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Ann Thorac Surg 1998;65:1685-1689
© 1998 The Society of Thoracic Surgeons

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Original articles: cardiovascular

Expression of Vascular Adhesion Molecules in Saphenous Vein Coronary Bypass Grafts

^a Department of Cardiothoracic Surgery, National Heart & Lung Institute, Imperial College of Science, Technology & Medicine, Heart Science Centre, Harefield Hospital, Middlesex, United Kingdom

Accepted for publication February 1, 1998.

Address reprint requests to Prof Yacoub, Harefield Hospital, Harefield, Middlesex UB9 6JH UK

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Adhesion of blood elements to the endothelium is an important step in the development of vein graft disease. This study examines the expression of vascular adhesion molecules on explanted saphenous vein bypass grafts.

Methods. Immunocytochemical staining was performed using explanted saphenous vein grafts from 28 patients. Antibodies against the endothelial markers CD31, von Willebrand factor, intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin were used.

Results. Staining for CD31 and von Willebrand factor demonstrated the presence of endothelial cells in the lumen and the vasa vasorum. Expression of intercellular adhesion molecule-1 was variable between grafts, whereas vascular adhesion molecule-1 and E-selectin were almost always absent on the luminal endothelium. In contrast, the endothelium of the vasa vasorum stained positively for intercellular adhesion molecule-1 and vascular adhesion molecule-1, and was also seen on nonendothelial cells within the vessel wall. Expression of these adhesion molecules did not vary with the severity of vein graft disease.

Conclusions. This study highlights the blood vessels in the adventitia as possible sites for the adhesion and migration of cells into the vessel wall.

	Introduction

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It is believed that the pathogenesis of vein graft disease is similar to that described for atherosclerosis, which is associated with the release of growth factors, cytokines, and chemoattractants from infiltrating leukocytes and monocytes [1–4]. The initial event in this cell-mediated damage to the vessel wall is upregulation of the expression of adhesion molecules, which allows the tethering and attachment of leukocytes and platelets. These events are governed by the interactions between molecules such as LFA-1, MAC-1, VLA-4, p150/95 integrins, and L-selectin, which are expressed on the surface of certain circulating cell types, with molecules that have been either induced or upregulated on the surface of endothelial cells. These endothelial vascular adhesion molecules include the selectin family of molecules (E-, L-, and P-selectin) and the immunoglobulin superfamily, which include platelet endothelial cell adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) [5].

It has been established that the expression of these adhesion molecules is very low or absent on segments of saphenous vein that have been removed at the time of operation before distention and grafting [6]. Thus, stimuli must exist to cause the induction of the postoperative expression of these molecules for cellular attachment and migration to feature in the pathogenesis of vein graft disease. Several possibilities exist for such a stimulus. These include perioperative cytokine release, trauma to the blood associated with cardiopulmonary bypass, and increased shear stress due to arterialization of the vein [7–10]. However, there is no direct evidence to suggest that failed vein grafts have an altered expression of endothelial vascular adhesion molecules concomitant with other atherosclerotic changes. We have attempted to characterize the distribution and quantify the expression of vascular adhesion molecules on saphenous veins that have been used as bypass conduits for between 3 and 12 years. It is hoped that this knowledge will aid our understanding of how these molecules play a role in the pathology of vein graft disease.

	Material and methods

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A total of 28 saphenous vein coronary artery bypass grafts were removed from 28 patients who had previously undergone coronary artery bypass grafting and were undergoing repeat coronary artery bypass grafting or orthotopic cardiac transplantation. Vessels were obtained from patients ranging in age from 52 to 79 years. After removal, vessel segments were cut into 5-mm long segments, mounted transversely on filter paper, embedded in OCT, snap frozen, and stored in liquid nitrogen until required.

