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Circulation. 1996;93:672-682

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(Circulation. 1996;93:672-682.)
© 1996 American Heart Association, Inc.

Articles

Neovascular Expression of E-Selectin, Intercellular Adhesion Molecule-1, and Vascular Cell Adhesion Molecule-1 in Human Atherosclerosis and Their Relation to Intimal Leukocyte Content

Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 15, 1993.

Kevin D. O'Brien, MD; Thomas O. McDonald, BS; Alan Chait, MD; Margaret D. Allen, MD; Charles E. Alpers, MD

From the Departments of Medicine (K.D.O., A.C.), Surgery (M.D.A.), and Pathology (T.O.M., C.E.A.), University of Washington, Seattle.


*    Abstract
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 *Abstract
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Background Leukocyte recruitment is an early event in atherogenesis, and the leukocyte adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) recently have been detected in human atherosclerosis. However, no previous study has evaluated either the distribution of these three molecules at different sites within the arterial intima or their relation to plaque leukocyte content.

Methods and Results Immunohistochemistry was performed on 99 coronary artery segments (34 controls and 65 with atherosclerotic plaque) to identify E-selectin, ICAM-1, VCAM-1, macrophages, smooth muscle cells, and T lymphocytes. For each segment, the presence or absence of adhesion molecule was determined at the arterial lumen, on intimal neovasculature, and on intimal nonendothelial cells. Each segment was scored for intimal macrophage and T-lymphocyte densities on a semiquantitative scale of 0 to 3. In atherosclerotic plaques, the prevalences of E-selectin, ICAM-1, and VCAM-1 on plaque neovasculature were twofold higher than their prevalences on arterial luminal endothelium. E-selectin was the only adhesion molecule for which expression on arterial luminal endothelial cells was more prevalent in plaques than in control segments. Increased plaque intimal macrophage density was associated with expression of VCAM-1 on neovasculature (P<.01) and on nonendothelial cells (P<.01). Increased plaque intimal T-lymphocyte density was associated with the presence of both ICAM-1 and VCAM-1 on neovasculature (both P<.01) and on nonendothelial cells (both P<.01).

Conclusions In atherosclerotic plaques, the expression of all three leukocyte adhesion molecules was more prevalent on intimal neovasculature than on arterial luminal endothelium. Further, the presence on neovasculature and nonendothelial cells of VCAM-1 and ICAM-1 was strongly associated with increased intimal leukocyte accumulation. These findings suggest that leukocyte recruitment through and/or activation of intimal neovasculature may play important roles in the pathogenesis of human atherosclerosis.


Key Words: arteriosclerosis • adhesion molecules • immunohistochemistry • endothelium • vasculature


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowDiscussion
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Leukocyte recruitment into the arterial wall is a fundamental component of atherogenesis,1 2 3 and several recent studies have examined the expression of leukocyte adhesion molecules in atherosclerosis both in animal models4 5 and in humans.6 7 8 9 The presence of several adhesion molecules has been documented in human atherosclerosis, including two members of the immunoglobulin superfamily class of adhesion molecules, ICAM-17 8 and VCAM-1,9 as well as a member of the selectin family of adhesion molecules, E-selectin.8 In general, previous studies have concentrated on the expression of adhesion molecules on arterial luminal endothelial cells. However, most human atherosclerotic plaques also contain neovasculature,10 11 12 which consists of small vessels arising primarily from the adventitial vasa vasorum.13 The possibility that neovasculature might play a role in the pathogenesis of atherosclerosis was hypothesized by Winternitz et al in the 1930s,10 followed by Geiringer in the late 1940s11 and by Barger et al in 1984.12 However, the potential participation of these vessels in atherogenesis has not been considered in animal models of this process.3

Recently, several observations from studies of human atherosclerotic plaques have refocused attention on the potential role of intimal neovasculature in atherogenesis. These include (1) identification of the mononuclear cell–specific adhesion molecule VCAM-1 on neovasculature of a large proportion of atherosclerotic plaques, suggesting that leukocytes may be recruited into the plaque through neovessels9 ; (2) demonstration that plasma proteins are present in intimal tissue surrounding plaque neovasculature, suggesting that these vessels could serve as a route for deposition of plasma proteins13 ; and (3) demonstration that macrophages surrounding plaque neovessels may express a molecule implicated in angiogenesis, aFGF, also referred to as FGF-1, suggesting that inflammatory cells might influence the growth of these vessels.14 In addition, the study that documented a high prevalence of neovasculature VCAM-1 in atherosclerotic segments further demonstrated that this adhesion molecule was detected with a much higher prevalence on neovasculature than at the arterial luminal surface of atherosclerotic plaques.9 This latter finding raised the possibility that plaque neovasculature might be a particularly important site for leukocyte recruitment into plaques. Although both ICAM-16 8 and E-selectin8 have been detected on arterial luminal endothelial cells in human atherosclerosis, no studies to date have examined systematically whether or not they also are expressed on plaque neovasculature. In addition, the relation of adhesion molecule expression at various sites in the arterial intima to intimal leukocyte content has not been evaluated.

Thus, the present study was undertaken to determine, in human coronary arterial segments, (1) the relative prevalence of each adhesion molecule in atherosclerotic compared with control coronary segments; (2) whether, like VCAM-1, the adhesion molecules ICAM-1 and E-selectin also are present on neovasculature of atherosclerotic plaques; and (3) whether adhesion molecule expression is associated with increased intimal macrophage and T-lymphocyte content.


