Adhesion and migration of smooth muscle cells during
atherosclerotic plaque formation
Name:
Claudia Tersteeg
Location:
UMC Utrecht, department of Experimental Cardiology
Supervisor: Dominique de Kleijn
Date:
August 2008
1
Abstract
During the initiation and progression of atherosclerosis, migration and proliferation of
vascular smooth muscle cells (VSMCs) play important roles in lesion formation. Contractile
VSMCs are adhesive and play a role in the contractile function of the artery. These cells can
differentiate into synthetic VSMCs upon injury, gaining the ability to migrate and proliferate.
This phenotype switch is accompanied by differential expression of several adhesion
molecules that are involved in cell-cell binding or cell-extracellular matrix interactions. The
adhesion molecules discussed in this thesis include the four major families of adhesion
molecules: integrins, cadherins, selectins and immunoglobulin superfamily cell adhesion
molecules. Members of these families of adhesion molecules were shown to play a role in
VSMCs migration, and thereby the formation of an atherosclerotic lesion. This thesis focuses
on the role of adhesion molecules during phenotype switching, and the migration of VSMCs
during arterial injury.
2
Table of contents
Abstract
2
Table of contents
3
1. Atherosclerosis
4
2. Smooth muscle cells in atherosclerosis
5
3. Integrins
6
3.1VSMC adhesion to the ECM requires integrins
3.2 Integrin transition during phenotype switching
3.3 Integrin signaling
4. Cadherins
4.1 N-cadherin
4.2 T-cadherin
5. Immunoglobulin superfamily cell adhesion molecules
5.1 Cytokine regulated expression
5.2 Soluble forms of cell adhesion molecules
6. Selectins
6.1 P-selectin
6
6
8
8
9
10
11
12
12
13
13
7. Phenotype switching upon injury and inflammation
14
8. Clinical interventions
15
9. Concluding remarks
15
References
17
3
1. Atherosclerosis
Atherosclerosis describes the process of plaque formation inside the artery. Starting at
a young onset and progressing during the years of life makes it a slow but complex disease.
Because of today’s life style in high-income countries, many people suffer from high blood
pressure, diabetes and high cholesterol as a results of smoking and high fat diet; all risk
factors for atherosclerosis. Its increasing prevalence makes it the main causes of death
worldwide1.
Inflammation plays an important role in the initiation and progression of
atherosclerosis. In a healthy artery, a layer of endothelial cells forms the inner lining; the
intima. Already in 1976, a theory was proposed by Ross where injured endothelium was held
responsible for its increased susceptibility to lipid accumulation and thrombus deposition,
leading to atherosclerosis2. Currently, it is thought that oxidized lipoproteins (oxLDL) initiate
an inflammatory response by activation of the endothelium, leading to the recruitment of
monocytes3. Activated endothelial cells express several leukocyte adhesion molecules at their
cell surface that can bind the endothelial adhesion molecules expressed on monocytes.
Binding results in rolling of the monocytes along the endothelium, towards the area of injury.
Upon attachment, the monocytes migrate into the subendothelial space of the intima upon
chemokine stimulation secreted by the intima4. Once inside the intima, monocytes
differentiate into macrophages that will take up oxLDL particles with their scavenger
receptors. Accumulation of lipid due to disturbed cholesterol efflux, leads to the formation of
foam cells, which will eventually form the lipid core of the plaque4. Next to monocytes,
vascular smooth muscle cells migrated from the media were shown to be able to
transdifferentiate into macrophages, to take up LDL and to form foam cells5.
Growth factors and cytokines produced by the endothelial and inflammatory cells
induce a phenotypic change of vascular smooth muscle cells (VSMCs) from a contractile to a
synthetic state, that allows them to migrate from the media into the intima6. Together with the
migration of VSMCs, new extracellular matrix (ECM) is formed.leading to a fibrous cap
containing VSMCs and collagen that separates the lipid core from the lumen. Inside the lipid
core, apoptosis and necrosis occurs and foam cells start to die, contributing their contents to
the necrotic core of the lesion, further propagating the inflammatory process. The lesion
expands by continuing monocyte entrance, lipid accumulation, apoptosis and necrosis7.
Macrophages keep getting activated and start to release MMP and other proteolytic enzymes
that break down the ECM and thereby thinning the fibrous cap8. Rupture of the cap will lead
to thrombus formation and hemorrhages that can eventually lead arterial occlusion and
subsequent myocardial infarction or stroke.
Figure 1: Plaque formation. A: Leukocytes adhere and migrate trough the endothelium. B: Macrophages
transform into foam cells, fatty streak forms. C: Formation of the necrotic core covered by a fibrous cap 2.
