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Cardiovascular Research 1998 40(1):191-205; doi:10.1016/S0008-6363(98)00141-2
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

The cell adhesion molecule E-cadherin is widely expressed in human atherosclerotic lesions

Yuri V. Bobrysheva,b,*, Reginald S.A. Lorda, Teruo Watanabec and Tsuyoshi Ikezawac

aSurgical Professorial Unit, St. Vincent's Hospital, Darlinghurst NSW 2010, Australia
bSchool of Anatomy, University of New South Wales, Sydney NSW 2052, Australia
cDepartment of Pathology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305, Japan

* Corresponding author. Tel.: +61-2-9-3612354; fax: +61-2-9-3604424.

Received 30 January 1998; accepted 17 April 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Various cell adhesion molecules are expressed in atherogenesis and the significance of their involvement in atherosclerotic lesion formation is well appreciated. In the present work, we examined whether the Ca2+-dependent cell adhesion molecule E-cadherin is also involved in atherogenesis. Methods: Specimens of carotid artery and aorta were obtained at operation. Expression of E-cadherin was studied by an immunohistochemical method. The nature of E-cadherin-expressing cells was examined by comparative analysis of consecutive sections and by a double immunostaining procedure. An immunohistochemical approach was also applied to examine how the accumulation of oxidised low density lipoproteins (LDL) by intimal cells is associated with E-cadherin expression. Results: No E-cadherin+ cells were found in normal non-atherosclerotic intima but E-cadherin+ cells were present in 96% of the atherosclerotic lesions. In atherosclerotic intima, E-cadherin was expressed by intimal cells showing varying degrees of transformation into foam cells. These E-cadherin+ cells also contained oxidised LDL in their cytoplasm. Differing numbers of CD68+ foam cells (15% to 60%) expressed E-cadherin but all the CD68+ macrophages without signs of transformation into foam cells were negative for E-cadherin. Neither smooth muscle cells nor foam cells of smooth muscle cell origin (smooth muscle alpha-actin+) were found to be positive for E-cadherin. T-cells (CD3+) and endothelial cells (von Willebrand factor+) were also negative for E-cadherin. Only a few vascular dendritic cells (S-100+) expressed E-cadherin and their expression was weak. We also found that a large proportion (40% to 85%) of E-cadherin+ cells did not stain with any cell-type specific markers. Conclusions: The finding that E-cadherin is expressed in atherosclerotic lesions expands our knowledge of cell adhesion molecules involved in atherogenesis. That E-cadherin is expressed in intimal cells transforming into foam cells suggests that lipid accumulation might be associated with the alteration and reorganisation of cell-to-cell interactions in atherogenesis. The present observations might assist in understanding the mechanisms associated with intracellular lipid accumulation.

KEYWORDS Arteries; Atherosclerosis; Cell adhesion molecules; E-cadherin; Foam cells; Dendritic cells


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The importance of leukocyte and endothelial cell adhesion molecules (CAMs) in the formation of intercellular contacts has been widely appreciated [1]. Expression of inflammatory CAMs such as ICAM-1, VCAM-1, P-selectin, and E-selectin has been intensely studied in atherosclerotic lesions and the increased expression of various inflammatory CAMs in atherogenesis is well documented [2–6]. The availability of mice with mutations for CD18, ICAM-1 and the selectins, as well as the development of mouse models with an increased susceptibility to atherosclerosis, promoted investigations on the role of inflammatory CAMs in the pathogenesis of atherosclerosis [7]. These genetic based studies provided strong evidence that the level of expression of CAMs can determine susceptibility to the formation of atherosclerotic lesions, while a deficiency of inflammatory CAMs can protect against the development of atherosclerosis [7].

Inflammatory CAMs responsible for heterotypical cell-to-cell interactions have received considerable attention [1–7], but CAMs determining mostly homotypical cell interactions have not yet been studied in human atherosclerosis or in models simulating human atherogenesis. In the present work we investigated whether the Ca2+-dependent cell adhesion molecule E-cadherin is expressed in normal and atherosclerotic human arteries. Our interest was stimulated by studies indicating that E-cadherin is expressed by Langerhans cells which are part of the family of dendritic cells [8, 9].

