Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Free Radic Biol Med. 2021 Nov 27;178:360–368. doi: 10.1016/j.freeradbiomed.2021.11.026

Discovery of sterically-hindered phenol compounds with potent cytoprotective activities against ox-LDL–induced retinal pigment epithelial cell death as a potential pharmacotherapy

Gopalan Gnanaguru 1, Ashley Mackey 1, Eun Young Choi 1, Anthoula Arta 1,2, Franco Aparecido Rossato 1, Thomas W Gero 3, Andrew J Urquhart 2, David A Scott 3, Patricia A D’Amore 1,*, Yin Shan E Ng 1,*
PMCID: PMC8758799  NIHMSID: NIHMS1762240  PMID: 34843917

Abstract

Late-stage dry age-related macular degeneration (AMD) or geographic atrophy (GA) is an irreversible blinding condition characterized by degeneration of retinal pigment epithelium (RPE) and the associated photoreceptors. Clinical and genetic evidence supports a role for dysfunctional lipid processing and accumulation of harmful oxidized lipids in the pathogenesis of GA. Using an oxidized low-density lipoprotein (ox-LDL)-induced RPE death assay, we screened and identified sterically-hindered phenol compounds with potent protective activities for RPE. The phenol-containing PPARγ agonist, troglitazone, protected against ox-LDL–induced RPE cell death, whereas other more potent PPARγ agonists did not protect RPE cells. Knockdown of PPARγ did not affect the protective activity of troglitazone in RPE, confirming the protective function is not due to the thiazolidine (TZD) group of troglitazone. Prototypical hindered phenol trolox and its analogs potently protected against ox-LDL–induced RPE cell death whereas potent antioxidants without the phenol group failed to protect RPE. Hindered phenols preserved lysosomal integrity against ox-LDL–induced damage and FITC-labeled trolox was localized to the lysosomes in RPE cells. Analogs of trolox inhibited reactive oxygen species (ROS) formation induced by ox-LDL uptake in a dose-dependent fashion and were effective at sub-micromolar concentrations. Treatment with trolox analog 2,2,5,7,8-pentamethyl-6-chromanol (PMC) significantly induced the expression of the lysosomal protein NPC-1 and reduced intracellular cholesterol level upon ox-LDL uptake. Our data indicate that the lysosomal-localized hindered phenols are uniquely potent in protecting the RPE against the toxic effects of ox-LDL, and may represent a novel pharmacotherapy to preserve the vision in patients with GA.

Graphical Abstract

graphic file with name nihms-1762240-f0007.jpg

Introduction

Geographic atrophy (GA), or late-stage dry age-related macular degeneration (AMD), is characterized by degeneration and death of the retinal pigment epithelium (RPE). Severe, irreversible vision loss results when RPE atrophy and the resulting death of photoreceptors extends into the macula. Drusen are an early sign of AMD 1; hypercholesterolemia is a risk factor for the development of AMD 2; and, cholesterol-lowering statins may provide some benefit in individuals aged 68 and older 3, suggesting that dysfunctional lipid metabolism plays a role in drusen formation and development of dry AMD. While drusen may be an early indication of RPE dysfunction, their presence can also exacerbate RPE pathology by disrupting the extracellular matrix and, along with the thickened Bruch’s membrane, alter the stiffness of the RPE substrate and initiate/exaggerate local inflammation. As there is no established treatment for dry AMD, new therapies are needed, and protection of the RPE from degeneration is a promising area of focus.

RPE cells are vital for the health and function of photoreceptor cells and the underlying choroidal vasculature 4. In AMD, the mechanism (s) through which RPE cells degenerate leading to vision loss is not clearly understood. There is compelling clinical and experimental evidence pointing to a role for age-dependent accumulation of unoxidized and oxidized forms of lipid-containing drusen beneath the RPE, linking RPE atrophy and AMD pathogenesis 5–9. In particular, exposure to oxidized lipoproteins has been shown to be detrimental to RPE cells 10–12. The scavenger receptor CD36 mediates the uptake of oxidized lipoproteins, which then accumulate in lysosomes leading to lysosomal destabilization and cell death 12–14.

Oxidized-LDL (ox-LDL) has been shown to induce pathologies associated with AMD such as RPE senescence, break-down of RPE barrier properties, RPE degeneration, basement membrane thickening, and inflammatory response 11–13, 15. As such, we used ox-LDL-induced RPE cell death as a reflection of dysregulated lipid metabolism in dry AMD to screen for potential therapeutics. Using the in vitro drug screen approach, we discovered that compounds containing a “sterically-hindered phenol,” a functional antioxidant group, display very potent protection against ox-LDL–induced death of RPE cells.

Methods

Cell culture

Primary human fetal RPE (hf-RPE) cells were isolated and cultured as described previously (Gnanaguru et al 2016). Human ARPE-19 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM-F12 medium supplemented with 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2. Cells were plated at a density of 5.0 x 104 cells/well for a 48-well plate, 1.0 x 105 cells/well for a 24-well plate, and 2.5-3.0 x 105 cells/well for a six-well plate. Confluent monolayers of cells were maintained in media containing 1% fetal bovine serum (FBS) for two to four weeks and then incubated in a serum-free media overnight before using for subsequent experiments. Reagents: DMEM-F12/L-glutamine media, penicillin, streptomycin (Lonza, Basel, Switzerland), FBS (Atlanta Biologicals, Flowery Branch, GA, USA).

Lipoprotein and drug treatment

Hf-RPE or ARPE-19 cells, grown at post confluence for two to four weeks, were serum-starved overnight and then treated with ox-LDL (500 μg/ml for Hf-RPE and 200 μg/ml for ARPE-19 cells) (Biomedical Technologies, Alfa Aesar, LLC, Ward Hill, MA, USA) with or without the indicated concentrations of the different compounds (below), or with DMSO vehicle for up to 36 hr. Conditioned media and cell lysates were collected from the treatment groups for subsequent studies.

Drugs used:

Peroxisome proliferator-activated receptor (PPAR) agonists fenofibrate, troglitazone, bezafibrate, 15-deoxy-Δ12,14-Prostaglandin J2 (PGJ2), ciglitazone, rosiglitazone, pioglitazone, and MCC-555 were from Cayman Chemical (Ann Arbor, MI, USA). Antioxidants 2,2,5,7,8-Pentamethyl-6-chromanol (PMC), 4-hydroxy-TEMPO (TEMPO, a superoxide dismutase mimic) and Tiron were from Sigma (St. Louis, MO), MCI-186 (MCI), Trolox and DL-a-lipoic acid (lipoic acid) were from Cayman Chemical, N-Acetyl-L-cysteine (NAC) was from Abcam (Cambridge, MA), and elamipretide (ELAM) was from Muse Chem (Fairfield, NJ). Trolox amide analogs (669, 738, 829) were from ChemBridge (San Diego, CA). Inflammasome inhibitors MCC950 and Isoliquiritigenin were from Cayman Chemical. The NADPH-oxidases inhibitor apocynin (APO) was from Abcam and diphenyleneiodonium (DPI) was from Sigma.

