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(Stroke. 1997;28:77-82.)
© 1997 American Heart Association, Inc.

Articles

Focal Expression of Intercellular Adhesion Molecule-1 in the Human Carotid Bifurcation

Matthias Endres, MD; Ulrich Laufs, MD; Hartmut Merz, MD Manfred Kaps, MD

the Klinik fur Neurologie (M.E., M.K.) and Institut fur Pathologie (H.M.), Med Universitat zu Lubeck, and Klinik III fur Innere Medizin, Universitat Koln (U.L.) (Germany).

Correspondence to Matthias Endres, MD, Stroke and Neurovascular Regulation Laboratory, Massachusetts General Hospital, Harvard Medical School, 149 13th St, Room 6403, Charlestown, MA 02129. E-mail endres@helix.mgh.harvard.edu.

	Abstract

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Background and Purpose In the carotid bifurcation, atherosclerotic plaques usually develop in the outer wall of the internal carotid artery. The aim of this study was to analyze the expression of the cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in the carotid bifurcation. These molecules play a role in inflammatory cell recruitment and atherosclerosis.

Methods We examined the expression of ICAM-1, VCAM-1, and E-selectin in 22 human carotid bifurcation specimens by means of immunohistochemistry. Double immunostaining was performed with antibodies against CD-3, CD-31, CD-68, and -smooth muscle actin combined with each of the cell adhesion molecule antibodies. In situ hybridization for ICAM-1 was performed in selected specimens.

Results A profound focal expression of ICAM-1 in the outer wall of the internal carotid artery could be demonstrated (86% of all specimens). This focal expression could be shown in histologically normal appearing bifurcations of young adults. ICAM-1 was expressed by subsets of macrophages and smooth muscle cells and by endothelial cells. VCAM-1 and E-selectin showed no focal expression and were not found in normal carotids. In advanced plaques all three adhesion molecules—ICAM-1, VCAM-1, and E-selectin—were expressed.

Conclusions We were able to demonstrate a focal expression of ICAM-1 in the outer lateral wall of the internal carotid artery, which is a high-risk region for the development of atherosclerotic lesions.

Key Words: atherosclerosis • carotid arteries • cell adhesion molecules • immunohistochemistry

	Introduction

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Although atherosclerotic lesions can be observed throughout the body, certain areas of the arterial tree are particularly prone to the development of lesions. Usually these sites are subject to increased hemodynamic stress.¹ The human carotid bifurcation is a high-risk region for the development of atherosclerotic plaques, which may lead to complications such as thrombosis, arterio-arterial embolism, and stroke.² The atherosclerotic plaques usually develop in the outer wall just opposite the carotid bifurcation.³

It is currently accepted that atherosclerotic lesions contain an immune-mediated inflammatory reaction,^{4 5} an observation that was first made by Virchow.⁶ Recently, active expression of several genes has been reported in developing atherosclerotic lesions. Among these, the CAMs may have an important role in the selective recruitment of inflammatory cells from the circulation into the lesion.^{7 8 9 10 11 12 13 14 15 16}

ICAM-1 and VCAM-1 belong to the immunoglobulin gene superfamily, while E-selectin (previously endothelial leukocyte adhesion molecule-1) belongs to the selectin family.¹¹ The binding of leukocytes to CAMs is mediated by selective counterreceptors (ß-integrins such as LFA-1 and Mac-1 for ICAM-1, VLA-4 for VCAM-1, and sialylated Lewis antigen for E-selectin).¹¹ Adhesion molecule expression can be induced selectively by the actions of various cytokines on endothelial cells. Interleukin-1 and tumor necrosis factor induce ICAM-1, VCAM-1, and E-selectin, whereas interferon gamma induces ICAM-1 alone.¹¹ Interleukin-1 mRNA and tumor necrosis factor protein have been demonstrated in macrophages and smooth muscle cells of atherosclerotic plaques in vivo.^{17 18 19}

In this study we investigated the expression of the CAMs ICAM-1, VCAM-1, and E-selectin in the human carotid bifurcation on the protein level as well as on the mRNA level in situ. CAM expression was studied in the different anatomic regions of the bifurcation. We particularly wanted to compare the expression in the outer lateral wall to that of the inner flow dividing wall. The carotid bifurcation appeared especially appropriate for our study because the well-studied hemodynamic condition phenomena^{20 21 22} allow sophisticated in vivo analysis of local hemodynamics (Fig 1).

