The role of smooth muscle cells and vascular dendritic cells in the inflammatory

response in atherosclerosis
Irena Tanaskovi1, Tatjana Kastratovi2, Neboja Arsenijevi3, Slobodan
Arsenijevi2, Aleksandra Mladenovi-Mihailovi4, Vesna Stankovi5, Vera Todorovi6,
Vesna Lakovi7
1

Department of histology and embryology, Medical Faculty, University of Kragujevac

Department of gynecology and obstetrics, Medical Faculty, University of Kragujevac
3
Department of microbiology and immunology, Medical Faculty, University of Kragujevac
4
Department of gynecology and obstetrics, Clinical hospital centre Zvezdara, Belgrade
5
Department of pathology, Medical Faculty, University of Kragujevac
6
Institute for medical research, Belgrade
7
Institute of histology and embryology, Medical Faculty, University of Belgrade
2

Correspondence:
Irena Tanaskovic, M.D., Ph.D.
Department of Histology and Embryology Faculty of Medicine University of
Kragujevac
Email: irena.vuk@gmail.com

Introduction: Atherosclerosis is a very complex disease. A complex interaction

exists between the critical cellular elements of the atherosclerotic lesion. These cellular
elements are macrophages, lymphocytes, neutrophils, endothelial cells, vascular smooth
muscle cells, vascular dendritic cells, platelets and bone marrow circulating endothelial
stem cells.
Objectives. The purpose of this work was determination of the role of vascular
dendritic cells and the role and phenotype state of smooth muscle cells during the
pathogenesis of atherosclerotic lesion.
Materials and Methods. During the course of this study, 30 samples of
atherosclerotic aortic aneurysms have been analyzed, 20 of them excised during surgery
and 10 obtained during autopsies. Sections 5m thick were stained by DAKO
LSAB+/HRP technique to identify -smooth muscle actin--SMA, vimentin, myosin
heavy chains-MHC, desmin, CD3, CD45, CD68, S100 protein and PCNA (DAKO
specification). Sections were also stained for electron microscopy.
Results. The results of this study have shown that aortic atherosclerosis is
characterized by the presence of a huge number of CD68-immunoreactive cells with lipid
inclusions in the cytoplasm. This finding indicates the process of monocytes transition
into foam cells. The finding of vimentin-immunoreactive foam cells (which points to
their smooth muscle origin) suggest that these cells express scavenger receptors and
competitively take part with macrophages in the accumulation of lipids and creation of
foam cells. In the atherosclerotic lesion there are also a huge number of cells which are
immunoreactive to S-100 protein, which is generally characteristic of vascular dendritic
cells.
Conclusions. Foam cells originate from macrophages (express CD68) and smooth
muscle cells (express vimentin). At the earliest stage of atherosclerosis, monocytes and
macrophages represent the main precursors of foam cells. From the stage of fatty streak,
in parallel with their synthetic activity, smooth muscle cells start to accumulate lipids.
Antigen presenting dendritic cells in atherosclerotic aorta could play an important role in
immune mechanisms during atherosclerotic lesion formation.

