Download 2009 - Waddensymposium

2nd Waddensymposium
Vascular regeneration
- from biology to clinical use -
June 28 – July 1, 2009, Grand Hotel Opduin, Texel, the Netherlands
nd
2 Waddensymposium
Vascular regeneration
- from biology to clinical use -
This symposium is organized in collaboration with:
www.waddensymposium.eu
Location: Grand Hotel Opduin, Texel
www.opduin.nl
Hereby the final program for our second “Waddensymposium” organized by the department
of Immunohematology and Blood Transfusion of the LUMC.
Location:
Grand Hotel Opduin
Ruijslaan 22
1796 AD De Koog (Texel)
The Netherlands
Department of Immunohematology and Blood Transfusion
Symposium secretariat: Amber Günthardt
Mail: [email protected]
Phone: +31 71 526 38 27 cell phone: +31.6.53527185
Organizing committee:
Wim Fibbe
Paul Quax
Peter van den Elsen
Jaap Jan Zwaginga cell phone +31.6.50655116
Sunday 28th of June
Final program 28th of June till 1st of July 2009
18.00
Arrival Hotel Opduin, Texel
20.00
Dinner
Monday 29th of June
07.30 – 08.15 Breakfast
08.15 – 08.30 Welcome, symposium in perspective: Willem Fibbe, Leiden [The Netherlands]
Vascular remodeling
Chair: Jaap Jan Zwaginga
08.30 – 09.05 Erik Biessen, Maastricht [The Netherlands]
“Role of mast cells in plaque instability “
09.05 – 09.40 Robert Kleemann, Leiden [The Netherlands]
“Pharmacological modulation of atherosclerotic plaques:
effects on lesion progression and regression”
09.40 – 10.15 Peter van den Elsen, Leiden [The Netherlands]
“Epigenetics and vascular disease”
10.15 – 10.45 Quick abstracts: R. Wierda, J. Karper, H. Roelofs, M. Ewing and L. Seghers
10.45 – 11.00 Coffee break
11.00 – 11.25 Christian Weber, Aachen [Germany]
“Chemokines in atherosclerotic vascular remodeling“
11.25 – 12.30 Abstracts
Marco de Ruiter, Leiden [The Netherlands]
“Maternal Transmission of Risk for Atherosclerosis”
Karolina Janeczek, Twente [The Netherlands]
“Mesenchymal Stem Cells as Source for Endothelial Progenitors”
Diana Ploeger, Groningen [The Netherlands]
“Modulated monocyte adherence: implications for peripheral vascular disease”
Margreet de Vries, Leiden [The Netherlands]
“Timp-1 modulates vein graft thickening and intra-plaque hemorrhages”
12.30 – 13.15 Lunch
13.15 – 18.00 Social event
18.00 – 20.00 Diner
Chair: Anton Jan van Zonneveld
20.00 – 21.30 Douglas Losordo, Chicago [USA]
“Intramyocardial autologous CD34 cell therapy for refractory angina”
Tuesday 30th of June
Biology of arteriogenesis and neovascularization
Chair: Peter van den Elsen
08.30 – 09.05 Paul Quax, Leiden [The Netherlands]
“NK cells and their role in modulating arteriogenesis”
09.05 – 09.40 Mervin Yoder, Indianapolis [USA]
“Circulating cord blood endothelial colony forming cells (ECFC) can be
specified to an arterial endothelial gene expression fate in vitro”
09.40 – 10.15 Victor van Hinsbergh, Amsterdam [The Netherlands]
“Hypoxia and angiogenesis”
10.15 – 10.45 Coffee break
10.45 – 11.20 Marco Harmsen, Groningen [The Netherlands]
“Stem cells in angiogenesis”
11.20 – 11.55 Imo Hoefer, Utrecht [The Netherlands]
“Modulation of arteriogenic response by monocytes”
11.55 – 12.30 Mat Daemen, Maastricht [The Netherlands]
“Mechanisms of Plaque Instability: role of circulating cells”
12.30 – 13.15 Lunch
13.15 – 18.00 Social event
18.00 – 20.00 Diner
Chair: Wim Fibbe
20.00 – 21.30 Toyoaki Murohara, Kumamoto [Japan]
“Implantation of Adipose-Derived Regenerative Cells Enhances
Ischemia-induced Angiogenesis”
Wednesday 1st of July
Therapeutic vascularization
Chair: Paul Quax
08.30 – 09.05 Anton Jan van Zonneveld, Leiden [The Netherlands]
“Mechanisms in endothelial maintenance and repair”
09.05 – 09.40 Jamie Case, Indianapolis [USA]
“Application of Poly-Chromatic Flow Cytometry Analysis
and Cell Sorting Techniques to Identify Novel Subsets of
Rare Circulating Cells with Angiogenic Potential”
09.40 – 10.15 Alain Tedgui, Paris [France]
“Immune-regulatory Pathways in Atherosclerosis”
10.15 – 11.15 Abstracts
Sacha Geutskens, Leiden [The Netherlands]
“Monocytes as cellular therapy for vascular regeneration”
Marie-Jose Goumans, Leiden [The Netherlands]
“CXCR4/ CD26 modulated recruitment of HHT-1 mononuclear cells to the
ischemic heart”
Eric van der Veer, Leiden [The Netherlands]
“The mRNA-binding protein Quaking is essential for vascular smooth
muscle cell function”
11.15 – 11.35 Coffee break
11.35 – 13.00 Position paper
13.00 – 13.30 Lunch
END OF MEETING
ABSTRACTS
Role of mast cells in plaque instability
MONDAY
Erik A.L. Biessen, Saskia C..A. de Jager , Theo J.C. Van Berkel, Ilze Bot
Manifest atherosclerosis may present as myocardial infarction and stroke and is generally
caused by thrombotic plaque rupture. The processes underlying plaque rupture are only
poorly understood, but may amongst others involve a fulminant inflammation. Pathology
studies indicated the increased presence of mast cells, important inflammatory effector cells
in allergy responses, in (peri)vascular tissue during plaque progression, suggestive of a causal
role in disease progression. Recent experimental data in mouse models for atherosclerosis
have shown that mast cells and derived mediators can profoundly impact plaque progression
and reduce plaque stability. In this presentation, we will discuss recent advances in
experimental research on mast cells in atherosclerosis and the therapeutic potential of
modulation of mast cell function in cardiovascular disease. We will delineate the
multitethered effects of acute and chronic focal mast cell activation, discuss the relevant
players in these processes and speculate on the endogenous triggers of mast cell mobilization
and activation in the adventitia.
MONDAY
Pharmacological modulation of atherosclerotic plaques: effects on lesion progression
and regression
Robert Kleemann
Atherosclerosis was previously thought to be a disease primarily involving lipid
accumulation in the arterial wall. Current concepts of the disease include involvement of the
immune system and chronic inflammation as crucial factors in all stages of the
atherosclerotic process: the initiation of endothelial dysfunction, fatty streak formation and
lesion progression and complication. This central role of inflammation and immunity in
atherogenesis suggests that anti-inflammatory therapies might have a beneficial role in
management of the disease. Because lipid lowering clearly causes inflammation associated
with atherosclerosis to diminish, it is difficult to assess whether pharmaceuticals used in
clinical practice have direct anti-inflammatory effects (ie, independent of their cholesterollowering effects). Furthermore, the set-up of many animal studies studying intervention in
the inflammatory component of atherogenesis also deviates from the clinical norm because
intervention is started simultaneously with onset of the experimentally controlled disease
(ie long before first lesions are formed). This translational discrepancy is mainly due to the
lack of appropriate (drug-sensitive) animal models that allow studying regression. Using a
humanized and drug-sensitive animal model, ApoE3Leiden mouse, evidence is provided for
an additional health benefit by quenching the inflammatory component of disease. Newly
developed experimental conditions enabling the study of lesion regression reveal that not all
compounds that reduce lesion progression are also effective in promoting lesion regression.
