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Keywords:

  • angiogenesis;
  • anti-angiogenesis;
  • chorioallantoic membrane;
  • fibroblast growth factor-2;
  • intussusceptive growth

Abstract

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that is commonly used in vivo to study both angiogenesis and anti-angiogenesis. This review 1) summarizes the current knowledge about the structure of the CAM's capillary bed; 2) discusses the controversy about the existence of a single blood sinus or a capillary plexus underlying the chorionic epithelium; 3) describes a new model of the CAM vascular growth, namely the intussusceptive mode; 4) reports findings regarding the role played by endogenous fibroblast growth factor-2 in CAM vascularization; and 5) addresses the use and limitations of the CAM as a model for studying angiogenesis and anti-angiogenesis. Anat Rec 264:317–324, 2001. © 2001 Wiley-Liss, Inc.


EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

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, and the enlarged distal portion as the allantoic vesicle. Fluid accumulation distends the allantois such that its terminal portion resembles a balloon in embryos.

The allantoic vesicle enlarges very rapidly from days 4–10 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 which is connected to 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).

This circulation and the position of the allantois immediately subjacent to the porous shell confer a respiratory function on the highly vascularized CAM. 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.

MORPHOLOGY OF THE CAM BLOOD VESSELS

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

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).

By day 8, the CAM displays small, thin-walled capillaries with a lumenal 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. The mesodermal vessels are now distinct arterioles and venules. In addition to the endothelium, the walls of arterioles (10–85 μm in 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 mesenchymal cells, and connective tissue has also accumulated within their walls. The mesenchymal 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).

INTERENDOTHELIAL JUNCTIONS

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

Between days 4–8 the endothelial cells form punctuate junctional appositions, and a few plasmalemmal vesicles are observed (Shumko et al., 1988).

Between days 9–13 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 14–18 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).

CAM EXTRACELLULAR MATRIX

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

The extracellular matrix of the CAM modifies its composition in terms of expression of fibronectin, laminin, and collagen type IV, and in 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., 1998).

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 (Ribatti et al., 1998). Moreover, fibronectin overexpression in extracellular matrix parallels the vasoproliferative processes induced by angiogenic stimuli in the CAM (Ribatti et al., 1997b). 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., 1998) in keeping with its role in the early formation and later differentiation of the subendothelial basement membrane (Risau and Lemmon, 1988).

Type IV collagen appears in the late stages of CAM vascular development concomitantly with the terminal differentation of endothelial cells and maturation of the basement membrane (Ribatti et al., 1998). 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).

Ausprunk (1986) demonstrated that yaluronic acid plays a crucial role in the formation, alignment, and 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.

CAM LYMPHATIC VESSELS

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

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 (Oh et al., 1987). Lymph is drained by trunks of the umbilical stalk into the coccygeal lymphatics and the lymph hearts of the embryo (Wilting et al., 1999). Ultrathin sections of the endothelium of the CAM lymphatic capillaries have an extremely thin endothelial lining and no basal lamina (Oh et al., 1997). The lymphatic endothelial cells of the differentiated CAM specifically express vascular endothelial growth factor receptor (VEGFR)-3 (Quek 2, flt4), whereas expression of VEGFR-2 (Quek 1, kdr, flk1) is found in both its blood vascular and its lymphatic endothelial cells (Wilting et al., 1996).

CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

Fulleborn (1895) described a “great blood sinus interrupted by strands of tissue.” Other authors (Narbaitz, 1977; McCormick et al., 1984; Schoefl, 1984) maintain that there is a vascular sinus and that the CAM vascular bed is 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). Apparently, in the chick CAM active migration of the chorionic epithelium rather than endothelial cells is 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 which is formed during the early stages of incubation, and eventually is 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., 1998). 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) 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 basement membrane and the lack of phagocytic cells intermingled with the endothelial cells provide the main morphological evidence of the existence of a capillary plexus.

NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

A widely accepted view is that blood vessels arise during development by two mechanisms: 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 postcapillary venules.

Intussusceptive microvascular growth (IMG) (“intussusception or nonsprouting angiogenesis”) is a new concept of microvascular growth which is relevant for many vascular systems, and which constitutes an additional and/or alternative mechanism for endothelial sprouting (Patan et al., 1992). The first reports on IMG were published by Caduff et al. (1986) and Burri and Tarek (1990), who investigated the lung vasculature in postnatal rats 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: 1) creation of a zone of contact between opposite capillary walls; 2) reorganization of the intercellular junctions of the endothelium, with central perforation of the endothelial bilayer; 3) formation of an interstitial pillar core; and 4) subsequent invasion of the pillar by cytoplasmic extensions of myofibroblasts and pericytes, and by collagen fibrils. It is thought that the pillars then increase in diameter and become a capillary mesh.

