Abstract
Abstract: :
Purpose: To determine the potential role of p53 in retinal neovascularization. Methods: In immediate harvest experiments, HUVEC and RF/6A rhesus monkey choroid retinal endothelial cells were challenged with hypoxia for 1, 2, 4, and 8 hours before harvesting of nuclear extracts and cell medium. In post-hypoxic experiments, cells were subjected to 8 hours hypoxia, after which harvesting was done at 0, 24, 48, and 72 hours after hypoxia challenge. Samples taken before hypoxic challenge or at 24-72 hours normoxia were used as normoxic controls. Nuclear extracts were used for detection of p53, mouse double minute-2 (MDM2), and histone deacetylase-1 (HDAC1) proteins by Western Blot. The release of vascular endothelial growth factor (VEGF) and interleukin-6 (IL6) in culture medium was determined by enzyme-linked immunoabsorbent assay (ELISA). Results: In immediate harvest experiments, RF/6A cells incubated for 4-8 h under hypoxic conditions exhibited p53 levels fourfold lower than those of the normoxic control, while in HUVEC cells, only 8 hours of hypoxia was necessary to observe p53 expression. During the same time frame, MDM2 levels increased, while HDAC1 expression remained constant. After 1-4 hours hypoxia, VEGF levels in both cell lines increased by 40-80% above baseline normoxic levels; however, a 40-50% decrease was seen at 8 hours hypoxia. A 50-80% reduction in normoxic IL6 levels was seen at all hypoxic timepoints in both cell lines. In post-hypoxic experiments, an increase in p53 at 24-72 h post-hypoxia was observed in HUVECs when compared to baseline (0 h). A biphasic alteration in VEGF release was seen in the same experiment in which a 20% reduction preceded a six-fold increase at 48 h and 72 h post-hypoxia, respectively, as compared to the baseline level. Lastly, while p53 levels were similar at 72 h under normoxic vs. post-hypoxic conditions, VEGF release in the post-hypoxic sample was five-fold greater. No difference was seen in IL6 release during the above time course, where 72 h normoxic and post-hypoxic medium samples displayed similar IL6 levels. Conclusions: P53 expression decreases in RF/6A cells but increases in HUVECs in response to longer hypoxic time points. P53 levels and VEGF/IL6 release are inversely correlated in HUVECs but not in RF/6A cells. P53 regulation of VEGF/IL6 release in RF/6A cells may be due to the activity of MDM2 but not HDAC1, yet the mechanism by which it negatively regulates each gene may be distinct. In our post-hypoxia studies, VEGF release in HUVECs is higher in spite of an increase in p53, which suggests that other counter acting transcription factor(s) may play a regulatory role in VEGF release.
Keywords: 483 neovascularization • 554 retina • 605 transcription factors