Immunophenotypic analysis of the aortic aneurysm wall suggests that vascular dendritic cells are involved in immune responses

Cardiovascular Surgery, Vol. 6, No. 3, pp. 240–249, 1998  1998 The International Society for Cardiovascular Surgery. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0967–2109/98 $19.00 + 0.00

PII: S0967-2109(97)00168-3

Immunophenotypic analysis of the aortic aneurysm wall suggests that vascular dendritic cells are involved in immune responses Y. V. Bobryshev, R. S. A. Lord and H. Pa¨rsson Surgical Professorial Unit, St Vincent’s Hospital, University of New South Wales, Sydney, New South Wales, Australia Objective: Inflammatory infiltrates similar to those in atherosclerotic plaques are prominent in the abdominal aortic aneurysm wall. Antigen presenting vascular dendritic cells are present in both early and advanced atherosclerotic lesions but their possible participation in abdominal aortic aneurysms has not been previously examined. This study reports the presence of vascular dendritic cells in abdominal aortic aneurysms and their participation in immune responses. Methods: Samples of the anterior wall were collected from 18 atherosclerotic infrarenal abdominal aortic aneurysms ranging in diameter from 5–8 cm. All the patients were operated upon electively and no ruptured or rapidly expanding abdominal aortic aneurysms were included. Specimens were immediately frozen or fixed in 10% buffered formalin. Vascular dendritic cells were identified with anti-CD1a or with S-100. T cells and T cell subpopulations were identified with anti-CD3, anti-CD4 and anti-CD8. B cells were studied with anti-CD20. Analyses were carried out in sets of consecutive parallel sections immunostained with these antibodies and double immunostaining included different combinations of antigens such as CD1a/CD3, S-100/CD4, S-100/CD8. Results: Most inflammatory infiltrates were found in the adventitia. These infiltrates contained B cells (CD20 + ) and T cells (CD3 + ) with their CD4 + and CD3 + cell subpopulations. In the aneurysm wall, CD1a + /S-100 + cells exhibiting dendritic appearance were detected and double immunostaining demonstrated that these vascular dendritic cells contained different lymphocyte populations including CD3 + , CD4 + , CD8 + and CD20 + cells. In some inflammatory infiltrates, B cells (CD20 + ) represented the predominant cell population (60–80%). Double immunostaining demonstrated that, in these infiltrates, vascular dendritic cells contacted CD20 + cells. Conclusions: vascular dendritic cells are involved in immune reactions in the aneurysm wall, and this process mostly occurs in the adventitia. Vascular dendritic cells contact both T cells and B cells, suggesting that these vascular dendritic cells differ from other dendritic cells, subtypes of which associate with T cells (Langerhans cells, interdigitating cells) and B cells (follicular dendritic cells).  1998 The International Society for Cardiovascular Surgery. Published by Elsevier Science Ltd. All rights reserved Keywords: abdominal aortic aneurysm, aorta, T cells, B cells, vascular dendritic cells

Introduction The mechanisms responsible for the weakening and destruction of the artery wall, which lead to the forCorrespondence to: Dr Yuri V. Bobryshev, Surgical Professorial Unit, Level 17, O’Brien Building, St Vincent’s Hospital, Darlinghurst, NSW 2010, Australia

240

mation of abdominal aortic aneurysms, have been extensively studied during the last decade, with changes in the media thought to be mainly responsible [1, 2]. Abdominal aortic aneurysms are also characterized by degradation of the extracellular matrix with an increase in collagen content and a decrease in elastin [3–6], these alterations being also associated with increased expression of metalloprotCARDIOVASCULAR SURGERY

JUNE 1998 VOL 6 NO 3

Immunophenotypic analysis of the aortic aneurysm wall: Y. V. Bobryshev et al.

