Identification of Potential Therapeutic Targets for Myopic Choroidal Neovascularization via Discovery-Driven Data Mining

Purpose: Myopic choroidal neovascularization (mCNV) is a prevalent cause of vision loss. However, the development of effective therapeutic targets for mCNV has been hindered by the paucity of suitable animal models. Therefore, the aim of this study is to identify potential genes and pathways associated with mCNV and to unearth prospective therapeutic targets that can be utilized to devise efficacious treatments. Methods: Text data mining was used to identify genes linked to choroid, neovascularization, and myopia. g: Profiler was utilized to analyze the biological processes of gene ontology and the Reactome pathways. Protein interaction network analysis was performed using strings and visualized in Cytoscape. MCODE and cytoHubba were used for further screening. Results: Discovery-driven text data mining identified 55 potential genes related to choroid, neovascularization, and myopia. Gene enrichment analysis revealed 11 biological processes and seven Reactome pathways. A protein-protein interaction network with 47 nodes was constructed and analyzed using centrality ranking. Key clusters were identified through algorithm tools. Finally, 14 genes (IL6, FGF2, MMP9, IL10, TNF, MMP2, HGF, MMP3, IGF1, CCL2, CTNNB1, BDNF, NGF, and EDN1), in addition to VEGFA, were evaluated as targets with potential as future therapeutics. Conclusions: This study provides new potential therapeutic targets for mCNV, including IL6, FGF2, MMP9, IL10, TNF, MMP2, HGF, MMP3, IGF1, CCL2, CTNNB1, BDNF, NGF, and EDN1, which correspond to seven potential enriched pathways. These findings provide a basis for further research and offer new possibilities for developing therapeutic interventions for this condition.</p

Changes in hypoxia-inducible factor-1α (HIF-1α) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) expressions by halofuginone treatment in a 661W photoreceptor cell line.

(A) A quantitative analysis of the HIF-reporter luciferase assay using 661W cells showed that CoCl2-induced HIF activity was suppressed by halofuginone treatment. The data were analyzed using one-way ANOVA followed by a Bonferroni post-hoc test. N = 3–9 per group. (B) A western blot image showed that halofuginone treatment reduced CoCl2-induced HIF-1α and BNIP3 protein expressions in 661W cells. (C) A quantitative analysis showed that CoCl2-induced Bnip3 mRNA expression was reduced by halofuginone treatment in 661W cells while Hif-1α mRNA expression was not changed. The data were analyzed using one-way ANOVA followed by a Bonferroni post-hoc test. N = 3–6 per group. HF: halofuginone. **P < 0.01, ***P < 0.001, ###P < 0.001.</p

Image_3_Lactoferrin Has a Therapeutic Effect via HIF Inhibition in a Murine Model of Choroidal Neovascularization.tiff

BackgroundLactoferrin, a type of glycoprotein, is contained in exocrine fluids such as tears, breast milk, sweat, and saliva, and is known to have anti-microbial, antioxidant, and anti-cancer effects. In the ophthalmological field, topical administration of lactoferrin has been reported to have a therapeutic effect in a murine dry eye model. Hypoxia-inducible factor (HIF) regulates various gene expressions under hypoxia, including vascular endothelial growth factor (VEGF), and is considered as an alternative target for neovascular ocular diseases such as age-related macular degeneration (AMD). We previously screened natural products and identified lactoferrin as a novel HIF inhibitor. In this study, we confirmed that lactoferrin has an HIF inhibitory effect and a therapeutic effect in a murine model of neovascular AMD.MethodsHIF inhibitory effects of lactoferrin were evaluated using a luciferase assay and western blotting in vitro. The quantified volume of choroidal neovascularization (CNV) induced by laser irradiation was compared with oral lactoferrin administration or conditional tissue specific Hif1a knockout mice.ResultsLactoferrin administration showed a significant HIF inhibitory effect in the retinal neuronal cells. Oral administration of lactoferrin or conditional Hif1a gene deletion significantly reduced CNV volume compared to controls.ConclusionsLactoferrin has a therapeutic effect in a laser CNV model by suppressing the retinal HIF activity.</p

Histological alterations by halofuginone treatment in a murine model of light induced retinopathy.

