The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis: The CAM assay in the study of angiogenesis and metastasis

The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis

Domenico Ribatti

The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis

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Prof. Domenico Ribatti Dipartimento di Anatomia Umana e Istologia Piazza G. Cesare, 11 Policlinico Università degli Studi di Bari 70124 Bari Italy [email protected]

ISBN 978-90-481-3843-2 e-ISBN 978-90-481-3845-6 DOI 10.1007/978-90-481-3845-6 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2010920248 © Springer Science+Business Media B.V. 2010 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Acknowledgments

I express my gratitude to my wife Beatrice Nico and all the colleagues and friends involved in the researches performed in the last twenty years concerning the study of several aspects of angiogenesis and antiangiogenesis by using the CAM assay. In particular, Angelo Vacca (Bari), Marco Presta (Brescia), Enrico Crivellato (Udine), Mirco Ponzoni and Vito Pistoia (Genova), Gastone G. Nussdorfer and Diego Guidolin (Padua), Valentin Djonov (Friburg), Marius Raica and Anca Maria Cimpean (Timisoara), Sandra Liekens (Leuvan).

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Contents

1 Chorioallantoic Membrane Vasculature . . . . . . . . . . . 1.1 Chorioallantoic Membrane and Its Embryological Origin 1.2 Morphology of Chorioallantoic Membrane Blood Vessels 1.3 Morphology of Chorioallantoic Membrane Lymphatic Vessels . . . . . . . . . . . . . . . . . . . . . 1.4 A Single Blood Sinus or a Capillary Plexus Beneath the Chorionic Epithelium? . . . . . . . . . . . . . . . . 1.5 A New Model of Choriollantoic Membrane Vascular Growth, the Intussusceptive Mode . . . . . . . . . . . . 1.6 Chorioallantoic Membrane Vascular Growth . . . . . . .

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2 Chorioallantoic Membrane in the Study of Angiogenesis, Antiangiogenesis, and the Vascularization of Grafted Tissues . 2.1 Use of Chorioallantoic Membrane in the Study of Angiogenic Molecules . . . . . . . . . . . . . . . . . . . 2.2 Role of FGF-2 in Chorioallantoic Membrane Vascularization 2.3 Use of Chorioallantoic Membrane in the Study of Antiangiogenic Molecules . . . . . . . . . . . . . . . . . 2.4 Use of Chorioallantoic Membrane in the Study of Vascularization of Grafted Tissues . . . . . . . . . . . . .

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3 Chorioallantoic Membrane in the Study of Tumor Angiogenesis 3.1 Use of Chorioallantoic Membrane in the Study of Tumor Angiogenesis . . . . . . . . . . . . . . . . . . . . . 3.2 Angiogenesis and Antiangiogenesis in Multiple Myeloma . . . 3.2.1 Biological and Clinical Studies . . . . . . . . . . . . . 3.2.2 Use of the Chorioallantoic Membrane . . . . . . . . . 3.3 Angiogenesis and Antiangiogenesis in Human Neuroblastoma 3.3.1 Biological and Clinical Studies . . . . . . . . . . . . . 3.3.2 Use of Chorioallantoic Membrane . . . . . . . . . . .

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4 Chorioallantoic Membrane in the Study of Tumor Metastasis . . . . 4.1 Use of Chorioallantoic Membrane in the Study of Tumor Metastasis 4.2 Spontaneous Metastasis Models . . . . . . . . . . . . . . . . . . 4.3 Experimental Metastasis Studies . . . . . . . . . . . . . . . . . .

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5 Other Applications of Chorioallantoic Membrane . . . . . . . . . . 5.1 In Ovo and Ex Ovo Methods . . . . . . . . . . . . . . . . . . . . 5.2 Use of Chorioallantoic Membrane in the Study of Tumor Lymphangiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Use of Chorioallantoic Membrane in the Study of Angiogenesis Associated with Wound Healing and Inflammation 5.4 Use of Chorioallantoic Membrane in the Study of Angiogenic Properties of Biomaterials and of the Evaluation of Drug Delivery Systems . . . . . . . . . . . . . . . . 6 Different Morphological Techniques and Methods of Quantifying the Angiogenic Response Used in the Study of Vascularization in the Chorioallantoic Membrane . . . . . . . . . . 6.1 Different Morphological Techniques That Can be Used to Study Vascularization of the CAM and the Genes Involved . . . 6.2 Methods of Quantifying the Angiogenic Response . . . . . . . . . 7 Advantages and Limitations of Chorioallantoic Membrane in Comparison with Other Classical In Vivo Angiogenesis Assays . 7.1 Advantages and Limitations of Chorioallantoic Membrane Assay 7.2 Other Classical In Vivo Assays in Comparison (Advantages, Disadvantages, and Limitations) with Chorioallantoic Membrane . . . . . . . . . . . . . . . . . . . . 7.2.1 The Corneal Micropocket Assay . . . . . . . . . . . . . 7.2.2 The Sponge/Matrix Implant . . . . . . . . . . . . . . . . 7.2.3 The Disk Angiogenesis System (DAS) . . . . . . . . . . 7.2.4 The Matrigel Plug . . . . . . . . . . . . . . . . . . . . . 7.2.5 The Dorsal Air Sac Model . . . . . . . . . . . . . . . . 7.2.6 The Chamber Assays . . . . . . . . . . . . . . . . . . . 7.2.7 The Zebrafish . . . . . . . . . . . . . . . . . . . . . . . 7.2.8 The Tumor Models . . . . . . . . . . . . . . . . . . . .

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Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction

As pointed out by Auerbach in 1991 “Perhaps the most consistent limitation to progress in angiogenesis research has been the availability of simple, reliable, reproducible, quantitative assays of the angiogenesis response.” In vitro angiogenesis assays, based on endothelial cell cultures or tissue explant, focus on isolated endothelial cell functions (e.g., endothelial cell proliferation, migration, or invasion) and do not examine the coordination of cell functions required for a successful angiogenic response (Jain et al., 1997; Auerbach et al., 2000). Although in vitro angiogenesis assays have been useful for identification of potential molecular targets to block endothelial cell responses and preliminary screening of novel pharmacological agents, they frequently cannot be correlated with in vivo angiogenesis measurements. This is most likely the result of the complex and multiple cellular mechanism evoked during new blood vessel formation in vivo. In vitro assays cannot be considered conclusive and the activity of a compound must be confirmed in other assays of increasing complexity, including in vivo assays of angiogenesis, angiogenic-dependent tumor growth, and metastasis. In vivo angiogenesis assays examine the entire spectrum of molecular and cellular processes. However, these in vivo assays are not only expensive and technically difficult to perform but also require substantial amounts of test compound and mostly rely on selective morphometric analysis for quantification (Jain et al., 1997; Auerbach et al., 2000). Because of these limitations, current drug development strategies for identification and testing angiogenesis inhibitors depend principally on the use of in vitro systems. Currently, novel angiogenesis-targeted therapies lack in vivo screening models suitable for objective, quantitative preclinical testing, making it difficult to obtain a dose–response analysis and estimate therapeutic doses before initiating clinical trials. The development of inhibitors of angiogenesis relies on a range of preclinical assays that mimic the various steps of the angiogenic cascade. Knowledge of the mechanism of action of the tested compound will dictate the choice of assay. Alternatively, the behavior of the compound in different assays may indicate the mechanism of action.

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In vivo assays are usually unsuitable for the quantitative screening of a large number of compounds, as they are often complex, expensive, and may require specific surgical skills. Nonetheless, they are always required to confirm ultimately the activity of a potential drug. The classical assays for studying angiogenesis in vivo include the hamster check pouch, the rabbit ear chamber, the rodent dorsal skin and air sac, the iris and avascular cornea of the rodent eye (Ribatti and Vacca, 1999), and the chick embryo chorioallantoic membrane (CAM). Several models have been introduced, including subcutaneous implantation in rodents of various three-dimensional substrates, including a polymer sponge (Andrade et al., 1987); Matrigel, a laminin-rich mixture of basement membrane components (Passaniti et al., 1992); and a polyvinyl alcohol foam disk covered on both sides with a Millipore filter (disk angiogenesis system) (Fajardo et al., 1998). Finally, zebrafish (Danio rerio) embryos may represent a suitable model to study the mechanisms of angiogenesis and angiosuppression during development (Nicoli and Presta, 2007). The CAM of the developing chick embryo is an extraembryonic membrane mediating gas and nutrient exchanges until hatching. The main function of the CAM is to serve as the respiratory organ for the embryo. It also plays a role in the storage of excretions, electrolyte transport (sodium and chloride) from the allantoic sac, and mobilization of calcium from the shell to start bone mineralization. Since the CAM has a very dense capillary network, it is commonly used to study in vivo both angiogenesis and antiangiogenesis in response to different factors. During 2000–2009, over 700 publications have used the chick embryo CAM as a model system to study angiogenesis and antiangiogenesis (NCBI, Pub Med). The CAM, particularly that of the White Leghorn, is the most widely used. The CAM of the Japanese quail has also been used. The quail-derived endothelium expresses a unique marker which can be identified using the QH1 antibody.

Chapter 1

Chorioallantoic Membrane Vasculature

1.1 Chorioallantoic Membrane and Its Embryological Origin Chick embryo development lasts 21 days before hatching. There are four extraembryonic membranes of the chick: the yolk sac, the amnion, the serosa, and the allantois. The serosa of the chick is occasionally called chorion; the term chorion, however, is more frequently applied to the composite layer formed by the fusion of the allantois and the serosa. The allantois of the chick embryo appears at about 3.5 days of incubation as an evagination from the ventral wall of the endodermal hind gut. During the fourth day, it pushes out of the body of the embryo into the extraembryonic celom. Its proximal portion lies parallel and just caudal to the yolk sac. When the distal portion grows clear of the embryo it becomes enlarged. The narrow proximal portion is known as the allantoic stalk and the enlarged distal portion as the allantoic vesicle. Fluid accumulation distends the allantois so that its terminal portion resembles a balloon in entire embryos. The allantoic vesicle enlarges very rapidly from the fourth to the tenth day of incubation. In this process, the mesodermal layer of the allantois becomes fused with the adjacent mesodermal layer of the chorion to form the CAM. A double layer of mesoderm is thus created: its chorionic component is somatic mesoderm and its allantoic component is splanchnic mesoderm. In this double layer an extremely rich vascular network develops and is connected with the embryonic circulation by the allantoic arteries and veins. Immature blood vessels, lacking a complete basal lamina and smooth muscle cells, scattered in the mesoderm grow very rapidly until day 8 and give rise to a capillary plexus, which comes to be intimately associated with the overlying chorionic epithelial cells and mediates gas exchange with the outer environment. At day 14, the capillary plexus is located at the surface of the ectoderm adjacent to the shell membrane. Rapid capillary proliferation continues until day 11; thereafter, the endothelial cell mitotic index declines rapidly, and the vascular system attains its final arrangement on day 18, just before hatching (Ausprunk et al., 1974). The allantoic (umbilical) artery after emerging from the embryonic abdominal wall branches into two primary chorioallantoic arteries and the CAM is drained by D. Ribatti, The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis, DOI 10.1007/978-90-481-3845-6_1,  C Springer Science+Business Media B.V. 2010

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Fig. 1.1 Allantoic sac of a 5-day embryo showing in ovo distribution pattern of allantoic vessels (reproduced from Ribatti, 2008)

a single chorioallantoic vein (Fig. 1.1). The allantoic artery passes out along the stalk of the allantois to the inner wall of the allantoic sac, where it divides into two strong branches, one running cephalic and the outer caudal to the margins of the sac where they pass over to the outer wall. The allantoic vein runs in the inner wall and passes over the CAM near to the sero-amniotic connection. Fuchs and Lindenbaum (1988) described six or seven generations of branches of the allantoic artery. The first five or six are located in a plane parallel to the CAM surface and deep to the vein, which has a similar distribution. The fifth and sixth generations of blood vessels change the direction, passing almost vertically in the two-dimensional capillary plexus. In the outer wall the arteries and veins branch and interdigitate in the deeper portions of the mesoderm, and end in an extraordinary fine-meshed capillary network interspersed with the ectodermal cells (Fig. 1.2). From day 6 to day 14, the third-order vessels do not increase significantly in number, while the number of first- and second-order vessels is increased. Moreover, between day 6 and day 10 intercapillary distances are substantially reduced, while between days 10 and 14, they remain constant. Finally, the average length of the first-, second-, and third-order microvessels are significantly reduced by day 14: this finding is consonant with the interpretation that consecutive branching of respective vessel order might serve to increase the total number of vessels, while simultaneously the length of each microvessel with the expanding network decreases (De Fouw et al., 1989). This circulation and the position of the allantois immediately subjacent to the porous shell confer a respiratory function on the highly vascularized CAM. Threedimensional analyses of the CAM microcirculation have revealed an architecture

1.2

Morphology of Chorioallantoic Membrane Blood Vessels

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Fig. 1.2 A macroscopic picture of the CAM vascular tree after intravenous India ink injection at 14 days of incubation. Note the extreme complexity of the vascular architecture (reproduced from Ribatti, 2008)

similar to that of the mature pulmonary microvasculature (Fuchs and Lindenbaum, 1988). In addition to the respiratory interchange of oxygen and carbon dioxide, the allantois also serves as a reservoir for the waste products excreted by the embryo, mostly urea at first and chiefly uric acid, later. CAM is also involved in the mobilization of calcium from the shell to start bone mineralization.

1.2 Morphology of Chorioallantoic Membrane Blood Vessels On day 4, all CAM vessels have the appearance of undifferentiated capillaries. Their walls consist of a single layer of endothelial cells lacking a basal lamina (Ausprunk et al., 1974). Between days 4.5 and 5.0 CAM microvascular endothelial cells are thicker, have few plasmalemmal vesicles, and contain large vacuoles (Fig. 1.3) (Rizzo et al., 1995a). By day 8, the CAM displays small thin-walled capillaries with a luminal diameter of 10–15 μm beneath the chorionic epithelium and other vessels with a diameter of 10–115 μm in the mesodermal layer, whose walls have a layer of mesenchymal cells surrounding the endothelium and are completely wrapped by a basal lamina together with the endothelial cells (Ausprunk et al., 1974). On days 10–12, the capillaries resemble those in the 8-day membrane and are now near the surface of the chorionic epithelium (Fig. 1.4). The capillary walls remain simple in structure, containing endothelial cells and a few mesenchymal cells

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Fig. 1.3 Scanning electron microscopy micrograph of a CAM at day 5 of incubation. The chorionic epithelium (CH) consists of a single layer of flat cells. Undifferentiated vessels (VE), consisting of a single layer of endothelial cells, are localized in the mesoderm. Abundant amorphous substance and random groups of collagen fibers are present between the chorion and the mesodermal blood vessels (reproduced from Ribatti et al., 1998a)

(presumptive pericytes) which are flattened and closely applied to the endothelial cells. The mesodermal vessels are now distinct arterioles and venules. In addition to the endothelium, the walls of arterioles (10–85 μm diameter) contain one or two layers of mesenchymal cells and increased amounts of connective tissue surrounding them. Venules (10–115 μm in diameter) are surrounded by an incomplete investment of

Fig. 1.4 A confocal laser scanner microscopy picture obtained by using an antibody anti-factor VIII-related antigen to detect endothelial cells and showing three sprouts arising from the CAM mesodermal blood vessels and invading the chorionic epithelium, resulting in a capillary meshwork (reproduced from Ribatti, 2008)

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Morphology of Chorioallantoic Membrane Blood Vessels

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mesenchymal cells and connective tissue has also accumulated within their walls. Microfilaments appear in the mesenchymal cells and gradually increase in number. These cells are presumed to be developing smooth muscle cells and the walls of CAM arterioles also develop a distinct adventitia containing fibroblast-like cells (Ausprunk et al., 1974). The ultrastructural alterations associated with the focal microvascular histodifferentiation are in line with the changes in the vascular pattern (Fig. 1.5). Small differences between CAM arterioles, capillaries, and venules are noted during the early phase. During the intermediate phase, the interstitial perivascular spaces increase their collagen content and cell volume density. During the late phase a circular tunic containing layers of presumptive smooth muscle cells surrounds the endothelium of the arterioles and not that of the venules (Shumko et al., 1988). Between days 12 and 18 in capillaries, as well as larger vessels, endothelial cells acquire endocytotic vesicles and Weibel–Palade bodies (Ausprunk, 1986). Smooth muscle cells increase their content of microfilaments and frequently have a contracted appearance. Finally, the basal lamina of endothelial cells, pericytes, and smooth muscle cells become continuous (Ausprunk, 1986). Between days 4 and 8 the endothelial cells form punctuate junctional appositions (Shumko et al., 1988). Junctional clefts between the adjacent CAM endothelial cells met the ultrastructural characteristics of tight junctions as early as day 5.0 of development (Rizzo et al., 1995b). CAM endothelial glycocalyx expresses a

Fig. 1.5 Scanning electron microscopy micrograph of a CAM at day 15 of incubation. Numerous capillaries are located between the chorionic (CH) epithelial cells. A large mesodermal blood vessel (arrow) is recognizable in the intermediate mesenchyme (reproduced from Ribatti et al., 1998a)

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continuous anionic barrier (comprised in part of sialic acid, which is the major contributor to the negative charge associated with the glycocalyx) from days 4.5 to 14 of development (Henry and De Fouw, 1996). By day 18, this barrier becomes discontinuous. The evidence that the expression of anionic domains remains constant despite changes in CAM microvascular permeability in early development suggests that anionic domains play a minimal role in the development of microvascular permselectively during normal angiogenesis. De Fouw and De Fouw (1999) tested the hypothesis that activation of the cyclic adenosine monophosphate (cAMP) signaling pathway at day 4.5 would increase permselectively prior to normal differentiation of CAM endothelial barrier properties at day 5.0 and demonstrated that exogenous cAMP activation decreases the permeability of the angiogenic CAM endothelium at day 4.5 without concomitant ultrastructural changes in the transendothelial macromolecular exchange pathways. Between days 9 and 13 the arteriolar endothelium displays more extensive junctional complexes with multiple membrane contact points (Shumko et al., 1988). In contrast to the arterioles, endothelial junctional appositions of the CAM venules remain punctuate (Shumko et al., 1988). Between days 14 and 18 these appositions remain as simple punctuate configurations (Shumko et al., 1988). The venules possess multiple sites of interendothelial contact with areas of junctional dilations, while the arterioles display complex interdigitating cell junctions (Shumko et al., 1988). Rizzo and De Fouw (1993) and Rizzo et al. (1993 b; 1995a) demonstrated that ultrastructural cytodifferentiation between days 6 and 14 is characterized by progressive reduction of endothelial cytoplasmic thickness and progressive increase of vesicular densities and showed an increase in endothelial permselectively to graded molecular weight fluorescein isothiocyanate (FITC) dextran between days 4.5 and 5.0. Inhibition of tyrosine phosphorylation reduces FITC dextran extravasation at day 4.5 (De Fouw and De Fouw, 2001). Moreover, anti-vascular endothelial growth factor (VEGF), but not anti-fibroblast growth factor-2 (FGF-2) antibodies, abolishes the temporal endothelial hyperpermeability and this finding is consistent with the established permeability-enhancing function of VEGF (De Fouw and De Fouw, 2000). Cruz et al. (1997) investigated the temporal restriction of chicken serum albumin (CSA), conjugated to FITC, by the CAM microvascular endothelium and demonstrated that FITC-CSA extravasation from the CAM pre-capillaries, capillaries, and post-capillaries is uniformly negligible. They concluded that the tight junctional clefts and the paucity of plasmalemmal vesicles provide an ultrastructural correlate to the negligible rate of CSA extravasation across the CAM endothelium and such restriction of endogenous macromolecules is consistent with principal respiratory function of the CAM and would serve to prevent impedance of gas exchange. Cruz and De Fouw (1999) evaluated the expression of vascular endothelial (VE) and neural (N) cadherins in the CAM between days 4.5 and 6 of incubation and demonstrated that the amount of N-cadherin remains uniform, while the amount of VE-cadherin increases between days 4.5 and 5.0 and this correlates with differentiation of the restrictive endothelial barrier. Furthermore, Cruz et al. (2000)

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Morphology of Chorioallantoic Membrane Blood Vessels

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identified a CAM endothelial VE-cadherin/β-catenin complex and demonstrated that phosphotyrosine labeling of β-catenin decreases concurrently with the increase in CAM endothelium selectively between days 4.5 and 5.0 and inhibition of protein tyrosine phosphatase impedes tyrosine dephosphorylation of β-catenin and this serves to partially restore macromolecular extravasation to elevated levels normally present at day 4.5. Oh et al. (1998) identified the cytoskeletal protein desmin and alpha smooth muscle actin in cells of the CAM microvasculature and connective tissue and showed that platelet-derived growth factor B (PDGF-B) stimulates myofibroblast formation and formation of non-capillary microvessels. These cells are positive for alpha smooth muscle actin and fibronectin and are negative for desmin. Subsequently, Kurz et al. (2002) demonstrated the presence of desmin-positive periendothelial cells in the capillary plexus and in the subintimal layer of larger microvessels. The cells located near the capillary plexus were most frequently ramified, but did not form continuous layers or tubes, were alpha smooth muscle actin negative, and were identified as pericytes. The wall of arteries showed less intense desmin staining, as compared to alpha smooth muscle actin. Moreover, confocal microscopy showed that the delicate, elongated cells in the innermost muscular layer of the arterial wall were desmin-positive. Nico et al. (2004) confirmed that desmin but not alpha smooth actin was expressed in such pericytes, whereas vascular smooth muscle cells in non-capillary microvessels expressed both alpha smooth actin and desmin (Fig. 1.6). More recently, Kurz et al. (2008), using triple labeling with fluorochrome-conjugate markers Sambucus nigra agglutinin, desmin or alpha smooth actin, and DNA-specific YoProx-1, reported the presence of a delicate, filamentous, circumferentially

Fig. 1.6 Immunohistochemical detection of factor VIII-related antigen and desmin in a CAM of a 12-day-old chick embryo by confocal (a) and differential interference optics microscopy (b). Note that in a, capillaries at the base of the chorion (CH) and a large vessel in the intermediate mesenchyme (asterisk) are positive to factor VIII and to desmin. Note in b a specific immunoreactivity to desmin in perivascular position (arrowheads) (reproduced from Nico et al., 2004)

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oriented alpha smooth actin pattern in periendothelial cells of the mature CAM capillary plexus, quite different from the coarser, axially oriented desmin pattern. and concluded that desmin and alpha smooth actin are not mutually exclusive in most pericytes. Ribatti et al. (2002 a) demonstrated that between days 12 and 15 of incubation, after postembedding immunogold treatment of ultrathin sections with the anti-VEGF receptor-2 (VEGFR-2) antibody, numerous gold particles, arranged singularly or in clusters, decorated the cytoplasm of the endothelial cells and the perivascular pericytes in the CAM vasculature (Fig. 1.7). The number of particles was higher in the abluminal side of the endothelial cells and decreased significantly on the abluminal side facing the pericytes, while it was unchanged in pericytes. Hlushchuk et al. (2007) demonstrated that 48 h after the application of PTK787, a potent inhibitor of the intrinsic kinase activity of VEGFRs, and of CPG53716, a highly specific inhibitor of the intrinsic kinase activity of the PDGF receptor (PDGFR), periendothelial cells of the CAM capillary plexus completely disappeared. The extracellular matrix of the CAM modifies its composition in terms of expression of fibronectin, laminin, collagen type IV and distribution of specific glycosaminoglycans, favoring the angiogenic process that occurs in the space between the chorionic epithelium and the mesodermal blood vessels (Ausprunk, 1986; Ribatti et al., 1998a). Fibronectin appears in the extracellular matrix beneath the chorion at early stages of development when the subepithelial capillary plexus is not yet formed and it may promote the migration of endothelial cells merging by sprouting from the mesodermal blood vessels (Fig. 1.8) (Ribatti et al., 1998a). Moreover, fibronectin

Fig. 1.7 Ultrastructural immunodetection of VEGFR-2 in CAM endothelial cells (E) and pericytes (P) at day 12 (a) and day 15 (b) of incubation. Immunogold particles, arranged singularly or in clusters, and more numerous in the endothelial cells at day 12, decorate the luminal and abluminal front of the endothelial cells and the cytoplasm of the pericytes (reproduced from Ribatti et al., 2002a)

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Morphology of Chorioallantoic Membrane Blood Vessels

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Fig. 1.8 CAM extracellular matrix immunohistochemistry. (a) Fibronectin immunoreactivity at day 6 of incubation is intense in the chorion (CH), in the extracellular matrix, and along the surface of the primitive blood vessels (arrowhead). (b) Laminin and (c) type IV collagen immunoreactivity at day 10 of incubation are detectable in the chorion (CH) and in the capillaries beneath the chorion (reproduced from Ribatti et al., 1997a)

overexpression in extracellular matrix parallels the vasoproliferative processes induced by angiogenic stimuli in the CAM (Ribatti et al., 1997a). Accordingly, a close relationship in vivo between fibronectin overexpression and angiogenesis has been demonstrated by others (Sariola et al., 1984; Risau and Lemmon, 1988). Laminin immunoreactivity is present during all stages of vessel formation in CAM development (Ribatti et al., 1998a) in keeping with its role in the early formation and later differentiation of the subendothelial basement membrane (Risau and Lemmon, 1988). Ingber et al. (1986) demonstrated that CAMs treated with combinations of angiostatic steroids and heparin exhibit capillary basement membrane fragmentation and eventually complete loss of fibronectin and laminin from regions of capillary involution and capillary basement membrane breakdown correlates with capillary retraction, endothelial rounding, and capillary regression. Type IV collagen appears in the late stages of CAM vascular development concomitantly with the terminal differentiation of endothelial cells and maturation of basement membrane (Ribatti et al., 1998a). It results in progressively slower microvascular endothelial cell proliferation and correlates with the formation of a lumen, gradual reduction in endothelial migration, establishment of cell polarity, and acquisition of a differentiated endothelial phenotype (Form et al., 1986; Nicosia and Madri, 1987). Ribatti et al. (1999 a) examined in the CAM the expression of the matrix metalloproteinase 2 (MMP-2) and correlated this parameter with the expansion of the CAM vasculature and with the expression of fibronectin, laminin, and type IV collagen. They demonstrated that in the early phases of CAM development, between days 6 and 8, when the increase of the CAM vasculature is maximal, higher values of MMP-2 and, respectively, of fibronectin immunoreactive area are detectable and suggested that MMP-2 activity and fibronectin expression are two strictly related components of angiogenesis occurring in vivo.

