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(Investigative Ophthalmology and Visual Science. 2000;41:1981-1989.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

The Lipofuscin Fluorophore A2E Mediates Blue Light–Induced Damage to Retinal Pigmented Epithelial Cells

Janet R. Sparrow1, Koji Nakanishi2 and Craig A. Parish2

From the Departments of 1 Ophthalmology and 2 Chemistry, Columbia University, New York, New York.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To determine whether the lipofuscin fluorophore A2E participates in blue light–induced damage to retinal pigmented epithelial (RPE) cells.

METHODS. Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 µM concentrations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/105 cells, as assayed by quantitative high-performance liquid chromatography (HPLC). Restricted zones (0.5-mm diameter spots) of confluent cultures were subsequently exposed to 480 ± 20-nm (blue) or 545 ± 1-nm (green) light for 15 to 60 seconds. Phototoxicity was quantified at various periods after exposure by fluorescence staining of the nuclei of membrane-compromised cells, by TdT-dUTP terminal nick-end labeling (TUNEL) of apoptotic cells and by Annexin V labeling for phosphatidylserine exposure.

RESULTS. Nonviable cells were located in blue light–exposed zones of A2E-containing RPE cells, whereas cells situated outside the illuminated areas remained viable. As shown by fluorescence labeling of the nuclei of membrane-damaged cells and by the presence of TUNEL-positive cells, the numbers of nonviable cells increased with exposure duration and as a function of the concentration of A2E used to load the cells before illumination. The numbers of blue light–induced TUNEL-positive cells also increased in advance of the increase in labeling of membrane-compromised cells, a finding that, together with Annexin V labeling, indicates an apoptotic form of cell death. Conversely, blue light–exposed RPE cells that did not contain A2E remained viable. In addition, illumination with green light resulted in the appearance of substantially fewer nonviable cells.

CONCLUSIONS. These studies implicate A2E as an initiator of blue light–induced apoptosis of RPE cells.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
It is well known that visible and UV radiation can produce photochemical lesions in the neural retina and retinal pigment epithelium (RPE).1 2 Indeed, RPE cells are particularly susceptible to wavelengths within the blue region of the spectrum.3 4 5 6 7 In addition to morphologic evidence of cell injury, blue light–induced damage to the RPE in vivo is manifest in blood–retinal barrier dysfunction8 and in substantial decreases in the apical-to-basal transport of leucine and chloride.9 Furthermore, studies in vivo have shown that the death of blue light–irradiated RPE cells occurs through apoptotic mechanisms.10 11 RPE cells in culture are also vulnerable to blue light irradiation, the phototoxicity elicited by 435-nm light being oxygen dependent.12 Blue light irradiation of RPE cell cultures containing autofluorescent pigment has also been reported to cause lysosomal membrane instability13 and, although cultures of porcine RPE cells exhibited no morphologic evidence of cellular damage after an 18-hour exposure to blue light, the growth of the RPE cells, in addition to that of endothelial cells and fibroblasts, was reduced significantly.14

The chromophore responsible for blue light–induced damage has been debated for some time, with candidates for this role including melanin and mitochondrial respiratory enzymes such as cytochrome-c oxidase.15 16 Indirect evidence also implicates component(s) of RPE lipofuscin as initiators of blue light damage.15 16 17 18 For instance, it has been shown that lipofuscin isolated from RPE can serve as photoinducible generators of singlet oxygen, superoxide anion, and H2O2.19 20 21 22 Moreover, in the presence of light and isolated lipofuscin granules, catalase is inactivated, and suspensions of RPE cells and rod outer segments undergo lipid peroxidation.23 Nevertheless, RPE lipofuscin is a heterogeneous mixture of fluorophores, and the identity of the species responsible for this photoreactivity remains to be determined.

A major hydrophobic component of RPE lipofuscin is the fluorophore A2E, a quaternary pyridinium salt that is the product of the reaction of all-trans retinal and ethanolamine.24 25 26 Under standard room light or monochromatic blue light (430 nm), A2E is known to interconvert with its double-bond isomer, iso-A2E, with equilibrium reached at an A2E:iso-A2E ratio of 4:1.27 Because we have detected both A2E and iso-A2E in RPE isolated in the dark from donor human eyes, we assume that this photoisomerization also occurs in vivo.27 By taking advantage of our ability to synthesize A2E,27 we have recently demonstrated that when A2E is accumulated by RPE cells in culture, it becomes localized to acidic organelles having a perinuclear distribution and can exert detergent-like activity when present in critical concentrations.28 The uptake of A2E complexed to low density lipoprotein (LDL) has also been shown to result in the alkalinization of lysosomes.29

Given that A2E strongly absorbs in the blue region of the spectrum, together with the known susceptibility of RPE to blue light damage, we undertook to investigate A2E as a fluorophore involved in blue light toxicity. To this end, we constructed a cell culture model that allows us to study the effects of blue light on RPE in the presence and absence of intracellular A2E.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
RPE Cell Cultures
A human adult RPE cell line (ARPE-19; American Type Culture Collection, Manassas, VA) that is devoid of endogenous A2E28 was maintained as stock cultures in 75-cm2 flasks with passage weekly. The cells exhibited no evidence of melanin pigmentation. For experimentation, ARPE-19 cells were grown on eight-well plastic chamber slides (Laboratory-Tek; Nunc, Napierville, IL), the bottoms of which were imprinted with an 8 x 8-mm ruled grid, with each square measuring 1 x 1 mm (64 squares). The latter grid was used to record and relocate cell populations according to the variable light conditions to which they had been exposed. The cells were grown as previously described,28 and all experiments were performed at confluence and within 3 weeks of plating.

A2E Synthesis and Cellular Uptake
A2E was synthesized from all-trans-retinal and ethanolamine (2:1 molar ratio), as previously described,27 and was stored as a stock solution (25 mM) in dimethyl sulfoxide (DMSO).28 For uptake into confluent cultures of ARPE-19 cells, A2E was delivered in 10-, 50- and 100-µM concentrations in culture media. All experiments included untreated cells. The autofluorescence of cell-associated A2E was detected by epifluorescence microscopy (Axiovert S100; Carl Zeiss, Thornwood, NY) using standard fluorescein isothiocyanate (FITC) filters (460–500-nm excitation, 510–560-nm emission). Internalized A2E was quantified by high-performance liquid chromatography (HPLC), as previously described.27 28

Excitation and Emission Spectra of A2E
UV/visible (Vis) radiation spectra of A2E in methanol (6 µM), were measured on a Lambda 40 spectrometer (Perkin–Elmer, Norwalk, CT). Fluorescence emission and excitation spectra of A2E in methanol were measured on a FluoroMax-2 spectrometer (Spex, Edison, NJ). Spectra were obtained using a 1-cm2 cuvette. Emission spectra were obtained using 400-nm excitation and excitation spectra were obtained while detecting the emission at 600 nm. Maximum absorbance was approximately 0.2.

