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Basic Fibroblast Growth Factor-Induced Angiogenesis in the Chick Embryo Chorioallantoic Membrane: An Electron Microscopy Study
Microvascular Research 53, 187 – 190 (1997) Article No. MR961993
BRIEF COMMUNICATION Basic Fibroblast Growth Factor-Induced Angiogenesis in the Chick Embryo Chorioallantoic Membrane: An Electron Microscopy Study Domenico Ribatti, Beatrice Nico, Mirella Bertossi, Luisa Roncali, and Marco Presta* Institute of Human Anatomy, Histology and Embryology, University of Bari, Bari 70124; and *Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia 25123, Italy Received May 23, 1996
INTRODUCTION Basic fibroblast growth factor (bFGF), one of the most potent angiogenic peptides (Baird and Bohlen, 1990), is a pleiotropic factor that interacts with various cell types. For instance, it is a mitogen for fibroblasts and smooth muscle cells and for some epithelial cells (Basilico and Moscatelli, 1992). The chick embryo chorioallantoic membrane (CAM) is widely used for the evaluation of angiogenic factors (Auerbach et al., 1991). We have shown previously that a bFGF-like molecule is present in the CAM (Ribatti et al., 1995). In this study, we have ultrastructurally investigated the effects of human recombinant bFGF and of neutralizing anti-bFGF antibody on CAM structure and vascularization.
MATERIALS AND METHODS Embryonic CAMs were treated on Day 8 with 4 mg of bFGF (30 embryos) or 400 ng of neutralizing poly-
clonal anti-bFGF antibody (30 embryos) absorbed on methylcellulose discs (Ribatti et al., 1995). Discs containing irrelevant rabbit IgG or vehicle alone (PBS) were used as negative controls and were implanted on top of CAM of 20 embryos. At Day 12, CAMs were processed for electron microscopy (Ribatti et al., 1995).
RESULTS A stratified chorionic epithelium with single, scattered degenerating epithelial cells was observed under all the bFGF polymers (Fig. 1A). The number of blood vessels, predominantly capillaries, was increased. Small vascular tubes located near the chorion were also recognizable and mitotic figures were detectable in these vessel primordia (Figs. 1B and 1C). The mesenchymal stroma consisted of densely arranged fibroblasts. Numerous leukocytes and scattered mast cells were present in perivascular positions (Fig. 1D). The endoderm consisted of a single layer of cuboidal cells.
0026-2862/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
MVR 1993
/
6z0b$$$201
187
03-11-97 13:52:23
mvrg
AP: MVR
188
Brief Communication
FIG. 1. Particulars of CAM morphology after treatment with exogenous bFGF. (A) A trilayered chorionic epithelium beneath the methylcellulose polymer (p). (B) A small vascular tube is recognizable near the chorion. (C) A capillary endothelial cell in mitosis. (D) A mast cell and a fibroblast in the intermediate mesenchyme. Original magnifications, A, 14000; B, 118,000; C, 121,000; D, 122,000.
Chorion under the polymers containing anti-bFGF antibody consisted of two layers of epithelial cells. Beneath the chorion only isolated capillaries were still recognizable (Fig. 2). No blood vessels were present in the mesenchyme, where few fibroblasts were still detectable among wide intercellular spaces devoid of collagen fibrils (Fig. 2). The endoderm consisted of a single layer of cuboidal epithelial cells. In 90% of control embryos both the vascular pattern and the fine structure of the chorioallantoic membrane resembled those of normally developed CAMs. In the remaining 10% of control embryos, the chorion in the area beneath the disc was thickened.
DISCUSSION This ultrastructural study demonstrates that exogenous bFGF applied onto the surface of the CAM exerts a pleiotropic action consisting mainly of stimulation of angiogenesis associated with hyperplasia of the chorion and fibroblast cell proliferation. Accordingly, the block of the activity of endogenous bFGF following application of anti-bFGF antibody causes inhibition of CAM neovascularization as well as inhibition of fibroblast cell proliferation and deposition of interstitial collagen fibers.