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Ausprunk (1986) demonstrated that hyaluronic acid (or its degradation products) plays a crucial role in the formation, alignment, or migration of the capillary plexus of the CAM, while heparan sulfate, chondroitin sulfate, and dermatan sulfate are important in the differentiation and development of arterial and venous vessels of the CAM. Accordingly, West et al. (1985) demonstrated that degradation products of hyaluronic acid, when placed on the chick CAM, are capable of inducing angiogenesis. The disappearance of hyaluronic acid from the subectodermal region of the CAM associated with an increase in the local concentration of hyaluronic acid degradation products may be responsible for the movement of vessels into the subectodermal region of the CAM and the proliferation of these vessels to form the capillary network (Ausprunk, 1986).

1.3 Morphology of Chorioallantoic Membrane Lymphatic Vessels In chick embryos, the deep lymphatics are first detectable during days 4–5 of incubation (Cark and Clark, 1920). These deep lymphatics are situated adjacent to large embryonic veins. In the CAM, arteries and arterioles are accompanied by a pair of lymphatics and a dense plexus of lymphatics is located around the large veins (Oh et al., 1997). Lymph is drained by trunks of the umbilical stalk into the coccygeal lymphatics and the lymph hearts of the embryo (Wilting et al., 1999). At the ultrastructural level the endothelium of the CAM lymphatic capillaries has no basal lamina and an extremely thin endothelial lining (Oh et al., 1997). The lymphatic endothelial cells of the differentiated CAM specifically express VEGFR-3 whereas expression of VEGFR-2 is found in both its blood vascular and its lymphatic endothelial cells (Wilting et al., 1996). The ligand of VEGFR-3, VEGF-C, is expressed ubiquitously in the allantoic bud, and later predominantly in the allantoic epithelium and the wall of larger blood vessels (Papoutsi et al., 2001 a). The lymphatics of the CAM are located immediately adjacent to the larger blood vessels and the expression of VEGF-C in the blood vascular wall serves for the patterning of lymphatics. The application of VEGF-C on the differentiated CAM induces development of lymphatics, which are derived by proliferation of the preexisting lymphatics (Oh et al., 1997). The homeobox gene prospero-related 1 (Prox-1) which is specifically expressed in lymphatics has been demonstrated in the CAM (Papoutis et al., 2001 a). Parson-Wingerter et al. (2006) studied in the quail CAM lymphatics and blood vessels the expression of VEGFR-2, alpha smooth actin (specific to CAM blood vessels), Prox-1 (specific to lymphatics), and the quail hematopoietic marker, QH1 by confocal/fluorescence microscope. VEGFR-2 was expressed intensely in isolated cells and lymphatics, and moderately in blood vessels. Prox-1 was absent from isolated progenitor cells prior to lymphatic recruitment. Finally, exogenously administered VEGF increased lymphatic vessel diameter and density. According to the hypothesis of Sabin (1909), the lymphatics of the CAM would develop as sprouts from the posterior lymph sacs. Papoutsi et al. (2001a) performed

1.4

A Single Blood Sinus or a Capillary Plexus Beneath the Chorionic Epithelium?

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homotopic grafting of the allantois of 3-day-old quail embryos into chick embryos, approximately 2 days before the lymph sacs are visible. After 10 days of reincubation, they observed quail-derived lymphatics in the CAM of the chick host which are QH1 and VEGFR-3 double-positive and show that splanchnic mesoderm contains lymphangioblasts that are first Prox-1 negative and then become Prox-1 positive. Overall these data indicate that the lymphatics of the CAM are of endogenous origin, and only a small subset might be derived by sprouting from the posterior lymph sacs.

1.4 A Single Blood Sinus or a Capillary Plexus Beneath the Chorionic Epithelium? For some authors (Fulleborn, 1895; Narbaitz, 1977; Mc Cormick et al., 1984; Schoefl, 1984) the CAM vascular bed is formed by a single flat sinus, interrupted by a series of gaps. When the sinus comes close to the CAM surface the architecture of the chorionic epithelium changes from that of a double layer of flat cells to an intricate arrangement of highly differentiated cells, such as the sinus-covering cells (Narbaitz, 1977), which are adapted for gas exchange, and villus-cavity cells, which are thought to be involved in the absorption of calcium from the egg shell (Coleman and Terepka, 1972). In the chick CAM, therefore, active migration of the chorionic epithelium rather than endothelial cells is apparently involved and the intraepithelial positioning of the vascular sinus is largely due to growth and differentiation of the chorionic epithelium. Other authors have identified a capillary plexus formed during the early stages of incubation, and eventually intimately associated with the overlying chorionic epithelial cells (Danchakoff, 1917; Moscona, 1959; Ausprunk et al., 1974; Fanczi and Feher, 1979; Burton and Palmer, 1989; Ribatti et al., 1998a). Danchakoff (1917) described a multitude of sprouts arising from the mesenchymal blood vessels and invading the chorionic epithelium, resulting in a well-perfused capillary meshwork. Burton and Palmer (1989), by using microvascular corrosion casting technique to investigate the three-dimensional arrangement of the plexus at various stages of incubation, reported that short vascular buds invaded the mesenchyme at day 6 from the arterial and venous sides, culminating in capillary plexus formation. The presence of a complete basal lamina and the lack of phagocytic cells intermingled with the endothelial cells provide the main morphological evidence of the existence of a capillary plexus. Burton and Palmer (1989) avoided the use of the term “sinus” because this term is usually applied to vessels with irregular lumina that lack an adventia or basal lamina and which are lined by phagocytic macrophage-like cells interspersed among the endothelial cells, while the endothelial cells of the CAM’s capillary plexus are lined by a complete basal lamina, which becomes interposed between the endothelial and chorionic cells.

12

1 Chorioallantoic Membrane Vasculature

1.5 A New Model of Choriollantoic Membrane Vascular Growth, the Intussusceptive Mode A widely accepted view is that blood vessels arise through two mechanisms during development, vasculogenesis and angiogenesis (Risau, 1997). Vasculogenesis entails the direct formation of blood vessels by differentiation of angioblastic precursor cells in situ, while angiogenesis (“sprouting angiogenesis”) entails new vessel formation from preexisting vessels, capillaries, and post-capillary venules. Intussusceptive microvascular growth (IMG) (“intussusception or non-sprouting angiogenesis”) is a new concept of microvascular growth relevant for many vascular systems, which constitutes an additional and alternative mechanism to endothelial sprouting (Patan et al., 1992). The first reports on IMG were published by Burri et al., who investigated the lung vasculature in postnatal rats (Caduff et al., 1986; Burri and Tarek, 1990) and postulated that the capillary network primarily increased its complexity and vascular surface by insertion of a multitude of transcapillary pillars, a process they called “intussusception” (meaning “in-itself growth”). They described four consecutive steps in pillar formation: creation of a zone of contact between opposite capillary walls; reorganization of the intercellular junctions of the endothelium with central perforation of the endothelial bilayer; formation of an interstitial pillar core; and subsequently invasion of the pillar by cytoplasmic extensions of myofibroblasts and pericytes, and by collagen fibrils. Lastly, the pillars are thought to increase in diameter and become a capillary mesh. Patan et al. (1993) observed the same morphological transformation during IMG in the CAM. Pillar formation in the CAM occurs both as transcapillary interconnection of opposite capillary walls and folding of the capillary wall into the lumen, followed by progressive thinning of the meso-like fold resulting in pillar separation (Patan et al., 1996). In addition tissue pillars can arise by capillary fusion. The walls of neighboring vessels running in parallel fuse at several places and give rise to one or more tissue pillars (Fig. 1.9) (Patan et al., 1997). According to Schlatter et al. (1997) CAM vascularization undergoes three phases of development with both sprouting and IMG: in the early phase (days 5–7) multiple capillary sprouts invade the mesenchyme, fuse, and form the primary capillary plexus. During the second (intermediate) phase (days 8–12), sprouts are no longer present since they have been replaced by tissue pillars, with a maximal frequency at day 11. During the late phase (day 13 and older), the growing pillars increase in size to form intercapillary meshes more than 2.5 μm in diameter. Intravascular casting coupled with serial sectioning for light and electron microscopy and demonstration of pillars or use of confocal laser scanning microscopy have supported indubitable evidence for the presence of transluminal pillars (Djonov et al., 2000). We have demonstrated that after recombinant human erythropoietin (Epo) stimulation, the generation of new blood vessels in the CAM occurred more frequently following an IMG mechanism (Crivellato et al., 2004). We have identified three morphological structural changes in terms of expression of IMG: pillar formation by folding of the lateral vascular wall, fusion of pillars, and connection of intraluminal tissue folds with the opposite vascular wall. In some instances, a single endothelial

1.6

Chorioallantoic Membrane Vascular Growth

13

Fig. 1.9 Mercox cast of developing CAM vasculature at day 12 of incubation. Three-dimensional structure containing a capillary plexus and a layer of supplying and collecting vessels is recognizable (a, b). Numerous pillars of different sizes caused by intussusceptive microvascular growth are detectable (c, d) (reproduced from Ribatti et al., 2001f)

cell connected the opposite walls of a preexisting vessel, like a transluminal bridge, expression of the connection of intraluminal tissue folds with the opposite vascular wall. This process caused the splitting of the original vascular structure into two newly formed blood vessels. This response is peculiar of Epo, because it is abolished when an Epo-blocking antibody was coadministered with Epo.

1.6 Chorioallantoic Membrane Vascular Growth An extensive morphometric investigation by De Fouw et al. (1989) has shown rapid extension of the CAM surface from 6 cm2 at day 6 to 65 cm2 at day 14. During this period, the number of feed vessels increased (2.5- and 5-fold for pre-capillary and post-capillary vessels), predominantly due to growth and remodeling after day 10. Strick et al. (1991) performed morphometric measurements of the CAM vascularity between days 8 and 18 of incubation in randomly selected areas using a computerized image analysis system. The measurements were limited to the arterial and venous systems and included the vessel endpoint density (VED) corresponding to the points of connection of the arterioles and venules to the capillary plexus, the length density, the fractional image area, and the vascular density index. They demonstrated that the vascularization of the CAM increases rapidly from days 8 to 12 of incubation. The VED shows the greatest increase (3.5-fold) during this time: vascular density index and length density increases by only 2-fold. There are

14

1 Chorioallantoic Membrane Vasculature

negligible changes in vascular density index and length density after day 14, but the VED continues to increase throughout the period of the study. Dusseau and Hutchins (1989) studied the effects of hypoxia on the number of pre- and post-capillary vessels in the CAM and demonstrated that the 15% oxygen environment produced a 54% increase in overall vessel density, with arterioles increasing 78% and venules 34%; the low oxygen regimen stimulated a preferential increase in the number of arterioles and control vs 15% oxygen groups showed no statistical differences for the diameters and lengths of individual arterioles and venules. Parson-Wingerter et al. (1998; 2000) demonstrated that the contribution of the arterial tree to CAM vascular density appears to be larger than that of the venous tree. In fact, they demonstrated that FGF-2 increases the rate of angiogenesis by a maximum of 72%, whereas angiostatin decreases the rate of vascular growth by a maximum of 68% (Parson-Wingerter et al., 1998). Moreover, transforming growth factor beta 1 (TGF-β1) strongly inhibits angiogenesis in the arterial tree by a maximum of 105% by inhibition of the normal increase in the number of new, small blood vessels (Parson-Wingerter et al., 2000). The potent inhibition of angiogenesis by TGF-β1 after 24 h is largely overcome by 48 h. This early but transient perturbation of angiogenesis by TGF-β1 contrasts spatiotemporal with the inhibition of angiogenesis by angiostatin (Parson-Wingerter et al., 1998), which significantly decreases angiogenesis in the CAM after 48 h but not after 24 h. The CAM endothelium exhibits an intrinsically high mitotic rate (thymidine labeling index 23% for 5 h thymidine exposure) until day 10 (Ausprunk et al., 1974). At day 11, this falls to 2% and remains low throughout the remaining incubation period. Investigation of the presence of bromodeoxyuridine-labeled endothelial cells in the growing CAM from day 6 to day 15 by Kurz et al. (1995) revealed a significant (>50%) loss of proliferative activity at day 10 (intermediate phase) in comparison with day 6 (sprouting phase). After day 10, proliferative activity decreased further, and at days 14 and 15 (late phase), dividing cells were >10% of the value of day 6. The measurement of a basal rate of angiogenesis is a useful tool for the quantitative description of angiogenic perturbation by both positive and negative regulators. Blood vessel patterns in the CAM during normal development and in experimental conditions have been previously investigated with measurements of vessel length density, endothelial proliferation intensity, complexity, fractal dimension and extended counting method (Kurz et al., 1995; 1998; Kirchner et al., 1996). Although the growth of vessels does not seem to be fractal (Kurz et al., 1998), fractal dimension, a statistical descriptor of space filling pattern and density, was often considered as a useful statistical index characterizing the complexity of a space-filling network of vessels in studies performed by using the CAM assay (Kirchner et al., 1996; Vico et al., 1998). However, the vascular bed should be addressed as a highly complex structure, and fractal dimension alone may not fully characterize all the aspects of its complexity. Thus, to investigate the degree of spatial order of a structure other measures have to be considered besides fractal dimension. Lacunarity, however, as a global parameter, doesn’t allow a detailed

1.6

Chorioallantoic Membrane Vascular Growth

15

description of the variety of structural heterogeneities, characterizing the vascular pattern of the CAM. To address this point, we have developed automatic image analysis methods allowing a quantitative evaluation of several parameters (fractal dimension, lacunarity and non-fractal order-disorder parameters, such as positional, topological and orientational order) characterizing the level of spatial order/disorder exhibited by the vascular network of the CAM in basal conditions and after treatment for 96 hours with an angiogenic cytokine, such as FGF-2, or with an angiostatic molecule, such as vinblastine (Guidolin et al., 2004). Results demonstrate a significant 38% decrease in vessel density was observed in the group treated with vinblastine as compared to the control group, while a 29% increase of the same parameter was observed after FGF-2 treatment. Moderate changes in the overall complexity were also detected in both treated groups when compared to the control one. A 8% increase of the fractal dimension was observed in the FGF-2 treated group and a 11% decrease in the group treated with vinblastine. Furthermore, the applied treatments significantly influenced the vascular network spatial arrangement. An increase of more than 40% in lacunarity was observed following treatment with vinblastine, indicating a more heterogeneous spatial arrangement of the CAM vessels. On the contrary, following FGF-2 treatment they result to fill the CAM in a more homogeneous way, as indicated by the significant decrease of this parameter with respect to the value observed in the control group. The order-disorder parameters proposed in this study allowed a more detailed description of the changes in the spatial arrangement of the vascular tree induced by the treatment with angiogenic or angiostatic molecules. A significant increase in positional disorder was observed following treatment with vinblastine: the positional order resulted increased of more than 30% when compared to the value obtained in the control group. No changes in this parameters were detected following FGF-2 treatment. In this group, however, a more regular pattern of branching was observed, as indicated by the significant decrease of topological order (–31%) with respect to the control. On the contrary, the treatment with vinblastine lead to a more irregular pattern of branching, resulting in a significant increase (~20%) of topological order. Both treatment groups didn’t exhibit changes in orientational order when compared with the control: at this stage of development of the vascular network vessels were distributed among almost all the possible orientations and the observed values for orientational order resulted close to 1 in all groups.

Chapter 2

Chorioallantoic Membrane in the Study of Angiogenesis, Antiangiogenesis, and the Vascularization of Grafted Tissues

2.1 Use of Chorioallantoic Membrane in the Study of Angiogenic Molecules The CAM is used to study molecules with angiogenic activity (Ribatti et al., 1996a, 2000a). Fertilized White Leghorn chicken eggs staged according to Hamburger and Hamilton (1951) are placed in an incubator as soon as embryogenesis starts and are kept under constant humidity at 37◦ C. On day 3, a square window is opened in the shell after removal of 2–3 ml of albumen to detach the CAM from the shell itself and the underlying CAM vessels are disclosed. The window is sealed with a glass and incubation goes on until the day of experiment. This technique may preserve a more physiological environment; however, it limits the area for use and observation. An angiogenic response occurs 72–96 h after stimulation in the form of an increased vessel density around the implant, with the vessels radially converging toward the center like spokes in a wheel. A variety of substances, normal cells, their conditioned media, and normal tissues have been reported to induce CAM angiogenesis (Tables 2.1 and 2.2). Table 2.1 Stimulators of angiogenesis tested in the CAM assay Substance

Authors

Activated protein C ADAM-33, a disintegrin and metalloprotease Adenosine diphosphate Adenosine Advanced glycation and products (AGEs) Alpha-fetoprotein All-trans-retinoic acid (ATRA) Amyloid beta Angiogenin Angiopoietin 4 Angiotensin II Antibody anti-endoglin Anti-integrin antibody Aprotinin, a broad-spectrum protease inhibitor

Jackson et al. (2005) Puxeddu et al. (2008) Fraser et al. (1979) Dusseau et al. (1986) Okamoto et al. (2002) Liang et al. (2004) Gaetano et al. (2001) Boscolo et al. (2007) Fett et al. (1985) Le Jan et al. (2003) Le Noble et al. (1991) Raab et al. (1999) Escher et al. (2009) Koutsioumpa et al. (2009)

D. Ribatti, The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis, DOI 10.1007/978-90-481-3845-6_2,  C Springer Science+Business Media B.V. 2010

17

18

2 Chorioallantoic Membrane Table 2.1 (continued)

Substance

Authors

Arsenic Arsenite sodium A1 Adenosine receptor agonist Baicalein, a prolyl-4-hydroxylase 2 inhibitor Beta amyloid (Abeta 1–40, Abeta 1–42) 4-Beta PMA and Dic8 Beta-sitosterol, a plant sterol Beta-thymosin peptides Bone morphogenetic protein endothelial cell precursor-derived regulators (BMPER) Bone sialoprotein 1-Butyryl-glycerol Carboxyethylpyrrole proteins Carcinoembryonic antigen-related CAM1 Cathepsin D C-C chemokine I-309 C-C chemokine CCL11 (eotaxin) C-C chemokine CCL15 C-C chemokine CCL23 C-reactive protein Chinese herbal medicine Chrysin derivatives Chuanxiong (Rhizoma chuanxiong) Ciclopirox olamine, an antimycotic agent Connective tissue growth factor Copper C-X-CL1/macrophage inflammatory protein-2 (MIP-2) Cytochrome P450 2C9-derived epoxyeicosatrienoic acids Danggui (Radix angelicae sinensis) Danshen (Radix salvae miltionrrhizae) Dehydroepiandrosterone Del1, an extracellular matrix protein Drm-gremlin, a bone morphogenetic protein antagonist Epichlorohydrin Epidermal growth factor Epoxyeicosatrienoic acids (EETs), cytochrome P450 arachidonic acid (AA) epoxygenase metabolites Erucamide, a fatty acid isolated from mesentery Erythropoietin Ethanol Extracellular matrix protein-1 (ECM-1) Fibrin Fibrin degradation products Fibroblast growth factor-1 Fibroblast growth factor-2 Fibroblast growth factor-4 Formylmethyonil leucyl phenylalanine Frza, a frizzled-related protein Glycosaminoglycan mimetics RGTAs (regenerating agents)

Soucy et al. (2003) Mousa et al. (2007) Clark et al. (2007) Cho et al. (2008) Boscolo et al. (2007) Tsopanoglou et al. (1993) Moon et al. (1999) Koutrafouri et al. (2001) Heinke et al. (2008) Bellahcène et al. (2000) Dobson et al. (1990) Ebrahem et al. (2006) Wagner and Ergün, (2000) Hu et al., (2008a) Bernardini et al. (2000) Salcedo et al. (2002) Hwang et al. (2004) Hwang et al. (2005) Turu et al. (2008) Gao et al. (2005) Peng et al. (2009) Meng et al. (2006) Linden et al. (2003) Shimo et al. (1999) Mc Auslan et al., (1983) Scapini et al. (2004) Michaelis et al. (2003) Meng et al. (2006) Meng et al. (2006) Liu D et al. (2008) Penta et al. (1999) Stabile et al. (2007) Girolamo et al. (2006) Stewart et al. (1989) Wang et al. (2005) Wakamatsu et al. (1990) Ribatti et al., (1999d) Gu et al. (2001) Han et al. (2001) Barnhill and Ryan (1983) Thompson et al. (1985) Lobb et al. (1985) Esch et al. (1985) Yoshida et al., (1994) Mc Auslan et al., (1983) Dufourcq et al. (2002) Rouet et al. (2005)

2.1

Use of Chorioallantoic Membrane in the Study of Angiogenic Molecules

19

Table 2.1 (continued) Substance

Authors

Gonadotropin Grastrin Growth differentiation factor 5, a BMP member Human cathelicidin antimicrobial peptide LL-37 Human cysteine-rich protein 61 (CYR61/CCN1) Heparin Heparin affinity regulatory peptide (HARP) Heparin-binding domain of fibronectin Hepatoma-derived growth factor Histamine HIV-Tat Homeobox B3 Homeobox D3 Human chorionic gonadotropin Human-soluble intercellular adhesion molecule-1 Hyaluronic acid (degradation products) Hyperglycemia Hypoxia Testicular hyaluronidase Interleukin-6 IL-1 beta Insulin-like growth factor II Intercellular adhesion molecule-1 (ICAM-1) Kininogen Lactate Laminin Laminin 1-chain peptide Laser irradiation Leptin Leukotrienes C4 and D4 Lipoprotein-A Mast cell activator compound 48/80 Melanotransferrin Monocyte chemoattractant protein-1 (MCP-1) Neuregulin-1 Neuropeptide Y (NPY) Neutrophil-activating protein-2 Nicotine Nicotinamide N-terminal domain (NoC1) of thrombospondin-1 Oligosaccharide sulfated Opioid Opioid endogenous peptides Osteocalcin Osteogenic protein-1 Osteopontin Pentapeptide SFLLR, an agonist of the activated thrombin receptor

Berndt et al. (2006) Clarke et al. (2006) Yamashita et al. (1997) Koczulla et al. (2003) Schütze et al. (2005) Ribatti et al. (1987) Papadimitriov et al. (2001) Viji et al. (2008) Everett et al. (2004) Thompson and Brown (1987) Urbinati et al. (2005) Myers et al. (2000) Boudreau et al. (1997) Zygmunt et al. (2002) Gho et al. (1999) West et al. (1985) Di Marco et al. (2008) Dusseau and Hutchins (1989) Arnold et al. (1987) Henández-Rodríguez et al., (2003) Naldini et al. (2006) Bae et al. (1998) Deng et al. (2007) Colman (2004) Kumar et al. (2007) Kumar VB et al. (2008) Ponce et al. (1999) Mirsky et al. (2002) Ribatti et al., (2001c) Tsopanoglou et al. (1994) Ribatti et al., (1998d) Clinton et al. (1988) Sala et al. (2002) Hong et al. (2005) Iivanainen et al. (2007) Ekstrand et al. (2003) Powell and Mousa (2007) Mousa and Mousa (2006) Kull et al., (1987) Staniszewska et al. (2007) Mousa et al. (2006) Blebea et al. (2000) Dai et al. (2008) Cantatore et al. (2005) Ramoshebi and Ripamonti (2000) Leali et al. (2003) Maragoudakis et al. (2001)

