August 2004
Volume 45, Issue 8
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Retinal Cell Biology  |   August 2004
Cytotoxicity of Oxidized Low-Density Lipoprotein in Cultured RPE Cells Is Dependent on the Formation of 7-Ketocholesterol
Author Affiliations
  • Ignacio R. Rodriguez
    From the Laboratory of Retinal Cell and Molecular Biology, Section on Mechanisms of Retinal Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
  • Shahabuddin Alam
    From the Laboratory of Retinal Cell and Molecular Biology, Section on Mechanisms of Retinal Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
  • Jung Wha Lee
    From the Laboratory of Retinal Cell and Molecular Biology, Section on Mechanisms of Retinal Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
Investigative Ophthalmology & Visual Science August 2004, Vol.45, 2830-2837. doi:https://doi.org/10.1167/iovs.04-0075
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      Ignacio R. Rodriguez, Shahabuddin Alam, Jung Wha Lee; Cytotoxicity of Oxidized Low-Density Lipoprotein in Cultured RPE Cells Is Dependent on the Formation of 7-Ketocholesterol. Invest. Ophthalmol. Vis. Sci. 2004;45(8):2830-2837. doi: https://doi.org/10.1167/iovs.04-0075.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine which components present in oxidized LDL are responsible for the cytotoxicity associated with its internalization by culture ARPE19 cells.

methods. ARPE19 cells were grown in 24-well and 96-well plates. Cell viability was measured by MTT and/or adenosine triphosphate (ATP) content. LDL was oxidized with Cu+2 and oxysterol content analyzed by a novel HPLC method.

results. OxLDL showed increased cytotoxicity with prolonged oxidation. Analysis of the oxLDL showed a predominance of the 7-oxygenated products, 7α-hydroxycholesterol (7αHCh), 7β-hydroxycholesterol (7βHCh), and 7-ketocholesterol (7kCh). Addition of these oxysterols to the ARPE19 cell in free form indicated that 7kCh is the most cytotoxic of the oxysterols but at physiologically unrealistic concentrations. Partitioning of individual oxysterols into nonoxidized LDL at concentrations similar to those found in the oxLDL also indicated that 7kCh is the most cytotoxic of the oxysterols. Transition metals are tightly bound by LDL and play an important role in the oxidation of LDL, but do not seem to enhance its cytotoxicity directly.

conclusions. Prolonged oxidation of LDL increases the levels of 7kCh due to further oxidation of 7αHCh and 7βHCh. The formation of 7KCh seems to be responsible for most of the cytotoxicity associated with oxLDL internalization in ARPE19 cells.