Introduction
Atherosclerosis is a very complex disease. A complex interaction take place
among the critical cellular elements of an atherosclerotic lesion; these cellular elements
are macrophages, lymphocytes, neutrophils, endothelial cells, vascular smooth muscle
cells, vascular dendritic cells, platelets and bone marrow-derived, circulating endothelial
stem cells (1, 2).
The mechanisms of atherogenesis remain uncertain. The response-to-injury
theory is the most widely accepted. Endothelial injury causes vascular inflammation and
a fibroproliferative response ensues. The earliest pathologic lesion of atherosclerosis is
the activation of the endothelium in response to injury (3, 4, 5, 6). Circulating monocytes
infiltrate the intima of the vessel wall, and these tissue macrophages act as scavenger
cells, taking up low density lipoprotein (LDL) cholesterol and forming the characteristic
foam cell of early atherosclerosis. These activated macrophages produce numerous
factors that are injurious to the endothelium. T lymphocytes elaborate interferon-gamma,
an important cytokine that impairs vascular smooth muscle cell proliferation and collagen
synthesis. In addition, it is possible to achieve, through influence of interpheron gamma,
various cell types to express II class MHC molecules on its surface and to express antigen
to T lymphocytes.
Platelet-derived growth factor (PDGF), insulin like growth factor, transforming
growth factors alpha and beta, thrombin, and angiotensin II are potent mitogens that are
produced by activated platelets, macrophages, and dysfunctional endothelial cells; these
mitogens characterize early atherogenesis, vascular inflammation, and platelet-rich
thrombosis at sites of endothelial disruption. Furthermore, activated macrophages
produce matrix metalloproteinases that degrade collagen (3, 4).The relative deficiency of
endothelium-derived nitric oxide further potentates this proliferative stage of plaque
maturation (7).
Smooth muscle cell migration and proliferation, as well as cellular inflammation,
are complex and inter-related biological processes that contribute to atherogenesis and the
clinical manifestations of atherosclerosis (3, 4, 5).
In the wall of normal, unchanged aorta, smooth muscle cels express contractile
phenotype. They are localized in subendothelium of intima, where they are oriented
longitudinally, and in media of aorta, there they are oriented circularly (5, 6). But, during
the vascular remodeling, which is a characteristic of the atherosclerosis process, loss of
contractile characteristics of smooth muscle cells occurs, as well as their migration and
notable synthetic activity, which contributes to development of atherosclerotic plaque (712).
Vascular dendritic cells (13) have important role during the inflammatory process
in subendothelium of intima. It is assumed that vascular dendritic cells represent a type of
antigen-expressive dendritic cells which are localized in the intima of great arteries (13,
14). Vascular dendritic cells, in large numbers, are also found in atherosclerotic lesions,
while they are rarely encountered in mature form in normal intima (15-17). Their role in
the development of atherosclerotic plaque is still not completely known.
Vascular dendritic cell and smooth muscle cells represent important cell
components in the development of the atherosclerotic lesions, although their functional
significance during this process is not entirely explained. Determining their distribution

in aortic wall would contribute to better understanding of the role of these cell entities in
pathogenesis of inflammatory reaction during the development of atherosclerosis, which
would lead to enhancement of existing therapy procedures.
Objectives
The purpose of this work was determination of the role of vascular dendritic cells
and the role and phenotype state of smooth muscle cells during the pathogenesis of
atherosclerotic lesion.
Material and methods
30 samples of changed atherosclerotic segments of the thoracic aorta have been
analyzed, and they have been submitted for immunohistochemical and
electromicroscopical analysis.
20 samples of aortic wall have been used in immunohistochemical research,
obtained intraoperatively during the aortic wall reconstruction with setting of Dacron
graft, from the Second surgical clinic in Belgrade. These samples were obtained during
2007th, from both sexes, age from 42 to 68 years. Parts of the wall which appeared to
macroscopically normal were analyzed, as well as parts which were in different stages of
development of the atherosclerotic lesion.
10 samples of aortic wall have been used for electromicroscopical research,
selected during autopsies at the Institute for pathological anatomy and forensic medicine
of Faculty of medicine at University in Kragujevac. Obtained samples have been
gathered from 2006th to 2008th. Aortic segments have been taken during the first 5 hours
after death, from both sexes, died due to cardiovascular and accidental causes.
All samples were divided according to degree of development of atherosclerotic
lesion in six subgroups: initial lesion (activation of endothelium), fatty streak stage,
preatheroma, atheroma, fibroatheroma and complicated lesions (plaque rupture,
ulceration, development of parietal thrombus e.s.).
The material was collected in accordance with the ethical guidelines for consent
contained in the Helsinki Declaration and the Medical Research Councils statement on
responsibility in investigation on human subjects (British Medical Council, 1966).
All results are shown as histological photomicrographies.
For light microscopy, the specimens were dehydrated in graded ethanol (70100%), cleared in xylol and embedded in paraffin. Sections of 5m thick were cut on
Leica Reinhart Austria microtome and stained. Immunocytochemical staining was
performed on 5m sections from formaldehyde-fixed paraffin-embedded blocks, using a
Labeled Streptavidin-Biotin Method with an LSAB kit (DAKO). Sections were
deparaffinized and rehydrated. After microwave treatment of 21 minutes in citrate buffer
pH 6,0, endogenous peroxidase activity was blocked with 3% H2O2 for 15 minutes. The
sections were first incubated with the primary antibodies for 60 minutes (-smooth
muscle actin--SMA, vimentin, myosin heavy chains-MHC, desmin, CD3, CD45, CD68,
S-100 protein and PCNA) then with biotinylated link antibodies and finally with
peroxidase-labeled streptavidin. Slides were counter-stained with hematoxylin, washed in
water and mounted (DAKO specification).