*    Methods
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 *Methods
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Human Coronary Arterial Tissue
A total of 99 coronary artery segments were obtained from 15 hearts explanted at the time of cardiac transplantation and were placed in methanol Carnoy's solution (60% methanol/30% chloroform/10% acetic acid) within 2 hours of organ excision, fixed for at least 12 hours, and then processed and paraffin-embedded according to conventional techniques. Of the 15 patients, 9 (8 men, 1 woman) had cardiomyopathy due to atherosclerotic coronary artery disease (ISCM) and 6 (5 men, 1 woman) had IDCM. ISCM patients were slightly, but not substantially, older (age range, 45 to 66 years; median age, 54 years; mean age, 54.4 years) than IDCM patients (age range, 40 to 56 years; median age, 53.5 years; mean age, 51.5 years). The prevalences of typical atherosclerotic risk factors were surprisingly similar for the two groups, with the relative prevalences for the ISCM versus IDCM groups of 67% versus 50% for a documented lipid disorder, 17% versus 11% for current smoking, and 67% for both groups for past smoking. None of the patients had a history of type I diabetes mellitus, but the prevalence of type II diabetes was higher in the IDCM group (33%) than in the ISCM group (11%).

Six-micrometer–thick tissue sections of the arterial segments were used for immunohistochemical analyses. The 99 coronary artery segments were classified according to conventional histological criteria into (1) atherosclerotic plaque, defined by the presence of typical features of luminal narrowing due to regional accumulation of cholesterol, foam cell and non–foam cell macrophages, and the presence of fibrous caps or (2) control coronary segments with intimal accumulation of smooth muscle cells and matrix, which represents the characteristic morphology of nonatherosclerotic, adult, human coronary arteries.15 Of 34 coronary segments classified by morphological criteria as controls, the majority (20 [59%]) were from patients with IDCM, and the remainder (14 [41%]) were from patients with ISCM. Of 65 coronary segments classified by morphological criteria as atherosclerotic plaque, only 10 (15%) were from patients with IDCM, and the majority (55 [85%]) were from patients with ISCM.

Antibodies and Antisera
Polyclonal E-selectin antiserum. Goat polyclonal antiserum directed against E-selectin was a gift of Drs R. Lobb and C. Benjamin, Biogen, Cambridge, Mass.16 The immunohistochemical specificity and sensitivity of the antiserum were determined by the following criteria: (1) positive immunohistochemical staining of E-selectin–transfected but not of untransfected CHO cells; (2) positive immunohistochemical staining of tonsil and of myocardial biopsies with rejection; and (3) abolition of positive staining on tonsil, myocardium, and coronary arteries by absorption of the E-selectin antiserum against E-selectin–transfected CHO cells. This antiserum was used at a titer of 1:2000.

ICAM-1 monoclonal antibody. A monoclonal antibody against ICAM-1, 1D8, was a gift of Drs John Harlan, University of Washington, and Tim Carlos, University of Pittsburgh. This antibody was generated as described previously.17 The immunohistochemical specificity and sensitivity of the antibody were determined as follows: (1) positive immunohistochemical staining of ICAM-1–transfected but not of untransfected CHO cells; (2) positive immunohistochemical staining of tonsil and of myocardial biopsies with rejection; and (3) abolition of positive staining on tonsil, myocardium, and coronary arteries by absorption of the ICAM-1 antibody against ICAM-1–transfected CHO cells. This antibody was used at a titer of 1:500.

Polyclonal VCAM-1 antiserum. Rabbit polyclonal antiserum directed against VCAM-1 was a gift of Drs R. Lobb and C. Benjamin, Biogen.18 The immunohistochemical sensitivity and specificity of the VCAM-1 antiserum have been described extensively in a previous report.9 This antiserum was used at a titer of 1:4000.

Cell type–specific monoclonal antibodies. Cell-type identification was performed by use of the following commercially available antibodies: anti–{alpha}-actin19 (Boehringer Mannheim), used at a titer of 1:1000, which in this context is specific for smooth muscle cells, and anti-CD68 (Dako Corp), used at a titer of 1:1000, which recognizes macrophages.18

T-lymphocyte antiserum. T lymphocytes were identified with CD3 antiserum (titer, 1:100) generated in rabbits, which recognizes the common T-lymphocyte receptor CD3.20

Apolipoprotein B antibody. Apolipoprotein B was detected with antibody 9A, which recognizes an epitope located near the LDL receptor–binding region of apolipoprotein B-100. The antibody was used at a titer of 1:1000 and was a gift of Dr Santica Marcovina of the Northwest Lipid Research Laboratory, Seattle, Wash.

Lectins. The lectin Ulex europaeus I, which binds to a fucose moiety on, and thereby recognizes, endothelial cells,21 was used at a titer of 1:1000 as described previously.9

Single-Label Immunohistochemistry
Single-label immunocytochemistry was performed as described previously9 22 using the primary antisera, antibodies, or lectin. Briefly, tissue sections were deparaffinized with xylene and rehydrated with graded alcohols. The slides were blocked with 3% hydrogen peroxide; washed with PBS; incubated for 60 minutes with the primary antiserum, antibody, or lectin; and then washed again with PBS. A biotin-labeled secondary antibody, either anti-rabbit (for VCAM-1 or CD3 antiserum), anti-goat (for E-selectin antisera), anti-mouse (for anti–{alpha}-actin or anti-CD68), or anti-Ulex, then was applied for 30 minutes, followed by an avidin-biotin-peroxidase conjugate (ABC Elite, Vector Laboratories) for 30 minutes. Standard peroxidase enzyme substrate, 3,3'-diaminobenzidine with nickel chloride, then was added to yield a black reaction product. The slides were counterstained with methyl green.