Illumination
At least 7 days after A2E loading,28 the cultures were removed from the incubator and immediately exposed to 480 ± 20-nm (blue) or 545 ± 15-nm (green) illumination delivered from a 100-W mercury lamp for 15 to 60 seconds. A single spot of focused illumination (0.5-mm diameter) was applied to the cell population located in an individual square of the locator grid. The irradiance levels of the 480-nm and 545-nm illuminations at full output were measured (Astral AA30; Scientech, Boulder, CO) as 75 mW/mm2 and 200 mW/mm2, respectively. Irradiances measured with the addition of an infrared filter were reduced by 16% and 19%, respectively, indicating that these fractions of the radiant energy were delivered as heat. The cultures were exposed to ambient lighting (approximately 75 foot candles) during plating and feeding and while transporting the cells to and from the exposure apparatus. The pH of the media was 7.2 to 7.5. Control conditions included cells not incubated with A2E but exposed to 480-nm light and A2E-containing cells not exposed to 480-nm light.

Fluorescence Assay of Cell Viability
Cell viability was quantified at the indicated times by a two-color fluorescence assay (Molecular Probes, Eugene, OR) in which the nuclei of nonviable cells appear red due to staining by a membrane-impermeant dye (Dead Red nucleic acid stain; Molecular Probes), whereas the nuclei of all cells stained green by a membrane-permeant dye (Syto 10 green fluorescent nucleic acid stain; Molecular Probes). Briefly, the cultures were incubated with the fluorescent dyes (1/500 dilution in HEPES-buffered Hanks’ balanced salt solution) for 15 minutes, after which the cells were washed and fixed in 4% glutaraldehyde for 1 hour. For fluorescence detection, the fixed cultures were examined using a fluorescence microscope (Axiovert 100; Zeiss) with the red stain excited at {lambda} 545 ± 15 nm and visualized at 620 ± 30 nm, and the green stain excited at 480 ± 20 nm and visualized at 535 ± 25 nm. The numbers of red–labeled nuclei in the light-exposed fields (0.5 mm diameter) were counted in fluorescence photomicrographs, and background numbers of dead cells, determined by counting red-labeled nuclei in unexposed areas of the cultures, were subtracted. Data are based on counts performed on three to six replicates per experiment. Images were processed for publication using Photoshop 5.0 (Adobe, San Jose, CA).

Detection of Apoptosis-Associated DNA Fragmentation
Cells undergoing apoptosis were detected by labeling for DNA strand breaks by terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick-end labeling (TUNEL).30 Briefly, cultures were fixed in 2% paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (2 minutes, 4°C) and incubated in TdT together with dUTP-fluorescein (37°C, 60 minutes; Boehringer–Mannheim, Indianapolis IN). Subsequent incubations were in anti-fluorescein antibody conjugated to alkaline phosphatase (37°C, 30 minutes) followed by fast red (Boehringer Mannheim) as the substrate for alkaline phosphatase detection. The numbers of TUNEL-labeled nuclei in the light-exposed fields (0.5-mm diameter) were counted, and background numbers of labeled cells, determined by counting TUNEL-positive nuclei in unexposed areas of the cultures, were subtracted. Data are based on three to six replicates per experiment. Controls consisted of TUNEL assay of A2E-containing unexposed cells, TUNEL assay of untreated (without A2E), unexposed cells and the incubation of A2E-containing light-exposed cells in labeling solution without TdT.

Annexin V Labeling of Apoptotic Cells
To detect phosphatidylserine (PS) on the external leaflet of plasma membranes, the cultures were incubated for 15 minutes at room temperature in sterile buffer (140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES/NaOH [pH 7.4]) containing 5 µg/ml FITC-labeled Annexin V (human recombinant; Alexis Biochemicals, San Diego, CA) and Dead Red (1:500 dilution; Molecular Probes). Subsequently, the cultures were washed, and Annexin V–positive/red-negative cells were counted under the fluorescence microscope. Counting of Annexin V–positive cells, which simultaneously excluded the membrane-impermeant red dye, served to control for nonspecific Annexin V binding.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
We allowed ARPE-19 cells to accumulate A2E by incubating confluent cultures for 2 hours at 37°C with A2E prepared as 10-, 50-, and 100-µM concentrations in media. Cells in control wells were not loaded with A2E. By epifluorescence microscopy, the internalization of A2E was evidenced by the presence of autofluorescent granules (Fig. 1) that were not present in untreated cells. Moreover, the cells in a single culture varied in the extent to which they accumulated A2E during the 2-hour incubation period. By quantitative HPLC (UV)., we estimated that the overall levels (mean ± SD) of A2E in the cultured cells were 64 ± 6 ng/105 cells at the 100-µM concentration, 23 ± 1 ng/105 cells at 50 µM, and less than 5 ng/105 cells at 10 µM. These amounts are comparable to A2E levels measured in human RPE isolated from healthy donor eyes (34–134 ng/105 cells).28



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  		Figure 1. Internalization of A2E by RPE cells in culture. (A) Epifluorescence detection of A2E accumulation in ARPE-19 cells 7 days after incubation with 100 µM A2E for 2 hours. From inspection of the cells within the plane of focus (arrow), it is apparent that the autofluorescence of internalized A2E was associated with intracellular granules. When not in focus, these granules appeared soft-edged and could not be resolved as individual puncta. n, nucleus. (B) Control cells not exposed to A2E. Photomicrographs (A, B) were obtained with FITC-appropriate filters and an exposure duration of 2 seconds. Scale bar, 10 µm.

 
To determine whether A2E-containing RPE cells were susceptible to blue light–induced phototoxicity, 1 week after A2E loading, restricted zones (0.5-mm diameter spots) of confluent monolayers were exposed to 480-nm light. Accordingly, using a fluorescence assay in which the nuclei of nonviable cells fluoresce red, we observed the death of A2E-loaded RPE cells exposed to blue light for varying durations of time (15–60 seconds; Figs. 2 and 3 ). The labeled nuclei were concentrated in a 0.5-mm diameter zone which corresponded to the area of illumination (Fig. 2A 2B 2D 2E 2G 2H) . Furthermore, the nonviable cells were uniformly distributed across the diameter of the illuminated field, and A2E-containing cells flanking the exposed area remained viable. Of note, RPE cells that did not contain A2E and that were exposed to blue light of the same intensity for 60 seconds, also remained viable (Fig. 2F) . The data presented were obtained in cells maintained either in complete media or in a defined buffer–salt solution (Krebs–Ringer phosphate buffer [KRPG] containing 5.5 mM glucose) during light exposure. Because the phototoxic response to blue light was the same under both conditions, the data were pooled.



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  		Figure 2. ARPE-19 cells containing A2E underwent cell death when exposed to blue light. The numbers of nonviable cells, detected by nuclear labeling with a membrane-impermeant dye, varied with duration of light exposure, concentration of A2E, and time after light exposure. The zones (0.5-mm diameter) of nonviable cells corresponded to the illuminated fields. Viable A2E-loaded cells flanking the zone of illumination excluded red stain. (A, B, and C) A2E-treated (100 µM in media) RPE cells were exposed to 480-nm illumination for 30 or 60 seconds or were not exposed (no light), and nonviable cells were labeled after 18 hours. (D, E, and F) A2E-treated (50 and 100 µM in media) and untreated cultures were exposed to 480-nm illumination for 60 seconds, and nonviable cells were labeled after 18 hours. (G, H, and I) A2E-treated (100 µM) RPE cells were exposed to 480-nm (blue) or 545-nm (green) illumination for 60 seconds and nonviable cells were labeled after 6 or 18 hours, as indicated. Scale bar, 100 µM.

