type of pulmonary hypertension) should be
accelerated, as the latter now takes a lot of time.
For the scientific part of this project
Only a very brief concept of the project was available at the start
in August of 2010. A clear and structured research protocol,
including the rationale of the study, had to be made by the
investigator. The project started with an elaborate study of the
literature, including literature about pulmonary hypertension,
congenital heart disease, the embryology of the heart, the
anatomy of the heart, different imaging techniques and validation
of these techniques, prognosis in pulmonary hypertension and,
cardiopulmonary exercise testing.
Using the literature and the concept for the project, the
investigator made an elaborate research protocol, including
the 3 major goals of the study. Together with the study of
literature, this took approximately 2-3 months. The investigator
further extended this protocol into an application for approval
of the medical ethical committee. This application includes a
sample size calculation and a statistical analysis plan, patient
information and ABR-form. Also collaboration was sought with
other institutions, namely St. Antonius hospital and St. Radboud
university hospital, making this a multi-center project. The
protocol was filed with the medical ethical committee on the 6th
of January.
One of the most important investigations in this study is
echocardiography. As the investigator will be performing these
herself, a training in echocardiography was necessary. The
investigator has spent 1.5 months in the Rijnstate hospital in
Arnhem learning how to perform echocardiograms. After this
six-week training, the investigator has spent one day a week
performing echocardiograms in the functional department of the
UMCU to improve her skills.
Partnerships
St. Antonius Hospital:
- Dr. R. Snijder MD PhD, pulmonologist
- Dr. M. Post MD PhD, cardiologist
St. Radboud University Medical Center:
- A. van Dijk MD PhD, cardiologist
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Funding
Funding was provided by ‘stichting opsporing pulmonale
hypertensie’ (STOPH).
Staff
Drs. M.M.P. Driessen; investigator (coordinating), employed by
ICIN (research for promotion)
Dr. G. Tj. Sieswerda; project leader, employed by Heart&Lung
division of the University Medical Center Utrecht (1st copromotor)
Dr. T. Leiner; co-investigator, employed by Radiology department
of the University Medical Center Utrecht (2nd co-promotor)
Dr. F.J. Meijboom; co-investigator, employed by Heart&Lung
division of the University Medical Center Utrecht
Dr. R.J. Snijder; co-investigator, employed by Heart&Lung
division of the St. Antonius Hospital
Dr. A. van Dijk; co-investigator, employed by Heart&Lung
division of the St. Radboud University Medical Center
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Heart Institute
ICIN
Netherlands
Heart Institute
Research line - Heart failure
Dutch Programma on Tissue Engineering
Principal investigator: P.A.F.M. Doevendans
Aim and method of the project
Title: MicroRNA-155 prevents necrotic cell death in human
cardiomyocyte progenitor cells via targeting RIP1.
Regenerative medicine offers a potential novel approach to
repair the injured heart. For cardiac repair, different cell types are
considered for transplantation to replace damaged tissue. In this
regard, cardiac progenitor cells (CPCs) are potentially the best
cell source due to their capability of differentiating into functional
cardiomyocytes, endothelial cells and smooth muscle cells, all
required for heart repair. Previously, we reported the isolation of
cardiomyocyte progenitor cells (CMPCs) from human heart that
are able to proliferate and efficiently differentiate into functional
cardiomyocytes, smooth muscle cells, and endothelial cells.
3-Months after transplantation of CMPCs in a mouse model of
myocardial infarction, we observed less outward remodeling
and improved cardiac function as compared to control injections.
Although high numbers of cells are injected for cell therapy,
few implanted cells survive, limiting the potential contribution
for myocardial repair. Most of the engrafted cells perished in
the first 48h after transplantation, partially due to the hostile
microenvironment of the ischemic myocardium. Strategies are
therefore required to improve cell survival after implantation.
Progress in 2010
Accumulating evidence suggests that miRNAs play an important
role in cell survival. Recently, we observed that microRNA-155
(miR-155) is highly expressed in proliferating CMPCs. However,
miR-155 does not regulate or determine cellular proliferation.
miR-155 was shown to be involved in cell death in tumor cell
lines. We therefore tested if miR-155 was involved in CMPC
cell survival and stimulated the cells with oxidative-stress or
anisomycin, a protein synthesis inhibitor. Interestingly the
level of miR-155 increased 4-fold upon stimulation while the
dominant cell death that could be observed in CMPCs was
necrosis, Subsequently, we tested if increasing miR-155 levels
via transient transfection would efficiently enhance CMPCs
survival. After transfections and induction of oxidative stress,
we observed that miR-155 could efficiently prevent oxidativestress induced necrosis, but not apoptosis (figure 1). miR-155
targets RIP1, a death domain protein required for activation of
death-receptor induced necrosis. In addition, necrotic cell death
was also reduced after targeting RIP1, either via Nec1 (synthetic
inhibitor) or siRNA (reducing RIP1 mRNA levels). Interestingly,
increased miR-155 levels did not change apoptotic and cell
survival related gene expression. Via this study we observed
that necrosis is the main mechanism of cell death in CMPCs
in vitro. Although necrosis was previously documented to be
a non-regulatory process, we showed that the process could
efficiently be inhibited via miR-155 or specifically by targeting
RIP1 (figure 2).
These data suggest that miRNAs can potentially be used
to improve the efficiency of cell-based therapy for cardiac
regeneration by enhancing myogenic differentiation or survival
of injected cells. Currently, we are testing these miRNAs for
their effect on CMPC differentiation and survival in a murine
myocardial infarction model.
Figure1: CMPCs were transfected with pre-miR155, antimiR155 or scr-miR and challenged by oxidative stress. Live,
apoptotic and necrotic cells were detected by AnnV/7-AAD
staining using flow cytometric analysis. We observed an
increased number of surviving cells and a reduced necrotic cell
population upon miR-155 transfection. (Data are presented as
mean +/- sem, N= 4 and *p<0.05)
Figure 2 (A) RIP1 mRNA expression in miR-155
overexpressing CMPC;: pre-premiR155, anti: antagomiR;
SCR-scrambeled (B) RIP1 protein expression detected by
Western blot
Funding
DPTE
Staff
Jia Liu, MD (UMCU)
Alain van Mil (ICIN)
Steven Chamuleau, MD, PhD (UMCU)
Pieter Doevendans, MD, PhD (UMCU)
Joost Sluijter, PhD (UMCU)
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ICIN
Netherlands
Heart Institute
ICIN-NHS Cell therapy program, translational studies
Principal investigator: P.A.F.M. Doevendans
Aim and method of the project
Title: Cell therapy for cardiac regeneration and protection
Previously, it has been assumed that transplanted MSC, due to
the influence of the local microenvironment, trans-differentiate
into cells of the cardiomyogenic lineage or even cardiomyocytes.
These cells would integrate into the myocardium and help to
regenerate the damaged tissue. After transplantation, however,
only a minor percentage of MSC were detected. More recent
studies show that the paracrine factors produced by MSC have a
protective effect on heart tissue; anti-apoptotic, anti-fibrotic and
pro-angiogenic effects were observed. So MSC are of interest
for clinical applications in cardiology, however, their beneficial
effects on cardiac tissue after injury are only partially elucidated.
Another cell type of potential interest is the epicardium derived
cell (EPDC). Also these cells have beneficial effects on cardiac
function, through an unknown mechanism.
Role of epicardium-derived cells (EPDCs) in
cardiomyocyte differentiation
Over the last years, several studies have focused on the role
of adult epicardium and EPDCs in heart repair. Recently, we
demonstrated that adult human spindle-shaped EPDCs injected
into the infarcted myocardium, preserved cardiac function and
reduced remodelling both early and late after the onset of
infarction (Winter et al. Circulation 2007). Not by differentiating
into new cardiomyocytes, but rather by paracrine protection
of the surrounding tissue, reducing myocardial cell death and
stimulating angiogenesis. Interestingly, when we combined
adult human EPDCs with human CMPCs, a cell source that is
able to differentiate into cardiomyocytes, we observed a more
powerful protection and stimulation of the infarcted heart than
either of these cell types separately (figure 1). The favorable
effect of combined transplantation is at least partly explained
by stimulation of distinct paracrine cascades. The contribution
Figure 1 LV function synergistically improves when both
CMPCs and EPDCs are transplanted (winter et al.,2009).
61
of injected EPDCs was instructive rather than constructive.
Recently we observed that 4 days post MI, the endogenous
epicardium is reactivated of with re-expression of developmental
genes and renewed EMT (figure 2). The therapeutic potential
of the endogenous epicardium is therefore a new field of
investigation.
Figure 2 While 2 days post MI, the epicardium does not
express the embryonic marker WT1, this is abundantly present
4 days after myocardial infarction.
A suitable preclinical animal model for studying the effectiveness
of cell therapy after a myocardial infarction is the pig.
Unfortunately, cell therapy with human CMPC cells failed due
to rejection, despite immune-suppression, and thus autologous
cells are needed for follow-up studies. For this, we started to
set-up a protocol to isolated porcine MSC from bone marrow
(BM) and compared their characteristics with human MSC
(hMSC) by morphology, immunophenotypic characteristics and
multilineage potential. Culture-expanded pig BM cells express
several markers that are also characteristic for hMSC. They
only lacked expression of CD105, CD73, and CD166, due to
an absence of cross-reactivity with antibodies. With a CFU-F
assay, CD271+ selected porcine BM cells showed enrichment
in MSC frequency, as is also true for CD271+ cells selected from
human BM. Subsequently, multipotency of pMSC and hMSC
towards osteoblasts, adipocytes and chondrocytes were found
to be equal. For pMSC as well as for hMSC large variations
in differentiation potential were observed, which were donor
dependent. The induction-stimuli required are not different for
pMSC and hMSC. In conclusion, pMSC show similarities and
dissimilarities in phenotype, but equal potency in differentiation
towards osteoblasts and adipocytes.
Progress in 2010
Recently, we studied the immune-modulatory potential of pMSC
and their in vivo effect on cardiac function in a mouse model
for myocardial infarction as compared to hMSCs. Mononuclear
cells were obtained after ficoll separation from porcine and
human peripheral blood (pPBMNC and hPBMNC, respectively),
and stained with carboxyfluorescein diacetate succinimidyl ester
(CFSE). pPBMNC or hPBMNC were maximally stimulated with
phorbol-12-myristate-13-acetate (PMA) and IL-. Stimulation
was performed in the presence or absence of MSC. After 6
days of culture CFSE expression of PBMNC was measured
by flow cytometry. Stimulation with PMA and IL-2 resulted in a
significant number of dividing cells. However, in the presence
of hMSC or pMSC, significantly fewer cells had divided and the
percentage of cells which had divided 4 or 5 times, were much
lower than in absence of MSC (figure 1).
In addition to these in vitro comparison studies between
pMSC and hMSC we started to explore their in vivo potential.
We injected 500.000 MSCs in a mouse model of myocardial
infarction, after the permanent ligation of the LAD. We analyzed
cardiac function with MRI (9.4T) at baseline and after 28 days
to monitor the effect of the cell therapy. As can be observed in
figure 2, both human and porcine MSCs were able to reduce
detrimental cardiac remodeling at 28 days post infarction, since
end systolic and diastolic volumes are smaller in cell injected
groups compared to PBS injected groups. Currently, we are
performing histological analysis of the mice hearts to localize
transplanted cells and if they contributed by trans-differentiation
into cardiovascular cell types.
We have isolated porcine MSC to be able to study the effect of
autologous cell therapy in a preclinical porcine model of chronic
injury. We have demonstrated that isolated pMSC have similar
characteristics as their human counterparts, both in phenotype
as well as in function since their immune-regulatory potential
and effects on cardiac remodeling are similar. We believe
that these cells can be used to explore potential benefit in a
preclinical model to bring cell transplantation therapy to a higher
level for potential future clinical application.
Funding
ICIN-NHS
Figure 2: Left ventricle volumes, determined by MRI (9.4T), at
baseline and 28 days after ligation of the LAD and cell injection
in a mouse model for MI. Both hMSC and pMSC display
similar effects on cardiac dimensions by preventing cardiac
dilation. (human MSC=hMSC, porcine MSC=pMSC, * p<0.05
pMSA vs control, # p<0.05 hMSC vs PBS)
Staff
Willy Noort, PhD (ICIN)
Sridevi Jaksani (ICIN)
Steven Chamuleau, MD, PhD (UMCU)
Pieter Doevendans, MD, PhD (UMCU)
Joost Sluijter, PhD (UMCU)
Figure 1: pMSC suppress proliferation of T cells from pig. Non-stimulated PBMNC were small and CFSE staining
was high (A, B). Stimulation of PBMNC resulted in an increase in size and proliferation (C, D). In (E), the stimulated
cells were not stained. PMA combined with IL-2 and hPBMNC (F), or with pPBMNC (H) showed after 6 days
significant numbers of cells in 4th or 5th division, respectively. When hMSC or pMSC were present in hPBMNC or
pPBMNC cultures, respectively, cells had divided less and lower % of cells were found in 4th or 5th division (G, I).
62
ICIN
Netherlands
Heart Institute
The PRIMA Study
Principal investigator: Y.M. Pinto
Project
Management of Chronic Heart Failure Guided by Individual
N-Terminal Pro-B-Type Natriuretic Peptide Targets: the PRIMAstudy
Aim
The purpose of the main study was to assess whether
management of heart failure (HF) guided by an individualized
N-terminal pro-B-type natriuretic peptide (NT-proBNP)
target would lead to improved outcome compared with HF
management guided by clinical assessment alone.
Methods
A total of 345 patients hospitalized for decompensated,
symptomatic HF with elevated NT-proBNP levels at admission
were included. After discharge, patients were randomized to
either clinically-guided outpatient management (n=171) or
management guided by an individually set NT-proBNP (n=174),
defined by the lowest level at discharge or 2 weeks thereafter.
The primary end point was defined as number of days alive
outside the hospital after index admission.
Progress in 2010
Last year, the main outcome of the PRIMA-study has
been published in JACC1. HF management guided by the
aforementioned individually set NT-proBNP target did not
significantly improve the primary end point (685 vs 664 days,
P = 0.49). In the NT-proBNP guided group a trend was seen
towards lower mortality, as 46 patients died (26.5%) versus 57
(33.3%) in the clinically guided group, but this was not statistically
significant. HF management guided by this individualized NTproBNP target did increase the use of HF medication (P=0.006),
and 64% of the HF-related events were preceded by an
increase in NT-proBNP. Furthermore, time dependent analysis
demonstrated that the individualized NT-proBNP target level
was an effective prognostic marker.
Outpatient elevation of NT-proBNP levels above the
individualized target value indicated an increased risk for
major endpoints such as total mortality (hazard ratio (HR) 1.84,
P=0.007), cardiovascular mortality (HR: 2.53, P < 0.001), and
HF-related mortality (HR: 3.69, P < 0.001).
In 2010 a number of subanalysis of the PRIMA-trial have been
proposed and are currently being analyzed:
- Prognostic value of dynamic markers during admission and
at the outpatient clinic in patients with symptomatic HF
- Define what variance in NT-proBNP levels at the outpatient
63
clinic has no prognostic consequences.
- Differences in NT-proBNP levels and other markers between
patients admitted with ‘de novo’ HF and patients admitted
because of heart failure known with chronic HF.
- Relationship between NT-proBNP levels over time and
echoparameters.
Furthermore data of the PRIMA-study will be used in a meta
analyses assessing the effect of natriuretic guided therapy of
heart failure in a larger population.
Partnership
The following centers have participated in the PRIMA-study:
Academic Medical Center Amsterdam
Amphia Hospital Breda
Atrium Medical Center Heerlen
Deventer Hospital
Erasmus Medical Center Rotterdan
Maastricht University Medical Center
Meander Medical Center Amersfoort
Orbis Medical Center Sittard
Reinier de Graaf Gasthuis Delft
University Medical Center Utrecht
VieCuri Medical Center Venlo
VU University Medical Center Amsterdam
Funding
Main funding of the PRIMA study (> € 200,000) was provided
by the Interuniversity Cardiology Institute of the Netherlands,
the Netherlands Heart Foundation, Netherlands Organisation
for Scientific Research (NWO) and the Royal Netherlands
Academy of Arts and Sciences (KNAW)
Minor funding of an unrestricted research grant (< € 70,000 per
sponsor) was provided by Pfizer, Astra Zeneca, Medtronic, and
Roche Diagnostics.
Staff
Research nurses:
M. Spanjers
M. Blok-Hoos
D. Bekkering
A. Peeters
V. Visser
V. van Wegberg
Study Coördinator:
L. Eurlings
CTMM - Triumph
Principal investigator: L.J. de Windt
Aim and Methods
MicroRNAs (miRNAs) are small non-coding ~22-nucleotide
RNAs that have recently been described as important regulators
of virtually all cellular processes, including heart failure. We
performed microarray analysis of RNA isolated from biopsies
of heart failure patients, and manipulated microRNAs in cell
culture and in mouse models to establish models to discover
new biomarkers in proteomics screens.
Progress in 2010
In the past 6 months, we made a final choice of two microRNAs
dysregulated in human and rodent heart failure as a strategic
choice to focus on for future Milestones within CTMM
TRIUMPH. Initial bioinformatics screens for target genes
of both microRNAs have yielded a common target protein,
and an animal knockout model created from this target. This
knockout animal model displayed a severe congestive heart
failure following conditional gene targeting, delivering a new
and unique mouse model to the consortium to obtain new
biomarkers (proteomics and metabolomics). These findings
were submitted as patent application PCT/NL2009/050484, and
a search report has returned from the European Patent Office.
Discussions are now underway with members of the licensing
group how to pursue both experimental as well as potential
further protection approaches of this IP. Meanwhile, we have
initiated interactions with Andrew Stubbs (WP2) to set up the
“Knowledge Portal” data flow between WP1 and WP2 by reanalyzing the bioinformatics concerning the obtained microRNA
datasets in order to predict additional biomarkers for immediate
proteomic testing by partner BG Medicine in existing human
heart failure samples and for testing in large animal models in
WP3. This “Knowledge Portal” workflow will act as a platform for
the other partners within CTMM TRIUMPH and their datasets.
Partnership
Prof. Dr. Y. Pinto, Academic Medical Center, Amsterdam, the
Netherlands
Prof. Dr. L.J. de Windt, Maastricht University Medical Center,
Maastricht, the Netherlands
Prof. Dr. D. Ducker, Erasmus MC, Rotterdam, the Netherlands
Prof. Dr. H. Hillege, UMCG, Groningen, the Netherlands
Funding staff
PhD students: S. Leptidis (ICIN/CTMM).
Postdocs: H. el Azzouzi (ICIN/CTMM), P. da Costa Martins
(ICIN/CTMM)
64
ICIN
Netherlands
Heart Institute
SYNOP2L in sarcomere formation and stabilization
Principal investigator: C.L. Mummery
Aim and Method of the project
With recent advances in the field of stem cell research,
including reprogramming of somatic cells to pluripotent stem
cells (induced pluripotent stem cells), improved genetic
manipulation and defined cardiac differentiation protocols,
generation of human cardiac disease models are within reach.
In addition, these differentiation models can be used to identify
new molecules which may play important roles in cardiac
development and disease. We have previously identified CHAP,
a novel z-disk protein, by genome-wide transcriptome analysis
in differentiating cardiomyocytes from human embryonic stem
cells (hESC) (Beqqali et al., Stem Cells 2006) and showed its
importance during cardiac development. We further investigated
the role of CHAP in cardiac disease in transgenic mice and its
possible role in cardiac hypertrophy and cardiomyopathy.
Progress in 2010
Recent advances in genetic manipulation of hESC made it
possible to generate fluorescent transgenic hESC lines (Braam
et al. Nat Methods 2008). By homologous recombination GFP
was placed in the locus of Nkx2.5 (collaboration with Monash
University, Australia). This Nkx2.5-GFP hESC reporter line
has been characterized and faithfully marks differentiating
cardiomyocytes. In combination with newly developed
defined high-throughput differentiation protocols, efficiency
of cardiomyocyte differentiation increased to more than 50%
(among the highest efficiencies worldwide) (Figure 1). Higher
percentages of cardiomyocytes can be achieved by FACS
sorting. In that same period the stem cell field has made
impressive advances by the description and generation of
patient-derived induced pluripotent stem (iPS) cells. Recently,
Figure 1: hESC-derived cardiomyoctes from Nkx2.5-GFP
hESC line and stained for Troponin I and DAPI (nuclei)
65
an iPS cell facility was started in our department (Anatomy &
Embryology) in collaboration with the department of Molecular
& Cellular Biology at the LUMC, where iPS cells are generated
from patients with cardiovascular diseases (such as long QT
syndrome and cardiomyopathies). Since hESC and hiPS cells
are comparable regarding phenotype, cell growth, pluripotency
and differentiation, it is expected that the technology advances
in the embryonic stem cell field regarding genetic manipulation
and cardiac differentiation can be applied on iPS cells, which
eventually may lead to purified cardiac cells harboring diseaseassociated mutations. Studies on diseased cardiac cells from
human pluripotent stem cells may provide new insights on the
molecular mechanism of the disease and can be used for drug
screening with the aim to develop tailor-made treatments for
patients. In addition, studies on healthy cardiac cells from human
pluripotent cells may increase our knowledge on the molecular
events that are important for human cardiac development and
the generation of specific cardiovascular cell-types, which
ultimately may support the fields of tissue engineering and
regenerative medicine. Furthermore, it is not unusual that factors
and signaling pathways important for cardiac development also
play a role in cardiac disease. We have recently identified a
novel protein, named CHAP (Cytoskeletal Heart-enriched Actinassociated Protein), expressed in heart and skeletal muscle,
for which its role is studied during muscle development and
disease.
