February 2010
Volume 51, Issue 2
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Retinal Cell Biology  |   February 2010
Mitochondrial DNA Damage Induced by 7-Ketocholesterol in Human Retinal Pigment Epithelial Cells In Vitro
Author Affiliations & Notes
  • Ana L. Gramajo
    From the Department of Ophthalmology, University of California, Irvine, California;
    Departamento de Oftalmologia, Centro Privado de Ojos Romagosa-Fundación VER, Cordoba, Argentina;
  • Leandro C. Zacharias
    From the Department of Ophthalmology, University of California, Irvine, California;
  • Aneesh Neekhra
    Department of Ophthalmology, University of Wisconsin, Madison, Wisconsin; and
  • Saurabh Luthra
    Department of Ophthalmology, Drishti Eye Centre, Dehradun, India.
  • Shari R. Atilano
    From the Department of Ophthalmology, University of California, Irvine, California;
  • Marilyn Chwa
    From the Department of Ophthalmology, University of California, Irvine, California;
  • Donald J. Brown
    From the Department of Ophthalmology, University of California, Irvine, California;
  • Baruch D. Kuppermann
    From the Department of Ophthalmology, University of California, Irvine, California;
  • M. Cristina Kenney
    From the Department of Ophthalmology, University of California, Irvine, California;
  • Corresponding author: M. Cristina Kenney, Department of Ophthalmology, University of California, Irvine Medical Center, Building 55, Room 220, 101 The City Drive, Orange, CA 92868; mkenney@uci.edu
Investigative Ophthalmology & Visual Science February 2010, Vol.51, 1164-1170. doi:10.1167/iovs.09-3443
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      Ana L. Gramajo, Leandro C. Zacharias, Aneesh Neekhra, Saurabh Luthra, Shari R. Atilano, Marilyn Chwa, Donald J. Brown, Baruch D. Kuppermann, M. Cristina Kenney; Mitochondrial DNA Damage Induced by 7-Ketocholesterol in Human Retinal Pigment Epithelial Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2010;51(2):1164-1170. doi: 10.1167/iovs.09-3443.

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

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Abstract

Purpose.: To assess oxysterol-induced mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in cultured human retinal pigment epithelial cells (ARPE- 19).

Methods.: ARPE-19 cultures were exposed for 6 and 24 hours to 40 μg/mL 7-ketocholesterol (7kCh), and total DNA was extracted. Long-extension polymerase chain reaction was performed to amplify the full-length mtDNA genome. The products were separated by electrophoresis on a 0.8% agarose gel stained with ethidium bromide. Superoxide and reactive oxygen/nitrogen species (ROS/RNS; hydrogen peroxide, peroxynitrite anions, and peroxyl radicals) were measured with an amine-reactive green-dye assay and 2`,7`-dicholorodihydrofluorescein diacetate (H2DCFDA) dye assay, respectively. The changes in mitochondrial membrane potential (ΔΨm) were measured with a cationic (green) dye assay. Western blot analysis was used to assess porins, a structural protein of the mitochondrial membranes.

Results.: The 7kCh-treated cultures showed significant increase in ROS/RNS production (P < 0.001) compared with untreated controls, but the superoxide levels were unchanged. The 7kCh-treated ARPE-19 cultures had diminished levels of the full-length 16.2-kb mtDNA band, a 2.2-fold decrease of the ΔΨm compared with control cultures (P < 0.001), and decreased levels of porins.

Conclusions.: 7kCh causes significant damage to the full-length intact mtDNA and mitochondrial dysfunction in ARPE-19 cells. These observations suggest that the mitochondria and its DNA may be targets for oxysterol-induced oxidative stress and may play a role in the pathogenesis of retinal diseases.