 


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  		Figure 3. Quantitation of nonviable cells after the illumination of A2E-containing ARPE-19 cells in culture. ARPE-19 cells accumulated A2E from 50- and 100-µM concentrations in media. Control cells (without A2E) did not accumulate A2E. Fields of cells (0.5-mm diameter) were exposed to 480-nm (blue) or 545-nm (green) light for 60 seconds. After 6, 12, and 18 hours the nuclei of nonviable cells were fluorescently labeled. Values are means ± SEM. The numbers in parentheses indicate the total number of replicates pooled from one to four experiments. The values for untreated cultures are less than 0.5. Note that the value for 18 hours at 100 µM is underestimated, because in some experiments cells had begun to detach from the culture dish.

 
Counting of fluorescently labeled nuclei in the illuminated zone (0.5 mm) further revealed that the frequency of membrane-compromised cells increased with time after exposure (Fig. 3) . In addition, at 6 and 12 hours after light exposure, the nuclei labeled with red fluorescence were more numerous at the 100-µM concentration of A2E than at the 50-µM concentration, presumably because at the higher loading concentration, a greater number of cells acquired critical concentrations of A2E. Even within a single-culture well, variations in the rate at which cells underwent death may have been due, in part, to differences in the A2E content of individual cells. When cultures were loaded with A2E from a 100-µM concentration in media, exposed to 60 seconds of blue light, and then labeled 18 hours after illumination, the numbers of nonviable red-labeled nuclei reached approximately 75% of the total number of nuclei in the illuminated zone (red-labeled nuclei [mean ± SEM]: 141 ± 18; total nuclei: 189 ± 13, n = 10). This value for the proportions of nonviable cells is actually underestimated, however, because the time course of cell death was somewhat variable and in some experiments, cells had begun to detach from the culture dish by 18 hours after exposure. Indeed, the loss of illuminated cells loaded from a 100-µM concentration of A2E accounts for the observation that at 18 hours after illumination, the numbers of red-labeled nuclei at the 50- and 100-µM concentrations of A2E are similar (Fig. 3 , 18 hours after light exposure). When the cells had not been loaded with A2E but were exposed to blue light, the incidence of labeled nuclei was not greater than background levels (0–1 nuclei/0.5-mm diameter field) calculated by counting red-labeled nuclei in nonilluminated regions of the cultures.

For cells accumulating A2E from 100-µM concentrations in media, blue light induced death at exposure times of 15 to 60 seconds, with the numbers of nonviable A2E-containing cells within the illuminated region increasing as a function of the exposure time (Fig. 7) . Blue light was also significantly more effective in causing lethal damage than green light (Figs. 2 3) , even though with green light the cells were exposed to considerably more radiant energy (blue light, 75 mW/mm2; green light, 210 mW/mm2). The differences in cell loss observed with green and blue light can readily be accounted for by both the UV/Vis and excitation spectra of A2E: peak absorbance and excitation occur at approximately 430 nm (blue), with the absorption-emission elicited at 545 nm (green) being approximately 10% of that elicited at 480 nm (blue; Fig. 4 ).



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  		Figure 7. The incidence of DNA strand breaks in the nuclei of blue light–damaged A2E-containing RPE cells increased before changes in membrane permeability. ARPE-19 cells were loaded with A2E from 100-µM concentration in media, and the cells were exposed to 480-nm (blue) light for 15, 30, or 60 seconds. One, 3, and 6 hours after exposure, DNA strand breaks were assayed by the TUNEL method, and the nuclei of membrane-permeable cells were fluorescently labeled. Values are means ± SEM of the numbers of labeled nuclei in an illuminated field. The numbers in parentheses indicate the numbers of determinations pooled from one to four experiments. All values at 1 hour after exposure were less than 0.5. The data for TUNEL-positive nuclei 6 hours after light exposure are also presented in Figure 6B .

 


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  		Figure 4. UV/Vis, excitation, and emission spectra of A2E in methanol. The absorbance spectrum had a major peak at 435 nm and a lesser peak at 335 nm. The excitation spectrum, monitored at 600-nm emission, was similar in shape with a maximum at 418 nm. A 400-nm excitation wavelength generated a yellow emission centered around 602 nm. Inset, structure of A2E.

 
To ascertain whether cell death occurred by apoptotic mechanisms, we assayed for DNA fragmentation, a process that begins early in apoptosis31 and that can be detected by the presence of TUNEL-positive nuclei. As shown in Figure 5 , blue light–induced TUNEL-positive cells were first detected within the illuminated regions of A2E-loaded cultures 3 hours after light exposure, with the frequency of TUNEL-labeled nuclei increasing at 6 hours after exposure. At 6 hours after blue light exposure, the numbers of TUNEL-labeled nuclei were also greater when the cells had been loaded with A2E from a 100-µM concentration than from a 50-µM concentration (Fig. 6A ). Conversely, apoptosis, as detected by TUNEL, was not induced by blue light exposure (60 seconds), when the cells had accumulated A2E from a 10-µM concentration. With blue light exposure, the numbers of TUNEL-positive cells within the illuminated region also increased as a function of the exposure time (15–60 seconds; Figs. 6B and 7 ). Green light exposure for 60 seconds was, however, associated with the induction of considerably fewer apoptotic nuclei (8.0 ± 1.4 versus 68.8 ± 9.7, green and blue light, respectively; Fig. 6C ).



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  		Figure 5. Detection of DNA strand breaks in the nuclei of A2E-containing RPE cells exposed to blue light. ARPE-19 cells were untreated (A) or were incubated with 100 µM A2E for 2 hours (B, C). The fields (0.5-mm diameter) of cells were exposed to 480-nm (blue) light for 60 seconds, and after 3 hours (B) and 6 hours (A, C), nuclei exhibiting DNA fragmentation were labeled by the TUNEL method. Scale bar, 100 µM.

 


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  		Figure 6. Quantitation of TUNEL-positive nuclei 6 hours after exposure of A2E-containing RPE cells to blue light. (A) ARPE-19 cells were devoid of A2E (0) or had accumulated A2E from 10-, 50-, and 100-µM concentrations in media, and fields of cells (0.5-mm diameter) were exposed to 480-nm (blue) light for 60 seconds. (B) ARPE-19 cells were loaded with A2E from 100-µM concentrations in media, and fields of cells were illuminated with 480-nm (blue) light for 15, 30, or 60 seconds. (C) ARPE-19 cells were loaded with A2E from 100-µM concentration in media, and fields of cells were exposed to 545-nm (green) light for 60 seconds. Values are means ± SEM of the numbers of replicates presented in parentheses. The replicates were pooled from one to four experiments.