20

2 Chorioallantoic Membrane Table 2.1 (continued)

Substance

Authors

Pituitary tumor transforming gene (PTTG) Placental growth factor-1 Platelet-derived growth factor Platelet-derived membrane microparticles Phorbol esters Pregnancy-associated plasma protein A (PAPP-A) Prostaglandin E2 Pyruvic acid SB 202190, a p38 inhibitor Selenomethionine Sixth Ig-like domain of cell adhesion molecule L1 (L1 Ig6), a ligand for alphavbeta3-integrin SPARC Sokotrasterol sulfate Soluble intercellular adhesion molecule-1 Tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) Thrombin Thymidine phosphorylase (PDECGF) Thymosin beta-15 Thyroid hormone Thyroid hormone analogue GC-1 TNF-related activation-induced cytokine (TRANCE) Transferrin Transforming growth factor beta Trefoil peptides Tumor necrosis factor alpha Urotensin-II Vascular endothelial growth factor-A Vascular endothelial growth factor-D Visfatin, an adipokine produced by adipose tissue ZEB-1, an E-box transcription repressor

Ishikawa et al. (2001) Ziche et al. (1997) Wilting et al. (1992) Chen et al. (2007b) Morris et al. (1988) Jadlowiec et al. (2005) Form and Auerbach (1983) Lee et al. (2001) Matsumoto et al. (2002) McAuslan and Reilly (1986) Hall and Hulbell (2004) Iruela-Arispe et al. (1995) Murphy et al. (2006) Gho et al. (2001) Liu JM et al. (2003) Tsopanoglou et al. (1993) Ishikawa et al. (1989) Koutrafouri et al. (2003) Davis et al. (2004) Mousa et al. (2005b) Kim YM et al. (2002) Carlevaro et al. (1997) Yang and Moses (1990) Rodrigues et al. (2003) Leibovich et al. (1987) Spinazzi et al. (2006) Wilting et al. (1993) Chen et al. (2007a) Kim et al. (2007) Clarhaut et al. (2009)

Filter disks are used to test angiogenic factor to a defined area of the CAM (Beckers et al., 1997). Filter disks can be cut from nitrocellulose membrane or Whatman paper. The disks are pre-soaked in test compounds and are usually dried before grafting on the CAM. The test substance can be soaked in inert synthetic polymers laid upon the CAM: Elvax 40 (ethylene-vinyl acetate copolymer) and hydron (a poly-2-hydroxyethylmethacrylate polymer) are commonly used. The two polymers were first described and validated by Langer and Folkman (1976): both provided to be biologically inert when implanted onto the CAM and both were found to polymerize in the presence of the test substance, allowing its sustained release during the assay. However, hydron requires the test substance to be added to a solution of hydron and ethanol. When the test pellets are vacuum dried, ethanol is removed leaving a solid pellet that contains the test substance. If the test material is not compatible with ethanol, Elvax can be used instead. Elvax is dissolved in methylene chloride before the

2.1

Use of Chorioallantoic Membrane in the Study of Angiogenic Molecules

21

Table 2.2 Angiogenic response induced by normal cells, their conditioned media (CM), and normal tissues implanted onto the CAM Cells

Authors

Adipose tissue from visceral and subcutaneous sites CM from 3T3 adipocytes Human atrial appendage tissue CM from basophils Chinese hamster ovary cells transfected with endothelin-1 Cytotrophoblast Dendritic cells Endometrium Endothelial cells transfected with FGF-2 CM from eosinophils Fibroblasts CM from keratinocytes Lymphocytes from lymph node and spleen CM from T lymphocytes CM from macrophages Mast cells CM from mast cells Neutrophils Pancreatic duct cells Anterior pituitary gland Placenta cells Placental tissues Spleen cells activated with mitogens Mammalian retina

Ledoux et al. (2008) Castellot et al. (1982) Lewis et al. (2006) De Paulis et al. (2006) Cruz et al. (2001) Zhou et al. (2003) Riboldi et al. (2005) Maas et al. (1999) Ribatti et al. (1997b) Puxeddu et al. (2005) Pinney et al. (2000) Barnhill and Ryan (1983) Auberbach et al. (1976) Ribatti et al. (1991) Leibovich et al. (1987) Ribatti et al., (2001b) Detoraki et al. (2009) Ardi et al. (2007) Movahedi et al. (2008) Gould et al. (1995) Burgos, (1983) Reynolds et al. (1987) Pliskin et al. (1980) Glaser et al. (1980)

test material is suspended/dissolved in the polymer, after which methylene chloride is removed by vacuum drying. A more sustained release can be achieved by “sandwiching” the test substance between two Elvax layers. The polymers cause the substance to be released at constant rates (nanograms to micrograms) around the clock. Methylcellulose disks are more widely used and are prepared usually as a final concentration of 1% solution of methylcellulose and then the agent of interest is added to the solution. The disks can be sterilized using UV light and subsequently they are placed onto the CAM surface. The release of the agent is slow and minimal reaction to control disks was reported. Preparations of methylcellulose disks involve spreading and drying of the factor containing mixtures on Teflon surfaces (Yang and Moses, 1990), glass surfaces (Ribatti et al., 1995), or parafilm (Hagedorn et al., 2004), before their application on the CAM. Matrigel mixtures can be distributed in small volumes directly onto the CAM where rapidly polymerization occurs. Alternatively, defined aliquots of Matrigel supplemented with test compounds can be pre-gelated at 37◦ C on nylon meshes and then placed onto the CAM (Vazquez et al., 1999; Watanabe et al., 2004). The test components may be incorporated into alginate pellet (Riboldi et al., 2005). The pellets are prepared by mixing a solution of sodium alginate with

22

2 Chorioallantoic Membrane

cells or purified molecules, followed by a dropwise releasing of the mixture into a CaCl2 -containing solution. The calcium ions cause immediate gelling of the alginate droplets, which, after washing can be implanted onto the CAM. Wilting et al. (1991, 1992) used culture coverslide glasses (Thermanox) 4–5 mm in diameter, on which 5 μl of several angiogenic factors was placed. Glasses were turned over and placed onto the CAM on day 9 of incubation, and the angiogenic response was evaluated 96 h later. Alternatively, when testing a fluid substance, the latter is inoculated (20–50 μl) directly into the cavity of the allantoic vesicle so that its activity reaches the whole vascular area in a uniform manner (Fig. 2.1) (Ribatti et al., 1987; Gualandris et al., 1996). We have developed a new method for the quantification of angiogenesis and antiangiogenesis in the CAM. Gelatin sponges treated with a stimulator or an inhibitor of blood vessel formation are implanted on growing CAM on day 8 (Ribatti et al., 2006a). Blood vessels growing vertically into the sponge and at the boundary between sponge and surrounding mesenchyme are counted morphometrically on day 12. The newly formed blood vessels grow perpendicular to the plane of the CAM inside the sponge, which does not contain preexisting vessels. The gelatin sponge is also suitable for the delivery of tumor cell suspensions, as well as of any other cell type, onto the CAM surface and the evaluation of their angiogenic potential (Ribatti et al., 2006a). As compared with the application of large amounts of a recombinant angiogenic cytokine in a single bolus, the use of cell implants that overexpress angiogenic cytokines allows the continuous delivery of growth factors, which is produced by a limited number of cells (as low as 10,000–20,000 cells per implant), thus mimicking more closely the initial stages of tumor angiogenesis and metastasis (Fig. 2.2). Cells that overexpress FGF-2 and secrete approximately

Fig. 2.1 Photographical reconstruction of a CAM portion of a control (a) and a heparin-treated (b) 7-day chick embryo. In the control CAM the vessels run straight and interdigitate regularly, whereas in the treated specimen, the vessel course is irregular or sinuous, their branching is remarkably frequent, dilated vascular tracts alternate with narrow ones, and vascular loops are very numerous (reproduced from Ribatti et al., 1987)

2.2

Role of FGF-2 in Chorioallantoic Membrane Vascularization

23

Fig. 2.2 Effect of VEGF-overexpressing V-12-MCF-7 cells on CAM vascularization. Cells were delivered at 18,000 cells per embryo on top of the CAM using a gelatin sponge implant. (a) Macroscopic observation of the CAM, done on day 12, shows the gelatin sponge surrounded by allantoic vessels that develop radially toward the implant in a “spoked-wheel” pattern. (b, c) Histologic analysis. A highly vascularized tissue is recognizable among the sponge trabeculae, consisting of newly formed blood vessels (b, arrowheads). The vessels are absent among the sponge trabeculae in specimens treated with medium alone (b) (reproduced from Ribatti et al., 2006a)

2–3 pg of FGF-2 throughout the experimental period exert a proangiogenic response when applied onto the CAM that is similar to the one elicited by 1 μg of recombinant cytokine (Ribatti et al., 2001a). Dreesmann et al. (2007) reported the successful development of an insoluble matrix made from a cross-linked collagen hydrolysate, i.e., a gelatin fraction, which induces a strong angiogenic response in the CAM assay. Yao et al. (2008) demonstrated that cross-linked collagen matrices and matrices both cross-linked and heparinized appeared to show a significantly higher angiogenic potential than unmodified matrices in the CAM assay. A cylinder model has been designated to assess the vascularization potential of engineered tissues (Borges et al., 2003). In this model, cell-containing matrices are applied within specially constructed plastic cylinders, allowing for continual observation of graft vascularization using a light microscope. Many techniques can be applied within the constraints of paraffin and plastic embedding, including histochemistry and immunohistochemistry. Electron microscopy can also be used in combination with light microscopy. Moreover, unfixed sponges can be utilized for chemical studies, such as the determination of DNA, protein, and collagen content, as well as for RT-PCR analysis of gene expression by infiltrating cells, including endothelial cells.

2.2 Role of FGF-2 in Chorioallantoic Membrane Vascularization To evaluate the presence of an FGF-2-like molecule in CAM and in chorioallantoic fluid (CAF), different amounts of CAM extracts and CAF samples obtained from embryos at days 8, 10, 14, and 18 of incubation were assayed for their ability to stimulate plasminogen activator production in GM 7373 endothelial cells. Both CAM and CAF samples induce an increase in GM 7373 cell-associated plasminogen activator activity in a dose-dependent manner (Ribatti et al., 1995). The potency of the different samples in stimulating plasminogen activator

24

2 Chorioallantoic Membrane

production in GM7373 endothelial cells differs as a function of the age of the embryo, suggesting that the amounts of plasminogen activator-inducing activity present in CAM and CAF may vary during embryonic development (Ribatti et al., 1995). To confirm the presence of FGF-2 in CAM and CAF, samples were assayed for their ability to interact with heparin and to cross-react with neutralizing polyclonal anti-human FGF-2 antibody. In a first experiment, CAM extracts obtained from a 14-day embryo were run through a heparin-Sepharose column or were incubated with neutralizing polyclonal anti-human FGF-2 antibody or with non-immune rabbit serum. Then the plasminogen activator-inducing activity of these samples was evaluated on GM 7373 endothelial cells. The plasminogen activator-inducing activity of CAM extract is retained by the heparin-Sepharose column and it is specifically neutralized by an anti-FGF-2 antibody, while non-immune rabbit serum was uneffective, thus identifying this activity as an FGF-2-like activity (Ribatti et al., 1995). In another set of experiments, an aliquot of CAF was obtained from different embryos and loaded onto a heparin-Sepharose column. Fractions were collected and assayed for their ability to stimulate plasminogen activator production in GM 7373 endothelial cells. Most of the plasminogen activator-inducing activity present in CAF binds to the resin and is eluted with the 2 M NaCl wash. Moreover, preincubation of this fraction with neutralizing anti-FGF-2 antibody completely abolishes its plasminogen activator-inducing activity (Ribatti et al., 1995). Aliquots of CAM and CAF obtained from a 14-day embryo were partially purified on heparin-Sepharose columns and probed in a Western blot with the affinity-purified anti-human FGF-2 antibody, recognizing a heparin-binding Mr of 16,000 protein in both samples (Ribatti et al., 1995). We have also quantified the temporal changes of FGF-2 in CAM and CAF during embryonic development, evaluating the amount of total plasminogen activator-inducing activity present in the crude CAM extracts and CAF samples obtained from chick embryos between day 6 and day 18 of incubation. The levels of FGF-2 in CAM and CAF vary significantly during embryonic development; maximal concentrations are observed between days 10 and 14 of incubation, when the vascular density of the CAM also reaches its maximum. The absolute concentrations of FGF-2 appear to be much higher in CAM than in CAF (Ribatti et al., 1995). In a series of experiments performed in vivo FGF-2 or anti-FGF-2 antibody was adsorbed on methylcellulose disks and applied on top of the CAM of embryos at day 8 of incubation. Application of FGF-2 led to a positive angiogenic response in 85% of the animals, consisting of a spoke-wheel vascular pattern around the implant (Ribatti et al., 1995). Under light microscopy, blood vessels, predominantly capillaries, with a narrow lumen, were distributed in the upper portion of the CAM and their number was increased, while the intermediate mesenchyme contained numerous fibroblasts (Fig. 2.2) (Ribatti et al., 1995). In keeping with the capacity to exert a mitogenic activity for a variety of cell types of mesodermal and neuroectodermal origin, FGF-2 induces also fibroblast cell proliferation and hyperplasia of the chorionic epithelium. At the ultrastructural level, small vascular tubes with a very narrow lumen, located beneath the chorion, were recognizable. Application of antiFGF-2 antibody on the surface of the CAM resulted in a significant antiangiogenic

2.2

Role of FGF-2 in Chorioallantoic Membrane Vascularization

25

Fig. 2.3 Semithin sections of CAM after treatment with FGF-2 (a) or anti-FGF-2 antibody (b). Note in a an increased number of small blood vessels (arrowheads) beneath the ectoderm (EC) and in the upper portion of the intermediate mesenchyme (above the line); the mesenchyme (M) also contains numerous fibroblasts (EN, endoderm). Note in b few vessels (arrowheads) beneath the ectoderm (EC), loosely arranged fibroblasts, and wide intercellular spaces; no blood vessels are detectable in the intermediate mesenchyme (M) (reproduced from Ribatti et al. 1995)

effect in 75% of the embryos (Ribatti et al., 1995). An avascular zone free of vessels could be evidenced beneath the implant after intravascular injection of India ink. Microscopically, few blood vessels were still recognizable beneath the implant. Also, no blood vessels were detectable in the intermediate mesenchyme, where fibroblasts were less numerous than in control embryos (Fig. 2.3). Quantification of the angiogenic response performed at day 12 of incubation by using a morphometric method confirmed the morphologic observations. When FGF-2 was applied on the surface of the CAM, the microvessel density was 3.3 times higher than in control embryos. Conversely, application of anti-FGF-2 antibody resulted in 3 times reduction in the microvessel density (Ribatti et al., 1995). These findings indicate that endogenous FGF-2 is intrinsically involved in CAM vascularization on the basis of the evidence that FGF-2 is present in elevated amounts in the CAM from day 6 to day 18 of incubation, maximal concentrations being observed between day 10 and day 14. Apparently, this observation does not fully agree with the time course of the vasoproliferative processes taking place in the CAM. Ausprunk et al. (1974) have shown that CAM endothelial cells have a labeling index of approximately 23% prior to day 11 of incubation; this index decreases to 2.8% thereafter. Nevertheless, in agreement with the kinetics of expression of CAM FGF-2, vascular density of the membrane continues to increase until days 12–14, when it reaches a plateau (Maragoudakis et al., 1988b). This apparent discrepancy can be explained by considering that vascular sprouting may occur through migration and redistribution of existing endothelial cells, in the absence of cell proliferation (Sholley et al., 1984). Interestingly, FGF-2 can induce capillary endothelial cells in vitro to invade a three-dimensional collagen matrix and to form capillary-like tubules, without cell proliferation, but dependent upon cell movement and protease production (Montesano et al., 1986). Thus, it is possible to hypothesize that endogenous chick FGF-2 may play a rate-limiting role in CAM vascularization by affecting not only the proliferation of endothelial cells but also their migration, redistribution, and invasive behavior.

26

2 Chorioallantoic Membrane

Our in situ hybridization data strongly suggest that the action of FGF-2 during this process occurs in two steps: at early stages of development the major source of FGF-2 is chorionic epithelial cells. Even though FGF-2 is devoid of a signal sequence for secretion (Abraham et al., 1986), an alternative mechanism of exocytosis of FGF-2 has been proposed (Mignatti et al., 1991, 1992). Limited amounts of FGF-2 can be released from cellular sites of synthesis and then sequestered in the extracellular matrix. Dissociation of extracellular FGF-2 from the matrix and binding to surface receptors follow (Moscatelli, 1992) and trigger a paracrine loop of stimulation. Thus, FGF-2 released by chorionic epithelial cells may induce an angiogenic response in undifferentiated vessels in the CAM mesoderm by stimulating endothelial cell proliferation, movement, and protease production (Montesano et al., 1986). At later stages, FGF-2 mRNA expression predominates in endothelial cells forming the capillary plexus, suggesting that FGF-2 plays an autocrine role in further development of the endothelium. When mouse aortic endothelial cells stably transfected with a retroviral expression vector harboring a human FGF-2 cDNA (pZipFGF-2 MAE) were injected twice into the allantoic sac of the chick embryo at days 8 and 9 significant modifications of the developing vasculature of the CAM were observed: blood vessels with an irregular course and frequently branching were present 4 days later. In contrast, blood vessels run straight and interdigitate regularly in the CAM of embryos injected with parental cells or vehicle (Gualandris et al., 1996). Intravenous injection of India ink revealed the presence of ink-filled enlarged hemangioma-like scattered within the blood vessel network of transfected cell-treated CAM (Fig. 2.4) (Gualandris

Fig. 2.4 Alterations of the CAM vasculature following injection of pZipFGF-2 MAE cells into the allantoic sac of chick embryo. Photographic reconstructions of the CAM vasculature visualized after intravenous injection of India ink. Note the irregular course, branching, the high density of CAM vasculature, and the presence of numerous hemangiomas of various sizes (arrows) (reproduced from Ribatti et al., 1999e)

2.2

Role of FGF-2 in Chorioallantoic Membrane Vascularization

27

et al., 1996; Ribatti et al., 1999e). These lesions are characterized by enlarged bloodfilled sacs lined by a thin endothelial cell monolayer. Careful examination of serial sections showed no sign of thrombotic and/or hemorrhagic lesions. India ink was evident within the endothelial cells-lined enlarged cavernae and the surrounding small blood vessels, but was undetectable in the stroma. Transfected endothelial cells treated for 3 h with mitomycin before injection into the allantoic sac were still able to induce a vasoproliferative response and the formation of hemangiomas (Ribatti et al., 1999e). In contrast, fixation of the cells with glutaraldehyde completely abolished their angiogenic and hemangioma-inducing activity. These data indicate that the injection of live, non-proliferating FGF-2-transfected endothelial cells is sufficient to induce the observed modification of the CAM vasculature that are therefore due to alterations of the behavior of the endothelial cells at the host. When transfected endothelial cells were injected twice into the allantoic sac together with neutralizing anti-FGF-2 polyclonal antibody, this latter had no effects on the modification of CAM vasculature induced by transfected endothelial cells (Ribatti et al., 1999e). These data suggest that the angiogenic and hemangioma-inducing activity exerted by the transfected endothelial cells injected into the allantoic sac may not depend on the release of FGF-2 into the allantoic fluid. To evaluate whether the angiogenic activity of transfected endothelial cells is due to diffusible factor(s), we assessed the activity of serum-free transfected cell-conditioned medium. The concentrated conditioned medium was adsorbed into a gelatin sponge and applied on top of the CAM. Live transfected endothelial cells were delivered onto the CAM under the same experimental condition as positive controls (Ribatti et al., 1999e). After 4 days, macroscopic observation of the CAM showed that the sponges treated with transfected endothelial cells or their concentrated conditioned medium were surrounded by numerous allantoic vessels which developed radially toward the implant in a “spoked-wheel” pattern. Scattered hemangiomas were recognizable in close proximity to the sponge. Also in this condition, the angiogenic activity of transfected endothelial cells was not affected when cells were applied onto the CAM together with neutralizing anti-FGF-2 antibody. The data further indicate that the transfected endothelial cells release an angiogenic activity distinct from FGF-2. The CAM may represent an in vivo system to assess the hypothesis that exogenous urokinase plasminogen activator may affect neovascularization via an endogenous FGF-2-dependent mechanism of action. Suspensions of urokinase plasminogen activator-overexpressing endothelial cells or parental endothelial cells were delivered on top of day 8 CAM by using a gelatin sponge implant (Ribatti et al., 1999b). Macroscopic observation of the CAM at day 12 showed that the gelatin sponges adsorbed with urokinase plasminogen activator-transfected (uPA-R5) endothelial cells were surrounded by allantoic vessels that developed radially toward the implant in a “spoked-wheel” pattern (Fig. 2.5). The allantoic vessels were less numerous in the specimens treated with parental endothelial cells (Neo 2 cells), whereas no vascular reaction was detectable around the sponges treated with vehicle only (Fig. 2.5) (Ribatti et al., 1999b). At the microscopic level, a highly vascularized tissue was recognizable among the trabeculae of uPA-R5

28

2 Chorioallantoic Membrane

Fig. 2.5 Effect of uPA-R5 cells on CAM neovascularization. Gelatin sponge adsorbed with uPAR5 cells is surrounded by allantoic vessels that develop radially toward the implant (asterisk) (a). The vessels are less numerous in the specimen treated with Neo 2 cells (asterisk) (b), whereas no vascular reaction is detectable around the vehicle-treated sponge (asterisk) (c) (reproduced from Ribatti et al., 1999b)

endothelial cell-treated sponges. The tissue consisted of newly formed blood vessels growing perpendicular to the plane of the CAM and of infiltrating fibroblasts within an abundant network of collagen fibers. The vessels were less numerous in the parental endothelial cell-treated sponges and were absent among trabeculae of implants treated with vehicle (Ribatti et al., 1999b). A higher microvessel density was detectable within the sponges treated with uPA-R5 endothelial cells that in those treated with parental endothelial cells or vehicle, when the angiogenic response was quantified by a morphometric method (Ribatti et al., 1999b). To assess whether the stronger angiogenic response elicited by uPA-R5 endothelial cells was due to an increased mobilization of endogenous FGF-2, these cells were added to the CAM in the presence of anti-FGF-2 antibody. Anti-FGF-2 antibody reduced the angiogenic response elicited by uPA-R5 endothelial cells to value similar to those measured in control endothelial cell-treated CAM (Ribatti et al., 1999b). It is interesting to note that uPA-R5 endothelial cells added with anti-FGF-2 antibody retain a limited angiogenic activity that is more potent than that exerted by parental (Neo 2) cells tested under the same experimental conditions. This suggests that released urokinase plasminogen activator may induce the mobilization of endogenous angiogenic factor other than FGF-2 and/or that urokinase plasminogen activator may per se elicit a limited angiogenic response. To confirm this hypothesis, the angiogenic activity of purified human urokinase plasminogen activator was evaluated and it exerted a dose-dependent angiogenic response in the CAM (Ribatti et al., 1999b). Furthermore, we compared the angiogenic activity of enzymatically active and inactive human urokinase plasminogen activator. For this purpose, purified human urokinase plasminogen activator was preincubated with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), an irreversible urokinase plasminogen activator inhibitor, or vehicle. After incubation, urokinase plasminogen activator samples were dialyzed extensively to remove free PMSF and their angiogenic activity was evaluated in the CAM. Preincubation of urokinase plasminogen activator with 1 mM PMSF significantly inhibits the

2.3

Use of Chorioallantoic Membrane in the Study of Antiangiogenic Molecules

29

angiogenic activity of the enzyme. Moreover, the angiogenic activity was reduced significantly by anti-FGF-2 antibody (Ribatti et al., 1999b). These data indicate that purified urokinase plasminogen activator exerts an FGF-2-dependent angiogenic activity in the CAM and that this effect depends, at least in part, upon a catalytic activity of the enzyme. To further substantiate this hypothesis, we compared the angiogenic activity of purified human urokinase plasminogen activator to that exerted by human ATF, lacking enzymatic activity. Human ATF was ineffective when assayed for its angiogenic capacity in the CAM. The inability of ATF to induce angiogenesis in the CAM indicates that the proteolytic activity of urokinase plasminogen activator is of pivotal importance in mediating its angiogenic capacity in vivo (Ribatti et al., 1999b). By utilizing the CAM assay, we have shown that anti-FGF-2 antibody reduce significantly the angiogenic activity exerted by uPA-R5 cells and purified human urokinase plasminogen activator, thus implicating extracellular endogenous FGF-2 in the growth of newly formed blood vessels stimulated by urokinase plasminogen activator. However, the incapacity of anti-FGF-2 antibody to fully suppress the angiogenic ability of purified urokinase plasminogen activator and uPA-R5 endothelial cells suggests that more factors besides FGF-2 might be implicated in protease-triggered CAM neovascularization. It is interesting to note that both uPA-R5 endothelial cells and purified human urokinase plasminogen activator exert an angiogenic response in the CAM that is less potent than that exerted by exogenous FGF-2, suggesting that the levels of endogenous angiogenic growth factors available to the protease action may represent a limiting factor in this experimental system. Overall, our findings demonstrate that uPA-R5 endothelial cells and purified urokinase plasminogen activator exert a potent angiogenic effect on the CAM which depends on the catalytic activity of the enzyme and is reversed by neutralizing anti-FGF-2 antibody. We have compared the angiogenic activity of FGF-2- or VEGF-transfected cells adsorbed onto gelatin sponges and applied on top of the CAM (Ribatti et al., 2001a). Both cell lines induced a comparable vasoproliferative response, as demonstrated by the appearance of similar number of blood vessels within the sponge (Fig. 2.2). Electron microscopy demonstrated that the VEGF-overexpressing cells modified the phenotype of the endothelium of the CAM blood capillaries. In fact, the endothelium lining 30% of these vessels showed segmental attenuations, was frequently interrupted, and became fenestrated, mimicking what is observed in tumor vasculature (Ribatti et al., 2001b).