Our interest is in understanding the pathogenesis of age-related macular degeneration, the leading cause of blindness in elderly individuals. 1 This is a complex disease that involves the aging process as well as genetics and environmental factors. The accumulation of cholesterol in Bruch’s membrane as a process of aging 2 as well as the epidemiologic association to atherosclerosis 3 suggests a mechanistic relation between these two diseases. In atherosclerosis, the accumulation of low-density lipoproteins (LDL) in arteries and its subsequent oxidation and ingestion by macrophages is believed to be critical in the formation of atherosclerotic plaques. 4 The internalization of oxidized LDL (oxLDL) by macrophages leads to foam cell formation, and this process is thought to be one of the principle causes of atherosclerosis. 5 The cytotoxicity of oxLDL has also been reported in aortic endothelial cells 6 7 and retinal pigment epithelium (RPE) cells. 8 In aortic endothelial cells, oxLDL was found to increase the amount of reactive oxygen species 6 and reduce biological activity. 7 In RPE cells oxLDL inhibits phagocytosis of rod outer segment membranes. 8 The oxidation of the esterified and unesterified cholesterol within the LDL particle generates a series of cholesterol oxides known as oxysterols that have potent pharmacological activities. 9 10 These include the inhibition of cholesterol synthesis and the induction of apoptosis and necrosis in a variety of cells. 10 Direct oxysterol cytotoxicity has been demonstrated in several tissue culture cell systems. 10 These oxysterols are the main suspects in cytotoxicity and other adverse biological effects associated with oxLDL. 11  
Our hypothesis is that, as humans age, a slow accumulation of cholesterol occurs in Bruch’s membrane and choriocapillaris under the macula 2 and then gradually oxidizes. As this material oxidizes it becomes increasingly more toxic impairing both RPE and scavenging macrophage function leading to inflammatory responses similar to those in atherosclerotic plaques. 5 6 7 This could lead to the formation of drusen deposits, which further stress the RPE and generate additional toxic substances. Macrophages also release VEGF in response to oxLDL internalization, 12 which may contribute to the choroidal neovascularization observed in some of the more severe cases of AMD. 
In our accompanying study we have shown that rat RPE cells will internalize human rhodamine-labeled LDL and form deposits in Bruch’s membrane within 24 hours. 13 This internalization does not seem to occur homogenously throughout the retina, suggesting that the fenestrated choroidal endothelium may have some filtering capabilities, allowing LDL to enter some locations and not others. 13 This may explain why in humans cholesterol accumulation seems to be greater in the macula than in the peripheral retina. 2  
In this study, we used a novel approach to study the oxysterol cytotoxicity by partitioning different oxysterols into nonoxidized LDL. This allowed us to avoid the complexity of a full LDL oxidation and to present each oxysterol individually to the RPE cells in LDL at physiologically relevant concentrations. We also examined the effects of transition metals on oxysterol cytotoxicity, since they can have both beneficial and detrimental effects on retinal cells. 14 The effects of zinc are particularly interesting, since this metal may play a beneficial role in slowing the progression of AMD. 15  
For the purposes of this article and its companion, 13 the word “cytotoxicity” refers to the measurable nonviable cell fraction, as determined in our assays. 
Materials and Methods
Cholesterol and oxysterols were purchased from Steraloids Inc. (Newport, RI) and dissolved in 100% ethanol to make 10-mM stock solutions. Human LDL cholesterol was purchased from Calbiochem (San Diego, CA). Penicillin-streptomycin (10,000 I.U/mL-10,000 μg/mL) was purchased from Media Teck Inc. (Herndon, VA). 
Tissue Culture
ARPE19 cells were purchased from American Type Culture Collection (Manassas, VA). hTERT-RPE1 cells were purchased from BD Biosciences-Clontech (Palo Alto, CA). hTERT cells are telomerase-immortalized human RPE cells. Both cell types were cultured in DMEM/F12 containing 10% fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. 
In cytotoxicity, experiments cells were grown in 24- and/or 96-well plates in serum-containing medium until confluent. The cells were then changed to serum-free medium and treated with oxLDL and/or oxysterols at different concentrations and for different times (see figure legends for details). 
Cell Viability Assays
