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In vivo effects of vascular endothelial growth factor on the chicken chorioallantoic membrane

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Abstract

The effect of vascular endothelial growth factor (VEGF165) on the chorioallantoic membrane (CAM) of 13-day-old chick embryos was studied. The factor was applied in doses of 0.5–4 μg for a period of up to 4 days. Macroscopical, histological and immunohistological studies were carried out. The localization of the factor was examined with an anti-VEGF antibody. The mitogenicity of VEGF165 and basic fibroblast growth factor (bFGF) were studied by means of the BrdU-anti-BrdU method. Furthermore, the effect of heparin alone and in combination with VEGF165 was investigated. VEGF165 specifically induces angiogenesis in doses of 0.5 μg and more. A brush-like formation of blood vessels can be seen in the region of the precapillary vessels. Angiogenesis also takes place in the region of the capillaries and the venules. Histologically we found indications of sprouting as well as of intussusceptive capillary growth. The presence of the factor in the application area could be demonstrated with the anti-VEGF antibody for a period of 3 days. The factor is located in the chorionic epithelium and the intraepithelial capillaries. The BrdU-studies show that VEGF165 induces strong endothelial cell proliferation, whereas bFGF elicits fibrocyte proliferation and minor endothelial cell proliferation. Heparin induces squamous metaplasia of the chorionic and allantoic epithelium in combination with an aggregation of fibrocytes. We could not detect any enhancement of VEGF165 by heparin.

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References

  • Algire GH, Chalkley HW (1945) Vascular reactions of normal and malignant tissues in vitro: vascular reactions of mice to wounds and to normal and neoplastic transplants. J Natl Cancer Inst 6:73–81

    Google Scholar 

  • Auerbach R, Auerbach W, Polakowski J (1991) Assays for angiogenesis: a review. Pharmacol Ther 51:1–11

    Google Scholar 

  • Ausprunk DH, Folkman J (1977) Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res 14:53–65

    Google Scholar 

  • Burri PH, Tarek MR (1990) A novel mechanism of capillary growth in the rat pulmonary microcirculation. Anat Rec 228:35–45

    Google Scholar 

  • Christ B, Grim M, Wilting J, Kirschhofer K von, Wachtler F (1991) Differentiation of endothelial cells in avian embryos does not depend on gastrulation. Acta Histochem (Jena) 91:193–199

    Google Scholar 

  • Connolly DT, Heuvelman D, Nelson R, Olander JV, Eppler BL, Delfino JJ, Siegel NR, Leimgruber RM, Feder J (1989) Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 84:1470–1478

    Google Scholar 

  • Crum R, Szabo S, Folkman J (1985) A new class of steroids inhibits angiogenesis in the presence of heparin or a heparin fragment. Science 230:1375–1378

    Google Scholar 

  • DeFouw DO, Rizzo VJ, Steinfeld R, Feinberg RN (1989) Mapping of the microcirculation in the chick chorioallantoic membrane during normal angiogenesis. Microvasc Res 38:136–147

    Google Scholar 

  • Dvorak HF, Sioussat TM, Brown LF, Berse B, Nagy JA, Sotrel A, Manseau EJ, Van de Water L, Senger DR (1991) Distribution of vascular permeability factor (vascular endothelial growth factor) in tumours: concentration in tumor blood vessels. J Exp Med 174:1275–1278

    Google Scholar 

  • Ferrara N, Henzel WJ (1989) Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 161:851–858

    Google Scholar 

  • Fiebich BL, Jäger B, Schöllmann C, Weindel K, Wilting J, Kochs G, Marmé D, Hug H, Weich HA (1993) Synthesis and assembly of functionally active human vascular endothelial growth factor homodimers in insect cells. Eur J Biochem 11:19–26

    Google Scholar 

  • Folkman J (1974) Tumor angiogenesis factor. Cancer Res 34:2109–2113

    Google Scholar 

  • Folkman J (1987) Angiogenesis. In: Verstraete M, Vermylen J, Lijnan R, Arnout J (eds) Thrombosis and haemostasis. Leuven University Press, Leuven, pp 583–596

    Google Scholar 

  • Gitay-Goren H, Soker S, Vlodavsky I, Neufeld G (1992) The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules. J Biol Chem 267:6093–6098

    Google Scholar 

  • Goldmann E (1907) The growth of malignant disease in man and the lower animals with special reference to the vascular system. Lancet 2:1236–1244

    Google Scholar 

  • Gospodarowicz D, Neufeld G, Schweigerer L (1986) Molecular and biological characterization of fibroblast growth factor, an angiogenic factor which also controls the profiferation and differentiation of mesoderm and neuroectoderm derived cells. Cell Differ Dev 19:1–7