einases [7–10]. Imbalance in the fibre-forming extracellular matrix protein synthesis is thought to lead to the breakdown in the structural integrity of the arterial wall [1–10]. In abdominal aortic aneurysms, the synthetic activity of medial smooth muscle cells, which are responsible for extracellular matrix formation, undergoes marked changes that increase the likelihood of mechanical failure of the arterial wall [2]. In the media the density of smooth muscle cells is significantly decreased [11]. Apoptosis with an increased expression of p53, which is itself a potential mediator of cell cycle arrest and programmed cell death, has been demonstrated in medial smooth muscle cells [11]. Another characteristic of abdominal aortic aneurysms is a change in the cellular composition of the outer aortic wall associated with massive infiltration of the media and adventitia by macrophages and lymphocytes [12–14]. In abdominal aortic aneurysms, macrophages secrete different cytokines and inflammatory infiltrate accumulation correlates with the production of tumour necrosis factor-␣ and interleukin-8 [15, 16]. Cytokines produced by macrophages might stimulate metalloproteinase expression [17] and, even more importantly, macrophages themselves express matrix metalloproteinase9 and metalloproteinase-3 [8, 9]. Macrophages may well be the main source of enhanced proteolytic activity in abdominal aortic aneurysms [8, 9]. We previously reported that cells from the family of antigen-presenting dendritic cells reside in the intima of large arteries [18, 19] and that these vascular dendritic cells are common in atherosclerotic lesions [19, 20]. Dendritic cells are known to be involved in various disease processes where they represent only a minor cell population [21–23]. Even though their numbers are small, dendritic cells are the most potent antigen-presenting cells known [21–23]. Vascular dendritic cells, similar to Langerhans cells and interdigitating cells [21–23] express both CD1a and S-100 protein [18], [20], which are markers for their immunohistochemical identification. Like other dendritic cells [21–23], vascular dendritic cells express HLA-DR [20], VCAM-1 [24] and ICAM-1[25]. Vascular dendritic cells often form clusters with T cells in primary atherosclerotic lesions in the aorta and carotid arteries [20]. Since VCAM-1/VLA-4 interactions are critical in T cell activation [26, 27], our previous observations [24, 25] that vascular dendritic cells display ICAM-1 and VCAM-1 in the arterial wall suggest that interactions between vascular dendritic cells and T cells might lead to the activation of T cells. In contrast with atherosclerotic plaques where the intimal layer is mainly affected, abdominal aortic aneurysms are characterized by the formation of inflammatory infiltrates, predominantly in the media CARDIOVASCULAR SURGERY

JUNE 1998 VOL 6 NO 3

and adventitia [12–14]. In contrast with primary atherosclerosis, where T lymphocytes are exclusively present, the adventitial infiltrates in abdominal aortic aneurysms contain a large number of both B cells and T cells [12]. The possible participation of vascular dendritic cells in aortic aneurysm has not been previously examined. We now report that vascular dendritic cells are constantly present in the inflammatory infiltrates in abdominal aortic aneurysm, which suggests their involvement in immune reactions in abdominal aortic aneurysm. The present work describes the distribution of vascular dendritic cells in abdominal aortic aneurysms and the co-localization of vascular dendritic cells with other cell types within inflammatory infiltrates.

Methods Tissue specimens and routine histology Material was collected in accordance with the principles outlined in the Declaration of Helsinki [28] and the present study was approved by the institutional review board of St Vincent’s Hospital, Sydney. Samples of anterior aneurysmal wall were collected from 18 typical atherosclerotic infrarenal abdominal aortic aneurysms ranging in diameter from 5 to 8 cm. All the patients were operated upon electively and no ruptured, rapidly expanding, or inflammatory type aneurysms were included. For immunohistochemistry, some aortic samples were processed by standard formalin fixation and paraffin embedding. Other unfixed samples were immediately embedded in OCT compound, rapidly frozen in liquid nitrogen and stored at ⫺ 70°C until cryostat sectioning. Paraffin and frozen sections were cut at 5–7 ␮m thickness and air dried for 45 min. Sections were stained for analysis with Mayer’s haematoxylin. Identification of intimal cells Vascular dendritic cells in the arterial intima were identified with anti-CD1a and S-100. 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 antiCD8. B cells were identified with anti-CD20. Macrophages were identified with anti-CD68 antibody. Endothelial cells were identified by von Willebrand factor. Smooth muscle cells in the arterial intima were identified with antibody to alphasmooth muscle actin or antibody to muscle actin. The sources and working concentrations of the antibodies used are given in Table 1. 241

Immunophenotypic analysis of the aortic aneurysm wall: Y. V. Bobryshev et al. Table 1 Antibodies used in the study Designation

