Abstract
Objectives
To determine the phenotypical state of smooth muscle cells during the pathogenesis of an atherosclerotic lesion, and to determine the morphological state of the endothelium and the origin of foam cells.Methods
Twenty-one samples of atherosclerotically changed right coronary arteries, which were divided into six subgroups based on the stage of atherosclerosis, were analyzed. The tissues were fixed in formalin and embedded in paraffin. Sections of 5 mum thickness were stained immunocytochemically using a labelled streptavidin-biotin/horse radish peroxidase kit (Dako, Denmark) for the identification of vimentin, alpha-smooth muscle actin, myosin heavy chains, desmin, S-100 protein, CD3, CD31, CD34, CD45, CD68 and proliferating cell nuclear antigen protein.Results
The present study showed that there is first functional and then morphological damage of the endothelium in the late stages of atherosclerosis. The preatheroma stage revealed the presence of intimal changes of smooth muscle cells, with expression of vimentin and alpha-smooth muscle actin and a lack of expression of desmin, which led to a switch to a synthetic phenotype. The described changes progressed into the later stages of atherosclerosis. Along with these changes, a large number of foam cells of variant origin were observed; some of the foam cells developed from monocyte-macrophage lineage (CD68-immunoreactive) and others originated from smooth muscle cells (vimentin- and S-100-immunoreactive). The late stages of atherosclerosis development, such as the atheroma stage, include intimal changes with the formation of a lipid core (S-100-immunoreactive cells and cell necrosis), while fibrosis in the lipid core and the accumulation of collagen fibres with extreme hypocellularity are characteristics of the fibroatheroma stage.Free full text
The origin and differentiation potential of smooth muscle cells in coronary atherosclerosis
Abstract
OBJECTIVES
To determine the phenotypical state of smooth muscle cells during the pathogenesis of an atherosclerotic lesion, and to determine the morphological state of the endothelium and the origin of foam cells.
METHODS
Twenty-one samples of atherosclerotically changed right coronary arteries, which were divided into six subgroups based on the stage of atherosclerosis, were analyzed. The tissues were fixed in formalin and embedded in paraffin. Sections of 5 μm thickness were stained immunocytochemically using a labelled streptavidin-biotin/horse radish peroxidase kit (Dako, Denmark) for the identification of vimentin, alpha-smooth muscle actin, myosin heavy chains, desmin, S-100 protein, CD3, CD31, CD34, CD45, CD68 and proliferating cell nuclear antigen protein.
RESULTS
The present study showed that there is first functional and then morphological damage of the endothelium in the late stages of atherosclerosis. The preatheroma stage revealed the presence of intimal changes of smooth muscle cells, with expression of vimentin and alpha-smooth muscle actin and a lack of expression of desmin, which led to a switch to a synthetic phenotype. The described changes progressed into the later stages of atherosclerosis. Along with these changes, a large number of foam cells of variant origin were observed; some of the foam cells developed from monocyte-macrophage lineage (CD68-immunoreactive) and others originated from smooth muscle cells (vimentin- and S-100-immunoreactive). The late stages of atherosclerosis development, such as the atheroma stage, include intimal changes with the formation of a lipid core (S-100-immunoreactive cells and cell necrosis), while fibrosis in the lipid core and the accumulation of collagen fibres with extreme hypocellularity are characteristics of the fibroatheroma stage.
Atherosclerosis is predominantly a disease of large- and medium-sized muscular arteries, and is characterized by endothelial dysfunction, vascular inflammation and the buildup of lipids, cholesterol, calcium and cellular debris within the intima of the vessel wall (1,2).
This buildup results in plaque formation, vascular remodelling, acute and chronic luminal obstruction, abnormalities of blood flow, and diminished oxygen supply to target organs. Atherosclerosis is a very complex disease. Complex interactions take place among the critical cellular elements of an atherosclerotic lesion; these cellular elements are macrophages, lymphocytes, neutrophils, endothelial cells, vascular smooth muscle cells, platelets and bone marrow-derived, circulating endothelial stem cells (2–4). In the processes of atherosclerosis, the blood vessel intima is thickened and neointima is created. Smooth muscle cells play the key role in this process. Smooth muscle cells originate from various sources, including media smooth muscle cells, endothelial cells, adventitial fibroblasts, circulating stem cells and neural crest cells. All of these cells in the intima are subject to phenotype modulation, that is, phenotype modification from the contractile to the synthetic phenotype as a response to microenvironmental changes.
