Our main focus is on the founding member of the vascular endothelial growth factor(VEGF) family, VEGF, which is a major angiogenic factor is human malignancies, including breast cancer. Recent findings suggest that VEGF supports cancer growth not only by stimulating angiogenesis, but also by acting as a survival factor for the cancer cells through neuropilin-1, a receptor for the 165 isoform of VEGF. A newer member of the VEGF family, VEGF-D, stimulates vascular and lymphatic endothelial cell growth but its function in the mammary gland is unknown. The goals of our project are:1) Characterize the role of VEGF in mammary gland development and carcinogenesis, using transgenic mouse models with either overexpression or conditional knockout of VEGF in mammary epithelial cells; 2)Understand the mechanism by which VEGF acts as a survival factor for breast carcinoma cells; 3) Define the role of VEGF-D in mammary gland biology. Findings: 1. Mammary carcinogenesis studies in the VEGF overexpressing mice showed marked reduction in the tumor latent period, increased tumor neovascularization,and induction of neuropilin-1 and its co-receptor plexin-A1 mRNA in the carcinomas.New findings were observed in mice with conditional knockout of VEGF at different stages of mammary gland development. Inactivation of the VEGF gene in mammary epithelial cells at puberty in VEGF-LoxP/ MMTV-Cre mice resulted in a severe delay in lobulo-alveolar development and almost complete shutdown of lactation. Inactivation of the VEGF gene in late pregnancy in VEGF-LoxP/WAP-Cre mice resulted in apoptosis of lobulo-alveolar epithelial cells, which does not occur normally until the pups are weaned and the mammary gland undergoes involution. These findings of premature apoptosis suggest that VEGF acts as a survival factor for mouse mammary epithelial cells. If VEGF also serves as a survival factor for human breast epithelial cells, which undergo frequent physiological cycles of apoptosis, it may play an important role in the pathogenesis of benign proliferative diseases of the breast. 2. Neuropilin-1 was expressed in different human breast carcinoma cell lines with the highest levels of both mRNA and protein expression in the poorly differentiated MDA-MB-231 cells. Confocal microscopic examination of immunostained human breast carcinomas showed that neuropilin-1 and plexin-A1 co-localize in the breast carcinoma cells. Based on these observations, together with data showing induction of neuropilin-1 and plexin-A1 in mammary carcinomas of the MMTV-VEGF transgenic mice, our working hypothesis is that neuropilin-1 and plexin-A1 form a functional receptor complex in breast carcinoma cells as has been described for neuronal cells. This receptor complex may therefore by important for cell motility and survival. We discovered a novel splice variant of neuropilin-1, which lacks the transmembrane and intracellular domains. Ongoing efforts examine if the soluble neuropilin-1 variant interferes with the binding of VEGF to the full-length neuropilin-1. The soluble neuropilin-1 may therefore have therapeutic significance, both as an inhibitor of angiogenesis and by blocking the autocrine effects of VEGF on cancer cells. 3. Our studies of VEGF-D have yielded two novel findings. First, that VEGF-D is expressed in vascular smooth muscle cells and ductal myoepithelial cells of normal human breast tissues but was not expressed in breast carcinomas. Second, that VEGF-D stimulates growth of vascular smooth muscle cells. Ongoing effeorts focus on identifying the receptor involved in the VEGF-D-mediated stimulation of smooth muscle cells replication. In vitro studies showed that VEGF-D mRNA is only detectable by RT-PCR and that it is not stimulated by hypoxia or cytokines known to regulate VEGF.