Mechanically altered embryonic chicken endothelial cells change their phenotype to an epithelioid phenotype

Monolayers of retracted endothelial cells exhibiting wounds or zones denuded of cells were obtained from aortic explants from 10- to 12-day-old chicken embryos. Using time-lapse videomicroscopy, we investigated the sequence of events that occurred both during and after closure of the monolayer wounds. Such wound closure (re-endothelialization process) occurred 4-12 hr after removing the explants, depending on wound width and presence of serum. The cells from along the wound edges appeared to move toward one another. We suggest an important role for bFGF and TGFbeta-2 and -3 during this process. Twenty-five hours after removal there were still some areas of retracted cells, and many of the cells displayed a weak von Willebrand's Factor (vWf) immunoreactivity. Surprisingly, after 63-65 hr many of the endothelial cells had become epithelioid in shape and the vWf immunoreactivity appeared increased. This epithelioid phenotype is currently considered typical of cultured vascular non-muscle-like cells and intimal thickening cells. By 5-7 days, the vast majority of cells in the monolayer had acquired an epithelioid morphology, showing a cobblestone appearance. These cells were significantly smaller than polygonal cells. Most importantly, they showed strong vWf immunoreactivity. At the edge of the monolayers we found that the majority of the cells had become epithelioid. Some of them detached from their neighbors and became round in shape and acquired mesenchymal characteristics, some expressing smooth muscle alpha-actin (SM alpha-actin). These findings demonstrate not only that embryonic endothelial cells that are transiently mechanically altered may change their phenotype to an epithelioid phenotype, but also that these cells may eventually transdifferentiate into mesenchymal cells expressing SM alpha-actin. Since some aspects of endothelial cell behavior have been shown to be regulated by locally released growth factors such as TGFbeta and FGF, we also investigated TGFbeta-2 and -3 and bFGF expression. Presence of TGFbeta-2 and -3 and bFGF-immunoreactive epithelioid and mesenchymal cells indicates that these growth factors may be involved in the changes described.