Cell viability was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) for mitochondrial dehydrogenase activity and/or by measuring adenosine triphosphate (ATP) levels (CellTiter-Gl Luminescent Cell viability assay; Promega, Madison, WI). The 24- and 96-well plates were read using a counter (Victor model 1420 multilabel counter; Wallac Inc., Gaithersburg, MD) with the appropriate filters. 
Preparation of Oxidized LDL
The LDL was oxidized using CuSO4, as previously described, 16 with some modifications. The LDL (1 mL, 1 mg/mL) was dialyzed in 500 mL of 1× PBS (pH 7.4) overnight. Sample for each time point was in separate dialysis tubing. Copper sulfate was added to a final concentration of 5 μM, and the LDL was allowed to oxidize at room temperature for 24, 48, and 72 hours. The oxidation was stopped by moving each individual sample to a dialysis chamber containing 1× PBS (pH 7.4) and 1 mM EDTA at the different time points. The oxLDL from each time point was then used for oxysterol analysis and cytotoxicity experiments. 
Sterol Analyses of oxLDL
The sterols were analyzed using a novel technique developed in this laboratory. Each oxLDL sample, 0.1 mL (1 mg/mL), was lyophilized and mixed with 0.2 mL of 60% KOH in methanol and 100 nM of β-sitosterol as the internal standard. The mixture was hydrolyzed at 37°C for 1 hour in a glass tube with a Teflon septum flushed with argon. The KOH was neutralized with 0.2 mL of 50% acetic acid, and the sterols extracted with 1 mL of a 50:50 mix of petroleum ether and dichloromethane. The organic phase was removed, evaporated with an argon stream, and dissolved in 100% ethanol. The cholesterol and oxysterol content was determined by HPLC analysis. The sterol analyses were performed using a HPLC system (model 2790, controlled with Empower Pro software; Waters Corp., Milford, MA). Sterols were detected using a photodiode array detector equipped with a 4-μL cell (model 996; Waters Corp.). The oxysterols were separated in a 4.6 × 250-mm column (X-terra RP-18; Waters Corp.) running a gradient, starting at 15% 1 mM phosphoric acid in water with 85% to 100% acetonitrile at 60°C in 15 minutes, flowing at 1 mL/min. The column was flushed with 100% methanol for 2 minutes and reequilibrated with 15:85 (vol/vol) 1 mM H3PO4 water-acetonitrile for 5 minutes between injections. Spectra were collected between 190 and 300 nm. A full description of this technique is in press in Biotechniques. 18  
Results
Cytotoxicity of oxLDL Versus Time Course of Oxidation by Cu+2
In our accompanying study, we have shown that LDL and oxLDL can be internalized by the cultured ARPE19 cells in large quantities. 13 It has been shown that oxLDL cytotoxicity was dependent on the content of lipid hydroperoxide (LPO) and the presence of transition metals such as copper and iron. 17 LDL is known to form complexes with transition metals, and this interaction has been reported to be necessary for its oxidation and toxicity. 16 17 To determine whether cytotoxicity was dependent on degree of oxidation in ARPE19 cells LDL was oxidized with Cu+2, as described earlier. The differentially oxidized LDL was given to the cultured ARPE19 cells at 0 to 50 μg/mL (total LDL) for 48 hours and cell viability measured by MTT (Fig. 1) . LDL cytotoxicity was observed to increase with prolonged exposure to Cu+2 oxidation. In this study, the amount of “cytotoxicity” refers to the amount of nonviable cells determined in our viability assays. 
Oxysterol Analyses of oxLDLs
The LDL used in these experiments is approximately 20% protein and 80% lipid, with cholesterol accounting for approximately 33% to 36% of the total mass of the LDL. The differentially oxidized oxLDLs were hydrolyzed, and the sterols were extracted, as described in the Methods section. The sterols were analyzed with a novel HPLC protocol. 18 We identified the presence of four main oxysterols identified by other investigators. 19 20 These were 25-hydroxycholesterol (25HCh), 7-α-hydroxycholesterol (7αHCh), 7-β-hydroxycholesterol (7βHCh), and 7-ketocholesterol (7kCh). The amounts were determined by peak area calculations relative to a known amount of authentic standards for each oxysterol. The plant-derived sterol β-sitosterol was used as an internal standard. In this HPLC protocol β-sitosterol separates cleanly from cholesterol and its oxidized derivatives. 