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. Circulating monocytes infiltrate the intima of the vessel wall, and these tissue macrophages act as scavenger cells, taking up low density lipoprotein cholesterol and forming the foam cells characteristic of early atherosclerosis. These activated macrophages produce numerous factors that are injurious to the endothelium. 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 (3–5).
The earliest pathological lesion of atherosclerosis is the activation of the endothelium in response to injury (3,6). The second lesion, the fatty streak, is the result of focal accumulation of serum lipoproteins within the intima of the vessel wall; at this lesion, microscopy reveals lipid-laden macrophages, T lymphocytes and smooth muscle cells in varying proportions. The fatty streak may progress to form a fibrous plaque, which is the result of progressive lipid accumulation, and the migration and proliferation of smooth muscle cells. 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. A relative deficiency in endothelium-derived nitric oxide further potentiates this proliferative stage of plaque maturation (7).
Smooth muscle cells are responsible for the deposition of the extracellular connective tissue matrix and form a fibrous cap that overlies a core of lipid-laden foam cells, extracellular lipids and necrotic cellular debris. Growth of the fibrous plaque results in vascular remodelling, progressive luminal narrowing, blood flow abnormalities, and compromised oxygen supply to the target organ (5).
Denudation of the overlying endothelium or rupture of the protective fibrous cap may result in exposure of the thrombogenic contents of the plaque core to the circulating blood. This exposure constitutes an advanced or complicated lesion. The plaque rupture occurs due to weakening of the fibrous cap. Inflammatory cells localize to the shoulder region of the vulnerable plaque. T lymphocytes produce interferon-gamma, an important cytokine that impairs vascular smooth muscle cell proliferation and collagen synthesis. Furthermore, activated macrophages produce matrix metalloproteinases that degrade collagen (3,4). These mechanisms explain the predisposition to plaque rupture and highlight the role of inflammation in the genesis of complications of the fibrous, atheromatous plaque. A plaque rupture may result in thrombus formation, partial or complete occlusion of the blood vessel, or progression of the atherosclerotic lesion due to organization of the thrombus and incorporation within the plaque (2,4).
Due to the fact that smooth muscle cells and other cells are cellularly heterogeneous, research into cytohistological neointima organization is crucial to determine the origin and progression of atherosclerotic changes.
OBJECTIVES
The purpose of the present study was to determine the phenotypical state of smooth muscle cells during the pathogenesis of atherosclerotic lesions, and to determine the morphological state of the endothelium and the origin of foam cells.
METHODS
Twenty-one samples of atherosclerotically changed right coronary arteries were obtained by the obduction of persons who died of either nonvascular or vascular causes. The samples were analyzed after being separated into the following six subgroups: early (initial) stage (I), fatty streak (II), preatheroma (III), atheroma (IV), fibroatheroma (V) and complicated lesions (VI) (8). The specimens were dehydrated in graded ethanol (70% to 100%), cleared in xylol and embedded in paraffin. Sections of 5 μm thickness were cut on a Leica SM 2000R microtome (Leica Microsystems, Austria) and stained with orcein and Alcian blue/periodic-acid Schiff at pH 1.0 and pH 2.5. Immunocytochemical staining was performed on 5 μm sections from formaldehyde-fixed paraffin-embedded blocks using a labelled streptavidin-biotin method (Dako, Denmark). Sections were deparaffinized and rehydrated. After 21 min of microwave treatment in citrate buffer pH 6.0, endogenous peroxidase activity was blocked with 3% H2O2 for 15 min. Immunocytochemical staining was performed to identify vimentin, alpha-smooth muscle actin (α-SMA), myosin heavy chains (MHC), desmin, S-100 protein, CD3, CD31, CD34, CD45, CD68 and proliferating cell nuclear antigen protein.