Type*

Clone

Specificity

Cell types identified Source

Working dilation

CD1a

M

NA1/34

CD1a

DAKO

1:50

S100

P

—

S-100A, S-100B

DAKO

1:700

CD3 CD4

P M

— MT310

CD3 CD4

DAKO DAKO

1:100 1:10

CD8

M

DK25

CD8

DAKO

1:50

CD20 EBM11 von Willebrand factor SMA

M M M

L26 PG-MI F8/86

Thymocytes, Langerhans cells, interdigitating cells, vascular dendritic cells Glial cells, ependyma, Schwann cells, Langerhans cells, interdigitating cells, vascular dendritic cells T cells Helper/inducer subtype of T cells Suppressor/cytotoxic subtype of T cells B cells Macrophages Endothelial cells

1:50 1:50 1:50

M

1A4

HMA

M

HHF35

CD20 DAKO CD68 DAKO Factor VIII-related DAKO antigen Smooth muscle Smooth muscle cells DAKO alpha-actin Human muscle actin Smooth muscle cells DAKO

1:50 1:50

*M, monoclonal antibody; P, polyclonal antibody.

Single immunostaining procedure The distribution of different cell types in abdominal aortic aneurysm was studied by a single staining immunoperoxidase procedure. An analysis was carried out using sets of consecutive parallel sections immunostained with antibodies to S-100, CD1a, CD3, CD4, CD8, CD68, muscle actin, smooth muscle ␣-actin, and von Willebrand factor. For single immunostaining, after eliminating endogenous peroxidase activity by 0.3% H2O2 for 5 min and treatment when necessary, by 0.1% trypsin in phosphate buffered saline for 5–10 min, the consecutive sections were pre-incubated with normal goat serum and then tested with avidin–biotin complex using the avidin–biotin–peroxidase complex method [29]. The sections were incubated for 30 min with each primary antibody. After washing in Tris–phosphate buffered saline, pH 7.6 (10 min), the sections were incubated for 20 min with the appropriate biotin-labelled secondary antibodies (horse Anti-mouse—Vector BA-2000, or goat Anti-rabbit—Vector BA1000). The sections were then washed in Tris–phosphate buffered saline for 5 min and treated with avidin–biotin complex (ELITE—avidin–biotin–peroxidase complex, Vector PK61000) for 30 min. After washing for 10 min in Tris–phosphate buffered saline, brown staining was produced by 5 min treatment with 3,3⬘-diaminobenzidine. All the incubations were completed at room temperature. For negative controls, the first antibodies were omitted or the sections were treated with an immunoglobulin 242

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 and sections were examined in an Olympus microscope at × 100 and × 400 magnifications. Double immunostaining procedure Analysis of the co-localization of vascular dendritic cells with other cell types was performed using a double immunostaining technique. Combinations of antigens including S-100/CD4, S-100/CD8, S100/CD20, S-100/CD68 and CD1a/CD3 were examined using Dako-Doublestain Kit System 40, K665. This kit allows simultaneous staining for detection of two different tissue markers on one section by a combination of mono- and polyclonal antibodies and of the peroxidase–antiperoxidase and alkaline phosphatase–antialkaline phosphatase technique. Using a rabbit primary antibody in the peroxidase–antiperoxidase system with a 3,3⬘diaminobezidine chromogen yields a brown reaction product at the site of the target antigen, while a mouse primary antibody in the alkaline phosphatase–antialkaline phosphatase system with a Fast Red chromogen results in a rose precipitate at the site of the identified antigen. This difference allows the topographical relationships between the two antigens to be observed. For double immunostaining, after eliminating endogenous peroxidase activity by 0.3% H2O2 CARDIOVASCULAR SURGERY

JUNE 1998 VOL 6 NO 3

Immunophenotypic analysis of the aortic aneurysm wall: Y. V. Bobryshev et al.

for 5 min and treatment, if necessary, by 0.1% trypsin in phosphate buffered saline for 5–10 min, the consecutive tissue sections were pre-incubated with normal swine serum and then tested according to the manufacturer’s instructions (DAKO). The sections were incubated with the 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 alkaline phosphatase–antialkaline phosphatase immune complex, followed by the rabbit peroxidase–antiperoxidase 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.

