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TSPO Ligands Promote Cholesterol Efflux and Suppress Oxidative Stress and Inflammation in Choroidal Endothelial Cells

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TSPO Ligands Promote Cholesterol Efflux and Suppress Oxidative Stress and Inflammation in Choroidal Endothelial Cells

Lincoln Biswas et al. Int J Mol Sci.

Abstract

Choroidal endothelial cells supply oxygen and nutrients to retinal pigment epithelial (RPE) cells and photoreceptors, recycle metabolites, and dispose of metabolic waste through the choroidal blood circulation. Death of the endothelial cells of the choroid may cause abnormal deposits including unesterified and esterified cholesterol beneath RPE cells and within Bruch's membrane that contribute to the progression of age-related macular degeneration (AMD), the most prevalent cause of blindness in older people. Translocator protein (TSPO) is a cholesterol-binding protein that is involved in mitochondrial cholesterol transport and other cellular functions. We have investigated the role of TSPO in choroidal endothelial cells. Immunocytochemistry showed that TSPO was localized to the mitochondria of choroidal endothelial cells. Choroidal endothelial cells exposed to TSPO ligands (Etifoxine or XBD-173) had significantly increased cholesterol efflux, higher expression of cholesterol homeostasis genes (LXRα, CYP27A1, CYP46A1, ABCA1 and ABCG1), and reduced biosynthesis of cholesterol and phospholipids from [14C]acetate, when compared to untreated controls. Treatment with TSPO ligands also resulted in reduced production of reactive oxygen species (ROS), increased antioxidant capacity, and reduced release of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and VEGF) induced by oxidized LDL. These data suggest TSPO ligands may offer promise for the treatment of AMD.

Keywords: TSPO; age-related macular degeneration; cholesterol efflux; choroidal endothelial cells; inflammation; oxidative stress.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) The expression and co-localisation of translocator protein (TSPO) and mitochondria in RF/6A cells. RF/6A cells were stained with Mito-tracker (a marker for functional mitochondria) following cell culture and then fixed with cold methanol. Subsequently, the slides were treated with anti-TSPO antibody. Images captured confocally show TSPO (Green), DAPI (Blue), mitochondria (Red), while the Merged image shows co-localisation of TSPO and mitochondria (630×). (B) Toxic effects of TSPO ligands Etifoxine and XBD173 on RF/6A cells. RF/6A cells were treated with different concentrations of Etifoxine for 24 h and assayed: a decline in cell viability at the concentration of 25 µM was noted compared to the untreated controls; similarly, 30 µM XBD173 had a significant toxic effect compared to the control. (C) Efflux of cholesterol initiated by acceptors (APOA1, ApoE, HDL and HS) and incubated with TSPO ligands Etifoxine (20 µM) and XBD173 (25 µM) was studied in RF6A cells. The percentage of [3H]cholesterol was calculated in the media and in the cells. Data for (B) and (C) are shown as mean ± SD. Statistical comparisons were done using One-way ANOVA (B) and Two-way ANOVA (C), followed by Bonferroni test. Each experiment was performed in triplicate and repeated twice. NS: non-significant, * p ≤ 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.
Figure 2
Figure 2
The effects of TSPO ligands on the expression of cholesterol efflux genes and metabolism genes in RF6A cells treated with Etifoxine (A) or XBD173 (B) were quantified by qRT-PCR. The RF/6A cells were cultured and treated with TSPO ligands Etifoxine (20 µM) and XBD173 (25 µM) and with 0.1% DMSO as a control for 24 h. All data were collected and analysed relative expression of β-actin and presented as mean ± SD. Statistical comparisons were done using Two-way ANOVA followed by Bonferroni test. NS: non-significant, * p ≤ 0.05; ** p ≤ 0.01; *** p < 0.001 and *** p ≤ 0.001.
Figure 3
Figure 3
TSPO ligands modified the lipid phenotypes in RF/6A cells. The effect of TSPO ligands ((A): Etifoxine, 20 µM; (B): XBD173, 25 µM) were studied on disintegrations per minute (dpm) incorporation/mg total protein of [14C]acetate (1 µCi/mL) into free cholesterol, cholesteryl ester, fatty acid, phospholipid and triglycerides compared with the 0.1% vehicle control. These TSPO ligands also were applied to RF/6A cells to quantify the total cellular cholesterol (C) after the excitation of oxLDL and with their vehicle control. Similarly, it was applied to quantify triglycerides level. All data was represented as mean ± SD and analysed by Two-way ANOVA (A,B) and one-way ANOVA (C,D) followed by Bonferroni test. Three independent experiments were performed and the value of statistical were pointed as * p ≤ 0.05; ** p < 0.01, **** p < 0.0001. NS: non-significant.
Figure 4
Figure 4
Lipid droplets were detected in RF6A cells by Oil Red O (ORO) staining after treatment with oxLDL and with oxLDL + TSPO ligands. The oxLDL-treated cells were compared to cells treated with Etifoxine (20 µM) + oxLDL or with XBD173 (25 µM) + oxLDL and to vehicle-treated controls. The cells were then fixed with 10% formalin and stained with ORO and images were captured by EVOS microscopy (A). Inserts show a 2.5× magnification of the selected regions. The lipid droplets were eluted with 100% isopropanol and optical density (OD) at 520 nm was measured using a plate absorbance reader. The OD was normalised with respect to total cell protein. The data were analysed using one-way ANOVA followed by Bonferroni test and presented as mean ± SD (B). * p ≤ 0.05, ** p < 0.01, and *** p < 0.001.
Figure 5
Figure 5
(A) TSPO ligands reduced the oxLDL-generated intra-cellular reactive oxygen species (ROS) level in RF6A cells. ROS levels in cells following treatment with oxLDL were compared to cells treated with Etifoxine (20 µM) + oxLDL, or with XBD173 (25 µM) + oxLDL for 24 h, and to vehicle-treated control cells. The mRNA expressions of oxidative genes (GPX1, Catalase and SOD1) were down-regulated following treatment with oxLDL compared to controls. Treatment with Etifoxine (B) or XBD173 (C) resulted in recovery of gene expression. The protein activity of Catalse (D), GSH (E) and MDA (F) were measured following treatment with oxLDL, with Etifoxine + oxLDL, and with XBD173 + oxLDL. The experiments were performed three times. All data are shown as mean ± SD and analysed using one-way ANOVA (A,DF) and Two-way ANOVA (B,C), followed by the Bonferroni test. * p ≤ 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.
Figure 6
Figure 6
The expression of inflammation genes was quantified in RF/6A cells following treatment with oxLDL and TSPO ligands. (A) mRNA expression of TNF-α, IL1-β, IL-6 and VEGF relative to β-ACTIN was measured following treatment with oxLDL and compared to that seen in cells treated additionally with Etifoxine (20 µM) or XBD173 (25 µM) for 24 h and in vehicle-treated control cells. (B) Following the same treatment, the protein concentrations of these genes were measured by ELISA. Data are represented as mean ± SD and were analysed by Two-way ANOVA followed by Bonferroni multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

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