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
MVR 1993
/
6z0b$$$202
03-11-97 13:52:23
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AP: MVR
189
Brief Communication
FIG. 2. CAM morphology after treatment with anti-bFGF antibody. An isolated capillary is present beneath the chorion. In the mesenchyme only two fibroblasts are present within wide intercellular spaces devoid of collagen fibers and blood vessels. Original magnification, 115,000.
Application of exogenous bFGF between Days 8 and 12 induces an angiogenic response in 85% of CAMs, and the microvessel density is 3.3 times higher in bFGFtreated than in control embryos (Ribatti et al., 1995). Transmission electron microscopy has confirmed that no mitotic endothelial cells or vascular sprouts are found in control CAMs at Day 12, while bFGF-treated CAMs show the presence of numerous newly formed and immature vessels at Day 12. In keeping with the capacity to exert a mitogenic activity for a variety of cell types of mesodermal and neuroectodermal origin (Burgess and Maciag, 1989; Gospodarowicz, 1991), bFGF induces also fibroblast cell proliferation and hyperplasia of the chorionic epithelium. An inflammatory infiltrate surrounding the newly formed blood vessels was observed in bFGF-treated CAMs, but not in control CAMs. The infiltrate, as demonstrated in other inflammatory-related angiogenesis processes (Folkman and Brem, 1992), may participate in the vasoproliferative response of the CAM to exogenous bFGF. Very few isolated capillaries are still recognizable be-
neath the chorion after application of anti-bFGF antibody, which prevents the formation of new blood vessels from Day 8 to Day 12, but also causes the regression of most of the vessels already present at the beginning of the antibody treatment at Day 8. Furthermore, we have observed that anti-bFGF antibody exerts an inhibitory effect on the intermediate mesenchyme where no blood vessels and only a few fibroblasts and collagen fibers are still detectable. This observation indicates that endogenous bFGF plays a role not only in the formation of the capillary plexus but also in the development of intermediate mesenchyme blood vessels. Also in keeping with its pleiotropic activity, endogenous bFGF is of importance for the physiological development of CAM mesodermal tissue by mediating fibroblast cell proliferation and deposition of collagen fibers.
ACKNOWLEDGMENTS This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro to D.R. and M.P. and from CNR to M.P. (Grants 94.00316.CT14 and 95.02925.CT14).
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
MVR 1993
/
6z0b$$$202
03-11-97 13:52:23
mvrg
AP: MVR
190
Brief Communication
REFERENCES
Auerbach, R., Auerbach, W., and Polakowski, I. (1991). Assays for angiogenesis. Pharmacol. Ther. 51, 1 – 11. Baird, A., and Bohlen, P. (1990). Fibroblasts growth factors. In Peptide Growth Factors (M. C. Sporn and A. B. Roberts, Eds.), pp. 369 – 417. Springer, New York. Basilico, C., and Moscatelli, D. (1992). The FGF family of growth factors and oncogenes. Adv. Cancer Res. 59, 115 – 165. Burgess, W. H., and Maciag, T. (1989). The heparin-binding (fibro-
blast) growth factor family of protein. Annu. Rev. Biochem. 58, 575 – 606. Folkman, J., and Brem, H. (1992). Angiogenesis and inflammation. In Inflammation: Basic Principles and Clinical Applications (J. I. Gallin, M., Goldtsein, and R. Snyderman, Eds.), pp. 821 – 839. Raven Press, New York. Gospodarowicz, D. (1991). Biological activities of fibroblast growth factor. Annu. N.Y. Acad. Sci. 638, 1 – 8. Ribatti, D., Urbinati, C., Nico, B., Rusnati, M., Roncali, L., and Presta, M. (1995). Endogenous basic fibroblast growth factor is implicated in the vascularization of the chick embryo chorioallantoic membrane. Dev. Biol. 170, 34 – 49.