Results Vascular dendritic cells were detected in all the samples of abdominal aortic aneurysm studied and they were distributed irregularly throughout the aneurysm wall. Vascular dendritic cells in the intima In all the samples, atherosclerotic alterations, mostly atherosclerotic plaques of the intima, were apparent. In atherosclerotic lesions, S100 + /CD1a + cells represented a minor cell population but more than 80% of inflammatory infiltrates contained dendritic shaped S-100 + /CD1a + cells. The distribution of vascular dendritic cells was irregular, and was similar to that in aortic samples with primary atherosclerosis, which have been previously reported [19], [20], [24], [25]. Vascular dendritic cells were frequently seen within inflammatory infiltrates associated with neovascularization and double immunohistochemical analysis demonstrated that the areas containing a large number of T cells were rich in vascular dendritic cells and macrophages. In these infiltrates, vascular dendritic cells were seen to be co-localized with T cells while B cells (CD20 + cells) were very rarely found. Vascular dendritic cells in the media Vascular dendritic cells and their clusters were detected between smooth muscle cells in the diminished medial layer (Figure 1A) but they were mostly associated with capillaries (Figure 1B). The areas around these capillaries contained large numbers of CD3 + cells and CD68 + cells. Double immunostaining demonstrated the co-localization of vascular dendritic cells with both T cells (Figure 1B) and CARDIOVASCULAR SURGERY

JUNE 1998 VOL 6 NO 3

macrophages, but B cells (CD20 + ) were not detected. Analysis of consecutive sections showed that the capillaries of the media were continuous with capillaries formed by neovascularization in the atherosclerotic intima as well as with vasa vasorum in the adventitia. Vascular dendritic cells in the adventitia All the specimens were characterized by the presence of massive inflammatory infiltrates, which mostly formed around the vasa vasorum. Analysis of consecutive parallel sections demonstrated that these massive inflammatory infiltrates all contained S-100 + /CD1a + cells (Figure 2A–B). Some vascular dendritic cells were present around the inflammatory infiltrates (Figure 2A) and they were also detected inside capillaries. Immunohistochemical analysis of the inflammatory infiltrates demonstrated large numbers of CD3 + T cells with both of their subpopulations, namely CD4 + cells and CD8 + cells (Figure 2C and D). The inflammatory infiltrates also contained many CD20 + cells and in some inflammatory infiltrates, B cells (CD20 + ) represented the predominant cell population (60–80%) (Figure 3A–D). Double immunostaining examination of the adventitial inflammatory infiltrates demonstrated that vascular dendritic cells were co-localized with T cells as well as with B cells (CD20 + cells) (Figure 2C–D, Figure 4A–B).

Discussion The present examination demonstrated the presence of S100 + /CD1a + vascular dendritic cells in abdominal aortic aneurysms. Vascular dendritic cells were found in both the media and adventitia but were predominantly within inflammatory infiltrates in the adventitia. In the media, vascular dendritic cells co-localized with T cells, while within inflammatory infiltrates in the adventitia, vascular dendritic cells were in contact with both T cells and B cells. This observation suggests that vascular dendritic cells might be different from other well-studied dendritic cells that are associated either with T cells (Langerhans cells, interdigitating cells) or with B cells (follicular dendritic cells) [21–23]. In contrast with intimal atherosclerosis, abdominal aortic aneurysms are characterized by a strikingly large number of B cells [12], which suggests that the co-localization of B cells with vascular dendritic cells observed in the present study serves an important immunological role. The co-localization of vascular dendritic cells with B cells might initiate or regulate the functional activity of B cell-dependent immune reactions in abdominal aortic aneurysms. To a large extent, vascular dendritic cells localize 243

Immunophenotypic analysis of the aortic aneurysm wall: Y. V. Bobryshev et al.

Figure 1 Vascular dendritic cells located in the deep layer of the media containing capillary network in an abdominal aortic aneurysm specimen. A: Clusters of S-100 + cells (arrows). B: S-100 + cells (brown) intermingled with CD4 + cells (rose) along capillaries. Note the association between some S-100 + cells and CD4 + cells (arrows). A: Paraffin section, avidin–biotin–peroxidase complex immunoperoxidase technique, counterstaining with Mayer’s haematoxylin. B: Frozen section, double immunostaining (S-100 + CD4: peroxidase–antiperoxidase + alkaline phosphatase–antialkaline phosphatase technique), counterstaining with Mayer’s haematoxylin. Original magnifications: × 400, × 400

within inflammatory infiltrates, the formation of which is associated with intensive neovascularization of the media and the adventitia. Our data agree with previous observations [14], [30], [31], that intensive neovascularization of the aortic wall is a prominent feature of abdominal aortic aneurysms. The density of medial microvessels in abdominal aortic aneurysms has been previously shown to increase by 15 times compared with non-atherosclerotic aorta and 244

more than three times compared with the aortic wall from patients with aortic occlusive disease [31]. Angiogenesis requires degradation of the extracellular matrix, a process that facilitates endothelial cell proliferation and migration from pre-existing vessels, and this process itself requires the release of proteolytic enzymes and specific angiogenic factors [32–34]. In particular, the initiation of neovascularization involves the dissolution of the capillary CARDIOVASCULAR SURGERY

JUNE 1998 VOL 6 NO 3

Immunophenotypic analysis of the aortic aneurysm wall: Y. V. Bobryshev et al.