18 The quantification of Ch, 7αHCh, 7βHCh, and 25HCh was performed at 200 nm, and 7kCh was quantified at 237 nm. The oxysterol 7kCh is sensitive to alkaline hydrolysis, forming a detectable yet unidentified derivative with an absorption maximum at 277 nm. The hydrolysis conditions used (under argon gas) minimized but did not completely eliminate the formation of this derivative. Thus, the 7kCh amounts reported may be slightly underestimated. The results of the analyses are shown in Table 1 and are presented as a percentage of the total sterols detected. Even after 72 hours, cholesterol remained the main sterol (74%) in the oxLDL. The oxidation conditions we used were considerably less harsh than those reported by Dzeletovic et al. 20 in which 52% to 76% of cholesterol was oxidized in 24 hours. We have found that heavily oxidized LDL becomes insoluble, and the ARPE19 cells have difficulty internalizing it effectively within the 48-hour experimental paradigm. Although this heavily oxidized LDL may be of physiological significance especially in plaque formation and macrophage toxicity, it was not used in these studies. 
Cytotoxicity of Individual Oxysterols
Previously published results have indicated that the concentration range for cytotoxicity of oxysterols is between 30 and150 μM. In ARPE19 cells, Ong et al. 21 used 25HCh and 7kCh at 25 to 155 μM to achieve 60% and 80% cell death in 48 hours, respectively. We found that determining the concentration at which oxysterols showed measurable and consistent cytotoxicity in cultured RPE cells was complicated by the extremely insoluble nature of oxysterols in aqueous medium. Measurements done in different types of culture flasks varied because of the differences in the ratio of media to plastic surface, where the insoluble oxysterols tend to adhere. The cytotoxicity results we are reporting were obtained in 24-well plates. We tested ARPE19 and hTERT cells with several oxysterols previously shown to be cytotoxic to ARPE19 cells 21 and others also present in oxLDL 19 20 (Table 1) . At the concentration of 50 μM (∼20 μg/mL) we found significant cytotoxicity between 24 and 72 hours (Fig. 2) . The results suggest that the oxysterols 25HCh, 7βHCh, and 7αHCh are markedly less cytotoxic than 20-α-hydroxycholesterol (20αHCh) and 7kCh when added directly to these cells. The presence of 20αHCh has not been reported in Cu+2 oxLDL, 19 20 but may be present in other forms of oxidized LDL. The hTERT cells seem to be slightly more resistant to the free oxysterols than ARPE19 cells (Fig. 2) but seem more susceptible to oxLDL (data not shown). 
Cytotoxicity of Oxysterols Partitioned in LDL
We were concerned that at the high-micromolar oxysterol concentrations used (50 μM), the cytotoxicity would be due to direct effects on the plasma membrane and not representative of the effects observed with the oxLDL (Fig. 1) . In addition, since Cu+2 oxidation of LDL creates a complex mixture of oxidized lipids that are too hydrophobic to study properly in aqueous media, we decided to take a different approach to determining which of the oxysterols were truly causing oxLDL cytotoxicity. We discovered that we could readily partition oxysterols into the hydrophobic lipid moiety of the LDL particle. Thus, we could analyze the cytotoxicity of each oxysterol by simply mixing the appropriate amount of the desired oxysterol with the nontoxic, nonoxidized LDL. In this manner, LDL can be used to transport individual and combinations of oxysterols at more physiologically relevant concentrations and without the complexity of a full oxidation. 
The LDL was diluted to a final concentration of 1 mg/mL LDL containing approximately 1 mM total cholesterol. The four main oxysterols 25HCh, 7αHCh, 7βHCh, and 7kCh were partitioned into LDL to achieve 5% and 10% oxysterol content relative to cholesterol. Different amounts of the oxysterol-laced LDLs (0–100 μg/mL) were given to the ARPE19 cells for 48 hours, and cell viability was measured by MTT hydrolysis (Fig. 3) . The results indicate that 7kCh is the most cytotoxic of the oxysterols tested, which correlates well with the results obtained with the free oxysterols (Fig. 2) . 7βHCh was markedly more cytotoxic when given with LDL (Fig. 3) than in free form (Fig. 2) and more cytotoxic than 25HCh and 7αHCh, but still considerably less cytotoxic than 7kCh. The 10% oxysterol-LDL was analyzed by HPLC to verify its oxysterol content (Table 2) . The results verify that our calculated values, which were based on cholesterol content, were approximately correct. 
Potentiation of LDL Cytotoxicity by Transition Metals
