The goals of this research are to determine the normal functions of type XVIII collagen and its endostatin domain, and to elucidate the molecular mechanisms by which they act. Endostatin has strong potential for use as an anti-angiogenic tumor therapy, however very little is known about the normal functions of it, or type XVIII collagen, from which it is derived. A type XVIII collagen homologue, cle-1, has been identified in the nematode C. elegans and the advantages of this model system will be exploited to analyze its function in development and to identify other molecules with which it may interact. Mutations in cle-1 can cause defects in cell and axon guidance, and may block apoptotic death of certain cells. Ectopic expression of the endostatin domain of cle-1 causes guidance defects and apoptotic death of particular cells. Whether mammalian and C. elegans endostatin domains are functionally equivalent will be tested by comparing the effects of recombinant proteins on migration and apoptosis of human endothelial cells, and on neovascularization of rat corneas. Human endostatin sequences will be expressed in C. elegans to determine if they can rescue cle-1 mutations and cause the expected ectopic phenotypes. The identities of specific cells affected by cle-1 mutations and ectopic expression will be determined using GFP markers and lineage analyses. By controlling the timing and location of ectopic endostatin expression, requirements for it to affect particular cells will be examined. Antibodies will be generated to determine the localization of the three CLE-1 isoforms and to assess how they are affected in mutants. The antibodies will also be used to analyze release of the endostatin domain from CLE-1 and other characteristics of the protein. Additional cle-1 mutations will be generated and characterized. Mutations that enhance or suppress cle-1 mutant or ectopic expression phenotypes will be generated and the genes that they affect identified. These genes may encode molecules that interact with type XVIII collagen and/or endostatin, e.g. other matrix proteins or receptors, or are necessary for their normal function, e.g. signaling pathway. Further studies of these genes may identify the mechanisms by which type XVIII collagen and endostatin exert their effects. The results of these studies may suggest possible side effects of endostatin treatment, that should be carefully monitored, and potential means to augment its effectiveness.
