Dataset Information

SLC7A11 Reduces Laser-Induced Choroidal Neovascularization by Inhibiting RPE Ferroptosis and VEGF Production.

ABSTRACT: In age-related macular degeneration (AMD), one of the principal sources of vascular endothelial growth factor (VEGF) is retinal pigment epithelium (RPE) cells under hypoxia or oxidative stress. Solute carrier family 7 member 11 (SLC7A11), a key component of cystine/glutamate transporter, regulates the level of cellular lipid peroxidation, and restrains ferroptosis. In our study, we assessed the role of SLC7A11 in laser-induced choroidal neovascularization (CNV) and explored the underlying mechanism. We established a mouse model of CNV to detect the expression level of SLC7A11 and VEGF during disease progression. We found the expression of the SLC7A11 protein in RPE cells peaked at 3 days after laser treatment, which was correlated with the expression of VEGF. Intraperitoneal injection of SLC7A11 inhibitor expanded the area of CNV. We examined functional proteins related to oxidative stress and Fe²⁺ and found laser-induced ferroptosis accompanied by increased Fe²⁺ content and GPX4 expression in the RPE-choroidal complex after laser treatment. We verified the expression of SLC7A11 in the ARPE19 cell line and the effects of its inhibitors on cell viability and lipid peroxidation in vitro. Application of SLC7A11 inhibitor and SLC7A11 knockdown increased the level of lipid peroxidation and reduced the cell viability of ARPE19 which can be rescued by ferroptosis inhibitors ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1). Conversely, SLC7A11 overexpression induced resistance to erastin or RSL3-induced ferroptosis. Moreover, we tested the possible regulatory transcription factor NF-E2-related factor 2 (NRF2) of SLC7A11 by Western blot. Knock-down of NRF2 decreased the expression of SLC7A11. Our study suggests that SLC7A11 plays a key role in the laser-induced CNV model by protecting RPE cells from ferroptosis. SLC7A11 provides a new therapeutic target for neovascular AMD patients.

SUBMITTER: Zhao X

PROVIDER: S-EPMC7930391 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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SLC7A11 Reduces Laser-Induced Choroidal Neovascularization by Inhibiting RPE Ferroptosis and VEGF Production.

Zhao Xiaohuan X Gao Min M Liang Jian J Chen Yuhong Y Wang Yimin Y Wang Yuwei Y Xiao Yushu Y Zhao Zhenzhen Z Wan Xiaoling X Jiang Mei M Luo Xueting X Wang Feng F Sun Xiaodong X

Frontiers in cell and developmental biology 20210218

In age-related macular degeneration (AMD), one of the principal sources of vascular endothelial growth factor (VEGF) is retinal pigment epithelium (RPE) cells under hypoxia or oxidative stress. Solute carrier family 7 member 11 (SLC7A11), a key component of cystine/glutamate transporter, regulates the level of cellular lipid peroxidation, and restrains ferroptosis. In our study, we assessed the role of SLC7A11 in laser-induced choroidal neovascularization (CNV) and explored the underlying mechan ...[more]

PMID: 33681224

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Project description:Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-?), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved.In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-? antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-? protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-? or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and ?-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-? and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of ?-catenin, VEGF, and p22phox and coimmunoprecipitation of ?-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-? following pretreatment with ?-catenin transcriptional inhibitors, XAV939 or JW67, or transfection with p22phox siRNA and compared to appropriate controls.Compared to the non-lasered control, TNF-? and VEGF protein were increased in the RPE/choroids in a murine laser-induced CNV model (p<0.05). An intravitreal neutralizing antibody to mouse TNF-? reduced CNV volume, and VEGF protein in the RPE/choroids (p<0.01) and oxidized phospholipids within CNV compared to IgG control (p<0.05). In cultured RPE cells and compared to controls, TNF-? induced ROS generation and increased activation of NOX4, an isoform of NADPH oxidase; both were prevented by pretreatment with the apocynin or VAS2870 or knockdown of p22phox, a subunit of NADPH oxidase. TNF-? treatment increased VEGF expression (p<0.001) and the formation of a transcriptional complex of ?-catenin and T-cell transcriptional factor; both were prevented by pretreatment with apocynin or knockdown of p22phox. Inhibition of ?-catenin by XAV939, but not the nuclear factor kappa B inhibitor, Bay 11-7082, prevented TNF-?-induced VEGF upregulation.Our results support the thinking that TNF-? contributes to CNV by upregulating VEGF production in RPE cells through ROS-dependent activation of ?-catenin signaling. These results provide mechanisms of crosstalk between inflammatory mediator, TNF-?, and ROS in RPE cells.

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.

Dataset Information

SLC7A11 Reduces Laser-Induced Choroidal Neovascularization by Inhibiting RPE Ferroptosis and VEGF Production.

Publications

SLC7A11 Reduces Laser-Induced Choroidal Neovascularization by Inhibiting RPE Ferroptosis and VEGF Production.

Similar Datasets

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