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Cardiovascular Research 1999 43(4):1003-1017; doi:10.1016/S0008-6363(99)00125-X
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Neovascular expression of VE-cadherin in human atherosclerotic arteries and its relation to intimal inflammation

Yuri V Bobrysheva,b,*, Sanjay M Cheriana, Stephanie J Indera and Reginald S.A Lorda

aSurgical Professorial Unit, Level 17, O’Brien Building, Victoria Street, St. Vincent’s Hospital, Darlinghurst NSW 2010, Australia
bSchool of Anatomy, University of New South Wales, Sydney NSW 2052, Australia

* Corresponding author. Tel.: +61-2-9361-2354; fax: +61-2-9360-4424

Received 22 January 1999; accepted 23 March 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The present work aimed to investigate how the Ca2+-dependent cell adhesion molecule vascular endothelial (VE)-cadherin might be involved in atherogenesis. Methods: Specimens of human carotid artery and aorta were obtained at operation. An immunohistochemical approach using cell-type specific antibodies examined how VE-cadherin expression in areas of neovascularisation related to the accumulation of immunocompetent and inflammatory cells within atherosclerotic plaque. Electron microscopy was used to examine the structural characteristics of neovessels and the cell composition in the surrounding intimal matrix. Results: In all the non-atherosclerotic aortic segments, VE-cadherin expression was observed only in the adventitia and the outer third of the media. Within the atherosclerotic arterial segments, VE-cadherin was expressed in all layers of the arterial wall including the intima where VE-cadherin was expressed by endothelial cells in areas of neovascularisation. In some neovessels, loss of VE-cadherin expression was associated with increased focal accumulation of T-cells, macrophages and dendritic cells. Electron-microscopic examination demonstrated varying degrees of endothelial continuity in the intimal neovessels. Within those neovessels which were surrounded by a large number of immunocompetent and inflammatory cells, some inter-endothelial cell contacts were open allowing the penetration of blood cells through patent intercellular zones. Conclusions: VE-cadherin is expressed in atherosclerotic lesions by endothelial cells associated with neovascularisation. Downregulation of VE-cadherin expression within some intimal neovessels is accompanied by increased entry of immunocompetent cells into the intimal matrix surrounding areas of neovascularisation which suggests that disorganising endothelial cell-to-cell interactions within neovessels is significant in atherogenesis.

KEYWORDS Arteries; Atherosclerosis; VE-cadherin; Cell adhesion molecules; Endothelial cells; Dendritic cells; Inflammation; Neovascularisation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Immuno-inflammatory processes are now an accepted component of atherosclerosis [1–5] with inflammatory cells accumulating within the wall of the affected vessel [6–8]. Specific molecules mediating the attachment to and migration of cells from the blood through the endothelium and into the intima have been identified including inflammatory cell adhesion molecules (CAMs) responsible for heterotypical cell-to-cell interactions [9–14]. In arterial segments with profound mononuclear leukocyte accumulation, the expression of ICAM-1 (CD54), VCAM-1(CD106) and E-selectin (CD62E) is increased, more so on endothelial cells lining the intimal neovasculature than on the endothelium of the arterial lumen [15–17].

Postmortem injection techniques and confocal microscopy confirm that intimal neovasculature is derived almost exclusively from the vasa vasorum of the adventitia [18–20]. Inflammatory cells enter well developed plaques almost entirely through new vessels, while the dense fibrous caps are practically impenetrable by blood cells [15,16,21]. Neovascularisation, therefore, appears to be the main route for the migration of leukocytes and immunocompetent cells into advanced atherosclerotic lesions [15,16,21] although the structural and molecular mechanisms involved in the formation and functions of neovascularisation remain poorly understood.

No previous reported studies have examined how vascular endothelial cadherin (VE-cadherin) expression contributes to neovascularisation and whether VE-cadherin expression may relate to the intimal leukocyte content within atherosclerotic plaques.