In previous publications we reported that dendritic cells reside in the intima of large arteries [10, 11], that their numbers increase in atherosclerotic lesions [11, 12], and that these vascular dendritic cells appear to be involved in local immunoinflammatory reactions [13]. Vascular dendritic cells share many of the immunogenic and structural characteristics typical of other dendritic cells [10–13]and a subtype of vascular dendritic cells might relate to Langerhans cells [14]. We were therefore interested to see whether vascular dendritic cells express E-cadherin.

Using anti-E-cadherin antibody we found that E-cadherin is widely expressed in human atherosclerotic lesions. The present report describes the distribution of E-cadherin expression in the arterial wall and the characteristics of E-cadherin-positive cells.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Tissue specimens and routine histology
The material was collected in accordance with the principles outlined in the Declaration of Helsinki [15].

Arterial wall segments from 27 carotid arteries and 20 aortas were obtained from patients whose ages ranged from 35 to 67 years. The carotid specimens were obtained by endarterectomy and the aortic specimens were collected during aortic reconstructions at St. Vincent's Hospital, Sydney. The characteristics of this material were previously reported [11, 16]. Because the specimens were relatively large (up to 5 cm), it was possible to find different atherosclerotic lesions, including early lesions, in the same specimen. The arterial specimens also included part of the adjacent normal-appearing arterial wall. According to their microscopic characteristics, the lesions were classified as diffuse intimal thickenings, fatty streaks, atheromatous plaques and complicated plaques as in our previous work [11].

For both routine histological examination and further immunohistochemical analysis, the arterial specimens were processed by standard formalin fixation and paraffin embedding. Sections were cut at 5-µm thickness. For routine histology they were stained with haematoxylin and eosin.

2.2 Investigating E-cadherin expression
E-cadherin antibody was purchased from Transduction Laboratories (Lexington, KY, USA). Expression of E-cadherin was examined in paraffin sections using a biotin–streptoavidin kit (Nichirei, Tokyo) and a 3-amino-9-ethylcarbazole (AEC) substrate–chromogen system (Dako, Carpinteria, CA, USA) which results in the red staining of antigens. After blocking endogenous peroxidase with 3% hydrogen peroxide in methanol for 20 min, microwave pre-treatment in 10 mM citrate buffer (pH 6.0) for 10 min and treatment with normal horse serum for 30 min, sections were incubated with E-cadherin antibody in a 5 µg/ml concentration for 45 min. The biotin–streptoavidin kit, which included biotinylated anti-mouse antibody and peroxidase-conjugated streptoavidin, was used according to the manufacturer's recommendations. The peroxidase–oxidase reaction was developed with the AEC substrate kit. As an alternative method for identifying E-cadherin expression, Envision-labelled polymer reagent (Dako, Kyoto, Japan) was used prior to the application of the AEC substrate kit. All immunohistochemical procedures were performed at room temperature. Duodenum sections were used for a positive control (Fig. 1A). For negative controls, the first antibody was omitted or the sections were treated with an immunoglobulin fraction of non-immune goat serum used as a substitute for the primary antibody. None of the negative control sections showed positive immune staining. Counterstaining was performed with Gill's haematoxylin. Sections were examined in an Olympus microscope at 10x10, 10x20 and 10x40 magnifications. The frequency of E-cadherin-positive cells in different areas was estimated by semi-quantitative analyses.


Figure 1
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  		Fig. 1 Different patterns of E-cadherin expression (A–D). (A) Immunostaining of duodenum epithelium with E-cadherin which was used as a positive control; note the typical basolateral staining of epithelial cells (red colour). (B–D) represent different patterns of E-cadherin expression in atherosclerotic intima: (B) scattered E-cadherin-positive cells; (C) a small cluster of E-cadherin-positive cells; (D) a large cluster of E-cadherin-positive cells. Note that all E-cadherin-positive cells contain ‘lipid droplets’ in their cytoplasm. In (D), the staining intensity of E-cadherin-positive foam cells varies with strongly E-cadherin-positive cells co-localising with weakly E-cadherin-positive cells. Immunoperoxidase technique; visualisation of E-cadherin expression was produced with AEC substrate kit which resulted in the formation of a red coloured product; counterstaining was with Gill's haematoxylin. Original magnifications: x400, x400, x400, x400.