View larger version (85K): [in this window] [in a new window]   Figure 1. Color-coded duplex ultrasound: axial (transverse) section of the human carotid bifurcation. Region of recirculation at the lateral wall of the internal carotid bifurcation (left) is coded in blue.

	Methods

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Human Carotid Bifurcation Specimens
A total of 22 autopsy specimens of carotid artery bifurcations from 17 individuals were obtained. The time interval from death to obtaining the specimens ranged from 12 to 24 hours; previous studies had shown good preservation of cell antigen for immunocytochemistry up to 3 days.^{9 10} The bifurcations were excised in toto 4 cm distal and proximal to the bifurcation, placed in 0.9% normal saline, frozen in liquid nitrogen, and stored at -80°C. Two surgical endarterectomy carotid artery specimens were obtained directly after excision, placed in normal saline, frozen, and stored at -80°C as well. Serial sections (6 µm) were cut on a cryostat at -20°C, collected on silane-coated slides, air dried overnight, and fixed in acetone for 10 minutes at room temperature before immunostaining. Transverse sections were cut at the level of the bifurcation and 1 and 2 cm proximal and distal of the bifurcation, respectively. In this way continuous sections and correct anatomic representation at the levels of the common carotid, the bulbus, the bifurcation, and the internal and external carotid arteries were obtained. The internal and external carotid arteries were prepared on the same slide.

For in situ hybridization experiments, additional sections were fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 20 minutes, washed twice with phosphate-buffered saline for 5 minutes each and once in 0.9% normal saline for 10 minutes, and then dehydrated by an ethanol series (40%, 60%, 85%, 90%, 95%, and 100%).

Sections were stained with hematoxylin and eosin as well as elastica van Gieson. The specimens were classified according to conventional histological criteria as follows: normal, diffuse intimal thickening, fatty streaks, fibro-fatty plaques, or advanced plaques.

Immunohistochemistry
We used the following mouse anti-human antibodies: anti–ICAM-1 (clone RR 1/1, Bender MedSystems); anti–VCAM-1 (Southern Biology Associates); anti–E-selectin (clone CL26CL0B7, Bender MedSystems); anti–-smooth muscle actin (clone 1A4), which in this context is specific for smooth muscle cells; anti–CD-68 (clone KP1), which recognizes macrophages; anti–CD-31 (clone JC/70A) for endothelial cells; anti–CD-3 (clone 4B5) for T-lymphocytes; and anti–CD-19 for B-lymphocytes (all antibodies by Dako A/S).

Single-label immunohistochemistry was performed according to the alkaline phosphatase/anti–alkaline phosphatase method with new fuchsin or fast-blue BB as substrate. Double immunostaining was performed according to a three-stage avidin-biotin method with fast-blue BB as substrate followed by the alkaline phosphatase/anti–alkaline phosphatase method with new fuchsin, enabling the identification of cell phenotypes associated with adhesion molecule expression. All sections were counterstained with hematoxylin. Antibodies were used at the following concentrations: anti–ICAM-1, anti–VCAM-1, and anti–E-selectin, 1:50; anti–-smooth muscle actin, 1:9000; anti–CD-68, 1:200; anti–CD-31, 1:60; anti–CD-3, 1:300; and anti–CD-19, 1:300.

Negative controls were performed by replacing antibodies with Tris-buffered saline or monoclonal mouse anti-rabbit immunoglobulin (Dako) (irrelevant antibody), as described elsewhere.¹⁴ Normal human tonsillar tissue and two surgery carotid artery specimens were used as positive controls.