Epigenetics and vascular disease
MONDAY
Peter J. van den Elsen
Recent insights into the pathogenesis of atherosclerosis reveal the importance of chronic
inflammation in the initiation and progression of vascular remodelling. It is now widely
appreciated that in addition to macrophages also NK, NKT and T cells play a critical role in
disease pathogenesis. Notably the unusual expression of molecules that play an important
role in immune regulation found on vascular wall components is thought to contribute to the
ongoing inflammatory process. It has become increasingly clear over the past years that
epigenetic mechanisms play an essential and fundamental role in the transcriptional control
of genes. These epigenetic mechanisms act to change the accessibility of chromatin by
modification of DNA and by modifications of histones. Epigenetic regulators, such as those
involved in histone acetylation and methylation modifications, are increasingly being
recognized as direct or indirect components in the regulation of expression of vascular,
immune and other tissue-specific genes. Deviations from these tightly controlled epigenetic
mechanisms therefore have a direct impact on the cellular portrait of expressed genes and
contribute to disease pathogenesis. Moreover, epigenetic modifications are influenced by
environmental factors and accumulate in time. Epigenetic processes are reversible and
therefore provide excellent targets for therapy. A small number of drugs directed against
epigenetic processes are already FDA approved. The basis of epigenetic regulation and the
contribution thereof in inflammatory processes, which contribute to the initiation and
progression of vascular remodelling, will be discussed.
Epigenetics and Atherosclerosis
MONDAY
Rutger J. Wierda, I.M. Rietveld, P.H. Quax, H.C. de Boer, J.H. Lindeman and P.J. van den
Elsen
Over the past decades it has become increasingly clear that gene regulation by epigenetic
mechanisms – such as histone methylation, histone acetylation and DNA methylation – plays
an important role in complex diseases – such as atherosclerosis – and inflammation.
Epigenetic processes modulate gene expression without modifying the actual genetic code,
whilst it can be inherited over multiple cell divisions.
Epigenetics also provides an attractive explanation how diet, environment and lifestyle may
contribute to disease development. In principle, epigenetics explains how external factors can
impose aberrant gene expression patterns in an individual life time and even transgenerationally. Epigenetic processes are reversible by nature and can be modulated by small
molecule inhibitors which target the enzymatic activities critical for histone methylation,
histone acetylation and DNA methylation. Because multiple inhibitors are already FDA
approved for usage in cancer therapy, this provides an excellent opportunity for therapeutic
intervention in vascular diseases.Using immunohistochemical staining of atherosclerotic
plaques in various stages of disease development we hope to identify epigenetic factors
which are involved in disease pathogenesis. Subsequently, we will indentify the genes which
are under the control of the disease-associated epigenetic factors by chromatin
immunoprecipitation (ChIP) and massive parallel sequencing. Using cell cultures of Human
Umbilical Vein Endothelial Cells (HUVECs) and vascular Smooth Muscle Cells (vSMCs)
we will investigate the influence of small molecule inhibitors on the expression patterns of
the identified genes. Preliminary data using IHC-P show the existence of differential histone
methylation modifications revealing the dynamic nature of these epigenetic processes in the
vascular wall. Furthermore, by using bisulfite sequencing and ChIP it was revealed that
several chemokine receptor genes display differential DNA methylation and histone
methylation patterns between HUVECs and SMCs which explains the observed differential
expression patterns of these genes upon stimulation with various inflammatory mediators.
MONDAY
TLR4 is involved in vein graft remodelling and can serve as a local therapeutic target.
J.C Karper, M.R de Vries, P.H.A Quax
Despite that the survival of auto-transplanted veins for revascularization is limited by
remodelling processes such as neointimal formation and atherotrombotic evolution, veins are
still widely used for bypass surgery. This is due to their easier accessibility than arterial
conduits. Toll like receptors are located on most of the cells in the vessel wall and are
capable of recognizing exogenous as well as endogenous ligands that are considered to be
involved in vascular remodelling. We hypothesize that TLR4 is an important mediator in the
initiation and development of vein graft disease. To explore this potential role of TLR4, a
venous bypass procedure was performed in Balb/c mice and in C3H/Hej mice. The latter
lacks proper TLR4 signalling. After 28 days the vein graft was removed and analysed.
C3H/Hej mice had 48% less vessel wall thickening than the Balb/c controls. To increase
venous graft survival, we hypothesize to interfere in the local TLR4 expression of the vein
graft in ApoE3Leiden mice using gene silencing approaches. These mice, when fed a high
cholesterolemic diet, are well known to develop massive vein graft disease due to neointimal
formation and accelerated atherosclerosis. Out of 5 lentiviral based constructs, all containing
a different short hairpin RNA vector against TLR4 for silencing TLR4 expression in vivo the
most potent vector for TLR4 protein knockdown was selected using infection of CHO cells
expressing murine TLR4 and subsequent measurement of TLR4/MD2 complex protein
expression by FACS analysis. This vector was used to infect vein graft segments using a
perivascular delivery procedure. At sacrifice at 28 days after surgery, the combination of
local lenti-shTLR4 delivery and subsequent TLR4 knockdown led to a 38% reduction of
vessel wall thickening in the vein graft segment and thus a superior graft patency. This
reduction of hyperplasia in vein graft remodelling after local interfering in the TLR4 pathway
indicates the potential of TLR4 as a local therapeutic target.
MSCs for vascular repair
MONDAY
Helene Roelofs
Multipotent stromal cells (MSC) are characterized by their capacity to differentiate into bone,
fat and cartilage and they are able to moderate immune responses and to enhance
engraftment. These characteristics have rendered them interesting candidates for various new
cell therapeutic approaches. We have already obtained good results with MSCs derived bone
marrow (BM-MSC) for treatment of graft versus host disease and engraftment enhancement
in clinical transplantation settings. MSCs have been found in a variety of tissues and can also
be isolated from more easily accessible adipose tissue (AT-MSC). Upon characterization of
MSCs from this interesting alternative source, we detected a CD34+ cell subset, that was not
present in BM-MSC populations. Recently, it was reported that pericytes (CD34+/CD31/CD144-) from the adipose stromal-vascular fraction coexpress mesenchymal markers
(CD10, CD13, and CD90) and are able to enhance the stability of endothelial networks and
produce angiogenic factors (VEGF, HGF, bFGF) and inflammatory factors (IL-6 and -8 and
MCP-1 and -2). We are currently investigating how the AT-MSC-specific CD34+
subpopulation is related to these pericytes and whether this subpopulation can be developed
into a cell therapeutic product with improved properties for vascular repair.
MONDAY
Therapeutic Effects of Annexin A5 on Accelerated Atherosclerosis in Vein Grafts
Mark M Ewing, M.R. de Vries, M. Nordzell, K. Pettersson, J.W. Jukema, P.H.A. Quax
Rationale
Venous bypass grafts can fail due to the occurrence of thrombosis, vein graft thickening and
accelerated atherosclerosis. Annexin A5 has antithrombotic, anti-inflammatory and antiapoptotic properties, making annexin A5 a promising therapeutic agent against accelerated
atherosclerosis. The aim of this study is to assess the therapeutic effects of annexin A5 on
post-interventional vascular remodeling, including inflammation and accelerated
atherosclerosis, in murine vein grafts.