 
Cell death by apoptosis typically proceeds through a programmed set of events, with DNA fragmentation beginning early, whereas the impermeability of the cell membrane is maintained until later stages.32 Thus, as part of our study we also compared the time course of appearance of TUNEL-positive nuclei with the increase in numbers of red-stained nuclei (Fig. 7) . Accordingly, when A2E-containing RPE cells were exposed to blue light of 15 to 60 seconds’ duration, neither TUNEL-positive nor red-stained nuclei were present 1 hour after light exposure. Subsequently, between 3 and 6 hours after light exposure, the numbers of TUNEL-positive and red-labeled nuclei increased, but at both intervals the numbers of TUNEL-positive cells were greater than the numbers of red-stained nuclei. This finding indicates that in the early stages of apoptosis, many cells that were undergoing DNA fragmentation had not yet exhibited altered membrane permeability. This pattern of events is consistent with an apoptotic form of cell death.

The redistribution of PS from the inner to the outer leaflet of the plasma membrane is also an early event in apoptotic cell death33 34 that occurs after the onset of DNA fragmentation and during the stage when membrane integrity is intact. Externally exposed PS was detected by Annexin V labeling. In addition, to establish that Annexin V was specifically binding to externalized PS rather than to PS within the inner leaflet of the plasma membrane, we simultaneously examined for membrane impermeability, indicated by the exclusion of the membrane-impermeant dye Dead Red. Accordingly, only nuclei that were both Annexin V positive and red negative were counted. Thus, in ARPE-19 cells loaded with A2E from a 100-µM concentration and illuminated with blue light for 60 seconds, Annexin V labeling was readily detectable as a bright membrane fluorescence at 3 hours after exposure. The mean number of Annexin V–positive and red-negative cells in blue light–illuminated fields of A2E-loaded cultures was 20.8 ± 1.9 (mean ± SEM; n = 24, three experiments). In contrast, no Annexin V–positive and red-negative cells were observed in untreated cultures exposed to blue light for 60 seconds (0 ± 0, n = 14, three experiments).

A2E-containing cells that were exposed to blue light also demonstrated nuclear condensation, a morphologic change that is ongoing through apoptosis. The progression of nuclear condensation was apparent when the sizes of the nuclei were compared at 6 hours and 18 hours after blue light irradiation (Figs. 8A 8B ). Moreover, at 18 hours after light exposure apoptotic bodies had also formed (Fig. 8C) .



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  		Figure 8. Morphologic evidence of apoptosis in blue light–exposed A2E-containing RPE cells. Nuclear condensation progressed between 6 (A) and 18 (B) hours after blue light exposure (60 second). (C) The fragmentation of some nuclei into multiple apoptotic bodies was apparent 18 hours after light exposure. ARPE-19 cells were loaded with A2E from 100-µM concentration in media. Nuclei of membrane-permeable cells were labeled with fluorescent red stain. Scale bar, (A, B) 25 µM; (C) 25 µM.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
These studies implicate A2E as a mediator of blue light damage in the RPE. Thus, using the criteria of the presence of TUNEL-positive cells, exposure of PS on the cell surface, late-onset membrane permeability, nuclear condensation, and apoptotic body formation, we deduce that healthy RPE cells that have amassed A2E at critical concentrations and are exposed to blue light can undergo an apoptotic form of cell death. Central to the conclusion that the photochemical damage is initiated by A2E is the finding that in the absence of internalized A2E, the RPE cells showed no loss of viability in response to blue light exposure. Furthermore, the extent of damage, measured as the numbers of nonviable cells at 6 and 12 hours after light exposure, was directly related to the A2E content of the cells. These results are consistent with previous observations of blue light–induced damage to RPE cells, in vivo.3 4 5 6 7 8 9 10 11 12 13 14 Additionally, although the present studies are pertinent to the question of whether blue light–induced injury can originate in the RPE cell rather than be triggered after primary damage to the photoreceptor cells,16 35 they do not preclude the possibility of direct blue light damage to the photoreceptor cells.

We have previously demonstrated by light and fluorescence confocal microscopy that the A2E that is internalized by cultured cells has a particulate appearance, indicating that it may be localized to spherical organelles.28 These intracellular granules of A2E also assume a perinuclear distribution, which is typical of lysosomes.36 Moreover, the colocalization of A2E with a fluorescent acidotropic probe revealed that A2E preferentially accumulates in acidic organelles and may indicate that the compartmentalization of A2E replicates that occurring in vivo. Given that lipofuscin is located within lysosomal storage bodies in the cell, it has been suggested that any adverse effects of its accumulation would be expressed, at least initially, within these organelles.23 24 Indeed, in light of the observation that lysosomal pH is elevated in cultured cells that have taken up A2E complexed to LDL,29 we have previously suggested28 that A2E may mediate a detergent-like disruption of the membrane-bound adenosine triphosphatase (ATPase) that otherwise maintains lysosomal acidity by pumping protons into the organelle.37 38 39 Oxidative damage to the lysosomal membrane may have also been a factor in the phototoxicity visited on A2E-containing cells in the present experiments.

It is said to be a feature of photochemically induced processes, that light intensity and exposure duration are interchangeable with respect to irradiant dose.5 8 35 There is, however, a departure from this relationship in exposures of long duration and low irradiance, because repair processes in the cell can balance damage mechanisms.35 In the present study the light intensity was held constant while exposure time was varied, and high irradiance levels with short exposure times were used. Accordingly, the changes observed in our tissue culture model reflected a response that was graded with respect to blue light exposure time. Further, the blue region of the spectrum had a marked ability to induce the death of A2E-containing cells, whereas the longer wavelength green light was considerably less effective. This wavelength dependence of the phototoxicity is consistent with the absorption and excitation spectra of A2E. Green light, however, appears to be more effective at inducing damage to the photoreceptor cell, with the action spectrum of the light damage coinciding with the absorption spectrum of rhodopsin.40 41 42 43

RPE cell death in animal models of light-induced damage16 and in cell culture models of chemical oxidant–induced injury44 has been shown to occur by means of apoptotic mechanisms. Although the photochemical events generating apoptosis under conditions of blue light injury are not fully understood, reactive oxygen intermediates are likely to serve as mediators. Evidence for the latter assertion is derived from a number of studies demonstrating that the addition of compounds with antioxidant capabilities blocks the onset of light damage.44 45 46 In addition, promoters of apoptosis, other than light, are known to elicit oxidative stress.47 Cellular DNA is considered to be a possible target of reactive oxygen species.47 For instance, when the death of photoreceptor cells is induced by high intensity light, oxidative damage to DNA, as witnessed by the presence of random single-strand DNA fragmentation, has been shown to precede the onset of apoptosis.48 49 In this context, it is also notable that lipofuscin has been shown to be a photogenerator of singlet oxygen, superoxide, and hydrogen peroxide, with blue light providing the most efficient radiation.20 21 22 It remains to be determined, however, whether oxidative damage is a feature of the blue light–induced RPE damage observed in the present experimental model.