 
2.3 Characterisation of cells expressing E-cadherin using sets of serial consecutive sections immunostained by single staining immunoperoxidase procedure
The analyses were carried out using sets of consecutive paraffin sections immunostained with antibodies to E-cadherin and cell type specific antibodies to macrophages (CD68, HAM56), smooth muscle cells (alpha-smooth muscle actin and human muscle actin), T-cells (CD3), B-cells (CD20), endothelial cells (von Willebrand factor) and mast cells (CD15) (Table 1). Vascular dendritic cells in the arterial intima were identified with S-100 antibody which is a convenient marker for their identification since the arterial intima does not contain neuronal or glial cells and other intimal cells do not stain positively with S-100 [11]. Oxidised low density lipoproteins (LDL) (o-LDL) were identified with anti-o-LDL antibody (a kind gift of Dr. Franz Tatzber) which allowed the formalin fixation of specimens and their routine embedding in paraffin [17].


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  		Table 1 Antibodies used

 
The sources and working concentrations of antibodies used are given in Table 1. The immunohistochemical procedures used for identifying different cell types were the same as described for E-cadherin identification. For blocking endogenous peroxidase, 3% H2O2 in methanol for 20 min was applied. To prevent lipid peroxidation in tissue sections in which the distribution of o-LDL was studied, the sections were pre-treated with 0.3% H2O2 in PBS containing 0.5 µM butylated hydroxytoluene (Sigma) for 10 min to block endogenous peroxidase. Pre-treatment of the sections with trypsin and microwave boiling in citrate buffer were used when necessary as indicated in Table 1. Comparison of the stained consecutive sections enabled the characteristics of E-cadherin-positive cells to be investigated and their cell nature to be determined. The same intimal areas were selected in consecutive sections, and the numbers of cells expressing two different antigens were estimated at a magnification of x400. The numbers of cells which displayed both antigens were expressed as a percentage of the total number of cells expressing only one of either antigens.

2.4 Characterisation of cells expressing E-cadherin using double immunostaining procedure
To reassess the data obtained from the comparison of the sections stained for E-cadherin and cell-type specific antibodies, a double immunostaining technique was used. Differing combinations of antigens including E-cadherin/CD68, E-cadherin/alpha-smooth muscle actin, E-cadherin/CD3 and E-cadherin/S-100 were analyzed using the approach previously described [18, 19]. The co-localisation of E-cadherin with o-LDL as well as the co-localisation of o-LDL with CD68 macrophage-associated antigen were also examined by double immunostaining. For double immunostaining, staining with the first primary antibody was performed using the same protocol as described above for a single immunostaining procedure. After visualisation of the first primary antibody with the AEC substrate kit, the slides with the tissue sections were washed several times during 60 min with 0.1 M glycine–hydrochloric buffer (pH 2.2) at 4°C. The sections were then treated, if necessary (Table 1), with 0.1% trypsin at 45°C for 30 min or a microwave pre-treatment in 10 mM citric buffer (pH 6.0) was applied for 10 min. The sections were further incubated overnight at 4°C with one of the second primary antibodies. After rinsing in PBS, the sections were incubated with an appropriate, anti-mouse or anti-rabbit biotinylated antibody (Nichirei, Tokyo) for 12 h at 4°C. After washing in PBS, the sections were incubated at room temperature with alkaline phosphatase-conjugated streptoavidin (Nichirei, Tokyo) for 30 min, and the reaction was then visualised with fast blue substrate kit (Nichirei, Tokyo). The double immunostaining procedure used for identifying antigens located in the same cells could give false negative results because visualisation of the first antigen can preclude visualisation of the second antigen. Therefore, the sequence of applying the first and second primary antibodies was reversed. Negative controls were performed as described above. None of the negative control sections showed positive immune staining.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 E-cadherin expression in atherosclerotic arterial wall
E-cadherin-positive cells were detected in 96% of atherosclerotic lesions taken from the aortas and carotid arteries but no E-cadherin expression was found in normal non-atherosclerotic intima (Table 2). In all the specimens, the media adjacent to non-atherosclerotic intima was also free of E-cadherin expression. In some segments of atherosclerotic wall adjacent to plaques, E-cadherin-positive cells were detected in the media but no E-cadherin-positive cells were found in the aortic adventitia (Table 2). The patterns of E-cadherin expression in carotid artery specimens were similar to those in aortic specimens. In the atherosclerotic lesions, almost all E-cadherin-positive cells exhibited signs of transformation into foam cells and some had the appearance of typical foam cells (Fig. 1 B–D).