4
2. Smooth muscle cells in atherosclerosis
Vascular smooth muscle cells (VSMCs) in the medial layer of an artery are
surrounded by elastic fibers and extracellular matrix (ECM) containing molecules such as
collagen and laminin9. These VSMCs are called contractile VSMCs, and their main function
is to sustain vascular tone and resistance. They have a more contractile function, expressing
proteins as smooth muscle (SM) α-actin10.During vasculogenesis and vascular injury,
however, they can differentiate in synthetic VSMCs, that have the capability to migrate and
proliferate. The function and morphology of contractile VSMCs is changing resulting in
proliferation and production of ECM components11. As a result, VSMCs that are found within
the same artery can differ in function as well as in morphology depending on their
location11,12. VSMCs adhere to the ECM to withstand tension due to contraction and
hemodynamic forces. Expression of several integrins and syndecans is found on contractile
VSMCs, together with muscle specific dystrophin-glycoprotein complex (DGPC) that
attaches the actin filaments to the ECM11. Upon vascular injury, VSMCs need to migrate
towards the injured site for repair. To obtain a migratory, or synthetic, phenotype, the cells
need to become more motile by disengagement of the adhesion molecules from the ECM, and
by expressing other adhesion molecules that allow them to migrate. Upon this phenotypic
change, these intimal VSMCs were shown to express lower levels of contractile proteins and
to have a larger ability to bind proteases and cytokines10.
Changing from a contractile to a synthetic state is important for the development of
many vascular diseases, including atherosclerosis. But to migrate towards the intima, other
very important complex processes and molecules are involved. Migration and adhesion are
very complex processes with many key molecules involved. Adhesion molecules mediate
adhesive interactions between cells of the same type (homophilic interactions) or to a different
class of cell adhesion molecules (heterophilic interactions)13. Figure 2 shows the four major
families of adhesion molecules that can be distinguished based on their function: cadherins,
immunoglobulin (Ig) superfamily, integrins and selectins13. Both the interaction of VSMCs
with the extracellular matrix and the interaction with adjacent cells play an important role in
the proliferation and migration of VSMCs during vascular diseases. ECM adhesion molecules
play a major role in VSMC adhesion, but cell-cell adhesion molecules are also found on this
cell type. Interaction with the ECM is provided by Focal Adhesions (FAs) that provide a site
of attachment where signal transduction is initiated. These sites provide an important role in
cell proliferation and migration, coordinating several events as actin polymerization, actin
stress fiber attachment and stress fiber contraction14. Their formation is mainly mediated by
members of the integrin family. This most studied type of adhesion molecules on VSMCs is
thought to play a key role in adhesion
and migration, but recent studies showed
the importance of other adhesion
molecules
for
proper
VSMC
functioning. This thesis will focus on the
function of the four major families of
adhesion molecules during phenotype
switching in the adhesion, migration and
proliferation of VSMCs.
Figure 2: The four major families of
cell adhesion molecules. Cadherins,
Ig-superfamily
cell
adhesion
molecules, integrins and selectins13.
5
3. Integrins
Integrins are cell surface proteins that mediate adhesion to the
ECM, and in lesser extent to surface proteins of other cells. Integrins
are heterodimers consisting of an α and β chain9. Both these chains are
transmembrane proteins with a short cytoplasmic domain that is
connected to cytoskeleton proteins including α-actinin, keratin, talin
and vinculin15. 16 α- and 8 β-chains are known, being able to combine
and form 22 different integrins that can bind to different ligands. Most
of the integrin ligands are ECM molecules and will be bound with
high affinity upon activation of the integrin.
Figure 3: Integrin receptor. Structure of an
integrin receptor. α and β chains connected to
cytoskeleton proteins9.
3.1 VSMC adhesion to the ECM requires integrins
Adhesion of VSMCs to ECM proteins was shown to be dependent on several integrin
receptors. Integrin β1 is the most predominant integrin present on VSMCs, functioning in
facilitating their growth and migration, and in adhering to ECM molecules16. Integrin β1
mediates adhesion to collagen type I and IV, and laminin17. Integrin α1β1 and α2β1 were
shown to participate in the adhesion of cultured VSMCs to collagen18. Tubulins, associated
with elastic fibers and basement membranes, adhere to cultured VSMCs via integrin receptors
α5β1 and α4β114. Under hypoxic conditions, adhesion of cultured human VSMCs to ECM
proteins is enhanced by activation of β1 integrins compared to cells grown under normoxic
conditions19.
Not only β1 integrin receptors mediate the adhesion of VSMCs. Osteopontin receptors
αvβ1 and αvβ5 adhere VSMCs to osteopontin together with integrin αvβ3, that is also
responsible for the migration of the cells20. Another study also implicated a role for integrin
αvβ5 in the adhesion to vitronectin, as demonstrated by rat aortic VSMCs cultured on a
vitronectin rich layer21. The same was shown by Baron and colleagues. By antibody mediated
blocking of integrin αvβ3 and αvβ5, adhesion of VSMCs to vitronectin and osteopontin was
inhibited22. Only blocking integrin αvβ3 also reduced the migration of VSMCs to osteopontin,
vitronectin and PDGF.