Transition metals have been implicated in potentiating oxysterol cytotoxicity 16 17 although the mechanism by which this occurs is not well understood. We cultured ARP19 cells in 96-well plates at 80% to 90% confluence. The cells were transferred to serum-free medium containing six different transition metals (Zn+2, Cu+2, Cd+2, Fe+3, Co+2, Mn+2) at 25, 50, and 100 μM concentrations. We then added 25 μg/mL 10% oxysterols LDL (approximately 2.5 μM oxysterols) and incubated for 48 hours (Fig. 4) . At this concentration, most of the oxysterol-LDL mixtures caused little or no cytotoxicity in 48 hours, except 7kCh-LDL, which caused approximately 40% to 50% cytotoxicity. Relatively nontoxic metals such as copper (Fig. 4A) , iron (Fig. 4B) , and manganese (Fig. 4C) showed little or no potentiation of cytotoxicity, even at 100 μM metal. Cobalt also showed no potentiation, but obvious cytotoxicity at 100 μM (Fig. 4D) . Zinc was cytotoxic at all concentrations, but the addition of LDL, with or without oxysterols, protected the cells from the cytotoxic effects of the metal at 25 and 50 μM (Fig. 4E) . Cadmium was so toxic to ARPE19 cells that no protection was observed, even at the 25-μM concentration; these samples served as a control for total cell death (Fig. 4F)
Discussion
The goals of the present studies were to determine which of the main oxysterols found in oxLDL are responsible for the cytotoxicity observed in ARPE19 cells and to evaluate a possible potentiation of this cytotoxicity by transition metals. ARPE19 cells readily internalize LDL and oxLDL, reaching a plateau of 10 to 12 pg/cell for LDL and 14 to 16 pg/cell for oxLDL within 24 hours after exposure at concentrations of 25 μg/mL or greater (Ref. 13 , companion article). Previous studies demonstrating oxLDL 8 and oxysterol cytotoxicity in APRE19 cells 21 did not fully address the potential mechanism(s) for the observed effects. The very high oxysterol concentrations (>150 μM) used by Ong et al. 21 could have caused the cytotoxicity by simple replacement of the membrane cholesterol, resulting in plasma membrane instability, rather than by a specific effect caused by the oxysterol internalization. The studies with oxLDL showing the inhibition of rod outer segment phagocytosis also did not demonstrate whether this effect was due to competitive inhibition and/or depletion of the CD36 receptor and/or to direct oxLDL cytotoxicity. The CD36 receptor is known to internalize both oxLDL 13 22 and rod outer segments. 23 In this study, we took advantage of the observation that oxysterols could be carried into the ARPE19 cells by LDL. This allowed us to look at the cytotoxic effect of each individual oxysterol at physiologically relevant concentrations without the effects of oxidized phospholipids and/or fatty acids. We also examined mixtures of oxysterols but did not detect any significant synergism (data not shown). This was also noticed by other investigators 24 who observed an upregulation of the fibrogenic cytokine transforming growth factor (TGF)-β1 by oxysterols in macrophages. Other components of oxLDL are also known also to have pharmacological effects, such as the oxidized fatty acids 25 and the phospholipids. 26 The partitioning of hydrophobic molecules in LDL may also be helpful in studying the effects of the other types of oxidized lipids found in oxLDL. Although this study focused specifically on the cytotoxic effects of the most abundant oxysterols present in oxLDL on ARPE cells, the noncytotoxic effects associated with oxLDL internalization such as the NF-κB activation, 6 nitric oxide decrease, 7 and inflammatory responses 12 24 26 are also of great interest to us. The chronic effects of nonlethal doses of oxLDL and the contribution of the other oxidized components on RPE function should be investigated further. 
We found that LDL becomes increasingly cytotoxic with prolonged oxidation (Fig. 1) . LDL also has a tendency to become insoluble if allowed to oxidize with Cu+2 for longer than 72 hours. Although this increases the amounts of cytotoxic lipids, it also increases the amount of time the ARPE19 cells need to internalize it. This could lead to problems in interpreting the cytotoxicity results, because the cells are not only in serum-free medium for a longer period, but also are covered with insoluble particles. These conditions on their own could induce stress and enhance cytotoxicity. Thus, we restricted this study to soluble forms of oxLDL and incubation times of 48 hours. However, this insoluble, highly oxidized LDL is likely to have the most profound effects on macrophages and may be a factor in AMD. A new animal model for age-related macular degeneration deficient in the chemokine receptor-2 (Ccl-2) clearly demonstrates that choroidal macrophages are needed to clear RPE secretions and maintain a healthy Bruch’s membrane. 27 The effects of oxLDL on choroidal macrophages should be seriously considered in the pathogenesis mechanism of AMD. 