2.3 Use of Chorioallantoic Membrane in the Study of Antiangiogenic Molecules The CAM is also used to study macromolecules with antiangiogenic activity (Ribatti et al., 1996a; 2000a). In studies of angiogenesis inhibitors (Table 2.3), there are two approaches which differ in the target vessels, i.e., those which examine the response

30

2 Chorioallantoic Membrane

in the rapidly growing CAM and those that evaluate the inhibition of growth induced by an angiogenic cytokine, such as FGF-2. When an angiostatic compound is tested, the vessels become less dense around the implant after 72–96 h, and eventually disappear (Ribatti et al., 1995; Iurlaro et al., 1998; Vacca et al. 1999a; Minischetti et al., 2000). The development of an “avascular zone” was initially described by Taylor and Folkman (1982), who showed that protamine would produce an avascular zone when applied to the leading edge of the CAM of a day 4 embryo. Table 2.3 Angiogenesis inhibitors tested in the CAM assay Substances

Authors

AAV-mediated gene transfer of TIMP-1 AA98V (H)/L, an antibody anti-CD146 Abeta peptides, naturally occurring peptides Aeroplysinin-1, a brominated compound isolated from a marine sponge Adiponectin Ad-vasostatin Agkistin, a snake venom-derived glycoprotein Ib antagonist AGM-1470 Alliin, a compound derived from garlic Alpha4beta1 antagonists Alpha(v)-beta 3/alpha(v)-beta 5 integrin antagonist (EDM 478761) Alpha(v)-beta 3/alpha(v)-beta 5 integrin antagonist ST1646 Amifostine Amiloride Aminopeptidase-N antagonists Angioinhibins Angiopoietin-2 Angiostatin Angiotensinogen Angiotensinogen (cleaved derivatives) Anthracyclines and titanocene dichloride Anti-alpha (v) beta 3 monoclonal antibody (LM609) Antibacterial substance isolated from the flesh fly Antibiotics (concanamycin group) Antibody anti-FGF-2 Antibody anti-VEGF Anti-CD146 monoclonal antibody Anti-collagen IV humanized antibody D93 Antioxidant molecule (curcumin and quercetin) Antithrombin Apicidin, a histone deacetylase inhibitor Aplidine Apolipoprotein(a) Kringle V Apomorphine (a dopamine receptor inhibitor) Aquaporin-1 siRNA Arginine deaminase 2-Aroylindole derivatives, tubulin inhibitors Arresten

Zacchigna et al. (2004) Lin Y et al. (2007) Paris et al. (2004) Rodríguez-Nieto et al. (2002) Brakenhielm et al. (2004) Li et al. (2006) Yeh et al. (2000) Kusaka et al. (1991) Mousa and Mousa, (2005) Calzada et al. (2004) Fu et al. (2007) Belvisi et al. (2005) Giannopoulou et al. (2003) Knoll et al. (1999) Bhagwat et al. (2001) Ingber et al. (1990) Lee OK et al. (2006) O’ Reilly et al. (1994) Brand et al. (2007) Célérier et al. (2002) Maragoudakis et al. (1999) Drake et al. (1995) Nishikawa et al. (2006) Ishii et al. (1995) Ribatti et al. (1995) Vitaliti et al. (2000) Yan et al. (2003) Pernasetti et al. (2006) Jackson et al. (2006) Kisker et al. (2001b) Kim SH et al. (2004) Taraboletti et al. (2004) Kim JS et al. (2004) Kim HJ et al. (2001) Camerino et al. (2006) Park et al. (2003) Mahboobi et al. (2001) Zheng et al. (2006)

2.3

Use of Chorioallantoic Membrane in the Study of Antiangiogenic Molecules

31

Table 2.3 (continued) Substances

Authors

Artesunate, a semi-synthetic derivative of artemisin extracted from the Chinese herb Artemisia annua Ascorbic acid Atiprimod (belonging to the azaspirane class of cationic amphiphilic drugs) Aurintricarboxylic acid Azaspirine, a fungal product Bactericidal/permeability-increasing protein Bacterium (PB2) isolated from sponge primonorphs Baicalein and baicalin, two flavonoids Bleomycin Blockers of volume-regulated anion channels Beta-cyclodextrin tetradeca sulfate (TDS)+angiostatic steroids Beta-escin, an extract of the horse chestnut Aesculus hippocastanum seed Beta-hydroxyisovolerylshikonin (beta-HISV) from Lithospermum erythrorhizon Bone morphogenetic protein-9 (BMP-9) Bortezomib Butyric acid CAI (carboxy-amidotriazole), an inhibitor of ligand-stimulated calcium influx Campesterol, a plant sterol Canstatin Capsaicin Carbon materials (graphite, multiwalled carbon nanotubes, fullerenes) Carrageenan depolymerization products Cartilage Catechins, green tea polyphenols Cerivastatin Cheiradone, a natural product isolated from a Euphorbia species Chemically sulfated Escherichia coli K5 polysaccharide derivatives Chemokine antagonist M3 Chondrocyte-derived inhibitor Chondromodulin-1 Chrysin Cigarette smoke condensate Clodronate Clotrimazole Contortrostatin Curcumin Curcumin analogues Curcumin (lipid microparticles) Cyclic peptide antagonists of alpha v beta 3 Cyclic peptide antagonists of alpha v beta 5 integrins

Huan-Huan et al. (2004) Ashino et al. (2003) Shailubhai et al. (2004) Gagliardi and Collins (1994) Asami et al. (2008) van der Schaftet et al., (2000) Thakur et al. (2005) Liu JJ et al. (2003) Oikawa et al. (1990a) Monolopoulos et al. (2000) Folkman et al. (1989) Wang et al. (2008) Komi et al. (2009) David et al. (2008) Roccaro et al. (2006b) Gururaj et al. (2003) Kohn et al. (1995) Choi et al. (2007) Hou et al. (2004) Min et al. (2004) Murugesan et al. (2007) Chen et al. (2007) Eisenstein et al. (1975) Maiti et al. (2003) Vincent et al. (2002) Hussain et al. (2008b) Presta et al. (2005) Andrés et al. (2008) Eisenstein et al. (1975) Hiraki et al. (1999) Lin et al. (2006c) Ejaz et al. (2009) Ribatti et al. (2008b) Thapa et al. (2008) Zhou et al. (1999) Gururaj et al. (2002) Hahm et al. (2004) Yadav et al. (2009) Brooks et al. (1994b) Friedlander et al. (1995)

32

2 Chorioallantoic Membrane Table 2.3 (continued)

Substances

Authors

Cyclooxygenase inhibitor Cyclooxygenase (COX)-5 LOX inhibitor Cyclopeptidic vascular endothelial growth inhibitor Cyclosporin Cytocholasin D 7-Deazaxanthine (inhibitor of thymidine phosphorylase) Deguelin, isolated from plants in the Mundulea sericea family Delphinidin, a vasoactive polyphenol Deoxycholic acid–heparin conjugate Deoxycytidine nucleoside analogue DFMO α-difluoromethylornithine (inhibitor of ornithine decarboxylase) Diaminoanthraquinone, a protein kinase C inhibitor Dichloropyridodithienenotriazine

Jung et al. (2007b) Park et al. (2009) Zilberg et al. (2003) Iurlaro et al. (1998) Melkonian et al., (2002 b) Balzarini et al. (1998) Kim JH et al. (2008)

Dihydroartemisinin, a metabolite of artemisinin derivatives Dihydrotanshinone I, a natural compound extracted from Salvia miltiorrhiza Bunge Digoxin Ditriazine derivative DTD DPTH-N10 Docetaxel Dominant-negative p65 PAK peptide Doxazosin, quinazoline-based alpha(1)-andrenoreceptor antagonist Doxycycline Doxorubicin Eclipta prostata, a Thai medicinal plant Emodin, an active component of the root and rhizome of Rheum palmatum Endocannabinoid anandamide Endorepellin, derived from the C-terminus of perlecan Endostar (recombinant human endostatin) Endostatin (recombinant) Enoic acanthonic acid (cyclooxygenase-2-inhibitor) Eponeomycin Epoxyeicosatrienoic acid antagonist Estrogen antagonists Ets-1 antisense Ets-1 antisense oligodeoxyribonucleotides Evodiamine, isolated from Chinese herbal drug Wu-Chu-Yu Fascaplysin, a selective CDK4 inhibitor Fenretinide Flavone acetic acid 19- and 20-fluorosynerazols Fractalkine, a CX3C chemokine

Favot et al. (2003) Lee et al. (2007) Roy et al. (2006) Takigawa et al. (1990) Takano et al. (1994) Martínez-Poveda et al. (2007) Chen et al. (2004) Bian et al. (2008) Svensson et al. (2005) Martínez-Poveda et al. (2008) Liu Y et al. (2008) Vacca et al. (2002b) Kiosses et al. (2002) Garrison et al. (2007) Richardson et al. (2005) Splawinski et al. (1988) Lirdprapamongkol et al. (2008) Kwak et al. (2006) Pisanti et al. (2007) Mongiat et al. (2002) Ling et al. (2007) Liu et al. (2007) Jung et al. (2007b) Oikawa et al. (1993a) Michaelis et al. (2005) Gagliardi and Collins (1993) Forough et al. (2006) Wernert et al. (1999) Shyu et al. (2006) Lin J et al. (2007) Ribatti et al. (2001a) Lindsay et al. (1996) Igarashi et al. (2004) Ryu et al. (2008)

2.3

Use of Chorioallantoic Membrane in the Study of Antiangiogenic Molecules

33

Table 2.3 (continued) Substances

Authors

Ganglioside GM3 Gastrodia elata Blume rhizome Genipin, an active principle of gardenia Ghrelin Gleditsia sinesis fruit extract (GSE) Glycine Goniodomin A (an antifungal polyester macrolide) Grateloupia longifolia polysaccharide isolated from the marine alga Green tea polyphenols (GTPs) Grifola frondosa (maitake mushroom) GRO-beta (CXC chemokine) GW654652, an indazolylpyrimidine acting as a VEGFRs tyrosine kinase inhibitor Heparan sulfate suleparoide Heparanase Heparin or heparin fragments+cortisone Heparin+11-hydrocortisone or 17-hydroxyprogesterone Heparin substitutes Heparins undersulfated and glycol split Hepatocyte growth factor (HGF)-like basic hexapeptides Herbamycin Histidine-proline-rich glycoprotein (HPRG) HIV-1 protease inhibitors Homeobox D10 (Hox D10) Homocysteine HST-1 protein Human prolactin phosphorylated Human neutrophil peptides (HNPs) 10-Hydroxycamptothecin Hydroxylated thalidomide metabolites Hyperforin, a phloroglucinol derivative found in St Johuíwort Hypertermia Hypoestoxide, a nonsteroidal antiinflammatory drug Hypoxic cytotoxin TX-402 Hypoxia cytotoxins Indinavir and saquinavir (HIV protease inhibitors) Indolin-2-ketone compound (Z24) Inhibitors of basement membrane biosynthesis

Chung et al. (2009) Ahn et al. (2007) Koo et al. (2004) Conconi et al., (2004b) Chow et al. (2003) Amin et al. (2003) Abe et al. (2002) Zhang et al. (2006)

Inhibitors of DNA methyltransferases (DNMT) Insulin-like growth factor (IGF)-binding protein (IGFBP)-3 Integrin alpha(v)beta3 antagonist Integrin alpha(v)beta5/alpha(v)beta3 antagonists Interleukin-12 Interleukin-18 Interleukin-21 Interleukin-27

Oak et al. (2005) Lee et al. (2008) Cao et al. (1995) Huh JI et al. (2005) Benelli et al. (1998) Sasisekharon et al. (1994) Folkman et al. (1983) Crum et al. (1985) Folkman et al. (1989) Casu et al. (2004) Fazekas et al. (2001) Oikawa et al. (1989a) Juarez et al. (2002) Sgadari et al. (2002) Myers et al. (2002) Nagai et al. (2001) Yoshida et al. (1994) Ueda et al. (2006) Chavakis et al. (2004) Xiao et al. (2001) Marks et al. (2002) Martínez-Poveda et al. (2005) Roca et al. (2003) Ojo-Amaize et al. (2002) Nagasawa et al. (2003) Nagasawa et al. (2002) Sgadari et al. (2002) Wang et al. (2004) Maragoudakis et al. (1988a), (1989), (1990) Hellebrekers et al. (2006) Oh et al. (2006) Friedlander et al. (1995) Kumar et al. (2001) Airoldi et al. (2007) Cao et al. (1999) Castermans et al. (2008) Shimizu et al. (2006)

34

2 Chorioallantoic Membrane Table 2.3 (continued)

Substances

Authors

Ionizing radiation Isoflavones isolated from a tempeh (fermented soyabean) extract Isoprostanes Isosorbide mononitrate and dinitrate, NO-releasing vasodilators JNI-17029259, an RTK inhibitor JNI-26076713, an orally bioavailable, non-peptide alpha(v) antagonist KIN-841, an hypoxia-dependent 2-nitroimidazole Kinin-free derivative of kininogen Kininogen and kininogen-derived polypeptides Kininostatin (domain 5 of high molecular weight kininogen) KV11, a novel peptide from human apolipoprotein(a) Lactacystin, a specific proteasome inhibitor Lambda-carragenan oligosaccharide Laminin-derived synthetic peptide Laminarin sulfate Larg-A, a polysaccharide extracted from the brown marine alga Sargassum stenophyllum Lebectin, a C-type lectin protein Lebestatin, a disintegrin from Macrovipera venom LMPAB, low molecular weight polysaccharide extracts from Agaricus blazei Lonicera japonica (Caprifoliaceae) Low molecular weight heparin sulfated Escherichia coli K5 polysaccharide derivatives Low molecular weight undersulfated glycol-split heparin Low-sulfated oligosaccharides derived from heparan sulfate Lysozyme Mainstream and sidestream cigarette smoke Marine-derived oligosaccharide sulfate (multiple receptor tyrosine inhibitor) Metastatin, a hyaluronan-binding complex from cartilage 2-Methoxyestradiol Methylene blue Methyltransferase inhibitors Microorganism fermentation extract Midkine (MDK), a heparin-binding growth factor Mitoxantrone Mixture containing ascorbic acid, lysine, proline, and green tea Monoclonal antibody to kininogen and kininostatin Motuporanines, isolated from marine sponge Multiple receptor tyrosine kinase (RTK) inhibitors Mustard essential oil containing allyl isothiocyanate Myo-inositol trispyrophosphate (ITPP) Neomycin

Karnabatidis et al. (2001) Kiriakidis et al. (2005) Benndorf et al. (2008) Pipìli-Synetos et al. (1995a) Emanuel et al. (2004) Santulli et al. (2008) Shimamura et al. (2003) Colman (2006) Zhang et al. (2002) Colman et al. (2000) Yi et al. (2009) Oikawa et al. (1998) Chen et al. (2007b) Sakamoto et al. (1991) Hoffman et al. (1996) Dias et al. (2005) Pilorget et al. (2007) Olfa et al. (2005) Niu et al. (2009) Yoo et al. (2008b) Presta et al. (2005) Casu et al. (2004) Hahnenberger et al. (1993) Ye et al. (2008) Melkonian ete al. (2002a) Ma et al. (2008) Liu et al. (2001) D’Amato et al. (1994) Zacharakis et al. (2006) Hellebrekers et al. (2006) Chui et al. (2006) van der Horst et al. (2008) Iigo et al. (1995) Roomi et al. (2005) Colman et al. (2002) Roskelly et al. (2001) Gangjee et al. (2008) Kumar et al. (2009) Sihn et al. (2007) Hu (1998)

2.3

Use of Chorioallantoic Membrane in the Study of Antiangiogenic Molecules

35

Table 2.3 (continued) Substances

Authors

Neridronate N-terminal region of neuregulin-2 Neurokinin-B (NK-B) NGR-SL-doxorubicin Nitric oxide donors Nitric oxide 2-Sodium, 5-hydroxy, -nitrotoluene sulfonate+angiostatic steroids Nonpeptide topomimetics Notch 4 Nucleolin antagonist Obtustatin, a disintegrin Octacosanol, a long-chain aliphatic alcohol Oncothanin, a peptide from the alpha 3 chain of type IV collagen 5 -O-Trityl nucleoside analogues Opioid peptides Oriental herbal cocktail Oxaliplatin Paclitaxel Plasminogen activator inhibitor-1 PAK1, a protein kinase downstream of the GTPases Rac and Cdc42 PE, a new sulfated saponin from sea cucumber Pedicularioside G 1,2,3,4,6,-Penta-O-galloyl-beta-D-glucose (PGG) Pentosan polysulfate (inhibitor of HIV-1 tat) Pentraxin-3 Peptide trivalent arsenical Perillyl alcohol Peroxisome proliferator-activated receptor-gamma (PPAR) agonists (pioglitazone and rosiglitazone) PEX, a noncatalytic metalloproteinase fragment Phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables Phenolic compounds (4-hydroxybenzyl alcohol) 1,4-Phenylenabis(methylene)selenocyanate Philinopside A, a novel sulfated saponin isolated from the sea cucumber Pentacta quadrangulari Phorbol esters Photodynamine therapy Piperazine derivative (SJ-8026) 1-Benzhydryl-sulfonyl-piperazine derivatives 3(a–e) Placental ribonuclease inhibitor Plasma hyaluronan-binding protein Plasmid encoding mouse endostatin Plasminogen activator inhibitor-1 Plasminogen-related protein B (PRP-B) Platelet factor 4

Ribatti et al., (2007b) Nakano et al. (2004) Pal et al. (2006) Pastorino et al. (2006) Powell et al. (2000) Pipìli-Synetos et al. (1995) Chen et al. (1988) Dings et al. (2006) Leong et al. (2002) Destouches et al. (2008) Marcinkiewicz et al. (2003) Thippeswamy et al. (2008) Shahan et al. (2004) Liekens et al. (2006) Dai et al. (2008) Lee HJ et al. (2006) Qian et al. (2007) Vacca et al. (2002b) Stefansson et al. (2001) Kiosses et al. (2002) Tian et al. (2005) Mu et al. (2008) Huh JE et al. (2005) Rusnati et al. (2001) Rusnati et al. (2004) Don et al. (2003) Loutrari et al. (2004) Aljada et al. (2008) Brooks et al. (1998) Xiao and Singh (2007) Lim et al. (2007) Schumacher et al. (2001) Tong et al. (2005) Tsopanoglou et al. (1993) Gottfried et al. (1992) Yi et al. (2005) Kumar CS et al. (2008) Shapiro and Vallee (1987) Jeon et al. (2006) Li et al. (2008) Stefansson et al. (2001) Morioka et al. (2003) Maione et al. (1990)

36

2 Chorioallantoic Membrane Table 2.3 (continued)

Substances

Authors

Platelet factor 4 (C-13 fragment) 16-kDa N-terminal fragment of human prolactin Poly(ADP-ribose) polymerase (PARP) inhibitor PJ-34 Poly-L-lysine/heparin Polysulfated derivative of the glucan laminarin Pomegranate 8-Prenylnaringenin, a phytoestrogen Prolactin Proline analogues, an inhibitor of proline hydroxylase and beta-aminopropionitrile Protamine Protease-activated receptor (PAR)-1 antagonists Prothrombin fragments 1 and 2 2,4-Diamino-5-substituted furo[2,3-D]pyrimidines with RTK and DHFR) inhibitor activity Purine analogue (6 methylmercaptopurine) Purine riboside derivative KIN 59 Pyracoumarin compounds decursin and decursinol angelate isolated from the herb Angelica gigas Pyrazine, a major chemical group in smoke p38 MAP kinase Quercetin, a dietary-derived flavonoid Quinoline-3-carboxiamide Radicicol RDG-peptidomimetic SCH221153 vs Tat-induced angiogenesis Recombinant human plasminogen kringle 1–3 (rk1–3) Recombinant human prothrombin kringles Recombinant kringle domain of tissue-type plasminogen activator Recombinant kringle domain of urokinase Recombinant kringle 5 domain of plasminogen Red wine polyphenolic compounds (RWPCs) Resveratrol Retinoids Rhodostomin, a snake venom disintegrin Ribavirin, antiviral drug used to treat hepatitis C virus Ribonuclease inhibitor Rosiglitazone, a PPAR-gamma ligand Ruthenium red-based compound (NAMI-A) Safrole oxide Salmosin, a snake venom-derived disintegrin Sangivamycin, an antibiotic with anti-tumor and anti-herpes virus activities Sanguinarine, a benzophenanthridine alkaloid Saurus chinensis Baill (ethanol extract of the dried aerial parts) SCH221153, a dual alpha(v)beta3 and alpha(v)beta5 integrin receptor antagonist

Li et al. (2007) Clapp et al. (1993) Pyriochou et al. (2008) Pacini et al. (2002) Hoffman et al. (1996) Toi et al. (2003) Pepper et al. (2004) Clapp et al. (1993) Ingber and Folkman (1988) Taylor and Folkman (1982) Zania et al. (2006) Rhim et al. (1998) Gangjee et al. (2005) Presta et al. (1999) Liekens et al. (2004) Jung et al. (2009) Melkonian et al. (2003) Matsumoto et al. (2002) Tan et al. (2003) Isaacs et al. (2006) Oikawa et al. (1993b) Urbinati et al. (2005) Youn et al. (2006) Kim TH et al. (2002) Kim HK et al. (2003) Kim KS et al. (2003) Jia and Bian (2007) Oak et al. (2005) Mousa SS et al. (2005) Oikawa et al. (1989b) Yeh et al. (2001) Michaelis et al. (2007) Chatterjee et al. (2006) Panigrahy et al. (2002) Vacca et al. (2002a) Zhao et al. (2005) Kang et al. (1999) Komi et al. (2007) Eun and Koh (2004) Yoo et al. (2008a) Kumar et al. (2001)

2.3

Use of Chorioallantoic Membrane in the Study of Antiangiogenic Molecules Table 2.3 (continued)

Substances

Authors

Sedum sarmentosum (a perennial herb) extract Serpin (Serp-1) Sesterterpenes, isolated from the Himalayan plant Leucosceptrum canum Short-COOH-terminal segment (PF-4) derived from platelet factor-4 Short heparin sequences spaced by glycol-split uronate residues Short peptide (NLLMAAS) Simvastatin, cholesterol-lowering agent SJ-8002, a new piperazine derivative S-Nitrosocaptopril Sodium caffeate, the sodium salt of caffeic acid Solanum nigrum Somatostatin Somocystinamide (lipopeptide) Soy isoflavones S-Phosphonate, antineoplastic ether lipid Spironolactone Squalamine Staurosporine Sulfated E. coli K5 polysaccharide Sulfated glycosaminoglycans Sulfated polysaccharide–peptidoglycan complex (DS-4152) Sulfated polysaccharide–peptidoglycan complex+cortisone acetate Sulfonated derivative of dystamycin Sulfonated dystomycin A derivatives Sulfonic acid polymers PAMS and related analogues Sulf-2, an endosulfatase

Jung et al. (2008a) Richardson et al. (2007) Hussain et al. (2008a)

Sulindac analogue-2 Sulindac metabolites Suramin Suramin analogues Suramin+angiostatic steroids Synthetic growth factor receptor-bound protein 2 (Grb2)-Src homology 2 (SH2)-binding domain antagonists Synthetic inhibitor of arylsulfatase TA138, a alpha v beta3 antagonist Taraxacum officinale (ethanol extract) Taspine, isolated from Radix et Rhizoma Leonticis TAU 1120 Taxol Temozolomide Tenasum-C peptide Terbinafine (TB), an oral antifungal agent Terpenoids from Bletilla striata

Hagedorn et al. (2002) Casu et al. (2002) Tournaire et al. (2004) Park et al. (2002) Yi et al. (2004) Jia et al. (2000) Xu et al. (2004) Xu et al. (2008) Woltering et al. (1991) Wrasidlo et al. (2008) Su et al. (2005) Jackson et al. (1998) Klauber et al. (1996) Sills et al. (1998) Oikawa et al. (1992) Leali et al. (2001) Jakobson et al. (1991) Tanaka et al. (1989) Inoue et al. (1988) Sola et al. (1995) Ciomei et al. (1994) Liekens et al. (1997) Morimoto-Tomita et al. (2005) Pyriochou et al., (2007) Elwich-Fils et al. (2003) Danesi et al. (1993) Gagliardi et al. (1998) Wilks et al. (1991) Soriano et al. (2004)

Chen et al. (1988) Mousa et al. (2005a) Jeon et al. (2008) Zhang et al. (2008b) Nozaki et al. (1993) Dardunoo et al. (1995) Kurzen et al. (2003) Saito et al. (2008) Ho et al. (2004) Liu MZ et al. (2008)