Figure 2 Inflammatory infiltrates in the adventitia containing vascular dendritic cells and T cells. A: S-100 + cells located around and within inflammatory infiltrates. B: S-100 + cells located in the area around vasa vasora, which are surrounded by a massive inflammatory infiltrate. C: CD1a + cells (brown) co-localizing with CD3 + cells (rose) within a massive inflammatory infiltrate. D: S-100 + cells (brown) co-localizing with CD4 + cells (rose) within a massive inflammatory infiltrate. A, B: Paraffin sections, avidin–biotin–peroxidase complex immunoperoxidase technique, counterstaining with Mayer’s haematoxylin. C, D: Frozen sections, double immunostaining (CD1a + CD3: peroxidase–antiperoxidase + alkaline phosphatase–antialkaline phosphatase, S-100 + CD4: peroxidase–antiperoxidase + alkaline phosphatase–antialkaline phosphatase technique), counterstaining with Mayer’s haematoxylin. Original magnifications: × 500, × 400, × 400, × 400

Immunophenotypic analysis of the aortic aneurysm wall: Y. V. Bobryshev et al.

Figure 3 S-100 + cells (A, B), CD3 + cells (C) and CD20 + cells (D) in a heavy inflammatory infiltrate in the adventitia from an abdominal aortic aneurysm specimen. Note that B cells predominate in this infiltrate. A–C: consecutive paraffin sections. B: detail of figure A. A–D: avidin–biotin–peroxidase complex immunoperoxidase technique, counterstaining with Mayer’s haematoxylin. Original magnifications: × 100, × 400, × 400, × 400

246

CARDIOVASCULAR SURGERY

JUNE 1998 VOL 6 NO 3

Immunophenotypic analysis of the aortic aneurysm wall: Y. V. Bobryshev et al.

Figure 4 Association of vascular dendritic cells with B cells in the adventitia from abdominal aortic aneurysm specimens (A, B). S100 + cells (brown) are shown by arrows. B cells are identified with anti-CD20 (rose). In A, an area containing mostly CD20 + cells is marked by an asterisk. Small stars mark vasa vasorum while the large star indicates an S-100 + nerve twig (brown). A, B: Frozen sections, double immunostaining (S-100 + CD20: peroxidase– antiperoxidase + alkaline phosphatase–antialkaline phosphatase) technique, counterstaining with Mayer’s haematoxylin. Original magnifications: × 400, × 400

basement membrane during which proteases are released into extracellular space with the potential to weaken the arterial wall [32], [34]. Neovascularization in abdominal aortic aneurysm strongly correlates with the accumulation of inflammatory infiltrates in the media and the adventitia [31]. Various factors, including fibroblast growth factor, epithelial growth factor, transforming factor-␤, tumour necrosis factor-␣, angiogenin and vascular endothelial cell growth factor, are involved in the regulation of angiogenesis [33]. These factors influence endothelial cell proliferation and their migration during neovascularization [34–37]. In abdominal aortic aneurysms, both macrophages and endothelial cells are involved in the local release of these biologically active substances [2], [14]. Our present observations that vascular dendritic cells reside within inflammatory infiltrates suggest that vascular dendritic cells might contribute to cytokine production in abdominal aortic aneurysms. The secretory activity of vascular dendritic cells has not CARDIOVASCULAR SURGERY

JUNE 1998 VOL 6 NO 3

yet been studied but it is known that in other pathological processes, other dendritic cells produce various cytokines [21–23]. We cannot exclude the possibility that vascular dendritic cells might contribute to the production of cytokines in abdominal aortic aneurysms as well, and clearly, the peculiarities of vascular dendritic cell synthetic activity require further study. Vascular dendritic cells were found within the inflammatory infiltrates that surrounded capillaries, and some vascular dendritic cells were detected inside capillaries, which suggests that the migration of vascular dendritic cells into areas containing inflammatory infiltrates might occur through the capillary network. In abdominal aortic aneurysm, this network connects capillaries of the intimal layer with the vasa vasorum of the adventitia, which might allow easy migration of vascular dendritic cells from the intimal layer to the adventitia as well as from the adventitia to the intima. In other tissues, dendritic cells are involved in reg247