Sections were also stained for electron microscopy. The primary fixative consisted
of 2,5% glutaraldehyde in 0,1 M sodium cacodylate-HCl buffer (pH 7,4) for 24h at 4C.
The specimens were postfixed for 1h at 4C in 1% osmium tetraoxide in 0,1 M
cacodylate buffer and 4,8% uranyl acetate for 24h at 4C. The samples were dehydrated
in graded ethanol (70-100%) and embedded in Epon 812. The samples were cut with a
diamond knife on an LKB Ultrotome. Ultra-thin sections were stained with 2% uranyl
acetate and alkaline lead citrate.
Results
The results of samples analyzed in this study showed that there were no visible
morphological changes in the structure of aortic wall at the stage of the initial lesion. The
individual foam cells (CD68-immunoreactive) and T lymphocytes (CD3immunoreactive) were present in the intima. Smooth muscle cells in intima and media
reacted to the contractile phenotype markers -SMA, MHC and desmin. Vascular
dendritic cells (S-100 immunoreactive) were localized directly below endothelium and
give impression that they are in contact with endothelial and smooth muscle cells. Also,
presence of a large number of S-100 immunoreactive SMCs has been noted in all parts of
vascular wall (Fig. 1).
During the fatty streak stage there was an increase in the number of foam cells
(CD68-immunoreactive) (Fig 2) and there was intense infiltration of T lymphocytes i
leukocita (CD3- and CD45-immunoreactive cells) (Fig 3). Vascular dendritic cells (S-100
immunoreactive) are localized all over the intima and they are not in contact with
endothelial cells any more, but looks like they express numerous cellular interactions
with monocyte-macrophage cell line, as well as with lymphocytes (Fig.4). This finding is
confirmed with electromicroscopical analysis (Fig. 5a, b). At this stage there was also the
presence of -SMA-immunoreactive, vimentin-immunoreactive and S-100immunoreactive SMCs (Fig. 6 a, b). Also at this stage, the adventitial myofibroblasts
started to show the characteristics of contractile smooth muscle cells (expression of MHC
and desmin) (Fig. 7).
The preatheroma stage showed the presence of intimal smooth muscle cells with
expression of vimentin and -SMA and a lack of expression of desmin. Visible smooth
muscle cells were longitudinally oriented, PCNA-immunoreactive and without noticeable
lipid inclusions. It is determined, with electronic-microscopic analysis, that there is a
presence of SMCs synthetic phenotype in intima, with well developed organelles of
synthetic way (Fig. 8). Also, a number of SMCs in apoptosis is noted (Fig.9). A large
number of vascular dendritic cells is present (S-100 immunoreactive), especially on the
border of intima and media, which make contacts with large number of lymphocytes
present in the plaque. Entire atherosclerotic plaque at the stage of preatheroma is marked
by notable proliferatory activity (large number of PCNA immunoreactive cells). On the
opposite part of the wall, a large number of PCNA- and S-100 positive cells was noted in
the inner media while one layer of MHC- and desmin-positive cells was noted in the
outer media.
At the atheroma stage, the intima showed focal thickening due to the
subendothelial lipid core, which consisted of cholesterol crystals, protheoglicans and
collagen fibres. Smooth muscle cells present in this focal thickening showed