The VE-cadherin gene encodes a Ca2+-dependent cell adhesion molecule that is responsible for the organisation of inter-endothelial junctions [22–24]. VE-cadherin (CD144) is an endothelium-specific, homophilic adhesion molecule concentrated at inter-endothelial cell contacts and is presumed to play an important role in the maintenance and regulation of the integrity of the endothelial cell layer [25–31]. In vitro and in vivo studies have demonstrated that anti-CD144 antibody interferes with VE-cadherin mediated inter-endothelial adhesion [32,33].

The present work was undertaken to examine how VE-cadherin is expressed in neovessels within atherosclerotic lesions and how its expression may be associated with the accumulation of intramural inflammatory infiltrates.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Tissue specimens
Arterial wall segments from 26 carotid arteries and 19 aortas were obtained from patients whose ages ranged from 32 to 71 years. The carotid specimens were obtained at endarterectomy and the aortic specimens were collected during aortic reconstructions at St. Vincent’s Hospital, Sydney. Material was collected in accordance with the principles outlined in the World Medical Association Declaration of Helsinki [34] and the present study was approved by the institutional review board of St. Vincent’s Hospital, Sydney. The characteristics of this material have been previously reported [21]. 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 the microscopic characteristics which were specified in our previous work [35], the lesions were classified as diffuse intimal thickenings, fatty streaks, non-complicated atherosclerotic plaques without fibrous caps and complicated atheromatous plaques containing fibrous caps.

The arterial specimens were prepared for immunohistochemical and electron-microscopic examinations. For immunohistochemistry, the unfixed specimens were immediately embedded in OCT compound, rapidly frozen in liquid nitrogen and stored at –70°C until cryostat sectioning. Frozen sections were cut at 5–7 µm thickness and air dried for 45 min. For electron-microscopic examination, small tissue pieces were cut from each specimen and fixed in 2.5% glutaraldehyde in phosphate buffered saline (PBS) (pH 7.2).

2.2 Immunohistochemical investigation of VE-cadherin expression
After eliminating endogenous peroxidase activity by 0.3% H2O2 for 5 min, the sections were preincubated with normal goat serum, then incubated for 30 min with monoclonal antibody to VE-cadherin (Cadherin 5; Clone TEA1/31, Immunotech, Beckman Coulter Australia Pty., A.C.N.) with a concentration of 10 µg/ml and tested by avidin–biotin complex using the ABC immunoperoxidase method [36]. After washing in tris-phosphate buffered saline, pH 7.6 (TPBS, 10 min), the sections were incubated for 20 min with the secondary horse anti-mouse antibody (VECTOR BA-2000). The sections were then washed in TPBS for 5 min and treated with avidin–biotin complex (ELITE-ABC, VECTOR PK61000) for 30 min. After washing for 10 min in TPBS, a brown staining was produced by treating the sections with 3,3'-diaminobenzidine (DAB). All the incubations were performed at room temperature. For negative control, the primary antibody was omitted or the sections were treated with an immunoglobulin fraction of non-immune goat serum (VECTOR S-1000) as a substitute for the primary antibody. None of the negative control sections showed positive immune staining. Counterstaining was performed with Mayer’s haematoxylin. Entire sections of the arterial segments were examined using an Olympus microscope at 10x10 and 10x40 magnifications to quantify the atherosclerotic lesions containing VE-cadherin expressing cells.

2.3 Immunohistochemical identification of intimal cells
Endothelial cells were identified by von Willebrand factor. T-lymphocytes in the arterial intima were identified with anti-CD3 and the T-helper/inducer and suppressor/cytotoxic T-cell subsets were identified with anti-CD4 and anti-CD8 antibodies respectively. Macrophages were identified with anti-CD68 antibody. Dendritic cells in the arterial wall were identified with S-100 antibody. S-100 antibody is a convenient marker for the identification of dendritic cells in the arterial wall since the arterial intima does not contain neuronal or glial cells and other intimal cells do not stain positively with S-100 [21,35]. Smooth muscle cells were identified with antibody to alpha-smooth muscle actin. The sources and working concentrations of antibodies used are given in Table 1.


