Apoptosis of the hyaloid vessels in vivo and in vitro in the newborn rat eye

Abstract

Purpose: In the newborn rat eye, the hyaloid vasculature persists after birth and regresses postnatally. Several studies have shown that apoptosis plays a role in this regression although the mechanism is unknown. Apoptosis may result due to a decrease in blood flow to the hyaloid vessels after retinal vascularization because cells are deprived of nutrients. After retinal vasculature development, a dramatic increase in the rate of apoptosis might be expected due to decreased hyaloid blood flow. In order to test this hypothesis, we evaluated the number of apoptotic cells in vivo at varying postnatal times and subjected eye endothelial cells to serum starvation as well as treatment with apoptosis inducers such as staurosporine, camptothecin, and actinomycin D. Methods: TREE (Transformed Rat Eye Endothelial) cells from a 7 day old rat were immortalized using an adenovirus E1A containing vector. Apoptotic cells in vivo and in vitro were identified by the TUNEL staining method, by Hoechst's and propidium iodide staining followed by observation of apoptotic bodies using the fluorescence and the confocal microscope. Results: In vivo results showed small numbers of apoptotic vascular eye cells in days 1–12 following birth with no increase occuring after retinal vascularization. Studies of the TREE cell line showed serum withdrawal to be a poor inducer of apoptosis with less than 5% apoptotic cells present after two days of serum withdrawal and about 30% after one week of serum deprivation. In order to test whether the TREE cell line was resistant to apoptosis, staurosporine, actimomycin D or camptothecin was added. Each of these known apoptotic inducers was able to cause 90–100% apoptosis in the TREE cell line within 1–2 days. Conclusions: These results suggest that the TREE cell line is not resistant to inducers of apoptosis; it just does not respond to serum withdrawal by undergoing apoptosis. These results may explain the lack of an apoptosis burst in hyaloid endothelial cells in vivo even when such cells are subjected to growth factor deprivation due to decreased blood flow. The results are also consistent with the hypothesis that some endothelial cells undergo apoptosis when deprived of serum and nutrients, but only a small fraction of the cell population undergoes nuclear fragmentation and death as blood flow decreases. It may take one month for the complete regression of the hyaloid vessels in the rat. This slow regression may be due to nutrient deprivation being a poor inducer of apoptotic death in eye endothelial cells.