The pathogenesis of age-related macular degeneration (AMD) is still not completely elucidated. AMD is a multifactorial disease in which aging and genetic, environmental, and nutritional factors are involved. Among these factors, cholesterol metabolism is believed to contribute since cholesterol accumulation in Bruch's membrane is implicated in the etiology and progression of AMD. 14 However, there is controversy as to whether hypercholesterolemia is a risk factor for AMD. Some reports show hypercholesterolemia, along with higher intake of specific types of fat, as a possible risk factor for macular degeneration. 58 Klaver et al. 9 report an association between apolipoprotein E (ApoE), a key regulator of cholesterol and lipid metabolism, and AMD. Although Dashti et al. 10 suggest no relationship between total plasma cholesterol levels and AMD, their study is based mainly on measurements of plasma ApoB and ApoA-I. However, native human retinal pigment epithelial (RPE) cells express mRNA transcripts for ApoA-I, ApoB, ApoC-I, ApoCII, and ApoE, and the main differences observed between AMD and controls patients are in ApoE, ApoC-III, LpC-III non-B, and LpE non-B. 11 Furthermore, there is a reported association between ApoE and AMD 12 ; an ApoE-deficient mouse model fed a high-cholesterol diet showed significant toxicity in retinal function and cellular morphology, 13,14 and excess cholesterol induced ultrastructural changes in the rabbit retina similar to those in human AMD. 6  
Furthermore, some cholesterol oxidation products (oxysterols) lead to the generation of reactive oxygen/nitrogen species (ROS/RNS) that have been implicated in the process of aging and degenerative diseases of the human eye. 1517 ROS/RNS are produced by mitochondria and, under optimal conditions, are neutralized by the mitochondrial cellular defense mechanisms. When overproduced, ROS/RNS can cause deterioration of mitochondrial oxidative stress defenses, induce irreparable damage at the molecular and cellular levels in susceptible cells, such as RPE cells, and contribute to pathologic conditions such as AMD. 18 ROS/RNS can also damage DNA, proteins, and cellular lipids, giving rise to lipid peroxidation, a situation that can further promote mtDNA damage. 19,20  
Oxidative stress is a cellular injury secondary to ROS/RNS production. Several oxysterols, such as 7-ketocholesterol (7kCh), 7-β-OH-cholesterol (7βHCh), 19-OH-, 22(R)-OH-cholesterol, 22(S)-OH-cholesterol, and 25-OH-cholesterol cause oxidative stress, lipid peroxidation, and finally cytotoxicity in retinal cells in vitro. 21 The production of oxidatively modified low-density lipoprotein (ox-LDL) is recognized as an early event in the development of atherosclerosis. 22 It has been hypothesized that the process of plaque formation that occurs in the coronary vessels can be similar to events that occur in retinal vasculature. 23,24 Plasma ox-LDL levels are significantly elevated in patients with AMD, and elevated levels of plasma ox-LDL may predict the risk for AMD more accurately than do other serum factors. 25  
7kCh, the predominant oxysterol in ox-LDL, is responsible for most of the cytotoxicity associated with ox-LDL–treated ARPE-19 cells. 26 Prolonged oxidation of LDL gradually raises the levels of 7kCh as a result of increased oxidation of 7-α-OH-cholesterol (7αHCh) and 7βHCh. 26,27 The retina has CYP450 enzymes that can modify oxysterols. The sterol 27-hydroxylase (CYP27A1), a mitochondrial P-450 enzyme that has substrate specificity for C27 sterols (including 7kCh), is expressed in the RPE cells, Müller cells, and choriocapillaris, and its major role appears to be to process 7kCh. 28 Animal models indicate that the increase in intermediates in cholesterol synthesis or their metabolites are pathogenetic and can lead to significant phenotypic changes. 29 Finally, the oxysterol-binding protein OSBP2, expressed mainly in the retina, binds to 7kCh and has little affinity for cholesterol or other oxysterols. 30 Based on the high cytotoxicity profile of 7-kCh, it was decided to further examine the effects of this oxysterol on ARPE-19 cells. 
Mitochondria and oxidative stress are significant areas of research related to diseases and aging. Mitochondria are unique because this organelle has its own DNA (mitochondrial DNA [mtDNA]). Human mtDNA is a double-stranded, circular molecule of 16,569 bp that encodes for 13 proteins and 24 RNAs involved with oxidative phosphorylation (OXPHOS). 31 mtDNA is more susceptible to oxidative damage than nuclear DNA (nDNA) because of its proximity to the inner mitochondrial membrane (source of endogenously produced oxygen radicals), lack of histone proteins, absence of introns, and high transcription rate. 3237 Furthermore, when mtDNA is damaged, changes in the encoding subunits of respiratory chain complexes can occur, decreasing OXPHOS, which in turn leads to higher ROS production. 35,36 This vicious circle of mtDNA damage and ROS/RNS production can lead to apoptosis and cell death. Moreover, mitochondria have replicative segregation during cell division, meaning that normal and mutant mtDNA combine when mutation takes place, 35,38,39 which can lead to cells with larger numbers of mtDNA mutations and further mitochondrial dysfunction. 