3.2 Integrin transition during phenotype switching
VSMCs need to obtain a synthetic phenotype to be able to migrate. During phenotype
switching, it has been shown that an altered set of integrins is expressed on the cell surface.
Integrins that are heavily expressed during the contractile state of the cell are downregulated
upon phenotype transition, and different integrins become upregulated.
Integrin αvβ3 and αvβ5 were shown to be upregulated during neointima formation in
21
rats . The authors also showed inhibited migration of cultured VSMCs by antibody mediated
blocking of these integrins. Previous studies already showed the increase of integrin β3
expression in atherosclerotic coronary arteries and balloon injured baboon brachial arteries
throughout the neointima23. In the balloon injured arteries, co-localization was observed
between integrin β3 positive cells and SM α-actin positive cells, indicating the expression of
integrin β3 in VSMCs after injury. More recently, the importance of integrin αvβ3 in
migration was again demonstrated24. Reduced neointima formation was observed in integrin
β3 deficient mice after carotid artery ligation. This was accompanied by decreased migration
and fewer intimal smooth muscle cells but without a difference in VSMC apoptosis or
6
proliferation. To determine if integrin αvβ3 was responsible for this effect, and not integrin
αIIbβ3, transplantation of bone marrow from integrin β3 wild type into integrin β3 deficient
mice was performed. These bone marrow derived cells express αIIbβ3 on platelets, whereas
αvβ3 is expressed on other cells like VSMCs, endothelial cells and inflammatory cells.
Transplantation resulted in reduced neointima formation after ligation, showing that integrin
αvβ3 mediated VSMCs migration contributing to intimal thickening.
Increased expression of SM α-actin and accumulation of α8 positive cells was
observed after arterial injury followed by neointima formation and luminal narrowing25.
Colocalization was observed for SM α-actin and integrin α8 with ECM proteins, showing that
α8 positive cells were embedded in a site of constrictive remodeling (figure 4).
Figure 4: Integrin α8, SM αactin, collagen and fibronectin
localization in the neointima.
Immunostaining of integrin α8
(A) and SM α-actin (B) reveal
co-localization in the neointima
(C).
Collagen
(D)
and
fibronectin (E) staining in the
neointima. (F) shows merged
image of SM α-actin (red) and
nuclei (blue) in the neointima.
Arrows indicate internal elastic
lamina25.
Actin Paxillin
Integrin β1 Paxillin
In a different study, the authors overexpressed α8β1 integrin leading to suppressed ability of
VSMCs to migrate26. Using passage-5 VSMCs that have a synthetic phenotype,
overexpression of this specific integrin induced differentiation into a contractile phenotype.
This was shown by upregulation of contractile markers as calponin SM α-actin and the
assembly of longer focal adhesions.
Migration of cultured human and rat aortic VSMCs towards collagen 1 was shown to
be inhibited by up-regulation of α2β1 integrin receptors27. Recent work by Abraham and
colleagues showed the importance
Control
Itgb1KO
of integrin
β1 in
VSMC
28
migration
.
Integrin
β1
knockout
A
mice showed poor spreading of
VSMCs
leading
to
loosely
organized vessel walls. In the
absence of integrin β1, focal
adhesions were formed though
short and disorganized and ECM
Figure 5: Characterization of cultured
integrin β1-knockout VSMCs. (A)
Anti-integrin β1 in red, anti-paxillin in
green. Control and integrin β1-knockout
VSMCs as indicated. (B) SM α -actin in
green, focal adhesions (paxillin) in red.
Disorganized cytoskeleton of integrin
β1-knockout VSMCs. Arrows indicate
cortical actin in KO mice28.
7
proteins as fibronectin and laminin were expressed but fibril formation, as found in the
presence of integrin β1, was absent (figure 5). Proliferation was upregulated, and transcription
factors playing a role in the regulation of VSMC differentiation were reduced, showing that in
the absence of integrin β1, VSMCs failed to acquire a fully differentiated and functional
phenotype28.
Embryonic loss of integrin α7 was shown to results in vascular defects, whereas this
loss in adult mice resulted in VSMC hyperplasia29. The authors showed that integrin α7β1
promotes the contractile phenotype of VSMCs and continued to investigate the cellular
mechanisms underlying this effect. By culturing VSMCs, they showed that upon loss of
integrin β7, the cells proliferated continuously30. These cells had a reduced capacity to
express contractile proteins after serum withdrawal. The MAP kinase pathway was activated,
leading to the activation of ERK, which may promote continuous cellular proliferation. In
integrin α7 deficient mice, increased neointima formation was observed, as well as thickening
of the medial layer and narrowing of the lumen after ligation injury, indicating that loss of
integrin α7β1 contributes to dramatic vascular remodeling and neointimal development.