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  		Table 1 Antibodies used

 
An analysis was carried out using sets of consecutive parallel sections immunostained with antibodies to VE-cadherin, von Willebrand factor (vWf), CD3, CD4, CD8, CD68, S-100 and alpha-smooth muscle actin (SMA). Comparison of the consecutive sections enabled the composition and co-localisation of different cell types to be investigated in different areas of atherosclerotic lesions. Immunohistochemical identification of cell types was performed using the ABC immunoperoxidase method as described in Section 2.2. In some staining, biotin–streptoavidin kit (Nichirei, Tokyo) and 3-amino-9 ethylcarbazole (AEC) substrate–chromogen system (Dako, Carpinteria, CA) which results in the red staining of antigens were also applied. For this staining, after blocking the endogenous peroxidase with 0.3% hydrogen peroxide in methanol for 20 min and treatment with normal horse serum for 30 min, sections were incubated with one of the cell-type specific antibodies for 45 min. Biotin–streptoavidin kit, which included biotinylated anti-mouse or anti-rabbit antibody and peroxidase-conjugated streptoavidin, was used according to the manufacturer’s recommendations. The peroxidase–oxidase reaction was developed with the AEC substrate kit. All immunohistochemical procedures were performed at room temperature. For negative controls, the primary antibodies were 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 Mayer’s haematoxylin.

2.4 Investigation of the co-localisation of VE-cadherin with von Willebrand factor
The co-localisation of VE-cadherin with von Willebrand factor was examined by double immunostaining. For double immunostaining, staining with VE-cadherin was performed using the same protocol as described above for the single immunostaining procedure utilising the AEC substrate kit (Section 2.3). After visualisation of VE-cadherin with the AEC substrate kit, 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 further incubated overnight at 4°C with the antibody to von Willebrand factor. After rinsing in PBS, the sections were incubated with an anti-mouse biotinylated antibody (Nichirei) for 12 h at 4°C. After washing in PBS, the sections were incubated at room temperature with alkaline phosphatase-conjugated streptoavidin (Nichirei) for 30 min, and the reaction was then visualised with a fast blue substrate kit (Nichirei). Negative controls were performed as described above by omitting the primary antibodies. None of the negative control sections showed positive immunostaining.

2.5 Double immunostaining procedure for investigating co-localisation of different cell types
Differing combinations of antigens were analysed (S-100/CD68, S-100/CD4, S-100/CD68, S-100/SMA) by a double immunostaining technique (DOUBLESTAIN Kit System). This kit allowed simultaneous staining for the detection of two different tissue markers on the same section by a combination of mono- and polyclonal antibodies and of the peroxidase–antiperoxidase (PAP) and alkaline phosphatase–antialkaline phosphatase (APAAP) technique. Using a mouse primary antibody in the PAP system with DAB chromogen yielded a brown reaction product at the site of the target antigen while a rabbit primary antibody in the APAAP system with Fast Red chromogen resulted in a rose coloured precipitate at the site of the identified antigen. This difference allowed the topographical relationships between the two antigens to be observed. For double immunostaining, after eliminating the endogenous peroxidase activity by 0.3% H2O2 for 5 min, the consecutive tissue sections were pre-incubated with normal swine serum and tested according to the manufacturer’s instructions (DOUBLESTAIN Kit System). The sections were incubated with a working solution of the two primary antibodies prepared by mixing equal volumes of each antiserum diluted to one-half of the established optimal dilution appropriate for single immunostaining procedure. The sections were then sequentially incubated with the mixture of anti-rabbit and anti-mouse link antibodies, the mouse APAAP immune complex, followed by the rabbit PAP immune complex. Positive and negative controls were carried out according to the DOUBLESTAIN Kit System manufacturer’s instructions. None of the negative control sections showed positive immune staining. Counterstaining was performed with Mayer’s haematoxylin.

2.6 Preparation of specimens for electron microscopic analysis. Ultrastructural identification of different cell types and their degenerative alterations in atherosclerotic lesions
After fixation of arterial samples in 2.5% glutaraldehyde in PBS (pH 7.2), specimens were postfixed in 1% osmium tetroxide, dehydrated in graded ethanol and propylene oxide and then were embedded in Araldite resin. Serial ultrathin sections were cut on a LKB-III ultratome. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with the aid of a Hitachi H7000 electron microscope at an accelerating voltage of 100 kV.

