VEGF121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of the chorioallantoic membrane

We have studied the effect of VEGF121 homodimer and VEGF121/165 heterodimer on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. The factors were applied in doses of 2-4 μg and the effects were evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was studied according to the BrdU - anti-BrdU method on whole mounts and sections. The labeling density was quantified in whole mounts. The fractal dimension, D, of the vascular tree was assessed as a value for vascular bifurcation density. Both forms of VEGF induce brush-like vessel formation in the precapillary region. New capillaries are found in the stroma of the CAM, which normally does not contain capillaries. Our results show that VEGF121 is a specific endothelial cell mitogen. A fourfold increase of BrdU-labeled endothelial cells is found after VEGF121 application. The fractal dimension of the vascular tree increases from 1.26 in the controls to 1.44 (VEGF121) and 1.41 (VEGF121/165). The endothelial cells of the newly formed capillaries possess many mitochondria and micropinocytotic vesicles, but no fenestrations. These capillaries are obviously formed by intussusceptive microvascular growth. Signs of sprouting are almost absent. An effect on the lymphatic vessels of the CAM is not detectable. Compared to VEGF165 and VEGF121/165, VEGF121 diffuses over a slightly greater distance. Using in situ hybridization, VEGF receptor-2 (flk-1/Quek1) and the homologous flt-4 (Quek2) receptor were studied in the CAM of normal quail embryos and after VEGF121 application on the CAM of 11-day-old quail embryos. During normal development, flk-1 expression becomes restricted to vascular endothelial cells of large vessels in the stroma of the CAM. VEGF121 application induces expression of flk-1 in capillaries that normally do not express the receptor. In the normal development of the CAM, flt-4 becomes restricted to endothelial cells of vessels that appear to be lymphatic vessels. Application of VEGF121 does not alter flt-4 expression, © 1996 Academic Press, Inc.