Preservation of differentiated phenotypes in cultured aortic endothelial cells by malotilate and phosphoascorbic acid

Confluent bovine aortic endothelial cells (BAECs) formed a cobblestone-shaped cell monolayer when cultured on a collagen gel in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's-F12 supplemented with 5% newborn calf serum. Within a few days, however, they lost cell-cell attachment and became fibroblastic. When BAECs were cultured in the same culture medium but further supplemented with either 10 micrograms/ml malotilate or 1 mM phosphoascorbic acid, the monolayer organization and the cobblestone-like cell morphology were maintained for more than 2 weeks although many sprout cells were observed underneath the monolayer. In contrast, if both malotilate and phosphoascorbic acid were present in the culture medium, a tight monolayer without underlying sprout cells was maintained for at least 4 months and the cells expressed factor VIII-related antigens and massively internalized acetylated low density lipoprotein. By electron microscopy, we observed well-developed gap and adherence junctions, Golgi apparatuses and vesicles many of which were open to the outside by fusing with either the apical or the basal surface, indicating high metabolic activity of the cells cultured for weeks in the same dish. Although malotilate-treated BAEC monolayers secreted increased levels of prostacyclin (PGI2), the drug did not appear to directly affect the PGI2 production pathway since the similarly increased PGI2 production was noted in tight monolayers formed without the use of malotilate. Our results indicate that malotilate and phosphoascorbic acid together preserve differentiated phenotypes in cultured endothelial cells.