Multiple factors are considered to contribute to the onset of age-related macular degeneration (AMD). Although controversial,50 51 it has been reported that a subset of patients with nonexudative AMD exhibit heterozygous mutations in the gene encoding for ABCR, the rod-photoreceptor-specific ATP-binding cassette transporter.52 53 The substrate transported by ABCR is hypothesized to be a phosphatidylethanolamine(PE)-all-trans-retinal Schiff-base adduct.54 It is suggested that the function of ABCR is to flip this Schiff-base adduct from the luminal to the cytosolic face of the outer segment disc membrane, thereby facilitating the reduction of all-trans-retinal by all-trans-retinol dehydrogenase.54 55 Significantly, we have previously proposed that this same PE-all-trans-retinal Schiff-base adduct is generated as the first step in the biogenesis of A2E,27 the second step being Schiff base formation with a second molecule of retinal to form a phosphatidyl-pyridinium bisretinoid (A2-PE) that would eventually be hydrolyzed to A2E.27 56 57 In light of this, it is reasonable to suppose that the consequences of reduced ABCR transport may be an accumulation of the PE-all-trans-retinal Schiff-base adduct54 55 and A2-PE, within the outer segment. This situation would ultimately promote the deposition of A2E in RPE cells because of the role of the RPE cell in phagocytosing packets of outer segment membrane. Mutations in both alleles of ABCR are also known to cause Stargardt disease,58 59 a macular degeneration of juvenile onset that is characterized by a pronounced accumulation of lipofuscin60 and progressive atrophy of the RPE.61 It is notable that mice with a null mutation in ABCR have recently been shown to exhibit accentuated levels of A2E in the RPE.55 Nevertheless, A2E has not been quantitated in patients with Stargardt disease, nor in individuals with an ABCR-associated autosomal recessive form of retinitis pigmentosa.62 63

The amassing of A2E and possibly other components of lipofuscin by the RPE cell is not a benign consequence of aging and disease. In addition to the observations made in the present study, we have previously shown that when A2E accumulates to critical concentrations in cultured ARPE-19 cells, it can behave as an amphiphilic detergent and perturb membrane integrity.28 The finding in the present work that blue light damage exhibited a dependence on A2E concentration is compatible with the concept that RPE lipofuscin must reach critical levels, above which disease is manifest. Nevertheless, although the accumulation of lipofuscin by aging RPE cells is greatest in the macula,64 65 evidence for a causal link between lipofuscin and AMD has not been found. Similarly, whether chronic or acute light damage contributes to the pathogenesis of AMD remains to be determined, because epidemiologic studies concerned with the relationship between light exposure (UV and visible radiation) and AMD have been inconclusive.66 67


   Footnotes
 
Supported by Fight for Sight, unrestricted funds from Research to Prevent Blindness and Grants EY-12951 (JRS) and EY-34509 (KN) from the National Institutes of Health.

Submitted for publication September 27, 1999; revised January 18, 2000; accepted January 31, 2000.

Commercial relationships policy: N.

Corresponding author: Janet R. Sparrow, Department of Ophthalmology, Columbia University, 630 W. 168th Street, New York, NY 10032. jrs88{at}columbia.edu