3.3 Integrin signaling
The importance of integrins during adhesion to the ECM and migration towards
injured endothelium is discussed above, but the presence of these integrins is not only
important for migration. Integrins are involved in several signaling pathways, including
protection from apoptosis, IGF-1 signaling and FAK activation.
Apoptosis of VSMCs in atherosclerotic plaques can accelerate the formation of an
unstable plaque leading to plaque rupture, calcification or increased stenosis31. Integrin αvβ3
was shown to protect VSMCs from apoptosis24. In a study by Cheng and colleagues, an
increase in integrin β3 expression was found after subjecting VSMCs to mechanical stress32.
A negative correlation with TUNEL-positive staining was found for this expression,
indicating that αvβ3 expression is associated with survival and not with apoptosis. The effect
of Ox-LDL induced apoptosis on VSMCs was protected by integrin β3, resulting from
stabilization of the cytoskeleton.
Insuline-like growth factor-1 (IGF-1) was shown to inhibit inflammatory responses
and to decrease plaque progression in atherosclerotic prone ApoE-/- mice33. VSMCs were
shown to express IGF-1 receptors, and their proliferation and migration is increased upon
increased expression of IGF-134, 35. Integrin αVβ3 was shown to be involved in the IGF-1
signaling cascade. Upon blocking the αvβ3 receptor, inhibition of IGF-1 signaling occurs in
VSMCs36. Exposing VSMCs to high glucose was shown to activate integrin αvβ3 ligands,
like ECM protein vitronectin37. This leaded to an increase in IGF-1 expression, followed by
Shc phosphorylation, eventually leading to MAP kinase activation, being essential for cell
proliferation.
Focal adhesion kinase (FAK) acts as a scaffold protein that is activated by integrin
binding to fibronectin. It was shown that FAK activation leads to cell migration by the
promotion of cell elongation that is followed by changes in the actin cytoskeleton38. Migration
is also induced by FAK activation as a results of the recruitment of signaling proteins like Src
that can activate the Ras/MAPkinase signaling pathway38, 39. A recent study showed that not
only integrin binding to fibronectin, but also to osteopontin results in increased migration of
VSMCs40. Integrin β3 expression was increased by high levels of osteopontin, leading to FAK
phosphorylation and increased migration.
8
4. Cadherins
The cadherin family consists of transmembrane adhesion proteins that form cell-cell
connections through a zipper like structure. A contractile bundle of actin filaments in each cell
binds to the lateral membrane by anchor proteins, including catenins, vinculin and α-actinin13.
These anchor proteins bind to cadherin in a homophilic interaction. Anchor proteins are
essential for strong adhesion, showed by disruption of α-catenin41. Another anchor protein,
p120-catenin binds to cadherins and is required to keep cadherins at the cell surface42.
Epithelial adherens junctions represent the typical form of cadherins. In the epithelium, they
form a continuous zonula adherens, or an adhesion belt9.
Cadherins contain a single transmembrane domain, a short C-terminal cytosolic
domain, and five extracellular cadherin domains that are necessary for Ca2+ binding and cellcell adhesion9. E-cadherin is named after epithelial cells, the main cell-type in which it is
expressed43. Increased expression of E-cadherin was found in atherosclerotic lesions, but was
not expressed by VSMCs44. N-cadherin is named after nerve cells and high expression was
also found in fibroblasts45. These are the
classical cadherins and their sequence is
related throughout their extracellular and
intracellular domains. Besides classical
cadherins, there are also a large number of
nonclassical cadherens. The nonclassical
cadherens include proteins with known
adhesive functions like the desmosomal
cadherins, but also proteins without adhesive
Figure 6: Classical
functions like T-cadherin, which lacks a
cadherins. Linkage of
46
transmembrane domain . The two cadherin
cadherin to the actin
mainly found on VSMCs are N-cadherin and
filaments
through
anchor proteins9
T-cadherin and are discussed below.
4.1 N-cadherin
N-cadherin expression was first discovered on nerve cells45, and was later shown to be
important in adherence junction formation between endothelial cells and pericytes during
angiogenic vessel formation in brain tissue47. VSMCs were shown to express N-cadherin48-50,
but its role on VSMC migration is not fully understood and studies show conflicting results.
Jones and colleagues showed increased levels of N-cadherin, β-catenin and plakoglobin in the
neointima of injured rat carotid arteries48. In vitro experiments showed a reduction in wound
repair probably due to a decrease in migration of VSMCs, after antibody mediated blocking of
N-cadherin. Furthermore, characterization of cell adhesion junctions present in VSMCs
showed the presence of N-cadherin, combined with α- and β- catenin, and plakoglobin as
anchor proteins, but the absence of E-cadherin. Studies in cancer cell lines support this data,
showing high expression levels of N-cadherin during migration and invasion of different cell
types51, 52.
