Hartman Gabriella D, Sishtla Kamakshi, Kpenu Eyram K, Mijit Mahmut, Muniyandi Anbukkarasi, Jo Ha-Neul, Junge Harald J, Shaw Aaron, Bischof Daniela, Liu Sheng, Wan Jun, Kelley Mark R, Corson Timothy W
Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, IN, USA; Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA.
Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, IN, USA; Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, USA; Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada.
Redox Biol. 2025 Jun;83:103646. doi: 10.1016/j.redox.2025.103646. Epub 2025 Apr 18.
Ischemic retinopathies, including proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP), are characterized by abnormal retinal neovascularization and can lead to blindness in children and adults. Current treatments, such as intravitreal anti-VEGF injections, face limitations due to high treatment burden and variable efficacy, as multiple signaling pathways, beyond VEGF, contribute to retinal neovascularization. Previous studies demonstrate that targeting the redox-mediated transcriptional regulatory function of APE1/Ref-1 reduces pathological neovascularization. We aimed to identify novel signaling pathways regulated by Ref-1 redox activity utilizing RNA sequencing of human retinal endothelial cells (HRECs) treated with a Ref-1 redox inhibitor. We found that Wnt/β-catenin signaling was significantly downregulated after Ref-1 inhibition. Given the role of Wnt signaling in vascular pathologies, we investigated how Ref-1 regulates Wnt/β-catenin signaling. Ref-1 inhibition downregulated Wnt co-receptors LRP5/6 at both the mRNA and protein levels in endothelial cells, suggesting transcriptional regulation. Ref-1 redox inhibitors APX3330 and APX2009 reduced Wnt3a-induced nuclear β-catenin levels, decreased Wnt transcriptional activity by TOPFlash luciferase assay, and blocked hypoxia-induced Wnt/β-catenin activation in HRECs. In the oxygen-induced retinopathy mouse model of retinal neovascularization, Ref-1 specific inhibitor APX2009 reduced the expression of Wnt-related genes at sites of neovascularization. These findings reveal a novel role for Ref-1 redox activity in modulating Wnt/β-catenin signaling in endothelial cells and highlight the potential of Ref-1 redox activity targeted inhibitors as a novel therapeutic approach for retinal neovascular diseases by modulating multiple disease-relevant pathways.
缺血性视网膜病变，包括增殖性糖尿病视网膜病变（PDR）和早产儿视网膜病变（ROP），其特征是视网膜新生血管异常，可导致儿童和成人失明。目前的治疗方法，如玻璃体内注射抗VEGF药物，由于治疗负担高和疗效不一而面临局限性，因为除VEGF外，多种信号通路都参与视网膜新生血管形成。先前的研究表明，靶向APE1/Ref-1的氧化还原介导的转录调节功能可减少病理性新生血管形成。我们旨在利用RNA测序技术，对用Ref-1氧化还原抑制剂处理的人视网膜内皮细胞（HREC）进行研究，以确定受Ref-1氧化还原活性调节的新信号通路。我们发现，Ref-1抑制后Wnt/β-连环蛋白信号通路显著下调。鉴于Wnt信号通路在血管病变中的作用，我们研究了Ref-1如何调节Wnt/β-连环蛋白信号通路。Ref-1抑制在内皮细胞的mRNA和蛋白质水平下调Wnt共受体LRP5/6，提示存在转录调节。Ref-1氧化还原抑制剂APX3330和APX2009降低Wnt3a诱导的核β-连环蛋白水平，通过TOPFlash荧光素酶测定法降低Wnt转录活性，并阻断缺氧诱导的HREC中Wnt/β-连环蛋白激活。在视网膜新生血管形成的氧诱导视网膜病变小鼠模型中，Ref-1特异性抑制剂APX2009降低了新生血管形成部位Wnt相关基因的表达。这些发现揭示了Ref-1氧化还原活性在内皮细胞中调节Wnt/β-连环蛋白信号通路的新作用，并突出了靶向Ref-1氧化还原活性的抑制剂作为一种通过调节多种疾病相关途径治疗视网膜新生血管疾病的新治疗方法的潜力。