Analyses of the oxLDL at different time points detected the presence of many oxysterols, although the composition was dominated by 7αHCh, 7βHCh, and 7kCh. This is in agreement with previously published analyses of Cu+2- and lipoxygenase-oxidized LDL which showed that 80% of the oxysterols made were oxidized at the 7-carbon position. 20 Our oxysterol analysis of the oxLDL found a strong correlation between cytotoxicity and the emergence of 7kCh. We observed the levels of 7αHCh and 7βHCh decrease with oxidation time, whereas the formation of 7kCh increased. We suspect this is caused by further oxidation of the 7αHCh and 7βHCh. We have also observed that authentic standards of 7αHCh and 7βHCh gradually form 7kCh by prolonged exposure to ambient light and temperature (data not shown). This suspected oxidation of 7αHCh and 7βHCh to 7kCh had also been reported. 20 The results suggest that 7αHCh and 7βHCh may be gradually oxidized to 7kCh, and the increasing levels of 7kCh seem to be responsible for the increase in cell death observed with prolonged oxidation. 
A recently published study showed that R28 and AREP19 cells are susceptible to 25HCh and 7kCh cytotoxicity. 21 These investigators used concentrations ranging from 25 to 155 μM (10–50 μg/mL) to achieve 60% to 80% cytotoxicity with 25HCh and 7kCh, respectively, in 48 hours. We used 50-μM oxysterol concentrations and found similar results for 7kCh but not 25HCh. In our experiments 25HCh is significantly less cytotoxic, achieving only 40% cytotoxicity in 72 hours (Fig. 2) . We also found that another oxysterol, 20αHCh, is possibly more cytotoxic than 7kCh, although it is not present in our oxLDL preparations. To our knowledge 20αHCh has not been reported as a component of oxLDL. In any event, the extreme insolubility of oxysterols in aqueous media made it difficult to determine the appropriate cytotoxic concentration accurately, because most of the oxysterol precipitated in the culture medium and/or bound to the plastic dish in which the cells are grown. In addition, our oxLDL analyses indicate that only 15% to 20% of its cholesterol (3%–5% of the total LDL) is in the form of oxysterols. This means that, assuming oxysterols are the main cytotoxic substances in oxLDL, they are cytotoxic at concentrations of 5 to 10 μM when incorporated in the oxLDL particle. This is less than one tenth of the concentration reported by Ong et al. 21 Our experiments using 7kCh-laced LDL showed 7kCH to be the most cytotoxic. We achieved 60% to 80% cytotoxicity with concentrations ranging from 2.5 to 5 μM 7kCh (Fig. 3) . This experiment conclusively demonstrated that 7kCh is responsible for most of the cytotoxicity associated with oxLDL in ARPE19 cells, with 7βHCh a distant second. These results may differ from other cell types. Additional cells should be examined to see whether this is a general effect. ARPE19 cells can internalize LDL in amounts averaging 10 to 12 pg per cell, 13 and this means it still takes roughly 6 × 108 molecules of 7kCh to kill an average ARPE19 cell. 
Transition metals play an important role in the oxidative process of lipoprotein. Metals can have both positive and negative effects on cells, depending on the specific metal and the concentrations used. LDL can very effectively bind transition metals, and this binding can only be partially reversed by chelators. 17 Because metals like zinc and copper have received considerable attention concerning the retina and RPE 14 as well AMD, 15 we decided to look at the effect they may have on oxysterol cytotoxicity. We used 10% oxysterol-LDL at 25 μg/mL, because, at this concentration, 7kCh causes approximately 40% to 50% cytotoxicity, but other oxysterols have no effect. We found that none of the metals tested significantly enhanced oxysterol cytotoxicity (Fig. 4) . Cytotoxicity with cobalt was only seen at 100 μM (Fig. 4D) . LDL with or without oxysterols protected the cells from zinc cytotoxicity (Fig. 4E) . Thus, although LDL could protect cells from acute metal exposure by binding and preventing the metals from directly interacting with the cells, it could also serve to bring small amounts of metals into cells, with good or bad consequences, depending on the nature of the metal and concentration already present. In any event, the data suggest that oxysterol cytotoxicity is independent of metal cytotoxicity although transition metals may play an important role in catalyzing the oxidation of LDL. This also suggests that transition metals may play an important role in atherosclerosis and AMD. 
 