This study used ARPE-19 and HMVEC cultures to examine the effect 7kCh has on mtDNA integrity. Our findings showed that 7kCh causes mtDNA damage and mitochondrial dysfunction in ARPE-19 cultures. 
Materials and Methods
Cell Culture
The human RPE cell line (ARPE-19) was obtained from ATCC (Manassas, VA). Cells were grown in a 1:1 mixture (vol/vol) of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 medium (DMEM F-12; Invitrogen-Gibco, Carlsbad, CA), 0.058% l-glutamine, 10% bovine growth serum, and antibiotics (penicillin G 100 U/mL, streptomycin sulfate 0.1 mg/mL, gentamicin 10 μg/mL, and fungizone-Amphotericin B 2.5 μg/mL). ARPE-19 cells have numerous morphologic features characteristic of differentiated RPE cells, including basolateral infoldings, apical microvilli, transepithelial resistance, and polarized distribution of cellular organelles. 40 Northern blot analysis analyses showed that ARPE-19 cells express CRALBP and RPE-65, both of which are synthesized by differentiated RPE cells in vivo. The ability of these cells to form tight junctions has been confirmed by the identification of junctional complexes in the apical region of the plasma membranes. 40 In this study we exposed the apical side of the ARPE-19 cells to 7kCh because previous studies have reported cell responses using this method 41 and because cells express receptors essential for the internalization of oxysterols. 42  
Cells in 60-mm tissue culture dishes (1.5 × 106 cells/dish) were treated for 6 and 24 hours with 7kCh (Sigma-Aldrich, St. Louis, MO) at a concentration of 20 μg/mL or 40 μg/mL. Stocks of 7kCh (5 mg/mL) were prepared in 100% ethanol and added to cell media before treatment. Control cultures were cells treated for 6 and 24 hours, with the comparable amounts of ethanol used to achieve 40 or 20 μg/mL 7kCh (ETOH equivalent 40 μg/mL or ETOH equivalent 20 μg/mL, respectively). In the same cases, untreated cells (not exposed to ethanol or 7kCh) were also examined. 
To contrast the highly sensitive responses observed by the ARPE-19 cells, we also examined some parameters of human microvascular endothelial cells (HMVECs). HMVECs and their tissue culture reagents were obtained from Cascade Biologics, Inc. (Portland, OR). Cells were acquired as proliferating quaternary cultures established from normal adult human dermis microvascular endothelial cells. HMVECs were grown in medium 131 supplemented with microvascular growth supplement containing 5% fetal bovine serum, hydrocortisone, recombinant human fibroblast growth factor, heparin, recombinant human epidermal growth factor, and dibutyryl cyclic AMP, as described previously. 43 This medium does not contain antibiotics or antimycotics. Before use, the tissue culture surfaces were coated with a commercially available sterile 0.1% gelatin solution (Attachment Factor; Cascade Biologics, Portland, OR). Cultures were treated for 24 hours with either 20 μg/mL or 40 μg/mL 7kCh or the ethanol equivalent (ETOH). 
Detection of ROS/RNS Production
ROS/RNS production was measured as described previously, 44 using the fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate kit (H2DCFDA; Molecular Probes, Eugene, OR), which detects hydrogen peroxide, peroxyl radicals, and peroxynitrite anions. Cells were washed with D-PBS and incubated with 10 μM H2DCFDA in D-PBS for 30 minutes at 37°C. ROS/RNS production was measured with the FMBIO III scanning unit (excitation λ, 488 nm; emission λ, 520 nm). The summarized data were from triplicate cultures of ETOH-treated or 7kCh-treated cultures. Data were analyzed using unpaired Student's t-test. 
Superoxide Anion Detection with Dye
Superoxide anion production was measured using the reagent (Molecular Probes). ARPE-19 cells were plated in either 24-well plates or 2-well chamber slides. After 24 hours, media were removed, and the cells were washed with D-PBS (Invitrogen) and incubated for 30 minutes with 10 μM dye (OxyBurst; Molecular Probes). Fluorescence intensity was measured with a laser scanning unit (excitation λ, 488 nm; emission λ, 520 nm; FMBIO III; Hitachi, South San Francisco, CA). The entire experiment was repeated twice. In this article we use the terms ROS/RNS to refer to the elements measured by the H2DCFDA methods and superoxide to refer to the elements measured by the dye (OxyBurst; Molecular Probes) assay. 
Mitochondrial Membrane Potential (ΔΨm) Measurements