Sample collection

Following ox-LDL treatment for 48 hr, conditioned media were collected to measure cell death. Lysotracker assay was performed at 36 hr to observe changes in the lysosomal number prior to cell death. RPE cell lysates were collected at eight or 18 hr for ROS assays, western blot, and total cholesterol measurements.

siRNA gene silencing

ARPE-19 cells were plated at a density of 50-75% (6.0 x 104 cells/well for 24-well format or 2.5 x 105 cells/well for six-well format). The following day, siRNA for PPARG and scrambled siRNA control were transfected into the cells using lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Cell lysates were collected 24 and 48 hr post transfection for RNA and protein analysis.

To study the effect of troglitazone or rosiglitazone on the survival of ARPE-19 cells depleted of PPARγ, cells were serum-starved for 24 hr post transfection and then incubated with ox-LDL in the presence or absence of troglitazone or rosiglitazone for 48 hr. Conditioned media were collected and the percentage of cell death was quantified by measuring the release of lactate dehydrogenase (LDH).

siRNA Pool Target Sequences
PPARG No. 1: 5′-GGGCGAUCUUGACAGGAATT-3′
No. 2: 5′-UUUCCUGUCAAGAUCGCCCTC-3′
Open in a new tab

Cell death measurement

LDH was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). Maximum LDH release was measured from cells that were maintained in parallel and lysed by freeze–thaw. The level of spontaneous LDH release was measured from negative control cells that received no lipoprotein or drug treatment. The percentage of cell death was calculated as: % cytotoxicity = 100% × (experimental LDH – spontaneous LDH) / (maximum LDH – spontaneous LDH).

Real Time PCR (RT-PCR)

Total RNA was isolated using RNA-Bee solution (CS-105B; Amsbio LLC, Cambridge, MA, USA) and reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Real-time PCR reactions were performed using the SYBR Green master mix (Roche, Basel, Switzerland) and PCR platform (Light Cycler 480 II; Roche). The relative gene expression level was quantified by normalizing to reference HPRT1 gene. Primer sequences used were: PPARG, forward primer: 5′-GGATTCAGCTGGTCGATATCAC-3′, reverse primer: 5′-GTTTCAGAAATGCCTTGCAGT-3′; HPRT1, forward primer: 5′-GCGATGTCAATAGGACTCCAG-3′, reverse primer: 5′-TTGTTGTAGGATATGCCCTTGA-3′.

Western blot

Cell lysates were collected using RIPA buffer containing proteinase inhibitor cocktail (Cell Signaling, Danvers, MA, USA). Protein lysates were separated using 4-20% gradient SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to nitrocellulose membrane. After blocking the membranes for one hr with Odyssey® blocking buffer (Li-COR Biosciences, Lincoln, NE, USA), the membranes were incubated overnight with primary antibodies (prepared in Odyssey blocking buffer with 0.1% tween) at 4°C on a shaker. The membranes were thoroughly washed in 1x PBST and then probed with appropriate secondary antibodies for 45 min at room temperature. After washes in 1X PBS, the membranes were scanned and the fluorescent intensity of the protein bands were quantified using Odyssey® Imaging System (Li-COR Biosciences, Lincoln, NE, USA). Primary antibodies: PPARγ and vinculin (catalog # 2443S and 13901S, Cell Signaling, Danvers, MA), NPC1 (Catalog # MAB10105, R&D systems, Inc. Minneapolis, MN), alpha-tubulin (catalog # CP06, Calbiochem, MilliporeSigma, Burlington, MA). Secondary antibodies: goat anti-rabbit 680 IRDye® and goat anti-mouse 800 IRDye® (Li-COR Biosciences, Lincoln, NE).

Assessment of lysosomal integrity

ARPE-19 cells were seeded on 12 mm cover slips at a density of 7.5 x 104 cells and maintained for two weeks. Following overnight serum starvation, cells treated with ox-LDL in the presence or absence of troglitazone, rosiglitazone, or trolox at different doses (0.1625, 1.3, and 10.4 μM) for 18 and 36 hr. Cells were then incubated with 500 nM of LysoTracker Red DND-99 (Life Technologies, Carlsbad, CA) and Hoechst 33,342 nuclear stain (Immunochemistry Technologies, Bloomington, MN) to visualize the lysosomes and nucleus, respectively. After washing in serum-free media, cells were fixed with 4% paraformaldehyde for five min at room temperature and washed with PBS. The cover slips were mounted onto slides using Prolong Gold Antifade Reagent (Life Technologies). Images were taken of five randomly selected fields using a fluorescent microscope (Axioscope Mot 2; Carl Zeiss Meditec, Inc., Dublin, CA) and analyzed as below.

Image processing

Images taken of the LysoTracker stained cells were processed using the using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD) to quantify the number of LysoTracker Red DND-99 stained lysosomal particles. After applying a median filter to remove background noise, the image was inverted, converted to 8-bit, and adjusted to a common threshold. LysoTracker-stained particles along the edges were excluded from quantification. Particles larger than 0 and smaller than 100 pixels were counted and normalized to the number of nuclei.

Lysosomal localization of FITC-labeled Trolox

The FITC-labeled Trolox was produced by the Medicinal Chemistry Core at Dana-Farber Cancer Institute (Boston, MA) (Supplementary Fig. S1B). ARPE-19 cells were plated at a density of 5.0 x 104 cells/well (24-well format). Cells were transduced with CellLight® BacMam (25 particle per cell) (Thermo Fisher Scientific, Waltham, MA) overnight in DMEM-F12 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2. After maintenance in serum free media for up to 24 hr, the cells were incubated with 25 μM of FITC-labeled trolox for 36 hr and then imaged using EVOS FL Auto inverted live imaging microscope (Thermo Fisher Scientific, Waltham, MA).

Total cellular cholesterol measurement

The intracellular cholesterol accumulation due to ox-LDL exposure was measured using the Amplex™ Red Cholesterol Assay Kit (ThermoFisher) according to manufacturer’s instruction. Briefly, ARPE-19 cells in 12-well plates were serum-starved overnight, and then incubated for 8 hours with 200 μg/mL ox-LDL. The ox-LDL was removed, the cells were washed once with PBS and were treated with or without 1.3 μM of PMC for 24 hours. Following the treatment, the cells were lysed in 300μl of buffer with 100 mM Tris–HCl, pH 7.4, 0.2% Triton X-100. Then 50 μl of cell lysate and cholesterol reference standards were aliquoted in duplicates into a 96-well plate and Amplex™ Red reaction buffer was added, the plates were incubated for 30 minutes at 37°C in the dark and fluorescence was measured at 530nm excitation and 590nm emission using a microplate spectrophotometer (Biotek Synergy Mx, Winooski, VT). Cholesterol concentration was normalized to the amount of total protein present in the lysate, which was measured by the BCA assay kit (Pierce, ThermoFisher).

