Abstract
Purpose :
Current intravitreal injections available for treatment of proliferative diabetic retinopathy (PDR) face variability in patient response, necessitating the exploration of alternative therapeutic targets. A promising candidate is APE1/Ref-1, a multifunctional protein that regulates transcription factors implicated in neovascular eye diseases by modulating redox status. An oral Ref-1 inhibitor APX3330 will be entering phase III clinical trials for DR and diabetic macular edema (DME). But the mechanism of Ref-1 in retinal neovascularization remains unclear. Canonical Wnt signaling activation is observed in several mouse models of retinal neovascularization, with anti-angiogenic treatments downregulating Wnt signaling components. We previously identified several downregulated Wnt genes after treatment of human retinal endothelial cells (HRECs) with Ref-1 redox inhibitors, including FZD1, FZD4, TCF7, and LRP5. We aimed to assess the potential of Ref-1 redox inhibitors to modulate retinal neovascularization via inhibition of the Wnt signaling pathway.
Methods :
HRECs were transfected with siRNA for Ref-1 or scrambled siRNA, and expression of Wnt signaling genes was assessed by qPCR. HRECs were treated with Ref-1 redox inhibitors APX3330 or APX2009, and protein expression of Wnt signaling components was assessed by immunoblot. HRECs were stimulated with Wnt3a and treated with Ref-1 redox inhibitors, scrambled siRNA, or siRNA for Ref-1. Nuclear localization of β-catenin was assessed by immunocytochemistry (ICC) and by immunoblot following sub-cellular fractionation.
Results :
Ref-1 knockdown downregulated several Wnt3a receptors in HRECs, while treatment with Ref-1 redox inhibitor APX2009 decreased protein expression of LRP5. HRECs stimulated with Wnt3a and treated with Ref-1 redox inhibitors APX2009 or APX3330 exhibited a decrease in nuclear localization of β-catenin and Ref-1 knockdown also decreased nuclear localization of β-catenin.
Conclusions :
Ref-1 redox activity modulates canonical Wnt signaling in HRECs via regulating levels of Wnt3a receptors and nuclear localization of β-catenin. These results provide evidence of novel targets regulated by Ref-1 redox activity, building a foundation of knowledge of new molecular targets modulating retinal neovascular disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.