Cells were plated onto 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) at 100,000 cells per well and incubated at 37°C in 5% CO2. After 24-hour incubation with 7kCh, cells were incubated with the cationic dye 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1; Cell Technology, Minneapolis, MN) at 37°C for 15 minutes and rinsed once with 1× phosphate-buffered saline (PBS), and the fluorescent signal was measured with the scanning unit (FMBio III; Hitachi) set to detect green (510–525 nm) and red (590 nm) emissions. Ratios of red to green fluorescence were calculated, and the data were analyzed by unpaired Student's t-test. 
Extraction of Total DNA
The extraction of total DNA was performed by phenol/chloroform, as previously described by Atilano et al. 45 Briefly, cells collected at 6 and 24 hours were homogenized in 1 mL of 1× STE (100 mM NaCl, 25 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0). Then the proteins were digested overnight at 50°C in the presence of 0.5% SDS and 15 μg/mL proteinase K (Invitrogen). Cellular RNAs were digested for 1 hour at 37°C with 5 μg/mL RNase A followed by phenol/chloroform extraction (Invitrogen). DNA was precipitated with 0.5 vol of 7.5 M ammonium acetate and 2 vol ethanol. The DNA pellet was resuspended in TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA-NaOH, pH 8.0) and quantified by spectrophotometric analysis at a 260/280-nm wavelength spectrophotometer (BioPhotometer; Eppendorf, Westbury, NY). 
LX-PCR
Long-extension (LX) PCR was performed according to the modified methods of Jessie et al. 46 and Melov et al. 47 Briefly, LX-PCR was performed with 50 ng extracted DNA, 0.2 μM each primer, 25 μL PCR mix (Premix D; Epicenter, Madison, WI), and 1μL enzyme mix (Failsafe; Epicenter), in a total volume of 50 μL. Samples were denatured for 2 minutes at 90°C, followed by 35 cycles of 10 seconds at 90°C, 16 minutes at 68°C, and a final elongation for 10 minutes at 72°C (forward primer, TGA GGC CAA ATA TCA TTC TGA GGG GC [15,148–15,174 bp]; reverse primer, TTC ATC ATG CGG AGA TGT TGG ATGG [14,841–14,816 bp]; GenBank accession number AC000021.2). These PCR primers were designed to specifically amplify 16,262 bp of the mtDNA, skipping only 334 bp of the cytb gene. LX-PCR products were separated by electrophoresis on a 0.8% agarose gel stained with ethidium bromide. Gel images were captured on a fluorescence imaging system (FMBio III; Hitachi) under identical conditions and were analyzed. The experiment was conducted in duplicate and repeated three times. With intact mtDNA, the expected amplification product of the LX-PCR is 16,262 bp. If the template mtDNA has deletions or rearrangements, then the LX-PCR product results in smaller bands. Therefore, the appearance of smaller sized bands or loss of the 16.2-kb mtDNA band represents damage to the intact mtDNA genome. 
LX-PCR has been demonstrated to be reliable and to represent mtDNA through verification by Southern blot analysis using a full-length digoxigenin-labeled mtDNA probe by our laboratory and that of others. 45,48,49 The LX-PCR technique was further substantiated by sequencing individual bands showing they were indeed mtDNA and not artifacts. 49  
Western Blot Analyses
ARPE-19 cells were collected and lysed with 0.1 M potassium phosphate with 0.2% Triton X-100. Protein concentrations were measured using the BCA protein assay (Pierce, Rockford, IL). Then 20 μg of each sample was fractionated on precast gradient SDS polyacrylamide gels (4%–20%; NuPAGE; Invitrogen). Some gels were stained with Coomassie Brilliant Blue to verify the equivalence of the protein concentrations. Other gels were transferred overnight at 4°C to polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked for 1 hour in Tris-buffered saline (100 mM Tris, 0.9% sodium chloride) containing 3% bovine serum albumin and 0.1% Tween 20. The membrane was incubated overnight at 4°C in primary antibody, a mouse anti–human monoclonal porin (1 μg/mL; Invitrogen). The secondary antibody was an alkaline-phosphatase–conjugated donkey anti–mouse IgG (1:2500 dilution; Chemicon, Temecula, CA). The protein signal was detected with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium phosphatase substrate tablets (Sigma Chemical). Band densities were scanned and quantified using an image analyses system (FMBio III; Hitachi). 
Results
Detection of ROS/RNS Production
In 7kCh-treated cultures, ROS/RNS production was significantly increased (Fig. 1). ROS/RNS levels increased significantly with 20 μg/mL 7kCh (22,910.66 ± 893.63 vs. 19,265.16 ± 405.35; P < 0.001) and 40 μg/mL (22,110 ± 449.57 vs. 18,646.90 ± 68.707 P < 0.001) compared with the ETOH equivalent–treated cultures. Cells exposed to 7kCh at both concentrations also had more ROS/RNS production than untreated control cells (18,865.08 ± 68.71; P < 0.001). There was no statistically significant difference between ETOH equivalent and untreated controls. For cells exposed to 7kCh, no differences in ROS/RNS production were observed at the concentrations tested. 
Figure 1.
 
