Capillaries in the Chicken Chorioallantoic Membrane

1Domenico Ribatti and 2Marco Presta

1Department of Human Anatomy and Histology, University of Bari 2Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy

The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane. It serves as a gas exchange surface through the eggshell and its function is supported by a dense capillary network. Because of its extensive vascu-larization and easy accessibility, the CAM has been broadly used to study the morphofunctional aspects of the angio-genesis process in vivo and to investigate the efficacy and mechanisms of action of pro-angiogenic and anti-angiogenic natural and synthetic molecules. Also, because of the lack of a fully developed immunocompetence system in the chick embryo, the CAM represents a host tissue for tumor engrafting suitable to study various aspects of the angiogenic and metastatic potential that characterizes human malignancies.

Developmental Anatomy of the Blood Vessels of the CAM

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 coelom. 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, the enlarged distal portion as the allantoic vesicle. Fluid accumulation distends the allan-tois so that its terminal portion resembles a balloon in complete embryos (Figure 1).

The allantoic vesicle enlarges very rapidly from days 4 to 10: an extensive morphometric investigation has shown rapid extension of the CAM surface from 6 cm2 at day 6 to 65 cm2 at day 14. In this process the mesodermal layer of the allantois fuses with the adjacent mesodermal layer of the chorion to form the CAM. An extremely rich vascular network connected to embryonic circulation by the allantoic arteries and veins develops between the two layers. Immature blood vessels scattered in the mesoderm and lacking a complete basal lamina and smooth muscle cells grow very rapidly until day 8, giving rise to a capillary plexus. The plexus associates with the overlaying chorionic epithelium and mediates gas exchange with the outer environment.

Capillary proliferation continues rapidly until day 10 (mitotic index equal to 23 percent). Then, the endothelial cell mitotic index declines rapidly to 2 percent and the vascular system attains its final arrangement on day 18, just before hatching. Besides sprouting angiogenesis that characterizes the early phases of CAM development, late CAM vascularization is supported by intussusceptive microvascu-lar growth in which the capillary network increases its complexity and vascular surface by insertion of transcapillary pillars. At day 14, the capillary plexus is located at the surface of the ectoderm adjacent to the shell membrane. The blood circulation and the position of the allantois immediately adjacent to the porous shell confer a respiratory function to the highly vascularized CAM. In addition to the respiratory interchange of oxygen and carbon dioxide, the allantois also serves as reservoir for the waste products excreted by the embryo—mostly urea at first, and chiefly uric acid later.

Ovo Embryo

Figure 1 Allantoic sac (a) of a 5-day embryo (e) showing in ovo distribution pattern of allantoic vessels (av). Original magnification: x25.

Structure of the Blood Vessels of the CAM

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. By day 8, the CAM displays small (10 to 15 mm in diameter), thin-walled capillaries beneath the chorionic epithelium and larger vessels (10 to 115 mm in diameter) in the mesodermal layer. Large vessels have a layer of mesenchymal cells surrounding the endothelium and are completely wrapped by a basal lamina. Starting from day 12, capillaries contain endothelial cells and a few mesenchymal cells (presumptive pericytes) closely applied to the endothelial abluminal side. Desmin-positive cells are evenly distributed all over the CAM and are located in close association with the capillary plexus. The mesodermal vessels are now distinct arterioles and venules (Figure 2). In addition to the endothelium, the walls of arterioles (10 to 85 mm in diameter) contain one or two layers of mesenchymal cells and increased amounts of connective tissue. Venules (10 to 115 mm in diameter) are surrounded by an incomplete investment of mesenchymal cells. Mesenchymal cells are presumed to be developing smooth muscle cells, and the walls of arterioles also develop a distinct adventitia containing fibroblast-like cells.

At days 4 to 8 the endothelial cells form punctuate junctional appositions and a few plasmalemmal vesicles are observed. Between days 9 and 13 the arteriolar endothelium displays more extensive junctional complexes with multiple membrane contact points. In contrast to the arterioles, endothelial junctional appositions of the CAM venules remains punctuate. The venules possess multiple sites of interendothelial contact with areas of junctional dilations, while the arterioles display complex interdigitating cell junctions.

Figure 2 Image of the CAM arteriovenous and capillary systems after casting of the vasculature (provided by Dr. V. Djonov, University of Berne, Switzerland). Original magnification: x100.

