REF-1 Overexpression in Neovascular Eye Diseases and Therapeutic Targeting with a Novel Inhibitor

Academic Background

Neovascular age-related macular degeneration (nAMD) is a common blinding eye disease predominantly affecting individuals aged 60 years and older. nAMD is characterized by the formation of subretinal neovascularization, leading to retinal hemorrhage, exudation, and vision loss. Currently, anti-vascular endothelial growth factor (VEGF) drugs are the mainstay treatment for nAMD. However, some patients exhibit poor response to these therapies, and long-term use can result in drug resistance and adverse side effects. Hence, identifying new therapeutic targets and drugs has become an important focus of ongoing research.

Reduction-oxidation factor-1 (REF-1), also known as apurinic/apyrimidinic endonuclease 1 (APE1), is a bifunctional protein with a dual role. It participates in DNA repair and also regulates various transcription factors (e.g., NF-κB, HIF-1α, and STAT-3) through its redox activity. This regulation influences processes such as inflammation, angiogenesis, and cell survival. While REF-1’s role in cancers and inflammatory diseases has been extensively studied, its function in eye diseases remains inadequately understood. This study aims to investigate REF-1’s role in nAMD and explore the therapeutic potential of a novel REF-1 inhibitor, APX2009.

Source of the Paper

This paper is a collaborative work by a team from multiple research institutions. The primary authors include Anbukkarasi Muniyandi, Gabriella D. Hartman, Kamakshi Sishtla, among others. The research team is affiliated with Indiana University School of Medicine, University of Alabama at Birmingham, University of Toronto, and other institutes. The paper was published in 2025 in the journal Angiogenesis under the title, “REF-1 is overexpressed in neovascular eye disease and targetable with a novel inhibitor.”

Research Procedures and Findings

1. Expression of REF-1 in nAMD

The study used immunohistochemistry to detect REF-1 expression in retinal and choroidal tissues from nAMD patients and healthy controls. The results revealed significantly higher REF-1 expression in the retinal pigment epithelium (RPE) and various retinal layers, such as the inner nuclear layer (INL) and outer nuclear layer (ONL), of nAMD patients compared to controls. Furthermore, REF-1 colocalized with markers of several cell types, including RPE cells, photoreceptors, and Müller glial cells, indicating its potential role in multiple pathological mechanisms of nAMD.

2. REF-1 Expression in Mouse Models

To further investigate REF-1’s role in neovascular eye diseases, the researchers used two mouse models: the laser-induced choroidal neovascularization (L-CNV) model and the Vldlr-/- spontaneous subretinal neovascularization (SRN) model. In the L-CNV model, REF-1 was significantly overexpressed in and around neovascular lesions, colocalizing with the vascular marker GS-IB4. In the Vldlr-/- model, REF-1 expression was markedly elevated in SRN lesions and colocalized with the hypoxia marker Hypoxyprobe, suggesting REF-1’s key role in hypoxia-driven angiogenesis.

3. Interaction Between APX2009 and REF-1

The researchers studied the interaction between APX2009 and REF-1 using nuclear magnetic resonance (NMR) spectroscopy and molecular docking. NMR results demonstrated chemical shift perturbations in multiple REF-1 residues (e.g., Asp70, Gln137, Ser164) upon binding with APX2009, indicating direct binding at the redox-active site of REF-1. Molecular docking further elucidated the binding mode of APX2009 to REF-1, providing structural evidence for its potential as an inhibitor.

4. Anti-Angiogenic Effects of APX2009

In vitro experiments demonstrated that APX2009 significantly inhibited proliferation, migration, and tube formation in various endothelial cell types, including human choroidal endothelial cells, brain microvascular endothelial cells, and human umbilical vein endothelial cells. Furthermore, APX2009 blocked cell cycle progression at the G1/S phase, inhibiting endothelial cell proliferation. These findings indicate that APX2009 disrupts key steps of angiogenesis by targeting REF-1’s redox activity.

5. In Vivo Efficacy of APX2009

In the L-CNV mouse model, intravitreal injections of APX2009 significantly reduced lesion volume and fluorescein leakage, with effects comparable to those of anti-VEGF drugs. In the Vldlr-/- SRN model, intraperitoneal administration of APX2009 significantly reduced the number and area of neovascular lesions and downregulated the expression of carbonic anhydrase 9 (CA9), a hypoxia-induced factor. These results further confirmed APX2009’s potential to inhibit neovascularization.

6. Safety Profile of APX2009

The safety of APX2009 was evaluated in cultures of human pluripotent stem cell-derived retinal ganglion cells (hPSC-RGCs). Treatment with APX2009 at doses up to 1 µM showed no toxicity and no reduction in cell numbers over seven days. This suggests that APX2009 is safe and well-tolerated at therapeutic doses.

Conclusions and Implications

This study is the first to systematically reveal REF-1 overexpression in nAMD and its critical role in angiogenesis and inflammation related to neovascular eye diseases. Using NMR and molecular docking, the researchers confirmed the potential of APX2009 as a REF-1 inhibitor and validated its anti-angiogenic effects in both in vitro and in vivo experiments. APX2009 not only effectively suppressed neovascularization but also demonstrated a favorable safety profile, providing new avenues for the treatment of nAMD.

Research Highlights

1. REF-1 Overexpression in nAMD: This study systematically identifies REF-1 overexpression in human nAMD and relevant mouse models, implicating REF-1 in angiogenesis and inflammation.
2. Mechanistic Insights into APX2009: Structural evidence from NMR and molecular docking revealed how APX2009 binds to REF-1, establishing its potential as a targeted inhibitor.
3. Multi-Faceted Anti-Angiogenic Effects: APX2009 inhibits endothelial cell proliferation, migration, and tube formation by disrupting cell cycle progression.
4. In Vivo Efficacy and Safety: In mouse models, APX2009 significantly reduced neovascular lesions with a safety profile comparable to existing treatments.

Additional Valuable Insights

The findings of this study not only provide a novel therapeutic target and drug candidate for nAMD but also open new avenues for research into REF-1’s roles in other diseases, such as cancer and inflammatory disorders. Future clinical trials for APX2009 could establish its broader applications in ophthalmology and beyond.