In Situ Hybridization Techniques
³⁵S-labeled cRNA probes for in situ hybridization were produced by in vitro transcription. The full-length 1.9-kb EcoRI cDNA fragment of the human ICAM-1 gene in the expression vector pBluescript sk- (Stratagene Inc) was obtained from ATCC. For in vitro transcription, linearization of the plasmids was performed with Kpn I and HindIII. The cDNA was transcribed into an antisense riboprobe with the use of T7 RNA polymerase as described previously,²³ with ³⁵S-UTP (New England Nuclear) as the radioactive label. Labeled probes were purified from unincorporated nucleotides by gel filtration with DNA-grade Sephadex G-50 (Pharmacia LKB). Probes were stored at -70°C and used within 5 days after synthesis. As negative controls, ³⁵S-labeled cRNAs in sense orientation were produced by using the RNA polymerase promotor at the 5' end of the cDNA. Tissue sections were fixed with paraformaldehyde and then treated with proteinase K.²³

After prehybridization, in situ hybridization in the presence of 50% formamide and 10% dextran sulfate with 700 000 dpm/µL per section of denatured cRNA probe was performed with either sense or antisense ICAM-1 probes.²³ Finally, the hybridized sections were washed and exposed to Kodak SB5 x-ray film for 3 to 5 days and then coated with Kodak NTB-2 photoemulsion. After 30 days, the slides were developed and counterstained slightly with hematoxylin and eosin.

	Results

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The maximum grade of atherosclerosis was determined in the carotid artery specimens. Of 22 specimens, 8 (36%) were found to have advanced plaques, 4 (18%) had fibro-fatty plaques, 4 (18%) had fatty streaks, and 6 (27%) had diffuse intimal thickening or were normal. Typically, maximum atherosclerotic alteration was found in the outer wall of the internal carotid artery opposite the bifurcation (14 of 16 specimens with advanced plaques, fibro-fatty plaques, or fatty streaks). Two specimens showed severe atherosclerosis involving the entire vessel circumference. Atherosclerotic alteration of the external carotid and the common carotid arteries was sparse and always less pronounced than in the internal carotid artery.

Immunocytochemistry for ICAM-1, VCAM-1, and E-selectin in human reactive tonsillar tissue and two surgery endarterectomy carotid artery specimens showed profound expression of all three adhesion molecules.

Adhesion Molecule Expression in Advanced Atherosclerosis
Expression of all three adhesion molecules could be shown in advanced plaques. The expression of E-selectin and VCAM-1 was much less intense than that of ICAM-1. E-selectin was expressed by endothelial cells overlying the plaque as well as by adventitial vessels (8 of 8 specimens). VCAM-1 showed very low expression in the arterial endothelium. The strongest immunoreactivity could be shown by subsets of smooth muscle cells and macrophages at the base of the plaques and also by endothelial cells of adventitial vessels (8 of 8 specimens).

Although strong throughout the entire atherosclerotic area, ICAM-1 showed maximal immunoreactivity in macrophage-rich zones. ICAM-1 was expressed by macrophages, smooth muscle cells, and endothelial cells. In endothelial cells overlying an atherosclerotic plaque, expression showed increased intensity. Additional staining of lymphoid aggregates and adventitial vessels was found.

Adhesion Molecule Expression in Moderate and Weak Atherosclerotic Lesions
Immunoreactivity of E-selectin and VCAM-1 in specimens with weak atherosclerotic alteration was very low. No staining of E-selectin or VCAM-1 could be seen in normal arteries. E-selectin showed occasional endothelial staining as well as some diffuse staining of the vasa vasorum in diffuse intimal thickening (1 of 6), fatty streaks (1 of 4), and fibro-fatty plaques (1 of 4). VCAM-1 showed no staining in diffuse intimal thickening at all. In fatty streaks (2 of 4) and fibro-fatty plaques (2 of 4), diffuse staining of vasa vasorum and occasional staining of mononuclear cells and smooth muscle cells were seen. Rare staining of the intimal endothelium overlying an atherosclerotic plaque could be shown.