Detection of total reactive oxygen species (ROS)

Intracellular ROS were detected using the total ROS Detection kit (Catalog # C6827, ThermoFisher Scientific, Waltham, MA), according to the manufacturer’s instructions. ARPE-19 cells were seeded into 96-well plates at a density of 1.0 x 104 cells per well and maintained for two wk. Cells were treated with 200 μg/mL of oxLDL in the presence or absence of drug indicated concentration for 12 hr. The growth media were removed, and the cells were incubated with the ROS probe in warm 1X PBS, as recommended by the manufacturer, and the fluorescent emission level was measured using a plate reader and then the percentage of fluorescent intensity was quantified.

Statistical analysis

Data are presented as the mean ± SEM of three independent experiments. To evaluate for statistical significance, 2-tail unpaired t-test or one-way ANOVA followed by the Tukey-Kramer multiple comparisons test or the Dunnett’s multiple comparisons test were used (Prism 6 software package, GraphPad, La Jolla, CA). Values of P < 0.05 were considered statistically significant.

Results

Troglitazone protects against ox-LDL–induced RPE cell death by a PPAR-independent pathway

We have reported that the intracellular accumulation of ox-LDL induces lysosomal destabilization leading to RPE cell death 12. Therefore, we aimed to identify small molecule inhibitors that inhibit lysosomal destabilization and thus prevent RPE cell death. As PPARα and PPARγ are reported to suppress ox-LDL induced damage in conditions like atherosclerosis 16, 17, we investigated whether treatment of hf-RPE cells with PPAR selective agonists would protect RPE from ox-LDL–induced cell death. Of the PPAR agonists tested, only troglitazone (PPARγ agonist) significantly suppressed ox-LDL–induced primary human RPE cell death by nearly 70% (Fig. 1A). In contrast, the PPARα-selective agonist fenofibrate or the PPAR δ/α-selective agonist bezafibrate did not show any significant protective effect against ox-LDL induced RPE cell death (Fig. 1A). Based on this finding, we investigated other known PPARγ agonists, both with and without the thiazolidinedione (TZD) domain, which is responsible for the activation of PPARγ 18.

Figure 1: Troglitazone protection of RPE from ox-LDL-induced cell death is independent of PPARγ.

Figure 1:

Open in a new tab

(A) Primary human fetal RPE cells were treated with ox-LDL (500 μg/ml) for 48 hr in the presence or absence of PPARα agonist fenofibrate (30 μM), pan-PPAR agonist bezafibrate (60 μM) and PPARγ agonist troglitazone (0.55 μM), and LDH release was measured in the conditioned media as a measure of cell death. The dosages used were based on the known EC50 of the drugs.

(B) Confluent ARPE-19 cells cultured for 2 wk in media with reduced FBS (1%) were treated with ox-LDL (ox-L) (200 μg/ml) for 48 hr in the presence or absence of various PPARγ agonists at denoted concentrations (in μM) and LDH release was assessed in the conditioned media as a measure of cell death.

(C) ARPE-19 cells transfected with 50 nM of scrambled (Scr) siRNA or PPARγ siRNA were treated with or without ox-LDL (200 μg/ml) in the presence or absence of troglitazone (trog) for 48 hr, and the relative levels of PPARγ transcript were measured by qPCR.

(D) LDH levels were measured in the conditioned media of scr-siRNA or PPARγ siRNA transfected ARPE-19 cells that were treated with or without ox-LDL (200 μg/ml) in the presence or absence of troglitazone or rosiglitazone for 48 hr. All data = mean ± SEM, *P<0.05 by 2-tail unpaired t-test, **P<0.01, ***P<0.001 by one-way ANOVA compared to the corresponding ox-LDL groups. Bezafibrate (beza), ciglitazone (cig), fenofibrate (feno), troglitazone (trog), rosiglitazone (rosi), pioglitazone (piog), vehicle (Veh)

After validating that ARPE-19 cells were sensitive to ox-LDL-induced cell death and that troglitazone effectively protected the cells in a comparable manner to primary human RPE (data not shown), we used ARPE-19 cells for the subsequent drug screening and mechanism of action experiments. To our surprise, despite the fact that the TZD-containing rosiglitazone is much more potent than troglitazone in activating PPARγ (EC50 of 30 nM vs. 550 nM), only troglitazone suppressed ox-LDL induced RPE cell death in a dose-dependent manner (Fig 1B). These results led us to investigate if, in fact, troglitazone exerts its cytoprotective effect through a PPARγ-independent mechanism.

After validating that PPARG siRNA effectively suppressed PPARG transcript expression by over 80% in the presence or absence of ox-LDL and troglitazone (Fig. 1C), scrambled control siRNA or PPARG siRNA transfected RPE cells were treated with vehicle or ox-LDL in the presence or absence of troglitazone or rosiglitazone. Conditioned media were collected to determine released LDH levels as a measurement of cell death. Results showed that troglitazone significantly (P< 0.001) inhibited ox-LDL–induced RPE cell death in the control and PPARγ suppressed group (Fig 1D), whereas rosiglitazone showed no protective effect in the control and PPARγ suppressed group (Fig 1D). These data strongly support a PPARγ-independent cytoprotective mechanism of troglitazone.

Hindered phenol compounds protect against ox-LDL–induced cell death

After identifying that troglitazone protection of RPE cells from ox-LDL–induced damage is independent of PPARγ (Fig 1), we focused on further understanding the unique cytoprotective property of troglitazone. Analysis of troglitazone chemical structure revealed the presence of a sterically-hindered phenol group (Supplementary Fig. S1A) 19. As hindered phenols have been reported to have antioxidant properties 20, we hypothesized that compounds that carry this functional group might protect cells from ox-LDL induced cell death.

To test this possibility, we screened different small molecule antioxidants with and without phenolic groups for their protective effects against ox-LDL–induced RPE cell death. Our data clearly showed that hindered phenol-containing compounds such as troglitazone, PMC (2,2,5,7,8-pentamethyl-6-chromanol) and trolox (6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid) protected RPE by about 90% at the concentrations tested, whereas other potent antioxidants lacking a phenol group such as 4-hydroxy-TEMPO, MCI-186, tiron (a mitochondrial localized antioxidant 21), DL-a-lipoic acid and N-Acetyl-L-cysteine (NAC) 22–25 all failed to protect the RPE against ox-LDL even at higher concentrations (Fig. 2A and 2B).