Methods & Results
Post-interventional vascular remodeling was induced by either femoral artery cuff placement
or by grafting of a venous segment in the common carotid artery of APOE*3-Leiden
transgenic mice, fed a cholesterol-rich diet. Mice were treated daily by intraperitoneal
injections with (1 mg/kg/day) recombinant human annexin A5 or vehicle only. After three
days, the presence of inflammatory cells within the lesions of the femoral arteries was
evaluated. Vein graft thickening and cellular composition of the vascular wall were analyzed
after 28 days.
Three days after femoral cuff placement adhesion and infiltration of leukocytes and
macrophages was retarded in recombinant human annexin A5 treated mice by 51 and 71%,
also the expression of TNF α and MCP-1in the arterial wall was significantly reduced by
respectively 31% and 43%. Treatment with annexin A5 led to a significant reduction of vein
graft thickening of 48% and a reduced number of leukocytes in the vein graft wall of 46%.
Annexin A5 treatment resulted in stabilization of a rupture-prone phenotype of the lesions by
showing a trend to reduced macrophage content, an increase in smooth muscle cell number
and collagen deposition, a decrease in fibrin/fibrinogen deposition, and a decreased number
of apoptotic cells. Furthermore, plaque hemorrhage features such as leaky vessels and
dissections were also decreased in the annexin A5 treatment group.
Conclusion
Daily treatment with annexin A5 resulted in reduction of inflammation, thrombosis and
accelerated atherosclerosis in murine vein grafts, thus a more stabile plaque phenotype.
Clinical Relevance
Annexin A5 is currently already used safely in patients as a diagnostic tool to detect
atherosclerosis non-invasively and possible clinical applications include systemic
administration and local delivery through drug-eluting stents. This study showed pronounced
effects of annexin A5 in vivo on accelerated atherosclerosis and this provides a perspective
for further research into therapeutic application of annexin A5 in cardiovascular disease.
MONDAY
C57BL/6 NK cell gene complex crucially involved in general vascular remodelling
Leonard Seghers, V. van Weel, J. van Bergen, R. Toes, A. Hellingman, M. de Vries, V. van
Hinsbergh, P. Quax
Vascular remodelling is key in disease processes such as Peripheral Arterial Disease (PAD),
restenosis and vein graft disease. Over years, evidence is accumulating that the immune
system plays an important role in vascular remodelling. Inflammatory cells as monocytes,
CD4+ and CD8+ T cells were already proven to be modulators of vascular remodelling.
Recently, NK cells were indicated to contribute to vascular remodelling in processes of
revascularisation and atherosclerotic lesion development. Here we further wished to address
the role of NK cells in vascular remodelling in general. Interestingly, C57BL/6 and BALB/c
mice are different with respect to remodelling; C57BL/6 mice display profound remodelling,
whereas BALB/c mice display much less vascular remodelling. In addition, these two mouse
strains are different in their NK gene complex (NKC) alleles, which are encoding for
activating and inhibitory NK cell receptors. Subsequently, these two mouse strains and a
third congenic BALB/c mouse strain expressing the C57BL/6 NKC alleles, BALB.B6CMV1r (CMV1r), were used in two different animal models of vascular remodelling, the
mouse model for restenosis and the vein graft mouse model respectively. At first,
contribution of NK cells in neointimal thickening as a response to vascular injury upon nonconstrictive cuff placement around the femoral artery was assessed. To do so, C57BL/6 mice
were depleted for NK cells using a NK1.1 antibody, control mice received a matched isotype
control. Mice depleted for NK cells showed significantly less formation of neointima when
compared to control mice, indicating a contributory role for NK cells in the process of
neointima formation.
Given the differences in vascular remodelling between C57BL/6 and BALB/c mice and the
differences in NK cell receptor repertoire, C57BL/6, BALB/c mice and the congenic BALB/c
mouse strain CMV1r received a non-constrictive cuff around their femoral arteries.
Surprisingly, congenic CMV1r showed progressive formation of neointima, comparative to
C57BL/6 and significantly different from BALB/c mice that displayed very little formation
of neointima. To verify whether NK cell contribution could be applied to vascular
remodelling processes in general, the vein graft mouse model was performed in the aforementioned three mouse strains. In concert with our previous observations, CMV1r mice
responded similar to C57BL/6 mice by displaying intimal hyperplasia in the venous bypass
graft. BALB/c mice showed significantly less hyperplasia when compared to both CMV1r
and C57BL/6 mice. Importantly, CMV1r displayed inflammatory infiltrate similar to that of
the C57BL/6 mice, whereas BALB/c mice nearly show inflammatory cell influx into the
vessel wall. In summary, we showed that NK cells play an important role in the induction of
neointima formation. This was shown by inhibited formation of neointima when NK cells
were absent. Subsequently, it was demonstrated that the expression of the C57BL/6 NK cell
receptor repertoire leads to more profound vascular remodelling, i.e. formation of neointima
and vein graft thickening by intimal hyperplasia. Furthermore, the observed differences in
inflammatory infiltrate between the three mouse strains strongly suggest that the C57BL/6
NK receptor repertoire might be involved in the initiation of an immune response that is
associated with profound vascular remodelling. Taken together, these data provide the first
clue to involvement of C57BL/6 NK gene complex in vascular remodelling in general.
Maternal Transmission of Risk for Atherosclerosis
MONDAY
Marco C De Ruiter
In the last 20 years increasing amount of epidemiological and pathological evidence has
become available illustrating the relationship between an adverse in-utero environment and
increased risk of vascular disease in the offspring. The fetal origins hypothesis of Barker
states that adaptation to unfavorable aspects of the maternal environment is beneficial to the
developing embryo in utero. However, when the adult environment differs from the fetal
environment, these adaptations may lead to an increased risk for cardiovascular disease.
Question is how the adverse maternal environment can be translated into an increased atherosusceptibility of the offspring. We have developed an apolipoprotein E heterozygous
knockout mouse model showing that maternal hypercholesterolemia is associated with
endothelial damage in the fetal vasculature. After birth, neither difference in morphology, in
intima/media thickness nor in lipid profile could be demonstrated between the maternally
exposed and non-exposed groups. The maternally exposed group, however, develops
substantial intimal hyperplasia in response to reduced shear stress levels and/or an
atherogenic diet. This indicates that athero-susceptibility can be imprinted in fetuses with
arteries that do not as yet manifest atherosclerotic disease. Global gene expression analysis
using microarrays of adult non-compromised arteries demonstrates upregulation of genes
involved in immune pathways and fatty acid metabolism in the offspring from
hypercholesterolemic mothers. These changes coincide with specific local changes in histon
modifcations in the fetal endothelial and the smooth muscle cells. We conclude that maternal
apoE-deficiency affects chromatin modification marks in specific cells of the vasculature of
the offspring in a non-uniform manner and that these changes are associated with an
alteration in athero-susceptibility. Our observations also imply that epigenetic marks are
variable within specific cells of the same tissue and that global analyses of epigenetic marks
will thus underestimate the complexity of the process.
Mesenchymal Stem Cells as Source for Endothelial Progenitors
MONDAY
Karolina Janeczek, N. Groen, H. Fernandes, N. Rivron, J. Rouwkema, C. van Blitterswijjk
and J. de Boer
Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from the bone
marrow, expanded in the culture dish and differentiated into various cell types. Therefore
MSCs are increasingly used in regenerative medicine as a source of cells for restoring wornout or damaged tissues such as cartilage, cardiac muscle and bone. The issue that remains to
be improved in current implantation strategies is maintaining cell survival after implantation.