CHAP is localized at the Z-disc of sarcomeres and interacts and
co-localizes with other Z-disc proteins, α-actinin, calcineurin
and calsarcin-1 and -2. Two isoforms of CHAP exist, the longer
isoform CHAPa which contains a PDZ-domain and nuclear
localization signal (NLS) and the shorter isoform CHAPb, which
lacks the PDZ-domain. ChapB is predominantly expressed
during early cardiac and skeletal muscle development,
whereas ChapA is only expressed in adult heart and skeletal
muscle. Interestingly, in addition to its sarcomeric localization,
CHAP was also able to translocate to the nucleus. CHAP was
associated with filamentous actin in the cytoplasm and the
nucleus when expressed ectopically in vitro, but in rat neonatal
cardiomyocytes, CHAP disrupted the subcellular localization of
a-actinin, another Z-disc protein. We have shown by loss-offunction studies in zebrafish that CHAP plays an important role
in cardiac development and maintenance of muscle integrity
(Beqqali et al. JCS 2010) (Figure 2). To unravel the function
of CHAPb in adult mice, we generated transgenic (Tg) mice,
which express CHAPb under control of the heart-specific
alpha-myosin heavy chain promoter. Although at 1 month of
age no clear phenotype was observed in CHAPb Tg mice,
at 3 months of age hearts showed an enlargement of the left
atrium and the appearance of stress fibers, which co-localized
with α-actinin, aligned with cardiomyocytes. Furthermore, in
isolated cardiomyocytes a decrease in calcium sensitivity and
maximal contractility of myofilaments was observed. However,
no signs of cardiac hypertrophy were observed yet. Further
progression of the phenotype was observed in 6 months old
CHAPb Tg animals, which displayed cardiac hypertrophy
and an enlarged left atrium. The hypertrophic response was
indicated by increase in wall thickness, cardiomyocyte size and
expression of hypertrophic markers (Nppa, Nppb and Myh7). In
addition, interstitial cardiac fibrosis was observed by Sirius red
staining and increase of collagens I and III mRNA expression
in ventricles. In the enlarged left atrium expression of Connexin
40 and 43 was down-regulated. Functional analysis by MRI
showed that 6 months CHAPb Tg mice showed impaired heart
function and cardiac hypertrophy when compared to wild type
mice. In conclusion, overexpression of CHAPb in vivo causes a
phenotype that resembles hypertrophic cardiomyopathy (HCM)
in humans and therefore may represent a novel candidate gene
for screening mutations in patients with HCM.
Partnerships
Dr. Jeroen Bakkers, Hubrecht Insitute, Developmental Biology
and Stem Cell Research, Utrecht, the Netherlands.
Dr. Elisabeth Ehler, The Randall Division of Cell & Molecular
Biophysics and the Cardiovascular Division, King’s College
London, London, UK.
Dr. Jolanda van der Velden, Laboratory for Physiology, Institute
for Cardiovascular Research, VU University Medical Center,
Amsterdam, The Netherlands.
Dr. Ed Stanley and Dr. Andrew Elefanty, Monash Immunology
and Stem Cell Laboratories (MISCL), Monash University,
Clayton, Australia.
Funding
This study was supported by Bsik Programme “Stem Cells in
Development and Disease”, the Netherlands Heart Foundation
grant 2006B209 and by European Community’s Sixth Framework
Programme contract (“HeartRepair”) LSHM-CT-2005-018630.
Staff
Dr.Abdelaziz Beqqali (until 1-5-2010): post-doc (LUMC)
Willemijn van Eldik: appointed PhD-student (ICIN)
Jantine Monshouwer-Kloots: technician (LUMC)
Prof. Christine Mummery: co-projectleader (LUMC)
Dr. Robert Passier: co-projectleader (LUMC)
Figure 2: (A) Immunoprecipitation (IP) of actin from COS1 cells expressing FLAG-CHAPb. No interaction with
FLAGCHAPb could be detected. (B) Co-IP of CHAPb
with FLAG-a-actinin-2 using anti-FLAG for IP (left panel).
Conversely, FLAG-a-actinin-2 co-IPed with CHAPb using
CHAP antibody for IP (lower panel). (C) Immunostaining for
a-actinin-2 in neonatal rat cardiomyocytes overexpressing
CHAPb-GFP revealed disorganized aactinin-2 while myomesin
localization was unaffected. Untransfected adjacent cells
showed normal a-actinin-2 expression. Scale bar: 10 mm.
(D) Ultrastructural analysis of somites of chap1 morphants
at 3 dpf. Wild-type and control MO-injected fish have normal
sarcomere structure, whereas chap1 morphants have severely
disrupted Z-discs (arrowheads) and sarcomeres. Scale bars:
2 mm (upper panel); 500 nm (lower panel). Adapted from
Beqqali et al JCS 2010.
66
ICIN
Research line - Imaging
Netherlands
Heart Institute
Myocardial viability with MRI
Principal investigator: G. Pasterkamp
Study1: 23Na Chemical Shift Imaging of myocardial
edema
Distinction between viable and non-viable myocardium is
essential for diagnosis and treatment of patients with coronary
artery disease. Delayed contrast-enhanced (DCE) MRI has
become the gold standard to determine the size of chronic
infarcts. However, although T2-weigthed MRI shows great
promise to delineate the area at risk after the event related to
the formation of edema, the accuracy of DCE-MRI to determine
the size of acute infarcts has been questioned because of
that same edema. As an alternative approach we would like
to propose 23Na Chemical Shift Imaging (CSI) with the aid of
a shift reagent. In edematous, but viable tissue we expect the
extracellular Na+ signal (Na+e) to be increased but little change
in the intracellular Na+ signal (Na+i). In acute infarcts with cell
membranes still intact, we expect Na+i to be very high, whereas
in chronic infarcts Na+e should be very high and Na+i should
be absent. To further explore this methodology, we have created
an isolated, perfused heart model of extracellular edema based
on different perfusion pressures, which has been characterized
using 31P MRS. Subsequently, we have characterized the
formation of edema by both DCE 1H MRI and 23Na CSI.
Isolated rat hearts were perfused with modified Krebs-Henseleit
buffer according to Langendorff and MRI and MRS scans were
performed with a 9.4T scanner (Bruker, Germany). We executed
31P-MRS, 23Na CSI and DCE-MRI.
Figure 1 shows the PPA signal intensity normalized to heart
dry weight at 60 and 140mmHg. Data were significantly
(P<0.03) different from 50min onwards and the ratio of PPA at
140mmHg/60mmHg at 70min was 1.27 ±0.21. As can be readily
seen in the 23Na CSI and the 1H MRI (Fig 2), the cross sectional
area (CSA) of the hearts following perfusion at 140mmHg was
increased. The Nae images yield a WH CSA of 1.32±0.11cm
at 60mmHg, increasing to 1.89±0.16 at 140mmHg with a
140mmHg/60mmHg ratio of 1.44±0.09, in good agreement with
the PPA data. The data from the 1H images were 1.37±0.04,
1.86±0.14 and 1.34±0.09, respectively, demonstrating good
agreement between 23Na and 1H data. When analyzing the LV
wall, similar data were obtained. In addition to the increase in
CSA, we also found an increase in LV Nae signal intensity, going
from 1.04±0.20 (arbitrary units, a.u.) at 60mmHg to 1.37±0.20
at 140mmHg, with a 140/60 ratio of 1.33±0.07. Unexpectedly,
we also found an increase in LV Nai signal intensity, going from
1.57±0.83 (a.u.) at 60mmHg to 1.79±0.97 at 140mmHg, with a
140/60 ratio of 1.15±0.11. With DCE-MRI, LV signal intensity
increased from 1.39±0.14 (a.u.) at 60mmHg to 1.50±0.22 at 140
mmHg, with a 140/60 ratio of 1.08±0.08.
> Fig 2: 23Na-CSI of extracellular and intracellular Na and Gdenhanced 1H MRI at perfusion pressures of 60 and 140mmHg.
There are a few limitations to this study that need to be considered.
First, the DCE-MRI protocol uses continuous perfusion of
contrast agent without nulling of ‘remote’ myocardium, so direct
comparison of the contribution of edema in a clinical protocol is
difficult. The separation of intra- and extracellular Na+ signals
is incomplete. Furthermore, the spatial resolution in the 1H
images is much higher than in the 23Na images. Finally, both
the DCE MRI and Nae images show spatial heterogeneity, i.e.
a mid-myocardial rim of different signal intensity, which is lost in
the average signal, but may be related to this edema model and
requires further analysis.
We conclude from this study that perfusion of hearts with
crystalline buffers at high perfusion pressures leads to formation
of interstitial edema, as evidenced by a larger distribution
volume for PPA, Nae and Gd. Unexpectedly, images of Nai
also showed an, albeit smaller, increase in signal intensity,
which could be explained by altered ion homeostasis, but could
also reflect imperfect separation of intra- and extracellular
signals. Whether 23Na CSI is better capable than DCE-MRI to
distinguish between normal, edematous and infarcted tissue in
both acute and chronic myocardial infarction requires further
experiments in an infarct model.
Figure 1: 31P-MRS signal from PPA normalized to heart dry
weight as function of perfusion time at 60 and 140mmHg.
68
ICIN
Study 2: Longitudinal assessment of T2* changes in
mouse myocardium following ischemia-reperfusion
Background
Ischemic injury triggers a cascade of histopathological changes,
which may lead to a progressive decline in heart performance.
Noninvasive techniques to characterize infarct development
are essential to assess efficacy of novel therapeutics. Late
gadolinium enhancement (LGE) MRI is a useful technique to
quantify infarct size. However, LGE may overestimate acute
infarct size due to the presence of edema. The aim of this
study was to explore the utility of quantitative T2* mapping as
a noninvasive technique to characterize the myocardium in the
acute and chronic phases following ischemia/reperfusion injury
in the mouse.
Netherlands
Heart Institute
Corresponding T2* maps revealed dynamic change throughout
the follow up period. The presence of infarction was further
corroborated by the Cine images, which showed increased wall
thickness (WT) in the mid-anterior and mid-anterolateral wall at
day 1, followed by progressive thinning below baseline levels at
days 7 and 28.
Conclusion
In the acute phase, the infarct area exhibited a decrease in T2*
both in infarct and remote areas and it was best visualized by
LGE. In the chronic phase, T2* further decreased in the infarct
area during scar maturation whereas it remained constant in the
remote area. Thus, T2* may enable distinction between acute
and chronic myocardial infarctions, providing complementary
information to LGE.
Staff
Eissah Aguor (ICIN)
Marcel Nederhoff (ICIN)
Kees van de Kolk (UMCU)
Figure 3
Figure 3 represents a collection of images, consisting of LGE,
T2* maps, and Cine MRI for a typical mouse at day 1, 7 and
28 after induction of the myocardial infarction. On day 1, LGE
resulted in a homogeneous enhancement of the infarction in the
mid-anterior and mid-anterolateral wall, whereas on days 7 and
28 enhanced area was smaller, heterogeneous, and difficult to
detect.
69
Funding
NHS, ICIN
MARCC Study
Principal investigator: A.C. van Rossum
Project
Towards a non-invasive anatomical and functional diagnostic
work-up of patients with suspected coronary artery disease using
Cardiovascular Magnetic resonance imaging and Multidetector
CT: the MAgnetic Resonance and CT in suspected Coronary
artery disease (MARCC) study.
Aim and method of the project
Primary objectives are to determine diagnostic accuracy
of cardiovascular magnetic resonance imaging (CMR) and
multidetector computed tomography for detection of significant
coronary artery disease (CAD) and to develop the most optimal
diagnostic algorithm involving the sequential use of both
imaging techniques. Second objective is to develop quantitative
methods for analysis of CMR perfusion data.
A total of 210 patients with chest pain and low to intermediate
probability CAD will be included. These patients will undergo
CT coronary calcium scoring, 64-slice CTCA and CMR
imaging (involving assessment of left ventricular function,
adenosine stress and rest myocardial perfusion and delayed
contrast enhancement). These non-invasive parameters will
be compared to invasive coronary angiography and fractional
flow reserve measurements. All patients will be followed up
after 1 year by telephone interview for clinical status, an interval
diagnosis of significant CAD on invasive coronary angiography
and major adverse cardiovascular events. Primary endpoints
are the diagnostic performance of these imaging modalities for
detection of significant CAD on invasive coronary angiography
and 1-year clinical outcome. From these data a new diagnostic
algorithm (decision-tree model) will be developed involving the
sequential use of these imaging modalities. Finally, the costeffectiveness of the proposed algorithm will be compared to
conventional strategies involving exercise electrocardiography
and SPECT.
Progress in 2010
In 2010 patient inclusion and 1-year follow-up of all patients
has been completed. In a first study, we compared computed
tomography coronary angiography (CTCA) to adenosine stress
and rest first pass magnetic resonance myocardial perfusion
imaging in patients with low-to intermediate probability CAD.
In this study the complementary role of CTCA and CMR in
the evaluation of patients with suspected CAD was shown.
CTCA can be used to reliably rule out CAD, but its capability
to demonstrate hemodynamically significant CAD is limited.
Furthermore, we showed that the positive predictive value of
CTCA for detection of significant CAD in patients with low to
intermediate probability CAD that are clinically referred for
non-invasive evaluation of chest pain is markedly lower than
generally reported. In a third study comprising 210 patients, we
showed the advantages of the combination of CTCA and CMR
for the diagnostic evaluation of patients with suspected CAD in
clinical practice. The combined work-up significantly improved
specificity and overall diagnostic accuracy for the detection of
significant CAD, and allowed the detection of alternative (extra-)
cardiac disease in patients without significant CAD.
Figure: Computed tomography coronary angiography (CTCA)
and first pass magnetic resonance myocardial perfusion
imaging (MRMPI) images of a 42-year old male with atypical
chest pain. CTCA curved multiplanar reconstruction images of
left anterior descending (A), circumflex (B) and right coronary
artery (C) showing single vessel disease with obstructive
coronary artery disease in the proximal left anterior descending
artery. MRMPI images of the mid ventricular short axis view
during adenosine infusion (D-F) and at rest (G-I) showing a
reversible perfusion defect in the myocardial septum.
Partnerships
Software developments for image postprocessing and analysis
of CMR perfusion data is obtained through collaboration with
LKEB of the LUMC (J Reiber and R van der Geest).
Funding
This project is supported by a research grant from the
Netherlands organization for health research and development
(ZonMw grant number 80-82305-98-09029).
Staff
AM Beek (VUmc, dept. of Cardiology)
SL Brinckman (VUmc, dept. of Cardiology)
JGJ Groothuis (ICIN)
AC van Rossum. (VUmc, dept. of Cardiology)
MR Meijerink (VUmc, dept. of Radiology)
C van Kuijk (VUmc, dept. of Radiology)
MBM Hofman (VUmc, dept of Physics and medical techn.)
J Reiber (LUMC, LKEB)
R van der Geest (LUMC, LKEB)
70
ICIN
Netherlands
Heart Institute
Therapeutic application of ultrasound
Principal investigator: O. Kamp
Aim and method of the project
For the past few decades ultrasound is used in the cardiology as a
diagnostic tool with multiple applications. Recent developments
have made it possible to use ultrasound as a therapeutic tool,
especially in the field of coronary artery disease.
In the Sonolysis trial we are evaluating the effect of 3D
ultrasound in combination with microbubbles and thrombolytics
on the improvement of fibrinolysis in STEMI patients, epicardialy
and microvasculature.
Furthermore, a new non-invasive therapy based on ultrasound
was recently developed to create angiogenesis. We are studying
the effect of this treatment in ischemic cardiomyopathy patients
with end stage coronary artery disease.
Progress in 2010
In 2009 we have included the 10th patient in the Sonolysis study.
In this study we hypothesize that ultrasound in combination
with ultrasound contrast agents and thrombolytics will result
in a significant increase in percentage of patients with TIMI3 flow compared to the placebo group (thrombolytics alone).
In the 10 included patients there seems to be a trend towards
more TIMI-3 flow in the treated group, without any increase in
major adverse cardiologic events. However, more patients need
to be included. Unfortunately, this study has temporarily been
stopped, due to supply problems of the microbubbles. This
study will restart in 2011 after an extensive search for a new
supplier of the microbubbles.
In 2009 we started with treatment of ischemic cardiomyopathy
patients in the Shockwave study. The shockwave therapy
is based on a high energy pulsed wave, which induces
angiogenesis by means of different mechanical, metabolic
and chemical factors. This angiogenesis might possibly be a
new, valuable effect for patients with end stage coronary artery
disease.
Until now, we have included 14 patients with ischemic
cardiomyopathy who were not eligible for surgery of coronary
intervention. At baseline and 3 months after treatment we
perform either a cardiac MRI or a dobutamine stress echo to
observe the improvement in myocardial blood flow and reverse
remodelling after shockwave therapy. Furthermore, we also
evaluate the wall motion abnormalities, left ventricular function
and NYHA class.
71
The cooperation with the department of anaesthesiology also
started in 2009. This project aims to gain insight in anesthesiarelated effects towards autonomic control of coronary blood flow
(using myocardial contrast echocardiography) in healthy subjects
and in patients with cardiovascular autonomic neuropathy, by
quantitatively and qualitatively analyzing MCE. With the results
of the current study, we expect to show the relation between
anesthetic-induced alterations in cardiovascular autonomic
regulation and myocardial blood flow.
In 2010 we have included 14 patients so far.
Furthermore, we started a joint research with the department
of vascular surgery investigating the effect of ultrasound and
microbubbles on thrombotic occlusions of the a. iliaca in pigs.
In a pilot study in 6 pigs we have tested the application of
microbubbles in the anesthetized pigs. In 2011 we will start the
actual study.
Figure 1
Staff
PhD student: Jeroen Slikkerveer
Karin de Boer, MD (VUmc, dep. of cardiology)
Carolien S.E. Bulte, MD (VUmc, dep. of anaesthesiology)
R. Arthur Bouwman, MD, PhD (VUmc, dep. of anaesthesiology)
Stephan A. Loer, MD, PhD (VUmc, dep. of anaesthesiology)
Kakkhee K. Yeung, MD (VUmc, dep. of vascular surgery)
J.Hillian. Nederhoed, MD (VUmc, dep. of vascular surgery)
Willem Wisselink, MD, PhD (VUmc, dep. of vascular surgery)
Yolande Appelman, MD, PhD (VUmc/ICIN, dep. of cardiology)
Albert C. van Rossum, MD, PhD (VUmc/ICIN, dep. of cardiology)
Thomas R. Porter, MD, PhD (University of Nebraska Medical
Center, dep. of cardiology)
Otto Kamp, MD, PhD (Vumc/ICIN, dep. of cardiology)
Directing stem cells using targeted microbubbles
Principal investigator: O. Kamp
Directing adipose tissue derived stem cells to the area at
risk in the heart after myocardial infarction using targeted
microbubbles. - Development of new molecular technique –
Aim and method of the project
Stem cell therapy is a promising tool to restore contractile
function after myocardial infarction. However, recent clinical
trials show rather disappointing results with only minor
improvements in cardiac function. Therefore, stem cell research
needs to return from bed to bench.
The major problem with stem cell therapy is the lack of
persistence of sufficient numbers of stem cells at the site of
injury. Less than 3% of the cells remain at the infarction site
after injection, independent of the route of administration. It is
not known what exactly happens to the other cells, because it is
difficult to track these cells in vivo directly after injection.
This project aims to overcome this problem by specifically
targeting the stem cells to the area at risk after myocardial
infarction. Adipose-derived stem cells will be coupled to contrast
microbubbles, this stem cell-bubble complex will be targeted to
specific molecules on endothelium of the injured vessel wall,
illustrated in fig. 1. This will result in larger quantities of stem
cells in the area at risk, thereby improving regeneration of the
heart. Besides the possibility of carrying targeting ligands on
the microbubbles, the presence of microbubbles has two other
main functionalities: 1) Microbubbles can be pushed towards the
vessel wall using the radiation force of diagnostic ultrasound.
