Blocking or inhibiting blood vessel growth (angiogenesis) remains a critical focal point for anticancer drug development. Studies on Vascular Endothelial Growth Inhibitor (VEGI; TNFSF-15) secreted from human endothelial cells have demonstrated that it is a potent negative regulator of angiogenesis that induces growth arrest and apoptosis in endothelial cells. Unfortunately, because of a limitation in the quantity of VEGI obtained in this manner and the difficulty encountered in the production of the recombinant form of this protein, preclinical studies using systemically administered VEGI to assess its therapeutic potential as an anti-cancer or anti-angiogenesis agent have not been possible. In addition, despite a considerable effort, no commercial or academic laboratory has succeeded in producing large quantities of this valuable molecule. We have now developed a procedure to produce VEGI in E. coil. In this proposal, three specific objectives will be pursued: 1. In a small scale pilot study, a number of expression vector constructs encoding variants of the four isoforms of VEGI will be expressed in E. coil as inclusion bodies, purified, and refolded. They will then be tested in endothelial cell growth arrest assays to isolate refolded, biologically potent VEGI isoforms. 2. Expression, purification, and refolding of isoforms of VEGI already identified as biologically active in pilot studies will be pursued at a much larger scale. One hundred milligrams to gram quantities of highly purified biologically active VEGI isolated from E. coli inclusion bodies will be prepared for in vivo animal studies. 3. The active isoforms of VEGI will be utilized in rodent xenograft studies to determine if VEGI has a potent antiangiogenic or anticancer effect when systemically administered. The long term objective of this study is to develop VEGI into an effective drug in diseases involving angiogenesis - particularly cancer, but also atherosclerosis and blindness (macular degeneration).