The key parameter in this problem is the supply of nutrients and oxygen with diffusion as
rate limiting factor. To maintain cell survival in big grafts, a vascular structure needs to be
introduced within the graft which can rapidly hook up to the host’s blood vessels upon
implantation. Such attempts were already successfully performed, however only with
clinically irrelevant endothelial cell lines. We investigated the potential application of MSCs
as a source for endothelial progenitors that can be used to cerate vascular network within
grafts. We cultured cells on surfaces covered with extracellular matrix proteins (Matrigel,
collagen, fibronectin) to differentiate MSCs into endothelial progenitors. We used both
primary and immortalized MSC clones and utilized human umbilical vein endothelial cells
(HUVEC) as positive control. We examined the optimal culture conditions to gain the
highest possible differentiation efficiency. We investigated three basic characteristics of the
examined cells: their ability to form capillary-like structures (CSLs), ability to take up
acetylated LDL and finally expression of markers typical for endothelial cells such as CD31.
We were able to obtain CSLs with similar efficiency in both cell lines when cells were
cultured on Matrigel. These structures were not observed when MSCs were grown on tissue
culture plastic, collagen or fibronectin. The fact that iMSCs reacted in a similar way as
hMSCs allowed us to use the former in further research without depending on fresh hMSCs
of different donors. The crucial parameters in culture condition were cell seeding density
(minimum 12000 cells per cm2) and medium components (MSCs created CSLs in both basic
and EGM-2 medium but expressed CD31 only when cultured in EGM-2 medium). Some of
the cells we obtained took up AcLDL. We are currently investigating which medium
component is crucial in such differentiation and what are the signaling pathways responsible
for it. To confirm our results we will use siRNA to interfere with selected signaling
pathways. We will select MSC-derived CD31+ cells by cell sorting and further examine their
nature by investigating the expression of other endothelial markers. We will also perform
several functional assays associated with endothelial cells in order to determine further
potential use of obtained cells. Our results confirmed the ability of MSCs to differentiate into
cells that can possibly be used to create a vessel network in grafts before implantation.
Furthermore, MSC-derived CD31+ cells can be tested in other clinical application such as
blood vessel tissue engineering or treatment of stent-inflicted vascular damage.
MONDAY
Specific extracellular matrix and serum components decrease monocyte adherence:
implications for monocyte plasticity and function in peripheral vascular disease
Diana Ploeger, M.J.A.van Luyn, M.C.Harmsen
Introduction: Peripheral vascular disease (PVD) is a condition in which the blood vessels, in
particular arteries, are damaged or dysfunctional, often as the result of atherosclerosis. PVD
associates with limited perfusion of the extremities and may severely handicap patients.
Beside stenting and bypass grafting, novel cell-based therapies employing so-called
endothelial progenitor cells (EPC) are promising in animal models for arteriogenesis.
Peripheral blood mononuclear cells contain different types of EPC of which the CD14positive monocytes are most abundant (~10%). In vitro and in vivo and under angiogenic
conditions these monocytes differentiate into EPC that secrete a host of pro-angiogenic
proteinaceous factors such as VEGF-A and FGF-2. Remarkably, the monocytes in PVD
patients do not warrant adequate vascular maintenance and repair because of the chronic
nature of this disease. In PVD, the endothelial monolayer is compromised, exposing the
extracellular matrix (ECM) to leukocytes. We hypothesized that part of the presumed EPC
dysfunction relates to a disturbed adherence of monocytes to the ECM and the subsequent
cell fate. We initiated our studies by investigating the in vitro interaction of the CD14+
monocytes of healthy donors to selected ECM components.
Materials and Methods:
CD14+ monocytes were isolated from 0.5L buffy coats using immunomagnetic bead
isolation. To determine the influence of the ECM on the plasticity and cell function of CD14+
monocytes, we optimized ECM coating procedures first. Thereafter, cell seeding densities
and the influence of culture medium compounds (e.g. FCS) on cell adhesion were determined
in time.
Results:
For all ECM components tested, a stable coating was present within one hour incubation.
Cross- linking of ECM components with glutaraldehyde resulted in a decreased coating
efficiency, compared to their non-cross linked controls. The coating density of ECM
components strongly influenced adherence of the CD14+ monocytes. CD14+ monocytes were
capable of efficient binding to low coating densities of Collagen I but lost their adhesive
capacity to high coating densities of Collagen I. Adherence of CD14+ monocytes to ECM is
influenced by culture medium compounds, such as FCS, which caused a 40-fold reduction in
adherence.
Conclusion & Perspective:
In conclusion, we show that adherence of CD14+ monocytes is influenced by different ECM
components and common culture medium compounds. Cross-linking of ECM coatings, as
preformed in most cell adhesion studies, interferes with cell adhesion and may thus influence
plasticity and subsequent function of cells. Here, we have devised an optimal protocol which
will be used to study the influence of ECM components on cell plasticity and function in an
in vitro model for arteriogenesis.
MONDAY
Overexpression of Timp-1 Modulates Plasma Cytokine Levels which Positively Affect
Vein Graft Thickening and Spontaneous Intra-plaque Hemorrhages in Vein grafts
Margreet R de Vries, J. Wouter Jukema, Paul H.A. Quax
Introduction
MMPs and their inhibitors Timps are known for their modulation of the extracellular matrix
and are thought to be key players in plaque stability. MMPs can also activate or degradate
cytokines, chemokines, interleukins and growth factors. Smooth muscle cell proliferation,
apoptosis and inflammation can be stimulated by Timp-1 in a MMP-dependent and MMPindependent manner.
Methods
One day prior to the vein graft procedure pCDNA3.1Timp-1 and Luciferase plasmids were
introduced via electroporation into the calf muscles of the mice. A venous segment (caval
vein) was placed as an interposition in the carotid artery in ApoE3Leiden mice fed a high
cholesterol diet for 3 weeks. 28 days after engraftment the grafts were harvested and
analysed for morphologic changes and intraplaque hemorrhage.. At day 7 and day 28 plasma
samples were collected for a 23-plex cytokine assay.
Results
In these vein graft lesions, accelerated atherosclerosis with accumulation of lipid loaded
foam cells was observed. This accelerated atherosclerosis progresses in time and results in a
significant increase in vein graft thickening with foam cell rich lesions, calcification and
necrosis within 28 days after surgery. More importantly, in these atherosclerotic lesions
features of intraplaque hemorrhage due to dissection, leaky vessels and erosion were
observed. In plasmasamples of the Timp-1 group (n=4) at 7 days, both pro-inflammatory and
anti-inflammatory cytokines as IL-1α, IL-1β, GM-CSF, KC, TNFα, Rantes, MIP1α, IFNγ,
IL2, IL3, IL-10 and IL-12 were significantly elevated compared to the Luciferase
plasmasamples. After 28 days only Rantes and Eotaxin were significantly higher in the
Timp-1 group. At this timepoint Timp-1 overexpression resulted in a 47 % reduction of
vessel wall thickening compared to the control group. More importantly, only one of the
Timp-1 treated animals (n=13) showed intraplaque hemorrhage, whereas 66% of the control
animals (10 out of 15) displayed features of intraplaque hemorrhage.