Negative controls included substitution of the primary antiserum, antibody, or lectin with either PBS or irrelevant antibodies to abolish staining.

Characterization of Plaque Leukocyte Content
Each coronary artery segment was scored for macrophage and T-lymphocyte content on a semiquantitative scale ranging from 0 to 3 in which 0 indicated absence of the cell type; 1, occasional isolated cells; 2, small focal collections of the cell type; and 3, large foci of the cell type. Scoring was performed independently by two observers (K.D.O. and T.O.M.) blinded to the results of adhesion molecule immunohistochemistry, and agreement was nearly 100%.

Statistical Analyses
The significance of differences between plaque and control segments in prevalences of individual adhesion molecules in the intima, adventitia, arterial luminal endothelium, and nonendothelial cells was tested with Fisher's exact test if the expected value for any cell was <5 or with Yates' corrected {chi}2 test if the expected value was >=5. Neovasculature was not detected in any control segments, so adhesion molecule expression could not be compared between plaque and control segments at this site. The significance of associations between plaque macrophage or T-lymphocyte densities and the prevalence of expression of each adhesion molecule on arterial luminal endothelial cells, on neovasculature, or on nonendothelial cells were tested by the {chi}2 test for linear trend in proportions; however, because no plaque segments with a macrophage score of 1 had intimal ICAM-1 expression, formal testing of associations of macrophage score with prevalences of ICAM-1 expression on arterial luminal endothelial cells, neovasculature of nonendothelial cells could not be performed. Statistical analyses were performed with the SPSS+ statistical program, and values of P<.05 were considered significant.


*    Results
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 up arrowMethods
*Results
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All Three Adhesion Molecules Are More Prevalent in the Intima of Atherosclerotic Plaques Than in the Intima of Control Arteries
All three adhesion molecules were detected in atherosclerotic plaques. Adhesion molecules were significantly more prevalent in atherosclerotic plaques than in control segments (all, P<.01) (Table 1Down). Of the three adhesion molecules, E-selectin was detected with the lowest frequency in atherosclerotic intima (21 of 65, or 32%), but its presence was highly specific for atherosclerosis, since it was not detected in any control segments. ICAM-1 also was much more prevalent in the intima of atherosclerotic (30 of 65, or 46%) than in control (6 of 34, or 18%) coronary segments. Although VCAM-1 was found with the highest prevalence in atherosclerotic intima (53 of 65, or 82%), it also was detected in the intima of a substantial minority of control arteries with diffuse intimal thickening (16 of 34, or 47%).


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  		Table 1. Prevalence of Adhesion Molecules in Intima of Atherosclerotic and Control Coronary Segments

Further, of 9 patients with ISCM, intimal E-selectin was detected in 5 of 9 patients (56%), intimal ICAM-1 was detected in 8 of 9 patients (89%), and intimal VCAM-1 was detected in 9 of 9 patients (100%), indicating that expression of adhesion molecules was not restricted to the plaques from a few individuals.

Distribution of Adhesion Molecules Within the Intima
The presence or absence of adhesion molecules at three distinct anatomic locations within the intima of atherosclerotic and control coronary segments was determined (1) on endothelial cells at the arterial luminal surface, (2) in association with neovasculature of atherosclerotic segments, and (3) on nonendothelial cells (Table 2Down).


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  		Table 2. Distribution of Intimal Adhesion Molecules

Adhesion molecule expression on arterial luminal endothelial cells. At the arterial lumen, only E-selectin expression was specific for atherosclerosis, since E-selectin was present on arterial luminal endothelial cells in 9 of 65 atherosclerotic segments (14%) but in none of the control segments (P<.03). ICAM-1 was detected on arterial luminal endothelial cells with similar prevalences of 9 of 65 (14%) in atherosclerotic segments and 6 of 34 (18%) in control segments. Similarly, VCAM-1 was detected on arterial luminal endothelial cells in 28 of 65 atherosclerotic segments (43%) and in 14 of 34 control segments (40%). Thus, in these specimens, neither ICAM-1 nor VCAM-1 expression on arterial endothelial cells was associated with histologically identified atherosclerosis. E-selectin expression on arterial luminal endothelial cells was highly specific for atherosclerotic segments, but the prevalence of E-selectin expression at this site was quite low. Therefore, the increased prevalence of adhesion molecule expression in atherosclerotic compared with control intima was due primarily to adhesion molecule expression on neovasculature and on nonendothelial cells rather than at the arterial lumen.

Neovascular adhesion molecule expression. In human coronary arteries, neovasculature that infiltrates into the intima is a characteristic of atherosclerotic segments but is not found in control segments. Of the atherosclerotic segments included in this study, 59 of 65 (91%) had infiltration of neovessels into the intima, which could be identified by examination of hematoxylin-eosin–stained sections. The presence of neovascular endothelial cells was confirmed by immunohistochemistry with the lectin Ulex europaeus I on neighboring sections. In contrast, evaluation of hematoxylin and eosin–stained sections, as well as of Ulex-stained sections, confirmed that intimal neovessels were not present in any of the segments classified as controls by morphological criteria.

