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      Cell-Mediated Delivery of Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor onto the Chick Chorioallantoic Membrane: Endothelial Fenestration and Angiogenesis

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          Abstract

          Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) exert their angiogenic activity by interacting with endothelial cells in a distinct manner. In this study, we investigated the morphological features of endothelial cells of the chick embryo chorioallantoic membrane (CAM) microvasculature after stimulation with FGF2 or VEGF. In order to provide a continuous delivery of the growth factor, we utilized a recently developed gelatin sponge/CAM assay in which a limited number of FGF2- or VEGF-transfected cells were adsorbed onto gelatin sponges and applied on the top of the CAM on day 8 of development. Their angiogenic activity was compared to that exerted by a single bolus of the corresponding growth factor. All the angiogenic stimuli induced a comparable vasoproliferative response, as demonstrated by the appearance of similar numbers of immature blood vessels within the sponge on day 12. No angiogenic response was observed in CAMs implanted with the corresponding parental cell lines or vehicle. Electron microscopy demonstrated that VEGF-overexpressing cells modified the phenotype of the endothelium of the blood vessels at the boundary between the implant and the surrounding CAM mesenchyme. The endothelial lining of 30% of these vessels showed segmental attenuations, was frequently interrupted and became fenestrated, mimicking what is observed in tumor vasculature. In contrast, the vessels consisted of continuous endothelium sealed by tight junctions in all the other experimental conditions. These results indicate that FGF2 and VEGF interact with endothelial cells of the CAM in a distinct manner. Both growth factors induce a potent angiogenic response, but only VEGF delivered in a continuous manner by its transfectants can modify the phenotype of the otherwise quiescent endothelium of CAM blood microvessels. The gelatin sponge/CAM assay may constitute a new model to study the mechanisms leading to endothelial fenestration in tumor growth.

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          Most cited references 3

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          The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency.

          Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for endothelial cells. VEGF is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia. VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The higher molecular weight forms of VEGF can be cleaved by plasmin to release a diffusible form(s) of VEGF. We characterized the proteolysis of VEGF by plasmin and other proteases. Thrombin, elastase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with soluble VEGF receptors, and ability to promote endothelial cell mitogenesis. Plasmin yields two fragments of VEGF as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111. A heterodimeric form of VEGF165/110, was isolated from partial plasmin digests of VEGF165. The carboxyl-terminal polypeptide (111-165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors. The various isoforms of VEGF (165, 165/110, and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM). In contrast, soluble FLT-1 receptor differentiated VEGF isoforms (165, 165/110, 110, and 121) with apparent affinities of 10, 30, 120, and 200 pM, respectively. Endothelial cell mitogenic potencies of VEGF110 and VEGF121 were decreased more than 100-fold compared to that of VEGF165. The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity.
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            Endogenous basic fibroblast growth factor is implicated in the vascularization of the chick embryo chorioallantoic membrane.

            Chorioallantoic membrane (CAM) and chorioallantoic fluid (CAF) of the chick embryo were studied for the presence of immunoreactive and biologically active basic fibroblast growth factor (bFGF) from Day 6 to Day 18 of incubation. An immunoreactive M(r) 16,000 bFGF-like molecule was detected both in CAM and in CAF. This molecule was identified as bFGF on the basis of its molecular weight, its affinity for heparin, and its capacity to induce plasminogen activator production in cultured endothelial GM 7373 cells. The levels of biologically active and immunoreactive bFGF vary in CAM and CAF during embryonic development, maximal concentrations being observed between Days 10 and 14 of incubation. At all time points investigated, absolute concentrations of bFGF were significantly higher in CAM (ranging from 25 to 183 ng/g of wet tissue) than in CAF (ranging from 0.2 to 4 ng/ml). In a parallel series of experiments performed at Day 8 and evaluated at Day 12 of chick embryo development, human recombinant bFGF and neutralizing anti-bFGF antibody were investigated for their capacity to affect the vasoproliferative processes of the CAM. The two molecules either were applied onto the surface of the CAM or were injected into the allantoic sac. When bFGF or anti-bFGF antibodies were absorbed on methylcellulose discs and applied on the top of the CAM, they exerted a strong angiogenic or anti-angiogenic effect, respectively. On the contrary, when bFGF or the corresponding neutralizing antibody was injected into the allantoic sac, no modifications of the vasoproliferative processes of the CAM were observed at either the macroscopic or the microscopic level. These results provide evidence indicating that endogenous bFGF has a rate-limiting role in the vascularization of the CAM during chick embryogenesis. bFGF located within the CAM, rather than that present in the CAF, appears to be involved in this developmental process.
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              Ontogeny of microvascular permeability to macromolecules in the chick chorioallantoic membrane during normal angiogenesis.

              Chick embryos were incubated using standard shell-less techniques for microscopic observations at Days 4.5, 5.0, and 5.5 of the normal 21-day gestation. The chorioallantoic membrane (CAM) was prepared for intravital fluorescence confocal microscopy. A graded series of FITC-dextrans (20, 40, 70, and 150 kDa) was injected via vitelline vein. The changes in interstitial optical intensity due to FITC-dextran extravasation were evaluated by computer-assisted image analysis. Apparent permeability coefficients (Ps) were calculated for first order postcapillary vessels from the changes in intensity as a function of time. On Day 4.5, Ps values (means +/- SE x 10(-7) cm/sec) in the CAM microvessels for FITC-Dextran 20, 40, 70, and 150 were 11.8 +/- 1.0, 6.4 +/- 0.4, 3.1 +/- 0.5, and 1.5 +/- 0.5, respectively. The respective Ps values fell dramatically on Day 5.0 to 2.2 +/- 0.5, 0.7 +/- 0.2, 0.6 +/- 0.2, and 0.6 +/- 0.2. On Day 5.5, Ps values for all these FITC-dextrans were equal to 0.7 +/- 0.3. The evaluation of FITC-Dextran 10 on Day 5.5 yielded a Ps value of 1.9 +/- 0.3. Our data demonstrate a rapid reduction in microvascular permeability to macromolecules during normal angiogenesis in the early stages of CAM development. Our data also suggest that these changes in permeability may reflect functional adaptations of the CAM. A comparison of our data to those available in the literature for adult and tumoral tissues demonstrates that microvascular permeability properties in these tissues are different from those of the CAM.
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                Author and article information

                Journal
                JVR
                J Vasc Res
                10.1159/issn.1018-1172
                Journal of Vascular Research
                S. Karger AG
                1018-1172
                1423-0135
                2001
                August 2001
                11 July 2001
                : 38
                : 4
                : 389-397
                Affiliations
                aDepartment of Human Anatomy and Histology, University of Bari Medical School, Bari; bInstitute of Pharmacological Sciences, University of Siena Pharmacy School, Siena; cDepartment of Biomedical Sciences, Human Oncology, University of Bari Medical School, Bari, and dDepartment of Biomedical Sciences andBiotechnology, University of Brescia Medical School, Brescia, Italy
                Article
                51070 J Vasc Res 2001;38:389–397
                10.1159/000051070
                11455210
                © 2001 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 3, Tables: 3, References: 41, Pages: 9
                Categories
                Research Paper

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