Expression of Vascular Endothelial Growth Factor in aortocoronary saphenous vein bypass grafts

Cardiovascular Surgery, Vol. 9, No. 5, pp. 492–498, 2001  2001 The International Society for Cardiovascular Surgery. Published by Elsevier Science Ltd All rights reserved 0967-2109/01 $20.00

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Expression of Vascular Endothelial Growth Factor in aortocoronary saphenous vein bypass grafts Y. V. Bobryshev†, A. E. Farnsworth‡ and R. S. A. Lord† †Surgical Professorial Unit, St. Vincent’s Hospital, University of New South Wales, Darlinghurst, NSW 2010, Australia and ‡Department of Cardiothoracic Surgery, St. Vincent’s Hospital, Darlinghurst, NSW 2010, Australia Neovascularisation is a prominent feature of long-term aortocoronary saphenous vein bypass grafts but mechanisms involved in the formation of neovessels have not been previously studied. Vascular Endothelial Growth Factor (VEGF) is an important angiogenic factor that induces migration and proliferation of endothelial cells, enhances permeability and modulates thrombogenecity. This study investigated the expression of VEGF in aortocoronary saphenous vein bypass grafts. Aortocoronary saphenous vein bypass grafts with angiographic luminal stenosis of >75% were explanted from 14 patients at redo coronary artery bypass grafting. The grafts demonstrated two distinct forms of graft occlusion: four out of the 14 graft occlusions (29%) resulted from severe hyperplastic transformation of the intima complicated by thrombi attached to the degenerating liminal endothelium; the remaining graft occlusions (71%) were due to the development of atherosclerotic lesions associated with mural thrombosis. Hiperplastically altered intimal segments were practically free of neovascularisation while atherosclerotic-like lesions contained neovessels irregularly distributed throughout. Intimal neovessels were located exclusively in microzones enriched with VEGF-expressing cells and, furthermore, neovascular endothelial cells themselves also displayed VEGF immunopositivity. Doubleimmunostaining revealed that in areas of neovascularisation, the vast majority macrophages (CD68+) expressed VEGF. Some CD68+ foam cells that surrounded branches of neovascularisation were also VEGF-positive. These findings suggest that VEGF expressed by neovascular endothelial cells and by macrophages may act as a local regulator of endothelial cells functions and may induce intimal neovascularisation in aortocoronary saphenous vein bypass grafts affected by atherosclerosis.  2001 The International Society for Cardiovascular Surgery. Published by Elsevier Science Ltd. All rights reserved Keywords: vascular endothelial growth factor (VEGF), aortocoronary saphenous vein bypass grafts, intimal neovascularisation

Introduction The long-term usefulness of aortocoronary saphenous vein bypass grafts used as conduits in cor-

Correspondence to: Dr Yuri V. Bobryshev, Surgical Professorial Unit, Level 17, O’Brien Building, St. Vincent’s Hospital, Victoria Street, Darlinghurst, NSW 2010, Australia. Tel.: +61-2-8382-2643; Fax: +61-2-9360-4424; e-mail: [email protected]

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onary artery bypass grafting is limited by a disease complex comprised of intimal hyperplasia and atherosclerosis [1, 2]. In our previous investigations of aortocoronary saphenous vein bypass grafts explanted from man, we noted that atheroscleroticlike lesions contained a large number of neovessels [3, 4]. Mechanisms of neovessel formation and the impact of neovascularisation on the graft failure are unknown. Several studies have recently focused on the CARDIOVASCULAR SURGERY

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Vascular endothelial growth factor: Y. V. Bobryshev et al. Table 1

Antibodies used in the study

Antibody

Cell identified

Source

Working dilation

Anti-VEGF Anti-VCAM-1 Anti-von Willebrand factor CD34 EBM11 (CD68) CD3 SMA (IA4) S100 CD15

VEGF expressing cells VCAM-1 expressing cells Endothelial cells Endothelial cells Macrophages T-cells Smooth muscle cells Dendritic cells; nerve tissue cells Mast cells, granulocytes