Figure 1.
 
Cytotoxicity of OxLDL in culture RPE cells. ARPE19 cells were grown in 24-well plates until 80% to 90% confluent in serum-containing medium. LDL was oxidized using Cu+2 for 24, 48, and 72 hours. The oxLDL was given to ARPE19 cells for 48 hours in serum-free medium and cell viability measured by MTT. The concentrations reported indicate total amounts of LDL.
Figure 1.
 
Cytotoxicity of OxLDL in culture RPE cells. ARPE19 cells were grown in 24-well plates until 80% to 90% confluent in serum-containing medium. LDL was oxidized using Cu+2 for 24, 48, and 72 hours. The oxLDL was given to ARPE19 cells for 48 hours in serum-free medium and cell viability measured by MTT. The concentrations reported indicate total amounts of LDL.
Table 1.
 
Analysis of Differentially Oxidized LDL
Table 1.
 
Analysis of Differentially Oxidized LDL
25HCh 7αHCh 7βHCh 7KCh Ch Others Cytotoxicity (% of Dead Cells in 48 h)
LDL 0.5 0.6 97.9 1.0 0
24 h-oxLDL 3.5 1.8 5.7 86.3 2.7 7
48 h-oxLDL 1.3 0.9 5.0 7.3 82.7 2.8 20
72 h-oxLDL 2.1 0.8 2.3 16.0 74.3 4.5 82
Figure 2.
 
Cytotoxicity of different oxysterols to cultured RPE cells. ARPE19 and hTERT cells were grown in 24-well plates and exposed to 50 μM of each oxysterol. Left: results are the average of four individual measurements and the bars represent the standard deviation. Cell viability of ARPE19 and hTERT cells after (A) 24 and (B) 72 hours, as determined by MTT analysis. Right: the state of the ARPE19 cells before the MTT assay. Similar results were observed for the hTERT cells (pictures not shown) although overall they were less susceptible to individual oxysterol exposure.
Figure 2.
 
Cytotoxicity of different oxysterols to cultured RPE cells. ARPE19 and hTERT cells were grown in 24-well plates and exposed to 50 μM of each oxysterol. Left: results are the average of four individual measurements and the bars represent the standard deviation. Cell viability of ARPE19 and hTERT cells after (A) 24 and (B) 72 hours, as determined by MTT analysis. Right: the state of the ARPE19 cells before the MTT assay. Similar results were observed for the hTERT cells (pictures not shown) although overall they were less susceptible to individual oxysterol exposure.
Figure 3.
 
Cytotoxicity of nonoxidized LDL mixed with oxysterols. LDL was mixed with different oxysterols to a final concentration of 1 mg/mL LDL (total weight including protein, cholesterol, and other lipids) and 5% and 10% oxysterol. The oxysterol LDL was given to the ARPE19 cells at 25 μg/mL in serum-free medium for 48 hours, and cell viability measured by MTT. Error bars represent the average SD of two different experiments with two individual measurements (four separate values).
Figure 3.
 
Cytotoxicity of nonoxidized LDL mixed with oxysterols. LDL was mixed with different oxysterols to a final concentration of 1 mg/mL LDL (total weight including protein, cholesterol, and other lipids) and 5% and 10% oxysterol. The oxysterol LDL was given to the ARPE19 cells at 25 μg/mL in serum-free medium for 48 hours, and cell viability measured by MTT. Error bars represent the average SD of two different experiments with two individual measurements (four separate values).
Table 2.
 
Analysis of 10% Oxysterol-laced LDL
Table 2.
 
Analysis of 10% Oxysterol-laced LDL
10% LDLs 25HCh 7αHCh 7βHCh 7KCh Ch Others
7αHCh-LDL 13.4 85.8 0.8
7βHCh-LDL 8.3 88.1 3.6
7KCh-LDL 12.9 86.4 0.7
25HCh-LDL 13.3 86.6 0.1
Figure 4.
 
Effects of metals on LDL cytotoxicity. Nonoxidized LDL containing 10% oxysterols was added to ARPE19 cell at 25 μg/mL. Metals were added at 0-, 25-, 50-, and 100-μM concentrations. The cells were incubated for 48 hours in 96-well plates. Cell viability was measured by ATP quantification, using a luminescent cell viability assay. The results represent an average of two experiments and four independent measurements.
Figure 4.
 
Effects of metals on LDL cytotoxicity. Nonoxidized LDL containing 10% oxysterols was added to ARPE19 cell at 25 μg/mL. Metals were added at 0-, 25-, 50-, and 100-μM concentrations. The cells were incubated for 48 hours in 96-well plates. Cell viability was measured by ATP quantification, using a luminescent cell viability assay. The results represent an average of two experiments and four independent measurements.
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Figure 1.
 
Cytotoxicity of OxLDL in culture RPE cells. ARPE19 cells were grown in 24-well plates until 80% to 90% confluent in serum-containing medium. LDL was oxidized using Cu+2 for 24, 48, and 72 hours. The oxLDL was given to ARPE19 cells for 48 hours in serum-free medium and cell viability measured by MTT. The concentrations reported indicate total amounts of LDL.
Figure 1.
 
