The Mechanism of PMC (2,2,5,7,8-Pentamethyl-6-chromanol), a Sterically Hindered Phenol Antioxidant, in Rescuing Oxidized Low-Density-Lipoprotein-Induced Cytotoxicity in Human Retinal Pigment Epithelial Cells

Abstract

Geographic atrophy or late-stage dry age-related macular degeneration (AMD) is characterized by drusen deposition and progressive retinal pigment epithelium (RPE) degeneration, leading to irreversible vision loss. The formation of drusen leads to dyshomeostasis, oxidative stress, and irreversible damage to the RPE. In this study, we used an in vitro model of oxidized low-density lipoprotein (ox-LDL)-induced human RPE damage/death to investigate the mechanism through which a sterically hindered phenol antioxidant compound, PMC (2,2,5,7,8-pentamethyl-6-chromanol), protects the RPE against ox-LDL-induced damage. We show that PMC exerts its protective effect by preventing the upregulation of stress-responsive heme oxygenase-1 (HMOX1/HO-1) and NAD(P)H: quinone oxidoreductase (NQO1) at the mRNA and protein levels. This effect was due to PMC's blockade of ROS generation, which in turn blocked nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, ultimately preventing the upregulation of antioxidant response elements (AREs), including HMOX1 and NQO1. The key role of HO-1 was demonstrated when the protective effect of PMC was inhibited by the knockdown of HMOX1. Additionally, PMC treatment under different experimental conditions and at different time points revealed that the continuous presence of PMC is required for the optimal protection against ox-LDL-induced cytotoxicity, defining the cellular pharmacokinetics of this molecule. Our data demonstrate the involvement of a key antioxidant pathway through which PMC mitigates the oxidative stress induced by ox-LDL and provides a potential therapeutic strategy for suppressing RPE degeneration/damage during AMD progression.

Keywords: antioxidants; cytoprotection; geographic atrophy; oxidized low-density lipoproteins; retinal pigment epithelium; sterically hindered phenols.

Figure 1

PMC protects hRPE cells from ox-LDL-induced damage by modulating apoptotic and antioxidant pathways. (A) hRPE cells were treated with 200 µg/mL for 24 and 48 h in the absence or presence of PMC (1.3 µM). Cytotoxicity was also measured in the untreated (control) cells and those treated with PMC alone. Cell death was measured in the condition media using the LDH assay, n = 3. Values are expressed as the mean ± SEM. The statistical analysis was conducted using one-way ANOVA, **** p < 0.0001, ns—non-significant, n = 3. A heatmap of the top differentially expressed genes from bulk RNA sequencing in (B) apoptosis and (C) antioxidant pathways, n = 3. Colors show intensity in z-scored units, where red shows replicates with a high expression (z-score = +5) and blue shows replicates with a low expression (z-score = −5).

Figure 2

Heme oxygenase-1 upregulation in ox-LDL-treated RPE cells is suppressed by treatment with PMC. (A) hRPE cells matured for 4 weeks were treated with ox-LDL (200 µg/mL) with or without the presence of PMC (1.3 µM) in serum-free media. The relative HMOX1 transcript levels were measured using qPCR. (B) Western blotting of the hRPE cells with the above indicated treatments at 24 and 48 h was conducted to examine HO-1 levels. (C) Quantification was performed using densitometry after normalization with GAPDH, n = 3. (D) HMOX-1 mRNA levels were determined through PCR in serum-starved ARPE-19 cells treated with ox-LDL in the presence or absence of PMC and qPCR. (E) Cell lysates from the ARPE-19 cells treated as above were examined using Western blot to determine HO-1 levels. (F) Quantification of the HO-1 levels was conducted after normalization with α-tubulin. HMOX1/HO-1 was also assessed in hRPE and ARPE-19 cells treated with PMC alone. Serum-starved untreated cells were considered as a control for all of the experiments. Values are indicated as the mean ± SEM of n = 3. One-way ANOVA was used for statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

NQO1 upregulation in ox-LDL-treated RPE cells is transiently suppressed by treatment with PMC. (A) Serum-starved hRPE cells were treated with ox-LDL (200 µg/mL) in the presence or absence of PMC (1.3 µM). NQO1 levels were determined using qPCR at 24 and 48 h, n = 6. (B) Western blot was conducted on cell lysates from the hRPE cells treated with ox-LDL with or without PMC to determine the NQO1 levels. Same GAPDH loading control was used for Figure 2B and Figure 3B as the targets were probed on the same membrane (C) Densitometry was conducted after normalization with GAPDH, n = 3. (D) Serum-starved ARPE-19 cells treated under the same experimental treatment conditions as above were analyzed for their NQO1 levels using qPCR, n = 6. (E) NQO1 protein levels were measured in cell lysates from the ARPE-19 cells in the ox-LDL-treated groups with or without PMC. (F) A densitometry analysis was conducted after normalization with GAPDH to quantify the NQO1 levels, n = 3. NQO1 levels were also measured in the hRPE and ARPE-19 cells treated with PMC alone. The serum-starved untreated cells were considered as the control for all of the experiments. Values are indicated as the mean ± SEM of the indicated n. A one-way ANOVA was used for the statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns—non-significant.