This acoustic radiation force can also be applied to the stem
cell-bubble complex, thereby facilitating the binding of the
stem cell-bubble complex to the endothelium. 2) Imaging and
tracking of individual stem cell-bubble complexes with contrastenhanced ultrasound, to investigate the fate of the stem cells
after injection. We anticipate five main tasks in the project:
1. Construction of the dual-targeted microbubbles
2. Mechanical ultrasound – bubble – cell interactions
3. Evaluation of the stem cell-bubble complex in cell culture
4. Proof of principle in a small animal study
5. Large animal study and ultrasound imaging
Progress in 2010
Ad 1) Construction dual-targeted microbubbles. We successfully
created dual-targeted microbubbles (fig 2) that will bind to
the stem cells via cell surface marker CD90, and to activated
endothelial cells via ICAM-1. For now, the molecule we choose
as target is ICAM-1. We performed immunohistochemistry
on human slices of myocardial infarction for several markers,
i.e. P-selectin, VCAM-1 and ICAM-1. The best results were
obtained for ICAM-1. Although ICAM-1 seems to be specifically
upregulated in the infarcted area, it is also a general marker
of inflammation. Therefore, we performed a study to find a
marker that is specifically upregulated in the infarcted area.
In this study we occluded the left coronary artery for 40 min,
followed by reperfusion. Rats were sacrificed and a micro-array
was performed on mRNA from the infarcted heart. The results
of this micro-array will be analyzed and confirmed by PCR and
immunohistological stainings.
Ad 2) Mechanical ultrasound – bubble – cell interactions.
A setup including the ultrafast framing Brandaris128 camera
has been used to quantify the secondary acoustic radiation
force between two biotinylated microbubbles targeted to a
NeutrAvidin coated surface. This setup will be used to determine
the binding force of targeted microbubbles to a coated surface.
During insonification with ultrasound (2.25 MHz), the distance in
between two targeted microbubbles was shown to decrease with
several hundreds of nm (up to 700 nm), after which the bubbles
were still able to move back to their equilibrium position within
80 μs. However, when the secondary acoustic radiation force
exceeded a certain threshold, which was in between 200 nN 400 nN in these experiments, the microbubbles were detached
from the surface. A model including microbubble deformation
was proposed: the restoring force due to microbubble
deformation opposes the secondary acoustic radiation force.
In this situation the adhesion contact area remains unaltered.
However, when the restoring force (due to deformation) exceeds
the microbubble binding force to the target, the microbubble
will be detached. The point of unbinding (lipid pulled out of the
microbubble shell or breakage of the biotin-NeutrAvidin bond)
needs further investigation, for which fluorescent microbubbles
will be used. Using this model, the microbubble binding force
will be distilled from these data.
To study the effect of primary acoustic radiation force on the
targeting efficiency of microbubbles and eventually the targeting
efficiency of the stem cell-bubble complex, a new setup was
designed and constructed. In this setup a parallel plate flow
72
ICIN
cell chamber (provided by Philips) can be mounted on top of
a watertank holding an illumination fibre and 1 MHz ultrasound
transducer. The most efficient parameters (e.g. pressure,
number of cycles, duty cycle) for pushing the stem cell-bubble
complex under flow conditions from the centerline of the flow
to the target wall will be derived from these experiments. Goal
of these experiments is to enhance the targeting efficiency of
the stem cell-bubble complex under flow conditions. The results
obtained with this setup, in combination with calculations, will be
valuable for the in vivo experiments, scheduled at a later stage.
Ad 3) Evaluation of the stem cell-bubble complex in cell culture.
Stem cell culture. Most results in 2010 have been obtained with
the study on shifting stem cell culture completely from foetal
bovine serum (FBS) as a culture supplement to animal-free,
clinical grade platelet lysate (PL). We tested the effect of PL
on size, proliferation, rate of attachment, cell surface markers,
transmigration behavior and differentiation capacity. We found
that stem cells cultured on PL-supplemented medium performed
equal (differentiation) or better (smaller size, proliferation, rate
of attachment, transmigration) compared to cells grown on
FBS-supplemented medium. We now culture all our stem cells
on PL. Manuscript in preparation.
Flow experiments. The binding of the complexes will be studied
under flow conditions. Transmigration. We will investigate
whether the stem cells retain their capacity to extravasate while
they have numerous bubbles attached to their cell membrane
in a transwell assay. First experiments demonstrated that our
stem cells without microbubbles are able to migrate through
endothelial cells. In the following experiments the effect of
microbubbles will be tested.
Differentiation by ultrasound and microbubbles. The effect
of ultrasound and targeted bubbles on the differentiation and
viability of the stem cells will be investigated.
Ad 4) Proof-of-principle in a small animal study. For this we
will induce an acute myocardial infarction in rats via a ligation
on the LAD for 40 minutes, followed by reperfusion. First pilot
experiments are planned for May 2011.
Ad 5) Large animal study and ultrasound imaging. This study is
more specifically aiming to image and track the stem cell-bubble
complexes after injection. For this we will induce a myocardial
infarction in pigs, because a pig heart resembles a human
heart especially in size, and therefore a clinical echo machine
can be used for the detection of the targeted stem cell-bubble
complexes.
73
Netherlands
Heart Institute
This study is designed to investigate different 3D transducers
and software for optimal imaging quality of single cell-bubble
complexes, and is planned for the 4th year of this project.
Partnerships
This project is a collaboration between the dept. of Biomedical
Engineering, Thorax Centre at the Erasmus Medical Centre in
Rotterdam, and different depts. of the Institute for Cardiovascular
Research at the VU University Medical Center in Amsterdam.
Funding
The project is funded by STW, together with a financial
contribution by Oldelft, Targeson and Sanquin.
Staff
A. van Dijk, Dept. of Pathology, Institute for Cardiovascular
Research VU University Medical Center, Amsterdam, the
Netherlands
BA. Naaijkens (PhD student), Dept. of Pathology, Institute
for Cardiovascular Research VU University Medical Center,
Amsterdam, the Netherlands, ICIN
HWM. Niessen, Dept. of Pathology, Institute for Cardiovascular
Research VU University Medical Center, Amsterdam, the
Netherlands
VWM. van Hinsbergh, Dept. of Physiology, Institute for
Cardiovascular Research VU University Medical Center,
Amsterdam, the Netherlands
RJP. Musters, Dept. of Physiology, Institute for Cardiovascular
Research VU University Medical Center, Amsterdam, the
Netherlands
M. Helder, Dept. of Orthopaedic Surgery, VU University Medical
Center, Amsterdam, the Netherlands
O. Kamp, Dept. of Cardiology, Institute for Cardiovascular
Research VU University Medical Center, Amsterdam, the
Netherlands, ICIN
LJM. Juffermans (post-doc), Dept. of Physiology, Dept. of
Cardiology, Institute for Cardiovascular Research VU University
Medical Center, Amsterdam, the Netherlands, ICIN
TJA. Kokhuis (PhD student), Dept. of Biomedical Engineering,
Thorax Centre, Erasmus Medical Centre, Rotterdam, the
Netherlands, ICIN
M. Harteveld, Dept. of Biomedical Engineering, Thorax Centre,
Erasmus Medical Centre, Rotterdam, the Netherlands
K. Kooiman, Dept. of Biomedical Engineering, Thorax Centre,
Erasmus Medical Centre, Rotterdam, the Netherlands, ICIN
N. de Jong, Dept. of Biomedical Engineering, Thorax Centre,
Erasmus Medical Centre, Rotterdam, the Netherlands, ICIN
Implications of CTA Findings
Principal investigator: J.J. Bax
Study project
Implications of CTA findings for subsequent management:
Relationship between 64-slice CTA coronary angiography,
SPECT imaging and conventional coronary angiography
Aim of the project
Non-invasive imaging plays a key role in the evaluation of
coronary artery disease (CAD). Myocardial perfusion imaging
with single photon-emission computed tomography (SPECT)
is widely available and frequently used. In recent years, noninvasive assessment of cardiac anatomy has become available
with the introduction of multi-slice computed tomography
coronary angiography (CTA). The technique has matured
rapidly and its introduction has resulted in a shift from functional
imaging to non-invasive assessment of the coronary anatomy.
Diagnostic and prognostic comparison between anatomic and
functional imaging of CAD was performed.
Progress in 2010
The diagnostic and prognostic value of CTA was further
evaluated in dedicated populations. Data on diagnostic accuracy
of CTA have mostly been derived in patients with high pre-test
likelihood of CAD but not in patients with an intermediate pretest likelihood (which represent the target population for CTA).
61 patients (61% male, age 57±9 years) referred for invasive
coronary angiography underwent additional 64-slice CTA; 920
segments were identified on invasive coronary angiography
of which 885 (96%) were interpretable on CTA. Results are
summarized in Table 1. On a patient level, sensitivity, specificity,
positive predictive value, and negative predictive value were
respectively 100%, 89%, 76%, and 100%. These observations
confirm the good diagnostic accuracy in patients with an
intermediate pre-test likelihood. Notably, the high negative
predictive value allowed rule out of significant stenosis, and CTA
can be used as gatekeeper for invasive coronary angiography.
The prognostic value of CTA was evaluated in patients with
diabetes (DM). Patients with DM are known to be at elevated
risk for CAD and accurate risk stratification is therefore of
paramount importance. 313 patients with DM, group I (males
213, age 62±11 years), and 303 non DM patients, group II
(males 203, age 63±11 years), underwent clinical evaluation
with 64-row CTA. CTA scans were classified as normal, nonobstructive CAD (≤50% luminal narrowing, and obstructive CAD
(>50% luminal narrowing).
Patients were followed for a mean of 20±5 (range 6-44)
months. At multivariate analysis DM (p<0.001) and evidence
of obstructive CAD (p<0.001) were independent predictors of
outcome. Obstructive CAD remained a significant multivariate
predictor both in DM and non DM patients. Both in DM and non
DM patients, the event rate was 0% in the absence of CAD. The
event rate increased to 36% in non DM patients and 47% in DM
patients with obstructive CAD. These observations indicate that
both in DM and non DM patients CTA is clinically useful for risk
stratification.
Funding
Netherlands Society of Cardiology
Partnerships
The University Hospital Zurich, Switzerland (Prof. dr. P.A.
Kaufmann), the Cardiovascular Center, Aalst, Belgium (Prof. Dr.
W. Wijns) and the Liverpool Hospital, University of New South
Wales, Sydney, Australia (Prof. dr. D.Y. Leung); The University
of Parma (Dr. F. Cademartiri); The Medical Center Haaglanden
(Dr. J.H. Schreur).
Staff:
Drs. J.M. van Werkhoven
Dr. J.D. Schuijf
Prof. Dr. J.W. Jukema
Prof. Dr. J.J. Bax
Table 1: Diagnostic accuracy of CTA
Variable
Segmental Analysis
Vessel Analysis
Patient Analysis
Excluded
35/920 (3.8%)
0%
0%
Sensitivity
23/29 (79%, 64-94%)
22/26 (85%, 71-99%)
17/17 (100%, 100-100%)
Specificity
841/856 (98%, 97-99%)
148/157 (94%, 90-98%)
40/44 (98%, 83-100%)
Positive predictive value
23/38 (61%, 46-77%)
22/31 (71%, 55-87%)
17/21 (81%, 64-98%)
Negative predictive value
841/847 (99%, 99-100%)
148/152 (97 (94-100%)
40/40 (100%, 100-100%)
Diagnostic accuracy
864/885 (98%, 97%-99%)
170/183 (93%, 89-97%)
57/61 (93%, 86-99%)
74
ICIN
Netherlands
Heart Institute
Modification of Risk with CT Coronary Angiography
Principal investigator: J.J. Bax
Project
Modification of Risk with CT Coronary Angiography in cardiac
high-risk asymptomatic patients.
Aim and method
The aim of the multi-center study (Erasmus MC Rotterdam,
LUMC Leiden, UMC Utrecht) entitled “Modification of risk with
MSCT – coronary angiography in high-risk cardiac asymptomatic
patients”, which is funded by the Netherlands Heart Foundation,
is to assess whether CT coronary angiography can modify
the risk of adverse cardiovascular events of patients without
symptoms for coronary artery disease (CAD) but who are at
high-risk based on traditional risk factors. High-risk patients are
patients with diabetes mellitus, familial hypercholesterolemia,
peripheral vascular disease and elevated risk of CAD defined as
>10% risk on cardiovascular mortality and morbidity according
to the SCORE adjusted for the Netherlands. In these patients,
non-enhanced coronary calcium scan will be performed to
obtain the coronary artery calcium score (CACS) according
to the Agatston approach. In addition all patients will undergo
a contrast-enhanced CT scan to determine the presence
and degree of coronary atherosclerosis (extent, distribution
and severity of coronary plaques and plaque characteristics:
calcified, non-calcified and mixed). The severity of coronary
stenosis will initially be evaluated using a dichotomous approach
(50% stenosis as a cut-off). Adverse cardiovascular events (allcause mortality, cardiac mortality, non-fatal myocardial infarction
and stroke) will be documented for all patients during a 5-year
follow-up period. At present, enrollment is still ongoing.
Figure 1A:Volume Rendered Reconstruction of a chronic
total occlusion (CTO) (arrow) of the right coronary artery
(RCA) in this asymptomatic, 52 year old man with Diabetes.
The proximal part of the left anterior descending coronary
artery (LAD) shows additional calcified lesions. LM=left main
coronary artery.
Progress 2010
A total of 645 patients successfully underwent Calcium scanning
and CT coronary angiography. Preliminary collection of follow-up
data revealed cardiac death in 3 patients. Myocardial infarction
in 1 patient, and unstable angina requiring hospitalization in 3
patients.
Revascularization due to angina was necessary in 19 patients.
Follow-up data collection will be continued during the study.
Familial Hypercholesterolemia
In the Erasmus MC Rotterdam a total of 132 patients with
FH have been studied of which 101 patients have been fully
analysed.
One hundred and one cardiac asymptomatic FH patients (62
men; mean age 53±7 years) underwent CACS and CT coronary
angiography using a Dual Source CT scanner.
Results
The CACS was 0 in 20% of the patients. In total 485 plaques
were detected with CT coronary angiography, of which 10% were
obstructive. In 15% of the patients there was no CT-evidence of
any coronary artery disease while 27% had obstructive disease.
Men exhibited more advanced CAD at a younger age than
women. Fifty percent of all plaques were calcified, 28% noncalcified and 22% mixed. The extent of CAD was compared to
120 individuals with high-risk features and there was significant
75
B:Multi Planar Reconstruction (MPR) of right coronary artery
showing a chronic total occlusion (CTO) of the RCA over a
total length of 3,5 cm.
less coronary artery disease compared to the patients with FH.
Conclusion; the presence of CAD was accelerated in middleaged FH patients with more than a quarter having obstructive
CAD, despite ‘normalized’ cholesterol levels on intense statin
treatment. This study is submitted for publication.
Asymptomatic patients with Diabetes
In EMC 80 patients with DM were studied. The demographics
were: male 51%, age 57 years, smoking 31%, dyslipidemia
77%, hypertension 27%, family history 53% and total cholesterol
5.4 mmol/L.
Calcium score 0: 20%, 1-100: 32%, 100-400: 24%, >400: 24%.
CT CA obstructive CAD 26%, non-obstructive CAD 60% and no
CAD 24% (figure 1).
The segmental distribution of CAD in the coronary tree is shown
in figure 2 for patients with DM and FH. Overall the extent and
severity of CAD was comparable in both patient populations.
In LUMC 124 patients with DM were studied. The age was 61
±21 years and 57% were males. Calcium score of <10: 40%,
10-400: 40%, >400: 20% was found and
CT CA revealed obstructive CAD: 33% and non-obstructive
CAD: 50% of the patients.
High-Risk individuals
A total of 130 patients have been evaluated in EMC. The mean
age was 56 years and 65% were males. The majority had more
than 1 risk factor. Total cholesterol was 5.0 mol/L.
Calcium score of 0: 40%, 1-100: 35%, 100-44: 15% and >400:
10%.
CT CA, no CAD: 33%, non-obstructive CAD: 42% and
obstructive CAD: 24%.
A total of 55 patients, age 65±14 years, males 75% were
evaluated in LUMC.
Calcium score: <10: 37%, 10-400: 47%, >400 : 16%
CTCA: obstructive CAD: 18%, non-obstructive CAD: 60%.
Partnerships
Multicenter Study with Erasmus MC, LUMC and UMCU.
UMCU has started recruitment of patients in December 2010.
Funding
Netherlands Heart Foundation (NHS) grant number 2006T102
Figure 2: Distribition of significant coronary artery disease as
detected by CT CA in patients with both Diabetes Mellitus and
Hypercholesterolemia. DM=Diabetes Mellitus, FH= Familial
Hypercholesterolemia.
Staff
Drs. L.A. Neefjes, ICIN, EMC
Drs. G.J. ten Kate ICIN , EMC
Prof. P. J. de Feyter, EMC
Dr. E.J. Sybrands, EMC
Dr. J.G. Langendonk, EMC
Mrs. A.J. Galema-Boers, EMC
Drs. M. J. Boogers, ICIN
Dr. J. D. Schuijf, LUMC
Prof. J.J. Bax, LUMC
Drs H. El Aidi, ICIN , UMCU
Prof. P.A. Doevendans, UMCU
76
ICIN
Netherlands
Heart Institute
Cardiovascular screening of asymptomatic patients
Principal investigator: J.W. Jukema
Project
Cardiovascular screening of asymptomatic patients with
diabetes mellitus
Aim of the project
Worldwide, the prevalence of diabetes mellitus (DM), which
in turn is an important risk factor for cardiovascular disease,
is increasing. Importantly, coronary artery disease (CAD) in
diabetic patients progresses more often asymptomatic (silent
ischemia), leading to more advanced disease at time of
diagnosis. In these patients progression of CAD can possibly be
delayed by aggressive treatment and adverse events prevented
by early intervention. Therefore, it is highly desirable to identify
patients at higher risk for CAD before the development of overt
clinical disease.
Nevertheless, cardiovascular screening of asymptomatic
diabetic patients remains a controversial issue and evidently
more data are needed on this topic. The aim of the current
project is to identify predictors of cardiovascular disease in
asymptomatic diabetic patients. To this end we perform a
comprehensive cardiovascular screening including laboratory
analysis, computed tomography coronary angiography (CTA),
single-photon emission computed tomography (SPECT)
myocardial perfusion imaging (MPI) and measurement of
vascular stiffness.
Figure 1. Example of a CTA examination of an asymptomatic
patient with diabetes. Multi planar reconstruction of the left
anterior descending coronary artery (LAD) revealing a mixed
plaque with significant stenosis in the proximal LAD.
Methods
CTA is performed with a 320-row scanner to assess coronary
anatomy and coronary atherosclerosis (Figure 1). After
assessment of coronary artery calcium, CT angiography is
performed using prospective triggering.The reconstruction
with the best image quality is selected for evaluation of the
coronary arteries. The coronary arteries are divided into 17
segments and per segment the presence of atherosclerotic
plaque is assessed using axial images and curved multi planar
reconstructions. Finally, in all segments with plaque the degree
of luminal narrowing and plaque composition (non-calcified,
mixed or calcified) is determined.
77
SPECT MPI is used to detect myocardial ischemia (Figure 2).
After acquisition, images are processed to obtain the shortaxis, vertical long-axis, and horizontal long-axis sections, as
well as polar map formats, normalized to maximal myocardial
activity. Subsequently, semi-quantitative visual interpretation
is performed to evaluate the extent and severity of CAD
(ischemia).
Vascular stiffness is measured non-invasively by applanation
tonometry. To assess pulse wave velocity (PWV), pulse waves
are recorded at the common carotid artery and the femoral artery
by sequential tonometry with simultaneous electrocardiographic
gating. Aortic PWV (m/s) is defined as the distance between
the 2 recording sites traveled by the pulse wave, divided by
the transit time. Subsequently, to determine the augmentation
index (AIx), peripheral pressure waveforms are recorded from
the radial artery. The corresponding central aortic pressure
waveform is generated by a validated generalized transfer
function. Thereafter, from the central aortic pressure waveform
the first shoulder attributable to left ventricular ejection and
the merging point with the second shoulder from the reflected
wave are identified. The absolute augmented pressure is the
maximum systolic pressure minus pressure at the inflection
point. Finally, the AIx is defined as the absolute augmented
pressure divided by the pulse pressure and expressed as a
percentage.
Figure 2. Example of a normal SPECT MPI in an
asymptomatic patient with diabetes, showing no ischemia.
This extensive screening permits optimal risk stratification of
patients referred to the diabetes outpatient clinic. In 2010, the
analysis of epicardial adipose tissue (EAT) on CTA is started to
determine whether EAT (analyzed with dedicated software) is
related with biomarkers of CAD.
Staff
Drs. C. J. Roos
Prof. Dr. J.W. Jukema
Prof. Dr. J.J. Bax
2-dimensional speckle tracking echocardiography
Principal investigator: J.J. Bax
Project
Implications of 2-dimensional speckle tracking echocardiography
findings for subsequent management of patients with type 2
diabetes mellitus.