Conclusion
Overexpression of Timp-1 results in pronounced expression of pro- and anti-inflammatory
cytokines after 7 days which almost all declined to comparable levels as the Luciferase group
at day 28 except for Rantes. After 28 days reduced vessel wall thickening but more
importantly reduced intraplaque hemorrhage could be detected in the Timp-1 group.
Targeting the Microvasculature for Ischemic Tissue Repair
MONDAY
Douglas W. Losordo
As the population ages and the acute mortality from cardiovascular disease decreases, a large
population of patients is emerging who have symptomatic chronic ischemic vascular disease,
many of whom remain severely symptomatic despite exhausting conventional medical
therapy and mechanical revascularization. In addition, mounting evidence suggests that
microvascular insufficiency plays a significant role in the pathophysiology of ischemia. At
the present time, there are no therapies that directly address the needs of this patient
population. Pre-clinical and early clinical data indicate that a variety of growth factors and
stem/progenitor cells may be employed therapeutically for repair of ischemic tissue.
Preclinical studies documented the potential therapeutic potency of endothelial progenitor
cells, both as cultured and freshly isolated cells. Early phase clinical trials using a variety of
approaches have been completed providing data of feasibility, safety and bioactivity. Later
phase trials are under way. Accordingly, the goal of ischemic tissue repair appears feasible
and is being approached in human clinical trials. We performed a phase II randomized
controlled trial of autologous CD34 cell therapy in 167 patients with refractory, class 3 and 4
angina who were not candidates for revascularization. The results of this study will be
presented. The evolution of the strategy of ischemic tissue repair will require an ongoing
dialogue between clinicians, scientists, regulators and industry to take full advantage of
advances in our understanding of the biology of these processes and their appropriate
application to patients.
NK cells and their role in modulating arteriogenesis
TUESDAY
Leonard Seghers, Paul Quax
The involvement of inflammatory cells in the induction of neovascularisation and
arteriogenesis (collateral formation) is well established. It has been suggested for several
years that monocytes are involved in the regulation of arteriogenesis. Moreover, we and
others have demonstrated a key role for CD4+ T-cells and NK cells in arteriogenesis using
depletion studies and mice deficient in e.g. MHC class II, or NK cells. The exact mechanism
how these cells affect arteriogenesis is not completely understood. However, it is tempting to
suggest that the NK-cells might function as cytokine factories that upon activation start to
produce cytokines, chemokines and growth factors. Therefore we hypothesize that the NK
cell effector function in arteriogenesis relies on certain specific NK cell receptors.
The fact that NK cell receptors play a role in arteriogenesis is firstly confirmed by the
observation that mice lacking MHC class I molecule expression, necessary for NK cell
interactions with the host, show impaired arteriogenesis.
To map out the possible candidate receptors, we made use of the difference in NK cell
receptor repertoire between Balb/C and C57B6, which are also known to be two strains of
mice that are their opposites in their arteriogenic response. A congenic Balb/C mouse strain
which is carrying the C57B6 NK cell receptor repertoire shows a rescue of the poor
responding Balb/C (max. 50% flow recovery after 28 days) phenotype towards the rapid
arteriogenic response seen in C57B6 mice (100% flow recovery in 14 days).
This difference might be caused by the presence of the activating B6 NK cell receptor Ly49H, which is normally absent in Balb/C mice.
To verify the involvement of this candidate receptor in arteriogenesis, its natural ligand the
CMV protein m157 was overexpressed by non-viral electroporation mediated intramuscular
gene transfer one day prior to femoral artery ligation. Blood flow recovery was monitored up
to 28 days showing a significantly impaired arteriogenic response in mice overexpressing
m157 as compared to control mice which were overexpressing Luciferase (p<0.01).
These results show that, induction of hyposensitivity of NK cells via in vivo systemic
exposure of the Ly49H NK receptor to its specific ligand m157, leads to impaired
arteriogenesis. This indicates that the Ly49H receptor most likely plays a key role in
modulation of arteriogenesis. This might provide an interesting pathway for modulating the
arteriogenic response by modulation of the immune response, and therefore has impact for
cell therapy approaches to induce revascularization.
EPC plasticity and function in angiogenesis and inflammation
TUESDAY
Martin C. Harmsen
Endothelial progenitor cells (EPC) from bone marrow or peripheral blood have been celebrated
for their capacity to augment therapeutic neovascularisation over the past decade. The
phenotype of cultured EPC is dictated by culture time, growth factors and extracellular matrix
substratum. The appropriate choice of culture conditions may present a relatively easy
accessible model for the in vivo behavior of circulating EPC. In general, monocytes (CD14+
fraction of MNC) emerge as rapidly proliferating colonies of spindle-shaped cell in MNC
cultures, hence their name: early EPC (eEPC). These eEPC are shortlived, and harbor a marker
profile that would qualify these cells as endothelial-like monocytes. More importantly, eEPC
can transiently engraft vascular lesions and secrete a plethora of pro-angiogenic growth factors,
which renders eECP promising candidates for therapeutic angiogenesis. On the other hand,
endothelial colony forming cells (ECFC) emerge from CD34+ MNC after prolonged culture as
genuine stem cells with a typical endothelial phenotype and limited secretome. In vivo ECFC
contribute to neovascularisation. Unfortunately, ECFC appear refractory to culturing from
adult blood.
The concerted action of eEPC and ECFC warrants (pre)clinical studies to assess future cellbased therapy to improve perfusion in patients with PVD or CVD. We set out to dissect the
role of circulating EPC, i.e. CD34+ and CD14+ MNC, in ischemic tissue repair. The
subcutaneous in vivo administration of human CD34+ EPC embedded in Matrigel plugs,
showed these hCD34+ EPC to augment influx of host (murine) inflammatory monocytic cells.
Simultaneously, the presence of hCD34+ EPC enhanced host angiogenesis. The hCD34+
appeared to generate a pro-angiogenic niche through secretion of related growth factors
including IL-8, MCP-1 (monocyte attraction) and VEGF, FGF-2, and HGF (angiogenesis).
Remarkably, in vitro the presence of CD34+ EPC upregulated the proliferation of CD14+ EPC
in a paracrine fashion through secretion of HGF. Moreover, CD34+ and CD14+ mutually
interacted through secretion of HGF, VEGF, FGF-1, IL-8 and MCP-1 among others. In the
rodent subcutaneous Matrigelplug model, the inclusion of these five paracrine factors effected
influx of inflammatory cells and vascularisation as efficient as the combination of
CD34+/CD14+ cells.
Thus, the local microenvironment in vascular lesions or tissue lesions can be adjusted to a
regenerative state by administration of therapeutic cells or their paracrine mediators. However,
the ‘adjustable’ microenvironment by itself will also affect administered cells. Transforming
growth factor-beta is one the prominent mediators that is present in vascular pathologies. In
atherosclerosis, TGF can induce adverse dedifferentiation of vascular endothelial cells,
known as EnMT or endothelial to mesenchymal transdifferentiation. We have shown that
ECFC are also prone to TGF-driven EnMT. In vitro, after EnMT ECFC feature typical
smooth muscle cells characteristics such as contractibility, secretion of ECM components and
increase of thrombogenicity. Thus, in vivo administered ECFC may adopt a phenotype after
EnMT that augments vascular remodeling during revascularization therapy.
