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Protective role of glutathione peroxidase 4 in laser-induced choroidal neovascularization in mice.

Roggia MF, Imai H, Shiraya T, Noda Y, Ueta T - PLoS ONE (2014)

Bottom Line: Lipid peroxidation in RPE/choroid tissue was evaluated by immunohistochemistry using antibody against 4-hydroxy-2-nonenal.In addition, GPx4 reduced the CNV size in a dose-dependent manner in vivo.GPx4 suppresses the increase in the VEGF-A protein level, which occurs during the development of pathological CNV, thus partly explaining the protective effect of GPx4 against CNV.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Purpose: To evaluate the influence of glutathione peroxidase 4 (GPx4) expression in retinal pigment epithelium (RPE)/choroid tissue using a mouse model of laser-induced choroidal neovascularization (CNV).

Methods: In this study, GPx4+/-, GPx4+/+, and GPx4-overexpressing transgenic mice were created for comparison. The mRNA and protein expression of vascular endothelial growth factor (VEGF)-A in RPE/choroid tissue were evaluated before and after CNV induction by laser. Moreover, we investigated the changes in the VEGF-A mRNA level in RPE/choroid tissue in the CNV model that have not been clearly shown previously. Lipid peroxidation in RPE/choroid tissue was evaluated by immunohistochemistry using antibody against 4-hydroxy-2-nonenal. To investigate the protective role of GPx4, the size of laser-induced CNV was compared on day 7 among the mice expressing different levels of GPx4.

Results: In the laser-induced CNV mouse model, laser treatment reduced the VEGF-A mRNA level in RPE/choroid tissue, while it increased the VEGF-A protein level. Evaluation of VEGF-A expression in RPE/choroid tissue of the GPx4+/-, GPx4+/+, and GPx4 transgenic mice revealed that GPx4 increased the VEGF-A protein level under physiological conditions (i.e., without laser treatment), while GPx4 suppressed the increase in the VEGF-A protein level under pathological conditions (i.e., after CNV induction by laser). In addition, GPx4 reduced the CNV size in a dose-dependent manner in vivo.

Conclusions: GPx4 suppresses the increase in the VEGF-A protein level, which occurs during the development of pathological CNV, thus partly explaining the protective effect of GPx4 against CNV.

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Related in: MedlinePlus

GPx4 expression in RPE/choroid.(A) Similar appearance of GPx4+/−, GPx4+/+ and GPx4+/+:Tg (GPx4) mice at 3 months of age expressing different levels of GPx4. (B) Hematoxylin-eosin staining of the retina and RPE/choroid of the mice expressing different levels of GPx4. (ONL; outer nuclear layer, INL; inner nuclear layer, GCL; ganglionar cells layer, Scale bar; 30 µm). (C) Western blot analysis of β-actin and GPx4 protein expression in the RPE/choroid. (D) Statistical evaluation for the comparative difference in GPx4 protein in RPE/choroid (mean±SEM, n = 5)
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pone-0098864-g002: GPx4 expression in RPE/choroid.(A) Similar appearance of GPx4+/−, GPx4+/+ and GPx4+/+:Tg (GPx4) mice at 3 months of age expressing different levels of GPx4. (B) Hematoxylin-eosin staining of the retina and RPE/choroid of the mice expressing different levels of GPx4. (ONL; outer nuclear layer, INL; inner nuclear layer, GCL; ganglionar cells layer, Scale bar; 30 µm). (C) Western blot analysis of β-actin and GPx4 protein expression in the RPE/choroid. (D) Statistical evaluation for the comparative difference in GPx4 protein in RPE/choroid (mean±SEM, n = 5)

Mentions: Figure 2 shows the generation of 3 types of mice expressing different levels of GPx4. All these mice grow normally (Figure 2A) and display normal morphology of the retina and RPE/choroid tissue in all three genotypes (Figure 2B). However, western blot analysis revealed different expression levels of GPx4 protein in RPE/choroid tissue among the 3 types of mice (Figure 2C), which confirmed the validity of using these mice to test the role of GPx4 in RPE/choroid tissue. We also confirmed that GPx1 and GPx2 protein expression in RPE/choroid is similar in these mice (Figure S2). Using antibody against 4-HNE revealed that the accumulation of oxidized lipid in RPE/choroid tissue was most abundant in the GPx4+/− mice and least in the GPx4-overexpressing [i.e., GPx4+/+:Tg (GPx4)] mice (Figure 3).