Endothelial cells, intimal macrophages, lymphocytes, vascular dendritic cells, mast cells and smooth muscle cells were identified using the criteria described in detail in our previous publication [37]. Electron-microscopic criteria for identification of degenerative alterations have also been detailed previously [38].


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 VE-cadherin expression in non-atherosclerotic arterial segments
Aortic specimens included the adventitia, media and intima but the adventitia was not present in specimens taken at carotid endarterectomy. In all non-atherosclerotic aortic segments, VE-cadherin expression was observed in the adventitia and the outer one third of the media, while the intima and inner two thirds of the media were free of both VE-cadherin expression and von Willebrand factor. Similarly, no expression of VE-cadherin was detected in the intima of non-atherosclerotic carotid arteries. In the aortic adventitia, VE-cadherin was expressed by endothelial cells of the vasa vasora (Fig. 1A–C), the patterns of staining suggesting that VE-cadherin was localised to the inter-endothelial contact zones (Fig. 1A–C). Analyses of parallel sections stained with von Willebrand factor and VE-cadherin and double immunostaining showed that VE-cadherin expression was restricted to von Willebrand factor positive cells (Fig. 1D, E). Electron-microscopic examination demonstrated that endothelial cells of the vasa vasora formed typical contacts through adherence-type junctions. Variants of individual inter-endothelial contacts within the vasa vasorum are shown in Fig. 2A–C. Endothelial cells lining the microvessels of the media, which were continuous with microvessels of the adventitia, also expressed VE-cadherin and formed inter-endothelial contacts (Fig. 3A–C). The ultrastructure of these inter-endothelial contacts was analogous to that in the vasa vasorum (Fig. 3C).


Figure 1
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  		Fig. 1 VE-cadherin and von Willebrand factor expression in vasa vasorum in the adventitia (A–F). A–D: Patterns of distribution of VE-cadherin expression which appear different according to the angle of section of the vessels depicted in the illustration. Note that VE-cadherin expression is located exclusively along the inter-endothelial contacts. B: Shows a cross section of a microvessel in which the location of VE-cadherin is indicated by arrows. E: Immunostaining of endothelium with antibody to von Willebrand factor (arrows). Note continuous staining of the endothelium along the lumen of the microvessel. A–E: ABC immunoperoxidase technique; The expression of VE-cadherin expression and von Willebrand factor demonstrated using 3,3'-diaminobenzidine resulting in a brown coloured product; counterstaining with Mayer’s haematoxylin. F: Double immunostaining of the endothelium with antibodies to von Willebrand factor and to VE-cadherin to show the specific co-localisation of von Willebrand factor and VE-cadherin within endothelial cells. VE-cadherin was visualised with AEC substrate kit (red product of reaction) (solid arrows) while von Willebrand factor immunostaining was produced with Fast Blue substrate kit (blue product of reaction) (open arrows). In A–F, L=the lumen of microvessels. Original magnifications: x400, x400, x860, x860, x860, x980.

 

Figure 2
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  		Fig. 2 Ultrastructural appearance of the endothelium of adventitial vasa vasorum (A–D). A: Low magnification of a microvessel surrounded by collagen (C) and elastin fibres (E). B–D: Variants of inter-endothelial contacts (arrows). Note that contacts between endothelial cells are formed by interdigitating folds of contiguous plasmalemma. Note also that the cytoplasm of the endothelial cells is rich in filaments and contains vesicles and mitochondria. In A–D, the lumen of the microvessels is indicated by stars. A–D: Electron micrographs. Original magnifications: x6200, x20 800, x17 800, x17 800.

 

Figure 3
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  		Fig. 3 VE-cadherin expression and the appearance of inter-endothelial contacts in microvessels in the media (A–C). Fig. A demonstrates VE-cadherin expression (brown colour) in a cross section of microvessels (arrows) while figure B shows the distribution of VE-cadherin in a longitudinal section of a medial microvessel. A and B: ABC immunoperoxidase technique; counterstaining with Mayer’s haematoxylin; original magnifications: x400, x400. C: Shows a typical ultrastructural appearance of inter-endothelial contacts (arrows); electron micrograph; original magnification: x17 800.