Modulation of the arteriogenic response by monocytes
TUESDAY
Imo Hoefer
The growth of pre-existent arteriolar connections into functional collateral arteries is a multistep process involving mechanical, cellular and chemical factors. Nowadays, the pivotal role
of changes in shear stress within the collateral vessels in the initiation of arteriogenesis is
widely accepted. This primary event is followed by the adhesion and migration of
inflammatory cells into the collateral vessel wall and the perivascular space. Within this
space, these cells facilitate the remodeling and growth of the vasculature by providing growth
factors and proteases. Monocytes are known to be actively involved in vascular growth since
the late 1960s, but it took several decades until arteriogenesis was sought to be stimulated by
attracting circulating monocytes to the growing collateral network. This can be achieved by a
variety of cytokines, MCP-1 being the strongest single arteriogenic factor. However, due to
the mechanistic similarity between arteriogenesis and atherogenesis, a non-selective
attraction of inflammatory cells poses the risk of worsening the underlying atherosclerotic
disease. Recent evidence suggests differential effects of monocyte/ macrophage subsets on
vessel growth and tissue preservation. These insights and the ability to specifically target
these subsets might provide new approaches to positively modulate the arteriogenic response
by monocytes combined with neutral or even beneficial effects on atherosclerotic plaques
TUESDAY
Implantation of Adipose-Derived Regenerative Cells Enhances Ischemia-induced
Angiogenesis
Toyoaki Murohara
Therapeutic angiogenesis using autologous stem/progenitor cells represents a novel strategy
for severe ischemic diseases. Recent reports indicated that adipose tissues could supply
adipose-derived regenerative cells (ADRCs) or adipose-derived stem cells (ADSCs).
Accordingly, we examined whether implantation of ADRCs would augment ischemiainduced angiogenesis using a mouse model. Subcutaneous adipose tissue was obtained from
the inguinal fat pad of C57BL/6J mice, and ADRCs were isolated using standard methods
including collagenase digestion followed by centrifuge. Flow cytometry revealed that
ADRCs expressed Sca-1 but not other lineage markers or endothelial markers. ADRCs
expressed stromal cell-derived factor 1 (SDF-1) proteins. Hind limb ischemia was induced
and culture-expanded ADRCs, PBS or mature adipocytes (MAs) as control cells were
injected into the ischemic muscles. At 3 wks, the ADRC group had a greater laser Doppler
blood perfusion index and a higher capillary density compared to the controls. Interestingly
the MA group showed suppressed blood flow recovery and capillary density compared to the
control group. Implantation of ADRCs increased circulating endothelial progenitor cells
(EPCs) as judged by flow cytometry and culture assay of early outgrowth EPCs. SDF-1
mRNA abundance at ischemic tissues and serum SDF-1 protein levels were greater in the
ADRC group than in the control group. Finally, intraperitoneal injection of an anti-SDF-1
neutralizing monoclonal antibody reduced the number of circulating EPCs and overall
therapeutic efficacies of ADRCs. In conclusion, adipose tissue would be a valuable source
for cell-based therapeutic angiogenesis. Moreover, chemokine SDF-1 may play a pivotal role
in the ADRCs-mediated angiogenesis at least in part by facilitating mobilization of EPCs.
Mechanisms in endothelial maintenance and repair
WEDNESDAY
Anton Jan van Zonneveld.
Disruption of the integrity of the endothelial monolayer will lead to the immediate
establishment of an injury-specific micro-environment, that includes aggregated platelets and
platelet secretion products, deposited fibrin and other injury-associated components, such as
growth factors and chemokines. Circulating stem- and progenitor cells can contribute to
vascular regeneration after an injury. The contribution of these stem cells to vascular
regeneration depends on the actual homing to the injury and the differentiation towards a
more endothelial cell phenotype.
The factors that lead to homing of these cells and subsequent differentiation, are not known.
We developed models to characterize the injury-associated factors in stem cell-mediated
vascular repair under ex vivo conditions, such as flow and the presence of an injury-specific
micro-environment. This allows the detailed assessment of the initial steps that take place
when vascular progenitor cells (CD34+ cells purified from umbilical cord blood) home to
sites of vascular injury.Following homing, the injury-associated micro-environment mediates
differentiation of CD34+ progenitors toward a more endothelial cell phenotype. As it has
become apparent that microRNAs play a key role in cellular differentiation and vascular
development, we study the role of endothelial microRNAs in endothelial differentiation and
dedifferentiation. To that end, we perform microRNA profiling and set up the technology to
assess the function of microRNAs in vitro and in vivo.
WEDNESDAY
Application of Poly-Chromatic Flow Cytometry Analysis and Cell Sorting Techniques
to Identify Novel Subsets of Rare Circulating Cells with Angiogenic Potential
Jamie Case
Assay for circulating blood cell subsets that comprise the endothelial progenitor cell (EPC)
pool in human peripheral blood by flow cytometry is increasingly used as a biomarker to
determine cardiovascular disease risk and tumor angiogenesis since EPCs function in
vasculogenesis and angiogenesis. Despite analytical advances in poly-chromatic flow
cytometry (PFC), conventional flow cytometric approaches are exclusively utilized to
enumerate and isolate EPCs, which has led to varied results as a biomarker in clinical studies,
potential cellular misidentification, and thus a lack of a plausible biological explanation for
how purported EPCs function in vascular repair. Using a novel protocol for PFC data
acquisition and analysis, we discovered that purported circulating endothelial cells (CECs)
and progenitors previously identified by a conventional flow cytometry approach, are not
endothelial cells, but largely microvesicles and in vivo engrafting hematopoietic stem cells
and myeloblasts, respectively. Further, accurate application of PFC identifies a rare
circulating endothelial colony forming cell with proliferative potential. Thus, application of
optimal PFC technology challenges existing paradigms of previously reported CECs and
EPCs and provides a robust methodological platform for future definitive studies of these
cells in human clinical studies of vascular disease.
Immune-regulatory Pathways in Atherosclerosis
WEDNESDAY
Alain Tedgui
Atherosclerosis is a chronic disease of the arterial wall where both innate and adaptive
immuno-inflammatory mechanisms are involved. Inflammation is central at all stages of
atherosclerosis. It is implicated in the formation of early fatty streaks, when the endothelium
is activated and expresses chemokines and adhesion molecules leading to
monocyte/lymphocyte recruitment and infiltration into the subendothelium. It also acts at the
onset of adverse clinical vascular events, when activated cells within the plaque secrete
matrix proteases that degrade extracellular matrix proteins and weaken the fibrous cap,
leading to rupture and thrombus formation. Cells involved in the atherosclerotic process
secrete and are activated by pro-inflammatory cytokines. Recent advances in our
understanding of the mechanisms of atherosclerosis provided evidence that the immunoinflammatory response in atherosclerosis is modulated by regulatory pathways involving the
two anti-inflammatory cytokines IL-10 and TGF, which play a critical role in counterbalancing the effects of pro-inflammatory cytokines. Interestingly, IL-10 and TGF- are also
the two cytokines that mediate the immune regulatory functions of a sub-population of T
cells, named regulatory T (Treg) cells. We recently demonstrated that natural CD4+CD25+
Treg cells play an important role in the control of atherosclerosis in apoE-/- mice. In addition,
administration of ovalbumin-specific Tr1 cells, with its cognate antigen, to apoE-/- mice, can
induce a marked suppression of Th1 (and Th2)-mediated responses with increased IL-10
production by stimulated peripheral T cells. Tr1 responses are associated with reduction in
the accumulation of inflammatory macrophages and T lymphocytes in lesions, as well as
partial inhibition of plaque development. Modulation of the peripheral immune response is
achievable by transfer of regulatory T cells. It is therefore believed that atherosclerosis
results from an imbalance between pathogenic T cells, either Th1 or Th2, producing proatherogenic mediators, and Treg cells with immunosuppressive properties, and that
promotion, expansion or exogenous administration of Treg cells, ideally specific of plaquederived antigens, might limit disease development and progression.