ROS production in ARPE-19 cells exposed to 7KCh. There is an increased generation of ROS/RNS at 24 hours in cells exposed to 7KCh compared with ETOH-equivalent or untreated controls (***P < 0.001).
Figure 1.
 
ROS production in ARPE-19 cells exposed to 7KCh. There is an increased generation of ROS/RNS at 24 hours in cells exposed to 7KCh compared with ETOH-equivalent or untreated controls (***P < 0.001).
Superoxide Anion Detection with Dye
We also wanted to determine whether the levels of superoxide anions, which can be generated by mitochondria and are upstream of the ROS/RNS elements, were also increased in the ARPE-19 cultures. The dye kit (OxyBurst; Molecular Probes) detects only the superoxide anions, whereas the H2DCFDA kit measures hydrogen peroxide, peroxyl radicals, and peroxynitrite anions. Figure 2 shows that the levels of superoxide anions remained unchanged in the 7kCh-treated and control cultures. There was no statistically significant difference between ARPE-19 cells exposed to 40 μg/mL or 20 μg/mL 7kCh (11,627 ± 2685 and 11,082 ± 3169, respectively), the respective ETOH controls (10,661 ± 16,15 and 11,123 ± 1692, respectively), or the untreated controls (11,097 ± 936). 
Figure 2.
 
