Evidence for cell death in the vascular endothelium in vivo and in vitro

Focal, spontaneous cell death in the rat aortic endothelium was demonstrated by cytochemistry. Cells with intracellular calcium deposits, indicating cell death with mitochondrial calcification, were identified by chlorotetracycline fluorescence. The same cells also contained cytoplasmic IgG, which binds to cytoskeletal components of the dead cell. The immunocytochemical detection of IgG in en face preparations was used as a quantitative method for detecting cell death in the aortic endothelium. The use of an indirect immunoperoxidase technique and "Häutchens" of paraformaldehyde-fixed tissue provided high sensitivity and cellular recovery with low background. A cell death frequency of 0.19% +/- 0.04% was observed in 5-month-old Sprague-Dawley rats. When compared with the replication rate of aortic endothelium in these animals, the data suggest that dead cells remain in the endothelium for more than 24 hours. This conclusion was supported by in vitro studies. Confluent cultures of bovine aortic endothelium were pulsed with trypan blue, and the residence time of blue cells was 3.5-4 days in non-flow culture system. Time-lapse video microscopy showed a prolonged cell death process with a phase of rapid intracellular movements, followed by undermining by surrounding cells and fragmentation of the dead cell. Migration of surrounding cells rapidly covered partial detachments of the dead cell, so that no holes could be detected in the monolayer when the dead cell finally detached. It is concluded that the normal turnover of cells in the aortic endothelium involves a prolonged phase of in situ cell death and finally detachment with very little or no exposure of subendothelial structures.