Conflicting data were shown by Koutsouki and colleagues53. Inhibition of N-cadherin
in cultured VSMCs by antibodies and inhibitory peptides resulted in reduced migration of the
cells. These results are supported by a study performed by Blindt and colleagues 50. A porcine
restenosis model was used to evaluate N-cadherin expression in dilated vessels. They showed
that VSMC N-cadherin expression was decreased in the neointima of the dilated vessels
compared to the media that still showed strong expression of N-cadherin. Comparison of the
contractile with the synthetic phenotype showed a downregulated expression of N-cadherin in
VSMCs that obtained the synthetic phenotype (figure 7). E-cadherin and β-catenin were both
9
unchanged. Upon blockade of N-cadherin in contractile VSMCs, the cells obtained a synthetic
phenotype shown by increased migration capacity.
Also during VSMC proliferation, expression of N-cadherin was shown to be
decreased49. Cultured VSMCs from human saphenous veins were stimulated with fetal calf
serum and PDGF to activate cell proliferation, and a decrease in N-cadherin expression was
found, most likely due to modulation of β-catenin signaling.
Figure 7: E-cadherin, β-catenin and Ncadherin expression. Western blots of Ecadherin,
β-catenin
and
N-cadherin
expression in contractile (quiescent, Q) and
synthetic (migratory, M) VSMCs. Reduced
N-cadherin expression in synthetic VSMCs50.
It is clear that more insight is needed to understand the role of N-cadherin on
migration and proliferation of VSMCs. Possible mechanisms how N-cadherin can mediate
these processes can be suggested by regulation of Rho activity and β-catenin signaling.
Assembly of actin filaments, that bind the anchor proteins to the lateral membrane at focal
adhesion sites, regulate cell migration via the Rho family containing small GTPases including
RhoA and Rac154. The study performed by Blindt and colleagues showed decreased RhoA
activity in synthetic VSMCs, comparable to the observed decreased N-cadherin expression50.
Conflicting results are reported by Liu and colleagues where RhoA activity is increased in
VSMCs that are subjected to stretch55. These cells show decreased activity of another
GTPase, Rac1. Because Rho activity determines the morphology of the cell, and thereby its
ability to migrate, Rho might have a more important role in VSMC migration then was
previously thought.
β-catenin signaling might be another important aspect of N-cadherin mediated VSMC
migration. Besides its role in adhesion by acting as an anchor protein, it plays a role in Wnt
signaling where it can translocate to the nucleus and bind to the transcription factor T-cell
factor (TCF) (reviewed in56). Upon binding, β-catenin can regulate transcription by of a
number of genes, including genes that are involved in the cell cycle, like cyclin D1 57, 58, and
also MMP758.
4.2 T-cadherin
A nonclassical cadherin known to be expressed in VSMCs is T-cadherin59. This
protein can mediate weak cell-cell interactions, lacks the transmembrane and cytosolic
domain, and is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI)
anchor46.
T-cadherin was localized in the leading edge of VSMCs migrating towards an injured
area60. In the same study, the authors showed that elevation of T-cadherin in cultured L929
fibroblasts reduced the adhering capacity of HUVEC endothelial cells that were seeded onto
the fibroblast monolayer, indicating that T-cadherin is not required for adhesion, but might be
important as a signaling molecule for cell-cell recognition. In atherosclerotic lesions, Tcadherin expression was shown to be increased in synthetic VSMCs compared to healthy
10
tissue, indicating a role for this cadherin in the involvement of VSMC on plaque
progression61. In rat carotid arteries, a temporal increase in T-cadherin expression was found,
that correlated with increased cell migration and proliferation activity during formation of the
neointima after balloon injury62. In cultured VSMCs, the authors describe an increased
expression of T-cadherin in dividing cells compared to non-dividing cells, indicating a role
for T-cadherin in the synthetic, more proliferative, phenotype. This was confirmed by another
study showing an increased expression of T-cadherin in cell populations in the S- and G2/Mphase, and promoted proliferation of VSMCs in the presence of T-cadherin63. In a different
study, these authors showed decreased adhesion and spreading of VSMCs and HUVECs upon
expression of recombinant T-cadherin protein in cultured cells (figure 8)64, supporting
previous data60. Adenoviral vector mediated overexpression of T-cadherin in HUVECs
resulted in increased de-attachment and migration induced by T-cadherin. Overexpression of
T-cadherin in VSMCs was not performed.
Figure 8: Recombinant T-cadherin protein inhibits
adhesion and migration of VSMCs and HUVECs. Cells
seeded in gelatin, collagen or laminin coated wells without
(control) or with recombinant T-cadherin protein or BSA64.
All together, this indicates that increased T-cadherin expression may play a role in
atherosclerosis by decreasing VSMC adhesiveness resulting in an upregulated migration and
proliferation capacity.