Aim of the project
Diabetic cardiomyopathy is defined as left ventricular (LV)
dysfunction that occurs independently of coronary artery
disease and hypertension. The etiological mechanisms
underlying diabetic heart disease are likely to be multifactorial,
ranging from altered myocardial metabolism, endothelial
dysfunction, microvascular disease, autonomic neuropathy,
and altered myocardial structure with fibrosis. Patients with
early diabetic cardiomyopathy often have evidence of global
diastolic dysfunction but preserved systolic function as reflected
by a normal LV ejection fraction (EF). In addition, recent studies
have evaluated the role of myocardial triglyceride accumulation
(steatosis) and diastolic dysfunction in patients with type
2 diabetes. Novel imaging technologies such as magnetic
resonance spectroscopy and 2-dimensional (2D) speckle
tracking strain echocardiography may provide further into the
ultrastructural and functional changes of the left ventricle that
take place at early stages of diabetic cardiomyopathy.
Aims of the present project included a retrospective analysis of
data from the PYRAMID study:
1)to evaluate multidirectional LV myocardial systolic and
diastolic functions in uncomplicated, normotensive, diabetic
patients using 2D speckle tracking echocardiography .
2)to relate myocardial triglyceride content as determined
by magnetic resonance spectroscopy, with biventricular
myocardial strain and SR as determined by
echocardiographic 2D speckle tracking analysis.
Progress in 2010
In diabetic patients multidirectional LV myocardial systolic and
diastolic functions were evaluated using 2D speckle tracking
strain post-processing in 47 male patients (57±6 years) with type
2 diabetes and 53 male controls matched by age, body mass
index and body surface area. Conventional echocardiography
included M-mode, 2D and color, pulsed- and continuous-wave
Doppler imaging. LV radial and circumferential strain and strain
rate were determined in the mid-ventricular short-axis view, and
longitudinal strain and strain rate were determined in the 3 apical
views. There were no differences in LV end-diastolic volume
index (40.7±8.9 vs 44.1±7.8mL/m2, p=ns), end-systolic volume
index (16.0±4.8 vs 17.8±4.3mL/m2, p=ns), ejection fraction
(61.0±5.5 vs 59.8±5.3%, p=ns). Transmitral E/A (0.95±0.21 vs
1.12±0.32, p=0.007) and pulmonary S/D ratios (1.45±0.28 vs
1.25±0.27, p=0.001) were more impaired in diabetic patients.
These patients had impaired longitudinal strain (-18.3 ± 2.2%
vs. -19.9 ± 1.9, p<0.001), systolic strain rate (-0.99 ± 0.17 s-1
vs. -1.07 ± 0.13 s-1, p<0.001) and early diastolic strain rate
(1.04 ± 0.25 s-1 vs. 1.26 ± 0.26 s-1, p<0.001) but preserved
circumferential and radial systolic and diastolic function as
compared to healthy controls. Diabetes was an independent
predictor for longitudinal strain (β=0.508), systolic strain rate
(β=0.513) and early diastolic strain rate (β=-0.477) on multiple
linear regression analysis (all p<0.001). Therefore, despite
normal LV mass, volumes and EF, the diabetic population
showed impairments of LV longitudinal strain and strain rate but
preserved circumferential and radial strain and strain rate. The
presence of diabetes mellitus was an independent predictor of
LV longitudinal strain, systolic strain rate and diastolic strain
rate.
Myocardial triglyceride content as determined by magnetic
resonance spectroscopy was compared with biventricular
myocardial strain and strain rate as determined by
echocardiographic 2D speckle tracking analysis; 42 men with
type 2 diabetes were included and exclusion criteria were HbA1c
> 8.5%, known cardiovascular disease or diabetes-related
complications, blood pressure > 150/85 mmHg. Myocardial
ischemia was excluded by a negative dobutamine stress test. LV
and RV volumes and EF were quantified by magnetic resonance
imaging. LV global longitudinal and RV free wall longitudinal
strain, systolic strain rate and diastolic strain rate were quantified
by echocardiographic speckle tracking analyses. Myocardial
triglyceride content was quantified by magnetic resonance
spectroscopy and dichotomized based on the median value of
0.76%. The median age was 59 years (interquartile range [IQR]
54, 62 years). Median diabetes diagnosis duration was 4 years,
and median glycated hemoglobin level was 6.2% (IQR 5.9,
6.8%). There were no differences in LV and RV end-diastolic
and end-systolic volume indices and EF between patients with
high (≥ 0.76%) versus low (< 0.76%) myocardial triglyceride
content. However, patients with high myocardial triglyceride
content had greater impairment of LV and RV myocardial strain
and strain rate (see Table).
Staff
Dr. C.T.A. Ng
Partnerships
The retrospective analysis was performed in collaboration
with the Departments of Radiology and Endocrinology at
Leiden University Medical Center, Leiden, The Netherlands,
in collaboration with the Department of Endocrinology, Free
University Medical Center, Amsterdam.
Funding
Department of Cardiology, Leiden University Medical Center
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ICIN
Variable
Low myocardial
triglyceride
(n = 21)
High myocardial
triglyceride
(n = 21)
p value
73.2
(66.6, 80.8)
32.2
(30.0, 37.3)
54.0
(50.5, 56.8)
68.0
(63.4, 78.3)
31.1
(25.6, 36.3)
55.1
(52.8, 58.8)
0.31
-19.3
(-18.5, -20.1)
-1.02
(-0.96, -1.14)
1.11
(1.05, 1.23)
-17.1
(-16.3, -17.7)
-0.87
(-0.82, -0.98)
0.93
(0.77, 1.11)
< 0.001
71.1
(64.0, 76.7)
34.9
(30.2, 36.9)
52.0
(49.6, 53.4)
69.3
(63.6, 73.0)
31.9
(29.0, 35.8)
53.0
(48.8, 54.4)
0.43
LEFT VENTRICLE
Magnetic Resonance Imaging
LVEDVI (mL/m2)
LVESVI (mL/m2)
LVEF (%)
2D Speckle Tracking
LV global strain (%)
LV global systolic SR (s-1)
LV global diastolic SR (s-1)
RIGHT VENTRICLE
Magnetic Resonance Imaging
RVEDVI (mL/m2)
RVESVI (mL/m2)
RVEF (%)
2D Speckle Tracking
0.23
0.22
< 0.001
0.003
0.31
0.63
-27.7
-24.5
0.016
(-25.1, -30.2)
(-19.0, -27.7)
-1.74
0.005
-2.11
RV free wall systolic SR (s-1)
(-1.77, -2.46)
(-1.44, -1.85)
1.65
0.001
2.10
RV free wall diastolic SR (s-1)
(1.75, 2.61)
(1.12, 1.86)
LV = left ventricular; RV = right ventricular; EDVI = end-diastolic volume index; ESVI =
end-systolic volume index; EF = ejection fraction; 2D = 2-dimensional; SR = strain rate
RV free wall strain (%)
Table 1: The myocardial triglyceride content was an independent correlate of
LV and RV longitudinal strain, systolic strain rate and diastolic strain rate.
Variable
Global LV longitudinal
strain
Standardized
p
beta
value
Global LV longitudinal
systolic SR
Standardized
p
beta
value
Global LV longitudinal
diastolic SR
Standardized
p
beta
value
Age
-0.197
0.28
-0.092
0.62
-0.179
0.27
Systolic blood
pressure
Blood glucose
0.034
0.82
0.104
0.49
0.003
0.98
0.192
0.23
0.209
0.20
-0.455
0.003
High-density
-0.146
0.43
lipoprotein
cholesterol
Low-density
0.161
0.27
lipoprotein
cholesterol
Myocardial
0.373
0.036
triglyceride
content
LV = left ventricular; SR = strain rate
-0.043
0.82
-0.003
0.99
0.197
0.19
0.054
0.67
0.373
0.039
-0.326
0.038
Table 2: In conclusion, high myocardial triglyceride content is associated with more pronounced
impairment of LV and RV functions in men with uncomplicated type 2 diabetes.
79
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Heart Institute
Diagnostic value of advanced echo imaging
Principal investigator: J.J. Bax
Project
“Incremental diagnostic value of advanced echocardiographic
imaging modalities”
Aim and methods
Conventional echocardiography is the most commonly used
imaging technique in clinical cardiology, due to its non-invasive
approach and wide-availability. However, this imaging modality
is characterized by several important limitations: 1) for the
quantification of left ventricular (LV) volumes and ejection
fraction (EF), 2D echocardiography relies on significant
geometric assumptions, it might be affected by oblique or
“forshortened” views and showed modest reproducibility; 2)
LVEF may not be sensitive enough to detect subtle myocardial
systolic dysfunction that may occur in several cardiac conditions
and should be the target for an early therapeutic approach;
3) conventional echocardiography permits only limited crosssectional views, which make in some cases the interpretation
and diagnosis of cardiac pathologies extremely complex
and achievable only by experienced operators. Advances
in echocardiography have more recently provided novel
approaches, such as real-time 3D echocardiography (RT3DE)
and speckle tracking strain analysis, which may overcome the
abovementioned limitations.
Progress 2010
In patients with LV aneurysm, referred for surgical ventricular
reconstruction, RT3DE and CMR were performed for clinical
evaluation of scar tissue, LV volumes and LVEF, shape of
the LV, LV wall motion abnormalities, and quantification of
mitral regurgitation. Excellent correlation (r=0.97, p<0.001)
and agreement was observed between RT3DE and CMR
for quantification of LV volumes, EF and sphericity index; in
a segment-to-segment comparison, RT3DE showed to be
accurate also for the analysis of wall motion abnormalities
(k=0.62) and LV regional thickness (k=0.56) as a marker of
myocardial scar. Also, mitral regurgitant volume assessed by
RT3DE had a good correlation (r=0.93) with regurgitant volume
measured by CMR, without significant bias (= -0.7ml/beat).
Next, in patients with a first ST elevation acute myocardial
infarction (AMI) and primary percutaneous coronary
intervention, the clinical echocardiograpic data were used to
retrospectively analyze speckle tracking strain for assessment
of LV torsional mechanics as parameters of LV systolic
myocardial performance. These parameters were compared
with LV remodeling at 6-month follow-up Compared with normal
controls (normal echo), peak LV torsion in AMI patients was
significantly impaired (1.54+/-0.64 degrees /cm vs 2.07+/-0.27
degrees /cm, p<0.001). By multivariate analysis, only LVEF
(beta=0.36, p<0.001) and infarct size (beta=-0.47, p<0.001) were
independently associated with peak LV torsion. By multivariate
analysis, only peak LV torsion (odds ratio=0.77; 95% CI, 0.650.92; p=0.003) and infarct size (odds ratio=1.04; 95% CI, 1.011.07; p=0.021) were independently related to LV remodeling.
Peak LV torsion provided modest but significant incremental
value over clinical, echocardiographic, and myocardial contrast
echocardiography variables in predicting LV remodeling. These
findings show that LV torsion is significantly impaired early after
AMI and that the amount of impairment of LV torsion may help
to predict LV remodelling.
Staff
Drs. N Ajmone
Funding
Department of Cardiology, Leiden University Medical Center
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Netherlands
Heart Institute
Ultrasound imaging and therapy
Principal investigator: N. de Jong
Conference
The “Fifteenth European conference on ultrasound imaging”
was again very successful. At
the time of writing the Sixteenth
conference lies already behind
us and was evenly successful
and we are preparing for the
Seventeenth conference, to
be held on January 19nd and
20nd, 2012
General
In project 60 special ultrasound
imaging
modalities
are
studied, including real-time
3D, ultrasound contrast and
super harmonic imaging. These projects are co-sponsored by
SenterNovem, STW, Eureka program, and ICIN. Companies
directly participating in this research are Oldelft, Esaote Europe,
and Vermon. In the Netherlands the Universities of Delft, Twente,
Amsterdam (VU) and Leiden are involved in these projects.
Super harmonic imaging
bubbles under the microscope and a high frame rate camera
(Brandaris 128, figure B)
Figure 1: The amplitude of the subharmonic response of
a BR14 contrast agent microbubble measured at driving
frequency of 10 MHz a) acoustically and b) optically.
The results of the measurements indicated that:
- The subharmonic scattering of the phospholipid-coated
contrast agent microbubbles is sufficiently detectable in the
frequency range around 10 MHz at low acoustic pressures.
- The frequency in which the subharmonic response is
maximum increases with increasing the amplitude of
acoustic pressure; in contrary to what has been observed
for the fundamental response.
- The subharmonic scattering signal can be enhanced up to
20 dB by increasing the ambient pressure.
In the Super harmonic project sponsored by STW we develop
a system which uses the third to the fifth harmonic generated
by non linear wave propagation of the ultrasound. In 2010 the
hardware (electronics, transducer) and software has been
finalised. Final clinical results are expected early 2011.
In conclusion, we have shown from single bubble measurements
that the subharmonic imaging has a great potential to be
exploited in the frequency range mostly suited for carotid
imaging. However this study has to be complemented with
forthcoming in vivo studies.
3D Carotid imaging
Sonodrugs
The presence and properties of atherosclerotic plaques, such
as size, location and the presence of angiogenesis, can be
shown by two-dimensional (2D) ultrasound imaging techniques.
However, the 2D character of ultrasound gives a limitation in
carotid ultrasound imaging. In the current carotid ultrasound
(CARUS) project we will research novel three-dimensional (3D)
ultrasound imaging methods and systems for complete, noninvasive, fast and cost-effective diagnosis of carotid plaques
to achieve full vascular patient care and vascular treatment
monitoring.
A major new European project (FP7) to develop drug delivery
technologies that could significantly impact the treatment of
cancer and cardiovascular disease. By allowing drugs to be
delivered to disease sites via the patients bloodstream and then
activated by focused ultrasound pulses, the SonoDrugs project
aims to maximize the therapeutic efficiency and minimize the
side effects of drug treatments for cancer and cardiovascular
disease.
In the CARUS project, sponsored by SenterNovem, high
density ultrasound matrix sensors are developed as well as
3D ultrasound beam forming and methods of characterizing
atherosclerotic plaques. One method include the use of contrast
agents.
Subharmonic scattering of contrast agents (BR14, Bracco
Research S.A., Geneva, Switzerland) in the frequency range
preferred for carotid imaging (5-15 MHz) has been investigated
within this project. We used 2 methods; in the first one we
investigated an ensemble of bubbles acoustically figure A, and
in the second method we looked at the behavior of individual
81
Characterisation of drug loaded bubbles
Phospholipid bubbles can be loaded with drugs. Within
Sonodrugs an intensive cooperation exists between University
of Ghent and the ErasmusMC. Phospholipid bubbles loaded
with doxorubicin liposomes have been investigated and their
acoustic properties have been determined. We concluded
that there is no difference in elasticity between bubbles with
and without drug loading but there is a clear difference in the
viscosity. Drug loaded bubbles are more viscous and have a
higher damping which can have consequences in the way the
drug has to be released from the bubble.
Chicken embryo chorioallantoic membrane model development.
For sonoporation understanding the vibration of the bubble is
mandatory. It has been studied with high-speed cameras like
Brandaris 128 (Chin, Lancee et al. 2003) which allows to capture
up to 25 million images per second. For studying microbubbles
vibration in vivo the most important point is the amount of light
especially for very short exposures of 10 ns. Bright field imaging
is standard used and in that case the sample must be as much
as possible transparent. The most often used subject for this
type of research is cremaster muscle of rodents (Lindner, Song
et al. 2001). This muscle is located outside of abdominal cavity
and can be extracted from the body without huge damages of
important vessels. On the other hand difficulties during surgery
and especially microscopy in combination with ultrasound could
cause death of the animal and incredibly increase the amount
of animals involve in these experiments.
According to the 3R’s policy to reduce amount of animals and
to replace highly developed animals with lower developed yolkless chicken embryo chorioallantoic membrane model (yolkless CAM) was developed in Erasmus MC.
This model is based on explantation of CAM from the yolk
described in Flammes work (Flamme 1987). During this
procedure we take out chicken embryo with the yolk from the
egg shell on 5-7 days of incubation (stages 22-32 according
Hamburger-Hamilton criteria (Hamburger and Hamilton 1992)).
In this shell-less embryo intravenous injection of microbubbles
in one of vitelline veins could be easily done (Figure 1).
Then after injection of microbubbles we cut out vascularised
part of the embryo from the yolk and place it on the optically
and acoustically transparent pad. After this we put it into the
setup for live cell microscopy. Explanted embryos can be kept
alive more than two hours without detectable damages. During
Figure 2: Microbubbles in CAM vessels. Microbubbles
aggregates are indicated by arrows
microscopy single bubbles can be easily distinguish in bright
field illumination.
Sonoporation efficiency could be also studied for example with
propidium iodide incorporation after application of ultrasound,
as well as bubble behavior with high speed camera.
In conclusion, according to the above and data from literature
CAM and yolk-less CAM models seems to be good for studying
in vivo microbubbles behavior, drugs delivery (Vargas, ZeisserLabouebe et al. 2007), tumors (Knighton, Ausprunk et al. 1977;
De Magalhaes, Liaw et al. 2010) and angiogenesis (Ribatti,
Nico et al. 2001). These kind of studies can be done without
DEC protocol and will cause a decrease in animal studies.
Directing adipose derived stem cells to the area at
risk in the heart after myocardial infarction using
targeted microbubbles: Development of a new
molecular therapeutic technique.
This project aims to specifically targeting the stem cells to
the area at risk after myocardial infarction. Adipose-derived
stem cells will be coupled to contrast microbubbles, this stem
cell-bubble complex will be targeted to specific molecules on
endothelium of the injured vessel wall. This is an STW sponsored
project leaded by VU, Amsterdam en ErasmusMC Rotterdam.
In Rotterdam we focus on the targeting the microbubbles to
the stem cells and the use of ultrasound radiation force local
targeting of the stem cell bubble complex.
Binding force of the bubble to a surface
Targeted ultrasound contrast agents (UCA) are a promising tool
within the field of molecular imaging. Compared to their nontargeted counterparts, ligands to specific targets have been
added to the shell which typically encapsulates a heavy gas.
Because of the presence of these ligands, targeted contrast
agents will only adhere to regions of the vascular endothelium
expressing specific proteins (e.g. inflammatory markers),
facilitating disease-specific contrast enhancement. To be able
to bind to their target, the targeted contrast agent should be
in close proximity to the vessel wall. However, because of
the characteristics of the blood flow profile inside the vessels,
most contrast agents will be present around the center line,
making binding to their target impossible. Acoustic radiation
force has been used successfully to move circulating UCA
towards the target endothelium. However, it was also shown
that ultrasound insonification can cause clustering of targeted
microbubbles due to a mutual interaction known as secondary
acoustic radiation force. Secondary acoustic radiation force is
the force exerted on a microbubble that arises due to the sound
emitted by a neighbouring microbubble when both are pulsating
in an incident ultrasound field. The instantaneous secondary
acoustic radiation force over a microbubble is given by F=-V∇
p with V the volume and ∇ p the pressure gradient in the fluid
due to the pulsating neighbouring microbubble. Because the
pressure gradient over the microbubble oscillates in time, the
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ICIN
force exerted on this bubble is alternating between attractive
and repulsive. However, because the volume of this bubble also
oscillates in time, there is a resulting net force exerted on this
microbubble when averaged over one period. When two bubbles
oscillate in phase (i.e. both have a resonance frequency above
or below the insonifying frequency) the net force is attractive.
The objective of this study was to investigate the behaviour of
targeted microbubbles under influence of secondary acoustic
radiation force using ultra high speed optical imaging with the
Brandaris128 camera.
Figure 3 shows the effect of ultrasound insonification on the
distance between two targeted microbubbles (R0 = 2.0 μm),
initially 7.1 μm apart, imaged with a framerate of 10 million
frames per second (100 ns between subsequent frames).
The pressure was gradually increased in subsequent movies
(6 in total, indicated by the green lines). The time in between
subsequent movies was 80 ms. From the decrease in distance
between the two microbubbles during ultrasound insonification
it is clear that there is a net attraction between the two bubbles.
Figure 1 shows that the microbubbles move towards each
other during insonification, but by the start of the next movie,
the distance between the bubbles is again 7.1 μm. Moreover,
from figure 1 it can be concluded that the distance in between
the microbubbles can decrease by several hundreds of nm
(up to 600 nm) after which the bubbles are still able to move
back to their equilibrium position. This implies that the bubbles
should still be attached to the coated surface. Experiments with
biotinylated microbubbles in contact with a non-coated surface
did not show this behaviour (results not shown here). Because
stretching of molecular bonds can not be responsible for this
phenomenon, some bubble deformation is plausible, although
no such deformations were observed (top view) during the
experiments. 83
Netherlands
Heart Institute
Figure 3: Left panel Distance in between two biotinylated
microbubbles targeted to a NeutrAvidin surface during
insonification with ultrasound (20 cycles, 2.25 MHz). The
pressure was increased in subsequent movies (indicated by
the green lines). The inset shows the two microbubbles. Right
panel Detailed plot of the distance (μm) between the two
bubbles as a function of time (μs) during insonification with 180
kPa.