There was a strong focal staining of ICAM-1 in the lateral outer wall of the internal carotid artery opposite the bifurcation and less intense staining in the lateral wall of the external carotid artery. This focal staining pattern could be shown in 19 specimens (86%), with some variation in the degree of expression. This focal immunoreactivity could be demonstrated at the level of the bifurcation. At the level of the common carotid artery, the bulbus, the external carotid artery, or distal parts of the internal carotid artery, no focal staining could be shown. For VCAM-1 and E-selectin, no focal staining could be demonstrated (0 of 22 specimens) (Fig 2).

View larger version (409K): [in this window] [in a new window]   Figure 2. Strong focal expression of ICAM-1 in a human carotid bifurcation with low-grade atherosclerotic alteration. Internal carotid artery is on the left side. A, ICAM-1 immunostaining (blue); B, ICAM-1 (blue) and CD-68 (red) double immunostaining; C, ICAM-1 in situ hybridization. VCAM-1 and E-selectin immunostaining show no expression (data not shown).

ICAM-1 expression was localized in the intimal endothelium, intima, and adventitial vasa vasorum, even in vessels with no or low-grade atherosclerosis (Fig 3). This focal expression was more marked in specimens with more pronounced atherosclerotic alterations. Atherosclerotic lesions of vessel regions adjacent to the lateral wall did not show this focal expression.

View larger version (402K): [in this window] [in a new window]   Figure 3. Carotid bifurcation, obtained from a male patient who died at the age of 22 years of internal hemorrhage. Internal carotid artery is on the left side. A, ICAM-1 (blue) and -smooth muscle actin (red) double immunostaining at the level of the bifurcation. Note focal expression of ICAM-1 in the outer lateral wall of the internal carotid bifurcation. B (x40) and C (x200) show higher magnifications of the lateral wall of the internal carotid artery (indicated by inset).

By means of double-labeling immunohistochemistry, macrophages, smooth muscle cells, and endothelial cells were identified as the ICAM-1–expressing cells. Areas of strong focal staining were profoundly infiltrated by macrophages. The ICAM-1 expression by macrophages and smooth muscle cells was stronger in vessels with pronounced atherosclerosis than in specimens with only beginning or moderate atherosclerotic lesions. The entire vessel wall showed profound immunoreactivity in late progressive lesions.

In Situ Hybridization Experiments
ICAM-1 mRNA was localized by in situ hybridization in selected specimens with weak atherosclerotic alterations. The focal expression on the protein level in the lateral wall of the carotid artery could be shown on the level of mRNA as well. The pattern of ICAM-1 mRNA expression was more extended than protein expression, which could clearly be demonstrated in serial sections of the same specimens (Fig 2). Control hybridizations performed on adjacent sections with a sense riboprobe demonstrated absence of a specific hybridization signal.

	Discussion

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The vascular endothelium is a dynamically mutable interface that responds to a variety of endogenous mediators, including humoral factors such as cytokines, hormones, growth factors, and exogenous products.^{24 25 26 27} The expression of adhesion molecules on the endothelium and vessel wall in atherosclerotic lesions may reflect an immune-mediated mechanism in the pathogenesis of atherosclerosis.^{7 8 9 10 11 12 13 14 15 16 28 29}

In our study the expression of the CAMs VCAM-1, ICAM-1, and E-selectin in the human carotid bifurcation was analyzed. We particularly examined the differential expression of the CAMs in the different anatomic parts of the carotid bifurcation. We were looking for a difference in immunoreactivity between the lateral outer wall of the carotid bifurcation in contrast to the inner flow dividing wall.