5. Immunoglobulin superfamily cell adhesion molecules
The most important members of the
immunoglobulin superfamily of cell adhesion
molecules are intracellular adhesion molecule-1
(ICAM-1) and vascular cell adhesion molecule
(VCAM-1). These cellular adhesion molecules
are cell surface glycoproteins consisting of 5
Figure 9: Typical
immunoglobulin like domains65.
structure
of
Their function has well been characterized
Immunoglobulin
on endothelial cells, where they can adhere to
superfamily
cell
monocytes and lymphocytes, upon stimulation
adhesion molecules.
with inflammatory cytokines66. Expression of
Ig like domains
attached to cytosol9.
ICAM-1 and VCAM-1 was also detected on
VSMCs, but its role on adhesion of VSMCs is not
thoroughly investigated67, 68.
Increased expression of both ICAM-1 and VCAM-1 was found in human
atherosclerotic lesions, but not in healthy medial VSMCs67, 69, 70, implicating a role for these
adhesion molecules in atherosclerosis. It is known that VCAM-1 on monocytes binds to
integrin α4β1, expressed on the surface of endothelial cells71. Integrin α4β1 was shown also to
be expressed on VSMCs72. Antibody mediated blocking of integrin α4β1 and VCAM-1
11
reduced the expression of VSMC-specific differentiation markers, indicating a role for the
interaction in the differentiation of VSMCs. More recently, it was shown that expression of
VCAM-1 is required for VSMC accumulation in plaques of ApoE-/- mice after arterial
injury73. That VCAM-1 acts directly on integrin α4β1 on VSMCs, was indicated by Petersen
and colleagues74. This study showed that VCAM-1 expression is necessary for the migration
of VSMCs, using siRNA against VCAM-1 in cultured VSMCs. Reduced VCAM-1
expression by siRNA resulted in decreased migration of VSMCs as shown with a scratch
assay (figure 10). A decreased proliferation was also observed after VCAM-1 reduction, but
this decrease was not as big as the decrease in migration. The authors indicate that the
interaction between integrin α4β1 and VCAM-1 can enhance the proliferative response of
VSMCs to different stimuli, but the exact mechanism remains unclear.
Figure 10: Decreased migration after wounding in
the absence of VCAM-1. After 24h and 48h,
decreased migration observed in the number of
VSMCs that migrated towards wound area. Cells
transfected with VCAM-1 siRNA or control siRNA74.
Other studies indicate a role for VSMCs VCAM-1 in the protection of monocytes
from apoptosis75. In vitro serum depletion leads to induced monocyte apoptosis, but this effect
was shown to be reversed upon VCAM-1 mediated binding to VSMCs.
ICAM-1 was found to be constitutively present in the contractile phenotype of
VSMCs, and to be increased upon differentiation into the synthetic phenotype76. This increase
was accompanied by an increase in monocyte chemoattractant protein-1 (MCP-1), another
member of the immunoglobulin superfamily cell adhesion molecules. Recently, both ICAM-1
and VCAM-1 were shown to be increased in the synthetic VSMCs compared to the
contractile phenotype77.
5.1 Cytokine regulated expression
Expression of ICAM-1 and VCAM-1 can be regulated by cytokines as TNFα and
IFNγ78, 79. TNFα was shown to induce expression of both ICAM-1 and VCAM-1 in VSMCs
whereas IFNγ specifically stimulated ICAM-179. A study from Zhang and colleagues showed
that atherosclerosis is promoted after TNFα stimulation of murine carotid artery grafts80.
TNFα stimulation resulted in increased VSMC proliferation in the initiation of plaque
formation, but no evidence for promotion of cell proliferation was found in the advanced
lesion. Expression of VCAM-1 and ICAM-1 was increased in the early phase, as well as in
the advanced stage of lesion formation. A study performed by Kusaba and colleagues showed
that IFNγ induced neointima formation81. In balloon injured rat arteries, both ICAM-1
expression and VSMC proliferation were increased and this increase could be abrogated by
inhibition of the IFN protein. These studies show that increased expression of the cell
adhesion molecules ICAM-1 and VCAM-1 is associated with increased proliferation of
VSMCs, leading to increased neointima or lesion formation.
12
5.2 Soluble forms of cell adhesion molecules
Soluble forms of ICAM-1 and VCAM-1 can be found in human blood82 and, mainly
sICAM-1, can be used as markers to predict future cardiovascular events83.