Immunophenotypic analysis of the aortic aneurysm wall: Y. V. Bobryshev et al.

ulating the functional activity of macrophages and activating T cells [21–23]. Furthermore, dendritic cells themselves can release a large number of different cytokines, which might affect the formation of new capillaries. The present work, which shows that vascular dendritic cells are involved in abdominal aortic aneurysms, suggests that vascular dendritic cells might affect the functional activity of lymphocytes and macrophages in inflammatory infiltrates.

16.

17.

18.

Acknowledgements

19.

This research was supported by The St Vincent’s Clinic Foundation, Sydney.

20.

21.

References 1. Ernst, C. B., Abdominal aortic aneurysm. New England Journal of Medicine, 1993, 328, 1167–1172. 2. Thompson, R. W., Basic science of abdominal aortic aneurysms: Emerging therapeutic strategies for an unresolved clinical problem. Current Opinion in Cardiology, 1996, 11, 504–518. 3. Rizzo, R. J., McCarthy, W. J., Dixit, S. N. et al., Collagen types and matrix protein content in human abdominal aortic aneurysms. Journal of Vascular Surgery, 1989, 10, 365–373. 4. White, J. V, Haas, K., Phillips, S. and Comerota, A. J., Adventitial elastolysis is a primary event in aneurysm formation. Journal of Vascular Surgery, 1993, 17, 371–381. 5. Minion, D. J., Davis, V. A., Nejezchleb, P. A. et al., Elastin is increased in abdominal aortic aneurysms. Journal of Surgical Research, 1994, 57, 443–446. 6. Dobrin, P. B. and Mrkvicka, R., Failure of elastin or collagen as possible critical connective tissue alterations underlying aneurysmal dilatation. Cardiovascular Surgery, 1994, 2, 484–488. 7. Vine, N. and Powell, J. T., Metalloproteinases in degenerative aortic disease. Clinical Science, 1991, 81, 233–239. 8. Newman, K. M., Ogata, Y., Malon, A. M. et al., Identification of matrix metalloproteinases 3 and 9 in abdominal aortic aneurysm. Arteriosclerosis and Thrombosis, 1994, 14, 1315–1320. 9. Newman, K. M., Jean-Claude, J., Li, H. et al., Cellular localisation of matrix metalloproteinases in abdominal aortic aneurysm wall. Journal of Vascular Surgery, 1994, 20, 814–820. 10. Freestone, T., Turner, R. J., Coady, A. et al., Inflammation and matrix metalloproteinases in enlarging abdominal aortic aneurysm. Arteriosclerosis, Thrombosis and Vascular Biology, 1995, 15, 1145–1151. 11. Lopez-Candales, A., Holmes, D. R., Liao, S. et al., Decreased vascular smooth muscle cell density in medial degeneration of human abdominal aortic aneurysms. American Journal of Pathology, 1997, 150, 993–1007. 12. Koch, A. E., Haines, G. K., Rizzo, R. J. et al., Human abdominal aortic aneurysms: Immunophenotypic analysis suggesting an immune-mediated response. American Journal of Pathology, 1990, 137, 1199–1213. 13. Louwrens, H. and Peace, W. H., Role of inflammatory cells in aortic aneurusms. In Aneurysms: New Findings and Treatments, eds J. S.T. Yao and W. H. Pearce. Appleton and Lange, Norwalk, Connecticut, 1994, pp. 11–23. 14. Thompson, M. M., Jones, L., Nasim, A. et al., Angiogenesis in abdominal aortic aneurysms. European Journal of Endovascular Surgery, 1996, 11, 464–469. 15. Pearce, W. H., Sweis, I., Yao, I. S. et al., Interleukin-l beta and

248

22.

23. 24.

25.

26. 27. 28.

29.

30.

31.

32. 33. 34.

35.