37

38

2 Chorioallantoic Membrane Table 2.3 (continued)

Substances

Authors

Tetrac (tetraiodothyroacetic acid), a deaminated, non-agonist thyroid hormone Tetrameric tripeptide Transforming growth factor beta 1

Mousa et al. (2008)

6-Thioguanine (6-TG) Thrombospondin-1 Thrombospondin-1 type III repeats Thymidine phosphorylase inhibitors Thymosin peptides Tinzaparin, a low molecular weight heparin Tissue inhibitor of metalloproteinase-3 (TIMP-3) 23-Amino acid fragment of tissue factor pathway inhibitor (TFPI) Titanocene dichloride TNP470+interferon alpha Tocotrienol, an unsaturated version of vitamin E Tocotrienol (T3) Topoisomerase inhibitors Topoisomerase I inhibitors Topotecan Torilin, a sesquiterpene compound Trapidil Triamcinolone acetonide (TA) Tricyclodecan-9-yl-xanthate Triphenylmethane derivative (aurintricarboxylic acid) Tripterygium wilfordii Hook (extracts) Triptolide Triterpene acids (urosolic acid and oleanolic acid) Trypanosoma cruzi calreticulin Two-chain high molecular weight kininogen and kininogen-derived polypeptides Tyrosine phosphatase SHP-2 inhibition TZT-1027 (Soblidotin), a microtubule-depolymerizing agent Ulmus davidiana var. japonica Undersulfated, low-molecular weight glycol-split heparin Ursodeoxycholic acid Ursolic and oleanolic acid Valproic acid Vanillyl alcohol Vascular endothelial cell growth inhibitor (VEGI), a protein of the TNF superfamily VASH1B, the alternative splicing product of vasohinibin 1 Vasostatin (N-terminal amino acids 135–164) Vasostatin in combination with B7H3 expression plasmids VEGF-toxin conjugate VEG1 (a protein of the TNF superfamily) Vinblastine

Ponticelli et al. (2008) Parson-Wingerter et al. (2000) Presta et al. (2002) Chandrasekaran et al. (2000) Margosio et al. (2008) Liekens et al. (2004) Koutrafouri et al. (2001) Mousa and Mohamed (2004) Anand-Apte et al. (1997) Hembrough et al. (2004) Bastaki et al. (1994) Minischetti et al. (2000) Nakagawa et al. (2007) Miyazawa et al. (2004) Jackson et al. (2008) Yang et al. (2006) Puppo et al. (2008) Kim et al. (2000) Benelli et al. (1995) McKay et al. (2008) Maragoudakis et al. (1990) Gagliardi and Collins (1994) Zhu et al. (2007) Ding et al. (2005) Sohn et al. (1995) Molina et al. (2005) Zhang et al. (2002) Mannell et al. (2008) Watanabe et al. (2007) Jung et al. (2007a) Pisano et al. (2005) Suh et al. (1997) Sohn et al. (1995) Michaelis et al. (2004) Jung et al. (2008b) Zhai et al. (1999) Kern et al. (2008) Li et al. (2007) Ma et al. (2007) Ramakrishnan et al. (1996) Zhai et al. (1999) Vacca et al. (1999a)

2.4

Use of Chorioallantoic Membrane in the Study of Vascularization of Grafted Tissues

39

Table 2.3 (continued) Substances

Authors

Vinblastine+rapamycin Vitamin D-binding protein (DBP-maf) Vitamin D3 analogues Vitreous von Hippel–Lindau protein (pVHL) Wogonin, a flavonoid Zoledronic acid

Marimpietri et al. (2005) Kisker et al. (2003) Oikawa et al. (1990b) Lutty et al. (1983) Bae et al. (2005) Lin et al. (2006b) Scavelli et al. (2007)

2.4 Use of Chorioallantoic Membrane in the Study of Vascularization of Grafted Tissues The technique of grafting-isolated portions of embryos to the CAM has offered opportunity for an attack upon varied types of embryological problems and has been used more extensively than any other in vivo method. The method of transplanting tissues has been used successfully to study the development of all individual organs. In the CAM system, survival of grafted tissues is dependent on rapid neovascularization. In non-neoplastic tissues, which lack the ability to stimulate host angiogenesis (Folkman and Cotran, 1976) early vascularization can only occur by rescue of the intrinsic graft blood vessels. In the CAM, failure of revascularization to occur within the first 3 days ultimately led to the disruption and removal of implants (Ausprunk et al., 1975). This suggests that revascularization of implants occurring after this time must result from an ingrowth of host capillaries. The formation of peripheral anastomoses between host and donor vessels is the most common mechanism involved in the vascularization of grafted embryonic tissues on the CAM, whereas growth of CAM-derived vessels into transplants would be expected to take place in grafts of tumor tissues, and only in some embryonic grafts (Ausprunk et al., 1975). When adult tissues such as skeletal muscle, heart, kidney, and liver were transplanted onto the chick CAM, revascularization did not occur (Ausprunk et al., 1975). It is conceivable that diffusable molecular factor emanating from the transplanted tissue could stimulate undamaged host vessels to sprout and anastomose with the existing graft vasculature. One advantage of the avian system is the ability to construct quail/chick chimeric embryos using tissue grafting techniques (Sariola, 1985). The cells from the donor and host species can be distinguished from one another based upon the morphology of the nuclear heterochromatin (Le Douarin, 1973) or by the expression of antigenic determinants specifically expressed by quail endothelial (MB1) and hematopoietic (QH1) cells (Pardanaud et al., 1987; Peault et al., 1983). Because of the species specificity of these monoclonal antibodies, blood vessels that are MB1 or QH1 positive that appear within a grafted organ rudiment must be of quail origin. Therefore, if a quail embryonic organ that was grafted to a chicken embryo later contains MB1/QH1-positive endothelial cells, then vascularization of that organ must involve

40

2 Chorioallantoic Membrane

vasculogenesis. Likewise, if a chicken organ that was grafted to a quail embryo contains MB1/QH1-positive endothelial cells, then the organ must have undergone angiogenesis. These experiments demonstrate that particular organs such as brain, kidney, and limb bud are vascularized exclusively through angiogenesis (Stewart and Wiley, 1981; Ekblom et al., 1982; Pardanaud et al., 1989). In contrast, certain organs like lung and pancreas are vascularized exclusively through vasculogenesis (Pardanaud et al., 1989). Transplantation studies have demonstrated that host-derived blood vessel from non-neuronal sources will develop functional, structural, and histochemical features of capillaries with a blood–brain barrier once they have invaded and vascularized donor brain tissue. Therefore, brain tissue or the neural environment provides some signals or factors capable of inducing blood–brain specializations (Papoutsi et al., 2000a). Bertossi et al. (1998) implanted fragments of the adrenal gland of chick or quail embryo onto quail or chick CAM. They demonstrated that the grafting CAM induced the formation of peripheral anastomoses between the graft and the CAM original microvasculature and new growth of vessels from the CAM into grafted tissue and vice versa. Moreover, the CAM vessels that grow into the adrenal gland and the adrenal vessels that grow into the CAM maintained the original endothelial phenotype. Bertossi et al. (1999) have further investigated the vasculature of embryonic tissues grafted onto CAM performing single grafts of adrenal gland or cerebellum and double grafts of adrenal gland plus cerebellum. They demonstrated the presence of new microvessels growing from the CAM into grafted tissues, and vice versa, in both single and double transplants. Moreover, the adrenal, fenestrated sinusoids and the cerebellar, barrier-provided capillaries maintained their original phenotype when they grow within the non-native tissue.

Chapter 3

Chorioallantoic Membrane in the Study of Tumor Angiogenesis

3.1 Use of Chorioallantoic Membrane in the Study of Tumor Angiogenesis The CAM has long been a favored system for the study of tumor angiogenesis (Dagg et al., 1956; Auerbach et al., 1976; Weiss et al., 1979; Ribatti et al., 1996a; Ribatti, 2004), because at this stage the chick’s immunocompetent system is not fully developed and the conditions for rejection have not been established (Leene et al., 1973). In birds, in fact, the immunocompetence only develops after hatching (Weber and Mausner, 1977). As other vertebrates, chickens are protected by a dual immune system comprised of B and T cells, controlling the antibody and cell-mediated immunity, respectively. The B cells are differentiated in the bursa of Fabricius, the organ equivalent to the bone marrow in mammals, whereas T cells are differentiated in the thymus (Funk and Thompson, 1996; Davison, 2003). Until day 10, the chick embryo immune system is not completely developed. The presence of T cells can be first detected at day 11 and of B cells at day 12 (Janse and Jeurissen, 1991). By day 12, mononuclear phagocytes are found in the yolk sac, spleen, bursa, gut, thymus, and liver (Janse and Jeurissen, 1991). The two major inflammatory cell types present in day 10–15 embryos are heterophils and monocytes. Heterophils functionally serve as an avian analogue of mammalian neutrophils and represent a main source of MMP-9 in the chick embryo. Therefore, chicken heterophils could be identified by staining with a specific anti-chicken MMP-9 antibody (Zijlstra et al., 2006). On the other hand, monocytes are the major source of MMP-13 in the chick embryo and could be identified by immunostaining with an anti-MMP-13 antibody (Zijlstra et al., 2004). After day 15, the B-cell repertory begins to diversify and by day 18 chicken embryos become immunocompetent. The CAM is one of the earliest models used to grow tumor xenografts. In 1911, Rous and Murphy demonstrated the growth of the Rous 45 chicken sarcoma transplanted onto the CAM (Rous and Murphy, 1911). One year later, Murphy (1912) reported that mouse and rat tumors implanted onto the CAM could be maintained by continuous passage from egg to egg and described the effects of these heterologous transplantation on CAM and chick embryo. Murphy tried to culture and passage human tumors, but less successful. Later, the CAM assay was improved through D. Ribatti, The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis, DOI 10.1007/978-90-481-3845-6_3,  C Springer Science+Business Media B.V. 2010

41

42

3

Chorioallantoic Membrane in the Study of Tumor Angiogenesis

the removal of a square of the shell to expose the CAM surface (Clark, 1920). The choice of the 9-day-old embryo for tissue grafting and the selection of the junction point of two or more large blood vessels as a graft site was introduced by Willier in 1924 followed by the creation by Burnet in 1933 of an artificial air space above the CAM. In 1949, Taylor and his group reported an alternative method of implantation of mouse tumors on the yolk sac (Taylor et al., 1948). Karnofsky and co-workers compared the behavior of tumor cells and tissues derived from chicken, mouse, and humans when they were implanted on the CAM surface (Karnofsky et al., 1952). Several parameters were evaluated, such as tumor growth, histological features, the effects of continuous passage in eggs on tumor growth, viability after re-transplantation into its original host, and the effects on the chick embryo. On the basis of these parameters, the tumors were differently classified. When small fragments of Rous chicken sarcoma were implanted on CAM on day 8 of incubation, all embryos died 7–8 days after implantation, by massive hemorrhages. Large tumors were recognizable on the CAM surface, weighting up to 5 g. An entrapment of the CAM into the tumor was detectable and the tumor mass was hanging downward on the underside of the CAM. Microscopically, various shaped cells with mitotic figures were detectable and when tissues were removed from these embryos and re-implanted onto other CAM of day 8, they induced the formation of typical sarcoma. Mouse sarcomas were carried indefinitely by continuous passages on the CAM and had a similar rate of growth on mice and chicken. Human Hodgkin lymphoma implanted on CAM produced edema in the first 24 h after implant and into the embryo after 4 days, probably as a consequence of the effects of a toxic substance produced by the tumor. Moreover, some necrotic patches were recognizable in the liver of the embryo suggestive for a possible failed development of a metastasis. Korngold and Lipari (1955) demonstrated that human tumor transplanted and retransplanted on CAM retains and expresses human antigens after several passages. Harris implanted human squamous cell carcinoma, sarcoma, adenocarcinoma, and bronchogenic carcinoma samples on the CAM, evaluated the presence of metastases into the embryo after 10 days, and tested the inhibitory capacity of chemotherapeutic agents on their growth (Harris, 1958). Kaufman recommended that the grafts should be made within 3 h after the drawing of samples from the center of the tumor for a greater survival and was the first to describe the changes in the CAM adjacent to the tumor implantation site, consisting in the formation of epithelial pearls into the mesenchyme near to the implant, fibroblastic proliferation, keratinization, and stratification of the chorionic epithelium (Kaufman et al., 1955). All studies of mammalian neoplasms in the CAM have utilized solid tumors and cell suspensions derived from tumors (Tables 3.1, 3.2 and 3.3). Compared with mammal models, where tumor growth often takes between 3 and 6 weeks, assays using chick embryos are faster. Between 2 and 5 days after tumor cell inoculation, the tumor xenografts become visible and are supplied with vessels of CAM origin. Tumors grafted onto the CAM remain non-vascularized for a couple of days, after which they can be penetrated by new blood vessels and begin a phase of rapid

3.1

Use of Chorioallantoic Membrane in the Study of Tumor Angiogenesis

43

Table 3.1 Angiogenic response induced by tumor samples implanted onto the CAM Tumor

Authors

Adenocarcinoma of the endometrium Glioblastoma Head and neck squamous cell carcinoma Hepatocellular carcinoma Lipoma Lymphoma B-cell non-Hodgkin’s lymphoma Meningioma Neuroblastoma Ovarian endometrioma Mouse teratoma

Palczak and Splawinski (1989) Klagsbrun et al. (1976) Petruzzelli et al. (1993) Marzullo et al. (1998) Lucarelli et al. (1999) Mostafa et al. (1980) Ribatti et al. (1990) Klagsbrun et al. (1976) Ribatti et al. (2002b) Ria et al. (2002) Auerbach et al. (1976)

Table 3.2 Angiogenic response induced by tumor cells and their conditioned media (CM) implanted onto the CAM Tumor cells

Authors

Chinese hamster ovary cell aggregates stably transfected with endothelin-1 (CHO-ET-1) CM from IL-16 overexpressing basal cell carcinoma cell line CM from Kaposi’s sarcoma cell line CM of human anaplastic thyroid carcinoma Endothelial cells isolated from patients with multiple myeloma Friend erythroleukemia cells GM7373 endothelial cells overexpressing uPA Gynecologic tumor cell lines Hepatitis C virus-infected cells Lymphoblastoid cells Mammary tumor cells transfected with int-2 oncogene Mammary tumor cells transfected with VEGF Mouse C57 melanoma B-16 melanoma cells Neuroblastoma cell lines Neurofibroma Schwann cells Plasma cells isolated from patients with multiple myeloma Urothelial carcinoma cells with low MKP-1 expression Walker carcinoma 256 cells

Cruz et al. (2001) Jee et al. (2004) Masood et al. (2001) Ono et al. (1989) Vacca et al. (2003) Pacini et al. (2008) Ribatti et al. (1999a) Ishiwata et al. (1988) Nasimuzzaman et al. (2007) Vacca et al. (1998) Costa et al. (1994) Ribatti et al. (2001b) Auerbach et al. (1976) Takigawa et al. (1990) Ribatti et al. (2002b) Sheela et al. (1990) Ribatti et al. (2003a) Shimada et al. (2007) Klagsbrun et al. (1976)

growth. Tumor cells can be identified in the CAM, as well as in the internal organs of the embryo, such as lungs, liver, and brain (Gordon and Quigley, 1986; Bobek et al., 2004). Walker 256 carcinoma specimens implanted on the CAM do not exceed a mean diameter of 0.93 ± 0.29 mm during the prevascular phase (approximately 72 h). Rapid growth begins 24 h after vascularization and tumors reach a mean diameter of 8.0 ± 2.5 mm by 7 days (Knighton et al., 1977). When tumor grafts of increasing size (from 1 to 4 mm) are implanted on the 9-day CAM, grafts larger than

44

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Chorioallantoic Membrane in the Study of Tumor Angiogenesis

Table 3.3 Antiangiogenic molecules involved in the inhibition of angiogenic response induced by tumor cell suspensions of tumor bioptic specimen implants onto the CAM Breast cancer and fibrosarcoma cell lines treated with a CTGF-polyclonal antibody Breast cancer cell lines treated with E-peptide of the pro-insulin-like growth factor-I Breast cancer cell lines treated with COX-5 LOX inhibitor Carcinoembryonic antigen-expressing carcinoma cell lines treated with doxorubicin Colon cancer cells treated with silymarin and silibinin Colon adenocarcinoma cell line treated with anti-angiopoietin-2 antibody Fibrosarcoma cells transfected with soluble intercellular adhesion molecule-1 Glioma cell nodules treated with myo-inositol trispyrophosphate Hepatocellular carcinoma cell line treated with an antisense oligonucleotide targeting midkine Hepatocellular carcinoma bioptic specimens treated with vinblastine+rapamycin Hepatocellular carcinoma bioptic specimens treated with anti-leptin antibody Leukemia LIK cells treated with 6-thioguanine Mammary cells treated with octreotide acetate Mammary cells infected with a retroviral expression vector carrying the int-2 oncogene Melanoma cell line in which twist is down-regulated Melanoma cell line treated with obtustatin, a smoke venom HTS-disintegrin CM of human multiple myeloma cell line treated with dihydroartemisin Multiple myeloma endothelial cells treated with bortezomib Neuroblastoma bioptic specimens treated with fenretinide Neuroblastoma tumor xenografts treated with interferon gamma CM of neuroblastoma cell lines treated with bortezomib Neuroblastoma bioptic specimens and tumor xenografts treated with bortezomib CM of neuroblastoma cell lines treated with vinblastine+rapamycin Neuroblastoma bioptic specimens and tumor xenografts treated with vinblastine+rapamycin Prostate tumor cells treated with human uteroglobulin Murine sarcoma S-180 treated with 4’-thioguanosine Neuroblastoma cancer xenografts treated with bortezomib+fenretinide Neuroblastoma, ovarian, and lung cancer xenografts treated with targeted liposomal doxorubicin

Shimo et al. (2001) Chen et al. (2007) Park et al. (2009) Stan et al. (1999) Yang et al. (2005) Wang et al. (2007) Gho et al. (2001) Sihn et al. (2007) Dai et al. (2007) Ribatti et al. (2007a) Ribatti et al. (2008a) Presta et al. (2002) Danesi et al. (1997) Costa et al. (1994) Hu et al. (2008b) Brown et al. (2008) Wu et al. (2006) Roccaro et al. (2006a) Ribatti et al. (2001d) Ribatti et al. (2006b) Brignole et al. (2006) Brignole et al. (2006) Marimpietri et al. (2005) Marimpietri et al. (2005, 2007) Patierno et al. (2002) Miura et al. (2004) Pagnan et al. (2009) Pastorino et al. (2008)

1 mm undergo necrosis and autolysis during the 72-h prevascular phase. They shrink rapidly until the onset of vascularization, when rapid growth resumes (Knighton et al., 1977). Ausprunk and Folkman (1976) compared the behavior of tumor grafts to grafts of normal adult and embryo tissues. In tumor tissue, preexisting blood vessels in

3.2

Angiogenesis and Antiangiogenesis in Multiple Myeloma

45

the tumor graft disintegrated within 24 h after implantation and revascularization occurred by penetration of proliferating host vessels into the tumor tissue. By contrast, preexisting vessels did not disintegrate in the embryo graft and anastomosed to the host vessels with almost no neovascularization. In adult tissues, preexisting graft vessels disintegrated (although this process was slower than in tumor vessels) and did not stimulate capillary proliferation in the host. Lastly, tumor vessels did not reattach to those of the host. Hagedorn et al. (2005) have developed a glioblastoma multiforme tumor progression model on the CAM. They demonstrated that avascular tumors formed within 2 days, than progressed through VEGFR-2-dependent angiogenesis, are associated with hemorrhage, necrosis, and peritumoral edema. Blocking of VEGFR-2 and PDGFR signaling pathways by using small-molecule receptor tyrosine kinase inhibitors abrogated tumor development. Moreover, gene regulation during the angiogenic switch was analyzed by oligonucleotide microarrays, permitting identification of regulated genes whose functions are associated mainly with tumor vascularization and growth. CAM can also be used to study the effects of antiangiogenic treatments on the angiogenic response induced by tumor cell suspensions or tumor bioptic specimens implanted onto the CAM.

3.2 Angiogenesis and Antiangiogenesis in Multiple Myeloma 3.2.1 Biological and Clinical Studies Several years ago, for the first time in literature, we have demonstrated that in patients with monoclonal gammopathy of undetermined significance and multiple myeloma, angiogenesis correlates with plasma cell growth (S-phase fraction) (Vacca et al., 1994). Moreover, angiogenesis is paralleled by an increased angiogenic ability of bone marrow plasma cell-conditioned medium of patients with active multiple myeloma as compared with those with non-active multiple myeloma and monoclonal gammopathy of undetermined significance, and partly dependent FGF-2 production (Vacca et al., 1999b). Myeloma plasma cells induce angiogenesis directly via the secretion of angiogenic cytokines, such as VEGF and FGF-2, and indirectly by the induction of host inflammatory cell infiltration, and degrade the extracellular matrix with their matrix degrading enzymes, such as MMP-2 and MMP-9 and urokinase-type plasminogen activator (Vacca and Ribatti, 2006). Reciprocal positive and negative interactions between plasma cells and bone marrow stromal cells, namely, hematopoietic stem cells, fibroblasts, osteoblasts/osteoclasts, chondroclasts, endothelial cells, endothelial cell progenitor cells, T cells, macrophages, and mast cells, mediated by an array of cytokines, receptors, and adhesion molecules, modulate the angiogenic response in multiple myeloma (Ribatti et al., 2006c). We have recently demonstrated that macrophages and mast cells contribute to build neovessels in active multiple myeloma through vasculogenic mimicry, and this ability proceeds

46

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Chorioallantoic Membrane in the Study of Tumor Angiogenesis

parallel to the progression of the plasma cell tumors (Scavelli et al., 2008; Nico et al., 2008). The VEGF signaling pathway can be inhibited in multiple myeloma at various levels, that is, by blocking the activity of VEGF with monoclonal antibodies, blocking the VEGFR with specific inhibitors or interfering with the tyrosine kinases activated by the VEGF/VEGFR interactions (Ria et al., 2003). Since thalidomide possessed antiangiogenic properties (Ribatti and Vacca, 2005), Singhal et al. (1999) for the first time used thalidomide on compassionate basis to treat 84 patients with relapsed and refractory multiple myeloma. Thalidomide has also been combined with other active agents in the management of relapsed multiple myeloma, while the absence of myelosuppression and other important adverse effects suggests that it could be combined with chemotherapy (Ribatti and Vacca, 2005). Bortezomib (Velcade, formerly PS-341) is the first proteasome inhibitor to have shown anti-cancer activity in both solid tumors and hematological malignancies (Roccaro et al., 2006b). Multiple myeloma is the prototype cancer where bortezomib has shown marked in vitro activity, which was followed by rapid translation to phase I, II, and III clinical trails, and resulted in accelerated approval by the Food and Drug Administration for the treatment of patients with relapsed refractory disease (Roccaro et al., 2006a).