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Noell, WK (1980) Possible mechanisms of photoreceptor damage by light in mammalian eyes Vis Res 20,1163-1171[Medline][Order article via Infotrieve]
  2. Marshall, J, Greenstein, V, Kline, D, Owsley, C, Werner, JS (1986) Optical radiation and the aged eye Waxler, M Hitchins, VM eds. Optical Radiation and Visual Health ,103-124 CRC Press Boca Raton, FL.
  3. Ham, WT, Ruffolo, JJ, Mueller, HA, Clarke, AM, Moon, ME (1978) Histologic analysis of photochemical lesions produced in rhesus retina by short-wavelength light Invest Ophthalmol Vis Sci 17,1029-1035[Abstract/Free Full Text]
  4. Ham, WT, Mueller, HA, Ruffolo, JJ, et al (1984) Basic mechanisms underlying production of photochemical lesions in the mammalian retina Curr Eye Res 3,165-174[Medline][Order article via Infotrieve]
  5. Ham, WT, Allen, RG, Feeney–Burns, L, et al (1986) The involvement of the retinal pigment epithelium Waxler, M Hitchins, VM eds. Optical Radiation and Visual Health ,43-67 CRC Press Boca Raton, FL.
  6. Tso, MOM, Fine, BS (1979) Repair and late degeneration of the primate foveola after injury by argon laser Invest Ophthalmol Vis Sci 18,447-461[Abstract/Free Full Text]
  7. Busch, EM, Gorgels, TGMF, van Norren, D. (1999) Temporal sequence of changes in rat retina after UV-A and blue light exposure Vision Res 39,1233-1247[Medline][Order article via Infotrieve]
  8. Putting, BJ, van Best, JA, Vrensen, GF, Oosterhuis, JM (1994) Blue light-induced dysfunction of the blood-retinal barrier at the pigment epithelium in albino versus pigmented rabbits Exp Eye Res 58,31-40[Medline][Order article via Infotrieve]
  9. Pautler, EL, Morita, M, Beezley, D. (1989) Reversible and irreversible blue light damage to the isolated mammalian pigment epithelium LaVail, MM Anderson, RE Hollyfield, JG eds. Inherited and Environmental Induced Retinal Degenerations ,555-567 Alan R. Liss New York.
  10. Hafezi, F, Marti, A, Munz, K, Reme, CE (1997) Light-induced apoptosis: Differential timing in the retina and pigment epithelium Exp Eye Res 64,963-970[Medline][Order article via Infotrieve]
  11. Reme, CE, Weller, M, Szczesny, P, et al (1995) Light induced apoptosis in the rat retina in vivo. Morphological features, threshold and time course Anderson, RE LaVail, MM Hollyfield, JG eds. Degenerative Diseases of the Retina ,19-24 Plenum Press New York.
  12. Crockett, RS, Lawwill, T. (1984) Oxygen dependence of damage by 435 nm light in cultured retinal epithelium Curr Eye Res 3,209-215[Medline][Order article via Infotrieve]
  13. Wihlmark, U, Wrigstad, A, Roberg, K, Nilsson, SE, Brunk, UT (1997) Lipofuscin accumulation in cultured retinal pigment epithelial cells causes enhanced sensitivity to blue light irradiation Free Radic Biol Med 22,1229-1234[Medline][Order article via Infotrieve]
  14. Dorey, CK, Delori, FC, Akeo, K. (1990) Growth of cultured RPE and endothelial cells is inhibited by blue light but not green or red light Curr Eye Res 9,549-559[Medline][Order article via Infotrieve]
  15. Boulton, M. (1998) Melanin and the retinal pigment epithelium Marmor, MF Wolfensberger, TJ eds. The Retinal Pigment Epithelium ,68-85 Oxford University Press New York.
  16. Remé, CE, Hafez, F, Marti, A, Munz, K, Reinboth, JJ (1998) Light damage to the retina and retinal pigment epithelium Marmor, MF Wolfensberger, TJ eds. The Retinal Pigment Epithelium ,563-586 Oxford University Press New York.
  17. Eldred, GE (1998) Lipofuscin and other lysosomal storage deposits in the retinal pigment epithelium Marmor, MF Wolfensberger, TJ eds. The Retinal Pigment Epithelium ,651-668 Oxford University Press New York.
  18. Dillon, J. (1991) Photophysics and photobiology of the eye J Photochem Photobiol B 10,23-40[Medline][Order article via Infotrieve]
  19. Gaillard, ER, Atherton, SJ, Eldred, G, Dillon, J. (1995) Photophysical studies on human retinal lipofuscin Photochem Photobiol 61,448-453[Medline][Order article via Infotrieve]
  20. Boulton, M, Dontsov, A, Jarvis–Evans, J, Ostrovski, H, Svistunencko, D. (1993) Lipofuscin is a photoinducible free radical generator J Photochem Photobiol B 19,201-204[Medline][Order article via Infotrieve]
  21. Rozanowska, M, Jarvis–Evans, J, Korytowski, W, Booulton, ME, Burke, JM, Sarna, T. (1995) Blue light-induced reactivity of retinal age pigment J Biol Chem 270,18825-18830[Abstract/Free Full Text]
  22. Rozanowska, M, Wessels, J, Boulton, M, et al (1998) Blue light induced singlet oxygen generation by retinal lipofuscin in non-polar media Free Rad Biol Med 24,1107-1112[Medline][Order article via Infotrieve]
  23. Wassell, J, Davies, S, Bardsley, W, Boulton, M. (1999) The photoreactivity of the retinal age pigment lipofuscin J Biol Chem 34,23828-23832
  24. Eldred, G, Lasky, MR (1993) Retinal age pigments generated by self-assembling lysosomotropic detergents Nature 361,724-726[Medline][Order article via Infotrieve]
  25. Sakai, N, Decatur, J, Nakanishi, K. (1996) Ocular age pigment "A2E": an unprecedented pyridinium bisretinoid J Am Chem Soc 118,1559-1560
  26. Ren, RX-F, Sakai, N, Nakanishi, K. (1997) Total synthesis of the ocular age pigment A2E: a convergent pathway J Am Chem Soc 119,3619-3620
  27. Parish, CA, Hashimoto, M, Nakanishi, K, Dillon, J, Sparrow, JR (1998) Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium Proc Natl Acad Sci USA 95,14609-14613[Abstract/Free Full Text]
  28. Sparrow, JR, Parish, CA, Nakanishi, K. (1999) A2E. a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture Invest Ophthalmol Vis Sci 40,2988-2995[Abstract/Free Full Text]
  29. Holz, FG, Schutt, F, Kopitz, J, et al (1999) Inhibition of lyososomal degradative functions in RPE cells by a retinoid component of lipofuscin Invest Ophthalmol Vis Sci 40,737-743[Abstract/Free Full Text]
  30. Gavrieli, Y, Sherman, Y, Ben-Sasson, SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation J Cell Biol 119,493-501[Abstract/Free Full Text]
  31. Schwartzman, RA, Cidlowski, JA. (1993) Apoptosis: the biochemistry and molecular biology of programmed cell death Endocr Rev 14,133-151[Abstract/Free Full Text]
  32. Darzynkiewicz, Z, Bruno, S, Del Bino, G, et al (1992) Features of apoptotic cells measured by flow cytometry Cytometry 13,795-808[Medline][Order article via Infotrieve]
  33. Koopman, G, Reutelingsperger, CPM, Kuijten, GAM, Keehnen, RMJ, Pals, ST, van Oers, MHJ. (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis Blood 84,1415-1420[Abstract/Free Full Text]
  34. Boersma, AWM, Nooter, K, Oostrum, RG, Stoter, G. (1996) Quantification of apoptotic cells with fluorescein isothiocyanate-labeled Annexin V in Chinese hamster ovary cell cultures treated with cisplatin Cytometry 24,123-130[Medline][Order article via Infotrieve]
  35. Mellerio, J. (1994) Light effects on the retina Albert, DM Jakobiec, FA eds. Principles and Practice of Ophthalmology: Basic Sciences ,1326-1345 WB Saunders Philadelphia.
  36. Heuser, J. (1989) Changes in lysosome shape and distribution correlated with changes in cytoplasmic pH J Cell Biol 108,855-864[Abstract/Free Full Text]
  37. Hayashi, H, Niinobe, S, Matsumoto, Y, Suga, T. (1981) Effects of Triton WR-1339 on lipoprotein lipolytic activity and lipid content of rat liver lysosomes J Biochem (Tokyo) 89,573-579[Abstract/Free Full Text]
  38. Ohkuma, S, Moriyama, Y, Takano, T. (1982) Identification and characterization of a proton pump on lysosomes by fluorescein isothiocyanate-dextran fluorescence Proc Natl Acad Sci USA 79,2758-2762[Abstract/Free Full Text]
  39. Mellman, I, Fuchs, R, Helenius, A. (1986) Acidification of the endocytic and exocytic pathways Annu Rev Biochem 55,663-700[Medline][Order article via Infotrieve]
  40. Noell, WK, Walker, VS, Kang, BS, Berman, S. (1966) Retinal damage by light in rats Invest Ophthalmol 5,450-473[Abstract/Free Full Text]
  41. Shahinfar, S, Edward, DP, Tso, MOM (1991) A pathologic study of photoreceptor cell death in retinal photic injury Curr Eye Res 10,47-59[Medline][Order article via Infotrieve]
  42. Williams, TP, Howell, WL (1983) Action spectrum of retinal light damage in albino rats Invest Ophthalmol Vis Sci 24,285-287[Abstract/Free Full Text]
  43. Organisciak, DT, Darrow, RM, Bicknell, IR, Jiang, Y-L, Pickford, M, Blanks, JC (1991) Protection against retinal light damage by natural and synthetic antioxidants Hollyfield, JG Anderson, RE La Vail, MM eds. Retinal Degenerations ,189-199 CRC Press Boca Raton, FL.
  44. Cai, J, Wu, M, Nelson, KC, Sternberg, P, Jones, DP (1999) Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells Invest Ophthalmol Vis Sci 40,959-966[Abstract/Free Full Text]
  45. Organisciak, DT, Kutty, RK, Leffak, M, et al (1995) Oxidative damage and responses in retinal nuclei arising from intense light exposure Anderson, RE LaVail, MM Hollyfield, JG eds. Degenerative Diseases of the Retina ,9-17
  46. Organisciak, DT, Winkler, BS (1994) Retinal light damage: practical and theoretical considerations Osborne, NN Chader, GJ eds. Progress in Retinal and Eye Research ,1-29 Pergamon Press Oxford, UK.
  47. Buttke, TM, Sandstrom, PA. (1994) Oxidative stress as a mediator of apoptosis Immunol Today ,7-10
  48. Specht, S, Leffak, M, Darrow, RM, Organisciak, DT (1999) Damage to rat retinal DNA induced in vivo by visible light Photochem Photobiol 69,91-98[Medline][Order article via Infotrieve]
  49. Organisciak, DT, Darrow, RA, Barsalou, L, Darrow, RM, Lininger, LA (1999) Light-induced damage in the retina: differential effects of dimethylthiourea on photoreceptor survival, apoptosis and DNA oxidation Photchem Photobiol 70,261-268
  50. Stone, EM, Webster, AR, Vandernburgh, K, et al () Allelic variation in abcr associated with Stargardt-disease but not age-related macular degeneration Nat Genet 20,328-329
  51. De La Paz, M, Guy, VK, Abou–Donia, S, et al (1999) Analysis of the Stargardt disease gene (ABCR) in age-related macular degeneration Ophthalmology 106,1531-1536[Medline][Order article via Infotrieve]
  52. Allikmets, R, Schroyer, NF, Singh, N, et al () Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration Science 277,1805-1807
  53. Shroyer, NF, Lewis, RA, Alllikmets, R, et al (1999) The rod photoreceptor ATP-binding cassette transporter gene, ABCR, and retinal disease: from monogenic to multifactorial Vision Res 39,2537-2544[Medline][Order article via Infotrieve]
  54. Sun, H, Molday, RS, Nathans, J. (1999) Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt’s disease J Biol Chem 274,8269-8281[Abstract/Free Full Text]
  55. Weng, J, Mata, NL, Azarian, SM, Tzekov, RT, Birch, DG, Travis, GH (1999) Insights into the function of rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in abcr knockout mice Cell 98,13-23[Medline][Order article via Infotrieve]
  56. Eldred, GE (1995) Lipofuscin fluorophore inhibits lysosomal protein degradation and may cause early stages of macular degeneration Gerontology 41,15-28
  57. Katz, ML, Gao, C-L, Rice, LM (1996) Formation of lipofuscin-like fluorophores by reaction of retinal with photoreceptor outer segments and liposomes Mech Ageing Dev 92,159-174[Medline][Order article via Infotrieve]
  58. Allikmets, R, Singh, N, Su, H, et al (1997) A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt’s macular dystrophy Nat Genet 15,236-246[Medline][Order article via Infotrieve]
  59. Lewis, RA, Shroyer, NF, Singh, N, et al (1999) Genotype/phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt’s disease Am J Hum Genet 64,422-434[Medline][Order article via Infotrieve]
  60. Delori, FC, Staurenghi, G, Arend, O, Dorey, CK, Goger, DG, Weiter, JJ (1995) In vivo measurement of lipofuscin in Stargardt’s disease-fundus flavimaculatus Invest Ophthalmol Vis Sci 36,2327-2331[Abstract/Free Full Text]
  61. Lee, BL, Heckenlively, JR (1999) Stargardt’s disease and fundus flavimaculatus Guyer, DR Yannuzzi, LA Chang, S Shields, JA Green, GR eds. Retina-Vitreous-Macula ,978-988 WB Saunders Philadelphia.
  62. Martinez–Mir, A, Paloma, E, Allikmets, R, et al (1998) Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR Nat Genet 18,11-12[Medline][Order article via Infotrieve]
  63. Cremers, FP, van de Pol, DJ, van Driel, M, et al (1998) Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt’s disease gene ABCR Hum Mol Genet 7,355-362[Abstract/Free Full Text]
  64. Feeney–Burns, L, Hilderbrand, ES, Eldridge, S. (1984) Aging human RPE: morphometric analysis of macular, equatorial and peripheral cells Invest Ophthalmol Vis Sci 25,195-200[Abstract/Free Full Text]
  65. Dorey, CK, Wu, G, Ebenstein, D, Garsd, A, Weiter, JJ (1989) Cell loss in the aging retina Invest Ophthalmol Vis Sci 30,1691-1699[Abstract/Free Full Text]
  66. West, SK, Rosenthal, FS, Bressler, NM (1989) Exposure to sunlight and other risk factors for age-related macular degeneration Arch Ophthalmol 107,875-879[Abstract/Free Full Text]
  67. Taylor, H, West, S, Munoz, B. (1992) The long term effects of visible light on the eye Arch Ophthalmol 110,99-104[Abstract/Free Full Text]