36.

tumor necrosis factor-alpha release in normal and diseased human infrarenal aortas. Journal of Vascular Surgery, 1992, 16, 784–789. Koch, A. E., Kunkel, S. L., Pearce, W. H. et al., Enhanced production of the chemotactic cytokines interleukin-8 and monocyte chemoattractant protein-1 in human abdominal aortic aneurysms. American Journal of Pathology, 1993, 142, 1423–1431. Nolan, K. D., Mesh, C. L., Shively, V. P. et al., Cytokines modulate matrix mettaloprotease and TMP gene expression. Surgical Forum, 1992, 53, 346–348. Bobryshev, Y. V. and Lord, R. S. A., Ultrastructural recognition of cells with dendritic cell morphology in human aortic intima. Contacting interactions of vascular dendritic cells in atheroresistant and athero-prone areas of the normal aorta. Archives of Histology and Cytology, 1995, 58, 307–322. Bobryshev, Y. V. and Lord, R. S. A., S-100 positive cells in human arterial intima and in atherosclerotic lesions. Cardiovascular Research, 1995, 29, 689–696. Bobryshev, Y. V., Lord, R. S. A., Rainer, S. et al., Vascular dendritic cells and atherosclerosis. Pathology Research and Practice, 1996, 192, 462–467. King, P. D. and Katz, D. R., Mechanisms of dendritic cell function. Immunology Today, 1990, 11, 206–211. Steinman, R. M., The dendritic cell system and its role in immunogenicity. Annual Review of Immunology, 1991, 9, 271– 296. Sprecher, E. and Becker, Y., Role of Langerhan’s cells and other dendritic cells in disease states. In vivo, 1993, 7, 217–227. Bobryshev, Y. V., Lord, R. S. A., Rainer, S. and Munro, V. F., VCAM-1 expression and network of VCAM-1 positive vascular dendritic cells in advanced atherosclerotic lesions of carotid arteries and aortas. Acta Histochemica, 1996, 98, 185–194. Bobryshev, Y. V. and Lord, R. S. A., Vascular dendritic cells express intercellular adhesion molecule-1 in atherosclerotic plaques. Biomedical Research, 1997, 18, 179–182. Lbelda, S. M. and Buck, C. A., Integrins and other cell adhesion molecules. FASEB Journal, 1990, 4, 2868–2880. Springer, T. A., Adhesion receptors of the immune system. Nature, 1990, 346, 425–434. British Medical Council: Human Experimentation. Code of ethics of the World Medical Association and statement on responsibility in investigations on human subjects. British Medical Journal, 1964, 2, 177–180. Hsu, S.-M., Raine, L. and Fanger, H., Use of avidin–biotin– peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. Journal of Histochemistry and Cytochemistry, 1981, 29, 577–580. Herron, G. S., Unemori, E., Wong, M. et al., Connective tissue proteinases and inhibitors in abdominal aortic aneurysms. Involvement of the vasa vasorum in the pathogenesis of aortic aneurysms. Arteriosclerosis and Thrombosis, 1991, 11, 1667–1677. Holmes, D. R., Liao, S., Parkes, W. C. and Thompson, R. W., Medial neovascularisation in abdominal aortic aneurysms: A histopathological marker of aneurysmal degeneration with pathophysiological implications. Journal of Vascular Surgery, 1995, 21, 761–772. Ingber, D. E. and Folkman, J., How does extracellular matrix control capillary morphogenesis? Cell, 1989, 58, 803–805. Schott, R. J. and Morrow, L. A., Growth factors and angiogenesis. Cardiovascular Research, 1993, 27, 1155–1161. Ausprunk, D. H. and Folkman, J., Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumour angiogenesis. Microvascular Research, 1977, 14, 53–65. Sunderkotter, C., Steinbrink, K., Goebeler, M. et al., Macrophages and angiogenesis. Journal of Leukocyte Biology, 1994, 55, 410–422. Herron, G. S., Banda, M. J., Clark, E. J. et al., Secretion of metalloproteinases by stimulated capillary endothelial cells. II.

CARDIOVASCULAR SURGERY

JUNE 1998 VOL 6 NO 3

Immunophenotypic analysis of the aortic aneurysm wall: Y. V. Bobryshev et al. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. Journal of Biological Chemistry, 1986, 261, 2814–2818. 37. Szekanecz, Z., Shah, M. R., Harlow, L. A. et al., Interleukin-8

CARDIOVASCULAR SURGERY

JUNE 1998 VOL 6 NO 3

and tumour necrosis factor alpha are involved in human aortic endothelial cell migration. Pathobiology, 1994, 62, 134–139. Paper accepted 3 December 1997

249