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  		Table 2 E-cadherin expression in different types of atherosclerotic lesions

 
In different types of atherosclerotic lesions, numerical differences in E-cadherin expression were apparent between different layers of the intima within the wall of the same vessel as well as between different zones of the same layers. Within atherosclerotic plaques, the areas containing E-cadherin-positive cells bordered on areas which were free from E-cadherin expression. Furthermore, within the same plaques but in different areas containing E-cadherin-positive cells, the numbers of these E-cadherin-positive cells were found to vary from less than 1% up to 80% when microscopic fields were examined at a magnification of x400. The variations in the extent of E-cadherin expression cannot be related to the age of the patient or the duration of disease as within the same arterial segments there were intimal areas with different extents of E-cadherin expression.

Because the density and numbers of E-cadherin-positive cells varied considerably between different areas of the same lesion and because there were marked variations in the numbers and the distribution of E-cadherin-positive cells between specimens obtained from different individuals, we avoided estimating the precise overall numbers of E-cadherin-expressing cells in different lesions. We have, however, estimated the relative frequency of E-cadherin-expressing cells in different areas of several types of atherosclerotic lesions (Table 3). In intimal thickenings, the distribution of E-cadherin-positive cells was uneven with the highest proportion of E-cadherin-positive cells located in the middle and deeper sublayers. E-cadherin-positive cells were more regularly distributed in fatty streaks than in atherosclerotic plaques. In atheromatous plaques, the external parts of the lipid core were always rich in E-cadherin expression unlike the central parts in which E-cadherin expression was sparse. In plaques, the fibrous caps showed the lowest proportion of E-cadherin-expressing cells. In complicated atherosclerotic plaques, the areas of neovascularisation with large numbers of inflammatory cells were always poor in E-cadherin expression while the areas of neovascularisation which lacked inflammatory cells showed variable E-cadherin expression.


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  		Table 3 Relative frequency, micro-patterns of distribution and appearance of E-cadherin-positive cells in different atherosclerotic lesions

 
In all the atherosclerotic lesions studied, three common micro-patterns of distribution of E-cadherin-positive cells were identified: (i) isolated scattered E-cadherin-positive cells (Fig. 1B); (ii) small clusters consisting of 2 to 4 E-cadherin-positive cells (Fig. 1C); and (iii) aggregates of E-cadherin-positive cells of different sizes (Fig. 1D). The E-cadherin-positive cells were of different shapes and, from their appearance, two types of E-cadherin-positive cells were recognised. Type I E-cadherin-positive cells were represented by elongated, spindle-like cells which typically, were longitudinally orientated along the arterial lumen (Fig. 1B, C). The numbers of lipid vacuoles in their cytoplasm varied markedly, although cells containing only a few lipid vacuoles were prevalent (Fig. 1B, C). Type II E-cadherin-positive cells were represented by round, oval or irregularly shaped cells usually containing a large number of lipid vacuoles (Fig. 1D). In some of these, the cytoplasm was completely filled by vacuoles (Fig. 1D). The incidence of different micro-patterns of these cells in the same types of atherosclerotic lesions were similar between the carotid and aortic specimens (Table 3).

The intensity of E-cadherin immunostaining in different intimal cells varied (Fig. 1B–D). When the intensity of E-cadherin staining of duodenal epithelium was used for comparison (Fig. 1A), in more than 70% of all E-cadherin intimal cells, the intensity of the staining was found to be stronger, equal to, or approaching that of duodenal epithelium. In different atherosclerotic lesions, 45% to 60% of all E-cadherin-positive cells expressed E-cadherin more strongly than duodenum epithelium. Many weakly E-cadherin-positive cells showed ill-defined contours (Fig. 1D) and virtually all of them were seen to locate in cell aggregates or in the lipid core.

