The Nuclear Factor-kappa B p50 subunit is involved in flow-induced outward arterial remodeling.

Abstract

Outward arterial remodeling is a structural enlargement of the artery that is associated with unstable inflammatory atherosclerotic lesions. Toll-like receptor (Tlr) activation is known as a key pathway in outward arterial remodeling. Tlr activation results in nuclear translocation of the transcription factor Nuclear Factor-kappa B (NF-kappaB) that controls the transcription of many inflammatory genes. The NF-kappaB subunit p50 is generally considered to be an inhibitory subunit of the NF-kappaB complex. We therefore hypothesize that NF-kappaB p50 inhibits outward arterial remodeling.
Carotid artery ligation in mice, induced outward remodeling in contralateral arteries of NF-kappaB p50(-/-) (p50(-/-)) and wild type (WT) arteries. p50(-/-) arteries showed more outward arterial remodeling than WT arteries (19894.0+/-3136.7 microm(2) vs. 6120.7+/-2741.2 microm(2), respectively, P=0.006). In vitro, lipopolysaccharide induced higher cytokine expression levels in p50(-/-) cells compared to WT cells. In vivo, more outward remodeling in p50(-/-) arteries was associated with a decrease in collagen density and an increased influx of macrophages.
The NF-kappaB p50 subunit is involved in outward arterial remodeling. This is probably due to modulation of macrophage influx and adventitial collagen, leading to enhanced flow-induced outward arterial remodeling after targeted deletion of NF-kappaB subunit p50.

Full text

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Toll-like receptor 4 signaling in atherosclerotic disease
atherosclerosis, arterial remodeling, cross-talk
Karlijn van Keulen

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Toll-like receptor 4 signaling in atherosclerotic disease
atherosclerosis, arterial remodeling, cross-talk
van Keulen, Judith Karlijn
Utrecht, Universiteit Utrecht, Faculteit Geneeskunde
Proefschrift Universiteit Utrecht, met een samenvatting in het Nederlands
© JK van Keulen 2008
ISBN:
Cover-design: Hester Haars
Lay-out:
Printed by:
978-90-393-4985-4
Wendy Schoneveld
Gildeprint, Enschede

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Toll-like receptor 4 signaling in atherosclerotic disease
atherosclerosis, arterial remodeling, cross-talk
Het Toll-like receptor 4 signaal in atherosclerose
atherosclerose, arteriële remodellering, wisselwerking
(met een samenvatting in het Nederlands)
Proefschrift
ter verkrijging van de graad van doctor aan de Universiteit Utrecht
op gezag van de rector magnificus, prof.dr. J.C. Stoof,
ingevolge het besluit van het college voor promoties
in het openbaar te verdedigen op
dinsdag 27 januari 2009 des ochtends te 10.30 uur
door
Judith Karlijn van Keulen
geboren op 13 februari 1981 te Dordrecht

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Promotor
Prof.dr. G. Pasterkamp
Co-promotor
Dr. D.P.V. de Kleijn
The research described in this thesis was supported by a grant of the Netherlands Heart
Foundation (NHF-2001B162).
Publication of this thesis is financially supported by the Dutch Atherosclerosis Society
and the University Medical Center Utrecht.
Financial support by the Prof. R.L.J. van Ruyvenstichting and the Interuniversity
Cardiology Institute of the Netherlands for the publication of this thesis is gratefully
acknowledged.
Financial support by the Netherlands Heart Foundation for the publication of this thesis
is gratefully acknowledged.

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Contents
chapter 1
Introduction
7
chapter 2
Toll-like receptor 4 is involved in outward arterial remodeling
25
chapter 3
The nuclear factor-kappa B p50 subunit is involved in
flow-induced outward arterial remodeling
41
chapter 4
Extra domain A of fibronectin is involved in
outward arterial remodeling
57
chapter 5
Levels of extra domain A containing fibronectin in
human atherosclerotic plaques are associated
with a stable plaque phenotype
71
chapter 6
Human atherosclerotic plaque levels of extra domain A
of fibronectin are associated with TGF-beta signaling
molecules including Endoglin
91
chapter 7
Identification of gene expression regulated by
both Toll-like receptor and transforming growth factor
pathways in monocytes
105
chapter 8
Summary and discussion
123
Nederlandse samenvatting
137
Color supplement
147
Dankwoord
153
List of publications
158
Curriculum Vitae
159

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Introduction
Partly published in
Gerard Pasterkamp, J Karlijn van Keulen,
Dominique P de Kleijn
Role of Toll-like receptor 4 in the initiation and
progression of atherosclerotic disease
Eur J Clin Invest. 2004 May;34(5):328-34. Review.
and
Manon M Oude Nijhuis, J Karlijn van Keulen,
Gerard Pasterkamp, Paul H Qaux, Dominique P de Kleijn
Activation of the innate immune system in
atherosclerotic disease
Curr Pharm Des. 2007;13(10):983-94. Review.
Chapter1