After exceeding a certain threshold in pressure (in this case
270 kPa), the 2 bubbles didn’t returned to their original position
because the bond was broken caused by the acoustic radiation
force. We concluded from this experiment that the binding of the
targeted bubble was around 400 nN.
Investigators
Nico de Jong (ICIN), Valeria Garbin (ErasmusMC), Michel
Versluis (ErasmusMC), Detlef Lohse (ErasmusMC), Ton van
der Steen(ErasmusMC/ICIN), Ying Luan (ErasmusMC), Tom
Kokhuis (ICIN), Erik Gelderblom (ErasmusMC), Telli Faez
(ErasmusMC), Miranda Harteveld (ErasmusMC), Rik Vos
(ErasmusMC), Marcia Emmer (ICIN), Marlies Overvelde
(ErasmusMC), Paul van Neer (ErasmusMC), Guillaume Matte
(ErasmusMC), Benjamin Dollet (ErasmusMC), Hans Bosch
(ErasmusMC), Jeroen Sijl (ErasmusMC), and Klazina Kooiman
(ErasmusMC).
MRI screening for rupture-prone vulnerable plaques
Principal investigator: G. Pasterkamp
Project
Screening for rupture-prone atherosclerotic plaques with
molecular MRI
This project is part of a larger NHS consortium on MRI imaging
of atherosclerotic plaques. The projects that are executed within
the UMCU consist of several small substudies. Roughly they
can be divided into two parts; cardiac imaging and imaging of
atherosclerosis.
The first part of the project involves imaging of atherosclerotic
lesions.
In one study we have focused on a different approach of
identifying atherosclerotic lesions; with 1H-NMR spectroscopy. It
is thought that hypoxia and inflammatory cell infiltration promote
neovascularization. In these hypoxic areas new vasculature
is leaky and most likely the primary source of intra-plaque
hemorrhage, leading to increased risk of plaque rupture. One of
the metabolites associated with hypoxia is lactate. A universal
technique to detect and quantify metabolites, such as lactate, is
1H-NMR spectroscopy. We hypothesize that increased levels
of lactate are associated with increased plaque vessel density
as well as hypoxia markers and is consequently a marker for
rupture-prone plaques.
In femoral atherosclerotic plaques (n=50), obtained after
surgery, 1H NMR spectra analysis was performed. Frozen
samples were grinded, dissolved in PBS and filtered. The
filtered solution was analyzed by 1H-NMR-spectroscopy at
400 MHz. Spectra were analyzed and lactate peak integrals
(doublet present at 1.32ppm) used for analysis. Vessel density
in the plaque was determined in histological slides stained for
vascular endothelium (CD34). HIF-1α (hypoxia-inducible factor
1α) staining was graded as no, minor, moderate or heavy. We
observed that vessel density increased with increasing lactate
levels, the Spearman correlation plot showed a significant
correlation (p=0.006) with a moderate correlation coefficient
(r=0.383) (figure 1A). Additionally showed heavy HIF1α staining
a significant associated with high lactate levels (p=0.015) (figure
1B).
Diastolic dysfunction is an abnormality in the relaxation of the
ventricles. It is often found in people suffering from diabetes
and can lead to pulmonary edema and systolic heart failure.
Currently, Doppler ultrasound is considered the gold standard
to diagnose diastolic dysfunction. Mouse models are often used
to study (cardiovascular) diseases but Doppler ultrasound is
challenging due to the small size of the animals. Alternatively,
cardiac MRI can be used to assess heart function; therefore
we assessed the hypothesis that high temporal resolution
MRI can accurately detect diastolic dysfunction in a murine
diabetes model. We imaged streptozotocin-induced diabetic
BALB/c mice and healthy controls (n=18) at baseline and 4, 8
and 12 weeks after confirmed diabetes (glucose > 15mmol/L)
with high temporal resolution self-gated MRI. This resulted in
53±3 (controls) and 64±16 (diabetics) frames per cardiac cycle
at week 4 (mean±SD). Data was expressed as the percentile
contribution of the early (‘E’) and late (atrial ‘A’) filling phase. In
the E phase no differences where found between the controls
and diabetic mice at baseline and week 4. However, in the A
phase significant differences were found (figure 2) between
controls and diabetic mice at week 4 (p=0.003) and between
baseline and week 4 measurements in the diabetic group
(p=0.006). The data analysis of weeks 8 and 12 are currently
being processed. We aim to detect diastolic dysfunction in a
very early stage so to predict severe (systolic) heart failure.
Figure 2: percentile ‘E’ and ‘A’ contributions of total volume in
control and diabetic mice at baseline and 4 weeks diabetes. A
significant difference is observed between baseline and week
4 in diabetic mice as well as between control and diabetic mice
at week 4.
Staff
Sandra Bovens (ICIN)
Marcel Nederhoff (ICIN)
Kees van de Kolk (UMCU)
The second part involves cardiac imaging, one of the cardiac
imaging studies is aimed to detect diastolic dysfunction with
MRI.
Funding
NHS, ICIN
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Netherlands
Heart Institute
Imaging of Atherosclerotic Plaque and Neovascularization
Principal investigator: A.F.W. van der Steen
Project
Intravascular Photoacoustics and Intravascular Ultrasound
for imaging of Atherosclerotic Plaque Composition and
Neovascularization
vulnerability. This study was completed in September 2010 and
the results were reported at the IEEE Ultrasonics Symposium in
San Diego in October 2010.
Acoustic Sizing of an ultrasound contrast agent
A novel acoustic sizing method able to estimate the size
distribution of an ultrasound contrast agent noninvasively was
developed in the laboratory in 2009. A paper entitled “Acoustic
sizing of an ultrasound contrast agent” was published in
Ultrasound in Medicine and Biology in October 2010.
3D microvascular map
3D imaging is required to get a complete picture of a plaque
heterogeneity and vascularization (vasa vasorum). A 3D
dataset can be acquired during a mechanical pullback of the
catheter through a region of interest. Is has been proven that
the superior echogenecity of contrast microbbubles can make
plaque microvasculature visible on intravascular ultrasound
(IVUS) images. By tracing this specific echogenecity of contrast
microbubbles along an IVUS pullback, we expect that mapping
in 3D the microvasculature is feasible. In order to test this
approach a phantom study was conducted. A polyvinyl alcohol
based vasa vasorum model was manufactured, exhibiting a
vasa vasorum mimicking branch pattern with a diameter ranging
from 200 to 100 μm. After perfusion of the vasa vasorum model
with the ultrasound contrast agent Definity®, a manual pullback
consisting of 93 cross sectional IVUS images spaced every 200
μm was performed. Perfused areas were segmented in two
registered contrast-enhanced IVUS planes and compared to
coregistered 10 μm thick slices of the vasa vasorum model. The
vasa vasorum mimicking microchannel diameters measured
with contrast-enhanced IVUS agreed within 30% with the
slice diameters. As contrast-enhanced IVUS imaging can be
carried out in-vivo, this method could be used during clinical
IVUS investigations as an additional diagnostic for plaque
IVPA/IVUS imaging of an advanced human atherosclerotic
plaque. (a) Histology: Oil Red O stain shows the presence
of lipid rich plaque (*) as well as a calcified area (Ca); Lu:
lumen, Pf: peri-adventitial fat. (b) IVUS image, IVPA image at
(c) 1210 nm (high lipid absorption) and (d) 1230 nm (low lipid
absorption).
Intravascular photoacoustic imaging of human coronary
atherosclerosis
Intravascular photoacoustics (IVPA) is a tool for plaque
composition imaging. Like near infrared spectroscopy, it
capitalizes on the differences in the chemical absorption spectra
of various tissue types. By selecting the excitation wavelength,
a photoacoustic signal can be excited from specific tissue types
in the artery wall.
An integrated intravascular photoacoustics (IVPA) and
ultrasound (IVUS) catheter with an outer diameter of 1.25 mm
was developed. The catheter comprises an angle-polished
optical fiber adjacent to a 30 MHz single-element transducer.
The ultrasonic transducer was optically isolated to eliminate
artifacts in the PA image.
Imaging of human atherosclerotic arteries ex vivo using a hybrid
intravascular photoacoustic/intravascular ultrasound catheter
was demonstrated. Specific photoacoustic imaging of lipid
content, a key marker of plaque vulnerability, was achieved.
This study was presented at the IEEE Ultrasonics Symposium
in San Diego in October 2010 and at SPIE biomedical optics
in January 2011. A paper entitled “Intravascular photoacoustic
imaging of human coronary atherosclerosis” was published in
the journal Optics Letter on February 1st, 2011.
Figure 1: 3D reconstruction of the contrast-enhanced IVUS
pullback dataset. The upper plane corresponds to location 2,
the lowest plane to location 1. The red volume is the 3D VV
reconstruction of the model.
85
Investigators
David Maresca, Krista Jansen, Marcia Emmer, Gijs van Soest,
Ton van der Steen
Institution
Dept. Biomedical Engineering (Thoraxcenter), ErasmusMC
Comparison of plaque observation in CTA and VH IVUS
Principal investigator: J.J. Bax
Project
A systematic comparison of plaque observations and
their prognostic value between patients presenting with
acute coronary syndromes and with stable coronary artery
disease using non-invasive computed tomography coronary
angiography (CTA) and invasive virtual histology intravascular
ultrasound (VH IVUS).
Aims and methods
The aim is to evaluate atherosclerosis (stenosis and plaque
characteristics) and/or ischemia in patients with unstable and/
or stable coronary artery disease (CAD) using multidetector
computed tomography angiography (CTA) and/or intravascular
ultrasound (IVUS) and/or single photon emission computed
tomography (SPECT).
Progress in 2010
CTA provides insight into the severity and extent of anatomical
disease, but is unable to evaluate the hemodynamical relevance
of CAD. Previously, a large discrepancy has been shown
between atherosclerosis on CTA and ischemia on functional
testing, with only half of the obstructive stenoses (≥50% luminal
narrowing) on CTA showing abnormal perfusion. Accordingly,
anatomical CTA variables of atherosclerosis were identified
that were associated with ischemia on SPECT in patients
with stable CAD. On CTA, the calcium score, the degree of
stenosis (≥50% and ≥70% stenosis), plaque extent and location
were determined. Plaque composition was classified as noncalcified, mixed or calcified. Ischemia was defined as a summed
difference score ≥2 (between stress and rest SPECT images).
Multivariate analysis revealed that the degree of stenosis
(presence of ≥70% stenosis, odds ratio (OR) 3.5), plaque
extent and composition (mixed plaques ≥3, OR 1.7 and calcified
plaques ≥3, OR 2.0) and location (atherosclerotic disease in left
main coronary artery and/or proximal left anterior descending
coronary artery, OR 1.6) were independent predictors for
ischemia on SPECT (example Figure 1). In addition, CTA
variables of atherosclerosis such as plaque extent, composition
and location had significant incremental value for the prediction
of ischemia over the presence of ≥70% stenosis (Figure 2).
These observations indicate that the degree of stenosis but
also plaque extent, composition and location were predictive of
the presence of ischemia on MPI.
Figure 1. Patient example exhibiting all the significant
predictors on multidetector computed tomography
angiography (CTA) for prediction of ischemia on myocardial
perfusion imaging (SPECT). In panel A, a 3D volume
rendered reconstruction is provided, showing the left anterior
descending coronary artery (LAD). Panel B: A curved
multiplanar reconstruction (MPR) of the LAD is shown
demonstrating the presence of obstructive CAD (≥50%) in the
proximal segment (arrow). Panel C: Another curved MPR of
the LAD is shown in a different view, revealing the presence
of multiple diseased segments (cross sectional images a, b
and c), the presence of obstructive lesion in the LAD (arrow
c) and the presence of mixed plaque (cross sectional images
a, b and c). Panel D: Stress SPECT short-axis image showing
the presence of a perfusion defect, particularly evident in the
antero-septal region (arrow). Panel E: Rest SPECT short-axis
image demonstrating normal perfusion.
The diagnostic accuracy of CTA has extensively been tested
against invasive coronary angiography (ICA) for the detection
of significant coronary artery stenoses; but CT can also detect
atherosclerosis that is mainly located in the vessel wall, which
can be detected on intravascular ultrasound (IVUS). Indeed
when CTA was compared to ICA the relation was suboptimal
(with sensitivity, specificity, positive and negative predictive
values of 100%, 85%, 81% and 100%); but when compared
to IVUS, an excellent relation was observed, and patients with
positive CTA and negative ICA had indeed atherosclerosis
located in the vessel wall on IVUS.
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Funding
Netherlands Heart Foundation (NHS) grant 2007B223
Staff
Drs. J.E. van Velzen (ICIN)
Dr. J.D. Schuijf (LUMC)
Prof. Dr. J.W. Jukema (LUMC)
Prof. Dr. J.J. Bax (LUMC)
Figure 2. Bar graph representing the incremental predictive
value of angiographic multidetector computed tomography
(CTA) variables of atherosclerosis for the prediction of
ischemia on myocardial perfusion imaging (MPI) on a patient
basis. The addition of the degree of stenosis (presence of
≥70% stenosis) provided incremental predictive information
to baseline clinical variables for the prediction of ischemia on
SPECT. Furthermore, the addition of extent and composition
of atherosclerosis (≥3 calcified plaques) and location
(atherosclerotic disease in the left main coronary artery and/
or proximal left anterior descending coronary artery) on MSCT
resulted in further incremental predictive value over baseline
clinical variables, and degree of stenosis on MSCT.
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Vulnerability assessment of atherosclerotic plaques
Principal investigator J.J. Bax
Project
Vulnerability assessment of atherosclerotic plaques with noninvasive multimodality imaging.
Aim and methods
Atherosclerosis is a systemic disease of the arterial vessel
wall and is one of the leading causes of morbidity and
mortality in the industrialized world. Rupture of a fibrous cap is
considered to be the critical event in atherosclerotic coronary
and carotid artery disease that leads to myocardial infarction
and stroke or transient ischemic attack (TIA). Therefore, in
order to improve preventive strategies, there is an urgent
need for early identification of plaques with a high probability
to rupture. Histopathological studies have put forward that the
pathological characteristics of ruptured plaques include large
volume, large necrotic core and a thin ruptured fibrous cap. A
shortcoming of histopathological studies is that the conclusions
are based on a histopathogical finding from a single point in
time. Therefore, serial in-vivo examination of atherosclerotic
lesions would be preferred to better understand the process
involved in the development of the vulnerable plaque, with a
high risk to rupture. High-field Magnetic Resonance Imaging
(MRI) has emerged as a promising noninvasive imaging
modality for the serial assessment of carotid artery vessel wall.
Currently, it is technically challenging to quantify all plaque
components on high-field MRI. The aim of the current project is
to develop standardized high-field MRI scan sequences for the
identification and quantification of vulnerable atherosclerotic
plaque components in the carotid artery. A second aim is to
detect differences in carotid plaque morphology and long-term
plaque behavior in patients with TIA and CVA by high-field
MRI carotid plaque imaging. Therefore, patients with recent (<
2 weeks) TIA and patients with recent (< 2 weeks) CVA, are
evaluated including a standard clinical work-up (neurologic
analyses, laboratory analysis, duplex carotid arteries, CT
cerebrum) standard brain MRI, pre-contrast vessel wall highfield MRI, post-contrast high-field MRI at baseline and one year
follow-up.
Progress in 2010
While patient inclusion is ongoing, a high-field (3T) multi-contrast
MRI protocol, consisting of 5 MR-weightings is developed;
details of the developed scan protocol are provided in Table 1.
For clinical application, scan-rescan reproducibility is
paramount. Several studies reported interscan reproducibility
of quantitative plaque measurements using 1.5T scanners.
However, limited information is available on the new generation,
high-field MRI scanners. High-field MRI is suspected to
demonstrate a number of advantages due to an increased
signal-to-noise ratio (SNR), improved spatial resolution, and a
reduced scan time, and therefore, needs to be further evaluated
in the context of measurement reproducibility for prospective
serial clinical applications. An analysis to investigate scanrescan reproducibility of 5 MR weightings and to determine
which of the weightings best approximates the reference
standard (combination of the commonly used weightings)
is performed. Nine healthy volunteers (50% male, mean age
26 years) underwent repeated MR-examinations (3T-PhilipsMRI) of the left carotid artery vessel wall with the previously
stated 5 weightings (Table 1). A standard phased-array coil
with two flexible elements of 14×17cm was used to obtain
nine transverse imaging sections of the left carotid artery with
identical in-plane resolution (0.46×0.46mm2) (Table 1). Scanrescan analysis was
performed in a slice of the common carotid artery. Blinded
analysis of the vessel wall area and lumen area was performed
using dedicated software (Vessel Mass) for the 5 weightings
and compared with the rescan. An example of a scan-rescan
data is provided in Figure 1. Furthermore, the 5 weightings were
compared with the reference standard. The reference standard
was the combination of the 5 commonly used weightings.
Reproducibility of the 5 MR-weightings for both lumen area and
vessel wall areas are shown in table 2. T1-TFE approximated
best the reference standard (lumen, r=0.96; vessel wall,
r=0.78). The developed high field carotid vessel wall imaging
showed high scan-rescan reproducibility for the 5 different MRweightings.
T1-TFE showed highest correlation to the reference standard.
88
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Staff
Drs. E. S.J. Kröner
Dr. H.J. Siebelink
Dr. H.J. Lamb
Prof. Dr. J.J. Ba
Figure 1: Example of the co-registered contrast-weightings
and 3D time-of-flight obtained in two scanning sessions (Scan
and Rescan). From top to bottom: T1 weighted turbo field echo
(T1-TFE), T2 weighted turbo (segmented) spin echo (T2-TSE),
Proton density weighted turbo (segmented) spin echo (PDTSE), T1 weighted turbo (segmented) spin echo (T1-TSE) and
time of flight (TOF).
89
Netherlands
Heart Institute
Biomechanics of vulnerable plaque rupture
Principal investigator: F. Gijsen
Aim and method of the project
Rupture of atherosclerotic plaques in arteries is responsible
for life threatening cardiovascular events, including myocardial
infarction and stroke. In this project, vulnerable plaques in the
carotid arteries of genetically modified mice will be studied.
In-vitro imaging experiments will be carried out to measure
pressure induced deformation and rupture of plaques by means
of high resolution MRI. The in vitro experiments will be used to
validate a biomechanical finite element model of atherosclerotic
plaques.
Experimental work
A custom made set-up was designed and developed for the
fixation of murine carotid arteries that contain plaques. Flow
and pressure in and around the vessel can be controlled by
flow and pressure pumps. The vessel is fixed at the appropriate
axial stretch and the temperature was set at 37° C, to mimic
the in-vivo conditions. At different pressure steps, MRI data is
obtained.
High resolution MR imaging is performed with the aim of
creating biomechanical models. Unfortunately, the resolution of
this dataset was not sufficient for creating the models (in-plane
25 μm, slice thickness 50 μm), and alternative imaging was
considered. A new set-up was created, that can be used in the
MRI and in a multiphoton microscope (MPM) imaging set-up,
available at Leiden University. This new set-up was constructed
to obtain a better SNR and resolution with MRI and has been
matched to the MPM set-up. With the MPM, z-stacks can be
made throughout the vessel wall with an in-plane resolution of
less than 1 μm and a slice thickness of 2 μm. Due to limited
penetration depth, the vessel will be imaged from 4 sides. Using
iso-surfaces, vessel segments can be visualized and used to
create biomechanical models. Initial pilot results from a healthy
coronary artery are displayed below.
In future measurements, MPM imaging of six atherosclerotic
murine carotid arteries will be done and protocols will be
developed to create biomechanical models from the data. This
will be done as a feasibility study, as biomechanical models
were never constructed from MPM image data so far.
Based on these results, pressure experiments will be done until
the plaque ruptures, either with MR imaging or MPM imaging.
High resolution MRI will be made for the translational aspect to
clinical MRI data from human atherosclerotic carotid arteries.
Modeling work
As the biomechanical stress analyses in this project will be based
on pressurized geometries, we need to be accounted for the
initial stress that is present in the vessels at the time of imaging.
Adaptations to the finite element computational software were
required to evaluate the effect of neglecting the initial stress
state on the 2D plaque stress distribution. Twenty histological
sections, perfusion fixed at 100 mmHg, were segmented and
finite element models were created. Peak plaque and cap
stresses were computed with and without initial stress. When
initial stress was accounted for, the general relations between
peak cap stress and cap thickness, necrotic core thickness and
necrotic core angle, remained intact. However, on a plaquespecific basis, accounting for initial stress showed a different
effect on the absolute cap stress for each plaque. This suggests
that whenever stress analyses are performed on pressurized,
and thus pre-stressed structures, accounting for initial stress
is indispensible. We will use this in our future biomechanical
analyses, based on the MRI and/or MPM measurements.
