Angiogenesis, the formation of new blood vessels from existing vessels, is a process critical to many normal physiologic processes. However, uncontrolled capillary growth can have dramatic consequences, as seen in the rapid growth of well-vascularized tumors. Indeed, failure to induce a new blood supply will limit the size of a tumor to less than 1-2 mm in diameter. It is clear that controlling angiogenesis could have a major impact on treatment of solid tumors. Using the PCR-based subtraction hybridization technique of Representational Difference Analysis (RDA) we have identified ten genes that are expressed in cultured capillary endothelial cells (EC) forming tubes (neo-capillaries) in collagen gels, but not in EC growing in monolayers. We have isolated five known genes and five novel genes, one of which, A21, is a bHLH-zip transcription factor. Targeted reduction of A21 expression by antisense oligonucleotides blocked tube formation in vitro. Such differentially expressed genes are likely to be critical to EC differentiation into tubes and mature capillaries, and as such may be useful therapeutic targets. Our four aims are: (1) select genes that are differentially expressed in tube-forming EC in culture and in neovessels in vivo; (2) determine the biological function of the candidate genes; (3) design strategies to disrupt gene function and tube formation in vitro; and, (4) test these strategies for their ability to block angiogenesis in vivo. By identifying genes differentially expressed during angiogenesis, and designing effective molecular therapies that reduce their expression and thereby reduce capillary growth in vitro and in vivo, we hope to identify future targets for specific therapy aimed at blocking growth of solid tumors.