Santa Cruz Dako Dako Novocastra Laboratories Ltd Dako Dako Dako Dako Dako

1:700 1:100 1:50 1:50 1:50 1:100 1:100 1:700 1:50

mechanisms of neovessel formation occurring in primary atherosclerotic lesions in human large arteries, including the coronary arteries, carotid arteries and the aorta [5–10]. The importance of intimal neovascularisation in atherosclerosis has been suggested to relate the development of such complications as intimal haemorrhage, plaque rupture and the formation of occlusive thrombus [11– 15]. Despite the diversity of neovessel distribution in atherosclerotic plaques, the density of newly formed blood vessels in atherosclerotic intima in coronary arteries has been found to positively correlate with the degree of luminal stenosis [12]. The newly formed blood vessels are frequently associated with chronic inflammatory cell infiltrates and the formation of granulation tissue resulting from the chronic inflammation/repair process has been suggested to be important in vessel occlusion [12]. The mechanisms of intimal neovascularisation and the major stimuli inducing neovascularization in the atherosclerotic intima are poorly understood but recent findings suggest a crucial role of vascular endothelial growth factor (VEGF)/vascular permeability factor in this process [16–18]. VEGF is a multifunctional cytokine for endothelial cells expressing VEGF receptor-1 (flt-1) and 2 (flk1/KDR) [19, 20] that increases vascular permeability [21] and integrin expression [22] and that also modulates endothelial expression of fibrinolysis- and coagulation-related agents including plasminogen activators and plasminogen activator inhibitor-1 [23]. Interaction between VEGF isoform(s) and its receptors has been found to be the most important angiogenic event in both mammalian embryogenesis and in the physiological and pathological angiogenesis in adults [24]. VEGF also induces tissue factor expression and migration of monocytes expressing VEGF receptor-1 [25, 26]. In primary atherosclerotic plaques in human coronary plaques, VEGF has been found expressed by a variety of intimal cell types including smooth muscle cells and T lymphocytes [16–18]. Advanced atherosclerotic plaques containing a large number of VEGF-positive cells are much richer in neovascularisation than earlier CARDIOVASCULAR SURGERY

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atherosclerotic lesions containing fewer VEGFexpressing cells [16–18]. Our present study aimed to investigate the relationship between neovascularisation and VEGF expression in aortocoronary saphenous vein bypass grafts.

Materials and methods This study was approved by the Institutional Review Board of the St. Vincent’s Hospital, Sydney, and the materials were collected in accordance with the principles outlined in the Declaration of Helsinki [27]. Specimens Human aortocoronary saphenous vein bypass grafts with luminal stenosis of >75% as demonstrated by angiography, were explanted from 14 patients at redo coronary artery bypass graft operation at the St. Vincent’s Hospital, Sydney. The patients (11 male and 3 female) were aged between 51 and 75 yr (mean age 66.6 yr) with the graft implant time ranging from 8 to 22 yr (mean 12.9 yr). The clinical details of patients and some characteristics relating to cell composition and expression of apoptosisrelated proteins have been previously reported [4]. Every specimen was divided into two parts: one portion was fixed in 10% buffered formalin and embedded in paraffin while the other unfixed portion was 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 µm thickness and air dried. Single immunohistochemical staining An analysis was carried out using sets of consecutive parallel sections immunostained with antibodies summarised in Table 1. After eliminating endogenous peroxidase activity by 0.3% H2O2 for 5 min, the sections were preincubated with normal serum, and then were tested by avidin–biotin complex using the ABC immunoperoxidase method [28] as previously descibed [3, 4, 10]. In brief: after washing in Tris493

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phosphate buffered saline (TPBS), pH 7.6, the sections were incubated with the appropriate secondary antibody. The sections were then washed in TPBS for 5 min and treated with avidin–biotin complex (Elite -ABC, Vector PK6100). A red staining was produced by treatment with the AEC substrate kit or ABC substrate kit [3, 4]. All the incubations were performed at room temperature. For negative control, the primary antibody was omitted or the sections were treated with an immunoglobulin fraction of non-immune serum 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 using an Olympus microscope at 10×10 and 10×40 magnifications. Double immunohistochemistry For double immunostaining, combinations of antibody to VEGF with the various cell type specific antibodies (Table 1) were used. Double immunostaining procedures were carried out as previously described [10]. In brief: after visualisation of the first antigen with the AEC substrate kit or ABC substrate kit, the tissue sections were washed several times during 60 min with 0.1 M glycine-hydrochloric buffer (pH 2.2) at 4°C. The sections were further incubated overnight at 4°C with the secondary antibody. After rinsing in TPBS, the sections were incubated with a biotinylated antiobody and then were incubated with alkaline phosphatase-conjugated streptoavidin. Alternatively, differing combinations of antigens were analysed by using DakoDoublestain Kit System as previously used [10]. The kit allows simultaneous staining for the detection of two different tissue markers on the same section by a combination of the peroxidase-anti-peroxidase (PAP) and alkaline phosphatase-antialkaline phosphatase (APAAP) techniques. Negative controls were carried out according to the Doublestain Kit System manufacturer’s instructions (Dako). Counterstaining was performed with Mayer’s haematoxylin.