In atherosclerotic segments, the prevalence of expression of all three adhesion molecules was approximately twofold higher for neovasculature than at the arterial luminal surface. For all plaques, the percentages of atherosclerotic segments with adhesion molecule expression on neovasculature versus arterial luminal endothelial cells were as follows: for E-selectin, 28% versus 14%; for ICAM-1, 25% versus 14%; and for VCAM-1, 69% versus 43% (all, P<.01). If the 6 plaques without neovasculature are excluded from consideration, the relative prevalences of adhesion molecule expression on neovasculature versus at the arterial lumen are, for E-selectin, 31% versus 16%; for ICAM-1, 28% versus 14%; and for VCAM-1, 76% versus 41%. Therefore, all three leukocyte adhesion molecules may be detected on neovascular endothelial cells, consistent with the hypothesis that the neovasculature may serve as an important route for inflammatory cell entry into the atherosclerotic plaque. An example of E-selectin, ICAM-1, and VCAM-1 expression on plaque neovasculature in a region with infiltration of macrophages and T lymphocytes and apolipoprotein B deposition is shown in Fig 1Down, while Fig 2Down shows, at higher magnification, neighboring sections of another plaque, with neovascular expression of all three adhesion molecules in a region with prominent accumulation of both macrophages and T lymphocytes.



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  		Figure 1. Photomicrographs showing expression of E-selectin, ICAM-1, and VCAM-1 in a plaque with neovasculature, macrophage, and T-lymphocyte infiltration and apolipoprotein B accumulation. Expression of adhesion molecules (solid arrows) on neovascular endothelium of an atherosclerotic plaque is demonstrated by the black immunoreaction products of the polyclonal anti–E-selectin antiserum (a), the monoclonal anti–ICAM-1 antibody (b), and the polyclonal anti–VCAM-1 antiserum (c). High background staining noted with the polyclonal anti–E-selectin antiserum in this particular section (a) was not typical of the arterial sections of this study. Presence of endothelial cells in these vessels is confirmed by staining with Ulex lectin (d). Also, VCAM-1 is present on numerous nonendothelial cells, including both smooth muscle cells and macrophages, in the intima as well as on occasional nonendothelial cells in the media (c). Surrounding the neovessels is a prominent inflammatory cell infiltrate, composed of both macrophages, as identified with the anti-CD68 antibody (e), and T-lymphocytes, as identified by the polyclonal anti-CD3 antiserum (f). In addition, immunostaining for apolipoprotein B, an apolipoprotein present on the plasma lipoproteins, LDL, and lipoprotein (a), demonstrates that apolipoprotein B is present in the intima (g) at the borders of the perineovascular inflammatory cell infiltrate (e, f). Intimal and medial smooth muscle cells, as well as neovascular pericytes, are identified with the anti–smooth muscle {alpha}-actin antibody (h). Also, all three adhesion molecules (open arrows, a through c) are present on some endothelial cells of the adventitial vasa vasorum. Magnification x200, methyl green counterstain.




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  		Figure 2. Adhesion molecule expression on plaque neovasculature and associated leukocyte infiltration. Photomicrographs of neighboring sections demonstrate with a black label the presence of E-selectin (a), ICAM-1 (b), and VCAM-1 (c) on neovascular endothelium. Particularly prominent nonendothelial cell VCAM-1 also is demonstrated in c, as well as dense accumulation in this region of macrophages (identified with a black label to the CD68 antigen) (d) and T lymphocytes (identified with a black label recognizing the CD3 antigen) (e). Magnification x400, methyl green counterstain.

Intimal nonendothelial cell adhesion molecule expression. Both ICAM-1 and VCAM-1 were detected frequently on intimal nonendothelial cells. ICAM-1 was identified on intimal nonendothelial cells in 24 of 65 atherosclerotic segments (37%) but was not present on nonendothelial cells of control intima (P<.01). VCAM-1 was detected on intimal nonendothelial cells in 45 of 65 plaques (61%) but only 4 of 34 control segments (12%) (P<.01). ICAM-1 and VCAM-1 could be detected on subsets of both macrophages and smooth muscle cells. In contrast to VCAM-1 and ICAM-1, E-selectin expression was restricted to endothelial cells; it was not detected on nonendothelial cells in any of the 99 coronary artery segments studied.

Adventitial Adhesion Molecule Expression
Intimal neovasculature has been shown by postmortem injection techniques12 and by confocal microscopy13 to arise almost exclusively from the adventitial vasa vasorum. Although expression of all three adhesion molecules on adventitial vasa vasorum was more prevalent in plaques than in control segments (Table 3Down), the difference was significant only for ICAM-1. E-selectin was detected in 17% of plaques versus 6% of controls (P=.21), whereas ICAM-1 was detected in 34% of plaques versus 3% of controls (P<.01) and VCAM-1 was detected in 62% of plaques versus 47% of controls (P=.24). An example of E-selectin, ICAM-1, and VCAM-1 expression on adventitial endothelium is shown in Fig 1Up.


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  		Table 3. Prevalence of Adhesion Molecules on Adventitial Vasa Vasorum in Atherosclerotic and Control Coronary Segments

Relation of Intimal Endothelial Cell Adhesion Molecule Expression to Intimal Leukocyte Content
Arterial luminal endothelial cell adhesion molecule expression and intimal leukocyte content. Due to insufficient sample sizes, apparent trends between intimal macrophage content and the percentage of plaques with expression of E-selectin and ICAM-1 at the arterial lumen were not formally testable (Table 4Down and Fig 3Down). However, these trends are consistent with the findings of previous studies demonstrating (1) that positive ICAM-1 staining is present on a greater percentage of the arterial luminal endothelial circumference in lesion types with significant inflammatory cell content, ie, fatty streaks and fibrofatty lesions, than in nondiseased segments or fibrous caps without prominent inflammation6 and (2) that both E-selectin and ICAM-1 are present in higher percentages both of plaques and of nondiseased segments without inflammatory cells.8 In contrast, there was no clear correlation between expression of VCAM-1 at the arterial lumen and intimal content of either macrophages or T lymphocytes.