Monocytes as cellular therapy for vascular regeneration
WEDNESDAY
Sacha B. Geutskens, A.W. Hellingman, R.T. van Beem, L. Seghers, M.R. de Vries, AJ van
Zonneveld, P.J. van den Elsen, C.E. van der Schoot, P.H.A. Quax and JJ Zwaginga
Monocytes are multipotent hematopoietic progenitors that develop into immune cells, such as
macrophages and myeloid dendritic cells, when stimulated accordingly. We recently found
that CD14+ monocytes show a pro-angiogenic phenotype upon culture with CD4+ Tlymphocytes. Pro-angiogenic monocyte differentiation was characterized by the formation of
CFU-Hill colonies and the increased production of pro-angiogenic factors such as VEGF,
PDGF, TNF, TGF- and MCP-1. Initial monocyte-T-cell interactions were TCR-MHC
class II-mediated followed by the induction of pro-angiogenic monocyte differentiation via
medium-soluble factors. We now observe that transfusion of human monocytes that were
stimulated with activated CD4+ T-cell-conditioned medium significantly improve bloodflow
recovery after induction of ischemia in the hind limb of nude mice, as compared to
transfusion with unstimulated monocytes or PBS. Using -actin labelling we found that more
collaterals formed in the adductor muscles of mice transplanted with stimulated monocytes,
as well as in mice transplanted with unstimulated monocytes as compared to PBS-treated
mice. However, a significantly increased collateral size was only observed in mice
transplanted with stimulated monocytes, which is in agreement with the improved hind limb
perfusion observed. Thus, in addition to classic immune cell differentiation monocytes
develop into cells that efficiently promote collateral artery formation (arteriogenesis) upon
receiving the appropriate stimulation. These results support a role for alternative T-cellmonocyte mediated responses in vascular repair and render the pro-angiogenic monocyte an
interesting candidate for the development of a regenerative cellular therapy that promotes
adult re-vascularization in patients with arterial obstructive diseases.
WEDNESDAY
Impaired recruitment of HHT-1 mononuclear cells to the ischemic heart is due to an
altered CXCR4/CD26 balance
S Post, A Smits, A vdn Broek, J Sluijter, I Hoefer, B Janssen, R Snijder, J Mager, G
Pasterkamp, C Mummery, P Doevendans, Marie-José Goumans
Aim: Mononuclear cells (MNCs) from patients with hereditary hemorrhagic telangiectasia
type 1 (HHT1), a genetic disorder caused by mutations in endoglin, show a reduced ability to
home to infarcted mouse myocardium. Stromal-cell derived factor-1α (SDF-1α) and its
receptor CXCR4 are crucial for homing and negatively influenced by CD26. The aim of this
study was to gain insight into the impaired homing of HHT1-MNCs.
Methods: CXCR4 and CD26 expression on MNCs was determined by flow cytometry.
Transwell migration to SDF-1α was used to analyze in vitro migration. Experimentally
induced myocardial infarction in mice, followed by tail vein injection of MNCs, was used to
study homing in vivo.
Results: Although HHT1-MNCs express elevated levels of CXCR4, this was
counterbalanced by high levels of CD26, resulting in decreased migration towards a SDF-1α
gradient in vitro. Their migration was enhanced by inhibiting CD26 with Diprotin A.
Furthermore, while MNCs from healthy controls responded to TGFβ stimulation by
increasing CXCR4 and lowering CD26 expression levels, HHT1-MNCs did not react as
efficiently. In particular, CD26 expression remained high. Interestingly, homing of HHT1MNCs to the infarcted region of the murine heart was restored by pre-incubating the HHT1MNCs with Diprotin A before injection into the tail vein.
Conclusions: We show that impaired homing of HHT1-MNCs is caused by an impaired
ability of the cells to respond to SDF-1α. Our results suggest that modulating CD26 levels
using inhibitors like Diprotin A can restore homing in cases where increased CD26
contributes to the underlying pathological mechanism.
WEDNESDAY
The mRNA-binding protein Quaking is essential for proper vascular smooth muscle cell
function
Eric van der Veer, A.O. Kraaijeveld, M.R. de Vries, F. Segers, D. Pons, M. Doop,
S. Richard, P. Quax, AJ van Zonneveld, J.W. Jukema and E.A.L. Biessen
The mRNA-binding protein Quaking (QKI) has a well established role in the nervous system
during myelination. Recently, Quaking has also been implicated in embryonic vascular
development, where it plays a vital role in regulating vascular cell differentiation, such as
pericytes and vascular smooth muscle cells (VSMC). However, a role in adulthood remains
elusive. We hypothesized that decreased QKI expression could trigger aberrant VSMC
migration and proliferation, a frequent complication of percutaneous coronary intervention
(PCI) that increases restenotic risk. To establish a causal involvement of QKI in vascular
remodelling we focused on the consequences of decreased QKI expression on VSMC
phenotype and functionality. For this, we utilized primary VSMCs derived from aortas of Qk
viable mutant (Qkv) mice versus wild type (WT) littermates. VSMCs derived from Qkv mice
were characterized by a flattened, enlarged morphology with prominent stress-fibers as
opposed to WT cells which displayed a healthy phenotype. In keeping with this finding, Qk v
VSMC displayed a decreased capacity to deal with oxidative and serum withdrawal-induced
stress. Moreover, Qkv VSMCs displayed significantly decreased transwell cellular migration
(WT 116 vs. Qkv 40 migrated cells; N=5; P<0.001), cellular proliferation (WT 2,149 vs. Qkv
607 DPI; N=25; P<0.001), and extracellular matrix production (WT 36 vs. Qkv 18 µm/ml;
N=6; P<0.05). To gain insight into whether the levels of quaking expression could impact
restenotic risk, we screened the qki gene for single nucleotide polymorphisms (SNPs) that
could influence proper VSMC functioning. For this, we assessed the GENetic DEterminants
of Restenosis (GENDER) project, a multicenter, prospective study design that enrolled 3,104
consecutive patients after successful PCI. We set out to characterize the role of QKI in instent restenosis by analysis for association of 12 SNPs throughout the qki gene and the risk of
restenosis. As many as seven SNPs were found to significantly associate with the risk for
target vessel revascularization (TVR). The strongest associations were found close to the
transcription start site (2786 T/C: HR: 2.3, 95%CI: 1.3-3.9, P=0.002) and in intron 3 (57896
A/G: HR: 0.7, 95%CI: 0.6-0.9, P=0.005 and 65752 A/G: HR: 1.5, 95%CI: 1.1-1.9, P=0.006).
Ten percent of patients carried the 65752G risk allele but lacked the protective 57896G
allele. Importantly, these patients were at high risk to develop restenosis (HR: 1.8, 95%CI:
1.3-2.5, p<0.001). In conclusion, we have identified QKI as a central regulator of VMSC
function, with decreased expression of QKI leading to VSMC dysfunction. Furthermore,
these studies reveal a strong link between QKI and the risk for in-stent restenosis after PCI,
suggesting that Qk may serve as a marker for TVR risk prediction and a therapeutic target.
The natural history of aortic atherosclerosis: a systematic histopathological evaluation
R.A.van Dijk, R. Virmani, J.H. von der Thüsen, A.F. Schaapherder, J.H.N. Lindeman
Risk factor profiles for the different vascular beds (i.e. coronary, carotid, peripheral and
aortic) are remarkably different, suggesting that atherosclerosis is a heterogeneous disorder.