Results Histological characteristics The grafts demonstrated two distinct forms of graft occlusion: four out of the 14 graft occlusions (29%) resulted from severe hyperplastic transformation of the intima complicated by thrombi attached to the degenerating luminal endothelium; the remaining graft occlusions (71%) were due to the development of atherosclerotic lesions associated with mural thrombosis [4]. 494

VEGF expression in graft intima without signs of atherosclerotic transformation Immunostaining with anti-VEGF antibody demonstrated that, in hyperplastically altered intimal segments without signs of atherosclerotic transformation, only a few scattered cells in the deep portion of intima expressed VEGF and the intensity of this staining was low. Staining of parallel sections with anti-von Willebrand factor and anti-CD34 antibodies demonstrated that the intima in these grafts was practically free of neovascularisation and consisted of spindle-shaped alpha-smooth muscle actin+ cells which were encrusted in the extracellular matrix. VEGF expression in grafts affected by atherosclerosis All the 10 grafts affected by atherosclerosis contained VEGF-positive cells irregularly distributed throughout the lesions (Figure 1). VEGF-positive cells were most frequently observed in the areas where neovessels were present. It was evident that some neovessels themselves also displayed VEGF immunopositivity [Figure 1(A,C)]. Analysis of consecutive sections stained with anti-VCAM-1 demonstrated that neovascular endothelial cells expressed VCAM-1 [Figure 1(A,B)]. The co-occurrence of both neovascularisation and vegf expression was consistently observed in all graft atherosclerotic lesions but we were not able to detect any areas containing neovessels where there were no VEGF positive cells. Around neovessels, irregularly shaped VEGF-positive cells were often observed [Figure 1(C,D)] and some foam cells also displayed vegf immunopositivity [Figure 1(E)]. Identification of the nature of VEGF-positive cells Immunostaining of consecutive sections with cell type specific antibodies showed that neovascularisation areas contained large numbers of macrophages (CD68+) and T-cells (CD3+) intermingled with few B-cells (CD20+), dendritic cells (S-100+) and mast cells (CD15+). Some spindle-shaped smooth muscle cells (SMA+) were also consistently present in these areas. Branches of the inflamed neovascularisation were surrounded by foam cells mainly expressing CD68 antigen. Double-immunostaining revealed that in areas of neovascularisation, the vast majority macrophages (CD68+) expressed VEGF [Figure 2(A,B)]. Some CD68+ foam cells that surrounded branches of neovascularisation were also VEGF-positive. Furthermore, as noted above, endothelial cells of neovessels also displayed VEGF immunopositivity [Figure 1(A)]. Adventitial vasa vasora associated with neovessels were CARDIOVASCULAR SURGERY

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Figure 1 Expression of VEGF in aortocoronary saphenous vein bypass grafts. (A) and (B) represent consecutive sections demonstrating co-expression of VEGF (A) and VCAM-1 (B) by neovascular endothelial cells (arrows). Neovessels are marked by asterisks. In (C), small arrows show endothelial cells expressing VEGF and large arrows mark irregularly-shaped VEGF+ cells located around a neovessel (asterisk). (D) demonstrates VEGF expression in irregularly-shaped cells (arrows) while (E) shows VEGF immunopositivity displayed by foam cells (arrows). ABC immunoperoxidase technique. Counterstaining with Mayer’s haematoxylin. Magnification: ×400 (A–E)

Figure 2 Double immunostaing demonstrating expression of VEGF by macrophages (A) and by a cluster of macrophages (B) in areas of neovascularisation in a aortocoronary saphenous vein bypass graft (A,B). CD68+ macrophages (brown) expressing VEGF (rose) are shown by large arrows. Small arrows indicate neovascular endothelial cells expressing VEGF (rose). Neovessels are marked by asterisks. A combination of PAP and APAAP immunotechniques. Counterstaining with Mayer’s haematoxylin. Magnification: ×400 (A,B)

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intensely positive for VEGF. We were not able to detect VEGF in T-cells and smooth muscle cells.