The sections were incubated first with primary antibody for 60 min, then with biotinylated link antibody and finally with peroxidase-labelled streptavidin. Slides were counterstained with hematoxylin, washed in water and mounted.
RESULTS
Examination of morphological lesions of coronary atherosclerosis showed that at the stage of the initial lesion, there were no visible morphological changes in the structure of the coronary artery wall; moreover, endothelial continuity was preserved, and individual foam cells (CD68-immunoreactive) and T lymphocytes (CD3-immunoreactive) were present in the intima (Figure 1). Smooth muscle cells in the intima (longitudinally oriented) and media (circularly oriented) reacted to the contractile phenotype markers α-SMA, MHC and desmin (Figure 2).
Coronary atherosclerosis – the stage of the initial lesion. There are no visible morphological changes in the structure of the coronary artery wall (immunohistochemical staining of CD45, original magnification ×256)
Coronary atherosclerosis – the stage of the initial lesion. Smooth muscle cells in the intima (longitudinally oriented) and media (circularly oriented) react to the contractile phenotype markers: alpha-smooth muscle actin, myosin heavy chains and desmin (immunohistochemical staining of myosin heavy chains, original magnification ×16)
During the fatty streak stage, there was an increase in the number of foam cells (CD68-immunoreactive) (Figure 3), and there was intense infiltration of T lymphocytes (CD3-immunoreactive cells) (Figure 4). At this stage, there was also the presence of smooth muscle cells of the synthetic phenotype (vimentin-immunoreactive) and S-100-immunoreactive cells (from the neural crest) – but they were not yet foam cells. Also at this stage, the adventitial myofibroblasts started to show the characteristics of contractile smooth muscle cells (expression of MHC and desmin) (Figure 5). While these changes in the smooth muscle cells developed, the endothelium was still perserved and showed no significant changes.
Coronary atherosclerosis – the fatty streak stage. An increase in the number of foam cells (immunohistochemical staining of CD68, original magnification ×256)
Coronary atherosclerosis – the fatty streak stage. The intense infiltration of T lymphocytes (immunohistochemical staining of CD3, original magnification ×128)
Coronary atherosclerosis – the fatty streak stage. The adventitial myofibroblasts start to show the characteristics of contractile smooth muscle cells. Medial smooth muscle cells do not show signs of the contractile phenotype, with a lack of expression of myosin heavy chains (immunohistochemical staining of myosin heavy chains, original magnification ×32)
The preatheroma stage showed the presence of intimal dedifferentiation of smooth muscle cells, with the expression of vimentin and α-SMA, and a lack of expression of desmin. Visible smooth muscle cells were longitudinally oriented, proliferating cell nuclear antigen protein-immunoreactive and without noticable lipid inclusions. Endothelial continuity was perserved, but the cells showed the expression of CD31 and CD34 on their surface.
At the atheroma stage, the endothelium was discontinued, and CD31- and CD34-immunoreactive cells were present but in fewer numbers than in the previous stage. The intima showed focal thickening due to the subendothelial lipid core, which consisted of cholesterol crystals, proteoglycans and collagen fibres. Smooth muscle cells present in this focal thickening showed immunoreactivity to α-SMA and vimentin, and together with a large number of foam cells, formed the cell population at the intimal thickening. Foam cells of variant origin were noticed; some of the foam cells developed from monocyte-macrophages lineage (CD68-immunoreactive) and the others were of smooth muscle cell origin (vimentin- and S-100-immunoreactive) (Figures 6A and 6B).
A Foam cells of monocyte-macrophage lineage (immunohistochemical staining of CD68, original magnification ×256). B Foam cells of smooth muscle cell origin (immunohistochemical staining of alpha-smooth muscle actin, original magnification ×32)
Fibrosis in the lipid core and the accumulation of collagen fibres with extreme hypocellularity, but without any other changes in the structure of the vascular wall (other than those mentioned in the previous stage), are characteristics of the fibroatheroma stage (Figure 7).