Figure 2: Hindered phenol compounds protect RPE cells from ox-LDL–induced cell death.

Figure 2:

Open in a new tab

(A) Confluent ARPE-19 cells maintained in culture in low serum media over two wk were treated with ox-LDL (200 μg/ml) in the presence or absence of hindered-phenol compounds troglitazone (trog), Trolox, or PMC as well as anti-oxidants that do not contain hindered phenol structure (TEMPO, MCI, tiron, lipoic acid, and NAC). Conditioned media were collected after 48 hr of treatment to determine LDH level as a measure of cell death.

(B) LDH levels were measured in the conditioned media collected from ARPE-19 cells treated with ox-LDL (200 μg/ml) for 48 hr and with a dose curve of hindered phenol compounds Trolox or troglitazone to determine the dose response curves (IC50 for Trolox is 5.436 μM and IC50 for troglitazone is 0.740 μM). Data = mean ± SEM, ***P<0.001 by one-way ANOVA compared to the ox-LDL group.

As we have shown that ox-LDL induced RPE cell death is mediated through lysosomal destabilization 12, we also determined the effect of phenol antioxidants on lysosomal integrity in cells fed with ox-LDL. Examination of ox-LDL treated RPE lysosomes by LysoTracker® revealed that phenol compounds troglitazone and trolox significantly preserved RPE lysosomes in a dose-dependent manner compared to ox-LDL treated group, whereas the negative control rosiglitazone failed to protect RPE lysosomes from ox-LDL–induced lysosomal destabilization (Figure 3). These data show that hindered phenol compounds are uniquely effective in protecting against ox-LDL–induced RPE death, and that their mechanism of action is likely via their antioxidant function and lysosomal protective activity. Since troglitazone, trolox, and PMC all showed similar protective effects against ox-LDL–induced RPE cell death, we used trolox or PMC for further mechanistic characterization.

Figure 3: Hindered phenol compounds inhibit ox-LDL–induced lysosomal destabilization.

Figure 3:

Open in a new tab

Left panels, confluent ARPE-19 cells that were maintained in culture in low serum media for two wk were treated with ox-LDL (200 μg/ml) for 36 hr in the presence or absence of rosiglitazone (rosig), troglitazone (trog), or Trolox at indicated concentrations. Following treatment, cells were incubated with LysoTracker™ red DND-99 and DAPI to label the nuclei, and the cells were imaged. Scale bar: 25μm.

Graph below, the number of labeled lysosomal particles were counted using imageJ software and normalized to DAPI labeled nucleus. Data = mean ± SEM, *P<0.05 and ***P<0.001 by one-way ANOVA compared.

Hindered phenol compounds are localized to the lysosomes

Lysosomes are sensors of reactive oxygen species (ROS) 26, 27, and permeabilization of the lysosomal membrane can trigger ROS formation under pathological conditions 28, 29. As lysosomal uptake of ox-LDL induces ROS generation as well as lysosomal destabilization 12, 30–32, we next investigated if hindered phenol compounds localize to RPE lysosomes following uptake. We generated RPE cells that transiently express lysosomal membrane protein (LAMP) 1-RFP to visualize the lysosomes, followed by treatment with FITC-tagged trolox (Supplementary Fig. S1B) and live cell imaging to assess the intracellular localization of FITC-trolox. The results revealed that FITC-trolox co-localized with LAMP1-RFP-expressing lysosomes/lysosomal membrane in the RPE cells (Fig. 4A). We also confirmed that incorporating the FITC tag did not alter the function of trolox in protecting the RPE from ox-LDL–induced cell death and FITC-trolox displayed activity similar to native trolox (Fig. 4B).

Figure 4: Hindered phenol compound targets the lysosomes of RPE.

Figure 4:

Open in a new tab

(A) ARPE-19 cells were transduced with CellLight™ BacMam 2.0 lysosomal membrane protein (LAMP) 1-RFP to visualize lysosomes. The LAMP1-RFP expressing cells were then treated with FITC-labeled trolox (25 μM). After 36 hr, the cells were examined by live imaging to visualize FITC-labeled trolox localization in LAMP1-RFP expressing lysosomes. Arrows point to FITC-labeled Trolox localization within LAMP1-RFP. Scale bar: 50 μm.

(B) ARPE-19 cells were treated with ox-LDL (200 μg/ml) for 48 hr in the presence or absence of native or FITC-tagged Trolox at different concentrations to determine their cytoprotective effects. Data = mean ± SEM, ***P< 0.001 compared to the ox-LDL group by one-way ANOVA.

As ox-LDL is known to elicit ROS generation, we next examined if the hindered phenol compounds and some trolox amide derivatives (Supplementary Fig. S1C and Supplementary Table 1) influenced ROS levels and cell death in ox-LDL treatment treated cells. We hypothesized that the lipophilicity of the hindered phenol compound could possibly play a role in its accumulation in the lysosomes/lysosomal membrane and therefore contribute to the protective activity for the RPE against ox-LDL–induced lysosomal destabilization and cell death (Fig. 2). We found that PMC and trolox amide derivatives 669, 738, and 829 significantly (P<0.0001) suppressed ox-LDL induced ROS generation at a low concentration of 0.43 μM, whereas N-acetyl cysteine (NAC), an established and less lipophilic antioxidant that does not contain a hindered phenol structure, required an extremely high concentration of 1,000 μM to suppress ox-LDL induced ROS generation (Fig. 5A). These data are consistent with the RPE protective effects of hindered phenol PMC and analogs (669, 738, 829), which at 0.43 μM completely inhibited ox-LDL–induced cell death, whereas NAC at a higher concentration of 1.29 μM failed to protect the RPE (Supplementary Fig. S2). As we predicted in light of the lysosomal sub-cellular location for ox-LDL induced ROS generation, the MitoSOX assay did not indicate a significant increase in mitochondrial superoxide upon ox-LDL treatment (Fig. 5B). In addition, mitochondrially-targeted anti-oxidant elamipretide 33, even at a high concentration (10 μM), suppressed cell death by only 25 and 34% at 24 and 48 hr, respectively, whereas a low dose of PMC (1 μM) significantly suppressed ox-LDL–induced cell death at 83% and 87% at 24 and 48 hr, respectively (Fig. 5C). This observation supports the concept that mitochondria are not the primary site of ROS generation and cellular damage induced by ox-LDL.

Figure 5: Hindered phenol compounds suppress cytosolic ROS induced by ox-LDL uptake.

Figure 5:

Open in a new tab

(A) Total ROS levels were measured in ARPE-19 cells eight hr post treatment with ox-LDL (200 μg/ml) in the presence or absence of Trolox analog PMC, Trolox amide analogs (669, 738, 829), or N-acetyl cysteine (NAC) at various indicated concentrations.