Almost all E-cadherin-positive intimal cells showed signs of transformation into foam cells but not all foam cells expressed E-cadherin. The proportion of E-cadherin-expressing cells in the total foam cell population was estimated by examination at magnification x400. In fatty streaks, E-cadherin-positive foam cells constituted up to 80% of the total number of foam cells. In general, the proportion of E-cadherin-positive foam cells in plaques was lower than in fatty streaks. Mosaic clusters containing both E-cadherin-positive and E-cadherin negative foam cells were also seen in fatty streaks and in plaques. In different areas of the same plaque, differing numbers of foam cells expressed E-cadherin. In the outer parts of the lipid core, 20% to 80% of foam cells expressed E-cadherin. In the central part of the lipid core, 70% to 100% of foam cells were E-cadherin negative. Most of these E-cadherin negative cells had pyknotic nuclei and showed ill-defined contours consistent with cell destruction.

3.2 E-cadherin expression versus oxidised LDL accumulation
Single immunostaining showed an association between E-cadherin expression and foam cell formation. We therefore compared the patterns of E-cadherin expression with the patterns of accumulation of o-LDL. When the antibody against o-LDL (anti-o-LDL) was applied, two patterns of specific immunostaining, namely, extracellular and intracellular, were identified in the atherosclerotic lesions. Staining of the extracellular matrix was usually weak and the areas of anti-o-LDL immunopositivity were irregularly distributed throughout the atherosclerotic intima. In contrast, anti-o-LDL positive cells mostly exhibited intense staining and always conformed to the appearance of forming or mature foam cells. Anti-o-LDL positive cells were seen to locate separately and in groups. The areas populated by anti-o-LDL positive foam cells were contiguous with areas containing mainly anti-o-LDL negative cells.

Analysis of serial sections stained with E-cadherin antibody and anti-o-LDL demonstrated that in many areas of atherosclerotic intima, the patterns of distribution of both markers were similar, or even almost identical, but the extent of the accumulation of o-LDL was usually larger than that of E-cadherin expression (Fig. 2). Comparison of serial sections showed that virtually all E-cadherin-positive cells contained o-LDL in their cytoplasm. Double immunostaining confirmed that E-cadherin expression co-existed with the accumulation of o-LDL in the cytoplasm of foam cells but some foam cells containing o-LDL were free of E-cadherin expression (Fig. 3A–C).


Figure 2
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  		Fig. 2 E-cadherin expression and accumulation of o-LDL in atherosclerotic lesions (A–F). (A) and (D) represent the patterns of E-cadherin expression in a fatty streak (A) and an atherosclerotic plaque (D), while (B) and (E) show the patterns of accumulation of o-LDL (A and B as well as D and E are sets of consecutive sections). (F) represents a negative control which was produced in the section consecutive to that shown in (E). (C) shows the typical appearance of E-cadherin-positive cells in fatty streaks. Immunoperoxidase technique; visualisation of E-cadherin expression (red) was produced with AEC substrate kit; counterstaining was with Gill's haematoxylin. Original magnifications: x100, x100, x400, x100, x100, x100.

 

Figure 3
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  		Fig. 3 Double immunostaining demonstrating the association of the accumulation of o-LDL with E-cadherin expression in foam cells located separately (A, B) and in a massive group (C). In (A) and (B), o-LDL were visualised with AEC substrate kit (red product of reaction), (small arrows) while E-cadherin immunostaining was produced with a Fast Blue substrate kit (blue product of reaction) (large arrows). Note that these foam cells contain both E-cadherin and o-LDL. Note also that o-LDL are present in the extracellular matrix where they are irregularly distributed. In (C), E-cadherin expression was visualised with AEC substrate kit (red) while the location of o-LDL was identified using a Fast Blue substrate kit (blue). E-cadherin and o-LDL are irregularly distributed in the cytoplasm of foam cells. Some cytoplasmic areas contain a violet coloured product resulting from the combination of red and blue colours. A few cells containing o-LDL are E-cadherin negative (blue). Original magnifications: x400, x400, x400.