immunoreactivity to -SMA and vimentin, and together with a large number of foam
cells, formed the cell population at the intimal thickening. With electronic-microscopical
analysis, the presence of a large number of SMCs of synthetic phenotype in necrosis has
been discovered in the plaque (Fig. 10). Foam cells of variant origin were noticed; some
of the foam cells developed from monocyte-macrophages lineage (CD68immunoreactive) and the others were of smooth muscle cell origin (vimentin and S-100
immunoreactive). A smaller number (compared to the previous stage) of S-100
immunoreactive vascular dendritic cells has been noted.
At the fibroatheroma stage, all samples had focal intimal thickening with fibrosis
in the lipid core and the accumulation of collagen fibers with extreme hypocellularity.
Rare smooth muscle cells (SMCs) showed immunoreactivity on -SMA and vimentin,
but not on desmin or MHC. Vascular dendritic cells (S-100 immunoreactive) have been
observed only in the part of the aortic wall on the opposite side of atherosclerotic plaque,
in the thin layer on the border of the intima and media. The media is characterized by the
straightening, fragmentation, fenestration and reduplication of elastic lamellae and
synthetic phenotype of smooth muscle cells. The decay of the media occurs from the
internal to the external part of the media. The results of immunohistochemical staining to
CD3 and CD45 antigens have shown a large number of T lymphocyte and leukocyte
infiltration in all parts of the vascular wall.
Discussion
In all analyzed samples of the atherosclerotically changed parts of the aortic wall
the modified SMCs of the synthetic phenotype form the dominant cell population. It is
shown, with electronic-microscopical research, that the development of the synthetic
phenotype is followed by a reduction of myofilaments. With immunohistochemical
research, it is determined that SMCs of the synthetic phenotype expressed -SMA and
vimentin. According to the literature, the SMCs located in the tunica intima in
unmodified aorta are different from those found in atherosclerotic intima (2, 3). Even
though one type of smooth muscle cell accumulates in the intima in the early phases of
atherosclerotic development in the form of a resident longitudinal muscular layer, the
other type accumulates in the developed atheroma and, as mentioned, is probably derived
from cells migrating form the media to the intima, as well as from the existing intimal
layer. (3). The migration of the cells from the media occurs under the influence of a
strong hemoattractant, PDGF, which is secreted by activated macrophages, and is
intensively expressed in the atherosclerosis (4). SMCs respond to the action of PDGF by
proliferation in the atherosclerotic vessel intima. The literature also indicates that media
SMCs migrate through fenestras in the internal elastic lamina and, at the same time,
intracellular and extracellular lipid deposits are accumulated, thus creating fatty streaks
(2, 3, 4).
In the initial stages of atherosclerosis, SMCs express -SMA, MHC and desmin,
but after the preatheroma stage, they expressed only -SMA and vimentin. At the fatty
streak stage, in the media of the samples analyzed in the present study, there was a
substantial decrease of desmin expression with a parallel increase in the number of
vimentin-immunoreactive cells in the inner media layer. We also found a decrease in the
number of cells that expressed MHC. These results correspond to the literature (6-12).

The loss of desmin expression with concurrent vimentin expression is the first sign in the
process of the switching from the contractile to the synthetic phenotype (6, 9, 11, 12).
According to the existing literature, vimentin is an intermediary filament that can be
found in SMCs with contractile phenotype as well, but it is coexpressed there with
desmin. With the loss of the contractile phenotype and characteristic desmin expression
(with the switching of cells to the synthetic phenotype), the expression of vimentin
filaments can be noticed (18-21).
The results obtained from the analysis of all samples at the fatty streak stage show
intensive T lymphocyte infiltration, which corresponds to the studies of other authors
stating that this stage of lesion development is characterized by the presence of this type
of leukocyte in atherosclerotic plaque (12).
At the preatheroma stage, the results of immunohistochemical methods in the part
of the wall opposite to the atherosclerotic lesion show that the process of the losing of
contractile characteristics has spread to this side as well, but media on this side is more
preserved than the one directly below the atherosclerotic lesion, and that the process of
the losing of contractile characteristics is slower. The results of immunohistochemical
methods indicate that this process occurs from the internal to the external part of the
media. The observed immunoreactivity of the internal part of the media to -SMA and
vimentin points to, according to the available literature, the high degree of the losing of
contractile characteristics of SMCs (6, 8 and 11). More differentiated cells in the external
part of the media show immunoreactivity to -SMA and MHC, while the expression of
vimentin and desmin does not take place. Therefore, we can assume that they are in the
intermediary stage of differentiation. Literature shows that myosin expression is
connected to the phases of SMCs development but that it is not the sufficient evidence of
the differentiated contractile phenotype (6, 8). The results of this research have indicated
the complete differentiation of the contractile phenotype of (desmin-immunoreactive)
cells only in the thin layer in the external part of the media close to adventitia. The
coexpression of the vimentin and desmin filaments is shifted here towards the
contractile phenotype (6, 7). Even though the process of the losing of contractile
characteristics of SMCs, accompanied by the straightening of lamellae, covers only the
internal part of the media, while the external part is more preserved. All mentioned results
show that the process of atherosclerosis, although predominantly occurring in area where
the atherosclerotic plaque is formed, also causes changes on the opposite part of the wall,
which matches literary data regarding remodeling of the entire vascular wall, in the case
of abnormal blood flow through changed vascular wall (7, 8).
In stages of lesion after preatheroma stage, by the analysis of the SMCs
population in the intimal subendothelium, we observe substantial hypocellularity,
especially in the area of the atherosclerotic plaque, which corresponds to the available
literature data (4, 5 and 7). The small number of SMCs in the area of the focal thickening
and in the parts of the opposite wall as well, show the synthetic phenotype described in
the studies of other authors (4-8).
The results of the present study showed that the fatty streak stage is characterized
by the presence of a number of CD68-immunoreactive cells with a large number of lipid
inclusions in the cytoplasm. This finding shows the transition process of monocytes into
foam cells, on the basis of which we conclude that circulating monocytes in the initial
phases of lesions are the main precursors to foam cells. This result concurs with the