ICAM-1 can bind to lymphocyte function-associated antigen-1 (LFA-1). sICAM-1
was shown to be able to inhibit lymphocyte attachment to cerebral endothelial cells when
bound to LFA-184. A recent study in rat aortic VSMCs showed that these cells also express
LFA-185. In this study, rat aortic VSMCs were stimulated with sICAM-1 leading to increased
migration as shown with a Boyden chamber assay. Upon antibody mediated blocking of the
LFA-1 receptor, this effect was neutralized. This indicated that LFA-1 on VSMCs plays an
important role in binding sICAM-1. It was not shown if sICAM-1 also affected the
proliferation of VSMCs. The same authors showed in a different study the role for sVCAM-1
in VSMC migration and proliferation86. Cultured rat aortic VSMCs stimulated with sVCAM1 showed an increase in migration and also proliferation. By blocking integrin α4β1 with
antibodies, these effects were abolished, indicating that next to VCAM-1 also sVCAM-1
binds to this integrin receptor. No indication is given by the authors about the phenotype of
the VSMCs before or after stimulation with the soluble cell adhesion molecule. The results
suggest that soluble forms of ICAM-1 and VCAM-1 can stimulate the migration and
proliferation of VSMCs, but it might be interesting to know if this is a result from an induced
phenotypic switch into the synthetic phenotype.
6. Selectins
The fourth and last family of adhesion molecules consists of selectins. These are cellsurface carbohydrate-binding proteins that mediate a variety of calcium dependent cell-cell
adhesion interactions in the blood circulation9. L-selectin is found on leukocytes87, P-selectin
on platelets and activated endothelial cells88, 89, and E-selectin also on activated endothelial
cells90. The lectin domain of these transmembrane proteins bind
with a heterophilic interaction to specific oligosaccharide on
adjacent cells. Selectins were shown to have an important role at
sites of inflammation where leukocytes and platelets recognize
and bind to the damaged endothelium13. Only P-selectin is so far
shown to be expressed by VSMCs.
Figure 11: Selectin structure. Selectin
binds to actin filament trough anchor
proteins. Lectin domain of selectin binds
to oligosaccharide on adjacent cell9.
6.1 P-selectin
To date, the most important selectin known is P-selectin. This selectin is expressed
and stored in the Weibel-Palade bodies of endothelial cells88 and in α-granules of platelets89.
For many years, the role of P-selectin was mainly devoted to recruitment of leukocytes and
mediating interactions of platelets and leukocytes with damaged endothelium in the vessel
wall (reviewed in reference 91). More recent articles show the role of P-selectin on VSMCs
during atherosclerosis and arterial damage.
A study by Li and colleagues showed that not only macrophages and monocytes
express P-selectin during carotid artery injury in mice, but also SM α-actin positive VSMCs
13
were shown to express this specific selectin in the neointima and media of the artery92.
However, no expression of P-selectin was observed in atherosclerotic prone ApoE-/- mice.
These results are in conformation with studies from Kumar93 and Zeiffer94.
The study from Kumar and colleagues was the first that indicated a possible role for
VSMC and P-selectin using the carotid artery ligation model in mice to induce vascular
remodeling93. Arteries from P-selectin-/- mice were compared to those from wild type mice,
and increased P-selectin staining was observed in the media and neointima of wild type mice,
together with increased leukocyte and platelet infiltration. No stainings were performed for
VSMC markers, but the authors do suggest that VSMCs are influenced by P-selectin. Zeiffer
and colleagues showed that P-selectin is indeed associated with VSMCs94. The carotid artery
ligation model was used in ApoE-/- mice, and high expression levels of P-selectin were found
14 days after injury (figure 12). The authors showed furthermore that increased P-selectin
expression on VSMCs results in higher arrest of monocytes, demonstrated by laminar flow
assays.
Figure 12: P-selectin expression in
neointimal VSMCs after injury in
ApoE-/- mice. (A) P-selectin
expressing neointimal VSMCs in
non-reendothelialized
areas,
constituting the luminal lining of the
neointima. (B) In lesions covered
with endothelial cells, P-selectin is
expressed by the majority of
VSMCs. Arrows indicate internal
elastic lamina94.
The functional implications of P-selectin in VSMC migration and proliferation have
still not been elucidated, but these studies suggest increased adhesiveness of VSMCs that
express P-selectin to arrest monocytes during neointima formation. VSMCs seem to acquire
the ability to express P-selectin after differentiating into the synthetic phenotype, as a result of
arterial injury.
7. Phenotype switching upon injury and inflammation
Contractile properties and expression of specific smooth muscle proteins distinguish
contractile VSMCs from synthetic VSMCs10, 11. During phenotype switching, integrins were
shown to express an altered set on the cell surface21, 23-26, 28-30, whereas other cell adhesion
molecules show an increased or decreased expression50, 53, 62, 63, 77, 93, 94. The development of
atherosclerosis is associated with migration of VSMCs from the media towards to intima.
Evidence is provided that the synthetic VSMCs participate in plaque formation, but whether
this participation is good or bad is still open for debate. Stable atherosclerotic lesions are
associated with a high VSMCs content, but examples are shown in this thesis that the
migration of VSMCs towards lesions is accompanied with inflammation and increased
neointima formation leading to increased stenosis.