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N. E. Fishkin, J. R. Sparrow, R. Allikmets, and K. Nakanishi
Isolation and characterization of a retinal pigment epithelial cell fluorophore: An all-trans-retinal dimer conjugate
PNAS, May 17, 2005; 102(20): 7091 - 7096.
[Abstract] [Full Text] [PDF]

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 Arch OphthalmolHome page
R. E. Braunstein and J. R. Sparrow
A Blue-Blocking Intraocular Lens Should Be Used in Cataract Surgery
Arch Ophthalmol, April 1, 2005; 123(4): 547 - 549.
[Full Text] [PDF]

Home page
 Arch OphthalmolHome page
M. A. Mainster
Intraocular Lenses Should Block UV Radiation and Violet but Not Blue Light
Arch Ophthalmol, April 1, 2005; 123(4): 550 - 555.
[Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Beharry, M. Zhong, and R. S. Molday
N-Retinylidene-phosphatidylethanolamine Is the Preferred Retinoid Substrate for the Photoreceptor-specific ABC Transporter ABCA4 (ABCR)
J. Biol. Chem., December 24, 2004; 279(52): 53972 - 53979.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. R. Kim, N. Fishkin, J. Kong, K. Nakanishi, R. Allikmets, and J. R. Sparrow
Rpe65 Leu450Met variant is associated with reduced levels of the retinal pigment epithelium lipofuscin fluorophores A2E and iso-A2E
PNAS, August 10, 2004; 101(32): 11668 - 11672.
[Abstract] [Full Text] [PDF]

Home page
 J Biol RhythmsHome page
D. C. Klein
The 2004 Aschoff/Pittendrigh Lecture: Theory of the Origin of the Pineal Gland-- A Tale of Conflict and Resolution
J Biol Rhythms, August 1, 2004; 19(4): 264 - 279.
[Abstract] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
R. A. Radu, N. L. Mata, A. Bagla, and G. H. Travis
Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt's macular degeneration
PNAS, April 20, 2004; 101(16): 5928 - 5933.
[Abstract] [Full Text] [PDF]