Frozen sections, 6 µm thick, were cut from each specimen, mounted on glass slides, allowed to dry, and then fixed for 5 minutes in 10% neutral buffered formalin before staining with hematoxylin and eosin or elastic van Gieson. Vein grafts were then categorized into four groups according to their histologic appearance (Table 1). Additional 6-µm thick frozen sections were then cut, mounted onto glass slides, allowed to dry for 30 to 120 minutes, and then fixed for 15 minutes in acetone before immunohistochemical staining. Sections from each vein graft were incubated for 30 minutes with one of the following monoclonal mouse antibodies: anti-CD31 (Dako Ltd, High Wycombe, UK, at a dilution of 1:50), anti-ICAM-1 (CD54 [R&D Systems, Abingdon, UK] at a dilution of 1:250), anti-VCAM-1 (CD106 [R&D Systems] at a dilution of 1:250), and anti E-selectin (CD62E [R&D Systems] at a dilution of 1:50). Each antibody was diluted in 0.005 mol/L Tris-buffered saline solution, pH 7.6. Negative control sections were incubated with either antibody diluent only or an irrelevant IgG₁ monoclonal mouse antibody (product X0931, Dako). All sections were incubated subsequently for 30 minutes in biotinylated rabbit anti-mouse F(ab)₂ fragment (Dako) diluted 1:300 in a 5% solution of human AB serum in 0.005 mol/L Tris-buffered saline solution. Sections were then immersed for 30 minutes at room temperature in streptavidin biotin–horseradish peroxidase complexes (Dako), which had been prepared by diluting the components 1:200 in 0.05 mol/L Tris buffer, pH 7.6, at least 30 minutes before use. Sections were then treated for 5 minutes with a diaminobenzidine tetrahydrochloride (product D5905, Sigma, Poole, UK)/hydrogen peroxide substrate to visualize the antigenic sites. Each of the above stages was followed by three 5-minute washes in 0.005 mol/L Tris-buffered saline solution, pH 7.6. The sections were then rinsed in cold running tap water, counterstained with Mayer’s hematoxylin, washed again in cold tap water, and then dehydrated through graded alcohols, cleared in CNP30 (Merck, Lutterworth, UK), and finally mounted in DPX.

	Results

Top Abstract Introduction Material and methods Results Comment References

Generally, ICAM-1 staining of the luminal endothelium varied within groups with no obvious pattern. The VCAM-1 staining was almost always absent, with only a few luminal endothelial cells positive. When present, it was only seen in areas of endothelium that were also positive for ICAM-1 (Fig 1). E-selectin was only weakly positive in the areas that had stained for VCAM-1 (Table 1).

View larger version (96K): [in this window] [in a new window]   Fig 1. Photomicrographs of a moderately diseased vein graft showing (a) luminal endothelial cells strongly positive for CD31 (solid arrows) and some weaker stained foamy macrophages in the intima adjacent to the endothelial surface (open arrows); (b) luminal endothelial cells positive for intercellular adhesion molecule-1 (solid arrows) and some nonendothelial cells weakly positive within the intima, with those near the luminal surface resembling foamy macrophages and stronger staining of spindle-shaped cells lying deeper within the intima (open arrows); (c) vascular adhesion molecule-1 staining of a few weakly positive endothelial cells lining the lumen (solid arrows) and numerous strongly positive nonendothelial cells deeper within the intima (open arrows); and (d) a negative control with an irrelevant antibody showing no staining.

In contrast to the luminal endothelium, the majority, if not all, of the vasa vasorum were positive for ICAM-1, irrespective of the stage of the disease. In all but three veins VCAM-1-positive staining was identified in the vasa vasorum. The intensity of this staining was generally weak and was only seen in a small number of the total vasa vasorum present in each vessel section (Fig 2). This observation remained constant across all categories of the disease. Staining for E-selectin was a weaker reflection of the VCAM-1 staining, being present in only some of those vessels positive for VCAM-1 (Table 2).

View larger version (95K): [in this window] [in a new window]   Fig 2. Photomicrographs showing (a) numerous CD31-positive endothelial cells in vasa vasorum within the adventitia of a vein graft; (b) intercellular adhesion molecule-1-positive staining, which was evident in the majority of vasa vasorum in which CD31-positive cells were present; (c) some cells more weakly positive for vascular adhesion molecule-1 (solid arrows); and (d) a negative control with an irrelevant antibody showing no staining.

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
In this study we have demonstrated that saphenous veins used as coronary artery bypass grafts have a specific distribution of the expression of the adhesion molecules ICAM-1 and VCAM-1. These molecules, which belong to the immunoglobulin superfamily, mediate adhesion and transmigration of blood leukocytes through the endothelium. The adhesion of all leukocytes is mediated by the expression of ICAM-1, whereas VCAM-1 is specific for lymphocytes, monocytes, and eosinophils.