 
3.2 VE-cadherin expression in atherosclerotic lesions of aortas and carotid arteries
Within atherosclerotic arterial segments, VE-cadherin expression was present not only in the adventitia and media, but also in the intima where VE-cadherin expression was restricted to the endothelial cells in areas of neovascularisation (Table 2). In general, the patterns of distribution of VE-cadherin expression within atherosclerotic lesions were similar in the carotid and aortic specimens. VE-cadherin was expressed in 18% of lipid streaks in carotid arteries and 33% of lipid streaks in aortas (Table 2). In non-complicated atherosclerotic plaques, neovascularisation was more prominent and VE-cadherin expression was detected in 83% of carotid plaques and in 88% of aortic plaques (Table 2).


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  		Table 2 VE-cadherin expression in non-atherosclerotic arterial segments and in different types of atherosclerotic lesions

 
Neovascularisation was well developed in all complicated atheromatous plaques, namely, those displaying a typical structure with a necrotic core containing calcified deposits of varying sizes and the presence of a fibrous cap on the luminal side. Within atheromatous plaques, plexuses of neovascularisation were developed mainly beneath the necrotic core at the junction between the intima and media (Fig. 4A). Within each atheromatous plaque, VE-cadherin was expressed in all the microvessels formed by neovascularisation, although the patterns of VE-cadherin distribution differed within individual microvessels (Fig. 4A–E). The patterns of VE-cadherin distribution were dependent on the structural integrity of the endothelium within neovessels and their sprouts and were also related to the degree of accumulation of round-shaped cells having the appearance of inflammatory cells around microvessels (Fig. 4A–E; Fig. 5A, B). Immunohistochemical staining with various cell-type specific antibodies showed that most of these round-shaped cells stained positively with anti-CD3 antibody (Fig. 5C).


Figure 4
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  		Fig. 4 Variants of VE-cadherin expression in atheromatous plaques (A–E). A: The distribution of VE-cadherin in a neovessel located at the base of an atheromatous plaque, at the border between the media and intima. B: Shows the regular distribution of VE-cadherin within formed neovessels (small arrows) and their sprouts (large arrows). C: Neovessel surrounded by inflammatory cells shows disorganisation of VE-cadherin expression (small arrows); large arrows show sprouts of neovascularisation exhibiting intense staining with antibody to VE-cadherin. D and E: Loss of regularity of VE-cadherin expression within neovessels surrounded by large numbers of inflammatory cells. In figures D and E, note the decreased intensity of immunostaining with antibody to VE-cadherin. A–E: ABC immunoperoxidase technique; counterstaining with Mayer’s haematoxylin; original magnification: x400, x400, x520, x320, x320.

 

Figure 5
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  		Fig. 5 Disturbance in the regularity of VE-cadherin expression, disruption of the integrity of neovessels, and accumulation of immunocompetent cells around damaged neovessels within atheromatous plaques. A: VE-cadherin expression within neovessels surrounded by inflammatory cells. Note the uneven distribution of VE-cadherin expression along the neovascular endothelium. The neovessel lumen is marked by a star. B: Neocapillaries (stars) exhibiting different degrees of loss of von Willebrand factor in the intimal areas enriched with inflammatory cells. C: Accumulation of CD3+ cells around neovessels (stars). A–C: ABC immunoperoxidase technique; counterstaining with Mayer’s haematoxylin. D: Demonstrates the presence of S-100+ cells (rose) (arrows) attached to the luminal surface of the endothelium of neovessels (stars) surrounded by inflammatory cells; immunoperoxidase technique; visualisation of S-100+ cells was produced with AEC substrate kit resulting in the formation of a rose coloured product; counterstaining with Mayer’s haematoxylin. E: Double immunostaining showing the accumulation of S-100+ cells (rose) and CD4+ cells (brown) and their co-localisation (arrows) around intimal neovessels. The lumen of neovessels is marked by stars. Double immunostaining: S-100+CD4; APAAP+PAP technique; counterstaining with Mayer’s haematoxylin. A–E: Original magnifications: x400, x400, x400, x400, x400.

