Abstract
Purpose :
Anti-vascular endothelial growth factor intravitreal injections are the current mainstay treatments for retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR), but lack of universal response creates a need to identify new therapeutic targets. One promising target is the redox function of APE1/Ref-1, a bifunctional protein that regulates transcription factors implicated in neovascular eye diseases. We previously demonstrated an increase of Ref-1 expression in the oxygen-induced retinopathy (OIR) mouse model, but the expression of Ref-1 in other mouse models of retinal neovascularization and cell-type localization is unknown. Here, we aimed to assess Ref-1 expression in the Vldlr knock-out (VKO) genetic mouse model of subretinal neovascularization and Ref-1’s cell-type localization in OIR.
Methods :
VKO and wild-type mice underwent optical coherence tomography (OCT), fundus imaging, and fluorescein angiography (FA) before euthanasia at P14, P21, P28, 8 weeks, 3 months, and 6 months old, with pimonidazole injection 2 hours before euthanasia. Retinal and choroidal flatmounts were coimmunostained for Ref-1, vasculature (isolectin B4), and Hypoxyprobe to detect hypoxia via pimonidazole adducts. For OIR, neonatal mice and dams were exposed to hyperoxia (75% O2) or room air for 5 days (P7-P12), with enucleation at P12, P15, and P17. Frozen sections were coimmunostained for Ref-1, isolectin B4, RPE65 (RPE), cone arrestin (cones), rhodopsin (rods), vimentin (Müller glia), and Brn3a (retinal ganglion cells).
Results :
Ref-1 was overexpressed in the VKO mouse model compared to wild-type and colocalized with neovessels. OCT, fundus, and FA revealed that increased Ref-1 expression was associated with development of neovascularization. Mean fluorescence intensity of Ref-1 in VKO eyes was higher compared to age-matched control eyes (p<0.0001, n=8-18, t-test). In OIR, Ref-1 colocalized with endothelial cells and the nuclei of cones, horizontal cells, and retinal ganglion cells.
Conclusions :
Ref-1 is highly expressed in angiogenic tufts in the murine VKO and OIR models, suggesting a role for Ref-1 in retinal neovascularization. Ref-1 colocalizes with the nuclei of several cell types in both healthy and diseased eyes, suggesting regulation of DNA damage and oxidative stress. These results provide context for this potential therapeutic target for PDR and/or ROP.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.