    Google Scholar 

  • Gospodarowicz D, Abraham JA, Schilling J (1989) Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc Natl Acad Sci USA 86:7311–7315

    Google Scholar 

  • Greenblatt M, Shubik P (1968) Tumor angiogenesis: transfilterdiffusion studies in the hamster by the transparent chamber technique. J Natl Cancer Inst 41:111–124

    Google Scholar 

  • Herbert JM, Laplace MC, Maffrand JP (1988) Effect of heparin on the angiogenic potency of basic and acidic fibroblast grwoth factors in the rabbit cornea assay. Int J Tissue React 3:133–139

    Google Scholar 

  • Jakeman LB, Winer J, Bennett GL, Altar CA, Ferrara N (1992) Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues. J Clin Invest 89:244–253

    Google Scholar 

  • Jakob W, Jentzsch KD, Mauersberger B, Heder G (1978) The chick embryo chorioallantoic membrane as a bioassay for angiogenesis factors: reactions induced by carrier materials. Exp Pathol 15:241–249

    Google Scholar 

  • Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N (1989) Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246:1306–1309

    Google Scholar 

  • Norrby K, Sörbo J (1992) Heparin enhances angiogenesis by a systemic mode of action. Int J Exp Pathol 73:147–155

    Google Scholar 

  • Paku S, Paweletz N (1991) First steps of tumor-related angiogenesis. Lab Invest 65:334–346

    Google Scholar 

  • Patan S, Haenni B, Burri PH (1993) Evidence for intussusceptive capillary growth in the chicken chorio-allantoic membrane (CAM). Anat Embryol (Berl) 187:121–130

    Google Scholar 

  • Peek MJ, Norman TM, Morgan C, Markham R, Fraser JS (1988) The chick chorioallantoic membrane assay: an improved technique for the study of angiogenic activity. Exp Pathol 34:35–40

    Google Scholar 

  • Plate KH, Breier G, Weich HA, Risau W (1992) Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359:845–848

    Google Scholar 

  • Plouët J, Moukadiri H (1990) Characterization of the receptor to vasculotropin on bovine adrenal cortex-derived capillary endothelial cells. J Biol Chem 265:22071–22074

    Google Scholar 

  • Plouët J, Schilling J, Gospodarowicz D (1989) Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cells. EMBO J 8:3801–3806

    Google Scholar 

  • Ribatti D, Roncali L, Nico B, Bertossi M (1987) Effects of exogenous heparin on the vasculogenesis of the chorioallantoic membrane. Acta Anat (Basel) 130:257–263

    Google Scholar 

  • Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF (1983) Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219:983–985

    Google Scholar 

  • Spanel-Borowski K, Schnapper U, Heymer B (1988) The chick chorioallantoic membrane assay in the assessment of angiogeneic factors. Biomed Res 9:253–261

    Google Scholar 

  • Tischer E, Gospodarowicz D, Mitchell R, Silva M, Schilling J, Lau K, Crisp T, Fiddes JC, Abraham JA (1989) Vascular endothelial growth factor: a new member of the platelet-derived growth factor gene family. Biochem Biophys Res Commun 165:1198–1206

    Google Scholar 

  • Usuki K, Saras J, Waltenberger J, Miyazono K, Pierce G, Thomason A, Heldin CH (1992) Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity. Biochem Biophys Res Commun 184:1311–1316

    Google Scholar 

  • Vaisman N, Gospodarowicz D, Neufeld G (1990) Characterization of the receptors for vascular endothelial growth factor. J Biol Chem 265:19461–19466

    Google Scholar 

  • Vallee BL, Riordan JF, Lobb RR, Higachi N, Fett JW, Crossley G, Bühler R, Budzik G, Breddam K, Bethune JL, Alderman EM (1985) Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review. Experientia 41:1–42

    Google Scholar 

  • Wilting J, Christ B, Bokeloh M (1991) A modified chorioallantoic membrane (CAM) assay for qualitative and quantitative study of growth factors. Studies on the effect of carriers, PBS, angiogenin and bFGF. Anat Embryol (Berl) 183:259–271

    Google Scholar 

  • Wilting J, Christ B, Weich HA (1992) The effects of growth factors on the day 13 chorioallantoic membrane (CAM): a study of VEGF165 and PDGF-BB. Anat Embryol (Berl) 186:251–257

    Google Scholar 

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This paper is dedicated to Professor R. Ortmann on the occasion of his 80th birthday

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Wilting, J., Christ, B., Bokeloh, M. et al. In vivo effects of vascular endothelial growth factor on the chicken chorioallantoic membrane. Cell Tissue Res 274, 163–172 (1993). https://doi.org/10.1007/BF00327997

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