 
Within areas of neovascularisation without signs of inflammation, a small number of CD68+ cells and S-100+ dendritic cells were regularly present, while T-cells were absent or seldom seen. The majority of cells surrounding areas of neovascularisation stained positively for alpha-smooth muscle actin. In these areas, the microvessels consistently exhibited a regular distribution of VE-cadherin expression localised between endothelial cells (Fig. 4A, B).

In contrast, within the microvessels surrounded by a large number of inflammatory cells, VE-cadherin expression was distributed irregularly and often its intensity was markedly reduced (Fig. 4C–E). Staining of parallel consecutive sections with antibodies to VE-cadherin, von Willebrand factor, CD3, CD4, CD8 and CD68 confirmed that the different patterns of VE-cadherin expression were related to varying degrees of inflammatory cell accumulation, especially CD3+ cells around microvessels. T-cell subpopulations were estimated in serial sections stained with anti-CD4 and anti-CD8 antibodies and their proportions within individual lesions were found to range from about equal numbers to three CD4+ cells for every one CD8+ cell. Immunohistochemical examination revealed that microvessels which displayed a decreased expression of VE-cadherin were surrounded by larger numbers of T-cells and macrophages. The loss of VE-cadherin within inter-endothelial contacts was found to relate to a breakdown in the continuity of the endothelial lining within capillaries which was evidenced by the staining of parallel sections with antibody to von Willebrand factor.

Of the inflammatory infiltrates associated with areas of neovascularisation, approximately 90% contained S-100+ cells (Fig. 5D, E). Analysis of parallel consecutive sections showed that S-100+ cells often co-localised with CD3+ cells. Double immunostaining demonstrated that within these inflammatory infiltrates, S-100+ cells formed contacts with CD4+ cells (Fig. 5E). S-100+ cells were also regularly observed within the lumen of microvessels formed by neovascularisation (Fig. 5D).

Electron-microscopic examination of areas of neovascularisation confirmed that the neovessels showed varying degrees of endothelial integrity (Fig. 6A–D; Fig. 7A–D). Within neovessels located in the intimal matrix which contained only few inflammatory cells as well as in early sprouts of neovessels located amongst inflammatory and foam cells, the endothelial lining was continuous and the inter-endothelial zones contained adherence-type junctions (Fig. 6A, B). In contrast, within neovessels which were surrounded by foam cells and a large number of inflammatory cells, some endothelial cells contained varying numbers of lipid inclusions and some endothelial cells appeared to be swollen (Fig. 7C, D).


Figure 6
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  		Fig. 6 Ultrastructural appearance of endothelial cells forming neovascular sprouts and neovessels within atheromatous plaques (A–D). A: Neovascular sprout lacking luminal space. Note that the neovascular sprout is located between intimal cells which are filled with ‘lipid droplets’. Within the neovascular sprout, the endothelial cells contacts are filled with material of high electron density. B: A detail of figure A showing the complexity of the inter-endothelial contacts. C: The appearance of endothelial cells within a neovessel, the lumen of which is filled with erythrocytes. The cytoplasm of the endothelial cells contains multiple lipid inclusions. D: Shows the penetration of a mononuclear cell (star) through the neovascular endothelium (E). The neovascular endothelium contains lipid inclusions and the cytoplasm of the migrating mononuclear cell contains cisterns of a tubulovesicular system (arrows) as well as mitochondria and free ribosomes while it lacks other cytoplasmic organelles. L=the lumen of the neovessel. A–D: Electron micrographs. Original magnifications: x11 400, x19 900, x10 000, x14 800.