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
MVR 1993
/
6z0b$$$202
03-11-97 13:52:23
mvrg
AP: MVR
BRIEF COMMUNICATION Basic Fibroblast Growth Factor-Induced Angiogenesis in the Chick Embryo Chorioallantoic Membrane: An Electron Microscopy Study Domenico Ribatti, Beatrice Nico, Mirella Bertossi, Luisa Roncali, and Marco Presta* Institute of Human Anatomy, Histology and Embryology, University of Bari, Bari 70124; and *Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia 25123, Italy Received May 23, 1996
INTRODUCTION Basic fibroblast growth factor (bFGF), one of the most potent angiogenic peptides (Baird and Bohlen, 1990), is a pleiotropic factor that interacts with various cell types. For instance, it is a mitogen for fibroblasts and smooth muscle cells and for some epithelial cells (Basilico and Moscatelli, 1992). The chick embryo chorioallantoic membrane (CAM) is widely used for the evaluation of angiogenic factors (Auerbach et al., 1991). We have shown previously that a bFGF-like molecule is present in the CAM (Ribatti et al., 1995). In this study, we have ultrastructurally investigated the effects of human recombinant bFGF and of neutralizing anti-bFGF antibody on CAM structure and vascularization.
MATERIALS AND METHODS Embryonic CAMs were treated on Day 8 with 4 mg of bFGF (30 embryos) or 400 ng of neutralizing poly-
clonal anti-bFGF antibody (30 embryos) absorbed on methylcellulose discs (Ribatti et al., 1995). Discs containing irrelevant rabbit IgG or vehicle alone (PBS) were used as negative controls and were implanted on top of CAM of 20 embryos. At Day 12, CAMs were processed for electron microscopy (Ribatti et al., 1995).
RESULTS A stratified chorionic epithelium with single, scattered degenerating epithelial cells was observed under all the bFGF polymers (Fig. 1A). The number of blood vessels, predominantly capillaries, was increased. Small vascular tubes located near the chorion were also recognizable and mitotic figures were detectable in these vessel primordia (Figs. 1B and 1C). The mesenchymal stroma consisted of densely arranged fibroblasts. Numerous leukocytes and scattered mast cells were present in perivascular positions (Fig. 1D). The endoderm consisted of a single layer of cuboidal cells.
0026-2862/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
MVR 1993
/
6z0b$$$201
187
03-11-97 13:52:23
mvrg
AP: MVR
188
Brief Communication
FIG. 1. Particulars of CAM morphology after treatment with exogenous bFGF. (A) A trilayered chorionic epithelium beneath the methylcellulose polymer (p). (B) A small vascular tube is recognizable near the chorion. (C) A capillary endothelial cell in mitosis. (D) A mast cell and a fibroblast in the intermediate mesenchyme. Original magnifications, A, 14000; B, 118,000; C, 121,000; D, 122,000.
Chorion under the polymers containing anti-bFGF antibody consisted of two layers of epithelial cells. Beneath the chorion only isolated capillaries were still recognizable (Fig. 2). No blood vessels were present in the mesenchyme, where few fibroblasts were still detectable among wide intercellular spaces devoid of collagen fibrils (Fig. 2). The endoderm consisted of a single layer of cuboidal epithelial cells. In 90% of control embryos both the vascular pattern and the fine structure of the chorioallantoic membrane resembled those of normally developed CAMs. In the remaining 10% of control embryos, the chorion in the area beneath the disc was thickened.
DISCUSSION This ultrastructural study demonstrates that exogenous bFGF applied onto the surface of the CAM exerts a pleiotropic action consisting mainly of stimulation of angiogenesis associated with hyperplasia of the chorion and fibroblast cell proliferation. Accordingly, the block of the activity of endogenous bFGF following application of anti-bFGF antibody causes inhibition of CAM neovascularization as well as inhibition of fibroblast cell proliferation and deposition of interstitial collagen fibers.