results of other authors (7, 12 and 22). Except CD68-immunoreactive foam cells at the
fatty streak stage, we noticed a substantial number of SMCs that showed
immunoreactivity to -SMA and vimentin with lipid inclusions in the cytoplasm. In some
of the SMCs the content of the lipid inclusions was so large that they looked similar to
foam cells. These cells can be spindle-shaped or star-shaped and they have short thick
extensions. The number of lipid inclusions in the cells varies, and therefore, they look as
if they are at different stages of phenotype transformations to foam cells. It is possible
that intimal vimentin-immunoreactive foam cells originate from the existing
longitudinally oriented contractile cells, which are primarily found in the intima. In the
early stages of atherosclerosis, in the intimal subendothelium, there are still a small
number of differentiated longitudinally oriented cells of the contractile phenotype. This
layer of cells primarily shows immunoreactivity to the markers specific for the
differentiation of SMCs of the contractile phenotype, namely, -SMA, MHC and desmin.
In the earliest phase, at the stage of the endothelium activation, these cells are
immunoreactive to desmin; however, at the fatty streak stage, they lose desmin
expression. It is also possible that vimentin- and -SMA-immunoreactive foam cells
primarily originate from the circularly oriented medial SMCs that migrate to the intima.
This hypothesis corresponds to the literature on the pathogenesis of atherosclerotic
plaque (21). Other authors have also indicated that SMCs with synthetic phenotype
partially originate from the original intimal muscular layer; moreover, it has been
definitively shown that these cells also originate from the media (22). The finding of
vimentin-immunoreactive foam cells (which points to their smooth muscle origin)
corresponds to the above described results of other authors, according to which a number
of SMCs express scavenger receptors and play a role, competitively with macrophages, in
the accumulation of lipids and creation of foam cells (21, 22, 23). The fact that only some
SMCs express scavenger receptors is possibly related to the different embryonic origins
of SMCs in different parts of the circulatory system. SMCs of large arteries in the upper
parts of the body can have a neuroectodermal origin (S-100-immunoreactive); in the
arteries of the lower parts of the body, they mainly originate from the mesoderm; and in
coronary arteries, they originate from a proepicardial organ (22, 24). Due to their
neuroectodermal origin, SMCs express S-100 protein (25, 26). It is possible that, exactly
due to this fact, SMCs can accumulate lipids in the aortic wall and modify themselves in
the foam cells.
Namely, in an atherosclerotic lesion there are also foam cells that are
immunoreactive to S-100 protein, which is generally characteristic of vascular dendritic
cells. According to the literature, on the margins of an atherosclerotic plaque, there are
vascular dendritic cells that express S-100 protein, which will be further discussed later.
However, according to the same data, vascular dendritic cells are located in the intima of
large arteries and they do not accumulate lipids (26). The observed S-100-imunoreactive
foam cells in the samples of the present study are most likely modified smooth muscle
cells. On the basis of the available literature, it has been established that cardiomyocytes
and some SMCs can express S-100 protein (26). It has been assumed that the SMCs of
the aorta, due to their neuroectodermal origin can express some types of S-100 protein
(26).
Results of this study have really shown that, in the fatty streak stage, in all parts of
the aortic wall there is a presence of the S-100 immunoreactive SMCs. This finding could