The most striking result was the strong focal expression of ICAM-1 in the lateral outer wall of the carotid bifurcation, mainly in the internal carotid artery and less pronounced in the external carotid artery. Interestingly, in vitro hemodynamic measurements and color-coded duplex ultrasound showed that at the lateral wall, where plaque formation occurs, shear stress is low and oscillates with regions of recirculation and vortex formation (References 3, 20, 30-32 and Fig 1). At the flow divider site, shear stress varies but remains relatively high and unidirectional. Atherosclerotic plaque development typically does not occur in the high-flow regions of arteries but rather downstream from the branches in regions of flow separation with relatively low wall shear stress.³³

Recently, it was shown that ICAM-1 is upregulated by shear stress in endothelial cells in vitro, in contrast to VCAM-1 and E-selectin.³⁴ The human ICAM-1 gene sequence contains a cis-acting transcriptional regulatory element, the so-called shear stress response element.^{35 36} The shear stress response element may represent a common pathway by which biomechanical forces influence gene expression.^{24 34} In that study a single shear stress regimen with laminar shear stress was used.³⁴ In vivo there is a complex mixture of pulsatile, turbulent, and disturbed laminar flow.⁴ In vitro studies with shear stress regimens other than laminar flow have shown that the endothelium responds to different shear stress parameters. For example, gene expression of c-fos is more effectively altered by pulsatile shear stress than by laminar shear stress,³⁷ whereas for platelet-derived growth factor-A and -B, pulsatile shear stress is less effective.^{38 39 40} Our results indicate that ICAM-1 expression and plaque formation in the carotid bifurcation correlate with low shear stress and oscillation in shear stress direction but not with laminar and unidirectional high shear stress. Interestingly, the focal ICAM-1 expression is not a phenomenon of endothelial cells alone: staining of macrophages and smooth muscle cells was found within the vessel wall.

Therefore, the question arises as to whether the ICAM-1 expression is induced first and the infiltration with leukocytes into the vessel wall follows or whether the ICAM-1 expression is a sign of an already existing atherosclerotic lesion. Since nonatherosclerotic specimens showed focal ICAM-1 staining, we believe that adhesion molecule expression, among other factors, precedes the first signs of atherosclerosis. The early expression of ICAM-1 in the high-risk region of the carotid bifurcation may represent pathogenetically an interface between hemodynamics (expression due to biomechanical forces) and morphology (expression as an early sign of atherosclerosis).

VCAM-1 and E-selectin showed no focal immunoreactivity in the human carotid bifurcation and were not expressed in normal arteries or low-grade atherosclerotic alterations. VCAM-1 and E-selectin lack the shear stress response element,³⁴ and in endothelial cells no shear stress–dependent expression of VCAM-1 and E-selectin could be shown.^{34 41 42}

In accordance with previous reports, a profound expression of all three CAMs was found in advanced atherosclerotic stages.^{9 10 12 13 14 16 29 43} VCAM-1 was expressed mainly by smooth muscle cells and vasa vasorum as well as by neovascularized vessels. E-selectin was expressed mainly by endothelial cells overlying an atherosclerotic plaque and vasa vasorum, which has been reported for coronary arteries previously.^{12 13 14}

In situ hybridization experiments revealed that the pattern of ICAM-1 mRNA expression was more extended than protein expression. This suggests a difference in transcriptional and translational activity.

In conclusion, our experiments demonstrate a localized expression of ICAM-1 but not VCAM-1 and E-selectin in the lateral wall of the internal carotid artery at the level of the bifurcation. For the first time a focal expression of ICAM-1 in this high-risk region for atherosclerotic lesions could be shown. These results may contribute to the understanding of the pathogenesis of atherosclerosis in the human carotid bifurcation.

	Selected Abbreviations and Acronyms

 

CAMs = cell adhesion molecules ICAM-1 = intercellular adhesion molecule-1 LFA-1 = lymphocyte function–associated antigen-1 VCAM-1 = vascular cell adhesion molecule-1 VLA-4 = very-late-activation antigen-4

	Acknowledgments

 
We would like to thank B. Kuhlmann for competent technical support, as well as Prof Dr A.C. Feller (Institute for Pathology, Medical University of Lubeck) and Prof Dr M. Bohm (Department of Cardiology, University of Cologne) for providing laboratory facilities and their personal assistance. We also thank Drs Klaus Fink and Michael Cutrer for critically reading the manuscript. Drs Matthias Endres and Ulrich Laufs are scholars of the Deutsche Forschungsgemeinschaft.

Received July 15, 1996; revision received September 23, 1996; accepted October 15, 1996.

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