 
3.3 Examination of the nature of E-cadherin-expressing cells
In some intimal areas, the patterns of CD68 positivity, E-cadherin expression and o-LDL accumulation were similar (Fig. 4A, B, D). Differing numbers of CD68+ foam cells (15% to 60%) expressed E-cadherin. However, all CD68+ macrophages which showed no signs of transformation into foam cells were negative for E-cadherin. Double immunostaining confirmed the co-localisation of CD68 macrophage-associated antigen with E-cadherin in the same cells (Fig. 5A, B). In the lipid core, E-cadherin-expressing foam cells usually showed weak immunopositivity for CD68 (Fig. 5A) while separately located E-cadherin-positive foam cells contained significant amounts of CD68 macrophage-associated antigen (Fig. 5B). In some lesions, the extent of E-cadherin expression markedly exceeded the expression of CD68 (Fig. 5C, D).


Figure 4
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  		Fig. 4 E-cadherin expression, accumulation of o-LDL and cell type specific markers in an atherosclerotic lesion. (A–D) show four consecutive sections of aortic atheromatous plaque immunostained for E-cadherin (A), o-LDL (B), smooth muscle alpha-actin (C) and CD68 macrophage-associated antigen (D). Some macrophage foam cells and their groups are positive for E-cadherin and contain o-LDL in their cytoplasm (A, B and D) while all the smooth muscle cells are E-cadherin negative (A and C). Immunoperoxidase technique; visualisation of E-cadherin expression was produced with AEC substrate kit (red); counterstaining was with Gill's haematoxylin. Original magnifications: x100, x100, x100, x100.

 

Figure 5
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  		Fig. 5 Co-localisation of E-cadherin expression and cell type specific markers identified in atherosclerotic lesions using double and single immunostaining procedures (A–F). (A) shows an area from atheromatous plaque, double-stained for E-cadherin and CD68 macrophage-associated antigen. CD68 was visualised with AEC substrate kit (red) and E-cadherin immunostaining was produced using the Fast Blue substrate kit (blue). (B) demonstrates a macrophage (blue) containing o-LDL (red). o-LDL were visualised with AEC substrate kit and E-cadherin immunostaining was produced with a Fast Blue substrate kit. (C–F) represent a set of consecutive sections of an area with neovascularisation from a complicated atheromatous plaque which were immunostained for E-cadherin (C), CD68 (D), CD3 (E) and alpha-smooth muscle actin (F). E-cadherin is expressed to a much larger extent than CD68 (C, D). This area does not show signs of inflammation and it is characterised by the absence of T-cells (CD3+) but does contain a large number of E-cadherin-positive cells (C, E). Note the difference between the pattern of E-cadherin expression and the pattern of distribution of smooth muscle cells (C, F). (C–F) Immunoperoxidase technique; visualisation of E-cadherin expression was produced with AEC substrate kit (red); counterstaining was with Gill's haematoxylin. Original magnifications: x400, x400, x100, x100, x100, x100.

 
Analyses of serial sections indicated that neither smooth muscle cells nor foam cells of smooth muscle cell origin (alpha-smooth muscle actin+) were positive for E-cadherin (Fig. 4A, C; Fig. 5C, F; Fig. 6A, B). Using double immunostaining we were not able to identify any cells simultaneously expressing alpha-smooth muscle actin and E-cadherin (Fig. 6D). In the areas where E-cadherin foam cells were present, the networks formed through smooth muscle cell processes were destroyed.


Figure 6
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  		Fig. 6 Immunostaining for E-cadherin, smooth muscle alpha-actin, and CD68 in a fatty streak in a carotid artery (A–D). Staining for E-cadherin (A), smooth muscle alpha-actin (B) and CD68 (C) produced in consecutive sections shows that in this fatty streak, the distribution of E-cadherin is different from that of smooth muscle cells and that macrophages are absent. (A–C) Immunoperoxidase technique; visualisation of E-cadherin expression (red) was produced with AEC substrate kit; counterstaining was with Gill's haematoxylin. (D) shows double immunostaining for smooth muscle alpha-actin (red) and E-cadherin (blue). Smooth muscle alpha-actin was visualised with AEC substrate kit and E-cadherin immunostaining was produced with the Fast Blue substrate kit. Note that smooth muscle cells are E-cadherin negative. Original magnifications: x100, x100, x100, x400.