Protective role of glutathione peroxidase 4 in laser-induced choroidal neovascularization in mice.

Roggia MF, Imai H, Shiraya T, Noda Y, Ueta T - PLoS ONE (2014)

GPx4 expression in RPE/choroid.(A) Similar appearance of GPx4+/−, GPx4+/+ and GPx4+/+:Tg (GPx4) mice at 3 months of age expressing different levels of GPx4. (B) Hematoxylin-eosin staining of the retina and RPE/choroid of the mice expressing different levels of GPx4. (ONL; outer nuclear layer, INL; inner nuclear layer, GCL; ganglionar cells layer, Scale bar; 30 µm). (C) Western blot analysis of β-actin and GPx4 protein expression in the RPE/choroid. (D) Statistical evaluation for the comparative difference in GPx4 protein in RPE/choroid (mean±SEM, n = 5)
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4045803&req=5

pone-0098864-g002: GPx4 expression in RPE/choroid.(A) Similar appearance of GPx4+/−, GPx4+/+ and GPx4+/+:Tg (GPx4) mice at 3 months of age expressing different levels of GPx4. (B) Hematoxylin-eosin staining of the retina and RPE/choroid of the mice expressing different levels of GPx4. (ONL; outer nuclear layer, INL; inner nuclear layer, GCL; ganglionar cells layer, Scale bar; 30 µm). (C) Western blot analysis of β-actin and GPx4 protein expression in the RPE/choroid. (D) Statistical evaluation for the comparative difference in GPx4 protein in RPE/choroid (mean±SEM, n = 5)
Mentions: Figure 2 shows the generation of 3 types of mice expressing different levels of GPx4. All these mice grow normally (Figure 2A) and display normal morphology of the retina and RPE/choroid tissue in all three genotypes (Figure 2B). However, western blot analysis revealed different expression levels of GPx4 protein in RPE/choroid tissue among the 3 types of mice (Figure 2C), which confirmed the validity of using these mice to test the role of GPx4 in RPE/choroid tissue. We also confirmed that GPx1 and GPx2 protein expression in RPE/choroid is similar in these mice (Figure S2). Using antibody against 4-HNE revealed that the accumulation of oxidized lipid in RPE/choroid tissue was most abundant in the GPx4+/− mice and least in the GPx4-overexpressing [i.e., GPx4+/+:Tg (GPx4)] mice (Figure 3).

Bottom Line: Lipid peroxidation in RPE/choroid tissue was evaluated by immunohistochemistry using antibody against 4-hydroxy-2-nonenal.In addition, GPx4 reduced the CNV size in a dose-dependent manner in vivo.GPx4 suppresses the increase in the VEGF-A protein level, which occurs during the development of pathological CNV, thus partly explaining the protective effect of GPx4 against CNV.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan.

ABSTRACT

Purpose: To evaluate the influence of glutathione peroxidase 4 (GPx4) expression in retinal pigment epithelium (RPE)/choroid tissue using a mouse model of laser-induced choroidal neovascularization (CNV).

Methods: In this study, GPx4+/-, GPx4+/+, and GPx4-overexpressing transgenic mice were created for comparison. The mRNA and protein expression of vascular endothelial growth factor (VEGF)-A in RPE/choroid tissue were evaluated before and after CNV induction by laser. Moreover, we investigated the changes in the VEGF-A mRNA level in RPE/choroid tissue in the CNV model that have not been clearly shown previously. Lipid peroxidation in RPE/choroid tissue was evaluated by immunohistochemistry using antibody against 4-hydroxy-2-nonenal. To investigate the protective role of GPx4, the size of laser-induced CNV was compared on day 7 among the mice expressing different levels of GPx4.

Results: In the laser-induced CNV mouse model, laser treatment reduced the VEGF-A mRNA level in RPE/choroid tissue, while it increased the VEGF-A protein level. Evaluation of VEGF-A expression in RPE/choroid tissue of the GPx4+/-, GPx4+/+, and GPx4 transgenic mice revealed that GPx4 increased the VEGF-A protein level under physiological conditions (i.e., without laser treatment), while GPx4 suppressed the increase in the VEGF-A protein level under pathological conditions (i.e., after CNV induction by laser). In addition, GPx4 reduced the CNV size in a dose-dependent manner in vivo.

Conclusions: GPx4 suppresses the increase in the VEGF-A protein level, which occurs during the development of pathological CNV, thus partly explaining the protective effect of GPx4 against CNV.

Show MeSH
Related in: MedlinePlus