Abstract
Purpose::
Retinopathy of prematurity (ROP) is a leading cause of blindness in children. The mouse model of oxygen induced retinopathy (OIR) is an invaluable tool for the study of this complex disease. Our group has previously identified novel human retinal specific isoforms of the transcription enhancer factor (RTEF-1). We have demonstrated that these isoforms are capable of modulating gene expression from the VEGF promoter using an in vitro reporter assay. Whether RTEF-1 plays a role in the retina in vivo is unknown. We aim to investigate whether RTEF-1 isoforms play a role within the eye during OIR in mice.
Methods::
Postnatal day 7 (P7) C57BL/6 (B6) mice were exposed to 75% oxygen for 5 days, after which they were removed from high oxygen and allowed to recover in room air. Both the hyperoxia exposed and normoxic control mice were sacrificed at time points P3, P7, P8, P10, P12, P14, P17, P21 and P24. Eyes were enucleated and the neural retina dissected. Protein and RNA were isolated and used for semi-quantitative RT-PCR and Western blot analysis. Amplified PCR products from P3, P7, P8, P17 controls and P17+O2 exposed were purified and sequenced. A mouse specific antibody was generated to the N-terminus of RTEF-1 and was used for immuno-precipitation and Western blotting.
Results::
Semi-quantitative RT-PCR demonstrated the presence of several RTEF-1 isoforms. Up regulation of the full length isoform was observed in OIR mice at P10, P12, and P21 compared to the control group. Sequence of 6 amplified products provided evidence of differential RTEF-1 splicing and identification of 4 novel isoforms. The 6 isoforms sequenced revealed exon splicing in which various predicted protein motifs are spliced or truncated; interestingly, nuclear localization signals and activation domains were among these processes. Immuno-precipitated RTEF-1 isoforms analyzed by Western analysis displayed changes in expression patterns of a 36kD isoform at P14 and P17. Proteins viewed by Western analysis appear to correlate in size to the predicted molecular weights of the sequenced isoforms.
Conclusions::
The events that lead to aberrant vessel growth and retinal pathology in this model are extremely complex and have not been fully elucidated. Up regulation of the VEGF gene is believed to be a major contributor to pathogenesis. We have previously determined that RTEF-1 isoforms are capable of influencing VEGF promoter activity in human cells and now show that various isoforms are present in the mouse retina in vivo. The study of the molecular mechanisms which controls these events is imperative to fully understanding this disease process.
Keywords: retinopathy of prematurity • ischemia • choroid: neovascularization