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8
Chapter 1

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Introduction
Atherosclerosis
Atherosclerosis is the underlying cause of major cardiovascular events such as
myocardial infarction and stroke, two of the most common causes of illness and
mortality in the Western world. During atherosclerosis, the arterial wall gradually
thickens to form an atherosclerotic plaque, which leads to narrowing of the arterial
lumen. The onset of atherosclerotic disease can be triggered by risk factors like
hypertension, diabetes mellitus, smoking, high fat diet, stress and high plasma
concentrations of low-density lipoprotein (LDL) cholesterol. These risk factors induce
endothelial dysfunction that leads to monocyte adherence and infiltration into the
endothelial layer. Fatty streaks develop already during early childhood [1] and may
progress into advanced atherosclerotic plaques. Plaque development is characterized by
migration and proliferation of smooth muscle cells and accumulation of lipids in the
sub-endothelial layer. Dependent on the composition of the advanced atherosclerotic
lesion, stable and unstable lesions can be discriminated. Unstable lesions or rupture-
prone lesions are characterized by a thin fibrous cap, a large atheroma, infiltrated and
activated inflammatory cells (predominantly macrophages), and low smooth muscle cell
content. Clinical symptoms appear in a later stage of life, when the lesions have
developed into unstable atherosclerotic plaques. When the plaque ruptures, the
lipidrich plaque content comes in contact with the blood and immediately initiates
thrombus formation, which often results in an acute occlusion of the artery (Figure 1).
In addition to plaque formation, the lumen of the atherosclerotic artery is also
determined by arterial remodeling (Figure 1). Inward remodeling accelerates the
process of luminal narrowing, while outward remodeling compensates for lumen loss
by lesion formation in the early phase of atherosclerosis. This seems a favorable
process; however, outward remodeling is considered to be a hallmark of vulnerable,
rupture-prone plaques, which cause clinical symptoms [2].
Arterial remodeling
An artery is not a rigid tube, only responsible for blood supply. In response to
hemodynamic, mechanical and biological stimuli the artery and the extracellular matrix
supporting the artery are capable to adapt its size and structure. Three different arterial
layers can be distinguished; the tunica intima, existing of connective tissue and a single
layer of endothelial cells lining up the artery at the luminal site. The endothelial cells are
able to sense differences in stretch or shear stress. The medial layer is the tunica media,
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Introduction

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which mainly consist of smooth muscle cells embedded in extracellular matrix
molecules like collagen, fibronectins and other proteoglycans and is involved in
adaptations to changes in blood pressure. The outer layer is called the tunica adventitia,
consisting of fibroblasts and extracellular matrix.
A change in shear stress by an acute change in blood flow leads to dilation or
constriction of the artery. If these changes in blood flow become chronic, structural
adaptations will be made. This process is called arterial remodeling and is defined as a
structural change in arterial size compared to a control or reference artery. An increase
in size is referred to as outward remodeling, while a decrease size is inward remodeling.
During arterial remodeling, the structural changes of the arterial wall involve matrix
turnover [3-6], cell migration [7] and inflammation [8].
10
Figure 1. Schematic representation of arterial remodeling together with atherosclerotic plaque
formation
Inward arterial remodeling accelerates luminal narrowing, while outward remodeling compensates for intima
formation. Outward remodeling is associated with unstable plaques, vulnerable for rupture, and when the
plaque ruptures the thrombogenic plaque contents come in contact with the blood and induce thrombus
formation.
Chapter 1