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  		Table 4. Correlation Between Adhesion Molecule Expression at Three Anatomic Sites in the Arterial Intima and Plaque Inflammatory Cell Density



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  		Figure 3. Bar graphs showing relation between arterial luminal endothelial adhesion molecule expression and intimal leukocyte content. For each of 65 atherosclerotic plaques, the presence or absence of each adhesion molecule on arterial luminal endothelial cells was determined. In independent analyses, intimal densities of macrophages (CD68) and of T lymphocytes (CD3) were scored on a semiquantitative scale in which 1=scattered cells, 2=small focal collections of cells, and 3=large focal collections of cells. By the {chi}2 test for linear trend in proportions, no significant relations were found between arterial luminal expression of any of the adhesion molecules and intimal macrophage or T-lymphocyte content. Because of insufficient sample sizes, apparent associations between arterial endothelial E-selectin expression and macrophage score and between arterial endothelial ICAM-1 and macrophage and T-lymphocyte scores could not be formally tested. ns indicates not significant; nt, not testable.

Neovascular adhesion molecule expression and intimal leukocyte content. No previous studies have examined whether adhesion molecule expression on neovasculature is related to intimal leukocyte content. This study found no association between E-selectin expression and intimal macrophage or T-lymphocyte content. In contrast, a strong association was found between neovascular expression of VCAM-1 and intimal macrophage content (P<.01). Although there appeared to be a relation between neovascular ICAM-1 and intimal macrophage content, it could not be tested for statistical significance because of small sample size (Table 4Up and Fig 4Down). Further, strong associations also were found between neovascular ICAM-1 or VCAM-1 and intimal T-lymphocyte content (both P<.01). These results suggest that neovascular expression of the immunoglobulin superfamily adhesion molecules ICAM-1 and VCAM-1 is associated with intimal accumulation of inflammatory cells.



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  		Figure 4. Bar graphs showing relation between neovascular adhesion molecule expression and intimal leukocyte content. For each of 65 atherosclerotic plaques, the presence or absence of each adhesion molecule on neovasculature was determined and compared with intimal densities of macrophages (CD68) and of T lymphocytes (CD3). By the {chi}2 test for linear trend in proportions, significant relations were found between the presence of neovascular VCAM-1 and both intimal macrophage and T-lymphocyte content as well as between the presence of neovascular ICAM-1 and intimal T-lymphocyte content. Because of insufficient sample size, an apparent association between neovascular ICAM-1 expression and macrophage score could not be formally tested. ns indicates not significant; nt, not testable.

Nonendothelial cell adhesion molecule expression and intimal leukocyte content. A highly significant association was found between nonendothelial cell VCAM-1 expression and intimal macrophage content (P<.01) (Table 4Up and Fig 5Down). Also, there was a highly significant association between nonendothelial cell expression of both ICAM-1 and VCAM-1 and intimal T-lymphocyte content (both, P<.01). Again, although there appeared to be a relation between nonendothelial cell ICAM-1 and intimal macrophage content, this association would not be tested for statistical significance (Table 4Up and Fig 5Down). E-selectin was not detected on any cell type other than endothelial cells.



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  		Figure 5. Bar graphs showing relation between nonendothelial cell adhesion molecule expression and intimal leukocyte content. The presence or absence of ICAM-1 and VCAM-1 on intimal nonendothelial cells was compared with the intimal densities of macrophages (CD68) and of T lymphocytes (CD3). By the {chi}2 test for linear trend in proportions, significant relations were found between the presence of nonendothelial cell VCAM-1 and both intimal macrophage and T-lymphocyte content as well as between the presence of nonendothelial ICAM-1 and intimal T-lymphocyte content. Because of insufficient sample size, an apparent association between nonendothelial cell ICAM-1 expression and macrophage score could not be formally tested. ns indicates not significant; nt, not testable.


*    Discussion
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*Discussion
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Because intimal leukocyte accumulation is a fundamental component of atherosclerosis,1 2 3 identification of the specific molecules that mediate leukocyte attachment to and migration across the endothelium recently has become an area of intense interest.4 5 6 7 8 9 At least three leukocyte adhesion molecules, E-selectin, ICAM-1, and VCAM-1, have been identified in human atherosclerosis.4 5 6 7 8 9 Expression of E-selectin8 and of ICAM-16 8 at the arterial lumen has been found to be increased in arterial segments with mononuclear leukocyte accumulation. A third adhesion molecule, VCAM-1, has been detected in animal models of atherosclerosis4 5 and also has been shown to be more prevalent in the intima of atherosclerotic plaques than in nonatherosclerotic segments of human coronary arteries.9 A striking finding of the latter study was that the increased VCAM-1 expression in atherosclerosis was due to higher prevalences of the molecule on intimal neovasculature and on nonendothelial cells rather than at the arterial lumen.9 This raised the possibilities that intimal neovasculature was a potentially important route for leukocyte recruitment into plaques and that the deep portion of the atherosclerotic intima was a site of "activation" by as yet unidentified stimuli, which might include cytokines, hypoxia, or growth factors. Interest in the neovasculature as a participant in atherosclerosis also has been renewed by studies demonstrating that plasma proteins are deposited around plaque neovessels13 and documentation of the expression of the angiogenic factor aFGF in macrophages surrounding plaque neovessels.14

This study is the first to demonstrate that E-selectin and ICAM-1 are expressed frequently by plaque neovessels. It also confirms that, in the coronary arterial segments studied, VCAM-1 is expressed frequently by plaque neovessels and suggests that, in these atherosclerotic coronary segments, all three adhesion molecules are twice as prevalent in neovessels as at the arterial lumen. This indicates that the neovasculature, by virtue of upregulated expression of leukocyte adhesion molecules, may be a very important route for inflammatory cell entry into the intima. Demonstration of the presence of adhesion molecules on plaque endothelial cells does not by itself prove that these molecules have actively participated in leukocyte recruitment, but it can, at the very least, be regarded as a marker of "activation"23 of these cells by cytokines or other molecules present in the surrounding intima.