correspond to the literature data, according to which one of nineteen sub-types of this
multigene, the S-100A6, is expressed in fibroblasts, vascular SMCs and cardiomyocites
(26).
As our previous studies have shown (12) it is obvious that there is a connection
between the accumulation of lipids and the expression of S-100 protein in the SMCs of
the tested samples. One of the subtypes of the S-100 protein is the Ca++ binding protein
S-100A8, which is a leukocyte hemoattractant, whose expression is induced by
interferon-gamma (INF-) and tumor necrosis factor (TNF) in macrophages, and whose
expression in macrophages and endothelial cells is partially decreased by interleukin-4
(IL-4) and interleukin-13 (IL-13) (27). It has also been shown that the heterocomplex
comprised of S-100A8 and S-100A9 Ca++-binding proteins shows a large affinity to
binding unsaturated lipid acids (28). It is also known that S-100 protein is expressed in
cells that are in the process of differentiation, protein phosphorylation and what is
particularly important here, proliferation events that are Ca++ -mediated (26, 29, 30).
Bearing in mind that proliferating cells increasingly express S-100 protein (26, 29), that
their proliferation is induced by the accumulation of lipids (28) and that SMCs can
express scavenger receptors (30), the finding of the S-100-immunoreactive foam cells in
the samples of the present study becomes clearer. Such result suggests that the origin of
the SMCs analyzed in this study represents their predetermined factor for accumulation
of lipids, which leads to assumption that it could be possible to research for new therapies
and procedures.
Nevertheless, the role of vascular myofribroblasts can not be left out, which due to
migration from adventitia in the media and expression of the markers of the contractile
phenotype, are hard to differentiate from SMCs. According to the literature, vascular
myofibroblasts can express S-100 protein as well. The proliferation of the myofibroblast
is the phenomenon of the early vascular response to the conditions of increased
pressure (31, 32). According to the same data, the increased expression of the
Transformating Growth Factor (TGF) in the adventitia (and other layers of the wall as
well) is the signal for differentiation of fibroblasts which start to express -smooth
muscle actin and then to migrate. Once activated fibroblasts express not only -smooth
muscle actin, but other markers of muscle differentiation as well, which makes it hard to
differentiate of smooth muscle media cells (33). At the same time they can express CD34
and S-100 proteins as well (34). Their recognition is based on the primary localization in
the adventitia. In parallel with this, arterial media shows a considerably lower level of
proliferation with high synthetic activity of resident SMCs (35).
In all samples analyzed in this study, except S-100 immunoreactive SMCs and
foam cells originating from them, the presence of a large number of S-100
immunoreactive vascular dendritic cells (VDCs) is also noted, which is confirmed with
the electronic-microscopical analysis. According to literary data from the other authors,
dendritic cells consist of a heterogenic cell population, regarding their localization
(lymphatic organs, but also cardiovascular system, Langerhans cells of skin, lungs,
kidneys, lymph, blood), and also their degree of maturation and ability to regenerate.
Besides having the ability to catch antigens, to internalize them, to decompose them
down to fragments and to, in a complex with class II MHC molecules, present them to
CD4+ T lymphocytes, dendritic cells show additional signals which stimulate
proliferation and differentiation of the lymphocytes (13, 14).