 

Figure 7
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  		Fig. 7 Destabilisation and degenerative alterations of neovascular endothelium in atheromatous plaques (A–D). A: An inter-endothelial contact showing sites in which the distance between the plasmalemma of neighbouring endothelial cells is increased (shown by arrows). B: The penetration of a mononuclear cell (marked by star) through the neovascular endothelium, the cytoplasm of which is oedematous. C: Dispersion of euchromatin, condensation of heterochromatin along the inner surface of the degenerating nuclear membrane, and oedema of the cytoplasm in a neovascular endothelial cell suggesting its necrotic death. D: Endothelial oedema (asterisks) and loss of integrity within the neovessel. Arrows indicate where the neovessel lacks a continuous lining by endothelial cells. Note the degenerative alterations in the nucleus and numerous ‘rest bodies’ in the cytoplasm of a cell (marked by star) located within the lumen of the degenerating neovessel. In figures A–D, L=the lumen of neovessels. A–D: Electron micrographs. Original magnifications: x17 800, x10 900, x17 500, x8100.

 
Within neovessels surrounded by inflammatory and foam cells, some inter-endothelial cell contacts were wider apart than normal (Fig. 7A). Some appeared to be open and blood cells passing through these open intercellular zones was observed (Fig. 6D; Fig. 7B). Some penetrating blood cells contained cisterns of the tubulovesicular system in their cytoplasm suggesting that these cells might relate to dendritic cells (Fig. 6D).

In some of the neovessels surrounded by inflammatory and foam cells, endothelial integrity had broken down (Fig. 7C, D). In these neovessels, the euchromatin within the nuclei of the endothelial cells was often dispersed and their cytoplasm was swollen implying necrosis of endothelial cells (Fig. 7C, D).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The present study is the first to demonstrate that VE-cadherin is expressed by intimal neovessels in atherosclerotic plaques. Despite the significance of neovascularisation for local immune-inflammatory reactions in atherosclerotic plaque [15,16,21], the mechanisms responsible for the formation of neovessels in the plaque are poorly understood and what triggers capillary endothelial cell growth from the outer part of the media into the intima is unknown. The present observation that VE-cadherin is consistently expressed in early sprouts of neocapillaries suggests that VE-cadherin is involved in the ingrowth of medial capillaries into the intima. Our observations that, in the arterial wall, VE-cadherin is expressed by von Willebrand factor positive cells, concurs with the well established fact that the VE-cadherin gene is exclusively and consistently expressed by endothelial cells [22–33]. VE-cadherin gene encodes a Ca2+-dependent cell adhesion molecule required for the organisation of inter-endothelial junctions [22–33].

How neovascular endothelium in atherosclerotic plaques is organised is unclear. In other tissues, adherence-type junctions act as a focal point for connections between the endothelial cell plasma membrane and its underlying actin–cytoskeleton complex [39–42]. Adherence-type junctions contain cadherins, a family of single-span transmembrane glycoproteins which directly associate with structural components of the cytoskeleton and which mediate Ca2+ dependent cell-to-cell adhesion in a homotypic fashion [22–24]. Vascular endothelial (VE)-cadherin is specific to the vascular endothelium and localises exclusively at lateral junctions of intact confluent endothelium [22–24]. VE-cadherin associates with the cytosolic proteins, alpha- and beta-catenins to form a complex at endothelial cell-to-cell contacts [22–24]. In vitro, VE-cadherin, alpha-catenin, and beta-catenin have been shown associated with each other at early stages of intercellular adhesion and become readily organised at nascent cell contacts. Gamma-catenin, also known as plakoglobin, becomes associated with cell-to-cell junctions only when endothelial cells approach confluence [22–24]. When endothelial cells migrate, this order is reversed, namely, plakoglobin dissociates first and then, VE-cadherin, alpha-catenin, and beta-catenin disassemble from the junctions [22–24]. The late association of plakoglobin with junctions suggests that while VE-cadherin/alpha-catenin/beta-catenin complex can function as an early recognition mechanism between endothelial cells, the formation of ‘mature’, cytoskeleton-bound junctions requires plakoglobin synthesis and organisation [22–24].

Vasa vasorum growing into the base of the plaque leads to intimal neovascularisation and provides an entry route for inflammatory cells and plasma proteins to access the intima [15,16,20]. Our study suggests that the degree of inflammatory cell accumulation within the atheromatous plaque relates to the expression of VE-cadherin. The present observations also suggest that downregulation of VE-cadherin allows inflammatory cells to enter the intima since the loss of VE-cadherin expression was found to be associated with a breakdown in the endothelial integrity within the neovasculature.