Figure 4

PMC prevents ox-LDL-induced oxidative stress. (A) ARPE-19 cells were treated with ox-LDL (200 µg/mL) in the presence/absence of PMC (1.3 µM) for 24 h. ROS levels were visualized using H2DCFDA staining (green), and the localization of ROS in the ER was estimated using the ER-Tracker™ (red). Changes in ROS levels in the ER were also examined in the cells treated with PMC. Untreated cells were considered as the controls. DAPI was used to stain the nucleus (blue); scale bar = 10 µm. Panel 4 (zoom) are higher-magnification images of the marked area in panel 3 (merge). (B) Quantification of colocalization of ROS in the ER was carried out using the Manders’ coefficient in the ImageJ 1.54g JaCoP plugin. (C) Quantification of the fluorescence intensity was conducted using ImageJ 1.54g. Values are indicated as the mean ± SEM of n = 6. A one-way ANOVA was used for the statistical analysis, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns—non-significant.

Figure 5

PMC prevents the nuclear translocation of Nrf2. (A) The nuclear translocation of Nrf2 (green) was visualized in APRE-19 cells treated with ox-LDL (200 µg/mL) in the presence/absence of PMC (1.3 µM) for 24 h. The nuclear translocation of Nrf2 was also imaged in the PMC-treated cells. Untreated cells were considered as the control. Nuclei were stained using DAPI (blue); scale bar = 10 µm. Panel 2 (zoom) are higher-magnification images of the area marked in panel 1 (Nrf2). Panel 3 shows the 3D reconstruction of the Z-stack images. (B) Quantification of the nuclear colocalization of Nrf2 was quantified using Manders’ coefficient in the ImageJ 1.54g JaCoP plugin. Values are indicated as the mean ± SEM of n = 10. A one-way ANOVA was used for the statistical analysis, * p < 0.05, ** p < 0.01, **** p < 0.0001, ns—non-significant.

Figure 6

PMC prevents ox-LDL-induced HO-1 upregulation and colocalization with the ER. (A) ARPE-19 cells were treated with ox-LDL (200 µg/mL) with or without PMC (1.3 µM) for 24 h. Following treatment, HO-1 (red) and ER localization with calreticulin (green) were visualized. Nuclei were visualized using DAPI (blue); scale bar = 25 µm. Panel 4 (zoom) is the higher-magnification images of the area marked in panel 3. (B) Quantification of HO-1’s colocalization with calreticulin was quantified based on the Manders’ coefficient in the ImageJ 1.54g JaCoP plugin. (C) Quantification of the fluorescence intensity was conducted using Image J. Values are indicated as the mean ± SEM of n = 8. A one-way ANOVA was used for the statistical analysis, *** p < 0.001, **** p < 0.0001, ns—non-significant.

Figure 7

HO-1 contributes to PMC protection against ox-LDL. (A) An illustration showing the timeline of 48 h siHMOX1 silencing and 24 h ox-LDL treatments in the presence or absence of PMC in the hRPE cells. (B) Western blotting was conducted to estimate the HO-1 levels in the hRPE cells subjected to siScr or siHMOX1 for 48 h followed by ox-LDL (200 µg/mL) treatments with or without PMC (1.3 µM). (C) Quantification of the HO-1 levels was performed using densitometry after normalization with GAPDH, n = 3. (D) The LDH levels were measured in the conditioned media from the siScr- or siHMOX-treated cells along with the siScr and siHMOX1 cells that were treated with ox-LDL with or without PMC, n = 14. (E) An illustration of the timeline indicating 48 h siHMOX1 silencing in the hRPE cells, followed by the additional 48 h treatment with ox-LDL with/without PMC. (F) HO-1 levels were detected in the hRPE cells treated with siScr or siHMOX1 for 48 h, followed by ox-LDL in the presence or absence of PMC using Western blotting. (G) A densitometry analysis was conducted to quantify the HO-1 levels after normalization with GAPDH, n = 3. (H) Cytotoxicity was analyzed by assaying LDH in the conditioned siScr and siHMOX1 media and siScr and siHMOX cells that were subjected to ox-LDL with/without PMC treatment, n = 6. Cells treated with ox-LDL in the presence or absence of PMC without siScr or siHMOX1 were used as the control for both 24 h and 48 h ox-LDL and ox-LDL + PMC treatments, n = 6. Values are indicated as the mean ± SEM of the indicated n. A one-way ANOVA was used for the statistical analysis, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns—non-significant.

Figure 8

Continuous presence of PMC is required for protection against ox-LDL. (A) Serum-starved hRPE cells were treated with either ox-LDL (200 µg/mL) and PMC (1.3 µM) alone, ox-LDL and PMC under different treatment conditions. ox-LDL + PMC denotes simultaneous treatment, while in the other treatment groups, either the cells were pretreated with PMC or with ox-LDL. Following these treatments, the media were replaced with ox-LDL + PMC, ox-LDL alone, or PMC alone. In another group, the media were not replaced, and treatments were added to the same media. Cells were pretreated with PMC and ox-LDL was added to the same media, or they were pretreated with ox-LDL and an approximately 10-fold higher PMC concentration (10 µM) was added to the same media. Untreated cells were considered as controls. Values are indicated as the mean ± SEM of n = 3. A one-way ANOVA was used for the statistical analysis, **** p < 0.0001, ns—non-significant. (B) A graphical summary of PMC-mediated protection against ox-LDL in the RPE cells. Uptake of ox-LDL via the CD36 receptor [28] causes lysosomal destabilization [29] and oxidative stress in the RPE, leading to ROS generation. This triggers Nrf2 dissociation from Keap1 and its translocation to the nucleus, where it interacts with the specific promoter region, ARE, leading to the upregulation of HMOX1 and NQO1. PMC prevents this upregulation of HMOX1/HO-1 and NQO1 levels by preventing ROS generation. Created using BioRender.