Cytotoxicity of OxLDL in culture RPE cells. ARPE19 cells were grown in 24-well plates until 80% to 90% confluent in serum-containing medium. LDL was oxidized using Cu+2 for 24, 48, and 72 hours. The oxLDL was given to ARPE19 cells for 48 hours in serum-free medium and cell viability measured by MTT. The concentrations reported indicate total amounts of LDL.
Figure 2.
 
Cytotoxicity of different oxysterols to cultured RPE cells. ARPE19 and hTERT cells were grown in 24-well plates and exposed to 50 μM of each oxysterol. Left: results are the average of four individual measurements and the bars represent the standard deviation. Cell viability of ARPE19 and hTERT cells after (A) 24 and (B) 72 hours, as determined by MTT analysis. Right: the state of the ARPE19 cells before the MTT assay. Similar results were observed for the hTERT cells (pictures not shown) although overall they were less susceptible to individual oxysterol exposure.
Figure 2.
 
Cytotoxicity of different oxysterols to cultured RPE cells. ARPE19 and hTERT cells were grown in 24-well plates and exposed to 50 μM of each oxysterol. Left: results are the average of four individual measurements and the bars represent the standard deviation. Cell viability of ARPE19 and hTERT cells after (A) 24 and (B) 72 hours, as determined by MTT analysis. Right: the state of the ARPE19 cells before the MTT assay. Similar results were observed for the hTERT cells (pictures not shown) although overall they were less susceptible to individual oxysterol exposure.
Figure 3.
 
Cytotoxicity of nonoxidized LDL mixed with oxysterols. LDL was mixed with different oxysterols to a final concentration of 1 mg/mL LDL (total weight including protein, cholesterol, and other lipids) and 5% and 10% oxysterol. The oxysterol LDL was given to the ARPE19 cells at 25 μg/mL in serum-free medium for 48 hours, and cell viability measured by MTT. Error bars represent the average SD of two different experiments with two individual measurements (four separate values).
Figure 3.
 
Cytotoxicity of nonoxidized LDL mixed with oxysterols. LDL was mixed with different oxysterols to a final concentration of 1 mg/mL LDL (total weight including protein, cholesterol, and other lipids) and 5% and 10% oxysterol. The oxysterol LDL was given to the ARPE19 cells at 25 μg/mL in serum-free medium for 48 hours, and cell viability measured by MTT. Error bars represent the average SD of two different experiments with two individual measurements (four separate values).
Figure 4.
 
Effects of metals on LDL cytotoxicity. Nonoxidized LDL containing 10% oxysterols was added to ARPE19 cell at 25 μg/mL. Metals were added at 0-, 25-, 50-, and 100-μM concentrations. The cells were incubated for 48 hours in 96-well plates. Cell viability was measured by ATP quantification, using a luminescent cell viability assay. The results represent an average of two experiments and four independent measurements.
Figure 4.
 
Effects of metals on LDL cytotoxicity. Nonoxidized LDL containing 10% oxysterols was added to ARPE19 cell at 25 μg/mL. Metals were added at 0-, 25-, 50-, and 100-μM concentrations. The cells were incubated for 48 hours in 96-well plates. Cell viability was measured by ATP quantification, using a luminescent cell viability assay. The results represent an average of two experiments and four independent measurements.
Table 1.
 
Analysis of Differentially Oxidized LDL
Table 1.
 
Analysis of Differentially Oxidized LDL
25HCh 7αHCh 7βHCh 7KCh Ch Others Cytotoxicity (% of Dead Cells in 48 h)
LDL 0.5 0.6 97.9 1.0 0
24 h-oxLDL 3.5 1.8 5.7 86.3 2.7 7
48 h-oxLDL 1.3 0.9 5.0 7.3 82.7 2.8 20
72 h-oxLDL 2.1 0.8 2.3 16.0 74.3 4.5 82
Table 2.
 
Analysis of 10% Oxysterol-laced LDL
Table 2.
 
Analysis of 10% Oxysterol-laced LDL
10% LDLs 25HCh 7αHCh 7βHCh 7KCh Ch Others
7αHCh-LDL 13.4 85.8 0.8
7βHCh-LDL 8.3 88.1 3.6
7KCh-LDL 12.9 86.4 0.7
25HCh-LDL 13.3 86.6 0.1
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