This study also is the first to correlate the degree of inflammatory cell accumulation in atherosclerotic plaques with the prevalence of expression of each of these three adhesion molecules at three different anatomic sites in the intima. Significant correlations were found between the degree of macrophage infiltration and the prevalence of E-selectin and ICAM-1 at the arterial luminal surface. These findings are consistent with the results of a study by Poston et al6 showing that the extent of arterial luminal ICAM-1 staining was greater in types of lesions with inflammatory infiltrates, ie, fatty streaks and fibrofatty lesions, than in types of lesions without inflammatory infiltrates, ie, nonatherosclerotic human arteries or fibrous plaques. Similarly, van der Wal et al8 showed that the prevalence of arterial luminal expression of both ICAM-1 and E-selectin was greater in both atherosclerotic plaques and nonatherosclerotic arterial segments with subendothelial inflammatory infiltrates than in nonatherosclerotic arterial segments without subendothelial inflammation. However, neither of these previous studies evaluated the expression of ICAM-1 or E-selectin on nonendothelial cells or on neovasculature. Finally, there was no correlation in the present study between VCAM-1 expression at the arterial lumen and leukocyte infiltration.

In the present study, a striking association was found between the degree of macrophage accumulation and expression of VCAM-1 on neovasculature and on nonendothelial cells of atherosclerotic plaques. In contrast, there was no relation between macrophage accumulation and E-selectin expression at these sites. The correlation between macrophage accumulation and neovascular VCAM-1 expression not only suggests that this molecule may indeed participate in entry of macrophages into plaques but also demonstrates the potential importance of the neovasculature as a portal for macrophage entry. In addition, the strong correlation between T-lymphocyte accumulation and neovascular ICAM-1 and VCAM-1 expression further underscores the potential importance of the neovasculature as a route for plaque growth. In contrast, the associations between macrophage accumulation and ICAM-1 expression on neovasculature or nonendothelial cells failed to reach statistical significance. Despite progressive increases in the percentage of segments with neovascular and nonendothelial cell ICAM-1 expression with increasing plaque macrophage scores, these associations could not be tested for statistical significance because of the lack of ICAM-1 expression in any segments with a macrophage score of 1.

The factors that mediate expression of adhesion molecules in atherosclerotic plaques are not known, but many have been proposed. These include diet-induced5 and genetic4 hypercholesterolemia, cytokines, minimally or extensively oxidized lipoproteins, and reactive oxygen species. Several studies have demonstrated that atherosclerotic tissue contains a variety of macrophage- and T lymphocyte–derived cytokines24 25 26 that may upregulate expression of one or all of the three adhesion molecules evaluated in the present study.23 27 Oxidized lipoproteins also are of particular interest, since lysophosphatidylcholine, a phospholipid generated during LDL oxidation, can increase the expression of VCAM-1 on endothelium both in vitro and in vivo28 and exposure of cultured human umbilical vein endothelial cells to oxidized LDL has been shown to increase cell surface expression of ICAM-1.29 The contention that these observations have physiological relevance is supported by the observations of several investigators that oxidation-specific epitopes are present in atherosclerotic tissue of animals30 31 32 33 34 35 36 and humans33 37 38 and by a recent study demonstrating expression by human aortic plaque macrophages of myeloperoxidase, which generates the reactive oxygen intermediate hypochlorous acid (HOCl).39 Finally, reactive oxygen species may themselves play a role in upregulation of VCAM-1 expression.36 40 Further study is needed to determine which of these many potential mediators are actually responsible for adhesion molecule expression in atherosclerosis.

The observation that both ICAM-1 and VCAM-1 frequently are expressed in atherosclerotic lesions has several implications for antiatherogenic strategies that target adhesion molecules. First, it suggests that several factors are likely to mediate expression of leukocyte-recruiting adhesion molecules, so that attempts to decrease adhesion molecule expression by targeting a specific cytokine are likely to fail. Second, because both adhesion molecules are common in atherosclerosis, targeting only one of them may still allow leukocyte recruitment by the other and thus be ineffective.

Determining the role of plaque neovasculature in atherogenesis and identification of factors that either promote or inhibit its growth may lead to the development of novel antiatherogenic strategies. Because plaque neovasculature could provide an important route for lipid deposition and inflammatory cell infiltration, it most likely results in plaque growth. Several potentially angiogenic cytokines have been detected in atherosclerotic plaques, including tumor necrosis factor-{alpha}41 42 and aFGF,14 and could represent targets for intervention. Vascular endothelial growth factor is another potent angiogenic factor43 that is of interest because it is expressed by vascular smooth muscle cells exposed to platelet-derived growth factor, transforming growth factor-ß, or hypoxia44 and can stimulate revascularization in a rabbit ischemic hind limb model.45 Alternatively, similarities between neovascular ingrowth in plaques and angiogenesis in tumors46 raises the possibility that antiangiogenic factors such as thalidomide47 or angiostatin48 might have applications in atherosclerosis as well.

