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(Investigative Ophthalmology and Visual Science. 2005;46:2587-2591.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0920
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Laser Induces Apoptosis and Ceramide Production in Human Retinal Pigment Epithelial Cells

Adiel Barak,1,2 Tzipora Goldkorn,3 and Lawrence S. Morse1

1From the Department of Ophthalmology and the 3Signal Transduction Laboratory, Department of Medicine, School of Medicine, University of California, Davis, California; and the 2Department of Ophthalmology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the cellular mechanisms involved in the cell death of human retinal pigment epithelial (hRPE) cells after their exposure to laser injury.

METHODS. Cultured human hRPE cells were irradiated for different lengths of time and at different levels of energy using diode laser photocoagulation coupled with an intraocular laser probe. Apoptosis was determined by TUNEL staining and annexin-V labeling of phosphatidylserine exposure. Ceramide levels were quantified by the diacylglycerol kinase assay using thin-layer chromatography.

RESULTS. Laser irradiation caused areas of apoptosis in the hRPE cells. These areas were detected around the ablated and necrotic laser scar and developed several hours after the laser irradiation. Laser irradiation concomitantly induced an increase in the intracellular production of ceramide, a lipid second messenger.

CONCLUSIONS. The results demonstrate that laser irradiation induces apoptosis in hRPE cells and suggest that the underlying signaling mechanism involves ceramide generation.

The widespread use of lasers in medical, industrial, and military fields has resulted in an increasing number of patients who have severe vision loss as a result of occupational eye injuries.1 2 3 Retinal destruction may occur in such injuries, resulting in extensive photoreceptor loss and severe visual impairment if the macula is involved.4 5 Laser photocoagulation therapy is also routinely used as treatment for many retinal disorders and may cause a sequela of either immediate or progressive vision loss.3 6 7 The biological tissue effect of laser-induced visual deterioration is due to nonselective tissue necrosis in the normal retina caused by the laser beam and by the thermal spread of the destructive effect to adjacent normal tissue (secondary degeneration).7 8 9 It has been reported that laser scars increase in size by up to 50% over a 24-month period.7 It is now well documented that much of the postinjury tissue damage results from delayed autodestructive mechanisms involving the formation of reactive oxygen intermediates, release of proinflammatory substances and the accumulation of intracellular calcium ion. All these mechanisms presumably lead to one final common pathway of cell death via the apoptotic cascade.

Apoptosis is a process of programmed cell death and an essential mechanism for the maintenance of homeostasis in multicellular organisms. The signal transduction cascades that activate apoptosis are present in all cells and are maintained in an inactive state under normal conditions. The apoptotic program can be activated by a wide variety of exogenous stimuli.10 11 Potential exogenous triggers of apoptosis range from growth factor withdrawal to ligand- or antibody-mediated engagement of specific cell-surface receptors capable of transducing lethal signals.12 13

Most of the signaling pathways that trigger apoptosis remain unknown. Increasing evidence demonstrating that sphingolipids may participate in these pathologic events has emerged over the past few years. The term "sphingomyelin pathway" has been coined to describe this pathway of cell signal transduction. This pathway is a signaling system that links specific cell-surface receptors and environmental stresses to the nucleus.14 It is initiated by hydrolysis of the sphingolipid sphingomyelin which is preferentially concentrated in the plasma membrane of mammalian cells.15 Sphingomyelin hydrolysis occurs within seconds to minutes after stimulation via the action of sphingomyelin-specific sphingomyelinases, to generate ceramide. Ceramide then serves as a second messenger in this system, leading to apoptotic DNA fragmentation. Agonists of the ceramide pathway include cytokines, such as tumor necrosis factor (TNF)-{alpha},16 17 interleukin-1,18 {gamma}-interferon,19 and antibodies directed against functional molecules, such as Fas/APO-1,20 21 22 or CD28 proteins,23 as well as stress-inducing agents, such as UV,24 ionizing radiation,25 26 and antileukemic agents.27 28