Superoxide activity in ARPE-19 cells exposed to 7KCh. There was no significant difference observed among 7KCh-treated cells, the ETOH-equivalent, or untreated controls.
Figure 2.
 
Superoxide activity in ARPE-19 cells exposed to 7KCh. There was no significant difference observed among 7KCh-treated cells, the ETOH-equivalent, or untreated controls.
Mitochondrial Membrane Potential (ΔΨm)
With a significant increase in ROS/RNS production in the ARPE-19 cultures, we wanted to determine whether 7kCh also had an effect on mitochondrial function; therefore, we analyzed for the loss of mitochondrial membrane potential (ΔΨm), which is an early sign of apoptosis (Fig. 3). Red fluorescence represents live cells, and green fluorescence represents cells undergoing apoptosis. The 7kCh-treated ARPE-19 cultures showed loss of ΔΨm compared with the ethanol-treated cells (Fig. 3). Quantification of the red-green fluorescence ratio showed ethanol-treated cells with an average fluorescence ratio of 3.887 ± 0.741, whereas the 7kCh-treated cells were 2.2-fold lower than control cultures (1.812 ± 0.247; P < 0.001). 
Figure 3.
 
ARPE-19 cells treated with 7kCh showed loss of ΔΨm compared with ethanol equivalent–treated cells. Twenty-four hours after 7kCh treatment, treated cells had a 2.2-fold loss of ΔΨm, as measured by the ratio of red fluorescence (live cells) to green fluorescence (apoptotic cells). The red-to-green average ratio in ethanol equivalent–treated cells was 3.887 ± 0.741 compared with 1.812 ± 0.247 in 7kCh-treated cells (***P < 0.001).
Figure 3.
 
ARPE-19 cells treated with 7kCh showed loss of ΔΨm compared with ethanol equivalent–treated cells. Twenty-four hours after 7kCh treatment, treated cells had a 2.2-fold loss of ΔΨm, as measured by the ratio of red fluorescence (live cells) to green fluorescence (apoptotic cells). The red-to-green average ratio in ethanol equivalent–treated cells was 3.887 ± 0.741 compared with 1.812 ± 0.247 in 7kCh-treated cells (***P < 0.001).
LX-PCR mtDNA Analysis
LX-PCR allows amplification of the total mitochondrial genome that contains the primer sites. The major LX-PCR product representing full-length mtDNA measures 16.2 kb. When mtDNA is damaged, LX-PCR yields smaller products. In ARPE-19 cultures treated with 7kCh for 24 hours, the LX-PCR mtDNA showed a significant loss in the 16.2-kb mtDNA band (Fig. 4A) compared with the ethanol-treated controls. Few smaller mtDNA bands were present in the 7kCh-treated samples. We were surprised by the extent of damage to mtDNA because ARPE-19 cells are considered to be scavengers and to be partially resistant to oxidative stressors. We then decided also to examine the HMVEC cultures, a cell line reported to be very sensitive to 7kCh and other drugs. 50,51 The HMVEC 7kCh-treated cultures retained the full-sized 16.2-kb mtDNA band compared with the ethanol-treated control group (Fig. 4B). 
Figure 4.
 
Ethidium-bromide–stained gels showing LX-PCR mtDNA from ARPE-19 and HMVEC cultures. (A) In ARPE-19 cultures, there was a significant decrease in the LX-PCR mtDNA 16.2-kb band after treatment with 7kCh. (B) In HMVEC cultures, 7kCh treatment did not eliminate the 16.2-kb LX-PCR mtDNA band. The 16.2-kb LX-PCR product corresponds to the full-length mtDNA. Lane M, molecular mass marker.
Figure 4.
 
Ethidium-bromide–stained gels showing LX-PCR mtDNA from ARPE-19 and HMVEC cultures. (A) In ARPE-19 cultures, there was a significant decrease in the LX-PCR mtDNA 16.2-kb band after treatment with 7kCh. (B) In HMVEC cultures, 7kCh treatment did not eliminate the 16.2-kb LX-PCR mtDNA band. The 16.2-kb LX-PCR product corresponds to the full-length mtDNA. Lane M, molecular mass marker.
Western Blot Analyses
Porins (also known as voltage-dependent anion channels) are important structural proteins of the mitochondrial membrane. 52 With the decreased levels of LX-PCR mtDNA, we wanted to determine whether the mitochondrial content was lower in the 7kCh-treated ARPE-19 cultures (Fig. 5A). Lane 3 shows the mitochondrial porin bands were present in ethanol equivalent–treated ARPE-19 cells but were decreased in 7kCh-treated cultures (lane 4). With this significant loss of mitochondrial porins after 7kCh treatment in ARPE-19 cells, we also examined the more sensitive HMVEC line. Lanes 1 and 2 show the porin bands at approximately 35.5 kDa in the ethanol equivalent–treated and 7kCh-treated HMVEC cultures, respectively. Porin levels were not significantly altered after 7kCh treatment in the HMVEC and were, in fact, 2.8-fold higher than the 7kCh-treated ARPE-19 cultures. Figure 5B shows a representative Coomassie Brilliant blue–stained SDS polyacrylamide gel of 20 μg protein aliquots from each sample to verify the equivalence of the protein concentrations. 
Figure 5.
 