CAM arterioles and venules are accompanied by a pair of interconnected lymphatics. Veins are also associated with lymphatics and larger veins are surrounded by a lymphatic plexus. Lymph is drained by trunks of the umbilical stalk into the coccygeal lymphatics and the lymph hearts of the embryo. CAM lymphatic capillaries have no basal lamina and an extremely thin endothelial lining that specifically expresses vascular endothelial growth factor receptor-3 (VEGFR-3, Quek-2, flt-4) whereas VEGFR-2 (Quek-1, kdr, flk-1) is expressed by both vascular and lymphatic endothelial cells.

Endogenous CAM Modulators of Blood Vessel Formation

The development of the vascular system of the CAM is a complex, highly regulated process that depends on genetic and epigenetic factors expressed by endothelial and non-endothelial cells.

Endothelial cell interaction with extracellular matrix (ECM) plays an important role in angiogenesis. Accordingly, ECM modifies its properties during CAM development in terms of expression of several components (e.g. fibronectin, laminin, type IV collagen, Secreted Protein Acidic Rich Cysteine (SPARC)), distribution of specific gly-cosaminoglycans, and production of lytic enzymes.

Fibronectin accumulates in the ECM beneath the chorion at early stages of development when the subepithelial capillary plexus is not yet formed, possibly promoting the migration of endothelial cells merging by sprouting from the mesodermal blood vessels. In contrast, type IV collagen appears in the late stages of CAM vascular development concomitantly with the terminal differentiation of endothe-lial cells and maturation of basement membrane. Laminin immunoreactivity is present instead during all stages of CAM vessel formation, in keeping with its role in the early formation and later differentiation of the subendothelial basement membrane. Finally, ialuronic acid plays a crucial role in the formation, alignment, or migration of the capillary plexus, while heparan sulfate, chondroitin sulfate, and dermatan sulfate are important in the differentiation and development of arterial and venous vessels.

In spite of the evidence that several growth factors, such as fibroblast growth factor-2 (FGF-2), VEGF, placenta growth factor (PlGF), platelet-derived growth factor (PDGF), transforming growth factor beta-1 (TGF b-1), angiogenin, and erythropoietin (Epo), stimulate blood vessel formation when applied to the CAM (see later discussion), the role of their endogenous counterparts in the development of the CAM vascular system is poorly understood. The expression of VEGFRs suggest that, in analogy with other developmental models, endogenous VEGF may play a role in CAM vascularization. Accordingly, neutralizing anti-VEGF antibodies prevent CAM vessel development. Also, direct experimental evidences implicates endogenous FGF-2 in this process.

FGF-2 protein levels change in the CAM during development and peak between days 10 and 14. At early stages of development, FGF-2 is expressed by chorionic epithelial cells and may trigger a paracrine loop of stimulation by inducing an angiogenic response in undifferentiated vessels of the CAM mesoderm. At later stages, FGF-2 mRNA expression predominates in endothelial cells forming the capillary plexus, suggesting an autocrine function in late vessel growth and maintenance. Accordingly, neutralizing anti-FGF-2 antibodies fully prevent neovascularization when applied to the CAM at day 8 of incubation. They also decrease fibroblast density within the mesoderm, but do not affect epithelial cells of the chorion and allantois. Thus, FGF-2 plays a rate-limiting role in the maturation of stroma and blood vessels during CAM development.

The proteolytic plasmin/plasminogen activator system is also involved in CAM vascularization, possibly affecting the invasive behavior of sprouting endothelium and by modulating the activity of endogenous modulators. For instance, endogenous urokinase plasminogen activator (uPA) mediates the formation of angiogenic SPARC-derived peptides, whereas exogenous uPA can induce neovascularization by increasing the mobilization of endogenous FGF-2 from ECM-associated reservoirs.

CAM as a Model to Study the Modulation of Angiogenesis

A. Tumor Angiogenesis and Metastasis

The CAM is a suitable site for transplanting tissues because the chick embryo immunocompetence system is not fully developed and the conditions for rejection have not been established. Transplants survive and develop by peripheral anastomoses between graft and original CAM vasculature or by new angiogenic vessels grown from the

CAM and invading the graft. The formation of peripheral anastomoses between host and preexisting donor vessels is the main, and the most common, mechanism involved in the revascularization of embryonic grafts. At variance, the growth of CAM-derived vessels into the graft is stimulated by tumor implants.

The CAM has long been a favored system for the study of tumor angiogenesis and metastasis. Tumor grafts remain avascular for 72 hours, after which they are penetrated by new blood vessels and a phase of rapid growth begins. The rate of growth during this vascular phase is greater for implants on days 5 and 6 to decrease at later days of implantation. The CAM may also be used to assess the ability of antiangiogenic molecules to inhibit growth and neovascu-larization of tumor xenografts.