Discussion The present study is the first to demonstrate that VEGF is expressed in aortocoronary saphenous vein bypass grafts. VEGF expression was found focally distributed in atherosclerotic-like lesions in the grafts and was associated with areas of neovascularisation. Practically no VEGF was detected in non-atherosclerotic hyperplastic intima. Similarly, a pilot examination of four normal saphenous veins showed that there was no VEGF expression in either the intimal or medial layers (unpublished data). In primary atherosclerotic lesions, the ingrowth of vasa vasorum through the media into the intimal lesions leads to the formation of nexuses of neovascularisation and thus provides an entry route for plasma proteins to access the intima. Neovascularisation appears also to be the main route for the migration of inflammatory and immunocompetent cells into the growing atherosclerotic lesions [5–10, 14] and thus may lead to lumen occlusion in aortocoronary saphenous vein bypass grafts. The present observation that neovessels in grafts were detected only in areas populated by cells expressing VEGF suggests that VEGF expression might contribute to graft occlusion. Recent in vivo and in vitro studies have demonstrated that the transformation of endothelial cells to an angiogenic phenotype is induced by various angiogenic factors including VEGF, basic fibroblast growth factor (bFGF), hepatocyte growth factor and interleukin-8 [29], but the interaction between the VEGF isoform(s) and its receptor types 1 and 2 appears to be crucial for angiogenesis [19–24]. This process is universal and occurs in embryogenesis [30, 31] as well as during tumour growth [32] and inflammation-repair processes [33]. The stimuli inducing VEGF expression (or overexpression) are not yet well understood but in vitro studies indicate that hypoxia [34, 35], growth factors including basic fibroblast growth factor [36], platelet-derived growth factor [37], transforming growth factor-ß [38] and cytokines including tumour necrosis factor alpha and -1beta [39], all of which participate in atherogenesis [40], stimulate smooth muscle cells, macrophages and other cell types to upregulate VEGF expression. Several potential binding sites for the transcriptional factors activator protein-1 (AP-1), AP-2, and Sp-151 and hypoxia regulatory elements have been identified in the VEGF gene promoter and in the 5⬘ and 3⬘ regions of the VEGF gene, respectively [41–43]. AP-1 activity has been found to participate in the enhancement of VEGF expression induced not only by the proinflammatory cytokine TNF-alpha but also by hypoxia in tumour cells [44]. 496

TNF-alpha can increase Sp-1-mediated VEGF expression as well [45]. In primary atherosclerosis, both hypoxia in the deep layer of atherosclerotic intima and proinflammatory cytokines TNF-alpha and IL-1beta can induce VEGF expression [46]. VEGF expression in aortocoronary saphenous vein bypass grafts might be the response to atherosclerosis related vessel wall hypoxia. Our study demonstrates that in aortocoronary saphenous vein bypass grafts, inflammatory cell accumulation within the atheromatous lesions occurs in the areas where cells express VEGF. The mononuclear cell adhesion molecule VCAM-1 was also found expressed by neovessels, which suggests that leukocytes and T-cells may be recruited into lesions through the neovessels. This is consistent with studies of primary atherosclerotic lesions [5, 8] which demonstrated that the expression of VCAM-1 on neovascular endothelium is associated with increased intimal leukocyte accumulation. Recently, we demonstrated that in aortocoronary saphenous vein bypass grafts, inflammatory infiltrates frequently contain antigenpresenting dendritic cells co-localising and forming clusters with T-cells [3] which may suggest that the initiation of primary immune responses may occur directly within the grafts. The present observation that macrophages surrounding neovessels express VEGF suggests that inflammatory cells might influence the growth of new vessels in the grafts.