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 IOVSHome page
M. Rozanowska, A. Pawlak, B. Rozanowski, C. Skumatz, M. Zareba, M. E. Boulton, J. M. Burke, T. Sarna, and J. D. Simon
Age-Related Changes in the Photoreactivity of Retinal Lipofuscin Granules: Role of Chloroform-Insoluble Components
Invest. Ophthalmol. Vis. Sci., April 1, 2004; 45(4): 1052 - 1060.
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 Hum Mol GenetHome page
A. V. Cideciyan, T. S. Aleman, M. Swider, S. B. Schwartz, J. D. Steinberg, A. J. Brucker, A. M. Maguire, J. Bennett, E. M. Stone, and S. G. Jacobson
Mutations in ABCA4 result in accumulation of lipofuscin before slowing of the retinoid cycle: a reappraisal of the human disease sequence
Hum. Mol. Genet., March 1, 2004; 13(5): 525 - 534.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Ophthalmol.Home page
M A Mainster and J R Sparrow
How much blue light should an IOL transmit?
Br. J. Ophthalmol., December 1, 2003; 87(12): 1523 - 1529.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
M Michaelides, D M Hunt, and A T Moore
The genetics of inherited macular dystrophies
J. Med. Genet., September 1, 2003; 40(9): 641 - 650.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. R. Sparrow, H. R. Vollmer-Snarr, J. Zhou, Y. P. Jang, S. Jockusch, Y. Itagaki, and K. Nakanishi
A2E-epoxides Damage DNA in Retinal Pigment Epithelial Cells. VITAMIN E AND OTHER ANTIOXIDANTS INHIBIT A2E-EPOXIDE FORMATION
J. Biol. Chem., May 9, 2003; 278(20): 18207 - 18213.
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 IOVSHome page
J. R. Sparrow, J. Zhou, and B. Cai
DNA Is a Target of the Photodynamic Effects Elicited in A2E-Laden RPE by Blue-Light Illumination
Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2245 - 2251.
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 Proc. Natl. Acad. Sci. USAHome page
J. R. Sparrow
Therapy for macular degeneration: Insights from acne
PNAS, April 15, 2003; 100(8): 4353 - 4354.
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 Proc. Natl. Acad. Sci. USAHome page
N. M. Haralampus-Grynaviski, L. E. Lamb, C. M. R. Clancy, C. Skumatz, J. M. Burke, T. Sarna, and J. D. Simon
Spectroscopic and morphological studies of human retinal lipofuscin granules
PNAS, March 18, 2003; 100(6): 3179 - 3184.
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 International Journal of ToxicologyHome page
P. Dayhaw-Barker
Retinal Pigment Epithelium Melanin and Ocular Toxicity
International Journal of Toxicology, November 1, 2002; 21(6): 451 - 454.
[Abstract] [PDF]

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 IOVSHome page
L. R. Thomson, Y. Toyoda, A. Langner, F. C. Delori, K. M. Garnett, N. Craft, C. R. Nichols, K. M. Cheng, and C. K. Dorey
Elevated Retinal Zeaxanthin and Prevention of Light-Induced Photoreceptor Cell Death in Quail
Invest. Ophthalmol. Vis. Sci., November 1, 2002; 43(11): 3538 - 3549.
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 IOVSHome page
C. S. Alge, S. G. Priglinger, A. S. Neubauer, A. Kampik, M. Zillig, H. Bloemendal, and U. Welge-Lussen
Retinal Pigment Epithelium Is Protected Against Apoptosis by {alpha}B-Crystallin
Invest. Ophthalmol. Vis. Sci., November 1, 2002; 43(11): 3575 - 3582.
[Abstract] [Full Text] [PDF]

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 JGPHome page
S. De and T. P. Sakmar
Interaction of A2E with Model Membranes. Implications to the Pathogenesis of Age-related Macular Degeneration
J. Gen. Physiol., July 30, 2002; 120(2): 147 - 157.
[Abstract] [Full Text] [PDF]

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 IOVSHome page
J. R. Sparrow, J. Zhou, S. Ben-Shabat, H. Vollmer, Y. Itagaki, and K. Nakanishi
Involvement of Oxidative Mechanisms in Blue-Light-Induced Damage to A2E-Laden RPE
Invest. Ophthalmol. Vis. Sci., April 1, 2002; 43(4): 1222 - 1227.
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 Mol. Cell. ProteomicsHome page
M. Miyagi, H. Sakaguchi, R. M. Darrow, L. Yan, K. A. West, K. S. Aulak, D. J. Stuehr, J. G. Hollyfield, D. T. Organisciak, and J. W. Crabb
Evidence That Light Modulates Protein Nitration in Rat Retina
Mol. Cell. Proteomics, April 1, 2002; 1(4): 293 - 303.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Ben-Shabat, C. A. Parish, H. R. Vollmer, Y. Itagaki, N. Fishkin, K. Nakanishi, and J. R. Sparrow
Biosynthetic Studies of A2E, a Major Fluorophore of Retinal Pigment Epithelial Lipofuscin
J. Biol. Chem., February 22, 2002; 277(9): 7183 - 7190.
[Abstract] [Full Text] [PDF]

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 Hum Mol GenetHome page
E. M. Stone, V. C. Sheffield, and G. S. Hageman
Molecular genetics of age-related macular degeneration
Hum. Mol. Genet., October 1, 2001; 10(20): 2285 - 2292.
[Abstract] [Full Text] [PDF]

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 IOVSHome page
G. Hoppe, A. D. Marmorstein, E. A. Pennock, and H. F. Hoff
Oxidized Low Density Lipoprotein-Induced Inhibition of Processing of Photoreceptor Outer Segments by RPE
Invest. Ophthalmol. Vis. Sci., October 1, 2001; 42(11): 2714 - 2720.
[Abstract] [Full Text] [PDF]

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 IOVSHome page
F. C. Delori, D. G. Goger, and C. K. Dorey
Age-Related Accumulation and Spatial Distribution of Lipofuscin in RPE of Normal Subjects
Invest. Ophthalmol. Vis. Sci., July 1, 2001; 42(8): 1855 - 1866.
[Abstract] [Full Text] [PDF]

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 Arch OphthalmolHome page
M. F. Marmor
Double Fault!: Ocular Hazards of a Tennis Sunglass
Arch Ophthalmol, July 1, 2001; 119(7): 1064 - 1066.
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 IOVSHome page
J. R. Sparrow and B. Cai
Blue Light-Induced Apoptosis of A2E-Containing RPE: Involvement of Caspase-3 and Protection by Bcl-2
Invest. Ophthalmol. Vis. Sci., May 1, 2001; 42(6): 1356 - 1362.
[Abstract] [Full Text]

Home page
 IOVSHome page
S. Beatty, I. J. Murray, D. B. Henson, D. Carden, H.-H. Koh, and M. E. Boulton
Macular Pigment and Risk for Age-Related Macular Degeneration in Subjects from a Northern European Population
Invest. Ophthalmol. Vis. Sci., February 1, 2001; 42(2): 439 - 446.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
M. Suter, C. Reme, C. Grimm, A. Wenzel, M. Jaattela, P. Esser, N. Kociok, M. Leist, and C. Richter
Age-related Macular Degeneration. THE LIPOFUSCIN COMPONENT N-RETINYL-N-RETINYLIDENE ETHANOLAMINE DETACHES PROAPOPTOTIC PROTEINS FROM MITOCHONDRIA AND INDUCES APOPTOSIS IN MAMMALIAN RETINAL PIGMENT EPITHELIAL CELLS
J. Biol. Chem., December 8, 2000; 275(50): 39625 - 39630.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Sun and J. Nathans
ABCR, the ATP-binding Cassette Transporter Responsible for Stargardt Macular Dystrophy, Is an Efficient Target of All-trans-retinal-mediated Photooxidative Damage in Vitro. IMPLICATIONS FOR RETINAL DISEASE
J. Biol. Chem., April 6, 2001; 276(15): 11766 - 11774.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Liu, Y. Itagaki, S. Ben-Shabat, K. Nakanishi, and J. R. Sparrow
The Biosynthesis of A2E, a Fluorophore of Aging Retina, Involves the Formation of the Precursor, A2-PE, in the Photoreceptor Outer Segment Membrane
J. Biol. Chem., September 15, 2000; 275(38): 29354 - 29360.
[Abstract] [Full Text] [PDF]

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