(A) Western blot analyses of mitochondrial porins from ARPE-19 cell and HMVEC cultures. Lane 1, protein from the ethanol equivalent–treated HMVEC cultures. Lane 2, protein from the 7kCh-treated HMVEC cultures. Lane 3, ethanol equivalent–treated ARPE-19 cells. Lane 4, 7kCh-treated ARPE-19 cell. In HMVECs, a strong staining pattern for total porins was seen by Western blot analysis. Similar levels of porins were even found in the ethanol-treated ARPE-19 and HMVEC cultures. In contrast, porin was barely detectable in the 7kCh-treated ARPE-19 cells. 7kCh-treated HMVECs had approximately 2.8-fold higher expression of total porins than did 7kCh-treated ARPE-19 cells. (B) Representative image of Coomassie Brilliant Blue–stained SDS polyacrylamide gel after electrophoresis of 20-μg protein aliquots from each sample to verify the equivalence of the protein concentrations. M, marker.
Figure 5.
 
(A) Western blot analyses of mitochondrial porins from ARPE-19 cell and HMVEC cultures. Lane 1, protein from the ethanol equivalent–treated HMVEC cultures. Lane 2, protein from the 7kCh-treated HMVEC cultures. Lane 3, ethanol equivalent–treated ARPE-19 cells. Lane 4, 7kCh-treated ARPE-19 cell. In HMVECs, a strong staining pattern for total porins was seen by Western blot analysis. Similar levels of porins were even found in the ethanol-treated ARPE-19 and HMVEC cultures. In contrast, porin was barely detectable in the 7kCh-treated ARPE-19 cells. 7kCh-treated HMVECs had approximately 2.8-fold higher expression of total porins than did 7kCh-treated ARPE-19 cells. (B) Representative image of Coomassie Brilliant Blue–stained SDS polyacrylamide gel after electrophoresis of 20-μg protein aliquots from each sample to verify the equivalence of the protein concentrations. M, marker.
Discussion
The relationship between mitochondrial abnormalities and retinal diseases is well recognized. Mutations and deletions in mtDNA can lead to pigmentary retinopathy, 5356 retinal dystrophy, 5759 and macular dystrophy. 60 In the present study, we found that 7kCh caused significant increases in ROS/RNS production, damage to the full-length 16.2-kb mtDNA, decreases in mitochondrial porin levels, and loss of the mitochondrial membrane potential (ΔΨm) in ARPE-19 cells, representing mitochondrial dysfunction. Our data support our hypothesis that oxidized cholesterols, such as 7kCh can play a significant role in generating mtDNA damage, oxidative stress, and loss of cell viability. 
In vivo studies have shown significant toxicity to retinal function and cellular morphology in ApoE-deficient mice fed a high-cholesterol diet. 14 Moreover, oxysterols, primarily 7kCh, are toxic in vitro to RPE cells 41 and cells derived from neuroretinas. 21,41 Human RPE cell degeneration is one of the initial events that occur in AMD. 6163 The present study used ARPE-19 cells to examine the effects of 7kCh on mitochondrial DNA integrity and function because ARPE-19 cells have structural and functional properties characteristic of human RPE cells in vivo 40 and can internalize LDL and ox-LDL in large quantities in vitro and in vivo. 42 There is controversy on whether 7kCh should be mixed with LDL in the in vitro experiments. Rodriguez et al. 42 studied cell toxicity using oxysterols mixed with LDL, which aided with the internalization of the complex. However, other studies show that 7kCh without LDL could induce caspase activities, apoptosis, and cell death. 41,64,65 It has been suggested that 7kCh could be internalized into the cells, perhaps through carriers in the serum. We have not evaluated the effects of 7kCh using primary RPE cells or other cell lines, but other investigators have. Joffre et al. 66 report increases in ROS levels and mitochondrial dysfunction in primary porcine RPE cells treated with 7kCh. Rodriguez et al. 26 show that 7kCh is the most cytotoxic of the oxysterols using the ARPE-19 cell model. Reports show that 7kCh induces apoptosis involving caspase-3, caspase-8, and caspase-12 pathways in rat R28 cells, HMVEC cells, and ARPE-19 cells. 50,67,68 In addition, 7kCh, localized to the choriocapillaris and Bruch's membrane, induces VEGF levels, which might play a role in neovascularization. 69  
Our data indicate that 7kCh-treated cultures showed significant signs of mtDNA damage, as demonstrated by LX-PCR. In ARPE-19 cells, significant loss of the full-length 16.2-kb LX-PCR mtDNA band and lower levels of porins, a structural mitochondrial protein, were noted. Interestingly, in HMVEC cultures, which are typically more sensitive to stress than are ARPE-19 cells, 50 the 16.2-kb LX-PCR mtDNA band remained unchanged. Our findings suggest that 7kCh can differentially damage mtDNA, which is in agreement with studies reporting dissimilar cellular responses in cell lines treated with oxysterols. 41 The appearance of smaller bands represents mtDNA rearrangements/deletions and has been associated with aging and diseases such as prostate cancer, Alzheimer disease, diabetes, Parkinson disease, and Down syndrome. 35,37,46,70,71  
The accumulation of oxidative damage to mtDNA can severely inhibit mtDNA biosynthetic capacity, inhibit mitochondrial function, and lead to apoptosis. It has been suggested that mtDNA rearrangements or mutations can alter the efficiency of mitochondrial energy production 70 by leading to a partially uncoupled OXPHOS, which then requires more calories to be consumed for the same amount of ATP produced, resulting in greater body heat production and higher ROS formation. 
Our study showed that 7kCh increased ROS/RNS production as measured by the H2DCFDA assay. Surprisingly, the superoxide levels were not changed after 7kCh treatment. Other in vitro studies have shown that mtDNA damage occurs after exposure to hydrogen peroxide (H2O2) and peroxynitrite. 33,34,72 These studies, along with our data, provide a link between oxidants and mtDNA damage that would ultimately alter gene expression, cellular energy production, and mitochondrial function. This is supported by findings of Miceli and Jazwinski 73 showing that in response to the loss of mitochondrial function, ARPE-19 cells undergo changes in nuclear gene expression similar to those found in AMD. Using p° cells, which are ARPE-19 cells depleted of their mitochondrial DNA, they showed an increased expression of genes encoding for drusen components, lipid transport, and inflammation. 73 This type of oxidant-related mtDNA damage within the human retina might play a significant role in disease pathogenesis. 
In ARPE-19 cultures, 7kCh treatment caused collapse of the ΔΨm, an early sign of apoptosis. The ΔΨm assay measures the opening of the mitochondrial permeability transition pore (mtPTP), which occurs in response to pro-oxidants and high levels of calcium within the mitochondria. This is the first report of 7kCh-induced changes of the mtPTP in human RPE cells. This change provides additional evidence that 7kCh affects mitochondria and likely alters its energy-producing function. As the intracellular ATP levels decrease, the cell may become less efficient, produce higher levels of ROS/RNS, and have a higher likelihood of cell death. Further studies are required to understand the relationship among oxysterols, mitochondrial dysfunction, and disease processes. 
Porin is an important structural mitochondrial transmembrane protein that is critical to mitochondrial stability. 52 When the ARPE-19 cells were treated with 7kCh, the cultures lacked the 35.5-kDa porin band. By comparison, we again examined the response of the more sensitive HMVEC cell line and found that porin levels did not decrease significantly after 7kCh treatment. These findings suggest that the ARPE-19 cells have mitochondria that are increasingly susceptible to oxidative injury and are more prone to extensive oxidative stress–related cell harm than HMVEC cultures, as suggested by the lower porin levels and mtDNA damage. This was surprising because we generally consider the ARPE-19 cells to be a hardier cell line that is easier to grow and is more resistant to drugs. 
In summary, our results show that 7kCh is cytotoxic to RPE cells 19,26,41 and support the hypothesis that a decline of mitochondrial function could play a major role in diseases of retinal degeneration. The significance of mtDNA damage within cells rests in the fact that once it occurs, it is perpetuated by ongoing mitochondrial replication over time. Given that there is no cure for AMD and that treatments to stop its progression have limited success, proper identification of its modifiable risk factors and understanding of its pathophysiology are mandatory. 
Footnotes
 Supported by Pan-American Association of Ophthalmology Foundation (David and Julianna Pyott Pan-American Retinal Research Fellowship), Discovery Eye Foundation, Iris and B. Gerald Cantor Foundation, Gilbert Foundation, Lincy Foundation, Ko Family Foundation, and Research to Prevent Blindness Foundation.
Footnotes
 Disclosure: A.L. Gramajo, None; L.C. Zacharias, None; A. Neekhra, None; S. Luthra, None; S.R. Atilano, None; M. Chwa, None; D.J. Brown, None; B.D. Kuppermann, None; M.C. Kenney, None
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Figure 1.
 