Patan et al. (1993) observed the same morphological transformation during IMG in the CAM (Fig. 1). 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 (Patan et al., 1997).

thumbnail image

Figure 1. Mercox cast of developing CAM vasculature at day 12 of incubation. A and B: A three-dimensional structure containing a capillary plexus and a layer of supplying and collecting vessels is recognizable. C and D: Numerous pillars of different sizes caused by intussusceptive angiogenesis processes. Original magnification: (A) ×100; (B and C) ×200; (D) ×400.

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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 and 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 >2.5 μm in diameter.

MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

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 fivefold for precapillary and postcapillary vessels, respectively), predominantly due to growth and remodeling after day 10. Rizzo and De Fouw (1993) found 50% of endothelial cells thinning out from day 10 to day 14 of incubation.

The CAM endothelium exhibits an intrinsically high mitotic rate (thymidine labeling index 23% for 5-hr thymidine exposure) until day 10 (Ausprunk et al., 1974). At day 11, this falls to 2% and remains low throughout the remaining incubation period. An investigation by Kurz et al. (1995) of the presence of bromodeoxyuridine-labeled endothelial cells in the growing CAM from day 6 to day 15 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 ultrastructural alterations associated with the focal microvascular histodifferentiation are in line with the changes in the vascular pattern. 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. Thus, their morphology is distinct (Shumko et al., 1988).

ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

We have previously demonstrated FGF-2 protein in extracts of chick CAM (Ribatti et al., 1995). Its levels change during development, and peak between days 10 and 14. These temporal changes correlate with the time course of CAM vasoproliferation. We have further demonstrated by in situ hybridization studies that FGF-2 mRNA is expressed in the CAM, and that the cellular source of this angiogenic factor changes during development. At day 5, chorionic epithelial cells express high levels of FGF-2 mRNA and only a faint FGF-2-related hybridization signal is apparent in blood vessels of the mesodermal layer and in the allantois. At day 10, high levels of FGF-2 mRNA are expressed by both chorionic cells and endothelial cells of the forming capillary plexus. At day 15, the endothelial cell-associated signal is further increased in the capillary plexus, whereas the chorion-associated signal is decreased.

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 (Ribatti et al., 1995). These findings point to a rate-limiting role for FGF-2 in the maturation of blood vessels and stroma during CAM development, and suggests that endogenous chick FGF-2 may affect the proliferation, migration, redistribution, and invasive behavior of endothelial cells.

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 for 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 follows (Moscatelli, 1992) and triggers 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.

EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

The CAM has long been a favored system for the study of tumor angiogenesis and metastasis (Dagg et al., 1956; Auerbach et al., 1976; Ribatti et al., 1996; Quigley and Armstrong, 1998), because at this stage the chick's immunocompetence system is not fully developed and the conditions for rejection have not been established (Leene et al., 1973). In fact, immunocompetence in birds develops only after hatching (Weber and Mausner, 1977). Tumors implanted on the CAM do not exceed a mean diameter of 0.93 ± 0.29 mm during the prevascular phase (approximately 72 hr). Rapid growth begins 24 hr 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 (1–4 mm) are implanted on the 9-day CAM, grafts larger than 1 mm undergo necrosis and autolysis during the 72-hr prevascular phase. They shrink rapidly until the onset of vascularization, when rapid growth resumes (Knighton et al., 1977).

Other studies using the tumor cell/CAM model have focused on the invasion of the chorionic epithelium and the blood vessels by tumor cells (Scher et al., 1976; Armstrong et al., 1982; Kim et al., 1998), i.e., the metastatic potency of tumors (Dagg et al., 1956). The cells invade the chorionic epithelium and the mesenchymal connective tissue below, where they are found in the form of a dense bed of blood vessels, which is a target for intravasation. Finally, the CAM is used to study inhibition of the induction of angiogenesis by tumor cells by anti-angiogenic drugs (Brooks et al., 1994; Presta et al., 1999).

EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

The CAM is also used to study macromolecules with angiogenic and anti-angiogenic activity (Ribatti et al., 1996, 2000). An angiogenic response occurs 72–96 hr after stimulation in the form of increased vessel density around the implant, with the vessels radially converging toward the center like spokes in a wheel (Fig. 2A) (Ribatti et al., 1995). Conversely, when an angiostatic compound is tested, the vessels become less dense around the implant and eventually disappear (Fig. 2B) (Ribatti et al., 1995; Iurlaro et al., 1998; Vacca et al., 1999a; Minischetti et al., 2000). 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 (Ribatti et al., 1987; Gualandris et al., 1996). We have developed a new method for the quantitation of angiogenesis and anti-angiogenesis in the CAM. Gelatin sponges treated with a stimulator or an inhibitor of blood vessel formation are implanted on growing CAMs on day 8. Blood vessels growing vertically into the sponge and at the boundary between sponge and surrounding mesenchyme are counted morphometrically on day 12. The gelatin sponge is also suitable for the delivery of cell suspensions onto the CAM surface, and the evaluation of their angiogenic potential (Ribatti et al., 1997a, 1999a; Vacca et al., 1999b). 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, and for reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of gene expression by infiltrating cells, including endothelial cells.