(B) ARPE-19 cells with and without ox-LDL (200 μg/ml) for eight hr and in the presence or absence of PMC, and then cells were labeled with MitoSOX™ to determine mitochondrial superoxide generation. Untreated cells and PMC alone treated cells were used as controls.

(C) ARPE-19 cells were treated with 200 μg/ml of ox-LDL for 48 hr in the presence and absence of PMC or the mitochondrially targeted antioxidant elamipretide (ELAM) at different concentrations. Conditioned media were collected at 24 and 48 hours and LDH levels were analyzed as a measure of cell death.

(D) ARPE-19 cells were treated with 200 μg/ml of ox-LDL for 48 hr in the presence and absence of PMC or different potent NADPH-oxidases inhibitors apocynin (APO) or diphenyleneiodonium (DPI) at different concentrations. Conditioned media were collected and LDH levels were analyzed as a measure of cell death. All data = mean ± SEM, **P<0.01, ***P< 0.001 and ****P<0.0001 by one-way ANOVA compared to the corresponding ox-LDL group.

Further corroborating our findings regarding the central role of the lysosomes in ox-LDL-induced cell death and protection, potent inhibitors of NADPH-oxidases (NOX) were not cytoprotective against ox-LDL induced RPE cell death in comparison to PMC (Fig. 5D), suggesting that NOX are not involved in the generation of ROS induced by ox-LDL in RPE. Lastly, because activation of the NLRP3 inflammasome pathway is involved in ox-LDL–induced RPE cell death 12, we tested if selective inhibitors of NLRP3 inflammasome protect the RPE against ox-LDL. While PMC at low concentration of 0.43 μM completely inhibited ox-LDL–induced RPE death, inflammasome-selective inhibitors failed to significantly suppress cell death at up to 10.4 μM (Supplementary Fig. S3).

Hindered phenol compound promoted lysosomal function and reduced intracellular ox-LDL accumulation

Lysosomal membrane protein Niemann-Pick C1 (NPC-1) is a key intracellular cholesterol transporter that help to maintain cellular cholesterol homeostasis 34–37. As our data show that hindered phenol compounds localized to the lysosomes, promoted lysosomal integrity, and suppressed ROS generation (Fig. 3, 4A and Fig. 5A), we examined if NPC-1 might be involved in the hindered phenol compound-mediated suppression of ROS generation. Treatment of serum-starved RPE cells with varying concentrations of PMC for 18 hr led to increased NPC-1 levels in a dose-dependent manner (Fig. 6A), with no change in NPC-2 level (data not shown). Moreover, ox-LDL treated cells that received PMC for 18 hr showed a significant two-fold increase in NPC-1 protein in comparison to ox-LDL treated cells without PMC (Fig, 6B). Finally, ox-LDL treatment of RPE for 8 hr significantly (P<0.01) increased the total cholesterol level, whereas PMC treatment significantly (P<0.001) reduced ox-LDL–mediated increase in intracellular cholesterol (Fig. 6C).

Figure 6: Hindered phenol compound increase NPC1 levels and reduce intracellular ox-LDL caused cholesterol accumulation.

Figure 6:

Open in a new tab

A. Serum starved ARPE-19 cells were treated with indicated doses of PMC for 18 hr, cell lysates were collected and NPC1 protein levels were examined by western blot and imaged Li-COR® infrared imaging system. The fluorescent intensity of NPC1 levels were normalized to the fluorescent intensity of α-tubulin to determine fold change.

B. ARPE-19 cells were treated with ox-LDL in the presence or absence of PMC, cell lysates were collected 18 hr post treatment and western blot was performed to determine the relative protein levels of NPC-1 using Li-COR® infrared imaging system. The fluorescent intensity of NPC1 levels were normalized to the fluorescent intensity of α-tubulin to determine the relative expression levels of NPC1.

C. ARPE-19 cells were treated with ox-LDL for 8hr, cells were washed and then incubated with or without PMC. Cell lysates were collected and the total cellular cholesterol content was determined by normalizing to total cellular protein content. Serum starved untreated cells served as control. * P<0.05, ** P<0.01, ***P<0.001.

Discussion

Our results demonstrate that hindered phenol compounds are localized to lysosomes/lysosomal membranes, suppressing lysosomally-derived ROS generation and facilitating the removal of ox-LDL, potentially through NPC-1 pathway, thereby inhibiting ox-LDL induced lysosomal destabilization and the subsequent cell death. The generation of free radicals is a component of normal metabolism, and cells possess a variety of mechanisms and systems to manage and respond to these by-products 38. Due to its high metabolic rate and relatively high local oxygen levels, the outer retina is predisposed to large fluctuations in levels of free radicals. A recent study of the early transcriptomic response to ox-LDL revealed upregulation of pathways that are designed to attenuate oxidative damage 15. However, in a number of pathological conditions, including AMD, variants in relevant genes, aging and/or smoking combine to overwhelm cells’ capacity to maintain homeostatic balance, resulting in pathology.

Dry AMD, and its late-stage counterpart, geographic atrophy, are characterized by the accumulation of sub-RPE deposits called drusen that have been shown to be comprised of a wide array of cells and plasma-associated proteins and lipids 1. Observations from genetic and epidemiological studies point to a multifactorial pathogenic process that includes the innate immune response (complement variants), altered lipid handling (high fat diet and variants of lipid handling genes) and oxidative stress (smoking and age-related injury). Many reports point to unchecked oxidative stress as the potential rate-limiting step in the development of AMD (reviewed in 39, 40), but precisely where these fit into the pathogenic process is a topic of significant research.

Rozing and co-authors hypothesize a “two-level model” that brings together a number of well-accepted variables. They suggest a first level of RPE damage due to oxidative stress, followed by a compensatory, but chronically unresolved, inflammatory response 41. In support of this idea, it is well documented that the process of aging also leaves the retina less able to offset both normal exposure to oxidative stress that is the result of light exposure and the elevated oxygen and metabolism that characterize the outer retina along with the pathologic reactive oxygen species associated with lifestyle choices such as smoking (reviewed in 42). Knowing the activity of PPARs as transcription factors whose activation increases lipid metabolism, suppresses the inflammatory responses, promotes lysosomal maturation and enhances degradation of oxidized lipids 43, we initially examined PPAR agonists as candidates for attenuating the pathogenesis of dry AMD. Protection of ox-LDL-induced RPE cell death by troglitazone, but not fenofibrate, directed us to PPARγ. However, the fact that rosiglitazone, another potent PPARγ agonist, was not protective, and that the knockdown of PPARγ in RPE did not attenuate the protection of RPE by troglitazone, indicated a mechanism of protection that was independent of PPARγ. Instead, the chemical structure of troglitazone revealed a hindered phenol group that was responsible for the RPE protective effect, as evidenced by the similar cytoprotective effects of other hindered phenol compounds such as trolox and PMC.