Hypocellularity at the fibroatheroma stage (immunohistochemical staining of alpha-smooth muscle actin, original magnification ×64)
DISCUSSION
The results of testing the phenotypical status of the smooth muscle cells of atherosclerotic lesions showed that the modified smooth muscle cells of the synthetic phenotype form the dominant cell population. The development of the synthetic phenotype was followed by a reduction of myofilaments. Smooth muscle cells of the synthetic phenotype expressed α-SMA and vimentin, as shown by immunohistochemical stainings.
According to the literature, the smooth muscle cells located in the tunica media in unmodified coronary arteries are different from those found in the 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 longitudinal muscular layer, the other type accumulates in the developed atheroma and, as mentioned, is probably derived from cells migrating from the media to the intima, as well as from the existing intimal layer (3). The migration of cells from the media occurs under the influence of the strong hemoattractant PDGF, which is secreted by activated macrophages and is intensively expressed in atherosclerosis (4). Smooth muscle cells respond to the action of PDGF by proliferation in the atherosclerotic vessel intima. The literature also indicates that dedifferentiated media cells migrate through fenestras in the internal elastic lamina and, at the same time, intracellular and extracellular lipid deposits are accumulated, thus creating fatty streaks (2–4).
In the media of the samples analyzed in the present study, at the fatty streak stage, there was a substantial decrease in 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. In the initial stages of atherosclerosis, smooth muscle cells express α-SMA, MHC and desmin, but after the preatheroma stage, they expressed only α-SMA and vimentin. These results correspond to the literature (9). The loss of desmin expression with concurrent vimentin expression is the first sign in the process of switching from the contractile to the synthetic phenotype (10). According to the existing literature, vimentin is an intermediary filament that can be found in differentiated smooth muscle cells as well, but it is coexpressed there with desmin. With the loss of the contractile phenotype and characteristic desmin expression (ie, with the switching of cells to the synthetic phenotype), the expression of vimentin filaments can be noticed (10–12).
In the samples analyzed in the present study, we observed that dedifferentiation spread to intimal and medial smooth muscle cells. By studying the switching process of media cells, we conclude that this process starts in the inner and spreads to the outer media. At the preatheroma stage, we observed the switching process of smooth muscle cells from the contractile to the synthetic phenotype, starting from the inner media layers.
Foam cells
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 (13).
Except CD68-immunoreactive foam cells at the fatty streak stage, we noticed a substantial number of smooth muscle cells that showed immunoreactivity to α-SMA and vimentin, and with lipid inclusions in the cytoplasm. In some of the smooth muscle cells, 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. The described dedifferentiated, longitudinally oriented smooth muscle cells and CD68-immunoreactive foam cells at the fatty streak stage are based in the luminary zone of the neointima.
It is possible that intimal, vimentin-immunoreactive foam cells originate from the dedifferentiation of the existing longitudinally oriented contractile cells, which are primarily found in the intima (in the layer below, directly above the internal elastic membrane). 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 (located directly above the internal elastic membrane). This layer of cells primarily shows immunoreactivity to the markers specific for the differentiation of smooth muscle cells of the contractile phenotype, namely, α-SMA, MHC and desmin. In the earliest phase, at the stage of 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 α-SMA-immunoreactive foam cells primarily originate from the circularly oriented, dedifferentiated medial cells that migrate to the intima. This hypothesis corresponds to the literature on the pathogenesis of atherosclerotic plaque (14). Other authors have also indicated that dedifferentiated muscle cells partially originate from the original intimal muscular layer; moreover, it has been definitively shown that these cells also originate from the media (15).
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 smooth muscle cells express scavenger receptors and play a role, competitively with macrophages, in the accumulation of lipids and the creation of foam cells (16). The fact that only some smooth muscle cells express scavenger receptors is possibly related to the different embryonic origins of smooth muscle cells in different parts of the circulatory system. Smooth muscle cells of large arteries in the upper parts of the body can have a neuroectodermal origin; in the arteries of the lower parts of the body, they mainly originate from the mesoderm; and in coronary arteries, they originate from the proepicardial organ (15). Others have hypothesized that the mesenchymal origin of coronary smooth muscle cells, that is, their differentiation from the proepicardial organ, is the predisposing factor for the accumulation of lipids (17,18).