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Figure 2. Image analysis reconstructions showing the effects on CAM vascularization at day 12 of incubation with (A) an angiogenic cytokine, fibroblast growth factor-2 (FGF-2); and (B) an angiostatic molecule, TNP 470. A: Numerous vessels develop radially towards the gelatin sponge (asterisk) soaked with FGF-2 in a “spoked-wheel” pattern. B: Very few vessels are recognizable around the sponge (asterisk) treated with TNP-470.

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A variety of growth factors have been reported to induce CAM angiogenesis (Table 1). However, the cellular mechanisms involved are not well defined. Activation of protein kinase c (PKC) was reported to induce CAM angiogenesis between days 9 and 11 (Tsopanoglou et al., 1993), whereas increased levels of intracellular cyclic adenosine monophosphate substantially downregulated this PKC-mediated angiogenic stimulus (Tsopanoglou et al., 1994).

Table 1. Studies demonstrating the angiogenic activity of several growth factors on the CAM
AuthorsGrowth factor
Esch et al. (1985)Basic fibroblast growth factor (bFGF) or
Wilting et al. (1991)Fibroblast growth factor-2 (FGF-2)
Olivo et al. (1992)
Ribatti et al. (1995)
Fett et al. (1985)Angiogenin
Wilting et al. (1991)
Olivo et al. (1992)
Leibovich et al. (1987)Tumor necrosis factor alpha (TNF-α)
Olivo et al. (1992)
Yang and Moses (1990)Transforming growth factor beta (TGF-β)
Wilting et al. (1992)Platelet derived growth factor (PDGF)
Wilting et al. (1992, 1993)Vascular endothelial growth factor (VEGF)
Ziche et al. (1997)Placenta growth factor-1 (PIGF-1)
Bouloumie et al. (1998)Leptin
Ribatti et al. (2001)
Ribatti et al. (1999b)Erythropoietin (EPO)
Ramoshebi and Ripamonti (2000)Osteogenic protein-1 (OP-1)
Bernardini et al. (2000)CC chemokine I-309

LIMITATION OF THE CAM ASSAY

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

The main limitation of CAM assays is the nonspecific inflammatory reactions that may develop as a result of grafting, inducing a secondary vasoproliferative response and impeding quantification of the primary response (Jakob et al., 1978; Spanel-Burowksi et al., 1988). 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 nonspecific 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 other 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 the rearrangement of existing vessels that follows contraction of the membrane (Knighton et al., 1991). Second, timing of the CAM angiogenic response is essential. Many studies determine angiogenesis after 24 hr, when there is no angiogenesis—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.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED

One of the most important technical problems facing us in the study of angiogenesis and anti-angiogenesis is the difficulty of obtaining meaningful assessments of efficacy. In vivo angiogenesis assays, such as those performed in the chick CAM, have made important progress in elucidating the mechanisms of action of several angiogenic factors and inhibitors. The main advantages of the in vivo assays are their low cost, simplicity, reproducibility, and reliability.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. EMBRYOLOGICAL ORIGIN OF THE CHORIOALLANTOIC MEMBRANE (CAM)
  4. MORPHOLOGY OF THE CAM BLOOD VESSELS
  5. INTERENDOTHELIAL JUNCTIONS
  6. CAM EXTRACELLULAR MATRIX
  7. CAM LYMPHATIC VESSELS
  8. CONTROVERSY ABOUT THE EXISTENCE OF A SINGLE BLOOD SINUS OR A CAPILLARY PLEXUS BENEATH THE CHORIONIC EPITHELIUM
  9. NEW MODEL OF CAM VASCULAR GROWTH: THE INTUSSUSCEPTIVE MODE
  10. MORPHOMETRIC EVALUATION OF CAM VASCULAR GROWTH
  11. ROLE OF ENDOGENOUS FGF-2 IN CAM VASCULARIZATION
  12. EMPLOYMENT OF THE CAM IN THE STUDY OF TUMOR ANGIOGENESIS AND METASTASIS
  13. EMPLOYMENT OF THE CAM IN THE STUDY OF ANGIOGENIC AND ANGIOSTATIC MOLECULES
  14. LIMITATION OF THE CAM ASSAY
  15. CONCLUSIONS
  16. LITERATURE CITED
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