3.2.2 Use of the Chorioallantoic Membrane Plasma cell culture medium obtained from patients with multiple myeloma was tested to their ability to induce angiogenesis in the CAM (Vacca et al., 1999b). The conditioned medium of 77% active multiple myeloma patients induced an angiogenic response (Fig. 3.1); by contrast, only 33 and 20% of conditioned medium

Fig. 3.1 CAM implanted with a gelatin sponge soaked with the conditioned medium of plasma cells of an active multiple myeloma patient shows macroscopically numerous blood vessels converging toward the sponge (a), whereas microscopically numerous blood vessels (arrowheads) are recognizable among the sponge trabeculae (b) (reproduced from Vacca et al., 1999b)

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from non-active multiple myeloma and patients with monoclonal gammopathy of undetermined significance, respectively, induced the response. Anti-FGF-2 antibody partly inhibited conditioned medium angiogenic response. In another work, we have attempted a fine characterization of the angiogenic response induced by plasma cells obtained from patients with active multiple myeloma, as compared to those obtained from patients with non-active multiple myeloma and, respectively, monoclonal gammopathies of undetermined significance in the CAM assay. To this purpose, we have investigated the time course of the angiogenic response induced by gelatin sponges soaked with the cell suspensions and implanted on the CAM surface from day 8 to day 12 of incubation by evaluating the number of vessels (Fig. 3.2), the vessel bifurcation, and the intervascular distance at 24, 48, 72, and 96 h after the implants (Ribatti et al., 2003a). Results demonstrated that plasma cell suspensions obtained from patients with active multiple myeloma induce a vasoproliferative response, significantly higher as compared to that induced by cell suspensions obtained from patients with non-active multiple myeloma and, respectively, with monoclonal gammopathies of undetermined significance. These responses are a function of the day of implantation. In fact, implants made from day 8 to day 10 are strongly angiogenic, while those made from day 11 to day 12 do not. This finding might depend on the fact that CAM endothelium exhibits an intrinsically high mitotic rate until day 10 (Ausprunk et al., 1974). Thereafter, the endothelial mitotic index declines rapidly and the vascular system attains its final arrangement on day 18, just before hatching (Ausprunk et al. 1974). Consequently, cell suspensions implanted on the CAM of successively older embryos are not able to induce a vasoproliferative response in parallel with the reduced rates of growth of CAM’s endothelial cells. We have isolated endothelial cells from bone marrow of patients with multiple myeloma (Vacca et al., 2003). They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo in the CAM assay (Vacca et al., 2003). More recently, we have attempted a fine characterization of the angiogenic response induced by multiple myeloma endothelial cells by using the CAM assay and by RT-PCR (Mangieri et al., 2008). Results showed that in the CAM assay multiple myeloma endothelial cells induced an angiogenic response comparable to that of FGF-2, while RT-PCR demonstrated that the expression of endostatin mRNA

Fig. 3.2 Time course of the macroscopic appearance of a CAM implanted at day 8 with a sponge loaded with plasma cells of an active multiple myeloma patient (a). Note that, whereas on day 9 no vascular reaction is detectable (b), on day 12 numerous allantoic vessels develop radially toward the implant in a spoke-wheel pattern (c) (reproduced from Ribatti et al., 2003a)

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Fig. 3.3 CAM treated with a gelatine sponge soaked with multiple myeloma endothelial cellconditioned medium shows numerous blood vessels converging toward the implant (a). Following treatment of the CAM with bortezomib, fewer blood vessels surround the sponge (b) (reproduced from Roccaro et al., 2006a)

detected in multiple myeloma-treated CAM was significantly lower with respect to control CAM. These data suggest that angiogenic switch in multiple myeloma may involve loss of an endogenous angiogenesis inhibitor, such as endostatin. As concerns the study of antiangiogenic molecules active in multiple myeloma by using the CAM assay, we have shown that the proteasome inhibitor bortezomib significantly inhibited the basal angiogenesis and the angiogenic response induced by culture medium for multiple myeloma endothelial cells (Fig. 3.3) (Roccaro et al., 2006a). Also zoledronic acid, a bisphosphonate used for multiple myeloma bone disease and hypercalcemia, significantly inhibited the angiogenic response induced by culture medium for multiple myeloma endothelial cells (Scavelli et al., 2007). More recently, we have demonstrated that gelatin sponges soaked with culture medium from multiple myeloma plasma cells or multiple myeloma endothelial cells triggered the formation of neovessels compared with vehicle alone, and that dasatinib, a novel orally bioactive tyrosine kinase inhibitor currently used for treating patients with hematologic and solid tumors, significantly reduced both plasma celland endothelial cell-induced angiogenesis (Fig. 3.4) (Coluccia et al., 2008). In another recent study, we have shown that human recombinant IL-12 was able to significantly inhibit the angiogenic response induced in the CAM assay by culture medium obtained from human multiple myeloma cell lines (Airoldi et al., 2008).

Fig. 3.4 CAMs treated with gelatin sponges soaked with multiple myeloma plasma cells (a) and endothelial cells (c) conditioned media show numerous blood vessels converging toward the implants. Following treatment of the CAMs with dasatinib, fewer blood vessels surround the sponges (b, d) (reproduced from Coluccia et al., 2008)

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3.3 Angiogenesis and Antiangiogenesis in Human Neuroblastoma 3.3.1 Biological and Clinical Studies Several studies implicate angiogenesis in the regulation of neuroblastoma growth and inhibition of angiogenesis is a promising approach in the treatment of neuroblastoma because of the high degree of vascularity of these tumors (Ribatti and Ponzoni, 2008). Kleinman et al. (1994) showed that human neuroblastoma cells induce angiogenesis in nude mouse during tumorigenesis. Meitar et al. (1996) evaluated the vascularity of primary untreated neuroblastoma from 50 patients. They found that the vascularity of neuroblastoma from patients with widely metastatic disease is significantly higher than in tumors from patients with local or regional disease. Canete et al. (2000) in a retrospective study showed that tumor vascularity was not predictive of survival of neuroblastoma patients and that neither disseminated nor local relapses were influenced by the angiogenic characteristics of the tumors. Eggert et al. (2000) performed a systematic analysis of expression of angiogenic factors in 22 neuroblastoma cell lines and in 37 tumor samples. They found that high expression levels of seven angiogenic factors correlated strongly with the advanced stage of neuroblastoma and this suggests that several angiogenic peptides set in concert in the regulation of neovascularization. Ara et al. (1998) found that increased expression of MMP-2, but not of MMP-9, in stromal tissues of neuroblastoma had significant association with advanced clinical stages. Sakakibara et al. (1999) have demonstrated that the higher gelatinases activation ratio resulting from high expression of a novel membrane-type matrix metalloproteinase-1 (MT-MMP-1) on neuroblastoma specimens is associated significantly with advanced stage and unfavorable outcome. Ribatti et al. (2001e) showed that the extent of angiogenesis and the expression of MMP-2 and MMP-9 were upregulated in advanced stages of neuroblastoma. Amplification of MYC-N is a frequent event in advanced stages of human neuroblastoma. MYC-N may regulate the growth of neuroblastoma vessels, because its amplification or overexpression is associated with angiogenesis in experimental (Schweigerer et al., 1990) and clinical settings (Meitar et al., 1996). MYCN amplification correlates with poor prognosis and enhanced vascularization of human neuroblastoma, suggesting that the MYC-N oncogene could stimulate tumor angiogenesis and thereby allow neuroblastoma progression (Ribatti et al., 2002b). Erdreich-Epstein et al. (2000) demonstrated by immunohistochemical analysis that αν β3 integrin was expressed by 61% of microvessels in high-risk neuroblastoma, but only by 18% of microvessels in low-risk tumors. It has been reported a very low tumor vascularity in Schwannian stroma-rich/stroma-dominant neuroblastoma tumors and that Schwann cells produce angiogenesis inhibitors, such as TIMP-2 and pigment epithelium-derived factor (PEDF), that are capable of inducing endothelial cell apoptosis (Huang et al., 2000; Crawford et al., 2001). Chlenski et al. (2002) isolated an angiogenic inhibitor in Schwann cell-conditioned medium, identified as SPARC, the expression of which is inversely correlated with the degree

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of malignant progression in neuroblastoma tumors. Furthermore, SPARC inhibited angiogenesis in vivo and impaired neuroblastoma tumor growth. Takahashi et al. (2002) demonstrated that osteopontin-transfected murine neuroblastoma cells significantly increased neovascularization in mice. Enforced expression of osteopontin in neuroblastoma cells significantly stimulated endothelial cells migration and induced angiogenesis in mice, as evaluated by dorsal air sac assay. Cells from the poorly differentiated human neuroblastoma cell line SHSY5Y were used as tumor xenografts in nude rats (Wassberg et al., 1997). One group of animals was treated with TNP-470 and the other group served as controls. TNP470 treatment induced a reduction in tumor growth rate, in microvascular density, and in the fraction of viable tumor cells. Nagabuchi et al. (1997) showed that TNP 470 treatment improved animal survival and reduced tumor growth of primary and metastatic murine neuroblastoma. Katzenstein et al. (1999), to investigate whether TNP-470 could more effectively inhibit neuroblastoma growth in animals with a low tumor burden, treated 30 nude mice with minimal disease with this angiogenesis inhibitor. Treatment initiated before tumor was clinically apparent either 12 h or 1 week after the mice were s.c. inoculated with cells from the human neuroblastoma cell line NBL-W-N. These authors also treated animals 3–9 weeks after tumor cell inoculation, once small (<400 mm3 ) or large (>400 mm3 ) tumors developed. They demonstrated that TNP-470 treatment does effectively inhibit neuroblastoma growth when the agent is administered in the setting of animal disease. Furthermore, when TNP-470 is administered to animals with small tumors, the rate of growth is reduced, while not significantly altering the tumor growth rate when it is administered to animals with large tumors. Wassberg et al. (1999) demonstrated that treatment with TNP-470 reduced the tumor growth and increased the tumor cell apoptotic fraction. Moreover, TNP-470-treated tumors exhibited striking chromaffin differentiation of neuroblastoma cells. The authors suggested that by inhibiting angiogenesis, TNP-470 induced metabolic stress, resulting in chromaffin differentiation and apoptosis in neuroblastoma. Shusterman et al. (2000) showed that TNP-470 significantly inhibited tumorigenicity when administered shortly after neuroblastoma xenograft inoculation and when administered following cyclophosphamide. Kaicker et al. (2003) investigated the antiangiogenic and anti-tumor properties of thalidomide in a xenograft model of human neuroblastoma. Intraperitoneal thalidomide or vehicle was administered beginning 1 week after implantation, an animal killed at 6 weeks. Thalidomide treatment did not significantly alter the tumor growth as compared with controls. However, thalidomide suppressed angiogenesis, as demonstrated by both fluorescein angiography and immunohistochemical staining, and induces apoptosis of endothelial cells in neuroblastoma xenografts. Kim, Moore et al. (2001) demonstrated in a murine model of human neuroblastoma that monoclonal antibody against VEGF partially suppresses tumor growth. In a further study, Kim et al. (2002a) have shown that topotecan either with or without anti-VEGF antibody significantly suppresses neuroblastoma xenograft growth in comparison with controls or anti-VEGF antibody alone. Combining topotecan with

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anti-VEGF antibody significantly inhibited rebound tumor growth in comparison with anti-VEGF antibody alone. Moreover, high-affinity blockade of VEGF, using the VEGF-TRAP, a composite decoy receptor based on VEGFR-1 and VEGFR-2 fused to an Fc segment of IgG1, abolished tumor vasculature in a xenograft model of neuroblastoma (Kim et al., 2002b). Klement et al. (2000) subjected two neuroblastoma cell lines to either continuous treatment with low dose of vinblastine, the monoclonal anti-VEGFR-2 called DC101, or both agents together. Both DC101 and low-dose vinblastine treatment individually resulted in significant, but ultimately transient, xenograft regression and diminished tumor vascularity. Remarkably, the combination therapy resulted in a full and sustained regression of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during the course of treatment which lasted more than 6 months. Davidoff et al. (2001) developed a gene therapy approach in which the genes for endostatin and the marker protein and potent immunogen green fluorescent protein (GFP) were delivered to murine neuroblastoma cells prior to inoculation of the tumor cells into syngeneic immunocompetent mice. Although the effect of either angiogenesis inhibition or immunomodulation alone resulted in only a modest delay in tumor growth, when these approaches were used in combination, prevention of the formation of appreciable tumors was effected in 63% mice. Jounneau et al. (2001) evaluated the efficacy of endostatin in a human neuroblastoma xenograft model in nude mice. Tumor growth was only slowed down in endostatin-treated mice when compared to control mice, and no statistically significant difference in serum levels of endostatin was observed between endostatin-treated and control groups. Streck et al. (2004) evaluated the influence of a preexisting primary neuroblastoma xenografts on the growth of a new second subcutaneous tumor, hypothesizing that an existing primary tumor could inhibit the growth of a secondary tumor, in part mediated by tumor release of endostatin. Decreased angiogenesis and increased apoptosis were seen in the secondary tumors of mice with preexisting tumors. Similarly, the weight of liver metastases was significantly less in mice in which the primary tumor was left in place as compared with those in which the primary tumor had been excised. Systemic endostatin levels in this model paralleled the status of the primary tumor; levels decreased with primary tumor excision but increased when the primary tumor was retained and allowed to grow. Although no difference in microvessel density was seen between groups in the liver metastasis, more tumor cell apoptosis was seen in liver metastasis when the primary tumor was retained. Pastorino et al. (2003) have described a way of achieving an anti-neuroblastoma response with an NGR peptide-targeted formulation of liposomal doxorubicin. This combination was active against both established primary tumors and earlyphase metastases by causing the selective apoptosis of tumor endothelial cells and destruction of the tumor vasculature. This strategy markedly enhanced the therapeutic index of doxorubicin and enabled metronomic administration of therapeutic doses. A dual mechanism of action was proposed: indirect tumor cell kill via the destruction of tumor endothelium by NGR-targeted liposomes and direct tumor cell kill via localization of liposomal doxorubicin to the tumor interstitial space.

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This combined strategy has the potential to overcome some major limitations of conventional chemotherapy. Pastorino et al. (2003, 2006) have shown neuroblastoma tumor regression, pronounced destruction of tumor vasculature, and increased lifespan in orthotopic neuroblastoma-bearing mice treated with doxorubicin-loaded liposomes and coupled at the external surface with an NGR-containing peptide, able to specifically recognize the angiogenic endothelial cell marker aminopeptidase N. More recently, Pastorino et al. (2008) validated the potential of this “vasculartargeting/vascular-disrupting agents” strategy, by evaluating NGR-targeted liposomal doxorubicin (TVT-DOX) in several murine xenografts of doxorubicin-resistant human cancer, including lung, ovarian, and, again, neuroblastoma.

3.3.2 Use of Chorioallantoic Membrane In a first paper published in 1998, we investigated two human neuroblastoma cell lines, LAN-5 and GI-LI-N, for their capacity to induce angiogenesis in the CAM assay (Ribatti et al., 1998b) and demonstrate that conditioned medium from both cell lines, LAN-5 cells more than GI-LI-N ones, induced angiogenesis (Fig. 3.5). The role that the oncogene MYCN plays in the regulation of angiogenesis in neuroblastoma remains controversial. With the aim to better elucidate this matter, we tested fresh biopsy samples from patients with MYCN-amplified and MYCN-nonamplified tumors for their angiogenic capacity by using the CAM assay (Fig. 3.6) (Ribatti et al., 2002b). Moreover, conditioned medium obtained from five different human neuroblastoma cell lines MYCN-amplified and MYCNnonamplified and bioptic fragments obtained from xenografts derived from four neuroblastoma cell lines injected in nude mice were assayed for their angiogenic potential (Fig. 3.6). Results clearly demonstrated that MYCN amplification parallels angiogenesis in neuroblastoma. When fresh biopsy samples from patients, conditioned medium derived from neuroblastoma cells lines, and bioptic fragments

Fig. 3.5 Histological sections of gelatin sponges soaked with a suspension of LAN-5 human neuroblastoma cells. Indirect immunoperoxidase using a monoclonal antibody against neuroblastoma cells and aminoethylcarbazole as chromogen. Note several neuroblastoma-positive cells in perivascular (a) and intravascular (b) positions (reproduced from Ribatti et al., 1998b)

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Fig. 3.6 Macroscopic pictures of two neuroblastoma biopsy fragments obtained from MYCNamplified (a) and MYCN-nonamplified (b) patients grafted on the CAM. The vessels converging radially in a spoked-wheel pattern are more numerous in MYCN-amplified specimen. Similar features are recognizable when CAMs are incubated with gelatin sponges soaked with the conditioned medium of an MYCN-amplified cell line (HTLA-230) (c) compared to a nonamplified cell line (ACN) (d). Fragments obtained from two xenografts derived from mice injected intravenously with HTLA-230 (e) or SH-SY5Y MYCN-nonamplified cells (f) grafted on the CAM induce a similar angiogenic response (reproduced from Ribatti et al., 2002b)

derived from xenografts of the same cell lines injected in nude mice were tested, the response was univocal: the angiogenic response was significantly higher in the MYCN-amplified specimens as compared to MYCN-nonamplified ones (Ribatti et al., 2002b). In 2001 we studied the effects of the synthetic retinoid fenretinide (HPR) in vivo by using the CAM assay (Ribatti et al., 2001a). Results showed that HPR inhibited VEGF- and FGF-2-induced angiogenesis in the CAM assay. A significant antiangiogenic potential of HPR has been observed also in neuroblastoma biopsies-induced angiogenesis in vivo in the CAM assay. Moreover, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGFR-2 and FGF2R-2 was reduced after implantation of HPR-loaded sponges, as compared to controls. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and

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an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors. We have investigated the antiangiogenic activity of interferon gamma (IFN-γ) by using an experimental model in which IFN-γ gene transfer clampens the tumorigenic and angiogenic activities of ACN neuroblastoma cell line in immunodeficient mice (Ribatti et al., 2006b). We demonstrated that ACN/IFN-γ xenografts had less in vivo angiogenic potential than the vector-transfected ACN/neo, when grafted onto the CAM (Fig. 3.7). In another study we evaluated the synergistic antiangiogenic effect of low dose of vinblastine and rapamycin in neuroblastoma (Marimpietri et al., 2005, 2007). The angiogenic responses induced by neuroblastoma cell-derived-conditioned medium, neuroblastoma tumor xenografts and human neuroblastoma biopsy specimens were inhibited in the CAM assay by each drug and more significantly by their combination. The observation that these well-known drugs display synergistic effects as antiangiogenics when administered frequently at very low dose may be of significance in the designing of new ways of treating neuroblastoma. Bortezomib is a selective and reversible inhibitor of the 26S proteasome that shows potent anti-tumor activity in vitro and in vivo against several human cancers of adulthood. No data are available on bortezomib activity against human

Fig. 3.7 CAM incubated with a bioptic specimen of ACN/IFN-γ tumor xenograft shows few vessels around the graft (a), whereas CAM incubated with a bioptic specimen of ACN/neo tumor xenograft shows numerous vessels around the graft (c). Immunohistochemical analysis of the grafts confirms this evidence, showing few (b) and, respectively, numerous (d) CD31-positive blood vessels (reproduced from Ribatti et al., 2006b)

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Fig. 3.8 CAM treated with a gelatin sponge soaked with HTLA-230 neuroblastoma cellconditioned medium shows macroscopically numerous blood vessels converging toward the implant (a) and microscopically numerous blood vessels are recognizable among the sponge trabeculae (b). Following treatment of the CAM with bortezomib, fewer blood vessels surround the sponge (c) and are recognizable among the sponge trabeculae (d) of the specimens treated with HTLA-230 neuroblastoma cell-conditioned medium. Similar features are observed when CAMs are treated with a neuroblastoma bioptic specimen (e) or with a tumor xenograft derived from mice injected intravenously with HTLA-230 cells (f). Following treatment with bortezomib, fewer vessels invade the bioptic specimen (g) or the xenograft (h) (reproduced from Brignole et al., 2006)

neuroblastoma and we demonstrated that bortezomib inhibited angiogenesis in CAM stimulated by conditioned medium from neuroblastoma cell lines, by neuroblastoma xenografts, and by primary neuroblastoma bioptic specimens (Fig. 3.8) (Brignole et al., 2006). More recently, we have further investigated the anti-tumor activity of bortezomib in combination with fenretinide against neuroblastoma cells (Pagnan et al., 2009). To this purpose, CAM was implanted with tumor xenografts derived from mice orthotopically injected with GI-LI-N neuroblastoma cells. After 96 h from the start of tumor specimen treatments, macroscopic observations unveiled fewer allantoic vessels that converged toward the implant in CAM treated with combined bortezomib plus fenretinide than in CAM treated with saline solution and in CAM treated with either drug alone (Fig. 3.9) (Pagnan et al., 2009). The therapeutic efficacy of cancer active targeting using doxorubicin (DOX)loaded immunoliposomes was evaluated with the CAM model (Pastorino et al., 2006). The DOX-loaded liposomes were coupled either to monoclonal antibodies targeting tumor cells (anti-GD2) or to NGR peptides that target tumor vessels. The antiangiogenic effects of these formulations were tested on xenografts derived from neuroblastoma cell lines grown on the CAM surface. When anti-GD2 or NGR liposomes were administered separately, 50–60% of vessel growth inhibition was achieved, whereas administering a combination of both types of liposomes increased vessel growth inhibition to 90%. We have further investigated in vivo evaluation of good manufacturing practice grade targeted liposomal doxorubicin

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Fig. 3.9 Effects of bortezomib (BTZ) and fenretinide (HPR) on angiogenesis in the CAM assay. CAMs were implanted with tumor xenografts derived from mice injected orthotopically with GILI-N human neuroblastoma cells (a). After treatment with BTZ and HPR, administered alone (b, c) or in association (d), fewer blood vessels invade the xenografts than those observed in the xenografts treated with saline solution (reproduced from Pagnan et al., 2009)

(TVT-DOX), bound to a CD13 isoform (aminopeptidase N) expressed on the vasculature of solid tumors, in human tumor xenografts of neuroblastoma (Pastorino et al., 2008). Neuroblastoma tumor xenografts were grafted onto CAM. CAMs were then incubated with vehicle alone or with DOX in either Caelyx (untargeted liposomes) of TVT-DOX. CAM incubated with Caelyx showed a decrease in the number of allontoic vessels radiating in a “spoked-wheel” pattern toward the xenografts when compared with those incubated with vehicle alone. However, incubation of the CAM with TVT-DOX significantly reduced the number of radiating vessels that invaded the implant compared with either specimens alone or CAM incubated with Caelyx as shown by morphometric assessment of microvessel area (Pastorino et al., 2008). We have investigated the effects of a topoisomerase I inhibitor (topotecan) on the angiogenic activity of hypoxic LAN-5 neuroblastoma cells in the CAM assay and demonstrated that topotecan was able to significantly inhibit this angiogenic activity (Puppo et al., 2008). More recently, we have purposed an alternative method to study the angiogenic and invasive potential of neuroblastoma cell suspensions implanted on the CAM surface (Mangieri et al., 2009). Neuroblastoma cells were seeded in Matrigel and thereafter the suspension was pipetted onto the CAM surface at day 8 of incubation inside a silicon ring previously loaded onto the CAM surface. Four days after implantation, the silicon ring was removed and the angiogenic and invasive responses were studied morphologically at macroscopic and microscopic levels and by RT-PCR by using human and chicken primers for several angiogenic cytokines, namely, VEGF-A, FGF-2, angiopoietin-1 (Ang-1), hypoxia inducible factor 2α (HIF-2α), and for an endogenous angiostatic molecule, namely, endostatin. Results showed that (i) neuroblastoma cells induced an angiogenic response in the CAM assay comparable to that induced by FGF-2; (ii) neuroblastoma cells are packed inside Matrigel or are recognizable in the CAM mesenchyme; (iii) angiogenic activity of neuroblastoma cells is associated with a high expression of the transcripts of human VEGF-A, FGF-2, Ang-1, and HIF-2α and a low expression in the transcript of a human endostatin while in the control specimens there is no expression of both angiogenic and angiostatic molecules; (iv) the expression of the transcripts of the

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same chicken angiogenesis stimulators and inhibitor is unmodified in treated and control specimens. Overall, these data indicate that neuroblastoma cell growth on the chick CAM expresses characteristics of the human disease. This experimental model could be employed for further research on human tumor progression and antiangiogenic molecules screening.

Chapter 4

Chorioallantoic Membrane in the Study of Tumor Metastasis

4.1 Use of Chorioallantoic Membrane in the Study of Tumor Metastasis The metastatic cascade involves a series of cellular events that are linked both temporally and spatially. Only a small percentage of malignant cells possess the required characteristics of the establishment of metastases (Liotta et al., 1991). Metastasis begins by an initial change in cell–cell adhesive interactions which allows dissociation of the tumor cells from the primary tumor and is followed by a local invasion and migration into the interstitial matrix. During hematogenous metastasis, the tumor cells undergo a process called intravasation by which they gain access to the host circulation. Eventually, the tumor cells arrest in the microvasculature and undergo extravasation and leave the circulation, which is followed by local invasion and the establishment of a secondary metastatic foci. Finally, these small foci may induce an angiogenic response. The chick embryo provides a model to study either spontaneous or experimental metastasis in a considerably shorter time, 7–8 days as compared to 4–10 weeks for most typical murine models. Furthermore, because the chick embryo is a closed system, the half-life of many experimental antagonists including small peptides tends to be much longer in the chick as compared to other animal models, allowing experimental evaluation of potential antimetastatic compounds that are limited in supply. Dagg et al. (1956) described the pattern of metastasis on CAM and chick embryo of three different human tumors (squamous cell carcinoma, sarcoma, and embryonal rhabdomyosarcoma) by using three different ways of submission (CAM surface, yolk sac inoculation, and subcutaneous implantation). They observed that the squamous cell carcinoma implanted on the CAM surface induced the formation of numerous small and large nodules at the site of implantation and had the most metastatic behavior into the chick embryo, forming nodules beneath the skin of the chick and metastatic foci into the eye, brain, liver, and myocardium, and demonstrated the intravascular pathway of metastasis and the spontaneous regression of tumors in few cases (Dagg et al., 1956).

D. Ribatti, The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis, DOI 10.1007/978-90-481-3845-6_4,  C Springer Science+Business Media B.V. 2010

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Locker et al. (1969) observed that hematogenous metastasis of Yoshida ascites hepatoma in the chick embryo liver shared ultrastructural changes, including aggregation of ribosomal particles and enlargement of mitochondria, accumulation of lipid vacuoles, and synthesis of glycogen. These changes probably reflected a more active metabolic state induced in the tumor cells by the chick environment. Many innovative studies of Ossowski (Ossowski and Reich, 1980; Yu et al., 1997; Kim et al., 1998) and Chambers (Chambers et al., 1982; Mac Donald et al., 1992; Koop et al., 1996) have consolidated the CAM as a useful model to study metastasis. Several methods for semiquantitative analysis of metastasis in the chick embryo have been developed including morphometric assessment of individual metastasized cells (Ossowski and Reich, 1980), detection of microscopic tumor colonies (Mac Donald et al., 1992), detection of human urokinase plasminogen activator within secondary organs of the embryo (Ossowski and Reich, 1980), the use of green fluorescent protein and in vivo videomicroscopy (Kops et al., 1996; Khoka et al., 1992; Brooks et al., 1993). Because the human genome is uniquely enriched in Alu sequences, PCR-mediated amplification of human-specific Alu sequences was used for semiquantitative detection of intravasated tumor cells in the CAM and within chicken tissues (Kim et al., 1998), followed by sensitive real-time Alu PCR assay (Zijlstra et al., 2002; Mira et al., 2002; Van der Horst et al., 2004).

