Ocular neovascular diseases like wet age-related macular degeneration are a major cause of blindness. Novel therapies are greatly needed for these diseases. One appealing antiangiogenic target is reduction-oxidation factor 1–apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1). This protein can act as a redox-sensitive transcriptional activator for nuclear factor (NF)-κB and other proangiogenic transcription factors. An existing inhibitor of Ref-1’s function, APX3330, previously showed antiangiogenic effects. Here, we developed improved APX3330 derivatives and assessed their antiangiogenic activity. We synthesized APX2009 and APX2014 and demonstrated enhanced inhibition of Ref-1 function in a DNA-binding assay compared with APX3330. Both compounds were antiproliferative against human retinal microvascular endothelial cells (HRECs; GI₅₀ APX2009: 1.1 μM, APX2014: 110 nM) and macaque choroidal endothelial cells (Rf/6a; GI₅₀ APX2009: 26 μM, APX2014: 5.0 μM). Both compounds significantly reduced the ability of HRECs and Rf/6a cells to form tubes at mid-nanomolar concentrations compared with control, and both significantly inhibited HREC and Rf/6a cell migration in a scratch wound assay, reducing NF-κB activation and downstream targets. Ex vivo, APX2009 and APX2014 inhibited choroidal sprouting at low micromolar and high nanomolar concentrations, respectively. In the laser-induced choroidal neovascularization mouse model, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4-fold compared with vehicle (P < 0.0001, ANOVA with Dunnett’s post-hoc tests), without obvious intraocular or systemic toxicity. Thus, Ref-1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo, and APX2009 is an effective systemic therapy for choroidal neovascularization in vivo, establishing Ref-1 inhibition as a promising therapeutic approach for ocular neovascularization.

Under healthy conditions, the cornea is an avascular structure which allows for transparency and optimal visual acuity. Its avascular nature is maintained by a balance of proangiogenic and antiangiogenic factors. An imbalance of these factors can result in abnormal blood vessel proliferation into the cornea. This corneal neovascularization (CoNV) can stem from a variety of insults including hypoxia and ocular surface inflammation caused by trauma, infection, chemical burns, and immunological diseases. CoNV threatens corneal transparency, resulting in permanent vision loss. Mainstay treatments of CoNV have partial efficacy and associated side effects, revealing the need for novel treatments. Numerous natural products and synthetic small molecules have shown potential in preclinical studies in vivo as antiangiogenic therapies for CoNV. Such small molecules include synthetic inhibitors of the vascular endothelial growth factor (VEGF) receptor and other tyrosine kinases, plus repurposed antimicrobials, as well as natural source-derived flavonoid and non-flavonoid phytochemicals, immunosuppressants, vitamins, and histone deacetylase inhibitors. They induce antiangiogenic and anti-inflammatory effects through inhibition of VEGF, NF-κB, and other growth factor receptor pathways. Here, we review the potential of small molecules, both synthetics and natural products, targeting these and other molecular mechanisms, as antiangiogenic agents in the treatment of CoNV.

Mitochondrial metabolism is a critical mechanism that is deregulated in numerous retinal diseases. Here, we elaborate a protocol to quantify oxygen consumption rate as a measure of mitochondrial respiration directly from mouse retinal tissue pieces. Our procedure combines the use of Seahorse extracellular flux technology and ex vivo retinal tissue isolation and is robustly reproducible under different treatment conditions. This protocol allows direct assessment of mitochondrial function in response to drug treatments or genetic manipulation in mouse models.

Nonenzymatic glycation (NEG) begins with the non-covalent binding of a glucopyranose to a protein. The bound glucopyranose must then undergo structural modification to generate a bound electrophile that can reversibly form a Schiff base, which can then lead to Amadori intermediates, and ultimately to glycated proteins. Inorganic phosphate (Pi) is known to accelerate the glycation of human hemoglobin (HbA), although the specific mechanism(s) of Pi as an effector reagent have not been determined. The aim of this study was to determine whether Pi and a glucopyranose can concomitantly bind to HbA and react while bound within the early, noncovalent stages to generate electrophilic species capable of progress in NEG. ³¹P and ¹HNMR of model reactions confirm that bimolecular reactions between Pi and glucopyranose occur generating modified glucose electrophiles. Computations of protein/substrate interactions predict that Pi can concomitantly bind with a glucopyranose in HbA pockets with geometries suitable for multiple acid/base mechanisms that can generate any of four transient electrophiles. Pi-facilitated mechanisms in the noncovalent stages predict that the glycation of β-Val1 of HbA to HbA1c is a “hot spot” because the β-Val1 pocket facilitates many more mechanisms than any other site. The mechanistic diversity of the Pi effect within the early noncovalent stages of NEG predicts well the overall site selectivity observed from the in vivo glycation of HbA in the presence of Pi. These insights extend our basic understanding of the NEG process and may have clinical implications for diabetes mellitus and even normal aging.

Neovascular or “wet” age-related macular degeneration (nAMD) is a leading cause of blindness among older adults. Choroidal neovascularization (CNV) is a major pathological feature of nAMD, in which abnormal new blood vessel growth from the choroid leads to irreversible vision loss. There is a critical need to develop novel therapeutic strategies to address limitations of the current anti-vascular endothelial growth factor biologics. Previously, we identified soluble epoxide hydrolase (sEH) as a possible therapeutic target for CNV through a forward chemical genetic approach. The purpose of this study was to validate sEH as a target by examining retinal expression of sEH protein and mRNA by immunohistochemistry and RNAscope in situ hybridization, respectively, and to assess the efficacy of an adeno-associated virus (AAV) vector designed to knock down the sEH gene, Ephx2, in the murine laser-induced (L-) CNV model. nAMD patient postmortem eye tissue and murine L-CNV showed overexpression of sEH in photoreceptors and retinal pigment epithelial cells. Ephx2 knockdown significantly reduced CNV and normalized mRNA expression levels of CNV-related inflammatory markers. Thus, this study further establishes sEH as a promising therapeutic target against CNV associated with nAMD.

The relationship between heme metabolism and angiogenesis is poorly understood. The final synthesis of heme occurs in mitochondria, where ferrochelatase (FECH) inserts Fe ²⁺ into protoporphyrin IX to produce proto-heme IX. We previously showed that FECH inhibition is antiangiogenic in human retinal microvascular endothelial cells (HRECs) and in animal models of ocular neovascularization. In the present study, we sought to understand the mechanism of how FECH and thus heme is involved in endothelial cell function. Mitochondria in endothelial cells had several defects in function after heme inhibition. FECH loss changed the shape and mass of mitochondria and led to significant oxidative stress. Oxidative phosphorylation and mitochondrial Complex IV were decreased in HRECs and in murine retina ex vivo after heme depletion. Supplementation with heme partially rescued phenotypes of FECH blockade. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis. Abstract Figure: Highlights: Heme synthesis inhibition changes mitochondrial morphology in endothelial cells Loss of heme causes a buildup of mitochondrial ROS and depolarized membrane potential Endothelial cells have defective oxidative phosphorylation and glycolysis on loss of heme Mitochondrial damage is caused by loss of functional heme-containing Complex IV and partially restored by exogenous heme