Synthesis and distribution of cytoskeletal elements in endothelial cells as a function of cell growth and...

Confluent cultures of adult bovine aortic endothelial (ABAE), corneal endothelial (BCE), and fetal bovine heart endothelial (FBHE) cells form a monolayer of highly flattened, closely apposed, and nonoverlapping cells. In ABAE and BCE cultures, this is associated with a 50-fold decrease in the rate of DNA sythesis and correlates with a 14-fold decrease in protein synthesis. In contrast, in confluent FBHE cultures only partial decreases in the rates of DNA synthesis (6-fold) and protein synthesis (3-fold) are observed. FBHE cells therefore fulfill the morphological, but not the biochemical, criteria for confluent cultured endothelial cell monolayers. The appearance of the cytoskeletal elements actin, tubulin, and vimentin in sparse and confluent cultures of endothelial cells has been analyzed by two-dimensional gel electrophoresis and immunofluorescence. Sparse versus confluent ABAE, FBHE, and BCE cultures showed no changes in their relative rates of synthesis or cellular content of tubulin. Actin behaved similarly to tubulin in FBHE and BCE cultures, while in ABAE cultures a small increase (3-fold) in its relative rate of synthesis was observed in confluent versus sparse cultures. BCE cultures showed no change in the rate of synthesis of vimentin, but the cellular content of vimentin was markedly increased when cultures reached confluence. When the distribution of vimentin in both sparse and confluent BCE cultures was analyzed by immunofluorescence, in both cases it appeared distributed throughout the cytoplasm as thin fibers and bundles of fibers. In confluent ABAE cultures, both the relative amount and biosynthetic rate of vimentin increased by 15-fold. This increase in the intracellular accumulation of vimentin correlated with its immunofluorescent distribution within the cells. While in sparse cultures, vimentin appeared to be distributed as thin fibers, in confluent cultures thick curl-like fibrous bundles could be seen distributed throughout the cytoplasm and organized in a perinuclear ring. In contrast, in FBHE cultures no significant changes in the distribution and organization or rate of synthesis of vimentin were observed.