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Inflammation and immunity in atherosclerosis and arterial remodeling
The immune system responds effectively to harmful agents by close interplay between
the innate and the adaptive immune recognition systems. The innate immune system
is limited to the recognition of evolutionary highly conserved pathogen motifs and
is considered to be the first line of defense. The adaptive immune system involves
dynamic adaptation to unique epitopes present on pathogens in the environment.
Atherosclerosis
Atherosclerosis is a chronic inflammatory disease of the vascular wall [9]. The immune
response is evident during initiation and progression of atherosclerotic disease [9,10].
Susceptibility to plaque formation is the greatest in vascular regions where
haemodynamic stress is changed [1], such as bifurcations and curvatures. At these low-
shear rate sites, the endothelial cells become activated and permeability, adhesion
molecule expression on the endothelial surface and chemokine production increases.
Atherosclerotic lesions mostly hide cells that play an important role in the first line of
defense against exogenous ligands, particularly macrophages and T-lymphocytes.
The monocytes and T-cells attach to the endothelial layer and home-in on the arterial
wall during the initiation of atherosclerosis. Oxidative processes and subsequent
endothelial dysfunction are recognized as aetiological factors in monocyte adherence
to the endothelial layer. In addition, monocytes/macrophages can be activated by
infectious agents, which have been suggested to play a role in atherosclerotic plaque
formation, but results are conflicting and causality is difficult to prove in humans [11-13].
Arterial remodeling
Arterial remodeling is a ‘wound healing’-like response of the arterial wall to sustained
changes in bloodflow, arterial injury after balloon angioplasty or de novo
atherosclerosis. During restructuring of the artery, inflammation and subsequent matrix
turnover and cell migration are involved. Matrix degradation in the arterial wall involves
matrix metalloproteinases (MMPs) [6,14-16]. MMPs are proteolytic enzymes capable of
degrading molecules in the extracellular matrix. MMP activity levels are increased
during remodeling [15] and without MMP-9, no structural adaptation can be induced
[6]. Next to matrix breakdown, also cell migration is hampered in absence of MMPs.
Genetic mouse models lacking MMP expression and studies using MMP inhibitors both
show a reduction in cell migration, and a decrease in arterial remodeling [6,17,18].
During arterial remodeling not only collagen breakdown, but also collagen synthesis is
increased. Collagen synthesis is essential for the maintenance of structure of an artery
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Introduction

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12
Figure 2. Schematic representation of Toll-like receptor signaling
TLRs can be activated by either exogenous or endogenous ligands. Upon ligand binding TLRs recruit
intracellular signaling adaptors to their receptor-signaling domain, resulting in activation and nuclear
translocation of NF-kB. NF-kB activates pro-inflammatory cytokines, like IL-6, IL-1b and TNF-a, increases
expression of adhesion molecules such as ICAM-1 and VCAM-1, and increases expression of
metalloproteinases and release of chemokines, e.g. MCP-1 and IL-8.
Chapter 1

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and is related to cell migration [19]. Active collagen synthesis is associated with cell
migration towards the neointima, and intact collagen can promote cell migration as a
chemotactic factor [20,21] or by providing a substrate for cell migration [22]. Collagen
synthesis and breakdown are well-balanced during outward remodeling, collagen
turnover is increased, while total collagen content in the arterial wall does not change
[23], to keep the integrity of the vessel wall but also allow cell migration.
The presence of macrophages in the atherosclerotic wall is associated with outward
arterial remodeling. These cells produce the MMPs capable of degrading the molecules
in the extracellular matrix.
Toll-like receptors
The innate immune system is the first line of defense against pathogens. Toll-like
receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) [24] and
activate the inflammatory cell via the NF-KB pathway (Figure 2) [25-27]. Not only
macrophages express these TLRs: cells commonly found in the vessel wall express TLR
proteins, such as endothelial cells [28], adventitial fibroblasts [29], and vascular
dendritic cells [30,31].
Toll-like receptor signaling and Nuclear Factor-kappa B
Ligand binding to TLRs transmits a transmembrane signal that activates nuclear
translocation of Nuclear Factor-kappa B (NF-kB) (Figure 2) [25,26,32]. NF-kB controls
the transcription of inflammatory genes and is composed of homo- and heterodimers
of Rel family proteins (p65 (RelA), RelB, c-Rel, p50 (NF-kB1) and p52 (NF-kB2)) [33]. In
non-activated state the NF-kB dimers are captured in the cytoplasm bound to
inhibitoryprotein B (IkB). NF-kB activation via Toll-like receptors or cytokines results in
translocation of predominantly p50/p65 heterodimers to the nucleus. The NF-kB p50
subunit lacks a transactivation domain [34] and forms heterodimers with subunits that
are transcriptionally active. Homodimers of p50 are capable to bind to the DNA and
thereby inhibit transcription activity by other NF-kB dimers. NF-kB p50 homodimers
therefore can act as a brake on NF-kB signaling [35,36]. Indeed, several studies report
that p50 has an inhibitory effect on TLR4 signaling. Upregulation of p50 homodimers
attenuates lipopolysaccharide (LPS) response and is essential for LPS tolerance [37], and
NF-kB p50 inhibits a LPS-induced shock [38]. The exact role and regulation of the
individual components of the NF-kB complex are not well-understood and are under
intense investigation.
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Introduction

