Retinopathy of prematurity (ROP) is a neovascularizing disease that causes blindness in premature infants. ROP is initiated by an arrest of normal retinal blood vessel development. Subsequently, the non-perfused peripheral retina becomes hypoxic leading to retinal neovascularization. Vascular endothelial growth factor (VEGF) has been implicated in playing a role in the pathogenesis of ROP. HIF-1 is a heterodimeric DNA binding protein consisting of an α and β subunit. HIF-1 protein expression and DNA binding increases in response to hypoxia. HIF-1 binding sites are present in several hypoxia inducible genes and HIF-1 has been implicated in the activation of VEGF transcription in response to hypoxia. It is therefore possible that HIF-1 plays a role in the pathogenesis of ROP.

We have shown that both HIF-1 α and β mRNA and HIF-1 α protein are expressed in the eyes of fetal and neonatal mice at various stages of gestation. In order to determine HIF-1 DNA binding in response to hypoxia in retinal cells, gel shift assays were done using human retinal pigment cells and bovine reticuloendothelial cells. Plates of confluent cells were exposed to either 21% or 1% oxygen for four hours and nuclear protein was extracted. Gel mobility shift assays were done using a P-32 labeled 18 bp oligonucleotide probe bearing the HIF-1 binding motif. HIF-1 DNA binding was seen specifically in hypoxic extracts of cells. HIF-1 DNA binding activity was inhibited by addition of a 20 fold molar excess of cold oligonucleotide. We hypothesize that during the vaso-obliterative phase in ROP an increase in HIF-1 activity in retinal cells occurs in response to hypoxia. This leads to an increase in VEGF transcription ultimately causing neovascularization.