Tree eye endothelial cells undergo apoptosis after serum starvation

Abstract

Purpose: Several explanations have been suggested for why endothelial cells undergo apoptosis in the regressing blood vessels found in the newborn rat eye. These explanations hypothesize that apoptosis occurs due to lack of blood flow causing nutrient and/or growth factor deprivation, that cell death is due to deficiency of survival/growth factors such as VEGF or that the endothelial cells are actively killed by macrophages or other cytotoxic cells. In order to investigate the role of apoptosis in blood vessel regression, we made a cell line from the eyes of seven day old rats. This TREE (Transformed Rat Eye Endothelial) cell line was subjected to deprivation of serum and growth factors and apoptotic cells enumerated to determine whether these conditions induced apoptosis. Methods: TREE (Transformed Rat Eye Endothelial) cells were isolated from the eyes of seven-day-old newborn rats. Eye tissue explants containing blood vessels were plated on tissue cultures plates and transformed using an adenovirus E1A containing vector. Endothelial cells were identified by uptake of and staining by di-I-LDL. The resulting cell line was cloned. Apoptotic cells were identified both by TUNEL staining and by observation of apoptotic bodies. Apoptotic cells were also observed for the presence of caspases by total caspase staining. Mitochondrial membrane permeability was measured using a Mito-Tag dye assay. Results: In TREE cells, deprived of serum for 1-5 days, the number of apoptotic cells increased linearly for days 1-3. By days 4 and 5 of serum deprivation, some cells became fragile and detached from the substrate making it difficult to quantitate apoptosis. Increasing amounts of activated caspases were detected by day 2 and caspase activity continued to increase on subsequent days. Collapse of the mitochondrial electrochemical gradient was also seen in serum-deprived cells using the Mito-Tag fluorescent dye assay. Conclusion: Apoptosis is known to play a significant role in regression of the hyaloid blood vessels in the neonatal rat. Thus it is important to determine what conditions signal the onset of apoptosis. Our results are consistent with the hypothesis that endothelial cells undergo apoptosis when deprived of serum and nutrients. Thus, lack of blood flow may cause endothelial cell apoptosis and this may eventually lead to regression of hyaloid blood vessels.