 
Vascular dendritic cells (S-100+) represented a minor cell population but when the sections were viewed at magnification x400, up to 5% of the total cell population were S-100 positive in some areas of atherosclerotic lesions. The peculiarities of their distribution in these specimens have been previously reported [11]. The present examination showed that only about 1 in 50 S-100-positive cells expressed E-cadherin and this expression was always weak. Some E-cadherin+/S-100+ cells were detected in the media (Fig. 7).


Figure 7
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  		Fig. 7 Visualisation of E-cadherin expression and S-100 positive cells in the arterial wall (A, B). (A) and (B) represent the results of double immunostaining in an intimal thickening (A) and in the media underlying a complicated atheromatous plaque in the aorta (B). E-cadherin (red) was visualised with AEC substrate kit and S-100 immunostaining (blue) was produced with the Fast Blue substrate kit. In (A), note that S-100 positive cells are negative for E-cadherin. In (B), note the presence of granules of both red and blue colours in the same cell which suggests the expression of E-cadherin by the S-100 positive cell. Original magnifications: 200, x400.

 
The comparison of consecutive sections stained for CD3 and E-cadherin indicated that T-cells (CD3+) did not express E-cadherin (Fig. 5C, E). When double immunostaining was applied, we also failed to detect the co-localisation of E-cadherin with CD3. Luminal endothelial cells as well as the endothelium of neovascularisation (von Willebrand factor+) were found to be negative for E-cadherin. In atherosclerotic lesions, mast cells (CD15+) were seen as less than 1% of the total cell population and were found to be negative for E-cadherin. B-cells (CD20+) were very seldom detected and we failed to find any B-cells expressing E-cadherin.

A comparison of consecutive sections stained for E-cadherin and cell type-specific markers showed that in all the specimens, 40% to 85% of all E-cadherin-positive cells did not stain with any cell type-specific markers including markers to macrophages, smooth muscle cells, endothelial cells, T-cells, B-cells, mast cells and vascular dendritic cells (Figs. 5–7GoGoA). Most E-cadherin-positive cells which were negative for any cell type-specific markers were found to contain vimentin. The results of double immunostaining agreed with that obtained from the comparative analysis of serial sections.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 E-cadherin: a new marker for atherosclerotic alteration of the arterial wall
The expression of E-cadherin is developmentally regulated [20–22]. In adults, E-cadherin expression is required for normal epithelial functions [20–22]. E-cadherin appears to be involved in a much wider spectrum of cell-to-cell interactions than was thought when E-cadherin was designated as epithelial (E)-cadherin [21, 23–26]. In the last decade, E-cadherin has been intensively studied, especially because the loss of E-cadherin has been attributed to a pathogenic role in tumour invasion [21, 26–28]. Our observation that E-cadherin is expressed by intimal cells transforming into foam cells suggests an even broader involvement of E-cadherin in pathological processes.

The observations that E-cadherin is not expressed in normal non-atherosclerotic intima and that patterns of its expression are different in different types of atherosclerotic lesions suggest that E-cadherin is involved in the formation and progression of lesions. In atherosclerotic intima, E-cadherin expression is mostly restricted to cells transforming into foam cells. All E-cadherin-positive cells (excluding vascular dendritic cells) were found to contain lipid vacuoles in their cytoplasm which suggests that E-cadherin gene activation might be associated with an alteration of the metabolism of intracellular lipids. The accumulation of o-LDL and the formation of foam cells are critical events in the development of atherosclerotic lesions [2, 5, 29]and the identification of cell adhesion molecules which are specifically associated with lipid transformation of intimal cells would assist in understanding the mechanisms involved.

The present study did not establish the functions of E-cadherin expression in atherogenesis but our observations that E-cadherin is expressed by scattered cells containing a few lipid vacuoles as well as by clustered foam cells in the early stages of lesion development might imply that E-cadherin is important for foam cell aggregation. If E-cadherin is involved in foam cell aggregation, it might be also involved in the development of the lipid core.