Little is known about the morphologic progression of atherosclerosis in the aorta, one of the
primary predilection sites of atherosclerosis.
Methods: A systematic analysis was performed in 260 consecutive peri-renal aortic patches
(stained with Movat Pentachrome and H&E) collected during organ transplantation (mean
donor age 46.5 (range 5-76) years; 54% ♂; mean BMI 24.9; 40% smokers; 20%
hypertensive). Plaque morphology was classified according to the modified AHA
classification scheme proposed by Virmani et al in 2000. Immunostaining against CD68 was
used to identify the distribution of intimal macrophages and monocytes in several predefined
locations
among
various
plaque
types
and
fibrous
cap
thickness.
Results: There was significant intimal thickening (p<0.002) and medial thinning (p<0.001)
with advancing age. The incidence of atherosclerotic plaques in the abdominal aorta
correlated with age (r=0.640, p=0.01). During the first three decades of life adaptive intimal
thickening and intimal xanthomas were the predominant lesions. In contrast, the fourth, fifth
and sixth decades hallmarked more complicated plaques of pathological intimal thickening,
early and late fibroatheromas (EFAs and LFAs), thin-cap FAs (TCFAs; cap thickness <155
µm), ruptured plaques (PRs), healed rupture and fibrotic calcified plaques. The mean
percentage of lesional macrophages increased significantly from LFAs to TCFAs (5% to 17
%; p<0,007). Macrophage infiltration of the fibrous cap was negatively correlated with
fibrous cap thickness (p<0.006); TCFAs and PRs (caps <100μm) contained significantly
more macrophages (19%) compared with caps 101-300μm (6%) and >300μm (2%).
Macrophages in shoulder regions were highest in early and late FAs (~45%) followed by
TCFAs (27%) and PR (20%). Further, intimal vasa vasorum were mostly seen adjacent to the
necrotic core of advanced atherosclerotic plaques and remained confined to the intimomedial border despite marked thickening of the intima.
Conclusion: This study shows that aortic atherosclerosis starts early in life. Gross plaque
morphologies of the peri-renal abdominal aorta are similar to coronary atherosclerosis yet
indications were found for site specific differences in macrophage distribution and
neovascularization.
PARTICIPANTS
Dr. Douwe E. Atsma
Leiden University Medical Center
Department of Cardiology
Albinusdreef 2, building 1, C5-P
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Prof. Erik A.L. Biessen
University of Maastricht
Department of Pathology
Cardiovasculair Research Instituut Maastricht
P. Debeyelaan 25
6229 HX Maastricht
[email protected]
Jamie Case, Ph.D.
Indiana University School of Medicine
Assistant Research Professor of Pediatrics
1044 West Walnut Street, R4-440
Indianapolis, IN, 46202-5525,
United States
[email protected]
Prof. Mat J.A.P. Daemen
University of Maastricht
Department of Medicine
Section Pathology, room 5.76
PO Box 616
6200 MD Maastricht
[email protected]
Prof. Peter J. van den Elsen
Leiden University Medical Center
Department of Immunohematology and
Blood Transfusion
Albinusdreef 2, building 1, E3-Q
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Drs. Mark Ewing
Leiden University Medical Center
Department of Cardiology
Albinusdreef 2, building 1, C5-P
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Prof. Wim E. Fibbe
Leiden University Medical Center
Department of Immunohematology and
Blood Transfusion
Albinusdreef 2, building 1, E3-Q
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Dr. Sacha Geutskens
Leiden University Medical Center
Department of Immunohematology and
Blood Transfusion
Albinusdreef 2, building 1, E3-Q
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Dr. Marie-José Goumans
Leiden University Medical Center
Department of Molecular Cell Biology
Albinusdreef 2, building 1
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Dr. Marco C. Harmsen
University Medical Centre Groningen
Department of Pathology and Medical Biology
Hanzeplein 1
9713 GZ Groningen
The Netherlands
[email protected]
Prof. Victor W.M. van Hinsbergh
VU University Medical Center
Laboratory for Physiology
Institute for Cardiovascular Research
Van der Boechorststraat 7
1081 BT Amsterdam
The Netherlands
[email protected]
Drs. Karolina Janeczek
University of Twente
Department of Tissue Regeneration
P.O. Box 271
7500 AE Enschede
The Netherlands
[email protected]
Dr. Imo E. Hoefer
University Medical Center Utrecht
Experimental Cardiology, G02.523
Heidelberglaan 100
3584 CX Utrecht
The Netherlands
[email protected]
Drs. Jacco Karper
Leiden University Medical Center
Department of Vascular Surgery, C11-15
Albinusdreef 2, building 1
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Dr. Robert Kleemann
Dr. Jan Lindeman
Leiden University Medical Center
Department of Surgery
PO Box 9600, K6-R
2300 RC Leiden
The Netherlands
[email protected]
TNO-Pharma, Gaubius-Laboratory
Department of Vascular and Metabolic
Diseases
PO Box 2215
2301 CE Leiden
The Netherlands
robert.kleemann@tno
Dr. Douglas W. Losordo
Feinberg Cardiovascular Research
Institute
Department of Medicine
303 E Chicago Avenue, Tarry 14-725
Chicago, IL 60611
United States
[email protected]
Prof. Toyoaki Murohara
University Graduate School of Medicine
Department of Cardiology,
Nagoya, 65 Tsurumai, Showa-ku,
Nagoya 466-8550
Japan
[email protected]
Ing. Diana Ploeger
University Medical Center Groningen
Department of Pathology & Medical Biology
Stem Cell and Tissue Engineering Research
Group
Hanzeplein 1 (EA11)
9713 GZ Groningen
The Netherlands
[email protected]
Prof. Paul H.A. Quax
Leiden University Medical Center
Department of Surgery
PO Box 9600, K6-R
2300 RC Leiden
The Netherlands
[email protected]
Prof. Ton J. Rabelink
Leiden University Medical Center
Department of Nephrology
Albinusdreef 2, building 1, C3-P
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Dr. Marco de Ruiter
Leiden University Medical Center
Department of Anatomy and Embryology
Albinusdreef 2, building 1
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Dr. Helene Roelofs
Leiden University Medical Center
Department of Immunohematology and
Blood Transfusion
Albinusdreef 2, building 1, E3-Q
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Drs. Leonard Seghers
Leiden University Medical Center
Department of Surgery, K-6
Albinusdreef 2, building 1
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Prof. Alain Tedgui
PARCC-INSERM
56 rue Leblanc
75010 Paris
France
[email protected]
Ir. Rutger Wierda
Leiden University Medical Center
Department of Immunohematology and
Blood Transfusion
Albinusdreef 2, building 1, E3-Q
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Mervin C. Yoder M.D.
IUSOM
1044 West Walnut, R4-402E
Indianapolis, IN 46202
United States
[email protected]
Ing. Margreet de Vries
Leiden University Medical Center
Department of Surgery
Albinusdreef 2, building 1
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Prof. D.C. (Christian) Weber
Institute for Molecular Cardiovascular Research
IMCAR
Pauwelsstraße 30
D-52074 Aachen
Germany
[email protected]
Prof. Anton Jan van Zonneveld
Leiden University Medical Center
Department of Nephrology
Albinusdreef 2, building 1, E3-Q
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]
Dr. Jaap Jan Zwaginga
Leiden University Medical Center
Department of IHB
Albinusdreef 2, building 1, E3-Q
P.O. Box 9600
2300 RC Leiden
The Netherlands
[email protected]