The main aim of this project is to understand the role of vascular endothelial growth factor (VEGF) and the recently discovered VEGF-D in mammary gland biology and tumor development. Both VEGF and VEGF-D are ligands for the mitogenic endothelial tyrosine kinase receptor, KDR. We found that VEGF-D, in contrast to VEGF, is not expressed in human breast cancer but is present in ductal myoepithelial cells and vascular smooth muscle cells of normal breast tissues. Currently, we use transgenic mouse models to characterize the function of VEGF in mammary gland development and carcinogenesis. These studies employ transgenic (MMTV-VEGF) mice and conditional mammary knockout (MMTV-Cre/VEGF-LoxP and WAP-Cre/VEGF-LoxP) mice, which were obtained from NIH and outside investigators as collaborative efforts. Preliminary findings show that VEGF gene inactivation in virgin mice (MMTV-Cre/VEGF-LoxP) results in severe delay in lobulo-alveolar development of the mammary glands and minimal milk production. Studies are ongoing to find out if lack of neovascularization is responsible for the delay in mammary gland development and if WAP-Cre/VEGF-LoxP mice, in which the VEGF gene is not inactivated until late pregnancy, display a different mammary phenotype. Ongoing carcinogenesis studies focus mainly on the early stages of mammary carcinoma development. We study three aspects of VEGF's functions in the transgenic models. First, the role in the switch to an angiogenic phenotype during mammary carcinogenesis. Second, the contribution to tumor stromal formation through its function as a vascular permeability factor. Third, the possibility that it acts as a survival factor for the breast carcinoma cells through an autocrine loop. There is recent evidence that neuropilin-1 is a receptor for VEGF165, and it enhances the mitogenic signal of VEGF through the endothelial KDR receptor. We found that human breast carcinoma cell lines express neuropilin-1, which raises the question if VEGF has autocrine effect on the cancer cells through the neuropilin-1 receptor. This is being addressed in oncogene transformed breast epithelial cells from the VEGF transgenic and knockout mammary glands. We have discovered a new splice variant of neuropilin-1, which lacks the transmembrane and intracellular domains. It will be important to determine if this soluble form of neuropilin-1 inactivates the full-length neuropilin-1, akin to the soluble endothelial flt-1 receptor, which is currently in clinical trials as an inhibitor of angiogenesis.