A manuscript describing this study was submitted to Journal of
Biomechanics and is currently under review. Future work on the
modeling will focus on 3D models and translation from the MRI
and/or MPM data to useable biomechanical models.
Partnerships This project is a corporation between the Erasmus
MC, Leiden University MC and TU/e. Funding The project is
fully funded by ICIN, with local financial support of the Erasmus
MC and LUMC.
Staff
Project leader: Frank Gijsen (Erasmus MC)
Researcher: Lambert Speelman (ICIN)
Researcher: Brigit den Adel (Leiden University MC)
MRI support: Louise van der Weerd (Leiden University MC)
Modeling support: Harald van Brummelen (TU/e)
General support
Jolanda Wentzel (Erasmus MC)
Wouter Jukema (Leiden University MC)
Ton van der Steen (Erasmus MC)
Rob Poelmann (Leiden University MC).
90
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Annexes
Netherlands
Heart Institute
ICIN
Netherlands
Heart Institute
Annex 1: ICIN Indicators
2004
2005
2006
2007
2008
2009
2010
47.5
53.7
74.1
83.1
86.1
89.4
89.7
17
22
30
31
38
46
40
126
139
119
273
402
454
402
Dissertations
4
6
11
13
15
21
20
ICIN professors
8
8
8
8
5
5
7
International fellowships
3
2
2
2
2
2
1
Annual turnover in million €
4.7
5.1
6.3
6.5
6.8
7.3
7.6
- of which KNAW contribution in million €
2.2
2.0
2.0
2.0
2.0
1.7
1.2
Portfolio of subsidies held in million €
5.2
6.8
9.3
9.5
11.4
12.7
10.4
Research staff in FTE
Active research projects
Articles in referenced international magazines
93
Annex 2: Doctoral Theses
Single microbubble imaging
Hendrik Jacob Vos
Promotores: Prof. dr. ir. A.F.W. van der Steen, Prof. dr. ir N. de
Jong
20 January 2010
Erasmus Universiteit Rotterdam
Clinical and functional outcomes after revascularization
strategies in acute coronary syndromes
Alexander Hirsch
Promotores: Prof. dr. J.J. Piek, Prof. dr. R.J. de Winter
Copromotor: Prof. dr. J.G.P. Tijssen
29 January 2010
Universiteit van Amsterdam
Ultrasound Contrast Agents: Dynamics of Coated Bubbles
Marlies Overvelde
Promotor: Prof. Dr. N. de Jong
09 April 2010
Universiteit Twente
Local atherosclerotic plaque biomarkers predict systemic
clinical outcome
Wouter Peeters
Promotores: Prof. dr. G. Pasterkamp, Prof. dr. F.L. Moll
Copromotores: Dr. D.P.V. de Kleijn Dr. J.P.P.M. de Vries
15 April 2010
Universiteit Utrecht
MRI of Atherosclerosis
Bernard te Boekhorst
Promotores: Prof. dr. G. Pasterkamp, Prof. dr. Van Echteld
Copromotor: Dr. M.J. Cramer
11 May 2010
Universiteit Utrecht
Genes, inflammation and age-related diseases
Stella Trompet
Promotores: Prof. dr. J.W. Jukema, Prof. dr. R.G.J. Westendorp
02 June 2010
Universiteit Leiden
Coronary atherosclerosis and wall shear stress
Alina van der Giessen
Promotores: Prof. dr. ir. A.F.W. van der Steen, Prof. dr. P.J. de
Feyter
Copromotor: Dr. ir. F.J.H. Gijsen
25 June 2010
Erasmus Universiteit Rotterdam
Ultrasonic Superharmonic Imaging
Paul van Neer
Promotores: Prof.dr.ir. A.F.W. van der Steen, Prof.dr.ir. N. de
Jong
Copromotores: Prof.dr.ir. A.P.G. Hoeks, Prof.dr.ir. A. Gisolf,
Prof.dr.ir. R. van Mastrigt
01 July 2010
Erasmus Universiteit Rotterdam
Drug eluting stent implantation for high risk patients and
novel technologies in percutaneous coronary intervention
Shuzou Tanimoto
Promotor: Prof. dr. W.J. van der Giessen
03 February 2010
Erasmus Universiteit Rotterdam
Endogenous & exogenous erythropoietin in left
ventricular dysfunction
Anne Belonje
Promotores: Prof. dr. D.J. van Veldhuisen, Prof. dr. W.H. van
Gilst
Copromotor: Prof. dr. A.A. Voors
15 September 2010
Universiteit Groningen
94
ICIN
Netherlands
Heart Institute
Further insights into inheritable arrhythmia syndromes:
Focus on electrocardiograms
Pieter Gerard Postema
Promotores: Prof. dr. A.A.M. Wilde, Prof. dr. ir. J.M.T. de Bakker
Copromotores: Dr. P.F.H.M. van Dessel, Dr. H.L. Tan
01 October 2010
Universiteit van Amsterdam
Novel cardiac imaging technologies: Implications in
clinical decision making
Victoria Delgado
Promotores: Prof. dr. Jeroen J Bax, Prof. dr. Martin J Schalij
Copromotor: Dr. J.D. Schuijf
11 November 2010
Universiteit Leiden
An update on amiodarone treatment for atrial fibrillation:
efficacy versus safety
Sheba Ahmed
Promotores: prof.dr. I.C. van Gelder, prof.dr. D.J. van Veldhuisen,
prof.dr. T.P. Links
13 October 2010
Universiteit Groningen
Cellular aging in cardiovascular diseases
Hisko Oeseburg
Promotor: Prof. dr. W.H. van Gilst
Copromotores: Dr. H.H.W. Silljé, Dr. R.A. de Boer, Dr. P. van
der Harst
24 November 2010
Universiteit Groningen
Exploiting Ultrasound Harmonics
Guillaume Matte
Promotores: Prof.dr.ir. N. de Jong, Prof.dr. A.F.W. van der Steen
25 November 2010
Erasmus Universiteit Rotterdam
Cryoballoon Pulmonary Vein Isolation for the Treatment of
Atrial Fibrillation and Issues in Follow-up Management
Yves van Belle
Promotor: Prof. dr. W. van der Giessen
25 November 2010
Erasmus Universiteit Rotterdam
The Systemic Right Ventricle
Michiel Winter
Promotor: Prof. dr. B.J.M. Mulder
26 November 2010
Universiteit van Amsterdam
Arrhythmogenesis in the remodeled heart, the rol of
spatially dispersed Cx43 expression
Mohamed Boulaksil
Promotores: Prof. dr. it. J.M.T. de Bakker, Prof. dr. M.A. Vos
Copromotores: Dr. H.V.M. van Rijen, Dr. T.A.B. van Veen
02 December 2010
Universiteit Utrecht
Atherosclerotic plaque and shear stress in carotid arteries
Harald Groen
Promotores: Prof. dr. ir. A.F.W. van der Steen
Copromotores: dr. ir. J.J. Wentzel, Dr. A. van der Lugt
10 November 2010
Erasmus Universiteit Rotterdam
95
Liver X receptor in the cardiovascular system
Irma Kuipers
Promotores: Prof. dr. W.H. van Gilst, Prof. dr. D.J. van Veldhuisen
Copromotores: Dr. R.A. de Boer
15 December 2010
Universiteit Groningen
Annex 3: Publications
1
Abdulrahman RM, Delgado V, Ng AC, Ewe SH, Bertini
M, Holman ER, Hovens GC, Pereira AM, Romijn JA,
Bax JJ, Smit JW; “Abnormal cardiac contractility in longterm exogenous subclinical hyperthyroid patients as
demonstrated by two-dimensional echocardiography
speckle tracking imaging.”; Eur. J. Endocrinol. 2010 Sep;
163 (3); 435-41
2 Adams R, Appelman Y, Bronzwaer JG, Slagboom T,
Amoroso G, van Exter P, Tijssen GP, de Winter RJ;
“Implementation of a prehospital triage system for patients
with chest pain and logistics for primary percutaneous
coronary intervention in the region of Amsterdam, the
Netherlands.”; Am. J. Cardiol. 2010 Oct 1; 106 (7); 931-5
3
Ahmed S, Ranchor AV, Crijns HJ, van Veldhuisen DJ,
Van Gelder IC; “Effect of continuous versus episodic
amiodarone treatment on quality of life in persistent atrial
fibrillation.”; Europace 2010 Jun; 12 (6); 785-91
4
Ajmone Marsan N, Bax JJ; “Cardiomyopathies: Myocardial
fibrosis assessed by CMR to predict events in HCM.”; Nat
Rev Cardiol 2010 Nov; 7 (11); 604-6
5 Akdim F, Stroes ES, Sijbrands EJ, Tribble DL, Trip
MD, Jukema JW, Flaim JD, Su J, Yu R, Baker BF,
Wedel MK, Kastelein JJ; “Efficacy and safety of
mipomersen, an antisense inhibitor of apolipoprotein B,
in hypercholesterolemic subjects receiving stable statin
therapy.”; J. Am. Coll. Cardiol. 2010 Apr 13; 55 (15); 16118
6
Alfonso F, Ambrosio G, Pinto FJ, van der Wall EE; “European
National Society Cardiovascular Journals: Background,
rationale and mission statement of the ‘Editors’ Club’ (Task
Force of the European Society of Cardiology).”; Neth Heart
J 2010 Apr; 18 (4); 202-8
7
Amin AS, Klemens CA, Verkerk AO, Meregalli PG, AsghariRoodsari A, de Bakker JM, January CT, Wilde AA, Tan
HL; “Fever-triggered ventricular arrhythmias in Brugada
syndrome and type 2 long-QT syndrome.”; Neth Heart J
2010 Mar; 18 (3); 165-9
8
Amin AS, Asghari-Roodsari A, Tan HL; “Cardiac sodium
channelopathies.”; Pflugers Arch. 2010 Jul; 460 (2); 22337
9
Antoni ML, Scherptong RW, Atary JZ, Boersma E, Holman
ER, van der Wall EE, Schalij MJ, Bax JJ; “Prognostic
value of right ventricular function in patients after acute
myocardial infarction treated with primary percutaneous
coronary intervention.”; Circ Cardiovasc Imaging 2010
May; 3 (3); 264-71
10 Antoni ML, Mollema SA, Delgado V, Atary JZ, Borleffs CJ,
Boersma E, Holman ER, van der Wall EE, Schalij MJ, Bax
JJ; “Prognostic importance of strain and strain rate after
acute myocardial infarction.”; Eur. Heart J. 2010 Jul; 31
(13); 1640-7
11 Antoni ML, Bertini M, Atary JZ, Delgado V, ten Brinke EA,
Boersma E, Holman ER, van der Wall EE, Schalij MJ,
Bax JJ, Van de Veire NR; “Predictive value of total atrial
conduction time estimated with tissue Doppler imaging for
the development of new-onset atrial fibrillation after acute
myocardial infarction.”; Am. J. Cardiol. 2010 Jul 15; 106
(2); 198-203
12 Antoni ML, Mollema SA, Atary JZ, Borleffs CJ, Boersma E,
Van de Veire NR, Holman ER, van der Wall EE, Schalij MJ,
Bax JJ; “Time course of global left ventricular strain after
acute myocardial infarction.”; Eur. Heart J. 2010 Aug; 31
(16); 2006-13
96
ICIN
13 Arslan F, Smeets MB, O’Neill LA, Keogh B, McGuirk
P, Timmers L, Tersteeg C, Hoefer IE, Doevendans PA,
Pasterkamp G, de Kleijn DP; “Myocardial ischemia/
reperfusion injury is mediated by leukocytic toll-like
receptor-2 and reduced by systemic administration of a
novel anti-toll-like receptor-2 antibody.”; Circulation 2010
Jan 5; 121 (1); 80-90
14 Arslan F, Keogh B, McGuirk P, Parker AE; “TLR2 and TLR4
in ischemia reperfusion injury.”; Mediators Inflamm. 2010;
2010 (0); 704202
15Asghari-Roodsari A, Lesani A, Javadi-Paydar M,
Tabatabaeefar L, Tavangar SM, Norouzi A, Dehpour AR;
“Tocolytic effect of delta9-tetrahydrocannabinol in mice
model of lipopolysaccharide--induced preterm delivery:
role of nitric oxide.”; Reprod Sci 2010 Apr; 17 (4); 391-400
16 Atary JZ, Bergheanu SC, van der Hoeven BL, Atsma DE,
Bootsma M, van der Kley F, Zeppenfeld K, Jukema JW,
Schalij MJ; “Impact of sirolimus-eluting stent implantation
compared to bare-metal stent implantation for acute
myocardial infarction on coronary plaque composition at
nine months follow-up: a Virtual Histology intravascular
ultrasound analysis. Results from the Leiden MISSION!
intervention study.”; EuroIntervention 2009 Nov; 5 (5); 56572
17 Atary JZ, Borleffs CJ, Liem SS, Bax JJ, van der Hoeven
BL, Bootsma M, van der Wall EE, van Erven L, Schalij
MJ; “Structured care for patients after acute myocardial
infarction: sudden cardiac death prevention--data from the
Leiden MISSION! AMI study.”; Europace 2010 Mar; 12 (3);
378-84
18 Atary JZ, van der Hoeven BL, Liem SS, Jukema JW, van
der Bom JG, Atsma DE, Bootsma M, Zeppenfeld K, van
der Wall EE, Schalij MJ; “Three-year outcome of sirolimuseluting versus bare-metal stents for the treatment of
ST-segment elevation myocardial infarction (from the
MISSION! Intervention Study).”; Am. J. Cardiol. 2010 Jul
1; 106 (1); 4-12
19 Atary JZ, de Visser M, van den Dijk R, Bosch J, Liem
SS, Antoni ML, Bootsma M, Viergever EP, Kirchhof CJ,
Padmos I, Sedney MI, van Exel HJ, Verwey HF, Atsma DE,
van der Wal EE, Jukema JW, Schalij MJ; “Standardised
pre-hospital care of acute myocardial infarction patients:
MISSION! guidelines applied in practice.”; Neth Heart J
2010 Sep; 18 (9); 408-15
20 Auger D, Van Bommel RJ, Bertini M, Delgado V, Ng AC,
Ewe SH, Shanks M, Marsan NA, Mooyaart EA, Witkowski
T, Poldermans D, Schalij MJ, Bax JJ; “Prevalence and
characteristics of patients with clinical improvement but not
significant left ventricular reverse remodeling after cardiac
resynchronization therapy.”; Am. Heart J. 2010 10 00; 160
(4); 737-43
97
Netherlands
Heart Institute
21 Bardy GH, Smith WM, Hood MA, Crozier IG, Melton IC,
Jordaens L, Theuns D, Park RE, Wright DJ, Connelly DT,
Fynn SP, Murgatroyd FD, Sperzel J, Neuzner J, Spitzer
SG, Ardashev AV, Oduro A, Boersma L, Maass AH, Van
Gelder IC, Wilde AA, van Dessel PF, Knops RE, Barr CS,
Lupo P, Cappato R, Grace AA; “An entirely subcutaneous
implantable cardioverter-defibrillator.”; N. Engl. J. Med.
2010 Jul 1; 363 (1); 36-44
22 Barlis P, Regar E, Serruys PW, Dimopoulos K, van
der Giessen WJ, van Geuns RJ, Ferrante G, Wandel
S, Windecker S, van Es GA, Eerdmans P, Jüni P, Di
Mario C; “An optical coherence tomography study of a
biodegradable vs. durable polymer-coated limus-eluting
stent: a LEADERS trial sub-study.”; Eur. Heart J. 2010 Jan;
31 (2); 165-76
23 Beek AM, Nijveldt R, van Rossum AC; “Intramyocardial
hemorrhage and microvascular obstruction after primary
percutaneous coronary intervention.”; Int J Cardiovasc
Imaging 2010 Jan; 26 (1); 49-55
24 Beenen LF, Adams R, Koster RW, Otto T; “Computed
tomography scanning during a traumatic resuscitation.”;
Am J Emerg Med 2010 Jul 30
25 Beijk MA, Boekholdt SM, Rittersma SZ, Pons D,
Zwinderman AH, Doevendans PA, Tio RA, Tijssen JG,
Jukema JW, de Winter RJ; “Toll-like receptor 4 gene
polymorphisms show no association with the risk of clinical
or angiographic restenosis after percutaneous coronary
intervention.”; Pharmacogenet. Genomics 2010 Sep; 20
(9); 544-52
26 Bell KJ, Hayen A, Macaskill P, Craig JC, Neal BC, Fox KM,
Remme WJ, Asselbergs FW, van Gilst WH, Macmahon S,
Remuzzi G, Ruggenenti P, Teo KK, Irwig L; “Monitoring
initial response to Angiotensin-converting enzyme inhibitorbased regimens: an individual patient data meta-analysis
from randomized, placebo-controlled trials.”; Hypertension
2010 Sep; 56 (3); 533-9
27 Belonje AM, Voors AA, van der Meer P, van Gilst WH,
Jaarsma T, van Veldhuisen DJ; “Endogenous erythropoietin
and outcome in heart failure.”; Circulation 2010 Jan 19;
121 (2); 245-51
28 Bentley JP, Asselbergs FW, Coffey CS, Hebert PR,
Moore JH, Hillege HL, van Gilst WH; “Cardiovascular
risk associated with interactions among polymorphisms
in genes from the renin-angiotensin, bradykinin, and
fibrinolytic systems.”; PLoS ONE 2010; 5 (9); e12757
29 Beqqali A, Monshouwer-Kloots J, Monteiro R, Welling M,
Bakkers J, Ehler E, Verkleij A, Mummery C, Passier R;
“CHAP is a newly identified Z-disc protein essential for
heart and skeletal muscle function.”; J. Cell. Sci. 2010 Apr
1; 123 (0); 1141-50
30 Bergheanu SC, van der Hoeven BL, Hassan AK, Dijkstra
J, Rosendaal FR, Liem SS, Schalij MJ, van der Bom JG,
Jukema JW; “Post-intervention IVUS is not predictive for
very late in-stent thrombosis in drug-eluting stents.”; Acta
Cardiol 2009 Oct; 64 (5); 611-6
31 Bergheanu SC, Pons D, van der Hoeven BL, Liem SS,
Siegerink B, Schalij MJ, van der Bom JG, Jukema JW;
“The 5352 A allele of the pro-inflammatory caspase-1
gene predicts late-acquired stent malapposition in STEMI
patients treated with sirolimus stents.”; Heart Vessels 2010
Oct 30
32 Bertini M, Sengupta PP, Nucifora G, Delgado V, Ng AC,
Marsan NA, Shanks M, Van Bommel RJ, Schalij MJ,
Narula J, Bax JJ; “Role of left ventricular twist mechanics in
the assessment of cardiac dyssynchrony in heart failure.”;
JACC Cardiovasc Imaging 2009 12 00; 2 (12); 1425-35
33 Bertini M, Delgado V, Den Uijl DW, Nucifora G, Ng AC, Van
Bommel RJ, Borleffs CJ, Boriani G, Schalij MJ, Bax JJ;
“Prediction of cardiac resynchronization therapy response:
value of calibrated integrated backscatter imaging.”; Circ
Cardiovasc Imaging 2010 Jan; 3 (1); 86-93
34 Bertini M, Delgado V, Nucifora G, Marsan NA, Ng AC,
Shanks M, Van Bommel RJ, Borleffs CJ, Ewe SH,
Boriani G, Biffi M, Schalij MJ, Bax JJ; “Effect of cardiac
resynchronization therapy on subendo- and subepicardial
left ventricular twist mechanics and relation to favorable
outcome.”; Am. J. Cardiol. 2010 Sep 1; 106 (5); 682-7
35 Bertini M, Borleffs CJ, Delgado V, Ng AC, Piers SR, Shanks
M, Antoni ML, Biffi M, Boriani G, Schalij MJ, Bax JJ, Van
de Veire NR; “Prediction of atrial fibrillation in patients with
an implantable cardioverter-defibrillator and heart failure.”;
Eur. J. Heart Fail. 2010 Oct; 12 (10); 1101-10
36 Bertini M, Ng AC, Borleffs CJ, Delgado V, Wijnmaalen AP,
Nucifora G, Ewe SH, Shanks M, Thijssen J, Zeppenfeld
K, Biffi M, Leung DY, Schalij MJ, Bax JJ; “Longitudinal
mechanics of the periinfarct zone and ventricular
tachycardia inducibility in patients with chronic ischemic
cardiomyopathy.”; Am. Heart J. 2010 Oct; 160 (4); 729-36
37 Bertini M, Delgado V, Nucifora G, Ajmone Marsan N, Ng
AC, Shanks M, Antoni ML, Van de Veire NR, Van Bommel
RJ, Rapezzi C, Schalij MJ, Bax JJ; “Left ventricular
rotational mechanics in patients with coronary artery
disease: differences in subendocardial and subepicardial
layers.”; Heart 2010 Nov; 96 (21); 1737-43
38 Bezzina CR, Pazoki R, Bardai A, Marsman RF, de Jong
JS, Blom MT, Scicluna BP, Jukema JW, Bindraban NR,
Lichtner P, Pfeufer A, Bishopric NH, Roden DM, Meitinger
T, Chugh SS, Myerburg RJ, Jouven X, Kääb S, Dekker
LR, Tan HL, Tanck MW, Wilde AA; “Genome-wide
association study identifies a susceptibility locus at 21q21
for ventricular fibrillation in acute myocardial infarction.”;
Nat. Genet. 2010 Aug; 42 (8); 688-91
39 Boink GJ, Rosen MR; “Regenerative therapies in
electrophysiology and pacing: introducing the next steps.”;
Archives of disease in childhood 2010 Dec 16
40 Boogerd CJ, Dooijes D, Ilgun A, Mathijssen IB, Hordijk R,
van de Laar IM, Rump P, Veenstra-Knol HE, Moorman AF,
Barnett P, Postma AV; “Functional analysis of novel TBX5
T-box mutations associated with Holt-Oram syndrome.”;
Cardiovasc. Res. 2010 Oct 1; 88 (1); 130-9
41 Boogers MJ, Borleffs CJ, Henneman MM, Van Bommel
RJ, van Ramshorst J, Boersma E, Dibbets-Schneider P,
Stokkel MP, van der Wall EE, Schalij MJ, Bax JJ; “Cardiac
sympathetic denervation assessed with 123-iodine
metaiodobenzylguanidine imaging predicts ventricular
arrhythmias in implantable cardioverter-defibrillator
patients.”; J. Am. Coll. Cardiol. 2010 Jun 15; 55 (24); 276977
42 Boogers MJ, Schuijf JD; “Quantification of stenosis
severity on multidetector row computed tomography.”;
EuroIntervention 2010 May; 6 (0); G57-64
43 Boogers MJ, Schuijf JD, Kitslaar PH, van Werkhoven JM,
de Graaf FR, Boersma E, van Velzen JE, Dijkstra J, Adame
IM, Kroft LJ, de Roos A, Schreur JH, Heijenbrok MW,
Jukema JW, Reiber JH, Bax JJ; “Automated quantification
of stenosis severity on 64-slice CT: a comparison with
quantitative coronary angiography.”; JACC Cardiovasc
Imaging 2010 Jul; 3 (7); 699-709
44 Boogers MJ, Fukushima K, Bengel FM, Bax JJ; “The role
of nuclear imaging in the failing heart: myocardial blood
flow, sympathetic innervation, and future applications.”;
Heart Fail Rev 2010 Oct 12
45 Borleffs CJ, van Rees JB, van Welsenes GH, van der
Velde ET, van Erven L, Bax JJ, Schalij MJ; “Prognostic
importance of atrial fibrillation in implantable cardioverterdefibrillator patients.”; J. Am. Coll. Cardiol. 2010 Mar 2; 55
(9); 879-85
46 Borleffs CJ, van Welsenes GH, Van Bommel RJ, van der
Velde ET, Bax JJ, van Erven L, Putter H, van der Bom JG,
Rosendaal FR, Schalij MJ; “Mortality risk score in primary
prevention implantable cardioverter defibrillator recipients
with non-ischaemic or ischaemic heart disease.”; Eur.