4.2 Spontaneous Metastasis Models Spontaneous metastasis model, based on grafting of human tumor cells on the CAM, has provided valuable information regarding key processes involved in human tumor progression, such as tumorigenesis, tumor-induced angiogenesis, and tumor cell intravasation. Histological observation of the CAM allows to study the migratory activity of tumor cells from the chorionic epithelium surface through the intermediate mesenchyme. For the first time, Ossowski found that the infiltration of the CAM mesenchyme by individual tumor cells was blocked when uPA tumor activity or production was inhibited and suggested that the increased invasive potential of tumor cells was correlated with cell surface-associated proteolytic activity (Ossowski 1988a, b). Specific serine proteases and MMPs, including tumor-derived MMP-9, were associated with the ability of the human tumor cells to intravasate following grafting onto the CAM (Kim et al., 1998). In addition, membrane type-1 MMP (MT1-MMP or MMP-14) has been reported to be a critical MMP for cell invasion of the CAM (Sabeh et al., 2004). Deryugina et al. (2005) demonstrated the existence of two variants of fibrosarcoma cells with different intravasation capacities in the CAM assay. They demonstrated that specific inhibition in the variant with higher invasive capacity of MMP-9 expression and activity with MMP-9 small interfering RNA and anti-MMP-9 monoclonal antibody resulted in an unexpected substantial increase of intravasation, indicating that targeting of certain MMPs may lead to enhanced malignancy (Deryugina et al., 2005). The requirement of MMP for blood vessel intravasation by tumor cells has also been studied using the migratory

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capability of tumor cells placed in the upper CAM via the embryonic vasculature to the lower CAM (Zijlstra et al., 2002).

4.3 Experimental Metastasis Studies CAM has been successfully used in experimental metastasis studies, providing comprehensive information about other critical steps of metastatic process including (i) survival of cancer cells in the systemic circulation and transport to target organs; (ii) arrest of cancer cells in the microcirculation; (iii) migration of cancer cells through the vessel wall into the interstitial space (extavasation); and (iv) proliferation of cancer cells in the target organs (colonization) (Scher et al., 1976; Armstrong et al., 1982; Chambers et al., 1982; Koop et al., 1996; Shioda et al., 1997; Kobayashi et al., 1998; Quigley and Armstrong, 1998; Bobek et al., 2004). Tumor cell suspensions or bioptic specimens, no greater than 0.2–0.5 mm, can be implanted on intact or scarified CAM surface. Ossowski introduced a new assay in which tumor cell suspensions, or fragments, were implanted on the CAM surface (Kim et al., 1998). The CAM surface was previously wounded and parts of the chorionic epithelium were damaged, exposing loose vascularized stroma. Tumor cells entered this tissue regardless of their invasive potential, but only cells capable of penetrating the blood vessel wall subsequently circulated and arrested in vessels of embryonic tissues. A major obstacle to investigate the in vivo gene expression of cancer cells during the early steps of metastasis has been that, when standard mouse models of experimental metastasis are used, most of the intravenously injected cancer cells perish rapidly in the microcirculation before extravasation (Fidler, 1975). On the contrary, most cancer cells arrested in the CAM microcirculation survive without significant cell damage, and a large number of them eventually complete extravasation. Chambers et al. (1982) compared the patterns of experimental metastasis in chick and mouse after intravenous injection of B16-F1 and B16-F10 murine melanoma cells and observed striking differences between the two models: (i) the number of tumors for a given number of cells injected is much higher in the chick than in the mouse; (ii) B16-F1 tumors grew in most embyronic chick organs while their growth in the mouse was restricted primarily to the lungs; and (iii) B16-F1 and B16-F10 formed a comparable number of tumors in embryonic organs after intravenous injection in the chick, whereas B16-F10 formed more tumors in the lung than B16-F1 after intravenous injections into mice (Chambers et al., 1992). Koop et al. (1994, 1995), by using B16-F10 melanoma cells injected intravenously into the CAM, demonstrated by intravital microscopy that more than 80% of cells arrested in the CAM microcirculation survived and extravasated within 24 h after injection. Shioda et al. (1997) observed that 10 min after the intravenous injection of fluorescent-labeled B16-F10 cells, the cells arrested into the capillary bed. Six hours later, tumor cells changed their shape and were spread in close contact with the capillary wall. They also found that mRNA levels of several metastasis-related genes in cancer cells increased temporarily during the early phases of this process.

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Moreover, the levels of VEGF mRNA increased 6 h after injection by favoring both angiogenesis and an increase in vascular permeability. Taizi et al. (2006) demonstrated that following injection into CAM blood vessels, human leukemia cell lines also engraft in the chick brain, similar to central nervous system involvement in human disease. Moreover, leukemic cells were detected in the embryos’ hematopoietic organs by PCR amplification of human-specific DNA sequences. Within a few days after inoculation of highly aggressive human tumor cells in the CAM, visible tumors are formed and tumor cells can be identified in distant portions of the CAM (lower CAM), as well as in the internal organs of the embryo such as lungs, liver, sternum, and brain (Kim et al., 1998; Zijlstra et al., 2002; Bobek et al., 2004; Gordon and Quigley, 1986). Lugassy and Barnhill (2007) have demonstrated by using the CAM assay that, while melanoma tumor cells close to the tumor inoculation were completely cuffing some vessels, further from the tumor, melanoma cells were observed in small groups of cells along the outside of the vessel and at distance from the tumor, isolated fluorescent tumor cells, as well as small tumor masses, were observed along the vessels. They named this process as angiotropism of melanoma cells, i.e., the capability of some melanoma cells to migrate along the outside of vessels in a pericyte-like location. Hagedorn et al. (2005) established a glioblastoma multiforme progression model by using the CAM assay and showed that glioma cells invasion into the CAM was clearly visible on immunostained sections and that blood vessels served as guiding structures for migration of tumor cells. Moreover, they demonstrated an upregulation of several cancer progression genes after 48 h from the implant. The majority of animal model systems to study cancer progression involve the use of immunocompromised mice and rats for hetero- or orthotopic transplantations of human tumor cells. Being naturally immunodeficient, the chick embryo accepts transplantation from various tissues and species without specific or non-specific immune responses. Other advantages are the following: (i) CAM is particularly rich in blood vessels and capillaries, allowing rapid vascularization, survival, and development of tumor cells or tissues placed on its surface; (ii) CAM is connected to the embryo through a continuous circulatory system that is readily accessible from experimental manipulations and observations; (iii) CAM allows to observe by in vivo microscopy the real-time changes in morphology of cancer cells arrested in its microcirculation; (iv) in contrast to standard mouse models, most cancer cells arrested in the CAM microcirculation survive without significant cell damage and a large number of them eventually complete extravasation; (v) the simplicity and low cost of the CAM assay strengthen the use of this experimental model. Metastatic cancer cells resemble stem cells in their ability to self-renew and to derive a diverse progeny. Embryonic microenvironments have been shown to inhibit the tumorigenicity of a variety of cancer cell lines. In this context, the embryonic microenvironment of the chick CAM could give an opportunity to study its influence on such metastatic properties of cancer cells.

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Experimental Metastasis Studies

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There are some limitations in the study of tumor metastasis by using the CAM assay. Non-specific inflammatory reactions represent the main limitation of the chick CAM assay. However, non-specific inflammatory reactions are much less frequent when implants are made relatively early in CAM development, when the host’s immune system is relatively immature. Because the duration of the assay is limited to a 7- to 9-day window available before the chick hatches, most tumor cells cannot produce macroscopically visible colonies in secondary organs before the termination of the assay. The reduced number of specific antibodies available with specificity for chicken tissues limits the fine characterization of the different steps of metastatic process by using this experimental model.

Chapter 5

Other Applications of Chorioallantoic Membrane

5.1 In Ovo and Ex Ovo Methods The embryo and its extraembryonic membranes are transferred to a Petri dish on day 3 or 4 of incubation and CAM develops at the top as a flat membrane and reaches the edge of the dish to provide a two-dimensional monolayer onto which multiple grafts can be placed (Auerbach et al., 1974; Jakobson et al., 1989). Because the entire membrane can be seen, rather than just a small portion through the shell window, multiple grafts can be placed on each CAM and photographs can be taken to document vascular changes over time. Subsequently, several modifications of this method have been described. Other types of containers used include plastic slings, plastic weigh boats, foam cups, and plastic dishes. Dugan et al. (1981) used an inert plastic container equipped with a “parafilm” ring (4–5 cm inside depth) to provide support for the embryo and its membranes. Advantages include somewhat longer viability and lower costs, though these are offset by the difficult of monitoring angiogenesis during incubation and by the fact that one cannot obtain two-dimensional photographs suitable for image analysis. Another method has been proposed by Nguyen et al. (1994): a collagen gel is conjugated with the testing substance or tumor cells and placed between two parallel pieces of meshes (bottom layer 4 × 4 mm, upper layer 2 × 2 mm). The resulting “sandwich” is then placed upon the CAM on day 8 of incubation. New blood vessels are induced to grow upright into collagen gels from the underlying CAM, and therefore can be clearly discriminated from the background vascular network. Seandel et al. (2001) proposed a modification of this assay, involving the use of threedimensional grafts, each of which consists of two native type I fibrillar collagens. The collagen mixture is distributed over the grid meshes and allowed to polymerize in a 37 ◦ C thermostat. Polymerization occurs within minutes, which ensures that the incorporated cells are distributed even throughout the three-dimensional collagen. Additional 30–45 min of incubation allows the collagen to completely solidify, after which time the collagen onplants are placed with forceps on the CAM of 10day-old shell-less embryos. The graft can be treated directly with chemicals, or the embryos can be systemically treated by intravenous injection through the allantoic vein. Within 2–4 days of incubation, the newly formed vessels, which are visualized D. Ribatti, The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis, DOI 10.1007/978-90-481-3845-6_5,  C Springer Science+Business Media B.V. 2010

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with a stereomicroscope above the lower nylon mesh, are regarded as angiogenic since only newly formed vessels infiltrate the collagen graft from the preexisting CAM vasculature, which is located below the nylon mesh of the graft. Survival rate of eggs cultured ex ovo is the major success-limiting step in this technique. In the original description of embryos cultured in Petri dishes there was a 50% loss in the first 3 days after cracking, with 80% of those which survive to day 7 continuing at least day 16 (Auerbach et al., 1974). Subsequently, improved survival rates were reported by the same laboratory, with 87% of the embryos surviving removing the shell and 68% alive on day 4 (Crum et al., 1985). Ex ovo method may be preferred to the in vivo method because (i) it allows the quantification of the response over a wider area of the CAM; (ii) large number of samples can be tested at any one time; and (iii) the time required for a response to occur is shorter (2–3 days).

5.2 Use of Chorioallantoic Membrane in the Study of Tumor Lymphangiogenesis Despite lymphatic vessels importance in tumors spread and metastasis, the growth pattern of lymphatics on normal CAM was less investigated due to the paucity of chick CAM-specific primary antibodies against lymphatic epitopes, except for QH1 antibody. Using QH1, VEGFR-3, and Prox-1 as markers, it was shown that tumor cells associated with lymphangiogenesis would stimulate proliferation of lymphatic vessels in the chick CAM (Papoutsi et al., 2000b, 2001). Papoutsi et al. (2001) implanted VEGF-C-expressing human A375 melanoma cells on the CAM, which formed solid tumors. They demonstrated that the tumors induced numerous lymphatics at the invasive front, positive to VEGFR-3 and Prox-1. Moreover, a great number of melanoma cells invaded the lymphatics and lymphangiogenesis was inhibited to some extent when melanoma cells were transfected with cDNA encoding soluble VEGFR-3. In another work, Papoutsi et al. (2000b) implanted two types of rat tumor cells, 10AS pancreatic carcinoma and C6 glioma cells, on the CAM surface. The tumor cells rapidly formed solid tumors which invaded the CAM and were vascularized by CAM vessels. Lymphatics, positive to VEGFR-3, were absent from C6 gliomas, whereas 10AS cells, which expressed high levels of VEGF-C, induced ingrowth of lymphatics into the tumors, with Brd-U-labeling rates about 9% of lymphatic endothelial cells.

5.3 Use of Chorioallantoic Membrane in the Study of Angiogenesis Associated with Wound Healing and Inflammation The CAM is an easily accessible in vivo model to study wound repair. It reproduces all the critical events observed in human wound healing, including reepithelialization, angiogenesis, macrophage infiltration, and fibronectin deposition, resulting in scar formation (Ribatti et al., 1996b). Using the CAM model of wound

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Use of Chorioallantoic Membrane in the Study of Wound Healing

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Fig. 5.1 (a–c) Light micrographs of an untreated CAM, 4 days after wounding. Note in (a), the hyerplasia of the chorionic epithelium; in (b), numerous vessels (arrowheads), fibroblasts, and mononuclear cells in the CAM mesenchyme; and in (c), naphthol-AS-D-chloroacetate esterasepositive cells (arrowheads) in perivascular position. (d, e) Light micrographs of a CAM treated with neutralizing anti-FGF-2 antibody, 6 days after wounding. Note in (d), two microvessels (arrowheads), scarce fibroblasts, and in (e), naphthol-AS-D-chloroacetate esterase-positive cells (arrowheads) inside a vessel. (f, g). Light micrographs of a CAM treated with human recombinant FGF-2, 3 days after wounding. Note in (f), a higher number of vessels (arrowheads), fibroblasts, and mononuclear cells and in (g) naphthol-AS-D-chloroacetate esterase-positive cells (arrowheads) in perivascular position (reproduced from Ribatti et al., 1999c)

healing, the role of specific growth factors can be investigated. For example, the inhibition of FGF-2 after CAM wounding by function blocking antibodies resulted in decreased wound healing by inhibiting microvessel and fibroblast density. Conversely, the application of FGF-2 to the wound greatly accelerated wound repair such that healing occurs 24 h earlier as compared to control wounds by stimulating angiogenesis, fibroblast proliferation, and macrophage infiltration (Fig. 5.1) (Ribatti Table 5.1 Stimulation of angiogenesis induced by human chronic inflammatory processes bioptic specimens tested in the CAM assay Tissue

Authors

Carotid atherosclerotic plaque Decidua basalis bioptic specimens of eclampsia Endometriosis Pyogenic granuloma Osteoarthritis cartilage Rheumatic arthritis tissue, sinovial and osteoarthritis cartilage Skin bioptic specimens of pyogenic granuloma Systemic sclerosis skin bioptic specimens Vitreous from patients with proliferative diabetic retinopathy

Bo et al. (1992) Selvaggi et al. (1995) Maas et al. (2001) Ribatti et al. (1996c) Smith et al. (2003) Ribatti et al. (2000b) Ribatti et al. (1996c) Ribatti et al. (1998c) Hill et al. (1983)

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et al., 1999c). Moreover, by supplementing wounded chick CAM with activated protein C, a serine protease which upregulates angiogenic growth factors, such as VEGF, IL-8, and monocyte chemotactic protein-1 (MCP-1) in vitro, an enhanced angiogenesis was observed (Jackson et al., 2005). The CAM can also be used to study the angiogenic response induced by bioptic specimens of chronic inflammatory processes (Table 5.1).

5.4 Use of Chorioallantoic Membrane in the Study of Angiogenic Properties of Biomaterials and of the Evaluation of Drug Delivery Systems CAM has been used as a screening assay for biological responses to biomaterials and as an alternative to traditional animal models and this assay will become an integral part of the testing process for developing potential biomaterials (Table 5.2) (Azzarello et al., 2007). Valdes et al. (2002) found that both acute and chronic inflammatory responses of the CAM to biomaterials are similar to those found in mammals. Acellular matrices, which are the noncellular part of a tissue, have been used in tissue regeneration studies. They can be transplanted without rejection and provide a matrix where cell growth, angiogenesis, and differentiation can occur. The angiogenic activity of brain, femoral, aortic, esophagus, and diaphragm acellular matrix has been investigated in the CAM assay (Figs. 5.2 and 5.3) (Ribatti et al. 2003b; Marzaro et al., 2006; Conconi et al., 2004a; 2005, 2009). Chick embryos can be used to evaluate the activity or toxicity of a drug on both the CAM and CAM-grafted tumors, as well as on the development of the body of the embryo (Vargas et al., 2007). Toxicity can be evaluated in terms of embryo death or adverse effects on the CAM, including inflammation and neovascularization.

Table 5.2 Stimulation of angiogenesis induced by biomaterials tested in the CAM assay Aortic acellular matrix Bioglass (derived glass ceramic scaffolds) Biomaterial for the controlled delivery of sphingosine 1-phosphate Brain scaffolds Degrapol scaffolds Diaphragmatic acellular matrix Femoral acellular matrix Porous biodegradable poly (DL-lactic) acid (PLA) scaffolds encapsulating VEGF

Conconi et al. (2004a) Vargas et al. (2009) Wacker et al. (2006) Ribatti et al. (2003b) Tan et al. (2007) Conconi et al. (2009) Conconi et al. (2005) Kanczler et al. (2007)

5.4

Use of Chorioallantoic Membrane in the Study of Angiogenic Properties

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Fig. 5.2 (a). Macroscopic picture of implant of an aortic acellular matrix surrounded by allantoic vessels developing radially toward it (I). The vasoproliferative response is comparable to that induced by a gelatin sponge (S) soaked with FGF-2 (b). Microscopically, acellular matrix (AM) is adherent to the chorion without invading the mesenchyme (c). At higher magnification, numerous newly formed blood vessels (arrows) are located at the interface between the AM and the CAM mesenchyme (d). At some points, blood vessels (arrows) are recognizable inside the acellular matrix (e) (reproduced from Conconi et al., 2004)

Fig. 5.3 At microscopic level, acellular brain scaffolds are adherent to the chorion without invading the mesenchyme (asterisk in a). At higher magnification, numerous newly formed blood vessels are recognizable radially arranged under the chorion (CH) and around large blood vessels of the CAM (b). At some points blood vessels (arrows) invade the chorion (CH) (c) or are recognizable upon it (d). A few naphthol-AS-D-chloroacetate esterase-positive mononuclear cells can be recognized in a perivascular position (e) (reproduced from Ribatti et al., 2003b)

Chapter 6

Different Morphological Techniques and Methods of Quantifying the Angiogenic Response Used in the Study of Vascularization in the Chorioallantoic Membrane

6.1 Different Morphological Techniques That Can be Used to Study Vascularization of the CAM and the Genes Involved Different morphological techniques can be used to study vascularization of CAM and the genes involved. The recent complete characterization of the chick embryo genome (www.nhgri.gov/11510730) will be helpful to synthesize a broad panel of antibodies with high specificity for chicken tissues, especially for blood and lymphatic endothelial cells and stroma components. This aspect could be useful to better characterize the interactions between implanted human and/or mouse tumors and chicken tissues. Comparison of the genomes of chickens, mice, and humans allows to get useful insights in the similarities and differences between these organisms (Burt, 2005). Comparative genomics revealed that the chick genome is three times smaller than the one of both human and mouse, but contains approximately the same number of genes. In the last years retroviral, lentiviral, and adenoviral vectors have been used to infect the CAM (as well as the whole-chick embryo), leading to the expression of the viral transgene. This allows the long-lasting presence of the gene product that is expressed directly by CAM cells. This makes feasible the study of the effects of intracellular or membrane-bound proteins as well as of dominant-negative gene products. This approach will shed new lights on the study of the metastatic process by using the CAM assay. When recombinant VEGF protein was applied to the CAM, protein was no longer detectable after 4 days. Using an adenovirus as expression vector, it allows a more extended presence of the gene product, which may be important for gene products which give a weaker response or which need to be present for an extended period to induce a biological response (Schughart and Accart, 2003). More sophisticated techniques have been designed recently to perform reliable quantitative evaluation of vascular density. These techniques include in ovo cell proliferation, layered expression scanning to visualize the protein of interest, and fluorescent confocal microscopy of new blood vessel formation in the CAM. Images of the vascular system of the CAM may be generated by injection of a contrast medium and digital subtraction angiography at days 8, 10, 12, and 14 of D. Ribatti, The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis, DOI 10.1007/978-90-481-3845-6_6,  C Springer Science+Business Media B.V. 2010

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incubation. The total vascular area of vessels per unit area is measured directly from the angiography images and the total vascular length is measured from skeletonized images (Siamblis et al., 1996; Nikiforidis et al., 1999). There are many attempts to evaluate the distribution of blood vessels according to a fractal pattern (Kirchner et al., 1996; Parson-Wingerter et al., 1998; Vico et al., 1998). This method of quantification involves “box counting” in which the image of the CAM is overlaid with series of grids of decreasing block size. For each grid the number of blocks which intersect with the structure is counted and from these data the fractal dimension is generated. If the chick embryo is injected with fluorescent-tagged lectin such as lens culinaris agglutinin (LCA), the intact vascular network of the CAM can be visualized by a top planar view of whole-mount preparations in a fluorescent microscope and highlighting the vasculature with Sambuco negro agglutinin, which specifically binds to chicken endothelium, results in a better discrimination of the ectoderm capillary plexus and blood vessels within CAM mesoderm (Deryugina and Quigley, 2008).

6.2 Methods of Quantifying the Angiogenic Response Several methods of quantifying the CAM angiogenic response have been developed. Quantification of angiogenesis was initially done by scoring the extent of vascularization on a graded scale of 0–4. Serial dilution assays were developed to score the number of positives at any particular dilution using four eggs per assay point. With dilution of the test sample and reduction in its concentration, the number of positives gradually decrease until an end point (0/4) is reached. Another method considers changes in the distribution and density of CAM vessels next to the implant which are evaluated in vivo by means of a stereomicroscope at regular intervals following the graft procedure. The score is 0 when no changes can be seen, +1 when few neovessels converge toward the implant, and +2 when a considerable change in the number and distribution of the converging neovessels is observed (Knighton et al., 1977). Angiogenesis was scored for each embryo in a double-blind procedure that analyzed the number and extent of branching of blood vessels within the area of each implant. The score ranges from +1 (low) to +4 (high) and the angiogenesis index was determined by subtracting a background score of 1 from all data (Friedlander et al., 1995). The density of branching blood vessels infiltrating under the implant was scored as follows: 0, negative; 0.5, change in vessel architecture but not directed to the point of sample application; 1, partial spoke wheel (one-third of the circumference exhibits directional angiogenesis); 2, spoke wheel; 3, strong and full spoke wheel (Chen et al., 2000). The number of vessel branch points contained in a circular region equal to the area of a filter disk was counted for each section. Percent inhibition data were expressed as the quotient of the experimental value minus the negative control value divided by the difference between the positive control and the negative control values (Powell et al., 2000).

6.2

Methods of Quantifying the Angiogenic Response

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The vasoproliferative response may be graded as a vascular index derived from photographic reconstructions. All converging neovessels contained inside a 1 mm diameter ring superimposed upon the CAM are counted: the ring is drawn around the implant in such a way that it will form an angle of less than 45◦ with respect to a straight line drawn from the implant’s center. Vessels branching dichotomically outside the ring are counted as 2, while those branching inside the ring are counted as 1 (Fig. 6.1) (Dusseau et al., 1986). Folkman and Cotran (1976) measured the degree of vasoproliferative response, as evaluated under the stereomicroscope, by an arbitrary 0–5 scale. Zero describes a condition of the vascular network that shows no change from the time of grafting; +1 marks a slight increase in the vessel density associated with occasional changes in the course of vessels converging toward the implant; +2, +3, +4, and +5 indicate a progressive increase in vessel density associated with more pronounced changes in their course, while a +5 score also highlights strong hyperemia. A coefficient describing the degree of angiogenesis can also be derived from the ratio of the calculated value to the highest attainable value. Therefore, the coefficient’s lowest value is 0 and the highest is 1 (Fig. 6.2). Strick et al. (1991) calculated the length of the vessels and express it in terms of index density, i.e., the vessel density relative to a fractional image area of the vasculature. Nguyen et al. (1994) expressed the vasoproliferative response after 72–216 h as a percentage of the squares in the upper mesh occupied by neovessels. The effect of the inhibitory substances (placed on the bottom mesh) is quantified by calculating the inhibition of the vasoproliferative response induced by an angiogenic factor. Use of a numerical grading scale allows the calculation of a coefficient of angiogenesis (Vu et al., 1985). Semiautomated image analysis techniques have also been developed (Voss et al., 1984; Jakob and Voss, 1984). Also fractal analysis has been used for evaluating the changes of CAM vasculature (Kirchner et al., 1996). The

Fig. 6.1 Evaluation of a proangiogenic response by macroscopic semiquantitative scoring of vessel branching. The drawing illustrates representative examples of different branching responses, with scores ranging from 0 to 2 (reproduced from Ribatti et al., 2006a)

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Fig. 6.2 Evaluation of proangiogenic response by macroscopic semiquantitative scoring. Drawings illustrate the representative examples of different responses (score ranging from 0 to 5) (reproduced from Ribatti et al., 2006a)

alterations in the branching pattern measured by fractal dimension and vessel density by grid intersection were evaluated to define the response to FGF-2 and to angiostatin (Parson-Wingerter et al., 1998). Quantitative evaluation of vessel density can be obtained by applying morphometric and planimetric methods to histologic observations of CAM specimen fixed at regular intervals after implantation. The number of vessels is evaluated as the total number of vessels present in six randomly chosen microscopic fields. Vessel density is evaluated by a planimetric method (Elias and Hyde, 1983) which utilizes a square reticule placed in the eyepiece of a photomicroscope. Six randomly chosen fields per section are observed and the total number of intersection points occupied by transversally sectioned vessels is counted. Vessel density equals this total number, expressed as a percent value of all intersection points. Evaluation of the number and density of vessels should be made by two independent observers and processed statistically. The uptake of 3 H thymidine into the whole CAM was evaluated using both autoradiography and scintillation counting and the rate of uptake was shown to be directly proportional to the concentration applied to the surface (Thompson et al., 1985). Angiogenesis was related to the total hemoglobin content which was proportional to the rate of 3 H thymidine uptake.