To gain further insight into the mechanism through which the hindered phenols were exerting their protective effect against ox-LDL–induced RPE cell death, we used fluorescently tagged trolox to track its subcellular localization. Results of these studies showed that trolox localized to the lysosomes. We speculate that the lipophilicity of hindered phenols can lead to their accumulation in the lysosomes/lysosomal membrane, where they protect against ox-LDL–induced lysosomal destabilization and cell death. This could explain our finding that the less lipophilic, but otherwise potent antioxidants such as TEMPO and NAC, are less effective compared to the more lipophilic hindered phenols in protecting the RPE.

We have previously shown that RPE cells take up ox-LDL via CD36 and are then trafficked to the lysosomes 12, and this study expands upon our prior work by showing that ox-LDL also generates ROS, a known intermediate to NLRP3 activation (reviewed in 38).

Consistent with this, we have previously demonstrated that the ultimate cause of RPE death due to ox-LDL is associated with activation of NLRP3 inflammasomes both in vitro and in vivo 44, and others have shown the involvement of Alu RNA 45, hyperglycemia 46, all-trans retinal 47 in NLRP3 inflammasome-mediated RPE toxicity. Recent reports have filled in some long-sought information on the steps that induce NLRP3 inflammasome assembly. A search for interacting partners with NLRP3 revealed NIMA-related kinase (NEK7), initially characterized for its role in mitosis 48 and which has now been shown to be activated by ROS (reviewed in 49), the target of the hindered phenol compounds that we have shown to be effective in protecting RPE from the cytotoxic effects of ox-LDL.

Thus, it seems likely that the hindered phenol compounds prevent RPE death by blocking or reducing the production or levels of ROS from internalized ox-LDL that would otherwise have gone onto mediate inflammasome activation. Consistent with this concept, N-acetylcysteine was shown to prevent inflammasome activation downstream of high glucose induced ROS production and puerarin inhibited NLRP3 activation by attenuating oxidative stress causes by ROS and ER stress 50, 51. Thus, blocking key upstream steps and in particular ROS generation in the processes that lead to NLRP3 inflammasome activation should be effective in preventing ox-LDL-induced and pyroptosis-mediated RPE cell death. Our data indicate that the class of hindered phenols described in this work are particularly effective antioxidants with respect to maintaining homeostasis in the lysosomal compartment against ox-LDL-induced toxicity, and are preventing RPE cell death upstream of inflammasome activation and that suppression of ROS generation is a rate limiting step in the process of inflammasome activation.

To harness the therapeutic potential of hindered phenols in animal models with AMD-like pathologies and especially RPE degeneration induced by aging and high fat diet 52, 53, a sustained-release formulation of hindered phenol, such as using a solid lipid nanoparticle (SLN) formulation should be a promising approach 54, 55. Our preliminary ocular pharmacokinetic data indicate that SLN depot formulation of hindered phenol compound delivered by periocular injection in the mouse will provide sustained release of the drug to achieve therapeutic dosage at the choroid.

Supplementary Material

1

Highlights.

  • Accumulation of oxidized and unoxidized forms of lipids contribute to RPE degeneration and AMD pathogenesis

  • Sterically-hindered phenol compounds suppressed ox-LDL-induced RPE cell death by inhibiting lysosomal destabilization and reducing ROS generation

  • Hindered phenol compounds increased RPE lysosomal NPC1 levels and decreased intracellular cholesterol accumulation

  • Hindered phenol compounds may represent a novel pharmacotherapy for AMD treatment

Acknowledgements

We thank Dhanesh Amarnani of the Schepens Eye Research Institute (SERI) for assisting with the LDH cell death assay, Lynn Zehbe for validating the ox-LDL–induced cell death assay in ARPE-19 cells. Dr. Magali Saint-Geniez for her assistance with human primary RPE cells isolation and culture.

G.G., A.M., E.Y.C., A.A., F.A.R., T.W.G, and Y.S.E.N. performed the experiments, collected and analyzed the data, and generated the figures. G.G., A.M., E.Y.C., A.A., F.A.R., T.W.G., D.S., and A.J.U. designed experiments and interpreted data, and performed literature searches. Y.S.E.N. and P.A.D. conceived the study, designed experiments, and interpreted data, generated figures, and performed literature searches. All authors wrote and approved the manuscript. Y.S.E.N. and P.A.D. are the guarantors of this work and, as such, had full access to all data in the study. They take responsibility for the integrity of the data and the accuracy of the data analysis.