(A and B) Retinal images of the optical coherence tomography (OCT) and quantitative analyses showed that halofuginone treatment suppressed outer retinal thinning in light-induced retinopathy, analyzed at 200, 400, 600, and 800 μm (dot marks were displayed at each point with a 200 μm interval) from optic nerve head (ONH, 0). N = 4 per group. The data were analyzed using two-way ANOVA followed by a Bonferroni post hoc test. Graphs were shown as mean  ±  standard deviation. (C) The enlarged representative retinal image of OCT to detect the outer nuclear layer. RNFL/GCL: the retinal nerve fiber layer and ganglion cell layer. IPL: the inner plexiform layer. INL: the inner nuclear layer. OPL: the outer plexiform layer. ONL: the outer nuclear layer. IS/OS: the junction between the photoreceptor outer and inner segments. RPE: the retinal pigment epithelium. (D) The TUNEL assay image showed that halofuginone treatment protected against outer retinal cell death (marked with white asterisks) in light-induced retinopathy. ONL: the outer nuclear layer. LIR: light-induced retinopathy. HF: halofuginone. ***P < 0.001, ###P < 0.001.</p

Changes in hypoxia-inducible factor-1α (HIF-1α) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) expressions in a murine model of light-induced retinopathy.

(A) Quantitative analyses showed that retinal Hif-1α and Bnip3 mRNA expressions increased after extensive light exposure. Graphs were illustrated as mean ± standard deviation. The data were analyzed using two-way Student’s t-test. N = 4 per group. h: hour(s). (B) Images and Quantitative analyses showed that the protein expressions of HIF-1α and BNIP3 (multiple bands in the black parentheses) increased after extensive light exposure. Graphs were shown as mean ± standard deviation. The data were analyzed using two-way Student’s t-test. N = 4 per group. LIR: light-induced retinopathy. *P < 0.05, **P < 0.01, ***P < 0.001.</p

Image_2_Lactoferrin Has a Therapeutic Effect via HIF Inhibition in a Murine Model of Choroidal Neovascularization.tiff

BackgroundLactoferrin, a type of glycoprotein, is contained in exocrine fluids such as tears, breast milk, sweat, and saliva, and is known to have anti-microbial, antioxidant, and anti-cancer effects. In the ophthalmological field, topical administration of lactoferrin has been reported to have a therapeutic effect in a murine dry eye model. Hypoxia-inducible factor (HIF) regulates various gene expressions under hypoxia, including vascular endothelial growth factor (VEGF), and is considered as an alternative target for neovascular ocular diseases such as age-related macular degeneration (AMD). We previously screened natural products and identified lactoferrin as a novel HIF inhibitor. In this study, we confirmed that lactoferrin has an HIF inhibitory effect and a therapeutic effect in a murine model of neovascular AMD.MethodsHIF inhibitory effects of lactoferrin were evaluated using a luciferase assay and western blotting in vitro. The quantified volume of choroidal neovascularization (CNV) induced by laser irradiation was compared with oral lactoferrin administration or conditional tissue specific Hif1a knockout mice.ResultsLactoferrin administration showed a significant HIF inhibitory effect in the retinal neuronal cells. Oral administration of lactoferrin or conditional Hif1a gene deletion significantly reduced CNV volume compared to controls.ConclusionsLactoferrin has a therapeutic effect in a laser CNV model by suppressing the retinal HIF activity.</p

Image_1_Lactoferrin Has a Therapeutic Effect via HIF Inhibition in a Murine Model of Choroidal Neovascularization.tiff