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

References 1. Motwani, J. G. and Topol, E. J., Aortocoronary saphenous vein graft disease: pathogenesis, predisposition, and prevention. Circulation, 1998, 97, 916–931. 2. Nielsen, T. G., Laursen, H., Gronholdt, M. L. et al., Histopathological features of in situ vein bypass stenoses. European Journal of Vascular and Endovascular Surgery, 1997, 14, 492–498. 3. Cherian, S. M., Bobryshev, Y. V., Inder, S. J. et al., Involvement of dendritic cells in long term aortocoronary saphenous vein bypass graft failure. Cardiovascular Surgery, 1999, 7, 508–518. 4. Wang, A. Y., Bobryshev, Y. V., Cherian S. M. et al. Expression of apoptosis-related proteins and structural features of cell death in explanted aortocoronary saphenous vein bypass grafts. Cardiovascular Surgery, 2001, in press. 5. O’Brien, K. D., Allen, M. D., McDonald, T. O. et al., Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plaques: implications for the mode of progression of advanced coronary atherosclerosis. Journal of Clinical Investigation, 1993, 92, 945–951. 6. Zhang, Y., Cliff, W. J., Schoefl, G. I. et al., Immunohistochemical study of intimal microvessels in coronary atherosclerosis. American Journal of Pathology, 1993, 143, 164–172. 7. O’Brien, E. R., Garvin, M. R., Dev, R. et al., Angiogenesis in human coronary atherosclerotic plaques. American Journal of Pathology, 1994, 145, 883–894. 8. Ignatescu, M. C., Gharehbaghi-Schnell, E., Hassan, A., et al.,

CARDIOVASCULAR SURGERY

OCTOBER 2001 VOL 9 NO 5

Vascular endothelial growth factor: Y. V. Bobryshev et al.

9.

10.

11.

12.

13. 14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24. 25.

Expression of the angiogenic protein, platelet-derived endothelial cell growth factor, in coronary atherosclerotic plaques: In vivo correlation of lesional microvessel density and constructive vascular remodeling. Arteriosclerosis, Thrombosis and Vascular Biology, 1999, 19, 2340–2347. O’Brien, K. D., McDonald, T. O., Chait, A. et al., 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. Circulation, 1996, 93, 672–682. Bobryshev, Y. V., Cherian, S. M. and Inder, S. J., Neovascular expression of VE-cadherin in human atherosclerotic arteries and its relation to intimal inflammation. Cardiovascular Research, 1999, 43, 1003–1017. Barger, A. C., Beeuwkes, R., Lainey, L. L. et al., Hypothesis: vasa vasorum and neovascularization of human coronary arteries: a possible role in the pathophysiology of atherosclerosis. New England Journal of Medicine, 1984, 310, 175–177. Kumamoto, M., Nakashima, Y. and Sueishi, K., Intimal neovascularization in human coronary atherosclerosis: its origin and pathophysiological significance. Human Pathology, 1995, 26, 450–456. Buja, L. M. and Willerson, J. T., Role of inflammation in coronary plaque disruption. Circulation, 1994, 89, 503–505. de Boer, O. J., van der Wal, A. C., Teeling, P. et al., Leukocyte recruitment in rupture prone regions of lipid-rich plaques: a prominent role for neovascularization? Cardiovascular Research, 1999, 41, 443–449. van der Wal, A. C. and Becker, A. E., Atherosclerotic plaque rupture-pathologic basis of plaque stability and instability. Cardiovascular Research, 1999, 41, 334–344. Couffinhal, T., Kearney, M., Witzenbichler, B. et al., Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries. American Journal of Pathology, 1997, 150, 1673–1685. Inoue, M., Itoh, H., Ueda, M. et al., Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: Possible pathophysiological significance of VEGF in progression of atherosclerosis. Circulation, 1998, 98, 2108–2116. Chen, Y. X., Nakashima, Y., Tanaka, K. et al., Immunohistochemical expression of vascular endothelial growth factor/vascular permeability factor in atherosclerotic intimas of human coronary arteries. Arteriosclerosis, Thrombosis and Vascular Biology, 1999, 19, 131–139. Klagsbrun, M. and D’Amore, P. A., Vascular endothelial growth factor and its receptors. Cytokine and Growth Factor Review, 1996, 7, 259–270. Stephan, C. C. and Brock, T. A., Vascular endothelial growth factor, a multifunctional polypeptide. P.R. Health Science Journal, 1996, 15, 169–178. Dvorak, H. F., Brown, L. F., Detmar, M. et al., Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. American Journal of Pathology, 1995, 146, 1029–1039. Senger, D. R., Claffey, K. P., Benes, J. E. et al., Angiogenesis promoted by vascular endothelial growth factor: regulation through 1ß1 integrins. Proceedings of National Academy of Science USA, 1997, 94, 13612–13617. Pepper, M. S., Ferrara, N., Orci, L. et al., Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells. Biochemical and Biophysical Research Communications, 1991, 181, 902–906. Ferrara, N. and Davis-Smyth, T., The biology of vascular endothelial growth factor. Endocrinology Review, 1997, 18, 4–25. Clauss, M., Weich, H., Breier, G. et al., The vascular endothelial growth factor receptor Flt-1 mediates biological activities: implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. Journal of Biological Chemistry, 1996, 271, 17629–17634.