We have shown in a prior study that the apoptotic cascade is activated in the retinal pigment epithelium (RPE) after oxidative stress and that the level of ceramide is elevated in those cells during the process.29 In the present study, we evaluated whether apoptotic mechanisms are activated after laser injury to the RPE and also sought to determine whether the ceramide pathway is involved in the apoptotic process.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
All materials in this report were obtained from Sigma-Aldrich (St. Louis, MO), unless stated otherwise. The immortal human RPE (hRPE) cell line ARPE-19 was used during all experiments, and cells were cultured as described elsewhere.30 Briefly, the cells were cultured in growth medium DMEM/F12 (Invitrogen-Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum. They were plated at a density of 5 x 104 cells/cm2 and incubated at 37°C with 10% CO2 atmosphere. The medium was changed every 3 days, and a final cell density of 0.7 to 1 x 105 cells/cm2 was obtained within 5 days of incubation. Subcultures were performed as follows: near-confluent cultures were treated with trypsin (0.05%)-EDTA (1 mM) in phosphate-buffered saline (PBS; pH 7.0). After the cells were detached from the plates, an equal volume of DMEM/F12 supplemented with 10% fetal bovine serum was added to stop the trypsinization. The cells were recovered by centrifugation and resuspended in culture medium for plating. Cell counts were determined with an improved Neubauer counter (American Optical Corp., Buffalo, NY). Cell viability was assessed by trypan blue exclusion.

Laser Ablation
A diode laser photocoagulation system (Oculite; Iridex, Mountain View, CA), coupled with an intraocular laser probe (which provided the endoprobe), was used in all cellular treatments. The endoprobe was made stationary by means of a titration stand, and increasing doses of laser irradiation were delivered to the ARPE-19 cells on the culture plates. A fixed distance of 2.0 mm was maintained between the endoprobe and the hRPE cells and was not changed throughout all the experiments. Laser photocoagulation was conducted on the ARPE-19 cells, which had been washed and briefly maintained in 1 mM PBS buffer. For each treatment, a six-well plate, each well containing 800,000 to 1000,000 cells, was irradiated with 20 laser burns that were carefully directed toward the treated cells. The endoprobe uses a 200-µm diameter fiber that, together with the infrared 810-nm laser beam, has a numerical aperture of 0.125. The spot diameter is 0.625 mm at a distance of 2 mm. The total area of cells directly damaged by the laser was 40 mm2, assuming 20 burns of laser irradiation. The laser irradiance was dependent on the actual laser spot size on the retina or RPE: irradiance (watts per square centimeter) = power (in watts)/spot area (in square centimeters). We used various power densities (from 0.5 to 2 W), and an endoprobe that uses a fiber 200 µm in diameter. Thus, the laser irradiance was 1591 W/cm2 for 1 W of laser power. The energy and duration of each set of treatments were predetermined. All experiments were performed in triplicate.

Apoptosis Assays
A terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay staining kit was used for the detection of cell death, and an annexin V staining kit (Annexin-V-Fluos; Roche Diagnostics, Indianapolis, IN) was used for the assay of phosphatidylserine (PS) exposure.

TUNEL Labeling
TUNEL staining was performed according to the manufacturer’s instructions. Briefly, after laser treatments, the attached cells were trypsinized as previously described in the cell culture subsection (above) and combined with the cells floating in the medium. The cells were washed twice in PBS, resuspended in 10% freshly prepared formaldehyde, and fixed to glass slides.31 The morphologic changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with the DNA-binding fluorochrome bis-benzamide, as previously described.32 Five hundred cells per slide were scored for the incidence of apoptotic chromatin changes. The slides were viewed under a fluorescence microscope (model SA; Nikon, Tokyo, Japan), and an imaging system (Scion, Frederick, MD) captured the view fields. Cells with positive brown staining were considered apoptotic.