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Toll-like receptor 4
TLR4 was identified as the first human homologue of the Drosophila Toll [39]. TLR4 is
well known as the receptor for lipopolysaccharide (LPS), a product of the outer
membrane of Gram-negative bacteria [39-41]. TLR4 is the only TLR for which
endogenous ligands have been identified. Furthermore, genetic variation in the human
TLR4 gene is associated with carotid intima thickness, and therefore an interesting
receptor to study in relation to atherosclerotic disease.
Arbour et al. [42] reported that a point mutation in the TLR4 gene cause
hyporesponsiveness to LPS in humans. There is increasing evidence supporting the role
of TLR4 in atherosclerotic lesion development. Toll-like receptor 4 expression has been
described in human atherosclerotic lesions [28]. Activation of TLR4 stimulates
mononuclear phagocytes to secrete chemokines which are recognized for their
involvement in the recruitment of monocytes and T lymphocytes in the arterial wall
[43]. The production of cytokines induced after TLR4 activation influences proliferation
and migration of vascular smooth muscle cells [11]. In addition, TLR4 activation leads
to higher expression levels of MMP-2, -9 and cathepsins; proteins involved in matrix
break down [44,45].
Toll-like receptor 4 and atherosclerosis
In human atherosclerotic plaques, elevated mRNA and protein levels of TLR1, TLR2 and
TLR4 have been found [10,46]. In those atherosclerotic plaques, expression of TLR2 and
TLR4 has been described in macrophages and endothelial cells. TLR2 and TLR4
expression by endothelial cells in normal arteries is low, but significantly increased
in endothelial cells lining atherosclerotic lesions [28]. Xu et al. showed that TLR4 is
expressed in lipid-rich, macrophage-infiltrated atherosclerotic lesions of mice and
humans [47].
Bone marrow transplantation of the atherosclerotic ApoE-/- or ApoE+/+ to
a C3H/HeJ host, which has a critical point mutation in the TLR4 molecule,
revealed no differences in atherosclerotic lesion formation [48]. However, a lower
number of macrophages was observed in the atherosclerotic lesions of the bone
marrow recipient C3H/HeJ mice compared
It could be suggested that the recruitment of monocytes into the arterial wall is
affected in TLR4-deficient C3H/HeJ mice. Macrophages and endothelial cells in
human atherosclerotic plaques express TLR4. Stimulation with ox-LDL, a trigger for
atherogenesis, induces an up-regulation of TLR4 expression in macrophages in vitro,
suggesting a role for TLR4 in lipid-mediated proinflammatory signaling [47].
with control mice [48].
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Chapter 1

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Injections of LPS in rabbits on hypercholesterolemic diets and in ApoE-/- mice increased
the rapid development of atherosclerotic disease [49,50]. Michelsen et al.
demonstrated that TLR4-/- mice crossed onto the ApoE-/- background show
significantly reduced atherosclerotic lesion development [51] with lesions containing
less lipids and macrophages. Furthermore, in hyperlipidemic mice lacking expression of
MyD88, a downstream adaptor molecule in TLR signaling, early atherosclerosis is
significantly reduced [51,52]. In those mice macrophage recruitment to the arterial wall
is decreased resulting in reduced chemokine levels.
The inflammatory response in the atherosclerotic artery is not limited to the luminal side
of the vessel wall. Inflammatory infiltrates are frequently present in the adventitia of
atherosclerotic vessels as well [53]. In the adventitial layer of the atherosclerotic
coronary artery, not only macrophages but also adventitial fibroblasts express the TLR4
[29]. Activation of adventitial fibroblasts by LPS in mice, using a cuff model, induces
formation of intimal lesions. In C3H/HeJ mice with the TLR4 point mutation, this intima
formation was reduced by 60% compared with wild-type mice [29].
Toll-like receptor 4 polymorphism and cardiovascular disease
Recently, also the first human studies confirmed the role of TLR4 in the progression of
atherosclerotic disease. Kiechl et al. [54] showed that humans with the Asp299Gly
TLR4 polymorphism had a lower risk of carotid atherosclerosis and less intima media
thickness in the common carotid artery. Their results were confirmed in a case–control
study in which acute coronary syndromes was the primary endpoint [55]. However, also
conflicting data have recently been published. In a large cohort of 1400 participants,
the presence of the Asp299Gly polymorphism was not associated with predisposition
or progression of atherosclerotic disease [56]. Moreover, longitudinal progression of
intima media thickness was not affected by this polymorphism in patients suffering
from familiar hypercholesterolemia [57]. The inclusion criteria and thereby the domain
of these studies merit careful consideration, as they differed substantially.
The TLR4 polymorphism Asp299Gly is also found to be associated with an increased
incidence of cardiovascular events. Boekholdt et al. showed that the Asp299Gly
polymorphism is also associated with a higher efficacy of statin therapy [58]. Statin
therapy resulted in an average reduction of cardiovascular events by 50%. Strikingly,
the risk on cardiovascular events in noncarriers was reduced from 18.1% to 11.5%,
while in carriers of the Asp299Gly allele, the risk was reduced from 29.6% to 2% [58].
Thus, an impressive reduction of cardiovascular events by statin therapy was mainly
observed when the TLR4 polymorphism was present. Edfeldt et al. studied the
15
Introduction