In summary, the present study confirms that neovessels may be detected in a substantial majority of advanced human atherosclerotic plaques and demonstrates not only that E-selectin, ICAM-1, and VCAM-1 are expressed on neovascular endothelium but also that their expression at this site is twice as prevalent as their expression at the arterial luminal surface. In addition, this study demonstrates that both neovascular and nonendothelial cell expression of ICAM-1 and of VCAM-1 are correlated strongly with increased intimal leukocyte content. Taken together, these data are consistent with the hypothesis that plaque neovasculature represents a significant route for infiltration of leukocytes into advanced atherosclerotic plaques, and they demonstrate that the neovasculature is a site of inflammatory activation. These findings have implications for the potential utility of antiatherogenic strategies that target adhesion molecule expression and also suggest that elucidation of factors that regulate the development of plaque neovasculature might lead to the development of novel antiatherogenic strategies. Finally, these studies raise the question of whether animal models of atherogenesis in which neovasculature is not a feature can provide sufficient insight into the disease in humans.


*    Selected Abbreviations and Acronyms
 
aFGF = acidic fibroblast growth factor
CHO = Chinese hamster ovary
ICAM-1 = intercellular adhesion molecule-1
IDCM = idiopathic cardiomyopathy
ISCM = ischemic cardiomyopathy
VCAM-1 = vascular cell adhesion molecule-1


*    Acknowledgments
 
This research was supported in part by grants HL-02788, HL-47151, and HL-30086 from the National Institutes of Health and by 94-WA-518R from the American Heart Association, Washington Affiliate. The authors wish to thank Carolyn Walden and John Hokansen for statistical advice and Lisa Anne Billings for expert assistance with manuscript preparation. We also gratefully acknowledge the contributions of Drs Salim Aziz and Daniel P. Fishbein of the University of Washington Cardiac Transplantation Program.


*    Footnotes
 
Reprint requests to Kevin D. O'Brien, MD, University of Washington, Division of Cardiology, Box 356422, 1959 NE Pacific St, Seattle, WA 98195-6422. E-mail cardiac@u.washington.edu.

Received July 26, 1995; revision received September 19, 1995; accepted October 4, 1995.