Synthetic VSMCs are less contractile, and have a larger ability to bind proteases and
cytokines10. The question arises if these cells acquire pro-inflammatory characteristics upon
14
differentiation. Increased expression was found of several adhesion molecules including
integrins, T-cadherin, ICAM-1, VCAM-1 and P-selectin, accompanied by increased migration
and in some studies also proliferation of VSMCs. These adhesion molecules were previously
shown to be able to bind inflammatory cells when present on activated endothelium. Several
recent studies indicate that these receptors are also able to bind inflammatory cells when
present on VSMCs. They were able to bind for example foam cells95, monocytes96 and
neutrophils97, mediated by several adhesion molecules and thereby suggesting a proinflammatory role for VSMCs.
It is now clear that VSMCs play an important role in the initiation phase, but
difficulties remain to demonstrate the exact role. Because of the inability to deplete this cell
type in an animal model, obviously leading to lethality in the embryonic stage, other options
are needed. Restricting the differentiation of VSMCs into a synthetic phenotype prior to
arterial damage, would give more insight in the involvement of these cells in neointima
formation. Also focusing on a combination of adhesion molecules in a single study should
give more information about the interactions between the different families of adhesion
molecules. As shown before, integrin α4β1 interacted with VCAM-1 on VSMCs74, but maybe
more adhesion molecules can activate each others expression. Another option would be to
find signals that induce the expression of certain adhesion molecules. TNF-α was already
shown to induce the expression of ICAM-1 and VCAM-179, but other inflammatory cytokines
could be able to activate expression of adhesion molecules that can thereby induce the
synthetic phenotype of VSMCs.
8. Clinical interventions
With more knowledge in the mechanisms of cell adhesion and migration, future
clinical therapies can be developed that can interfere with these events. During the initiation
of atherosclerosis, differentiation of VSMCs into the synthetic phenotype occurs, leading to
increased atherosclerosis. Preventing this differentiation might contribute to less migration
and inflammation during lesion formation. More insight into the role of different adhesion
molecules that are up- and downregulated during phenotype switching can give rise to
molecular targets that can be inhibited using blocking antibodies or small molecule inhibitors,
and that can thereby inhibit the migration of VSMCs. In the oncology field, small molecule
inhibitors were already shown to be able to inhibit tumor cell migration by intervention with
the integrin binding sites98-100. No studies have shown this for VSMCs in atherosclerotic
plaques so far. This knowledge can also lead to the development of drug eluting stents that
can prevent restenosis by inhibition of VSMCs migration. Hereby, it is possible to reduce
VSMC migration locally instead of systemically if necessary.
Another option would be blocking the receptors that are responsible for binding
inflammatory cytokines that induce adhesion molecule expression. Previous studies have
already shown that pro-inflammatory cytokines TNFα and IFNγ can regulate the expression
of ICAM-1 and VCAM-1 78, 79. Because these cytokines are not specifically involved in the
development of atherosclerosis, inhibiting its expression would not be the best option. With
more knowledge in the receptors binding these cytokines, therapies can be developed that
reduce cytokine binding and thereby reduce the expression of adhesion molecules that can
initiate phenotype switching of VSMCs.
Besides therapies to reduce phenotype switching and lesion formation, differential
expression of adhesion molecules might be a target for plaque imaging. Because of the
increased incidence of cardiovascular events as a result of atherosclerosis in the western
world, identification of a patient with a vulnerable plaque at high change to rupture would be
preferable. Nuclear imaging, using radio-labeled biomarkers would be an ideal method to
15
detect these vulnerable plaques. By radio-labeling adhesion molecules that are expressed by
synthetic VSMCs, an indication can be given about the stability of the plaque. Increased
expression of integrin αvβ3 was found in the neointima of rats21. The possibility to use this
integrin as an imaging target was shown by Lee and colleagues101. Enhanced uptake was
observed of radio-labeled RGD peptides that target integrin αvβ3 in ischemic hindlimbs of
mice, demonstrating the use of imaging in angiogenesis. No imaging studies have been
performed using adhesion molecules involved in VSMC migration so far, but these molecules
might be possible targets to image plaque progression.
9. Concluding remarks
The data summarized in this thesis shows the importance of cell adhesion molecules in
the physiology of VSMCs. It becomes clear that during both normal conditions as in
atherosclerosis, VSMC adhesion, migration and proliferation are complex processes requiring
several types of adhesion molecules. Many questions remain unanswered but progress has
been made over the last years in understanding the mechanism involved in cell adhesion and
VSMC behavior during the onset of atherosclerosis. Differentiation of VSMCs into the
synthetic phenotype was shown to be accompanied by a different expression pattern of
adhesion molecules, but the exact mechanism remains to be elucidated. It becomes clear that
using adhesion molecules on VSMCs as targets to reduce plaque formation could be
interesting in future clinical intervention.
16
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