The most significant expression of both ICAM-1 and VCAM-1 was seen in the vasa vasorum or in areas of neovascularization within the adventitia and neointima. In contrast, expression on the remaining luminal endothelium of the vein grafts was often absent. This pattern of expression on the luminal endothelium was independent of the severity of intimal smooth muscle cell growth and atherosclerotic changes. It has been shown that the remaining luminal endothelium of diseased vein grafts continues to show some expression of endothelial nitric oxide synthase [11]. Functional studies have confirmed the capacity of arterialized and diseased grafts to secrete varying amounts of nitric oxide [12]. However, it appears that transmigration of cells from the adventitial side of the vessel wall may be an important route whereby lymphocytes and monocytes can affect graft function. Indeed, this has also been suggested to be a significant route for cellular infiltration in human coronary artery atherosclerotic lesions, due to more prevalent expression of VCAM-1 in areas of neovascularization compared to the luminal endothelium [13]. Expression of VCAM-1 was demonstrated in areas of neovascularization at the base of atherosclerotic plaques, and is believed to represent areas of ongoing inflammatory cell recruitment after initiation of the atherosclerotic process [13]. Cellular rolling, attachment, and subsequent migration in these small vessels will be favored because of the lower flow and shear stress compared to that seen in the lumen of the saphenous vein, which is subjected to arterial pressures and high blood flow.

Neither the luminal endothelium nor the existing vasa vasorum have a strong expression of adhesion molecules in saphenous veins that are removed at the time of the initial operation before distention and grafting [6]. ICAM-1 is only moderately expressed on native veins, whereas the expression of VCAM-1 is much weaker, if not absent on the luminal endothelium. After operation there are a number of potential stimuli for the expression of adhesion molecules in veins. These include the action of cytokines released as a result of cardiopulmonary bypass, and increased stretch in the vessel wall because of mechanical distention and exposure to arterial pressure [7–10]. It has been demonstrated that both ICAM-1 and VCAM-1 can be upregulated on endothelial cells by a variety of different cytokines [14], and that increased shear stress can upregulate endothelial ICAM-1 and VCAM-1 [7, 9]. However, these mechanisms would be most likely to cause a global change of adhesion molecule expression on luminal endothelium as well as in vasa vasorum. For this reason it may be possible that regulation of adhesion molecule expression is under a local control mechanism, as has previously been suggested [13]. The possibility exists that vasa vasorum, which express ICAM-1 and VCAM-1, represent areas of activated endothelium due to neovascularization, which may occur as a result of the action of angiogenic factors released in response to hypoxia, possibly through the release of vascular endothelial growth factor [15]. Further studies are required to elucidate the precise role of adhesion molecule expression on the endothelium of the vasa vasorum, and their contribution to the migration of blood elements from the adventitial side of the vessel wall into the medial smooth muscle of saphenous vein bypass grafts.

Expression of ICAM-1 and VCAM-1 was also seen in nonendothelial cells present within the intima in some of the diseased vein grafts studied. Previous reports on atherosclerotic arteries have demonstrated ICAM-1 or VCAM-1 on subsets of macrophages and smooth muscle cells within the areas of diseased tissue [13, 16–18]. The function related to the expression of these molecules in the vessel wall is not understood. It has been suggested that smooth muscle expression of VCAM-1 slows down the migration of T cells causing them to remain within the vessel wall. Expression of ICAM-1 and VCAM-1 may therefore be associated with sites of inflammation and could be the result of the localized release of cytokines from activated T cells [19].

In conclusion, this study strongly suggests that the vasa vasorum are an important site for the interactions between blood elements and vascular endothelial cells in saphenous veins when these are used as bypass conduits. Hypoxia caused by harvesting, and changes in mechanical forces due to anatomic relocation of the vein could act as a trigger in altering the nature of the vessels that vascularize the vessel wall, with subsequent activation of the endothelium. These changes may contribute to the atherosclerotic process seen in vein graft disease, and therefore they warrant further study.

	References

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