Annexin V Binding to PS
The presence of apoptotic cells was evaluated by an early change in membrane phospholipid asymmetry of the cells during the early phases of apoptosis. The loss of cell membrane phospholipid asymmetry is accompanied by the exposure of PS to the outer membrane, as described elsewhere.33 Briefly, 106 cells attached to the culture dishes were washed with ice-cold PBS and incubated for 15 minutes at room temperature in the dark in a solution containing fluorescein-conjugated annexin V and propidium iodide (PI; 5 µg/mL) for fluorescence microscopy analysis. In this assay, cells negative for both PI and annexin V staining are live cells, PI negative- and annexin V-positive staining cells are early apoptotic cells, and PI positive- and annexin V-positive staining cells are primarily cells in the late stages of apoptosis. The red (PI) and green (fluorescein) fluorescent cells were counted.

Lipid Studies
Ceramide was quantified by the diacylglycerol kinase (DGK) assay, as described previously.34 35 In brief, after laser treatments, the cells were pelleted by centrifugation (300 g for 10 minutes), washed twice with ice-cold PBS, and extracted with 0.6 mL of chloroform-methanol-l N HCl (100:100:1, vol/vol/vol). Lipids in the organic-phase extract were dried under N2 and subjected to mild alkaline hydrolysis (0.1 N methanolic KOH for 1 hour at 37°C) to remove the glycerophospholipids. Samples were re-extracted, and the organic phase was dried under N2. The ceramide contained in each sample was resuspended in a 100-µL reaction mixture containing 150 µg of cardiolipin (Matreya, Pleasant Gap, PA), 280 µM diethylenetriaminepenta-acetic acid (DTPA), 51 mM octyl-15-D-glucopyranoside (Calbiochem-Novabiochem Corp., San Diego, CA.), 50 mM NaC1, 51 mM imidazole, 1 µM EDTA, 12.5 mM MgC12, 2 µM dithiothreitol, 0.7% glycerol, 70 µM [{gamma}-mercaptoethanol, 1 mM adenosine triphosphate (ATP), 10 µCi of [µ-P32]ATP (3000 Ci/mmol; Dupont New England Nuclear, Boston, MA), and 35 µg/mL Escherichia coli DGK (Calbiochem-Novabiochem Corp.) at pH 6.5. After 60 minutes at room temperature, the reaction was stopped by extraction of lipids with 1 mL of chloroform-methanol-l N HC1 (100:100:1), 170 µL of buffered saline solution (BSS; 135 mM NaC1, 1.5 mM CaC12, 0.5 mM MgC12, 5.6 mM glucose, and 10 mM HEPES [pH 7.2]) and 30 µL of 100 mM EDTA. The lower organic phase was dried under N2. Ceramide 1-phosphate was resolved by thin-layer chromatography on silica gel (60 plates; MCB Manufacturing Chemicals, Cincinnati, OH), with a solvent system of chloroform-methanol-acetic acid (65:15:5), and detected by autoradiography, and incorporated 32P was quantified by counting the liquid scintillation. The level of ceramide was determined by comparison with a concomitantly generated standard curve of known amounts of ceramide (ceramide type III; Sigma-Aldrich), and 200 picomoles ceramide was used as standard. The levels of ceramide recorded by autoradiography were normalized to account for the loss of cells due to laser ablation. This was accomplished by describing the results as pixels per milligram proteins.

Statistical Analysis
All experiments were performed in triplicate. The results were expressed as the mean ± SE. Statistical analyses were performed with Student’s t-test. Significance was defined as a two-tailed P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Diode Laser-Irradiation–Induced Apoptosis in hRPE Cells
hRPE cells were treated with the diode laser at increasing dosages and analyzed over time for phosphatidylserine exposure, as indicated by annexin V labeling, using fluorescence microscopy. Figure 1 demonstrates that positive annexin V staining was detected around the borders of laser ablation in the surrounding hRPE cells 4 hours after treatment. Temporal analysis showed an increase in the population of apoptotic cells and of dead cells, with maximum apoptosis occurring 8 hours after laser exposure, followed by a gradual increase in the amount of PI-positive cells (i.e., dead cells).