BackgroundLactoferrin, a type of glycoprotein, is contained in exocrine fluids such as tears, breast milk, sweat, and saliva, and is known to have anti-microbial, antioxidant, and anti-cancer effects. In the ophthalmological field, topical administration of lactoferrin has been reported to have a therapeutic effect in a murine dry eye model. Hypoxia-inducible factor (HIF) regulates various gene expressions under hypoxia, including vascular endothelial growth factor (VEGF), and is considered as an alternative target for neovascular ocular diseases such as age-related macular degeneration (AMD). We previously screened natural products and identified lactoferrin as a novel HIF inhibitor. In this study, we confirmed that lactoferrin has an HIF inhibitory effect and a therapeutic effect in a murine model of neovascular AMD.MethodsHIF inhibitory effects of lactoferrin were evaluated using a luciferase assay and western blotting in vitro. The quantified volume of choroidal neovascularization (CNV) induced by laser irradiation was compared with oral lactoferrin administration or conditional tissue specific Hif1a knockout mice.ResultsLactoferrin administration showed a significant HIF inhibitory effect in the retinal neuronal cells. Oral administration of lactoferrin or conditional Hif1a gene deletion significantly reduced CNV volume compared to controls.ConclusionsLactoferrin has a therapeutic effect in a laser CNV model by suppressing the retinal HIF activity.</p

Image_4_Lactoferrin Has a Therapeutic Effect via HIF Inhibition in a Murine Model of Choroidal Neovascularization.tiff

BackgroundLactoferrin, a type of glycoprotein, is contained in exocrine fluids such as tears, breast milk, sweat, and saliva, and is known to have anti-microbial, antioxidant, and anti-cancer effects. In the ophthalmological field, topical administration of lactoferrin has been reported to have a therapeutic effect in a murine dry eye model. Hypoxia-inducible factor (HIF) regulates various gene expressions under hypoxia, including vascular endothelial growth factor (VEGF), and is considered as an alternative target for neovascular ocular diseases such as age-related macular degeneration (AMD). We previously screened natural products and identified lactoferrin as a novel HIF inhibitor. In this study, we confirmed that lactoferrin has an HIF inhibitory effect and a therapeutic effect in a murine model of neovascular AMD.MethodsHIF inhibitory effects of lactoferrin were evaluated using a luciferase assay and western blotting in vitro. The quantified volume of choroidal neovascularization (CNV) induced by laser irradiation was compared with oral lactoferrin administration or conditional tissue specific Hif1a knockout mice.ResultsLactoferrin administration showed a significant HIF inhibitory effect in the retinal neuronal cells. Oral administration of lactoferrin or conditional Hif1a gene deletion significantly reduced CNV volume compared to controls.ConclusionsLactoferrin has a therapeutic effect in a laser CNV model by suppressing the retinal HIF activity.</p

Datasets are attached in this file.

Photoreceptor cell death can cause progressive and irreversible visual impairments. Still, effective therapies on retinal neuroprotection are not available. Hypoxia-inducible factors (HIFs) are transcriptional factors which strongly regulate angiogenesis, erythropoiesis, intracellular metabolism, and programed cell death under a hypoxic or an abnormal metabolic oxidative stress condition. Therefore, we aimed to unravel that inhibition of HIFs could prevent disease progression in photoreceptor cell death, as recent studies showed that HIFs might be pathologic factors in retinal diseases. Adult male balb/cAJcl (8 weeks old; BALB/c) were used to investigate preventive effects of a novel HIF inhibitor halofuginone (HF) on a murine model of light-induced retinopathy. After intraperitoneal injections of phosphate-buffered saline (PBS) or HF (0.4 mg/kg in PBS) for 5 days, male BALB/c mice were subjected to a dark-adaption to being exposed to a white LED light source at an intensity of 3,000 lux for 1 hour in order to induce light-induced retinal damage. After extensive light exposure, retinal damage was evaluated using electroretinography (ERG), optical coherence tomography (OCT), and TUNEL assay. Light-induced retinal dysfunction was suppressed by HF administration. The amplitudes of scotopic a-wave and b-wave as well as that of photopic b-wave were preserved in the HF-administered retina. Outer retinal thinning after extensive light exposure was suppressed by HF administration. Based on the TUNEL assay, cell death in the outer retina was seen after light exposure. However, its cell death was not detected in the HF-administered retina. Halofuginone was found to exert preventive effects on light-induced outer retinal cell death.</div

Uncropped western blot images are attached in this file.

Uncropped western blot images are attached in this file.</p