PROJECT SUMMARY Retinal neovascularization impairs visual function and is a hallmark of several neovascular eye diseases, including retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR). Diabetic retinopathy (DR) is the leading cause of vision-loss in working age adults, and ROP is the leading cause of preventable blindness in children. Current treatments include intravitreal (IVT) injections of anti-vascular endothelial growth factor (VEGF) biologics. However, these therapeutics are often accompanied by high treatment burden and resistance to therapy. Previous research indicates that VEGF alone is not sufficient for induction of neovascularization, suggesting that multiple disease-relevant pathways may be targeted to increase therapeutic response. Thus, there is a critical need to develop novel therapies that modulate multiple disease- relevant pathways and circumvent the issues with IVT injections. Prior studies indicate that APE1/Ref-1, a multifunctional protein with both endonuclease (APE1) and redox activity (Ref-1), regulates multiple transcription factors that are linked to retinal neovascularization. Preliminary data in the laser-induced choroidal neovascularization model demonstrated upregulation of Ref-1 during neovascularization, and oral administration of a small molecule Ref-1 redox inhibitor decreased disease severity. RNA-seq of human retinal endothelial cells revealed that a Ref-1 redox inhibitor downregulated Wnt signaling genes, implying that Ref-1 redox function modulates neovascularization through the Wnt signaling. But, the underlying molecular mechanisms of the novel Ref-1 target in retinal neovascularization have yet to be elucidated. Identifying the molecular mechanism underlying Ref-1 in retinal neovascularization is critical in developing novel therapies to reduce vision loss. The hypothesis of this proposal is that Ref-1 redox activity promotes induction of retinal neovascularization via activation of the Wnt signaling pathway, and reducing Ref-1 redox activity with a novel Ref-1 redox inhibitor will exhibit potent therapeutic effects via inhibition of the Wnt signaling pathway. Aim 1 will analyze the spatiotemporal expression of Ref-1 in retinal neovascularization in the oxygen-induced retinopathy (OIR) mouse model, the Vldlr-/- mouse model, and in human PDR tissue. Aim 2 will analyze the mechanism of Ref-1 regulation of the Wnt pathway in retinal endothelial cells through functional in vitro assays. Aim 3 will assess anti-angiogenic effects of a Ref-1 inhibitor in vivo by administering oral Ref-1 inhibitor in the OIR mouse model and evaluating changes in neovascularization. Effects on the Wnt signaling pathway will also be assessed using immunofluorescence. Completion of this study will bolster understanding of Ref-1 in neovascular eye diseases and provide a novel therapeutic strategy to combat vision loss and blindness due to these diseases.