ALTERATIONS IN GENE-EXPRESSION ASSOCIATED WITH CHANGES IN THE STATE OF ENDOTHELIAL DIFFERENTIATION

The endothelium maintains a developmental plasticity which allows rapi d phenotypic change in response to extracellular signals during normal processes, such as corpus luteum formation and wound healing, and in pathologic processes, such as tumor angiogenesis. Endothelial cells (E C) in culture have been very useful for investigating various aspects of endothelial growth and behavior. In spite of documented similaritie s between EC in vitro and the endothelium in vivo, many characteristic s of the vessel endothelium are lost when the cells are placed into cu lture. We have undertaken to identify differences in gene expression b etween differentiated vessel endothelium and dedifferentiated EC. We u tilized a new technique called differntial display which compares poly merase chain reaction (PCR)-amplified mRNA from two (or more) cell pop ulations. Endothelium scraped directly from freshly obtained aortas, a nd demonstrated to be free of contaminants, were used as the source of differentiated RNA, whereas proliferating, primary explanted EC grown for five days in the presence of basic fibroblast growth factor (bFGF ) provided a pool of 'dedifferentiated' RNA. Using differential displa y, we have observed numerous reproducible differences in gene expressi on. To confirm that the expression differences visualized by different ial display represented actual differences in gene expression, we isol ated vessel-specific and culture-specific cDNA tags for additional ana lysis. Three cDNA tags specific to vessel endothelium were cloned and sequenced, and compared to nucleotide and protein databases. Two of th e clones (A1 and 2.5) displayed no significant sequence similarity, wh ereas a third clone (A2) is nearly identical to a human expressed sequ ence tag (EST) and has significant sequence similarities to a plant an d Xenopus ubiquitin-like protein. Northern and/or in situ hybridizatio n analysis of the Al and A2 genes confirmed their restricted expressio n to the vessel endothelium. The expression of Al by the endothelium i n vivo is not simply a function of growth state, as cultured cells did not express Al even when grown to postconfluence. One other cDNA frag ment, selected as a culture-induced gene, was identified by sequence a nalysis as the bovine homologue of laminin B1, and Northern analysis c onfirmed that expression was induced upon culturing of EC. Use of diff erential display to study endothelial gene expression will allow us to investigate the molecular mechanisms that underlie initiation and:mai ntenance of endothelial differentiation.