A modified chorioallantoic membrane (CAM) assay for qualitative and quantitative study of growth factors

Abstract

The application of Thermanox tissue culture coverslips to the day 9 CAM of the chick causes constant effects beneath the carrier after 3 days, and these are associated with a change in the blood vessel pattern. Histological sections show enormous thickening of the CAM in the reactive areas. The stroma of the CAM shows fibrocyte proliferation, leucocyte infiltration, and clusters of dispersed ectodermal epithelial cells exhibiting signs of necrosis. The latter obviously cause a strong vascular response. The same effects are seen when the Thermanox discs are applied at day 11. Following application on day 12 a positive or negative response to the carrier is observed, whereas on day 13 no such carrier effects are seen. The only remaining effect is compression of the intra-ectodermal capillary plexus of the CAM. This can macroscopically be seen after peroxidase staining of the blood vessels. The effect of 5 microliters PBS dried on the Thermanox disc and applied to the day 13 CAM is to cause, after 3 days, hyperosmotic damage to the ectodermal epithelium, which becomes overgrown by fibrocytes. We found dose-dependent effects of salt-free human bFGF applied to the day 13 CAM. The first and main effect is fibrocyte proliferation (0.5 microgram). New capillaries appear with higher doses, but are not as frequent as would be expected for an angiogenic substance (1.25-2.5 micrograms). Also with higher doses additional hyperplasia of the endodermal (3.75 micrograms) and ectodermal (5 micrograms) epithelium can be seen. The latter might be a non-specific hyperosmotic effect. Leucocytes are regularly present within the reactive areas. When salt-free angiogenin is applied to the day 13 CAM, some effects appear with doses of 4.6 micrograms and more. The ectodermal epithelium of the reactive areas is discontinuous, exhibiting signs of necrosis. It is overgrown by parallel fibrocytes. Whether this is a non-specific hyperosmotic effect, or indicates enhancement of invasive growth, calls for further investigation.

Chorioallantoic membrane capillary bed: A useful target for studying angiogenesis and anti‐angiogenesis in vivo

“…Growth 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-␣) Olivo et al (1992) Yang andMoses (1990) Transforming growth factor beta (TGF-␤) Wilting et al (1992) Platelet derived growth factor (PDGF) Wilting et al (1992Wilting et al ( , 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 Erythropoietin (EPO) Ramoshebi and Ripamonti (2000) Osteogenic protein-1 (OP-1) Bernardini et al (2000) CC chemokine I-309…”

Induction of Neoangiogenesis in Ischemic Myocardium by Human Growth Factors

“…19,20 The growth of the allantoic systems can be directly observed by light microscopy. After incubation of 20 fertilized hen eggs for 13 days, the growth factor was applied to the membrane and covered with tissue culture coverslips.…”

Fibroblast Growth Factor 2 Activation of Stromal Cell Vascular Endothelial Growth Factor Expression and Angiogenesis

“…FGF-2 has long been known to be a potent activator of angiogenesis in several types of angiogenic models in vivo, including chick chorioallantoic membrane assay (Ribatti et al, 1997;Wilting et al, 1991), rabbit, mouse, and rat corneal pocket assay (BenEzra et al, 1993;DeLisser et al, 1997;Kenyon et al, 1996;Seghezzi et al, 1998;Ziche et al, 1997), and sponge or Matrigel subcutaneous implant models in rodents (Andrade et al, 1997;BenEzra et al, 1993;Fan, 1995a, 1995b;Passaniti et al, 1992). VEGF has also been described as an angiogenic factor in vivo (Asahara et al, 1995;Dellian et al, 1996;Wilting et al, 1993), yet its potency does not appear to equate with FGF-2 in many of the assays used.…”