Heart J. 2010 Mar; 31 (6); 712-8
47 Borleffs CJ, Thijssen J, de Bie MK, van Rees JB, van
Welsenes GH, van Erven L, Bax JJ, Cannegieter SC,
Schalij MJ; “Recurrent implantable cardioverter-defibrillator
replacement is associated with an increasing risk of
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53 Brouwer BG, Visseren FL, Algra A, van Bockel JH, Bollen
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58 Casini S, Tan HL, Demirayak I, Remme CA, Amin AS,
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59 Caslake MJ, Packard CJ, Robertson M, Cooney J, Nelson
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66 Coronel R, Lau DH, Sosunov EA, Janse MJ, Danilo
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67 Corsten MF, Dennert R, Jochems S, Kuznetsova T, Devaux
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69 da Costa Martins PA, Leptidis S, Salic K, De Windt LJ;
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70 da Costa Martins PA, De Windt LJ; “miR-21: a miRaculous
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71 da Costa Martins PA, Salic K, Gladka MM, Armand AS,
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72 de Bakker JM, Wittkampf FH; “The pathophysiologic
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73 de Boer TP, van Veen TA, Jonsson MK, Kok BG, Metz CH,
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74 de Graaf FR, Schuijf JD, van Velzen JE, Kroft LJ, de Roos
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75 de Graaf FR, Schuijf JD, Delgado V, van Velzen JE, Kroft
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76 de Graaf FR, Schuijf JD, van Velzen JE, Nucifora G, Kroft
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77 de Graaf FR, Schuijf JD, van Velzen JE, Boogers MJ, Kroft
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78 de Graaf FR, Schuijf JD, van Velzen JE, Kroft LJ, de Roos
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79 de Graaf FR, van Werkhoven JM, van Velzen JE, Antoni
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86 de Kleijn DP, Moll FL, Hellings WE, Ozsarlak-Sozer
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87 de Vries ST, Kleijn SA, van ‘t Hof AW, Snaak H, van Enst
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80 de Graaf FR, Schuijf JD, Scholte AJ, Djaberi R, van
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81 de Groot D, Haverslag RT, Pasterkamp G, de Kleijn DP,
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82 de Groot NM, Scholte AJ, Jongbloed MR, Schuijf JD,
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83 de Groot NM, Kirchhof CJ, Van Gelder IC, Meeder JG,
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84 De Jong AM, Maass AH, Oberdorf-Maass SU, van
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85 de Jong S, van Veen TA, van Rijen HV, de Bakker JM;
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88 Deelman LE, Declèves AE, Rychak JJ, Sharma K;
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89 Degreef LE, Opdam FL, Teepe-Twiss IM, Jukema JW,
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90 Delgado V, Ng AC, Van de Veire NR, van der Kley F, Schuijf
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91 Delgado V, Mooyaart EA, Ct Ng A, Auger D, Bertini M,
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92 Delgado V, Schuijf JD, Bax JJ; “Pre-operative aortic
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93 Delgado V, Ewe SH, Ng AC, van der Kley F, Marsan NA,
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94 Den Uijl DW, Delgado V, Tops LF, Ng AC, Boersma E,
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95 Derksen WJ, Gisbertz SS, Hellings WE, Vink A, de Kleijn
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96 Derksen WJ, de Vries JP, Vink A, Velema E, Vos JA, de
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97 Diercks GF, van Tintelen JP, Tio RA, Kerstjens-Frederikse
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98 Djaberi R, Schuijf JD, Jukema JW, Rabelink TJ, Stokkel
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99 Drenthen W, Boersma E, Balci A, Moons P, RoosHesselink JW, Mulder BJ, Vliegen HW, van Dijk AP, Voors
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100 Duffels MG, Mulder KM, Trip MD, de Groot E, Gort J, van
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101 Eefting D, Seghers L, Grimbergen JM, De Vries MR, de
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102Eschenhagen T, Force T, Ewer MS, de Keulenaer GW,
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103 Eurlings LW, van Pol PE, Kok WE, van Wijk S, Lodewijksvan der Bolt C, Balk AH, Lok DJ, Crijns HJ, van Kraaij DJ,
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104 Ewe SH, Ajmone Marsan N, Pepi M, Delgado V, Tamborini
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105 Ewing MM, De Vries MR, Nordzell M, Pettersson K, de
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106 Fernandes S, van Rijen HV, Forest V, Evain S, Leblond
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107 Folkeringa RJ, de Vos C, Pinto YM, Habets J, De Leeuw
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108 Gademan MG, Sun Y, Han L, Valk VJ, Schalij MJ, van Exel
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109 Garg S, Serruys PW, van der Ent M, Schultz C, Mastik
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110 Götte MJ, Rüssel IK, de Roest GJ, Germans T, Veldkamp
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111 Geleijnse JM, Giltay EJ, Schouten EG, de Goede J, Oude
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112 Germans T, Rüssel IK, Götte MJ, Spreeuwenberg MD,
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113 Germans T, Nijveldt R, Brouwer WP, Groothuis JG, Beek
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114 Girasis C, Schuurbiers JC, Onuma Y, Aben JP, Weijers B,
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115Girasis C, Schuurbiers JC, Onuma Y, Serruys PW,
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116 Gittenberger-de Groot AC, Winter EM, Poelmann RE;
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117 Goderie TP, van Soest G, Garcia-Garcia HM, Gonzalo
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118 Groen HC, van Walsum T, Rozie S, Klein S, van Gaalen
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119 Groen HC, Simons L, van den Bouwhuijsen QJ, Bosboom
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120Groothuis JG, Beek AM, Brinckman SL, Meijerink MR,
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122Groothuis JG, Kremers FP, Beek AM, Brinckman SL,
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123 Helbing WA, Luijnenburg SE, Moelker A, Robbers-Visser
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125 Hellings WE, Peeters W, Moll FL, Piers SR, van Setten
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126 Hendrix A, Vaartjes I, Mosterd A, Reitsma JB, Doevendans
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128Hirsch A, Nijveldt R, van der Vleuten PA, Tijssen JG,
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129Hobbs FD, Jukema JW, Da Silva PM, McCormack T,
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130 Hofman N, Tan HL, Alders M, van Langen IM, Wilde AA;
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131Hoogendijk MG, Potse M, Linnenbank AC, Verkerk AO,
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134 Huijbregts HJ, de Borst GJ, Veldhuis WB, Verhagen HJ,
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135 Hurks R, Hoefer IE, Vink A, Pasterkamp G, Schoneveld A,
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136Hurks R, Hoefer IE, Vink A, de Vries JP, Heijmen RH,
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137 Huzen J, van der Harst P, de Boer RA, Lesman-Leegte
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104
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139 Ionita MG, Arslan F, de Kleijn DP, Pasterkamp G;
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140Ionita MG, van den Borne P, Catanzariti LM, Moll FL,
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141 Ionita MG, Catanzariti LM, Bots ML, de Vries JP, Moll FL,
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144 Jukema JW, Chiang CW, Ferrières J, Santos RD, Verdejo
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145 Kaandorp TA, Bax JJ, Bleeker SE, Doornbos J, Viergever
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147 Kastelein JJ, Duivenvoorden R, Deanfield J, de Groot E,
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152 Koetsier M, Lutgers HL, de Jonge C, Links TP, Smit AJ,
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159 Lai RC, Arslan F, Tan SS, Tan B, Choo A, Lee MM, Chen
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160 Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS,
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161 Leenders JJ, Wijnen WJ, Hiller M, van der Made I, Lentink
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162Leeuwis JW, Nguyen TQ, Theunissen MG, Peeters W,
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153 Kooiman K, Emmer M, Foppen-Harteveld M, van Wamel A,
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154 Koopmann TT, Bezzina CR; “Genetics of lone atrial
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155 Kooter AJ, Ijzerman RG, Kamp O, Boonstra AB, Smulders
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156 Koyak Z, de Groot JR, Mulder BJ; “Interventional and
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157 Kuipers I, van der Harst P, Kuipers F, van Genne L, Goris
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158 Kuipers I, Li J, Vreeswijk-Baudoin I, Koster J, van der Harst
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163Leung KY, Danilouchkine MG, van Stralen M, de Jong
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164 Liu J, van Mil A, Vrijsen K, Zhao J, Gao L, Goumans MJ,
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169 Marcus FI, McKenna WJ, Sherrill D, Basso C, Bauce B,
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170 Marcus FI, McKenna WJ, Sherrill D, Basso C, Bauce B,
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180 Mills P, Timmis A, Huber K, Ector H, Masic I, Ivanusa M,
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272 Timmer SA, Germans T, Götte MJ, Rüssel IK, Dijkmans PA,
Lubberink M, Ten Berg JM, Ten Cate FJ, Lammertsma AA,
Knaapen P, van Rossum AC; “Determinants of myocardial
energetics and efficiency in symptomatic hypertrophic
cardiomyopathy.”; Eur. J. Nucl. Med. Mol. Imaging 2010
Apr; 37 (4); 779-88
273 Tolenaar JL, van Keulen JW, Leijdekkers VJ, Vonken EJ,
Moll FL, van Herwaarden JA; “A ruptured aneurysm after
stent graft puncture during computed tomography-guided
thrombin injection.”; J. Vasc. Surg. 2010 Oct; 52 (4); 10457
274 Tops LF, Schalij MJ, Bax JJ; “Imaging and atrial fibrillation:
the role of multimodality imaging in patient evaluation and
management of atrial fibrillation.”; Eur. Heart J. 2010 Mar;
31 (5); 542-51
275 Trompet S, Craen AJ, Jukema JW, Pons D, Slagboom PE,
Kremer D, Bollen EL, Westendorp RG; “Variation in the
CBP gene involved in epigenetic control associates with
cognitive function.”; Neurobiol. Aging 2011 Mar; 32 (3)
276 Tu S, Koning G, Jukema W, Reiber JH; “Assessment of
obstruction length and optimal viewing angle from biplane
X-ray angiograms.”; Int J Cardiovasc Imaging 2010 Jan;
26 (1); 5-17
277 Ulu N, Henning RH, Goris M, Schoemaker RG, van Gilst
WH; “Effects of ivabradine and metoprolol on cardiac
angiogenesis and endothelial dysfunction in rats with heart
failure.”; J. Cardiovasc. Pharmacol. 2009 Jan; 53 (1); 9-17
278 Umar S, Steendijk P, Ypey DL, Atsma DE, van der Wall EE,
Schalij MJ, van der Laarse A; “Novel approaches to treat
experimental pulmonary arterial hypertension: a review.”;
J. Biomed. Biotechnol. 2010; 2010 (0); 702836
279Vaartjes I, Hendrix A, Hertogh EM, Grobbee DE,
Doevendans PA, Mosterd A, Bots ML; “Sudden death
in persons younger than 40 years of age: incidence and
causes.”; Eur J Cardiovasc Prev Rehabil 2009 Oct; 16 (5);
592-6
280 van Bemmel T, Delgado V, Bax JJ, Gussekloo J, Blauw
GJ, Westendorp RG, Holman ER; “Impact of valvular heart
disease on activities of daily living of nonagenarians: the
Leiden 85-plus study a population based study.”; BMC
Geriatr 2010; 10 (0); 17
281 van Beusekom HM, Sorop O, van den Heuvel M, Onuma Y,
Duncker DJ, Danser AH, van der Giessen WJ; “Endothelial
function rather than endothelial restoration is altered in
paclitaxel- as compared to bare metal-, sirolimusand
tacrolimus-eluting stents.”; EuroIntervention 2010 May; 6
(1); 117-25
282Van Bommel RJ, Ypenburg C, Borleffs CJ, Delgado V,
Marsan NA, Bertini M, Holman ER, Schalij MJ, Bax JJ;
“Value of tissue Doppler echocardiography in predicting
response to cardiac resynchronization therapy in patients
with heart failure.”; Am. J. Cardiol. 2010 Apr 15; 105 (8);
1153-8
283Van Bommel RJ, Gorcsan J, Chung ES, Abraham WT,
Gjestvang FT, Leclercq C, Monaghan MJ, Nihoyannopoulos
P, Peraldo C, Yu CM, Demas M, Gerritse B, Bax JJ;
“Effects of cardiac resynchronisation therapy in patients
with heart failure having a narrow QRS Complex enrolled
in PROSPECT.”; Heart 2010 Jul; 96 (14); 1107-13
284Van Bommel RJ, Marsan NA, Koppen H, Delgado V,
Borleffs CJ, Ypenburg C, Bertini M, Schalij MJ, Bax JJ;
“Effect of cardiac resynchronization therapy on cerebral
blood flow.”; Am. J. Cardiol. 2010 Jul 1; 106 (1); 73-7
285 Van Bommel RJ, Delgado V, Schalij MJ, Bax JJ; “Critical
appraisal of the use of cardiac resynchronization therapy
beyond current guidelines.”; J. Am. Coll. Cardiol. 2010 Aug
31; 56 (10); 754-62
286 Van Bommel RJ, van Rijnsoever E, Borleffs CJ, Delgado
V, Marsan NA, Bertini M, Schalij MJ, Bax JJ; “Effect of
cardiac resynchronization therapy in patients with New
York Heart Association functional class IV heart failure.”;
Am. J. Cardiol. 2010 Oct 15; 106 (8); 1146-51
114
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287Van Bommel RJ, Borleffs CJ, Ypenburg C, Marsan NA,
Delgado V, Bertini M, van der Wall EE, Schalij MJ, Bax
JJ; “Morbidity and mortality in heart failure patients treated
with cardiac resynchronization therapy: influence of preimplantation characteristics on long-term outcome.”; Eur.
Heart J. 2010 Nov; 31 (22); 2783-90
288 Van Bommel RJ, Tanaka H, Delgado V, Bertini M, Borleffs
CJ, Ajmone Marsan N, Holzmeister J, Ruschitzka F, Schalij
MJ, Bax JJ, Gorcsan J; “Association of intraventricular
mechanical dyssynchrony with response to cardiac
resynchronization therapy in heart failure patients with a
narrow QRS complex.”; Eur. Heart J. 2010 Dec; 31 (24);
3054-62
289 Van de Veire NR, Djaberi R, Schuijf JD, Bax JJ; “Noninvasive imaging: Non-invasive assessment of coronary
artery disease in diabetes.”; Heart 2010 Apr; 96 (7); 560-72
290 Van de Veire NR, Braun J, Delgado V, Versteegh MI, Dion
RA, Klautz RJ, Bax JJ; “Tricuspid annuloplasty prevents
right ventricular dilatation and progression of tricuspid
regurgitation in patients with tricuspid annular dilatation
undergoing mitral valve repair.”; J. Thorac. Cardiovasc.
