DEVELOPMENT OF A HIGH-VOLUME SCREEN TO IDENTIFY INHIBITORS OF ENDOTHELIAL-CELL ACTIVATION

We have developed a high throughput screen to identify inhibitors of e ndothelial cell activation using E-selectin cell-surface expression as a marker. Endothelial cell activation is an important component of bo th acute and chronic inflammatory disease. Inhibitors of this process represent potential therapeutic agents, A cell culture system for prim ary human umbilical vein endothelial cells was generated, including an analysis of donor variablity, passage number, seeding density, and co st. The effects of IL-1 beta, TNF alpha, LPS, and an LPS-conditioned p lasma product on E-selectin expression were characterized. E-selectin expression on the surface of IL-1-stimulated endothelial cells was qua ntified with a direct ELISA on fixed cell monolayers. Automation of th e ELISA necessitated identification of methods for cell fixation, liqu id handling, and compound addition which would maintain the integrity of the cell monolayer. The ELISA is inexpensive, reproducible, and sut iable for high throughput primary cell assays, supporting a screening rate of 10,000 compounds/week. The method is compatible with a broad c hemical diversity, and the cellular format provides early information on the cellular uptake and cytotoxicity of compounds. We describe a sc reening paradigm which allowed us to identify inhibitors of endothelia l cell activation and simultaneously discriminate their activity from their cytotoxic effects. (C) 1996 Academic Press, Inc.