Further investigations are required to examine the molecular mechanisms responsible for the unzipping of inter-endothelial contacts within the plaque neovasculature. However, recent in vitro studies showing that leukocyte adhesion, and not transmigration, induces disruption of the VE-cadherin complex and its loss from lateral junction localisation [43,44], suggest that the downregulation of VE-cadherin in plaque neovascularisation may precede the structural disorganisation of inter-endothelial contacts. Leukocyte adhesion to TNF-alpha-activated endothelial monolayers has been found to significantly alter the molecular composition and organisation of the endothelial cell VE-cadherin complex, which has been implicated in the maintenance of endothelial cell-to-cell adhesion and cell-to-cytoskeleton integrity [43]. Leukocyte adhesion to confluent TNF-alpha-activated endothelial monolayers induces dissociation of beta-catenin and plakoglobin from VE-cadherin and loss of their lateral junctional co-localisation [43]. The engagement of adhesion molecules has also been implicated as a crucial factor in the triggering of endothelial cell-dependent events [43–46]. Endothelial cell-leukocyte adhesion initiates an inter-endothelial cell-dependent change that correlates with leukocyte transmigration [43–47]. Detailed studies are needed in order to determine the precise mediators responsible for the expression and downregulation of VE-cadherin in atherosclerosis and to analyse the correlation of the expression of VE-cadherin with the expression of other cell adhesion molecules.

The present work supports previous evidence that neovascularisation occurs where immuno-inflammatory reactions are most intense within plaques [15,16,21]. The mononuclear cell adhesion molecule VCAM-1 is expressed in human neovasularisation in a large proportion of atherosclerotic plaques, which suggests that leukocytes may be recruited into plaques through the neovessels [15,17]. In plaques, the expression of E-selectin and ICAM-1 is most prevalent within the neovasculature rather than on arterial luminal endothelium [16]. The expression of VCAM-1 and ICAM-1 in neovascular endothelium is also associated with increased intimal leukocyte accumulation [15,16]. Together, these findings strongly imply that leukocyte recruitment through intimal neovascularisation is important in atherogenesis [15,16]. Recently, we demonstrated that areas surrounding the neovasculature frequently contain antigen-presenting dendritic cells co-localising and forming clusters with T-cells [21,48] which may suggest the initiation of primary immune responses around zones of neovascularisation. Furthermore, macrophages surrounding neovessels express acidic fibroblast growth factor (aFGF, FGF-1) suggesting that inflammatory cells might influence the growth of new vessels [49].

Disorganisation of inter-endothelial contacts may allow antigen-presenting dendritic cells to infiltrate plaques via new vessels. Atherosclerotic lesions contain vascular dendritic cells which reside in their immature form within the normal intima and which become activated during atherogenesis [50–52]. Our observations also suggested that in addition to resident vascular dendritic cells, blood dendritic cells may infiltrate the lesions during plaque formation [21]. The frequent location of dendritic cells within areas of neovascularisation as well as the detection of S-100 positive cells within the lumen of the microvessels support the opinion that blood dendritic cells might penetrate into atherosclerotic plaques through neovascularisation.

During the last decade, inflammatory CAMs responsible for heterotypical cell-to-cell interactions have received considerable attention in studies on atherosclerosis and their significance in the accumulation of intramural inflammatory infiltrates has been appreciated [9–17]. In contrast, CAMs determining normally homotypical cell interactions have received only recent and limited attention [53]. The present work expands our knowledge of homotypical cell adhesion molecules involved in atherogenesis. Understanding the mechanisms responsible for neovascularisation may lead to strategies impending the growth of plaques occurring from an accumulation of intramural inflammatory infiltrates.

Time for primary review 32 days.


   Acknowledgements
 
We thank Dr Jennifer R. Allport, Brigham and Women’s Hospital, Boston, for a kind gift of the antibody to VE-cadherin which was used in our initial pilot study. We thank the St. Vincent’s Clinic Foundation, Sydney, Australia, for financial support.


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

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