Studies using the tumor cells/CAM model have also focused on the invasion of the chorionic epithelium and the blood vessels by tumor cells. The cells invade the epithelium and the mesenchymal connective tissue below, intravasate into the dense bed of blood vessels, and may invade the chick embryo.

Finally, delivery of tumor cells onto the CAM allows the fine study of the effects of tumor-derived angiogenic growth factors on blood vessel structure and functionality. For instance, tumor cell lines overexpressing FGF-2 or VEGF induce a quantitatively similar vasoproliferative response when grafted onto the CAM. However, in keeping with the different ability of FGF-2 and VEGF to modulate endothe-lial cell morphology and vascular permeability, an increased endothelial fenestration characterizes the blood vessels of the CAM stimulated by VEGF transfectants.

B. Molecules with Angiogenic and Anti-angiogenic Activity

The CAM is used to study molecules with angiogenic and anti-angiogenic activity following their delivery in ovo (Table I). Many protocols have been envisaged to deliver macromolecules and low-molecular-weight compounds onto the CAM by using silostatic rings, methylcellulose disks, silicon rings, filters, and plastic rings. Also, collagen and gelatin sponges treated with stimulators or inhibitors of blood vessel formation have been implanted on growing CAM. The gelatin sponge is also suitable for the delivery of cell suspensions onto the CAM surface and the evaluation of their angiogenic potential. This latter experimental condition allows the slow, continuous delivery of growth factors released by few implanted cells (20,000 cells per sponge or fewer). As compared with the application on the CAM of large amounts of a pure recombinant angiogenic cytokine in a single bolus, implants of cells overexpressing angiogenic cytokines enables the continuous delivery of growth factors, following a more "physiological" mode of interaction with the CAM vasculature.

Besides in ovo experimentation, a number of shell-less culture techniques have been devised, involving cultures of avian embryos with associated yolk and albumin outside

Table I Pro-Angiogenic and Anti-Angiogenic Molecules in the CAM Assay.

Pro-angiogenic molecules

Anti-angiogenic molecules

Fibroblast growth factor-2

Anti-FGF-2 antibodies

(FGF-2)

Vascular endothelial growth

Anti-VEGF antibodies

factor (VEGF)

Placenta growth factor-1

Anti-PlGF-1 antibodies

(PlGF-1)

Angiogenin

Anti-angiogenin antibodies

Tumor necrosis factor alpha

Interleukin-2 (IL-2)

(TNF-a)

Transforming growth factor

Angiostatin

beta (TGF-ß)

Platelet derived growth factor

Endostatin

(PDGF)

Erythropoietin (Epo)

Heparan sulfate

Osteogenic protein-1 (OP-1)

Modified heparins

Leptin

Heparanase inhibitors

Adrenomedullin

Protamine sulfate

CC chemokine I-309

Platelet factor-4 (PF-4)

Osteopontin/Eta-1

Pentosan polysulfate

Non- or low-sulfated saccharides

Arylsulfatase inhibitors

Sulfated polysaccharide peptidoglycan

a-, ß-, g-cyclodextrin

Suramin

Spironolactone

Tyrosine kinase receptor inhibitors

Adhesion molecule antagonists

Matrix metalloproteinase inhibitors

of the eggshell. Shell-less cultures facilitate experimental access and continuous observation of the growing embryo.

Typically, an angiogenic response occurs 72 to 96 hours after stimulation in the form of an increased density of vessels that converge radially toward the implant like spokes in a wheel. Conversely, when an angiostatic compound is tested, the vessels become less dense around the implant and eventually disappear (Figure 3). Alternatively, the molecules can be directly inoculated into the cavity of the allantoic vesicle so that their activity reaches the whole vascular area in a uniform manner.

Several semiquantitative and quantitative methods are used to evaluate the extent of vasoproliferative response or angiostatic activity at macroscopic and microscopic levels. Quantification of the CAM vasculature has been performed with the use of morphometric point-count methods, radiolabeled proline incorporation to measure collagenous protein synthesis, and fractal analysis of digital images.

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 CAM can be utilized for biochemical studies, such as the determination of DNA, protein, and collagen content, and for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression. Recently, studies of intracellular signaling pathways mediating the angiogenic response to growth factors and cytokines have been successfully performed. This has allowed the demonstration of the role of extracellular signal-regulated kinases (ERKs) and avb3 integrin engagement in FGF-2-mediated angiogenesis and of JAK2/STAT-3 pathway during neovascularization induced by GM-CSF.

Figure 3 Modulation of CAM vascularization: numerous vessels develop radially in a "spoked-wheel" pattern toward the stimulus triggered by an angiogenic cytokine (a); in contrast, very few vessels are recognizable around the implant of an angiostatic molecule (b). Original magnification: x50.

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