VEGF(121) 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 VEGF(121) homodimer and VEGF(121/165) he terodimer on the chorioallantoic membrane (CAM) of 13-day-old chick em bryos. The factors were applied in doses of 2-4 mu g and the effects w ere evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was stu died according to the BrdU-anti-BrdU method on whole mounts and sectio ns. The labeling density was quantified in whole mounts. The fractal d imension, D, of the vascular tree was assessed as a value for vascular bifurcation density. Both forms of VEGF induce brush-like vessel form ation in the precapillary region. New capillaries are found in the str oma of the CAM, which normally does not contain capillaries. Our resul ts show that VEGF(121) is a specific endothelial cell mitogen. A fourf old increase of BrdU-labeled endothelial cells is found after VEGF(121 ) application. The fractal dimension of the vascular tree increases fr om 1.26 in the controls to 1.44 (VEGF(121)) and 1.41 (VEGF(121/165)). The endothelial cells of the newly formed capillaries possess many mit ochondria and micropinocytotic vesicles, but no fenestrations. These c apillaries are obviously formed by intussusceptive microvascular growt h. Signs of sprouting are almost absent. An effect on the lymphatic ve ssels of the CAM is not detectable. Compared to VEGF(165) and VEGF(121 /165), VEGF(121) diffuses over a slightly greater distance. Using in s itu hybridization, VEGF receptor-2 (flk-1/Quek1) and the homologous fl t-4 (Quek2) receptor were studied in the CAM of normal quail embryos a nd after VEGF(121) application on the CAM of 11-day-old quail embryos. During normal development, flk-1 expression becomes restricted to vas cular endothelial cells of large vessels in the stroma of the CAM. VEG F(121) application induces expression of flk-1 in capillaries that nor mally do not express the receptor. In the normal development of the CA M, flt-4 becomes restricted to endothelial cells of vessels that appea r to be lymphatic vessels. Application of VEGF(121) does not alter flt -4 expression. (C) 1996 Academic Press, Inc.