Supported by May J. Wikstrom Fund via the Boston Foundation (P.A.D.), Edwin S. Webster Foundation research grant (Y.S.E.N.), Grimshaw-Gudewicz Foundation AMD research grant (Y.S.E.N.), and the NIH National Eye Institute Core grant P30EY003790.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • [1].Wang L, Clark ME, Crossman DK, Kojima K, Messinger JD, Mobley JA, Curcio CA: Abundant lipid and protein components of drusen. PloS one 2010, 5:e10329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].van Leeuwen R, Klaver CC, Vingerling JR, Hofman A, de Jong PT: Epidemiology of age-related maculopathy: a review. European journal of epidemiology 2003, 18:845–54. [DOI] [PubMed] [Google Scholar]
  • [3].Barbosa DT, Mendes TS, Cintron-Colon HR, Wang SY, Bhisitkul RB, Singh K, Lin SC: Age-related macular degeneration and protective effect of HMG Co-A reductase inhibitors (statins): results from the National Health and Nutrition Examination Survey 2005-2008. Eye 2014, 28:472–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Lakkaraju A, Umapathy A, Tan LX, Daniele L, Philp NJ, Boesze-Battaglia K, Williams DS: The cell biology of the retinal pigment epithelium. Progress in retinal and eye research 2020:100846. [DOI] [PMC free article] [PubMed]
  • [5].Ebrahimi KB, Handa JT: Lipids, lipoproteins, and age-related macular degeneration. Journal of lipids 2011, 2011:802059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Li CM, Clark ME, Rudolf M, Curcio CA: Distribution and composition of esterified and unesterified cholesterol in extra-macular drusen. Experimental eye research 2007, 85:192–201. [DOI] [PubMed] [Google Scholar]
  • [7].Storti F, Klee K, Todorova V, Steiner R, Othman A, van der Velde-Visser S, Samardzija M, Meneau I, Barben M, Karademir D, Pauzuolyte V, Boye SL, Blaser F, Ullmer C, Dunaief JL, Hornemann T, Rohrer L, den Hollander A, von Eckardstein A, Fingerle J, Maugeais C, Grimm C: Impaired ABCA1/ABCG1-mediated lipid efflux in the mouse retinal pigment epithelium (RPE) leads to retinal degeneration. Elife 2019, 8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Yamada Y, Tian J, Yang Y, Cutler RG, Wu T, Telljohann RS, Mattson MP, Handa JT: Oxidized low density lipoproteins induce a pathologic response by retinal pigmented epithelial cells. J Neurochem 2008, 105:1187–97. [DOI] [PubMed] [Google Scholar]
  • [9].Gnanaguru G, Wagschal A, Oh J, Saez-Torres KL, Li T, Temel RE, Kleinman ME, Naar AM, D’Amore PA: Targeting of miR-33 ameliorates phenotypes linked to age-related macular degeneration. Mol Ther 2021, 29:2281–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Yu AL, Lorenz RL, Haritoglou C, Kampik A, Welge-Lussen U: Biological effects of native and oxidized low-density lipoproteins in cultured human retinal pigment epithelial cells. Experimental eye research 2009, 88:495–503. [DOI] [PubMed] [Google Scholar]
  • [11].Kim JH, Lee SJ, Kim KW, Yu YS, Kim JH: Oxidized low density lipoprotein-induced senescence of retinal pigment epithelial cells is followed by outer blood-retinal barrier dysfunction. Int J Biochem Cell Biol 2012, 44:808–14. [DOI] [PubMed] [Google Scholar]
  • [12].Gnanaguru G, Choi AR, Amarnani D, D’Amore PA: Oxidized Lipoprotein Uptake Through the CD36 Receptor Activates the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells. Investigative ophthalmology & visual science 2016, 57:4704–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Picard E, Houssier M, Bujold K, Sapieha P, Lubell W, Dorfman A, Racine J, Hardy P, Febbraio M, Lachapelle P, Ong H, Sennlaub F, Chemtob S: CD36 plays an important role in the clearance of oxLDL and associated age-dependent sub-retinal deposits. Aging 2010, 2:981–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Gordiyenko N, Campos M, Lee JW, Fariss RN, Sztein J, Rodriguez IR: RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo. Investigative ophthalmology & visual science 2004, 45:2822–9. [DOI] [PubMed] [Google Scholar]
  • [15].Koirala D, Beranova-Giorgianni S, Giorgianni F: Early Transcriptomic Response to OxLDL in Human Retinal Pigment Epithelial Cells. Int J Mol Sci 2020, 21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Babaev VR, Ishiguro H, Ding L, Yancey PG, Dove DE, Kovacs WJ, Semenkovich CF, Fazio S, Linton MF: Macrophage expression of peroxisome proliferator-activated receptor-alpha reduces atherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation 2007, 116:1404–12. [DOI] [PubMed] [Google Scholar]
  • [17].Li AC, Brown KK, Silvestre MJ, Willson TM, Palinski W, Glass CK: Peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor-deficient mice. The Journal of clinical investigation 2000, 106:523–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA: An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). The Journal of biological chemistry 1995, 270:12953–6. [DOI] [PubMed] [Google Scholar]
  • [19].Barclay LRC, Vinqvist MR, Mukai K, Itoh S, & Morimoto H: Chain-breaking phenolic antioxidants: Steric and electronic effects in polyalkylchromanols, tocopherol analogs, hydroquinones, and superior antioxidants of the polyalkylbenzochromanol and naphthofuran class. Journal of Organic Chemistry 1993, 58(26):7416–20. [Google Scholar]
  • [20].Hope N, Bellare A: A comparison of the efficacy of various antioxidants on the oxidative stability of irradiated polyethylene. Clin Orthop Relat Res 2015, 473:936–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Oyewole AO, Wilmot MC, Fowler M, Birch-Machin MA: Comparing the effects of mitochondrial targeted and localized antioxidants with cellular antioxidants in human skin cells exposed to UVA and hydrogen peroxide. FASEB J 2014, 28:485–94. [DOI] [PubMed] [Google Scholar]
  • [22].Vorobjeva NV, Pinegin BV: Effects of the antioxidants Trolox, Tiron and Tempol on neutrophil extracellular trap formation. Immunobiology 2016, 221:208–19. [DOI] [PubMed] [Google Scholar]
  • [23].Kawai H, Nakai H, Suga M, Yuki S, Watanabe T, Saito KI: Effects of a novel free radical scavenger, MCl-186, on ischemic brain damage in the rat distal middle cerebral artery occlusion model. The Journal of pharmacology and experimental therapeutics 1997, 281:921–7. [PubMed] [Google Scholar]
  • [24].Arivazhagan P, Thilakavathy T, Ramanathan K, Kumaran S, Panneerselvam C: Effect of DL-alpha-lipoic acid on the status of lipid peroxidation and protein oxidation in various brain regions of aged rats. J Nutr Biochem 2002, 13:619–24. [DOI] [PubMed] [Google Scholar]
  • [25].Aldini G, Altomare A, Baron G, Vistoli G, Carini M, Borsani L, Sergio F: N-Acetylcysteine as an antioxidant and disulphide breaking agent: the reasons why. Free Radic Res 2018, 52:751–62. [DOI] [PubMed] [Google Scholar]
  • [26].Kubota C, Torii S, Hou N, Saito N, Yoshimoto Y, Imai H, Takeuchi T: Constitutive reactive oxygen species generation from autophagosome/lysosome in neuronal oxidative toxicity. The Journal of biological chemistry 2010, 285:667–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Zhang X, Cheng X, Yu L, Yang J, Calvo R, Patnaik S, Hu X, Gao Q, Yang M, Lawas M, Delling M, Marugan J, Ferrer M, Xu H: MCOLN1 is a ROS sensor in lysosomes that regulates autophagy. Nat Commun 2016, 7:12109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Dielschneider RF, Henson ES, Gibson SB: Lysosomes as Oxidative Targets for Cancer Therapy. Oxid Med Cell Longev 2017, 2017:3749157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Nohl H, Gille L: Lysosomal ROS formation. Redox Rep 2005, 10:199–205. [DOI] [PubMed] [Google Scholar]