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
MVR 1993
/
6z0b$$$202
03-11-97 13:52:23
mvrg
AP: MVR
189
Brief Communication
FIG. 2. CAM morphology after treatment with anti-bFGF antibody. An isolated capillary is present beneath the chorion. In the mesenchyme only two fibroblasts are present within wide intercellular spaces devoid of collagen fibers and blood vessels. Original magnification, 115,000.
Application of exogenous bFGF between Days 8 and 12 induces an angiogenic response in 85% of CAMs, and the microvessel density is 3.3 times higher in bFGFtreated than in control embryos (Ribatti et al., 1995). Transmission electron microscopy has confirmed that no mitotic endothelial cells or vascular sprouts are found in control CAMs at Day 12, while bFGF-treated CAMs show the presence of numerous newly formed and immature vessels at Day 12. In keeping with the capacity to exert a mitogenic activity for a variety of cell types of mesodermal and neuroectodermal origin (Burgess and Maciag, 1989; Gospodarowicz, 1991), bFGF induces also fibroblast cell proliferation and hyperplasia of the chorionic epithelium. An inflammatory infiltrate surrounding the newly formed blood vessels was observed in bFGF-treated CAMs, but not in control CAMs. The infiltrate, as demonstrated in other inflammatory-related angiogenesis processes (Folkman and Brem, 1992), may participate in the vasoproliferative response of the CAM to exogenous bFGF. Very few isolated capillaries are still recognizable be-
neath the chorion after application of anti-bFGF antibody, which prevents the formation of new blood vessels from Day 8 to Day 12, but also causes the regression of most of the vessels already present at the beginning of the antibody treatment at Day 8. Furthermore, we have observed that anti-bFGF antibody exerts an inhibitory effect on the intermediate mesenchyme where no blood vessels and only a few fibroblasts and collagen fibers are still detectable. This observation indicates that endogenous bFGF plays a role not only in the formation of the capillary plexus but also in the development of intermediate mesenchyme blood vessels. Also in keeping with its pleiotropic activity, endogenous bFGF is of importance for the physiological development of CAM mesodermal tissue by mediating fibroblast cell proliferation and deposition of collagen fibers.
ACKNOWLEDGMENTS This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro to D.R. and M.P. and from CNR to M.P. (Grants 94.00316.CT14 and 95.02925.CT14).
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
MVR 1993
/
6z0b$$$202
03-11-97 13:52:23
mvrg
AP: MVR
190
Brief Communication
REFERENCES
Auerbach, R., Auerbach, W., and Polakowski, I. (1991). Assays for angiogenesis. Pharmacol. Ther. 51, 1 – 11. Baird, A., and Bohlen, P. (1990). Fibroblasts growth factors. In Peptide Growth Factors (M. C. Sporn and A. B. Roberts, Eds.), pp. 369 – 417. Springer, New York. Basilico, C., and Moscatelli, D. (1992). The FGF family of growth factors and oncogenes. Adv. Cancer Res. 59, 115 – 165. Burgess, W. H., and Maciag, T. (1989). The heparin-binding (fibro-
blast) growth factor family of protein. Annu. Rev. Biochem. 58, 575 – 606. Folkman, J., and Brem, H. (1992). Angiogenesis and inflammation. In Inflammation: Basic Principles and Clinical Applications (J. I. Gallin, M., Goldtsein, and R. Snyderman, Eds.), pp. 821 – 839. Raven Press, New York. Gospodarowicz, D. (1991). Biological activities of fibroblast growth factor. Annu. N.Y. Acad. Sci. 638, 1 – 8. Ribatti, D., Urbinati, C., Nico, B., Rusnati, M., Roncali, L., and Presta, M. (1995). Endogenous basic fibroblast growth factor is implicated in the vascularization of the chick embryo chorioallantoic membrane. Dev. Biol. 170, 34 – 49.
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
MVR 1993
/
6z0b$$$202
03-11-97 13:52:23
mvrg
AP: MVR