Chapter 7

Advantages and Limitations of Chorioallantoic Membrane in Comparison with Other Classical In Vivo Angiogenesis Assays

7.1 Advantages and Limitations of Chorioallantoic Membrane Assay The main limitation of CAM assays is the non-specific inflammatory reactions that may develop as a result of grafting and induce a secondary vasoproliferative response, so quantification of the primary response is impeded (Jakob et al., 1978; Spanel-Burowksi et al., 1988) (Table 7.1). Inflammatory angiogenesis in which infiltrating macrophages or other leukocytes are the source of angiogenic factors cannot be distinguished from direct angiogenic activity of the test material without a detailed histological study of multiple positive and negative controls. Investigation of histological CAM sections would help to detect the presence of a perivascular inflammatory infiltrate, together with a hyperplastic reaction, if any, of the chorionic epithelium. However, a non-specific inflammatory response is much less likely when the test material is grafted as soon as the CAM begins to develop while the host’s immune system is relatively immature (Leene et al., 1973). In an extensive series of experiments by Jakob et al. (1978), a variety of carrier vehicle alone (Millipore filters, fiber glass disks, disks of filter paper, agarose, and polyacrylamide gels), as well as natural egg components (egg shell membrane, coagulated albumin, and coagulated yolk), produced a number of inflammatory reactions. Cortisone or angiostatic steroids have been included in the experimental protocol in order to prevent nonspecific inflammatory reactions. However, a non-specific inflammatory response is much less likely when the test material is grafted as soon as the CAM begins to develop while the host’s immune system is relatively immature (Leene et al., 1973). There are two more drawbacks to the CAM assay: first, the test material is placed on existing vessels, and newly formed blood vessels grow within the CAM mesenchyme. Real neovascularization can hardly be distinguished from a falsely increased vascular density due to rearrangement of existing vessels that follow contraction of the membrane (Knighton et al., 1991). On the contrary, in the rabbit of murine cornea assay, the presence of blood vessels that penetrate from the limbus into the avascular stroma of the cornea can be unambiguously interpreted as an index of angiogenic response. D. Ribatti, The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis, DOI 10.1007/978-90-481-3845-6_7,  C Springer Science+Business Media B.V. 2010

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76 Table 7.1 Advantages and disadvantages of chorioallantoic membrane assay

7

Advantages and Limitations of Chorioallantoic Membrane Advantages High embryo survival rate Easy methodology Sterility is not required Low cost Reproducibility Reliability Disadvantages Difficult monitoring Non-specific inflammatory reactions Preexisting vessels

Second, timing of the CAM angiogenic response is essential. Many studies determine angiogenesis after 24 h, when there is no angiogenesis, but only vasodilation. Measurements of vessel density are really measurements of visible vessel density, and vasodilation and neovascularization are not readily distinguishable. This drawback can be overcome by using sequential photography to document new vessel formation. Saline solutions should be avoided; in that hyperosmotic effect of crystal salts may damage the chorion epithelium and induce fibroblast proliferation (Wilting et al., 1991). This implies that the substance should be used at concentrations of picograms to milligrams: higher concentrations would indeed cause the hyperosmotic effect (Wilting et al., 1992). The CAM is also extremely sensitive to modification by environmental factors, such as changes in oxygen tension, which makes the sealing of the opening in the shell critical, pH, osmolarity, and the amount of keratinization (Auerbach et al., 2000).

7.2 Other Classical In Vivo Assays in Comparison (Advantages, Disadvantages, and Limitations) with Chorioallantoic Membrane To analyze the mechanisms underlying normal and pathological angiogenesis numerous in vivo angiogenic assays have been established employing different species of laboratory animals, including mammals (mouse, rat, hamster, and rabbit), birds (chick and quail), and fish (zebrafish) (Table 7.2). Table 7.2 Other in vivo angiogenesis assays

Corneal micropocket Sponge/matrix implant Disk angiogenesis assay (DAS) Matrigel plug Dorsal skin chamber Rabbit ear chamber Zebrafish

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Other Classical In Vivo Assays in Comparison with Chorioallantoic Membrane

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Table 7.3 Advantages and disadvantages of the other in vivo angiogenesis assays Corneal micropocket Advantages: New vessels are easily identified; immunologically privileged site; permits non-invasive and long-term monitoring Disadvantages: Atypical angiogenesis, as the normal cornea is avascular; technically demanding; traumatic technique; exposure to oxygen via surface can affect angiogenesis; non-specific inflammatory response with some compounds Sponge/matrix implant Advantages: Technically simple; inexpensive; well tolerated; suitable for study of tumor angiogenesis Disadvantages: Encapsulated by granulation tissue; time-consuming; variable retention of test compound within implant; non-specific inflammatory response Disk angiogenesis assay (DAS) Advantages: Technically simple; assesses wound healing and angiogenesis; quantitative analysis Disadvantages: Encapsulated by granulation tissue Matrigel plug Advantages: Technically simple; suitable for large-scale screening; rapid quantitative analysis Disadvantages: Matrigel is not chemically defined; analysis in plugs is time-consuming; expensive Dorsal skin chamber Advantages: Permits long-term monitoring; simple procedure Disadvantages: Non-specific inflammatory response Rabbit ear chamber Advantages: Clearest optical preparation for intravital microscopy; permits long-term monitoring Disadvantages: Technically demanding; non-specific inflammatory response; expensive Zebrafish Advantages: Intact whole animal; allows gene analysis of vessel development; large number of animals available for statistical analysis Disadvantages: Non-mammalian; embryonic

The variety of in vivo bioassays of angiogenesis have enabled investigators to make rapid progress in elucidating the mechanism of action of a variety of angiogenic factors and inhibitors. Cost, simplicity, reproducibility, and reliability are important determinants dictating the choice of methods. Each of the assays has its own advantages and disadvantages, and each one has an application for which it is best suited (Table 7.3). In choosing a particular assay, the investigator should consider the suitability of different model systems to answer specific questions. Ideally, two different assays should be performed in parallel to confirm the angiogenic or antiangiogenic activities of test substances.

7.2.1 The Corneal Micropocket Assay The corneal angiogenesis assay is still considered one of the best in vivo assays, inasmuch the cornea itself is avascular. Thus, any vessels seen in the cornea after stimulation are new vessels. The normally avascular cornea and anterior chamber

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Fig. 7.1 Schematic representation of the corneal micropocket assay. A micropocket is surgically produced in the corneal stroma of anesthetized animals by a surgical scalpel and a pliable spatula (a) and the test substance is inserted in the micropocket (b). The newly formed vessels start from the limbal vasculature and progress toward the implanted the implanted stimulus (c) (reproduced from Morbidelli L, In “In vitro and in vivo models of angiogenesis”, Ribatti D, Vacca A, Dammacco F, eds, 2003, pp. 9–19)

have long served as a substrate for assessing angiogenesis. The original method was developed for rabbit eyes (Gimbrone et al., 1974), but it has been adapted to mice, now the most frequently used test animal (Fig. 7.1) (Muthukkaruppan and Auerbach, 1979). One of the first demonstrations that tumors could induce neovascularization was obtained by introducing tumor pieces in the aqueous humor of the anterior chamber of the rabbit eye (Gimbrone et al., 1972). Other assays directly implanted tissue fragments on the iris (Maiorana and Gullino, 1978). Subsequently, tumor cells were introduced in the stroma of the cornea (Gimbrone et al., 1974), and then by substituting slow-release materials, such as ELVAX or hydron, containing semipurified angiogenic growth factors from tumor cells. To test inhibitors of angiogenesis, one can monitor the effect of such inhibitors on the locally induced angiogenic reaction in the cornea. The test inhibitors can be administered orally or systemically, the latter either by bolus injection or by intraperitoneal implantation of osmotic pumps loaded with the test inhibitor (Kisker et al., 2001a). Methods for quantification include measuring the area of vessel penetration, the progress of vessels toward the angiogenic stimulus over time, or in the case of use of fluorochrome-labeled high molecular weight dextran, histogram analysis, or pixel counts above a specific background threshold (Fig. 7.2). In mice and rats it is possible to obtain time point results. Following the evolution of the angiogenic response in only one animal is not recommended because each time the cornea is observed the animal has to be anesthetized. Experiments are made with a large number of animals and the use of a slit lamp stereomicroscope to observe unanesthetized animals allows the observation of newly formed vessels for a period of up to 1–2 months. Computer-assisted image analysis of corneal vascularization partially overcomes the problem of subjective evolution by the operator and provides multiple data points from each animal. A major advantage of the corneal micropocket angiogenesis assay is that the measurement of background vessels is unnecessary because the vessels grow on an otherwise avascular tissue. This simplifies the quantification of the neovascular

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Fig. 7.2 Computerized image analysis of the angiogenic response induced by the implant of two adjacent pellets releasing two angiogenic stimuli in the corneal micropocket assay (a). Following digitalization, the new vessels are extracted from the background (b). The image, converted to black and white, is analyzed for the number of vessels, the area occupied by vessels, and the degree of branching (reproduced from Morbidelli L, In “In vitro and in vivo models of angiogenesis”, Ribatti D, Vacca A, Dammacco F, eds., 2003, pp. 9–19)

area, thus removing a source of variation. It also eliminates the possibility of vessel dilatation being mistaken for angiogenesis. The demanding surgical procedures involved in this assay limit the animal number for a given experiment and also the practical use of this assay model. In addition, the space available for introducing test material is limiting and the inflammatory reactions are difficult to avoid.

7.2.2 The Sponge/Matrix Implant The general goal of most subcutaneous implant models is to trap a putative angiogenic substance into a suitable carrier, mostly an avascular sponge-like structure, which slowly releases the factor at the site of implant causing the recruitment of new vessels into implant. In 1987, Andrade and co-workers described a method of quantifying angiogenesis in sponge implants. After putting the agonist or antagonist of angiogenesis in circular polyether sponge disks with central cannulae, the subsequent change in blood flow can be measured by using the 133 Xe clearance technique for repeated measurements of relative blood flow changes through the sponges over a period of weeks. As the sponges originally contained no blood vessels, the increase in the rate of 133 Xe loss from the sponges was considered to represent neovascularization. Sponge implants have also been used as a framework to host different tumor cell lines in rodents for studying tumor angiogenesis. The advantage of implantation technique is that the assessment of the relative contributions of the tumor cells to early changes in the implant blood flow can be detected even before visible growth of the tumor mass is evident. Biochemical determination of several components of the fibrovascular tissue, such as wet and dry weights, DNA, protein, extracellular matrix components, hemoglobin, and enzyme activity, can provide assessment of cellular proliferation

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kinetics and extracellular matrix components involved in the process. Finally, utilizing morphological or morphometric approaches, the sequence of histological change and vascular density can be determined.

7.2.3 The Disk Angiogenesis System (DAS) Fajardo et al. (1998) introduced the DAS implanted subcutaneously in the host animal through a distal skin incision and then evaluated for penetration by hostderived blood vessels and/or other cell infiltrates. Angiogenic factors or antagonists are placed in the center of the disk. The DAS has been modified to enable the introduction of live cells, i.e., tumor cells or inflammatory cells into the center of disk (Nelson et al., 1993). The disks are harvested after 1–3 weeks, fixed, sectioned, and stained. Histological examination of sections of the disk show a rich neovascularization and a distinctive cellular infiltration at the edges, including fibroblasts, endothelial cells, and leukocytes. The most important advantage of the disk assay is that the vascular growth can be quantified easily and reproducibly. The procedure is simple and inexpensive. As the disk is an avascular structure at onset, all vessels are new and the problem of differentiating new from preexisting vessels does not arise. The DAS is a mammalian system and therefore more relevant to human physiology and pathology. In the DAS there is always a moderate, well-characterized, and measurable spontaneous vascular growth. The major drawback of this method is the inability to perform continuous in vivo monitoring of the angiogenic response. External and gross inspection cannot be performed. Each disk provides information for only one time point. Histological embedding, sectioning, and staining are required.

7.2.4 The Matrigel Plug Matrigel is a laminin-rich mixture of basement membrane components. Matrigel has not fully defined chemically. It contains collagen IV, laminin, nidogen/entactin, heparan sulfate proteoglycan, and growth factors, such as epidermal growth factor (EGF), TGF-β, PDGF, insulin like growth factor-1 (ILGF-1), nerve growth factor (NGF), and FGF-2 (Vukicevic et al., 1992). This suggests that caution should be exercised in the interpretation of experiments. Matrigel was initially used to investigate capillary tube formation in vitro (Kubota et al., 1988). Matrigel in liquid form at 4◦ C is mixed with an angiogenic cytokine and injected in the subcutaneous tissues of mice. At body temperature Matrigel rapidly forms a solid plug, trapping the growth factor to allow slow release and prolonged exposure to surrounding tissues. Matrigel containing test cells or substances is injected subcutaneously, where it solidified to form a plug. The animals are killed after 7–21 days and the Matrigel plugs excised for histologic examination. Angiogenesis is quantified as vessel area in the plug section by image

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Fig. 7.3 FGF-2-induced neovascularization in Matrigel plug. Matrigel pellets containing 150 ng of FGF-2 were implanted subcutaneously in mice and examined at day 7 by immunohistochemical analysis. Note the presence of numerous CD31+ endothelial cells infiltrating the plug (courtesy of Marco Presta, University of Brescia Medical School, Brescia, Italy)

analysis (Fig. 7.3). The quantification of the angiogenic response may also be performed measuring the amount of hemoglobin content (Passaniti et al., 1992). However, the hemoglobin assay may be misleading because blood content is much affected by the size of vessels and the extent of stagnant pools of blood. The sponge/Matrigel assay is a modified version of the Matrigel plug (Akhtar et al., 2002). Matrigel alone is first introduced subcutaneously into the mouse. A sponge soaked with the test material or tumoral tissue fragment is then inserted into the plug and new vessels can then be measured by injection of fluorescein isothiocyanate (FITC)-labeled dextran. Differences in the mice may affect the background levels of blood vessel formation one observes. Vessel formation in young mice (6 months old) is reduced as compared to mice 12–24 months old. Additionally, if the Matrigel is injected into different sites in the mouse, variability can result. Lower angiogenic response is observed if the material is injected into the dorsal surface of the animal, while one of the best areas in terms of angiogenic response is the ventral side of the mouse in the groin area close to the dorsal midline.

7.2.5 The Dorsal Air Sac Model The dorsal air sac model was developed by Selye who showed that simple implantation of a chamber ring loaded with tumor cells causes angiogenic vessel formation on the murine skin attached to the ring. Both sides of a Millipore ring are covered by filters and the resultant chamber is filled with a tumor cell suspension and then it is implanted into the preformed dorsal

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air sac of an anesthetized mouse (Yonekura et al., 1999). Following treatment with the compound of interest, the chamber is carefully removed and rings of the same diameters placed directly upon the sites that were exposed to a direct contact with the chamber. The number of newly formed blood vessels that lie within the area marked by the ring is counted, using a dissecting microscope. Care must be taken to not irritate the surface upon which the chamber is placed, as this may itself induce angiogenesis.

7.2.6 The Chamber Assays Since 1924, when the first transparent chamber model was introduced by Sandison (Sandison, 1924), many other chamber models have been described for studying angiogenesis and microcirculation in a wide variety of neoplastic and non-neoplastic tissues by means of intravital microscopy. A piece of skin (ear and skinfold chambers) or part of the skull (cranial window chamber) is removed from an anesthetized animal. Tumor cells, or a gel containing angiogenic factors, are then placed on the exposed surface and covered by glass. The rabbit ear chamber has been used to quantify structural and functional changes in the neovasculature of tumors (Dudar and Jain, 1983). The dorsal skin chamber has been used to study tumor angiogenesis, including xenografts in immunodeficient rodents (Dellian et al., 1996). Other semitransparent preparations include the hamster cheek pouch (Klintworth, 1973) and the rat mesentery assay (Norrby et al., 1990). Chamber assays allow for the determination of three-dimensional vessel growth in one animal, typically over a period of 1–3 weeks. Separate groups of mice are not required at each measurement point, and hence the number of animals used is minimized. One of the major advantages of chamber models is the possibility of monitoring angiogenesis in vivo continuously up to several weeks. In addition, at the end of experiments, tissue samples can be excised and further examined by histology, immunohistochemistry, and molecular biology. Finally, transparent chamber enables the determination of whether a newly formed blood vessel is perfused and contributed to tissue oxygenation. However, all chamber assays are invasive and technically demanding. The surgical procedure can induce a secondary angiogenic response through wound healing that could be superimposed on the effects of the test substance themselves. The disadvantage of the skinfold chamber is that it is not an orthotopic site for many of the tumors studied. Therefore, the cranial window preparation was generated to provide an orthotopic brain tumor model (Yuan et al., 2004).

7.2.7 The Zebrafish The zebrafish (Danio rerio) is a small tropical freshwater fish proposed for screening molecules that affect blood vessel formation (Serbedzija et al., 1999). The

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development of blood vessels such as the dorsal aorta, posterior cardinal vein, subintestinal veins, and intersegmental vessels in early zebrafish embryos is well characterized through analysis of mutations affecting their formation. The dorsal aorta and the posterior cardinal veins are formed by vasculogenesis (Zhong et al., 2001), while intersegmental vessels are thought to be formed by angiogenesis. These vessels are easily monitored (Isogai et al., 2001), thus making them suitable for identification of angiogenesis inhibitors. An impressive repertoire of genetic tools is available to modify the zebrafish genome. Mutations that significantly impair cardiovascular development are readily identifiable in zebrafish because of the transparency and small size of the developing zebrafish embryo, which permits easy visualization of the heart and blood vessels and allows the animals to receive enough oxygen by passive diffusion to survive for 4–5 days in the absence of a functional circulation. Among these tools, morpholino “knockdown” technology for reverse genetic analysis of gene function has emerged as a powerful approach to understanding molecular events in vasculogenesis and angiogenesis in the zebrafish (Kajimura et al., 2006). Several complementary studies based on the use of in situ hybridization, confocal microangiography, lineage tracking, and transgenic strains that express enhanced green fluorescent protein in endothelial cells contributed to a better understanding as to how the vasculature develops in zebrafish. More recently, it has been described a method to study tumor angiogenesis in zebrafish based on the injection of proangiogenic mammalian tumor cells into the perivitelline space of zebrafish embryos at 48 h post-fertilization (Nicoli and Presta, 2007). Within 24–48 h, proangiogenic tumor grafts induce an angiogenic response originating from the developing subintestinal vessels, and angiogenesis inhibitors added to the injected cell suspension or to the fish water prevent tumor-induced neovascularization (Fig. 7.4).

Fig. 7.4 Angiogenic responses triggered by tumor cell grafts in the zebrafish embryo. Zebrafish embryos were injected with mammalian tumor cells resuspended in a Matrigel solution at 48-h post-fertilization. After 24 h, note the alkaline phosphatase-positive vessels (arrows) sprouting from the subintestinal vessel plexus and converging vs the graft (∗ ) (courtesy of Professor Marco Presta, University of Brescia Medical School, Brescia, Italy)

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Zebrafish are inexpensive to use and easy to maintain long term, making largescale screening experiments possible. Furthermore, as the embryos develop outside the mother and are transparent, the direct observation of blood vessel formation is straightforward using a low-power binocular microscope (Lawson and Weinstein, 2002). As the early embryos used in most experiments are about 1–2 mm in size, a number of embryos are usually put together and the compound of interest, if small and lipophilic, added to the water and absorbed by the fish, whereas peptide/proteins have to be injected into the yolk sacs of embryos.

7.2.8 The Tumor Models Many different in vivo tumor models have been developed to test the activity of potential anti-cancer treatments. Tumors can be grown syngeneically (e.g., subcutaneous), orthotopically (in the tissue of origin), or as xenografts in immunodeficient rodents, to test the effect of substance on tumor size and animal survival at regular intervals (Fig. 7.5). A major disadvantage of all the tumor models is that tumors are established within a few weeks after tumoral cell implantation, whereas human cancer develops over a period of several months or years.

Fig. 7.5 Orthotopic neuroblastoma xenograft model in SCID mice. a and b Adrenal gland tumors (arrows) in mice that were injected orthotopically with SH-SY5Y cells at 14 (a) and 21 (b) days before killing. (c) and (d) Representative right adrenal gland (c) and liver (d) samples at 3 and 4 weeks after injection of neuroblastoma cells, respectively. (e–h) Histological analysis of representative ovary (e), kidney (f), liver (g), and lung (h) samples. Forty days after cell injection, animals were killed, the organs were removed, fixed, paraffin embedded, sectioned at 5 μm, and stained with H&E. Arrows indicate metastatic tumor invasion in the lung. Arrowheads show the normal ovaric follicular structure surrounded by tumor neuroblastoma cells (reproduced from Pastorino et al., 2003)

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Tumor models have also been used specifically to investigate antiangiogenic drugs. It is also possible to use such models to determine whether a new drug is antiangiogenic or anti-vascular in action. In the former, the drug will prevent or greatly reduce the growth of new blood vessels to the tumor; in the latter, it will damage the endothelial cell lining of the existing tumor blood vessels. Histological analyses include measurements of necrosis and morphology of the tumors, thrombosis formation (MSB staining for the detection of fibrin), microvessel density (Cd31/Cd34 staining), actively proliferating endothelial cells (PCNA and/or endoglin staining), and apoptosis (TUNEL).

Concluding Remarks

Neovascularization plays a crucial role in several pathological conditions, including chronic inflammation and tumor growth. It is reasonable to reserve the term “angiogenic factor” for substances that produce new capillary growth in an in vivo assay. In fact, studies have shown that a compound affecting cell proliferation, migration, or differentiation in vitro may not necessarily regulate endothelial cell activity in vivo (Liekens et al., 2001). A single assay that is optimal for all situations has not yet described. No single model is able to elucidate the entire process of angiogenesis, as there are differences between species, microenvironments, organ sites, whether embryonic or adult tissue is used, and manner of administration of test substances. With our limited understanding of the mechanisms involved in angiogenesis, none of the currently used assay systems allows an objective evaluation of the various components of the process of angiogenesis. There has been an exponential increase in the sophistication of in vivo imaging techniques including the availability of MRI, CT, and PCT facilities for scanning small animals, and the advent of confocal and multi-photon microscopy enabling fine structure imaging in situ. Investigators from different fields can choose from several in vivo angiogenesis and antiangiogenesis assays and one of the most important technical problems is the difficulty of obtaining meaningful assessment of efficacy and the search for rapid and reproducible assay is an area of interest. It is therefore appropriate to use specific assays that reflect particular disease process, tumor angiogenesis models to assess antiangiogenic compounds, and ischemic models to assess pro-angiogenic agents. In vivo assays are difficult to quantify, but new methods for imaging vessels and for image analysis are emerging that may help provide quantification of in vivo experiments, which is essential to study angiogenic and antiangiogenic molecules. Animal models are extremely complex, difficult to interpret for routine screening, and relatively expensive. The experiments performed in the chick CAM have resulted in important progress in elucidating the mechanisms of action of several angiogenic factors and inhibitors. The main advantages of this assay are their low cost, simplicity, reproducibility, and reliability. On the other hand, there are only very few restrictions to using CAM, essentially due to (i) non-specific inflammatory reactions that D. Ribatti, The Chick Embryo Chorioallantoic Membrane in the Study of Angiogenesis and Metastasis, DOI 10.1007/978-90-481-3845-6,  C Springer Science+Business Media B.V. 2010

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may develop with an attending secondary stimulation of angiogenesis and (ii) preexisting vessels may be present which make it hard to distinguish the extent of angiogenesis and antiangiogenesis. In view of these limitations, two different assays should ideally be performed in parallel to confirm the angiogenic or antiangiogenic activity of test substances and the predictive value of all in vivo angiogenesis assays remains to be established and results must be interpreted with care.

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