A novel early chorioallantoic membrane assay demonstrates quantitative and qualitative changes caused by antiangiogenic substances

The chicken chorioallantoic membrane (CAM) has been extensively used in the study of angiogenesis. However, the CAM assay can be difficult and time-consuming to quantify, provides poor quality images of the results, and is not very reproducible. In this study, a novel early CAM assay was developed: It was found to be quantitative through relatively simple methods, enabled high-quality imaging of results, and was reproducible. Additionally, unique qualitative changes in vessel structure were observed, and it was possible to measure veins and arteries separately. Treatment of the CAM on days 4 and 5 with SU5614, suramin, fumagillin, amiloride, and PI-88 reduced blood-vessel growth. SU5614 (4 mug) resulted in significant reductions in artery but not vein length (60% and 111%, respectively, vs control). Suramin tended to increase CAM vasculature at 50 mug but caused dramatic reductions both in vessel length and CAM growth at 100 mug. As with SU5614, the effect was greater with regard to arterial compared with venous length (49% and 74%, respectively, vs control). PI-88 (20 mug) also decreased artery and vein length (66% and 80%, respectively, vs control). In contrast, fumagillin (5 mug) and amiloride (20 mug) both reduced arterial growth slightly less than venous growth (67% and 54% and 50% and 44%, respectively, vs control). Each antiangiogenic substance caused a different qualitative pattern of change in vessel branching and structure. The early CAM assay will be useful in the screening of antiangiogenic substances. Further study of the qualitative effects of antiangiogenic treatments may be a valuable tool to increase our understanding of the angiogenic process itself.