The result of this study which indicates the localization of S-100 immunoreactive
cells in the intima and in the internal part of the media and on the border with intima,
could correspond to the described usual distribution of VDCs (13, 14). According to
existing literature, the antigen presenting VDCs are described in large arteries intima and
to a less degree in normal intima, while they can be found in the incomparably bigger
number in the intima of atherosclerotic lesions (13-17).
Vascular dendritic cells in the samples analyzed in this study have shown typical
ultrastructural properties of all dendritic cells. They are characterized by the presence of
long, branched extensions which protrude from the body in different direction. In the cell
body and its extensions, presence of granular and non-granular material of medium
electronic density is noted, especially expressed in the extensions which are the furthest
from the cell body. Nucleus has irregular form, with notable nucleolus and peripheral
heterochromatin. Cytoplasm shows the presence of mitochondria and well developed
smooth endoplasmic reticulum. The number of organelles varies depending on functional
state of the VDCs and their maturity. All described results match with literature (15 17).
In the initial lesion stages, VDCs were localizes directly below endothelium where
they are in contact with the endothelial cells and the SMCs. Starting from fatty streak
stage, VDCs are localized all over the intima, and also in the internal parts of the media,
and are no longer in contact with endothelial cells but make numerous cellular interaction
with monocyte-macrophage cellular line and also with the lymphocytes. Such results of
the VDCs distribution during the development of the atherosclerotic lesion match the
results of other authors (13, 14).
Vascular dendritic cells analyzed in this study express S-100 protein which is,
according to literary data, their usual property (15 17). According to the same data,
VDCs also express HLA-DR, intercellular adhesion molecule (ICAN-1) and vascular cell
adhesion molecule-1 (VCAM-1) (13, 14). Results of this study have shown that, starting
from the fatty streak stage, VDCs are in close contact with the lymphocytes in the intimal
infiltrate which, according to the literary data, points that VDCs take part in the
regulation of the immune response (36 39). Still, functions of the VDCs during the
atherogenesis are not yet clear (14). Bearing in mind that, according to their
morphological properties, VDCs are a part of the dendritic cell family, it can be assumed
that their function is similar to that of the other dendritic cells, namely, that they partake u
antigen processing and presentation (14, 15).
The results of this study have shown that, in the initial lesion stages,
atherosclerotic plaque consists of the foam cells, deriving from the macrophages and
activated T lymphocytes. Antigen-specific activation of T lymphocytes is conditioned
with the interaction of T cell receptor (TCR) with the antigen which is processed and
presented as a part of the II class MHC, and so it is possible that this function of
presentation of the antigen is carried out by the VDCs. Since, as previously mentioned,
according to the data of other authors (14, 40) is already known that VDCs express
ICAM-1 and VCAM-1 in the atherosclerotic lesions, it is possible the VDCs interact with
T lymphocytes through VCAM-1VLA-4, which is, according ot literature, crucial
moment in the activation of T lymphocytes (41 - 43).
It is known that one of the most significant changes in the atherosclerosis is the
increased expression of the adhesion molecules on the endothelial cells, both in initial
and later stages of this condition (44, 45). While endothelial cells express two groups of

10

the leukocytal adhesion molecules (members of the immunoglobulin superfamily and

selektine (46)), it is proven for VDCs that they express only VCAM-1 and ICAM-1
which are the members of the immunoglobulin superfamily (47). Results of the other
authors studies have also shown that in the human endothelium above the atherosclerotic
plaque, on the sited of macrophage infiltration, there is an increase in the expression of
the ICAM-1 (48, 49).
According to literature, increased expression of the VCAM-1 is characteristically
linked with the initial stages of the atherosclerosis. VCAM-1 interacts with the specific
integrine VLA-4 (short from Very Late Antigen 4), which expresses only at monocytes
and T lymphocytes; causing that in the initial stages these leucocytes predominantly
adhere to the endothelium. Since in the initial stages of the lesion VDCs are situated
directly below endothelium, as shown in the results of this study, it is certain that they
contribute, through their adhesion molecules, to the adhesion of the leucocytes in the
endothelium, consequently to the development of the atherosclerotic lesion. Such
findings suggest that the modulation and control of adhesion molecules effect in the
initial stages of the development of the atherosclerotic lesion might contribute to the
upgrading of the therapies and procedures in treatment of the atherosclerosis.
Conclusions
Smooth muscle cells in the aortic wall at the early stages of atherosclerosis,
express -SMA, MHC and desmin, and from the preatheroma stage onward, they express
only -SMA and vimentin. The development of synthetic phenotype is followed by a
reduction in desmin filaments and an increase in the expression of vimentin filaments.
The present study also observed that the development of the synthetic phenotype spreads
from the intimal to the medial smooth muscle cells. Development of the synthetic
phenotypes of the SMCs of the tunica media (based on the loss of desmin expression and
the appearance of vimentin expression) begins at the fatty streak stage. Phenotypical
modification of the smooth muscle cells (from the synthetic into the contractile
phenotype) and their migration from the media to the intima represents one of the key
moments in the pathogenesis of the atherosclerotic plaque. Synthetically active smooth
muscle cells synthesize large amounts of extracellular matrix and contribute to creation of
the callus in the subendothelium if the intima. Beside that, a number of smooth muscle
cells partake, competitively with the macrophages, in the accumulation of lipids and
forming of the foam cells.
Foam cells that originate from monocyte-macrophage lineage express CD68, and
those that originate from SMCs express vimentin and S-100 protein. At the earliest stage
of atherosclerosis, monocytes and macrophages represent the main precursors of foam
cells. At the fatty streak stage, parallel with the loss of the contractile characteristics, a
number of SMCs of synthetic phenotype start to accumulate lipids, which contributes to
the further development of the plaque and the inflammatory reaction in the
subendothelium of the intima
Vascular dendritic cells in the initial stages of lesion were localized directly
bellow endothelium, where they are in contact with the endothelial cells and the smooth
muscle cells, in that way contributing to increased expression of the adhesion molecules
and consequential adhesion of the monocytes and T lymphocytes on the endothelium.