*    References
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*References
 

  1. Gerrity RG. The role of the monocyte in atherogenesis, I: transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol. 1981;103:181-190. [Abstract]
  2. Faggiotto A, Ross R, Harker L. Studies of hypercholesterolemia in the nonhuman primate, I: changes that lead to fatty streak formation. Arteriosclerosis. 1990;4:323-340. [Abstract/Free Full Text]
  3. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;62:801-809.
  4. Cybulsky MI, Gimbrone MA Jr. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science. 1991;251:788-791. [Abstract/Free Full Text]
  5. Li H, Cybulsky MI, Gimbrone MA Jr, Libby P. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Arterioscler Thromb. 1993;12:197-204.
  6. Poston RN, Haskard DO, Coucher JR, Gall NP, Johnson-Tidey RR. Expression of intercellular adhesion molecule-1 in atherosclerotic plaques. Am J Pathol. 1992;140:665-673. [Abstract]
  7. Printseva OY, Peclo MM, Gown AM. Various cell types in human atherosclerotic lesions express ICAM-1: further immunocytochemical and immunochemical studies employing monoclonal antibody 10F3. Am J Pathol. 1992;140:889-896. [Abstract]
  8. van der Wal AC, Das PK, Tigges AJ, Becker AE. Adhesion molecules on the endothelium and mononuclear cells in human atherosclerotic lesions. Am J Pathol. 1992;141:1427-1433. [Abstract]
  9. O'Brien KD, Allen MD, McDonald TO, Chait A, Harlan JM, Fishbein D, McCarty J, Ferguson M, Hudkins K, Benjamin CD, Lobb R, Alpers CE. Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plaques: implications for the mode of progression of advanced coronary atherosclerosis. J Clin Invest. 1993;92:945-951.
  10. Winternitz MC, Thomas RM, LeCompte PM. The Biology of Arteriosclerosis. Springfield, Ill: Charles C. Thomas; 1938.
  11. Geringer E. Intimal vascularization and atherosclerosis. J Pathol Bacteriol. 1951;63:201-211. [Medline] [Order article via Infotrieve]
  12. Barger AC, Beeuwkes R III, Lainey LL, Silverman KJ. Hypothesis: vasa vasorum and neovascularization of human coronary arteries. N Engl J Med. 1984;310:175-177. [Medline] [Order article via Infotrieve]
  13. Zhang Y, Cliff WJ, Schoefl GI, Higgins G. Immunohistochemical study of intimal microvessels in coronary atherosclerosis. Am J Pathol. 1993;143:164-172. [Abstract]
  14. Brogi E, Winkles JA, Underwood R, Clinton SK, Alberts GF, Libby P. Distinct patterns of expression of fibroblast growth factors and their receptors in human atheroma and nonatherosclerotic arteries: association of acidic FGF with plaque microvessels and macrophages. J Clin Invest. 1993;92:2408-2418.
  15. Gordon D, Reidy MA, Benditt EP, Schwartz SM. Cell proliferation in human coronary arteries. Proc Natl Acad Sci U S A. 1990;87:4600-4604. [Abstract/Free Full Text]
  16. Allen MD, McDonald TO, Himes VE, Fishbein DP, Aziz S, Reichenbach DD. E-selectin expression in human cardiac grafts with cellular rejection. Circulation. 1993;88(suppl II):II-243-II-247.
  17. Allen MD, McDonald TO, Carlos T, Himes VE, Fishbein DP, Aziz A, Gordon D. Endothelial adhesion molecules in heart transplantation. J Heart Lung Transplant. 1992;11:S8-S13. [Medline] [Order article via Infotrieve]
  18. Bacchi CE, Marsh CL, Perkins CD, Carithers RL, McVicar JP, Hudkins KL, Benjamin CD, Martin JM, Lobb R, Alpers CE. Expression of vascular cell adhesion molecule (VCAM-1) in liver and pancreas allograft rejection. Am J Pathol. 1993;142:579-591. [Abstract]
  19. Skalli O, Ropraz P, Trzeciak A, Benzonan G, Gillessen D, Gabbiani G. A monoclonal antibody against {alpha}-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol. 1986;103:2787-2796. [Abstract/Free Full Text]
  20. van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation. 1994;89:36-44. [Abstract/Free Full Text]
  21. Holthöfer H, Virtanen I, Kariniemi A-L, Hormia M, Linder R, Miettinen A. Ulex europaeus I lectin as a marker for vascular endothelium in human tissues. Lab Invest. 1982;47:60-66. [Medline] [Order article via Infotrieve]
  22. O'Brien KD, Gordon D, Deeb S, Ferguson M, Chait A. Lipoprotein lipase is synthesized by macrophage-derived foam cells in human coronary atherosclerotic plaques. J Clin Invest. 1992;89:1544-1550.
  23. Collins T, Palmer HJ, Whitley MZ, Neish AS, Williams AJ. A common theme in endothelial activation: insights from the structural analysis of the genes for E-selectin and VCAM-1. Trends Cardiovasc Med. 1993;3:92-97.
  24. Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989;86:9717-9721. [Abstract/Free Full Text]
  25. Salomon RN, Underwood R, Doyle MV, Wang A, Libby P. Increased apolipoprotein E and c-fms gene expression without elevated interleukin 1 or 6 mRNA levels indicates selective activation of macrophage function in advanced human atheroma. Proc Natl Acad Sci U S A. 1992;89:2814-2818. [Abstract/Free Full Text]
  26. Hansson GK, Holm J. Interferon-{gamma} inhibits arterial stenosis after injury. Circulation. 1991;84:1266-1272. [Abstract/Free Full Text]
  27. Springer TA. Adhesion receptors of the immune system. Nature. 1990;346:425-434. [Medline] [Order article via Infotrieve]
  28. Kume N, Cybulsky MI, Gimbrone MA Jr. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest. 1992;90:1138-1144.
  29. Jeng JR, Chang CH, Shieh SM, Chiu HC. Oxidized low-density lipoprotein enhances monocyte-endothelial cell binding against shear-stress-induced detachment. Biochim Biophys Acta. 1993;1178:221-227. [Medline] [Order article via Infotrieve]
  30. Haberland ME, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science. 1988;241:215-218. [Abstract/Free Full Text]
  31. Boyd HC, Gown AM, Wolfbauer G, Chait A. Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe heritable hyperlipidemic rabbit. Am J Pathol. 1989;135:815-825. [Abstract]
  32. Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A. 1989;86:1372-1376. [Abstract/Free Full Text]
  33. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.
  34. Rosenfeld ME, Palinski W, Yla-Herttuala S, Butler S, Witztum JL. Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis. 1990;10:336-349. [Abstract/Free Full Text]
  35. Palinski W, Yla-Herttuala S, Rosenfeld ME, Butler SW, Socher SA, Parthasarathy S, Curtiss LK, Witztum JL. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis. 1990;10:325-335. [Abstract/Free Full Text]
  36. O'Brien KD, Chait A, Gown AM, Nagano Y, Kita T. Probucol treatment affects the cellular composition but not anti-oxidized low density lipoprotein immunoreactivity of atherosclerotic plaques in Watanabe heritable hyperlipidemic rabbits. Arterioscler Thromb. 1991;11:751-759. [Abstract/Free Full Text]
  37. Yla-Herttuala S, Rosenfeld ME, Parthasarathy S, Glass CK, Sigal E, Witztum JL, Steinberg D. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Sci U S A. 1990;87:6959-6963. [Abstract/Free Full Text]
  38. Yla-Herttuala S, Rosenfeld ME, Parthasarathy S, Sigal E, Sarkioja T, Witztum JL, Steinberg D. Gene expression in macrophage-rich human atherosclerotic lesions: 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts. J Clin Invest. 1991;87:1146-1152.
  39. Daugherty A, Dunn JL, Rateri DL, Heiecke JW. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest. 1994;94:437-444.
  40. Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, Alexander RW, Medford RM. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest. 1993;92:1866-1874.
  41. Barath P, Fishbein MC, Cao J, Berenson J, Helfant RH, Forrester JS. Tumor necrosis factor gene expression in human vascular intimal smooth muscle cells detected by in situ hybridization. Am J Pathol. 1990;137:503-509. [Abstract]
  42. Rus HG, Niculescu F, Vlaicu R. Tumor necrosis factor-{alpha} in human arterial wall with atherosclerosis. Atherosclerosis. 1991;89:247-254. [Medline] [Order article via Infotrieve]
  43. Miller JW, Adamis AP, Shima DT, D'Amore PA, Moulton RS, O'Reilly MS, Folkman J, Dvorak HF, Brown LF, Berse B. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am J Pathol. 1994;145:574-584. [Abstract]
  44. Brogi E, Wu T, Namiki A, Isner JM. Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation. 1994;90:649-652. [Abstract/Free Full Text]
  45. Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis: a single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest. 1994;93:662-670.
  46. Folkman J. Diagnostic and therapeutic applications of angiogenesis research. C R Acad Sci III. 1993;316:909-918. [Medline] [Order article via Infotrieve]
  47. D'Amato RJ, Loughnan MS, Flynn E, Folkman J. Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci U S A. 1994;91:4082-4085. [Abstract/Free Full Text]
  48. O'Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of the metastases by a Lewis lung carcinoma. Cell. 1994;79:315-328.[Medline] [Order article via Infotrieve]



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