  • [30].Sheedy FJ, Grebe A, Rayner KJ, Kalantari P, Ramkhelawon B, Carpenter SB, Becker CE, Ediriweera HN, Mullick AE, Golenbock DT, Stuart LM, Latz E, Fitzgerald KA, Moore KJ: CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation. Nature immunology 2013, 14:812–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Lara-Guzman OJ, Gil-Izquierdo A, Medina S, Osorio E, Alvarez-Quintero R, Zuluaga N, Oger C, Galano JM, Durand T, Munoz-Durango K: Oxidized LDL triggers changes in oxidative stress and inflammatory biomarkers in human macrophages. Redox Biol 2018, 15:1–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Bae YS, Lee JH, Choi SH, Kim S, Almazan F, Witztum JL, Miller YI: Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2. Circulation research 2009, 104:210–8, 21p following 8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Chatfield KC, Sparagna GC, Chau S, Phillips EK, Ambardekar AV, Aftab M, Mitchell MB, Sucharov CC, Miyamoto SD, Stauffer BL: Elamipretide Improves Mitochondrial Function in the Failing Human Heart. JACC Basic Transl Sci 2019, 4:147–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Houben T, Oligschlaeger Y, Bitorina AV, Hendrikx T, Walenbergh SMA, Lenders MH, Gijbels MJJ, Verheyen F, Lutjohann D, Hofker MH, Binder CJ, Shiri-Sverdlov R: Blood-derived macrophages prone to accumulate lysosomal lipids trigger oxLDL-dependent murine hepatic inflammation. Scientific reports 2017, 7:12550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Kulinski A, Vance JE: Lipid homeostasis and lipoprotein secretion in Niemann-Pick C1-deficient hepatocytes. The Journal of biological chemistry 2007, 282:1627–37. [DOI] [PubMed] [Google Scholar]
  • [36].Pfeffer SR: NPC intracellular cholesterol transporter 1 (NPC1)-mediated cholesterol export from lysosomes. The Journal of biological chemistry 2019, 294:1706–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Zhang JR, Coleman T, Langmade SJ, Scherrer DE, Lane L, Lanier MH, Feng C, Sands MS, Schaffer JE, Semenkovich CF, Ory DS: Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking. The Journal of clinical investigation 2008, 118:2281–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Checa J, Aran JM: Reactive Oxygen Species: Drivers of Physiological and Pathological Processes. J Inflamm Res 2020, 13:1057–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Zhang ZY, Bao XL, Cong YY, Fan B, Li GY: Autophagy in Age-Related Macular Degeneration: A Regulatory Mechanism of Oxidative Stress. Oxid Med Cell Longev 2020, 2020:2896036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].E BD, Marfany G: The Relevance of Oxidative Stress in the Pathogenesis and Therapy of Retinal Dystrophies. Antioxidants (Basel) 2020, 9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Rozing MP, Durhuus JA, Krogh Nielsen M, Subhi Y, Kirkwood TB, Westendorp RG, Sorensen TL: Age-related macular degeneration: A two-level model hypothesis. Progress in retinal and eye research 2020, 76:100825. [DOI] [PubMed] [Google Scholar]
  • [42].Kaarniranta K, Koskela A, Felszeghy S, Kivinen N, Salminen A, Kauppinen A: Fatty acids and oxidized lipoproteins contribute to autophagy and innate immunity responses upon the degeneration of retinal pigment epithelium and development of age-related macular degeneration. Biochimie 2019, 159:49–54. [DOI] [PubMed] [Google Scholar]
  • [43].Gervois P, Torra IP, Fruchart JC, Staels B: Regulation of lipid and lipoprotein metabolism by PPAR activators. Clin Chem Lab Med 2000, 38:3–11. [DOI] [PubMed] [Google Scholar]
  • [44].Tseng WA, Thein T, Kinnunen K, Lashkari K, Gregory MS, D’Amore PA, Ksander BR: NLRP3 inflammasome activation in retinal pigment epithelial cells by lysosomal destabilization: implications for age-related macular degeneration. Investigative ophthalmology & visual science 2013, 54:110–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Kim Y, Tarallo V, Kerur N, Yasuma T, Gelfand BD, Bastos-Carvalho A, Hirano Y, Yasuma R, Mizutani T, Fowler BJ, Li S, Kaneko H, Bogdanovich S, Ambati BK, Hinton DR, Hauswirth WW, Hakem R, Wright C, Ambati J: DICER1/Alu RNA dysmetabolism induces Caspase-8-mediated cell death in age-related macular degeneration. Proceedings of the National Academy of Sciences of the United States of America 2014, 111:16082–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [46].Yang Q, Li S, Zhou Z, Fu M, Yang X, Hao K, Liu Y: HDAC6 inhibitor Cay10603 inhibits high glucose-induced oxidative stress, inflammation and apoptosis in retinal pigment epithelial cells via regulating NF-kappaB and NLRP3 inflammasome pathway. Gen Physiol Biophys 2020, 39:169–77. [DOI] [PubMed] [Google Scholar]
  • [47].Liao Y, Zhang H, He D, Wang Y, Cai B, Chen J, Ma J, Liu Z, Wu Y: Retinal Pigment Epithelium Cell Death Is Associated With NLRP3 Inflammasome Activation by All-trans Retinal. Investigative ophthalmology & visual science 2019, 60:3034–45. [DOI] [PubMed] [Google Scholar]
  • [48].He Y, Zeng MY, Yang D, Motro B, Nunez G: NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature 2016, 530:354–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Liu G, Chen X, Wang Q, Yuan L: NEK7: a potential therapy target for NLRP3-related diseases. Biosci Trends 2020, 14:74–82. [DOI] [PubMed] [Google Scholar]
  • [50].Wang P, Chen F, Wang W, Zhang XD: Hydrogen Sulfide Attenuates High Glucose-Induced Human Retinal Pigment Epithelial Cell Inflammation by Inhibiting ROS Formation and NLRP3 Inflammasome Activation. Mediators Inflamm 2019, 2019:8908960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [51].Wang K, Zhu X, Zhang K, Yao Y, Zhuang M, Tan C, Zhou F, Zhu L: Puerarin inhibits amyloid beta-induced NLRP3 inflammasome activation in retinal pigment epithelial cells via suppressing ROS-dependent oxidative and endoplasmic reticulum stresses. Exp Cell Res 2017, 357:335–40. [DOI] [PubMed] [Google Scholar]
  • [52].Miceli MV, Newsome DA, Tate DJ Jr., Sarphie TG: Pathologic changes in the retinal pigment epithelium and Bruch’s membrane of fat-fed atherogenic mice. Curr Eye Res 2000, 20:8–16. [PubMed] [Google Scholar]
  • [53].Roddy GW, Rosa RH, Viker KB, Holman BH, Hann CR, Krishnan A, Gores GJ, Bakri SJ, Fautsch MP: Diet Mimicking “Fast Food” Causes Structural Changes to the Retina Relevant to Age-Related Macular Degeneration. Curr Eye Res 2020, 45:726–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].Abrishami M, Abrishami M, Mahmoudi A, Mosallaei N, Vakili Ahrari Roodi M, Malaekeh-Nikouei B: Solid Lipid Nanoparticles Improve the Diclofenac Availability in Vitreous after Intraocular Injection. Journal of drug delivery 2016, 2016:1368481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [55].Freitas LGA, Isaac DLC, Lima EM, Souza LG, Abud MA, Reis RGD, Tannure WT, Avila MP: Retinal changes in rabbit after intravitreal injection of sunitinib encapsulated into solid lipid nanoparticles and polymeric nanocapsules. Arq Bras Oftalmol 2018, 81:408–13. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1762240-supplement-1.pdf (506.4KB, pdf)

RESOURCES