PROLIFERATION PATTERN OF CAPILLARY ENDOTHELIAL-CELLS IN CHORIOALLANTOIC MEMBRANE-DEVELOPMENT INDICATES LOCAL GROWTH-CONTROL, WHICH IS COUNTERACTED BY VASCULAR ENDOTHELIAL GROWTH-FACTOR APPLICATION

The density and distribution of whole mount BrdU-anti-BrdU labeled end othelial cells (days 6-15) in the chick chorioallantoic membrane (CAM) was analyzed with computer-assisted microscopy. A significant loss of proliferative activity was noted after day 10: the density of labeled nuclei (in 10(-2) mm(-2)) decreased from a median 7.78 (days 6, 8, 10 ) to 2.42 (days 12, 14, 15). CAMs initially showed random patterns of labeled endothelial cells, but changed to clearly focal patterns after day 12. A regular arrangement of labeled nuclei was never seen. After application of vascular endothelial growth factor (VEGF) to the day 1 3 CAM, a significant increase in proliferative activity (11.50) and a random distribution of labeled endothelial cells was observed on day 1 5. Development of CAM precapillary vessels was assessed in terms of le ngth density (in mm(-1), mean +/- standard deviation), which was augme nted threefold from day 6 (1.22 +/- 0.05) to day 14 (3.54 +/- 0.23) an d then remained nearly constant. VEGF application from day 13 to 15 ra ised arterial length per unit area to 4.53 +/- 0.77. It is concluded t hat normally a local regulation of endothelial proliferation and diffe rentiation develops in the CAM, which doubles capillary endothelial ce ll density but simultaneously adapts to the decreasing need for endoth elial cells, and thus maintains the quasi two-dimensional vessel patte rn. However, proliferative foci persist in the capillary layer after d ay 10, and precapillary vessel density continues to increase until day 14. VEGF enhances DNA synthesis in all capillary endothelial cells. T his leads to the disappearance of proliferative foci, to multiple capi llary layers, and to an excessive formation of precapillary vessels. W e propose that CAM endothelial cell proliferation is regulated by fact ors like endothelial cell density and extension. The proliferative pat tern in capillaries, and the length density of precapillary vessels sh ould be used for the evaluation of angiogenesis in the CAM assay. We r ecommend the CAM after day 12 for evaluating putative angiogenic mitog ens. (C) 1995 Wiley-Liss, Inc.