CARDIOVASCULAR SURGERY

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26. Barleon, B., Sozzani, S., Zhou, D. et al., Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood, 1996, 87, 3336–3343. 27. 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. 28. 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 Histochemestry and Cytochemistry, 1981, 29, 577–580. 29. Colville-Nash, P. R. and Willoughby, D. A., Growth factors in angiogenesis: current interest and therapeutic potential. Molecular Medicine Today, 1997, 3, 14–23. 30. Breier, G., Damert, A., Plate, K. H. et al., Angiogenesis in embryos and ischemic diseases. Thrombosis and Haemostasis, 1997, 78, 678–683. 31. Shalaby, F., Rossant, J., Yamaguchi, T. P. et al., Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature, 1995, 376, 62–66. 32. Hanahan, D. and Folkman, J., Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 1996, 86, 353–364. 33. Appleton, I., Brown, N. J., Willis, D. et al., The role of vascular endothelial growth factor in a murine chronic granulomatous tissue air pouch model of angiogenesis. Journal of Pathology, 1996, 180, 90–94. 34. Hata, Y., Nakagawa, K., Ishibashi, T. et al., Hypoxia-induced expression of vascular endothelial growth factor by retinal glial cells promotes in vitro angiogenesis. Virchows Archives, 1995, 426, 479–486. 35. Shweiki, D., Itin, A., Soffer, D. et al., Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature, 1992, 359, 843–845. 36. Stavri, G. T., Zachary, I. C., Baskerville, P. A. et al., Basic fibroblast growth factor upregulates the expression of vascular endothelial growth factor in vascular smooth muscle cells: synergistic interaction with hypoxia. Circulation, 1995, 92, 11–14. 37. Stavri, G. T., Hong, Y., Zachary, I. C. et al., Hypoxia and platelet-derived growth factor-BB synergistically upregulate the expression of vascular endothelial growth factor in vascular smooth muscle cells. FEBS Letters, 1995, 358, 311–315. 38. Pertovaara, L., Kaipainen, A., Mustonen, T. et al., Vascular endothelial growth factor is induced in response to transforming growth factor-ß in fibroblastic and epithelial cells. Journal of Biological Chemistry, 1994, 269, 6271–6274. 39. Jackson, J. R., Minton, J. A., Ho, M. L. et al., Expression of vascular endothelial growth factor in synovial fibroblasts is induced by hypoxia and interleukin 1ß. Journal of Rheumatology, 1997, 24, 1253–1259. 40. Ross, R., The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 1993, 362, 801–809. 41. Silins, G., Grimmond, S., Egerton, M. et al., Analysis of the promoter region of the human VEGF-related factor gene. Biochemical and Biophysical Research Communications, 1997, 230, 413– 418. 42. Liu, Y., Cox, S. R., Morita, T. et al., Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells: identification of a 5⬘ enhancer. Circulation Research, 1995, 77, 638–643. 43. Minchenko, A., Salceda, S., Bauer, T. et al., Hypoxia regulatory elements of the human vascular endothelial growth factor gene. Cell and Molecular Biology Research, 1994, 40, 35–39. 44. Damert, A., Ikeda, E. and Risau, W., Activator-protein-1 binding potentiates the hypoxia-inducible factor-1-mediated hypoxiainduced transcriptional activation of vascular-endothelial growth factor expression in C6 glioma cells. Biochemical Journal, 1997, 327, 419–423.

497

Vascular endothelial growth factor: Y. V. Bobryshev et al. 45. Ryuto, M., Ono, M., Izumi, H. et al., Induction of vascular endothelial growth factor by tumor necrosis factor in human glioma cells: possible roles of SP-1. Journal of Biological Chemistry, 1996, 271, 28220–28228. 46. Bjornheden, T., Evaldsson, M. and Wiklund, O., A method for

498

the assessment of hypoxia in the arterial wall, with potential application in vivo. Arteriosclerosis, Thrombosis and Vascular Biology, 1996, 16, 178–185. Paper accepted 22 March 2001

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