Other studies have also shown that some foam cells originate from smooth muscle cells, probably due to the fact that smooth muscle cells can also express scavenger receptors if they are properly activated (14,15).
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. However, according to the same data, vascular dendritic cells are located in the intima of large arteries, and they do not accumulate lipids (19). The observed S-100-immunoreactive 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 smooth muscle cells can express S-100 protein (19). It has been assumed that the smooth muscle cells of the coronary arteries, due to their origin from the proepicardial organ, can express some types of S-100 protein in certain conditions (18).
It is obvious that there is a connection between the accumulation of lipids and the expression of S-100 protein in the smooth muscle cells of the tested samples. Recent studies have shown that Ca2+-binding protein S-100A8 is a leukocyte hemoattractant whose expression is induced by interferon-gamma and tumour necrosis factor in macrophages, and whose expression in macrophages and endothelial cells is partially decreased by interleukin-4 and interleukin-13 (20). It has also been shown that the heterocomplex comprised of S-100A8 and S-100A9 Ca2+-binding proteins shows a large affinity to binding unsaturated lipid acids (21). It is also known that S-100 protein is expressed in cells that are in the process of differentiation, protein phosphorylation and proliferation – events that are Ca2+-mediated (19,22).
In addition, it is known that in the process of apoptosis, smooth muscle cells can express scavenger receptors and transform themselves into foam cells (23). However, from the present study, it is not possible to determine to what extent apoptotic cells participate in the accumulation of lipids.
Bearing in mind that proliferating cells express S-100 protein (19,22), that their proliferation is induced by the accumulation of lipids (21) and that smooth muscle cells can express scavenger receptors (23), the finding of S-100-immunoreactive foam cells in the samples of the present study becomes clearer.
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 the atherosclerotic plaque (13).
The results of this research have shown that the adventitia also participates in the vascular response, that is, in the remodelling of the blood vessel in conditions of hypertension and hypercholesteremia. Adventitial fibroblasts in the initial stages of atherosclerosis, except those that are CD34-immunoreactive, start to express α-SMA, which is a marker of muscular differentiation; medial smooth muscle cells, however, lose the same markers. The presented results correspond to the literature stating that in conditions of vascular remodelling, adventitial fibroblasts show the characteristics of smooth muscle cells of the synthetic phenotype, meaning they are phenotypically modified in myofibroblasts (14,15).
With regard to the changes occuring in the structure of the endothelium, in the initial and fatty streak stages of atherosclerosis, the endothelium is perserved (8). The immunoreactivity to CD31 that was observed at the preatheroma stage correlates with the literature (6). However, the finding of immunoreactivity to CD34 on the cells covering the intimal thickening in the late stages of atherosclerosis deserves disscusion. Although the expression of CD31 is a characteristic of the vascular endothelium, it was not expected to be expressed in the late stages of atherosclerosis (5); its expression was possibly due to re-endothelization or to adhesion of circulating cells (eg, circulating endothelium precursors) (5).
CONCLUSIONS
In the subendothelium of the intima of atherosclerotic coronary arteries at the early stage of lesions, there are smooth muscle cells, foam cells, macrophages, leukocytes and T lymphocytes. At the early stages of atherosclerosis, smooth muscle cells express α-SMA, MHC and desmin, and from the preatheroma stage onward, they express only α-SMA and vimentin.
The development of the 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 dedifferentiation spreads from the intimal to the medial smooth muscle cells.
Foam cells originate from macrophages and smooth muscle cells. Foam cells that originate from monocyte-macrophage lineage express CD68, and those that originate from smooth muscle cells 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, in parallel with dedifferentiation, smooth muscle cells start to accumulate lipids.
The dedifferentiation of smooth muscle cells of the tunica media (based on the loss of desmin expression and the appearance of vimentin expression) begins at the fatty streak stage. Adventitial fibroblasts at the fatty streak stage show activation, which is indicated by the expression of contractile filaments.
REFERENCES
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