ROS production in ARPE-19 cells exposed to 7KCh. There is an increased generation of ROS/RNS at 24 hours in cells exposed to 7KCh compared with ETOH-equivalent or untreated controls (***P < 0.001).
Figure 1.
 
ROS production in ARPE-19 cells exposed to 7KCh. There is an increased generation of ROS/RNS at 24 hours in cells exposed to 7KCh compared with ETOH-equivalent or untreated controls (***P < 0.001).
Figure 2.
 
Superoxide activity in ARPE-19 cells exposed to 7KCh. There was no significant difference observed among 7KCh-treated cells, the ETOH-equivalent, or untreated controls.
Figure 2.
 
Superoxide activity in ARPE-19 cells exposed to 7KCh. There was no significant difference observed among 7KCh-treated cells, the ETOH-equivalent, or untreated controls.
Figure 3.
 
ARPE-19 cells treated with 7kCh showed loss of ΔΨm compared with ethanol equivalent–treated cells. Twenty-four hours after 7kCh treatment, treated cells had a 2.2-fold loss of ΔΨm, as measured by the ratio of red fluorescence (live cells) to green fluorescence (apoptotic cells). The red-to-green average ratio in ethanol equivalent–treated cells was 3.887 ± 0.741 compared with 1.812 ± 0.247 in 7kCh-treated cells (***P < 0.001).
Figure 3.
 
ARPE-19 cells treated with 7kCh showed loss of ΔΨm compared with ethanol equivalent–treated cells. Twenty-four hours after 7kCh treatment, treated cells had a 2.2-fold loss of ΔΨm, as measured by the ratio of red fluorescence (live cells) to green fluorescence (apoptotic cells). The red-to-green average ratio in ethanol equivalent–treated cells was 3.887 ± 0.741 compared with 1.812 ± 0.247 in 7kCh-treated cells (***P < 0.001).
Figure 4.
 
Ethidium-bromide–stained gels showing LX-PCR mtDNA from ARPE-19 and HMVEC cultures. (A) In ARPE-19 cultures, there was a significant decrease in the LX-PCR mtDNA 16.2-kb band after treatment with 7kCh. (B) In HMVEC cultures, 7kCh treatment did not eliminate the 16.2-kb LX-PCR mtDNA band. The 16.2-kb LX-PCR product corresponds to the full-length mtDNA. Lane M, molecular mass marker.
Figure 4.
 
Ethidium-bromide–stained gels showing LX-PCR mtDNA from ARPE-19 and HMVEC cultures. (A) In ARPE-19 cultures, there was a significant decrease in the LX-PCR mtDNA 16.2-kb band after treatment with 7kCh. (B) In HMVEC cultures, 7kCh treatment did not eliminate the 16.2-kb LX-PCR mtDNA band. The 16.2-kb LX-PCR product corresponds to the full-length mtDNA. Lane M, molecular mass marker.
Figure 5.
 
(A) Western blot analyses of mitochondrial porins from ARPE-19 cell and HMVEC cultures. Lane 1, protein from the ethanol equivalent–treated HMVEC cultures. Lane 2, protein from the 7kCh-treated HMVEC cultures. Lane 3, ethanol equivalent–treated ARPE-19 cells. Lane 4, 7kCh-treated ARPE-19 cell. In HMVECs, a strong staining pattern for total porins was seen by Western blot analysis. Similar levels of porins were even found in the ethanol-treated ARPE-19 and HMVEC cultures. In contrast, porin was barely detectable in the 7kCh-treated ARPE-19 cells. 7kCh-treated HMVECs had approximately 2.8-fold higher expression of total porins than did 7kCh-treated ARPE-19 cells. (B) Representative image of Coomassie Brilliant Blue–stained SDS polyacrylamide gel after electrophoresis of 20-μg protein aliquots from each sample to verify the equivalence of the protein concentrations. M, marker.
Figure 5.
 
(A) Western blot analyses of mitochondrial porins from ARPE-19 cell and HMVEC cultures. Lane 1, protein from the ethanol equivalent–treated HMVEC cultures. Lane 2, protein from the 7kCh-treated HMVEC cultures. Lane 3, ethanol equivalent–treated ARPE-19 cells. Lane 4, 7kCh-treated ARPE-19 cell. In HMVECs, a strong staining pattern for total porins was seen by Western blot analysis. Similar levels of porins were even found in the ethanol-treated ARPE-19 and HMVEC cultures. In contrast, porin was barely detectable in the 7kCh-treated ARPE-19 cells. 7kCh-treated HMVECs had approximately 2.8-fold higher expression of total porins than did 7kCh-treated ARPE-19 cells. (B) Representative image of Coomassie Brilliant Blue–stained SDS polyacrylamide gel after electrophoresis of 20-μg protein aliquots from each sample to verify the equivalence of the protein concentrations. M, marker.
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