News and Publications

Combinatorial Search of Angiogenic Endothelium

C. Liu, J. Yao, E. Miyagi,* T. Edgington

* Yokohama City University, Yokohama, Japan

The patterns of differential gene transcription and cell-surface molecular display of the microvascular endothelia of tumors are minimally known. To address the repertoires of endothelia of different organs, different functional states, and different pathologic states, we developed methods for in vivo selection of phage carrying various linear and constrained combinatorial libraries on the N-terminus of protein III. Using 3 different types of combinatorial libraries, we have detected amino acid sequences that may indicate novel expressed vascular surface molecules. We anticipate that such molecules modify the functional properties of the endothelia and thus account for the differences between angiogenic and resting vascular endothelium.

We are using murine tumors in rats and mice and human tumors in mice with severe combined immunodeficiency to narrow down the set of phage-display sequences recovered from the microvasculature of tumors. We are selecting those novel sequences that are not species restricted and thus may indicate general differentiation patterns for angiogenesis.

Combinatorial Search of Atherosclerotic Vessels

C. Liu, J. Yao, T. Edgington

To explore the lumenal surface of the vascular endothelium in vivo, we adopted a phage-display technology in which a combinatorial set of nucleotides that encodes 7 amino acids was placed within a pair of bordering cysteines for constraint purposes. The library was administered to mice in which the gene for apolipoprotein E had been inactivated; the mice had also been fed a high-fat diet to accelerate the development and increase the severity of atherosclerotic lesions of the aorta. The mice were preloaded with wild-type phage and then were injected with the library phage. After equilibration of the library phage, the mice were sacrificed and perfused to remove free phage. Phage were recovered from the aortas of the atherosclerotic mice and amplified for the next round of sequential selection in vivo. In subsequent rounds, phage were recovered from the lesions and repeatedly selected in vivo.

Sequence analysis of the combinatorial library inserts revealed 93 motifs containing 7 amino acids; 4 of the motifs were repeated among 99 recovered phage clones. These motifs have interesting similarities to EGF domains and certain other domains found in proteins. These "probe" peptidyl sequences are being further characterized. We are determining their binding to endothelial surface proteins in vivo and the complementary proteins that differ between atherosclerotic and normal aortic endothelium.

PUBLICATIONS

Huang, M., Syed, R., Stura, E.A., Stone, M.J., Stefanko, R.S., Ruf, W., Edgington, T.S., Wilson, I.A. The mechanism of an inhibitory antibody on TF-initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF-5G9 complex. J. Mol. Biol. 275:873, 1998.

Shoji, M., Hancock, W.W., Abe, K., Micko, C., Casper, K.A., Baine, R.M., Wilcox, J.N., Danave, I., Dillehay, D.L., Matthews, E., Contrino, J., Morrissey, J.H., Gordon, S., Edgington, T.S., Kudryk, B., Kreutzer, D.L., Rickles, F.R. Activation of coagulation and angiogenesis in cancer: Immunohistologic localization in situ of clotting proteins and vascular endothelial growth factor in human cancer. Am. J. Pathol. 152:399, 1998.

Yao, J., Mackman, N., Edgington, T.S., Fan, S.-T. Lipopolysaccharide induction of the tumor necrosis factor- promoter in human monocytic cells: Regulation by Egr-1, c-Jun and NF-B transcription factors. J. Biol. Chem. 272:17795, 1997.