Purpose: Fluctuations in oxygen are associated with the development of severe retinopathy of prematurity (ROP) in humans. However, the causal relationships between oxygen variability and severe ROP remain unknown. We investigated whether isoforms of vascular endothelial growth factor (VEGF) were differentially stimulated by hypoxia and by repeated fluctuations between hypoxia and hyperoxia, and whether isoforms were differentially expressed in association with intravitreous neovascularization. We also determined whether pigment epithelium-derived factor (PEDF) was dysregulated by oxygen fluctuations perhaps contributing to a delay in normal retinal vascular development.
Methods: We used the 50/10 oxygen-induced retinopathy (50/10 OIR) model that exposes newborn rat pups to repeated cycles of 24 h of 50% oxygen alternating with 24 h of 10% oxygen to cause a condition similar to human ROP. Animals were euthanized at postnatal day 2 (P2; after one cycle of 50/10% oxygen), P7 (after 3.5 cycles of 50/10% oxygen), and P14 (after seven cycles of 50/10% oxygen). Room air raised control rat pups were also exposed to a single episode of 24 h of hypoxia at P7 and P14 and assayed immediately afterwards. Retinal VEGF isoforms and PEDF were measured by RT-PCR. Total VEGF protein was measured by ELISA.
Results: We found that repeated cycles of hyperoxia and hypoxia caused greater expression of VEGF protein compared to control than did a single cycle of hyperoxia and hypoxia. VEGF164 mRNA had a greater fold change over control after repeated oxygen fluctuations than after a single episode of hypoxia. However, the other isoforms, VEGF188 and VEGF120, were expressed to a similar degree regardless of whether the stimulus was a single episode of hypoxia or repeated fluctuations in oxygen. VEGF164 was the predominant isoform expressed at the time of maximal intravitreous neovascularization. Retinal PEDF expression was elevated in pups in the 50/10 OIR model compared to control at P7, immediately after 50% oxygen. PEDF expression in the experimental group was similar to control at P18, when intravitreous neovascularization occurred.
Conclusions: Repeated fluctuations in oxygen results in a greater expression of the pathologic isoform, VEGF164, than does hypoxia alone. However, the other isoforms were upregulated to an equivalent degree over control by repeated fluctuations in oxygen or a single episode of hypoxia. Total VEGF protein was increased to a greater degree by repeated fluctuations in oxygen compared to a single cycle of oxygen. PEDF was increased over control early in the 50/10 OIR model and may play a role in the observed delay in retinal vascularization. These findings provide insight into the effect of repeated oxygen fluctuations on the development of severe ROP in preterm infants.