Elucidating how foam cells selectively adhere to each other is an important issue for understanding lipid core formation, itself a crucial event in the progression of early atherosclerotic lesions [5, 30, 31]. Morphological observations indicate that the formation of the lipid core is associated with the accumulation and aggregation of foam cells in the deep layers of the intima [30, 31]. The present observations of intense expression of E-cadherin by foam cells forming clusters in early atherosclerotic lesions might suggest that E-cadherin is important for foam cell cohesion. Studies on embryogenesis and in vitro experiments indicate that the switching on and off of E-cadherin expression correlates with a variety of morphological events involving cell aggregation or disaggregation, and that closely aggregating cells show greater E-cadherin expression than non-aggregating cells [20, 21, 27, 28]. The expression of E-cadherin by forming and mature foam cells might also indicate that their motility is diminished which further facilitates cellular aggregation since in embryogenesis, the downregulation of cell migration depends on E-cadherin expression [20–22].

In the present study, a high variability in the expression of E-cadherin was found in atheromatous plaques. Atheromatous plaques are characterised by widespread destruction of intimal cells, in particular, foam cells in and around the lipid (necrotic) core [31–34]. According to our observations, loss of E-cadherin expression relates to foam cell destruction in atheromatous plaque.

4.2 Nature of E-cadherin-expressing cells in atherosclerotic lesions
E-cadherin was found to be expressed by different cell types. Firstly, some foam cells of macrophage origin displayed E-cadherin. We are not aware of other work showing that macrophages express E-cadherin either in adults or in embryogenesis, or under pathological conditions. E-cadherin, however, was not expressed by those macrophages in which there were no signs of transformation into foam cells. This finding might suggest the pathological activation of the E-cadherin gene in macrophages transforming into foam cells. We speculate that this process might be important since a large proportion of CD68 positive foam cells (15% to 60%) were found to display E-cadherin.

Secondly, some vascular dendritic cells were also found to express E-cadherin. We estimated that only about 2% of the total population of vascular dendritic cells displayed E-cadherin immunopositivity and this expression was markedly weaker than that found in E-cadherin-positive foam cells. The finding of E-cadherin expression in vascular dendritic cells is important (albeit the proportion is small) because this strengthens the similarities between vascular dendritic cells and Langerhans cells. Langerhans cells express E-cadherin [8, 9, 23, 35]and its expression is thought to control trafficking and other functions of Langerhans cells [23, 35].

In the present study, smooth muscle cells, endothelial cells, B-cells and mast cells did not express E-cadherin in atherosclerotic lesions. In other circumstances, T-cells are capable of expressing E-cadherin [36, 37]but we failed to find any T-cells expressing E-cadherin in atherosclerotic lesions.

In atherosclerotic lesions, 40% to 85% of E-cadherin-positive cells did not stain with any cell type specific marker. This might relate to loss of cell type specific features during transformation of intimal cells to foam cells and the observation that all E-cadherin-positive cells showed signs of lipid transformation supports this possibility. However, only some of the E-cadherin-positive cells looked like mature foam cells while many others contained only a few lipid vacuoles in their cytoplasm. This implies that the shedding of cell type specific markers during transformation of intimal cells into foam cells cannot be exclusively responsible for the appearance of E-cadherin-positive cells which did not co-label with any cell type specific marker in the present study.

The presence of undefined cell types in human atherosclerotic lesions which do not mark with cell type-specific antibodies such as HHF35 (smooth muscle cells), HAM-56 (macrophages) or antibodies to CD45 (lymphocytes) or factor VIII related antigen (endothelium) has been previously reported [38, 39]and these unidentified cells have been referred to as mesenchymal-appearing intimal cells [39]or null cells [38]. In some arterial specimens, null cells expressing ICAM-1 comprised approximately 50% of all ICAM-1-expressing cells [38]. We earlier speculated that some null cells might be vascular dendritic cells [40]. The present investigation, however, shows that apart from smooth muscle cells, macrophages, endothelial cells, T-cells, vascular dendritic cells, mast cells and B-cells, another cell type does reside in the intima and represents a significant proportion of the E-cadherin-positive cell population. The origin and nature of the E-cadherin-positive null cell population requires further clarification.

Time for primary review 24 days


   Acknowledgements
 
We thank Dr. Franz Tatzber, Biomedica, Austria, for a kind gift of antibodies to oxidised LDL. We thank St. Vincent's Clinic Foundation, Sydney, Australia, for financial support. We also acknowledge a contribution from the Ministry of Education, Science and Culture of Japan in this research.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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