11

Starting from the fatty streak stage, vascular dendritic cells are localized everywhere in
the intima, and no longer in contact with endothelial cells, but make numerous cellular
interactions with monocyte-macrophage cell line, and also with lymphocytes. Such
results suggest that the vascular dendritic cells in the aortic wall have the function of the
antigen-presenting cells in the inflammatory reaction during the development of the
atherosclerotic lesion.
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Legend
Fig. 1. Initial stage of the atherosclerotic aortic lesion. Vascular dendritic cells
directly below endothelium and smooth muscle cells in the media of the aorta.
(Immunohistochemical staining on S-100 protein, original magnification x 128)
Fig. 2. Atherosclerosis of the aorta, fatty streak stage. Foam cells in the
subendothelium of the intima.
(Immunohistochemical staining on CD68, original magnification x 256)
Fig. 3. Atherosclerosis of the aorta, fatty streak stage. Leukocytal infiltration in the
plaque.
(Immunohistochemical staining on CD45, original magnification x 256)
Fig. 4. Atherosclerosis of the aorta, fatty streak stage. Vascular dendritic cells in
the intima of the aorta in contact with the lymphocytes.
(Immunohistochemical staining on S-100 protein, original magnification x 256)
Fig. 5 b. Atherosclerosis of the aorta, fatty streak stage. Lymphocyte in the vicinity
of the extension of the vascular dendritic cell
(TEM, original magnification x 54000)
Fig. 6 a and b. Atherosclerosis of the aorta, fatty streak stage. Smooth muscle cells
in the intima and media of the aorta.
(a) Immunohistochemical staining on -SMA, original magnification x 64)
(b) Immunohistochemical staining on vimentin, original magnification x 128)
Fig. 7. Atherosclerosis of the aorta, fatty streak stage. Miofibroblasts in the
adventitia.
(Immunohistochemical staining on MHC, original magnification x 64)
Fig. 8. Atherosclerosis of the aorta, preatheroma stage. Smooth muscle cell of the
synthetic phenotype, with euhromatic nucleus, expressed nucleolus and granulated
endoplasmic reticulum.
(TEM, original magnification x 54000)
Fig. 9. Atherosclerosis of the aorta, preatheroma stage. Smooth muscle cell in
apoptosis, with condensed heterochromatin adjacent to internal membrane of the nucleus,
preserved cellular membrane and apoptotic bodies forming.
(TEM, original magnification x 54000)

15

Fig. 10. Atherosclerosis of the aorta, atheroma stage. Smooth muscle cell in the
necrosis, with swollen mitochondria and damages cell membrane.
(TEM, original magnification x 54000)
Fig. 5a. Atherosclerosis of the aorta, fatty streak stage. Vascular dendritic cells in
atherosclerotic aortic intima
(immunohistochemical staining on S-100 protein, original magnification x 256)

16