Dataset Information

Reduction of Laser-Induced Choroidal Neovascularization in Mice With Erythropoietin RNA Interference.

ABSTRACT:

Purpose

The purpose of this study was to evaluate the pathological involvement of erythropoietin (EPO) in experimental choroidal neovascularization (CNV) and its association with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in the Chinese population.

Methods

Treatment effect of recombinant EPO protein were assessed by human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation, and ex vivo choroid-sprouting ability. The effect of intravitreal injection of Epo siRNA against neovascularization was evaluated in the laser-induced CNV mouse model. In addition, the association of EPO variants with neovascular AMD and PCV was determined.

Results

Exogenous supplementation of EPO significantly enhanced the migration and tube formation of HUVECs and promoted ex vivo choroid sprouting in mouse retinal pigment epithelium (RPE)-choroid-sclera complex culture. In the experimental CNV mouse model, Epo expression was found to be significantly upregulated by 3.5-folds in RPE-choroid-sclera complex at day 10 after laser induction as compared to the baseline. Immunofluorescence analysis showed that Epo was mainly expressed around the vascular endothelial cells in the RPE-choroid-sclera complex. Intravitreal injection of siRNA targeting Epo reduced 40% Epo expression and 40% CNV lesion areas as compared to the scramble control. However, EPO variants were not associated with neovascular AMD nor PCV in the Chinese population.

Conclusions

This study revealed the promotion of human endothelial cell tube formation in vitro and choroid sprouting ex vivo by EPO, and the reduction of laser-induced CNV in vivo by Epo RNA interference.

Translational relevance

Targeting EPO could be a potential additional treatment for CNV-related diseases.

SUBMITTER: Lv W

PROVIDER: S-EPMC9351596 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Publications

Reduction of Laser-Induced Choroidal Neovascularization in Mice With Erythropoietin RNA Interference.

Lv Wenjuan W Chen Wen W Huang Shaofen S Xu Yanxuan Y Liang Jia-Jian JJ Zheng Yuqian Y Chen Shaowan S Chen Shao-Lang SL Ng Tsz Kin TK Chen Haoyu H

Translational vision science & technology 20220801 8

<h4>Purpose</h4>The purpose of this study was to evaluate the pathological involvement of erythropoietin (EPO) in experimental choroidal neovascularization (CNV) and its association with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in the Chinese population.<h4>Methods</h4>Treatment effect of recombinant EPO protein were assessed by human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation, and ex vivo choroid-spr ...[more]

PMID: 35913417

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Project description:Choroidal neovascularization (CNV) is a major cause of severe visual loss in patients with age-related macular degeneration (AMD). Recently, itraconazole has shown potent and dose-dependent inhibition of tumor-associated angiogenesis. We evaluated the anti-angiogenic effect of itraconazole in a rat model of laser-induced CNV. After laser photocoagulation in each eye to cause CNV, right eyes were administered intravitreal injections of itraconazole; left eyes received balanced salt solution (BSS) as controls. On day 14 after laser induction, fluorescein angiography (FA) was used to assess abnormal vascular leakage. Flattened retinal pigment epithelium (RPE)-choroid tissue complex was stained with Alexa Fluor 594-conjugated isolectin B4 to measure the CNV area and volume. Vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression was determined 1, 4, 7, and 14 days after intravitreal injection by quantitative RT-PCR or Western blot. VEGF levels were analyzed by enzyme-linked immunosorbent assay (ELISA). Intravitreal itraconazole significantly reduced leakage from CNV as assessed by FA and CNV area and volume on flat mounts compared with intravitreal BSS (p = 0.002 for CNV leakage, p<0.001 for CNV area and volume). Quantitative RT-PCR showed significantly lower expression of VEGFR2 mRNA in the RPE-choroid complexes of itraconazole-injected eyes than those of BSS-injected eyes on days 7 and 14 (p = 0.003 and p = 0.006). Western blots indicated that VEGFR2 was downregulated after itraconazole treatment. ELISA showed a significant difference in VEGF level between itraconazole-injected and BSS-injected eyes on days 7 and 14 (p = 0.04 and p = 0.001). Our study demonstrated that intravitreal itraconazole significantly inhibited the development of laser-induced CNV in rats. Itraconazole had anti-angiogenic activity along with the reduction of VEGFR2 and VEGF levels. Itraconazole may prove beneficial for treating CNV as an alternative or adjunct to other therapies.

Project description:PurposeTo evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV).MethodsNine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 μm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 μg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation.ResultsIMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1wt/gfp mice. In vitro IMD-0354 (10-25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity.ConclusionThe present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD.

Dataset Information

Reduction of Laser-Induced Choroidal Neovascularization in Mice With Erythropoietin RNA Interference.

Purpose

Methods

Results

Conclusions

Translational relevance

Publications

Reduction of Laser-Induced Choroidal Neovascularization in Mice With Erythropoietin RNA Interference.

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