Nutrient deprivation may be a poor inducer of apoptosis in the newborn rat eye

Abstract

Purpose: The hyaloid vasculature of the newborn rat eye persists after birth and regresses postnatally. One hypothesis is that regression occurs due to a decrease in blood flow to the hyaloid after retinal development and vascularization. This decrease in blood flow may deprive cells of nutrients causing endothelial cell apoptosis. Since the retinal vasculature develops in the week following birth, a dramatic increase in the rate of apoptosis might be expected if competition for blood flow occurs. 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. Methods: TREE (Transformed Rat Eye Endothelial) cells were used. Apoptotic cells in vivo were identified both by TUNEL staining and apoptotic bodies. Apoptotic cells in vitro were enumerated by TUNEL staining, apoptotic bodies, Mito-Tag dye assay and caspase staining. Results: While apoptotic cells were found in vivo, there was not the dramatic increase expected after one week when the retinal vasculature was functional and competing with the hyaloid for blood flow. Similarly in vitro, there was not a lot of apoptosis (as judged by apoptotic bodies) in TREE cells that were deprived of serum for 1-5 days. By days 4 and 5 of serum deprivation, cells were markedly elongated and some had undergone collapse of their mitochondrial electrochemical gradient. Although serum deprivation resulted in decreased viability, many cells were still viable after 14 days of serum deprivation as shown by their proliferation after serum was added back. Addition of staurosporine or camptothecin to TREE cells resulted in complete loss of the mitochondrial permeability barrier and 100% development of apoptotic bodies after 24 and 48 hours incubation respectively. Conclusions: Our results show that serum starvation /growth factor deprivation does not cause a great amount of apoptosis in the TREE cell line whereas staurosporine and camptothecin are excellent apoptotic inducers. 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.