Surg. 2010 Sep 8
291 van den Heuvel M, Sorop O, Batenburg WW, Bakker CL,
de Vries R, Koopmans SJ, van Beusekom HM, Duncker
DJ, Danser AH, van der Giessen WJ; “Specific coronary
drug-eluting stents interfere with distal microvascular
function after single stent implantation in pigs.”; JACC
Cardiovasc Interv 2010 Jul; 3 (7); 723-30
292van den Hurk K, Alssema M, Kamp O, Henry RM,
Stehouwer CD, Diamant M, Boomsma F, Heine RJ,
Nijpels G, Paulus WJ, Dekker JM; “Slightly elevated
B-type natriuretic peptide levels in a non-heart failure
range indicate a worse left ventricular diastolic function in
individuals with, as compared with individuals without, type
2 diabetes: the Hoorn Study.”; Eur. J. Heart Fail. 2010 Sep;
12 (9); 958-65
293 van der Bijl N, de Bruin PW, Geleijns J, Bax JJ, Schuijf
JD, de Roos A, Kroft LJ; “Assessment of coronary artery
calcium by using volumetric 320-row multi-detector
computed tomography: comparison of 0.5 mm with 3.0 mm
slice reconstructions.”; Int J Cardiovasc Imaging 2010 Apr;
26 (4); 473-82
294 van der Bijl N, Joemai RM, Geleijns J, Bax JJ, Schuijf JD,
de Roos A, Kroft LJ; “Assessment of Agatston coronary
artery calcium score using contrast-enhanced CT coronary
angiography.”; AJR Am J Roentgenol 2010 Dec; 195 (6);
1299-305
295van der Bom T, Winter MM, Bouma BJ, Groenink M,
Vliegen HW, Pieper PG, van Dijk AP, Sieswerda GT, RoosHesslink JW, Zwinderman AH, Mulder BJ; “Rationale and
design of a trial on the effect of angiotensin II receptor
blockers on the function of the systemic right ventricle.”;
Am. Heart J. 2010 Nov; 160 (5); 812-8
115
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Heart Institute
296van der Bom T, Zomer AC, Zwinderman AH, Meijboom
FJ, Bouma BJ, Mulder BJ; “The changing epidemiology of
congenital heart disease.”; Nat Rev Cardiol 2011 Jan; 8
(1); 50-60
297 van der Giessen AG, Schaap M, Gijsen FJ, Groen HC, van
Walsum T, Mollet NR, Dijkstra J, van de Vosse FN, Niessen
WJ, de Feyter PJ, van der Steen AF, Wentzel JJ; “3D
fusion of intravascular ultrasound and coronary computed
tomography for in-vivo wall shear stress analysis: a
feasibility study.”; Int J Cardiovasc Imaging 2010 Oct; 26
(7); 781-96
298 van der Harst P, de Boer RA, Samani NJ, Wong LS, Huzen
J, Codd V, Hillege HL, Voors AA, van Gilst WH, Jaarsma T,
van Veldhuisen DJ; “Telomere length and outcome in heart
failure.”; Ann. Med. 2010; 42 (1); 36-44
299van der Hulst AE, Westenberg JJ, Kroft LJ, Bax JJ,
Blom NA, de Roos A, Roest AA; “Tetralogy of fallot: 3D
velocity-encoded MR imaging for evaluation of right
ventricular valve flow and diastolic function in patients after
correction.”; Radiology 2010 Sep; 256 (3); 724-34
300 van der Hulst AE, Roest AA, Delgado V, Holman ER, de
Roos A, Blom NA, Bax JJ; “Relationship between temporal
sequence of right ventricular deformation and right
ventricular performance in patients with corrected tetralogy
of Fallot.”; Heart 2011 Feb; 97 (3); 231-6
301 van der Laarse A, Steendijk P, van der Wall EE;
“Evaluation of pulmonary arterial hypertension: invasive or
noninvasive?”; Int J Cardiovasc Imaging 2010 May 17
314 van der Wall EE; “Tissue characterization in Takotsubo
cardiomyopathy; a valuable approach?”; Int J Cardiovasc
Imaging 2010 Feb; 26 (2); 233-6
302van der Pouw Kraan TC, Schirmer SH, Fledderus JO,
Moerland PD, Baggen JM, Leyen TA, van der Laan AM,
Piek JJ, van Royen N, Horrevoets AJ; “Expression of
a retinoic acid signature in circulating CD34 cells from
coronary artery disease patients.”; BMC Genomics 2010;
11 (0); 388
315 van der Wall EE, Schalij MJ; “Mitral valve prolapse: a
source of arrhythmias?”; Int J Cardiovasc Imaging 2010
Feb; 26 (2); 147-9
303 van der Spoel TI, Lee JC, Vrijsen K, Sluijter JP, Cramer
MJ, Doevendans PA, van Belle E, Chamuleau SA; “Nonsurgical stem cell delivery strategies and in vivo cell
tracking to injured myocardium.”; Int J Cardiovasc Imaging
2010 Jun 25
304 van der Vleuten PA, Nijveldt R, Tan ES, Tio RA, van Rossum
AC, Zijlstra F; “Value and limitations of electromechanical
endocardial mapping in the assessment of global and
regional left ventricular function and transmural extent
of infarction: a comparison with cardiovascular magnetic
resonance.”; EuroIntervention 2010 Nov; 6 (5); 616-22
305 van der Wal MH, Jaarsma T, Moser DK, van Gilst WH, van
Veldhuisen DJ; “Qualitative examination of compliance
in heart failure patients in The Netherlands.”; Heart Lung
2010 Mar-Apr; 39 (2); 121-30
316 van der Wall EE, Bax JJ, Swenne CA, Steendijk P, Schalij
MJ; “Cardiovascular dynamics in ischemic cardiomyopathy
during exercise.”; Int J Cardiovasc Imaging 2010 Feb; 26
(2); 161-4
317 van der Wall EE; “Mate for the heart: pulsatile or
continuous?”; Neth Heart J 2010 Feb; 18 (2); 59
318 van der Wall EE, Schuijf JD, Bax JJ, Jukema JW, Schalij
MJ; “CT perfusion angiography; beware of artifacts!”; Int J
Cardiovasc Imaging 2010 Mar; 26 (3); 355-8
319 van der Wall EE, Schalij MJ, van der Laarse A, Bax JJ;
“Cardiac resynchronization therapy; the importance of
evaluating cardiac metabolism.”; Int J Cardiovasc Imaging
2010 Mar; 26 (3); 293-7
320 van der Wall EE, Siebelink HM, Bax JJ; “Left ventricular
mass assessment by CMR; how to define the optimal
index.”; Int J Cardiovasc Imaging 2010 Apr; 26 (4); 469-72
306 van der Wall EE; “Invasive treatment of patients with
cardiomyopathy: CRT-ICD better than ICD alone.”; Neth
Heart J 2009 Dec; 17 (12); 455
321 van der Wall EE, Siebelink HM, Bax JJ; “Evaluation of
hypertrophic cardiomyopathy: new horizons for CMR?”;
Neth Heart J 2010 Mar; 18 (3); 116-7
307 van der Wall EE, Bax JJ, Jukema JW, Schalij MJ; “Distal
protection beneficial?”; Int J Cardiovasc Imaging 2009 Nov
20
322 van der Wall EE; “Invasive treatment of ACS: early or later
on?”; Neth Heart J 2010 Mar; 18 (3); 115
308 van der Wall EE, Schalij MJ; “Mitral valve prolapse: a
source of arrhythmias?”; Int J Cardiovasc Imaging 2009
Nov 24
309 van der Wall EE; “Tissue characterization in Takotsubo
cardiomyopathy; a valuable approach?”; Int J Cardiovasc
Imaging 2009 Nov 18
310 van der Wall EE; “Smoke gets in your eyes: the benefits of
a ban?”; Neth Heart J 2010 Jan; 18 (1); 3
311 van der Wall EE, Schuijf JD, Bax JJ, Jukema JW, Schalij
MJ; “Fingertip digital thermal monitoring: a fingerprint for
cardiovascular disease?”; Int J Cardiovasc Imaging 2010
Feb; 26 (2); 249-52
312 van der Wall EE, Schalij MJ, Bax JJ; “Cardiac
resynchronization therapy; evaluation by advanced
imaging techniques.”; Int J Cardiovasc Imaging 2010 Feb;
26 (2); 199-202
313 van der Wall EE, Bax JJ, Jukema JW, Schalij MJ; “Distal
protection beneficial?”; Int J Cardiovasc Imaging 2010
Feb; 26 (2); 135-8
323 van der Wall EE, Bax JJ, Reiber JH; “Cardiac magnetic
resonance imaging analysis in STEMI: quantitative or still
visual?”; Int J Cardiovasc Imaging 2010 May 8
324 van der Wall EE, Scholte AJ, Holman ER, Bax JJ; “Stress
imaging in patients with diabetes; routine practice?”; Int J
Cardiovasc Imaging 2010 May 8
325 van der Wall EE, van Velzen JE, de Graaf FR, Boogers
MM, Schuijf JD, Bax JJ; “Increased accuracy in computed
tomography coronary angiography; a new body surface
area adapted protocol.”; Int J Cardiovasc Imaging 2010
Jun; 26 (5); 601-4
326 van der Wall EE, Holman ER, Scholte AJ, Bax JJ;
“Echocardiography in Takotsubo cardiomyopathy; a useful
approach?”; Int J Cardiovasc Imaging 2010 Jun; 26 (5);
537-40
327 van der Wall EE, Siebelink HM, Scholte AJ, Bax JJ;
“Positron emission tomography; viable tool in patients preCABG?”; Int J Cardiovasc Imaging 2010 Aug; 26 (6); 661-4
116
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Netherlands
Heart Institute
335van der Wall EE, Zeppenfeld K, Bax JJ, Siebelink HM,
Schalij MJ; “CMR-determined scar volume: predictive for
ventricular tachycardias?”; Int J Cardiovasc Imaging 2010
Nov 9
336 van der Wall EE, Scholte AJ, Bax JJ; “Gated myocardial
SPECT imaging; true additional value in AMI?”; Int J
Cardiovasc Imaging 2010 Dec; 26 (8); 893-6
337 van der Wall EE, Schuijf JD, Bax JJ; “Use of the anteriorposterior chest diameter in CT: reduction in radiation
dose?”; Int J Cardiovasc Imaging 2010 Dec; 26 (8); 941-5
338 van der Wall EE; “New guidelines for the management of
atrial fibrillation: what’s new?”; Neth Heart J 2010 Nov; 18
(11); 519
339 van der Wall EE, van Velzen JE, de Graaf FR, Jukema
JW, Schuijf JD, Bax JJ; “320-row CT scanning: reduction
in tube current parallels reduction in radiation exposure?”;
Int J Cardiovasc Imaging 2010 Dec 7
340 van der Wall EE, de Graaf FR, van Velzen JE, Jukema
JW, Schuijf JD, Bax JJ; “Functional analysis by 64-slice CT
scanning: prediction of left ventricular dysfunction together
with reduction in radiation exposure?”; Eur. Heart J. 2010
Dec 18
328 van der Wall EE, de Graaf FR, van Velzen JE, Jukema
JW, Bax JJ, Schuijf JD; “IVUS detects more coronary
calcifications than MSCT; matter of both resolution and
cross-sectional assessment?”; Int J Cardiovasc Imaging
2010 Jul 11
329 van der Wall EE; “Remote monitoring: coming close?”;
Neth Heart J 2010 Jun; 18 (6); 283
330 van der Wall EE; “GENDER issues: media news value?”;
Neth Heart J 2010 Aug; 18 (7); 343
331 van der Wall EE, Jukema JW, Schuijf JD, Bax JJ; “100 kV
versus 120 kV: effective reduction in radiation dose?”; Int J
Cardiovasc Imaging 2010 Sep 1
332 van der Wall EE; “Increasing recognition of NHJ: a firsttime impact factor of 1.4!”; Neth Heart J 2010 Sep; 18 (9);
399
333 van der Wall EE; “NHJ direct online in January 2011!”;
Neth Heart J 2010 Oct; 18 (10); 463
334 van der Wall EE, Reiber JH; “Assessment of left ventricular
function: visual or quantitative?”; Int J Cardiovasc Imaging
2010 Oct 28
117
341 van der Werf C, Hofman N, Tan HL, van Dessel PF, Alders
M, van der Wal AC, van Langen IM, Wilde AA; “Diagnostic
yield in sudden unexplained death and aborted cardiac
arrest in the young: the experience of a tertiary referral
center in The Netherlands.”; Heart Rhythm 2010 Oct; 7
(10); 1383-9
342 van der Zwaan HB, Helbing WA, McGhie JS, Geleijnse ML,
Luijnenburg SE, Roos-Hesselink JW, Meijboom FJ; “Clinical
value of real-time three-dimensional echocardiography for
right ventricular quantification in congenital heart disease:
validation with cardiac magnetic resonance imaging.”; J
Am Soc Echocardiogr 2010 Feb; 23 (2); 134-40
343 van Derwall EE, Scholte AJ, Siebelink HM, Bax JJ;
“Assessment of left ventricular volumes; reliable by gated
SPECT?”; Int J Cardiovasc Imaging 2010 Oct 6
344Niessen HW, Van Milligen FJ, Paulus WJ, Meischl C,
Rossum BC, Spreeuwenberg M, Hack E, Aarden LA,
Makker SP, Hahn NE, Juffermans LJ, Krijnen PA, Vermond
RA, van Dijk A; “Intravenous clusterin administration
reduces myocardial infarct size in rats.”; Eur. J. Clin. Invest.
2010 Jul 26
345Hack E, Aarden LA, Makker SP, Hahn NE, Juffermans
LJ, Krijnen PA, Vermond RA, van Dijk A, Niessen HW,
Van Milligen FJ, Paulus WJ, Meischl C, van Rossum BC,
Spreeuwenberg M; “Intravenous clusterin administration
reduces myocardial infarct size in rats.”; Eur. J. Clin. Invest.
2010 Oct; 40 (10); 893-902
346 van Elderen SG, de Roos A, de Craen AJ, Westendorp
RG, Blauw GJ, Jukema JW, Bollen EL, Middelkoop HA,
van Buchem MA, van der Grond J; “Progression of brain
atrophy and cognitive decline in diabetes mellitus: a 3-year
follow-up.”; Neurology 2010 Sep 14; 75 (11); 997-1002
347 van Engelen K, Topf A, Keavney BD, Goodship JA, van
der Velde ET, Baars MJ, Snijder S, Moorman AF, Postma
AV, Mulder BJ; “22q11.2 Deletion Syndrome is underrecognised in adult patients with tetralogy of Fallot and
pulmonary atresia.”; Heart 2010 Apr; 96 (8); 621-4
350 Van Gelder IC, Smit MD, Alings M, Crijns HJ; “Upstream
therapy in patients with early atrial fibrillation: The
relevance of the Routine versus Aggressive upstream
rhythm Control for prevention of Early atrial fibrillation in
heart failure (RACE 3) study.”; Neth Heart J 2010 Nov; 18
(11); 522-3
351 van Gilst WH, van der Wall EE; “Einthoven dissertation
prize 2010.”; Neth Heart J 2010 Jun; 18 (6); 333-5
352 van Keulen JW, Vincken KL, van Prehn J, Tolenaar JL,
Bartels LW, Viergever MA, Moll FL, van Herwaarden JA;
“The influence of different types of stent grafts on aneurysm
neck dynamics after endovascular aneurysm repair.”; Eur
J Vasc Endovasc Surg 2010 Feb; 39 (2); 193-9
353 van Keulen JW, Moll FL, Tolenaar JL, Verhagen HJ, van
Herwaarden JA; “Validation of a new standardized method
to measure proximal aneurysm neck angulation.”; J. Vasc.
Surg. 2010 Apr; 51 (4); 821-8
354 van Keulen JW, Moll FL, Verhagen HJ, van Herwaarden
JA; “Commentary: DynaCT and its use in patients with
ruptured abdominal aortic aneurysm.”; J. Endovasc. Ther.
2010 Apr; 17 (2); 190-1
355 van Keulen JW, van Prehn J, Moll FL, van Herwaarden JA;
“Recurrent stent-graft disintegration caused by cardiacinduced aortoiliac movements.”; J. Endovasc. Ther. 2010
Jun; 17 (3); 354-5
356 van Keulen JW, Moll FL, Barwegen GK, Vonken EP, van
Herwaarden JA; “Pulsatile distension of the proximal
aneurysm neck is larger in patients with stent graft
migration.”; Eur J Vasc Endovasc Surg 2010 Sep; 40 (3);
326-31
357 van Keulen JW, Moll FL, van Herwaarden JA; “Tips and
techniques for optimal stent graft placement in angulated
aneurysm necks.”; J. Vasc. Surg. 2010 Oct; 52 (4); 1081-6
348van Es AC, van der Grond J, ten Dam VH, de Craen
AJ, Blauw GJ, Westendorp RG, Admiraal-Behloul F, van
Buchem MA; “Associations between total cerebral blood
flow and age related changes of the brain.”; PLoS ONE
2010; 5 (3); e9825
349Van Gelder IC, Groenveld HF, Crijns HJ, Tuininga YS,
Tijssen JG, Alings AM, Hillege HL, Bergsma-Kadijk JA,
Cornel JH, Kamp O, Tukkie R, Bosker HA, van Veldhuisen
DJ, Van den Berg MP; “Lenient versus strict rate control in
patients with atrial fibrillation.”; N. Engl. J. Med. 2010 Apr
15; 362 (15); 1363-73
358 van Keulen JW, Moll FL, Arts J, Vonken EJ, van Herwaarden
JA; “Aortic neck angulations decrease during and after
endovascular aneurysm repair.”; J. Endovasc. Ther. 2010
Oct; 17 (5); 594-8
359van Lammeren GW, de Vries JP, Vink A, de Kleijn DP,
Moll FL, Pasterkamp G; “New predictors of adverse
cardiovascular events following vascular surgery.”; Semin
Cardiothorac Vasc Anesth 2010 Jun; 14 (2); 148-53
360 van Leuven SI, van Wijk DF, Volger OL, de Vries JP, van
der Loos CM, de Kleijn DV, Horrevoets AJ, Tak PP, van der
Wal AC, de Boer OJ, Pasterkamp G, Hayden MR, Kastelein
JJ, Stroes ES; “Mycophenolate mofetil attenuates plaque
inflammation in patients with symptomatic carotid artery
stenosis.”; Atherosclerosis 2010 Jul; 211 (1); 231-6
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361 van Neer PL, Matte G, Danilouchkine MG, Prins C, van den
Adel F, de Jong N; “Super-harmonic imaging: development
of an interleaved phased-array transducer.”; IEEE Trans
Ultrason Ferroelectr Freq Control 2010; 57 (2); 455-68
362 van Neer PL, Vos HJ, de Jong N; “Reflector-based phase
calibration of ultrasound transducers.”; Ultrasonics 2011
Jan; 51 (1); 1-6
363van Nies JA, Marques RB, Trompet S, de Jong Z,
Kurreeman FA, Toes RE, Jukema JW, Huizinga TW, van
der Helm-van Mil AH; “TRAF1/C5 polymorphism is not
associated with increased mortality in rheumatoid arthritis:
two large longitudinal studies.”; Arthritis Res. Ther. 2010;
12 (2); R38
364 van Rees JB, Borleffs CJ, Bax JJ, Nagtegaal EM, van der
Velde ET, van Erven L, Schalij MJ; “Implementation of
lead safety recommendations.”; Pacing Clin Electrophysiol
2010 Apr; 33 (4); 431-6
365 van Rijen HV, de Bakker JM, van Veen TA; “Exciting
treatment of reentrant arrhythmias.”; Cardiovasc. Res.
2011 Jan 1; 89 (1); 4-5
366 van Rijsingen IA, Hermans-van Ast JF, Arens YH, Schalla
SM, de Die-Smulders CE, van den Wijngaard A, Pinto
YM; “Hypertrophic cardiomyopathy family with doubleheterozygous mutations; does disease severity suggest
doubleheterozygosity?”; Neth Heart J 2009 Dec; 17 (12);
458-63
367van Soest G, Goderie T, Regar E, Koljenović S, van
Leenders GL, Gonzalo N, van Noorden S, Okamura T,
Bouma BE, Tearney GJ, Oosterhuis JW, Serruys PW,
van der Steen AF; “Atherosclerotic tissue characterization
in vivo by optical coherence tomography attenuation
imaging.”; J Biomed Opt 2010 Jan-Feb; 15 (1); 011105
368 van Veldhuisen DJ, Voors AA, van Gilst WH; “The
European Journal of Heart Failure in 2010: current impact
factor, time-to-first decision, and number of submissions.”;
Eur. J. Heart Fail. 2010 Sep; 12 (9); 895-7
369van Velzen JE, Schuijf JD, de Graaf FR, Boersma E,
Pundziute G, Spanó F, Boogers MJ, Schalij MJ, Kroft LJ, de
Roos A, Jukema JW, van der Wall EE, Bax JJ; “Diagnostic
performance of non-invasive multidetector computed
tomography coronary angiography to detect coronary
artery disease using different endpoints: detection of
significant stenosis vs. detection of atherosclerosis.”; Eur.
Heart J. 2011 Mar; 32 (5)
370 van Velzen JE, Schuijf JD, van Werkhoven JM, Herzog
BA, Pazhenkottil AP, Boersma E, de Graaf FR, Scholte AJ,
Kroft LJ, de Roos A, Stokkel MP, Jukema JW, Kaufmann
PA, van der Wall EE, Bax JJ; “Predictive value of multislice
computed tomography variables of atherosclerosis for
ischemia on stress-rest single-photon emission computed
tomography.”; Circ Cardiovasc Imaging 2010 Nov 1; 3 (6);
718-26
119
Netherlands
Heart Institute
371 van Vliet P, de Boer TP, van der Heyden MA, El Tamer
MK, Sluijter JP, Doevendans PA, Goumans MJ;
“Hyperpolarization induces differentiation in human
cardiomyocyte progenitor cells.”; Stem Cell Rev 2010 Jun;
6 (2); 178-85
372van Vliet P, Smits AM, de Boer TP, Korfage TH, Metz
CH, Roccio M, van der Heyden MA, van Veen TA,
Sluijter JP, Doevendans PA, Goumans MJ; “Foetal and
adult cardiomyocyte progenitor cells have different
developmental potential.”; J. Cell. Mol. Med. 2010 Apr; 14
(4); 861-70
373 van Werkhoven JM, Heijenbrok MW, Schuijf JD, Jukema
JW, Boogers MM, van der Wall EE, Schreur JH, Bax JJ;
“Diagnostic accuracy of 64-slice multislice computed
tomographic coronary angiography in patients with
an intermediate pretest likelihood for coronary artery
disease.”; Am. J. Cardiol. 2010 Feb 1; 105 (3); 302-5
374 van Werkhoven JM, Heijenbrok MW, Schuijf JD, Jukema
JW, van der Wall EE, Schreur JH, Bax JJ; “Combined
non-invasive anatomical and functional assessment with
MSCT and MRI for the detection of significant coronary
artery disease in patients with an intermediate pre-test
likelihood.”; Heart 2010 Mar; 96 (6); 425-31
375van Werkhoven JM, Cademartiri F, Seitun S, Maffei E,
Palumbo A, Martini C, Tarantini G, Kroft LJ, de Roos A,
Weustink AC, Jukema JW, Ardissino D, Mollet NR, Schuijf
JD, Bax JJ; “Diabetes: prognostic value of CT coronary
angiography--comparison with a nondiabetic population.”;
Radiology 2010 Jul; 256 (1); 83-92
376 van Werkhoven JM, de Boer SM, Schuijf JD, Cademartiri
F, Maffei E, Jukema JW, Boogers MJ, Kroft LJ, de Roos
A, Bax JJ; “Impact of clinical presentation and pretest
likelihood on the relation between calcium score and
computed tomographic coronary angiography.”; Am. J.
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377 Veeger NJ, Zijlstra F, Hillege HL, van der Meer J; “Fourteenyear follow-up from CABADAS: vitamin K antagonists or
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