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  		FIGURE 1. Effect of laser irradiation on apoptosis induction in human retinal pigment epithelial (hRPE) cells, as seen by fluorescence microscopic analysis of fluorescein-conjugated annexin V and propidium iodine (PI). Areas of annexin V- and PI-positive cells surrounded the central ablated area 4 hours after laser irradiation of hRPE cells with a 1-W diode laser for a duration of 1 second. Untreated cells showed less than 1% annexin V staining. The image is a merger of separate images of annexin V and propidium iodine fluorescence.

 
Apoptosis was also detected by TUNEL assay. Cells treated with the diode laser at increasing dosages showed an intense brown staining of the nuclei, indicating the incorporation of dUTP onto nicked DNA ends. Again, the time course of the nuclear fragmentation demonstrated an increase in the number of apoptotic cells over time, with maximum apoptotic changes detected by TUNEL staining 16 hours after treatment. At 4 hours after laser irradiation, 3.4 of 500 examined cells showed intense brown staining of the nuclei, the same as untreated (control) cells. At 8 hours after treatment, an average of 9 of 500 treated cells that were examined per slide showed apoptotic changes, compared with 3.4 of 500 untreated cells. At 16 hours after laser irradiation, 14 of 500 treated cells showed apoptotic markers compared with 3.4 of 500 untreated cells. At 24 hours, the treated plate showed 5 of 500 apoptotic cells compared with 4 of 500 untreated cells. On direct examination of the plates, the laser ablation zone was now filled with hRPE cells, presumably growing and migrating from the margins of the laser ablation zone (Fig. 2) .



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  		FIGURE 2. Temporal effect of laser irradiation on apoptosis induction in human retinal pigment epithelial (hRPE) cells. TUNEL assay of hRPE cells irradiated with a 1-W, 1-second diode laser application, performed immediately (A), 16 hours (B) and 24 hours (C) after irradiation. No TUNEL-positive cells are detectable immediately after irradiation. Maximum apoptotic changes are detected by TUNEL staining 16 hours after treatment (B). No apoptotic cells were found, and the laser ablation zone was filled with hRPE cells at 24 hours after treatment.

 
Laser-Ablation–Induced Increases in Intracellular Ceramide Levels
Treatment of hRPE cells with laser irradiation in increasing dosages produced an increase in the ceramide levels, which were observed several hours after treatment. The increase in ceramide was proportional to the amount of laser energy applied, with a maximum threefold increase with 2-W pulses. Prolonged and persistent accumulation of ceramide occurred several hours after laser irradiation; thus, no increase in ceramide concentration was detected immediately after irradiation. A nonsignificant increase (less than double) in ceramide concentration was noted 4 hours after irradiation. Peak elevation of ceramide concentration was detected 8 hours after irradiation, with a maximum of a threefold increase with a 2-W pulse (data not shown). The ceramide levels decreased at 16 hours after irradiation, to double the ceramide concentration and returned to normal values at 24 hours (Fig. 3) .



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  		FIGURE 3. Top: effects of laser irradiation on ceramide levels in human retinal pigment epithelial (hRPE) cells. The cells were irradiated with diode laser at increasing dosages (0–2000 mW) for a duration of 1 second. After laser irradiation, the cells were incubated for 8 hours, after which the incubation was terminated and ceramide quantification was performed. Bottom: representative thin-layer chromatographic results of the ceramide band. The data are expressed as the change (x-fold) relative to time-matched untreated controls. The thin-layer chromatographic results were normalized using pixels/mg protein to overcome the cell destruction during laser ablation. A maximum of a two-fold increase of the original ceramide levels was detected when using 1000 mW of laser energy, 8 hours after laser ablation. The ceramide levels returned to normal at 24 hours after laser ablation. The data are shown as the mean ± SE. The mean resting untreated control level of ceramide was 200 picomoles.

 
The DGK assay failed to yield any conclusive results.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated the induction of apoptosis in hRPE cells after diode laser irradiation. The induction of apoptosis was accompanied by an increase in ceramide levels, presumably a second messenger that participates in the apoptotic cascade. The importance of the current findings lies in our ability to understand the mechanism of tissue degeneration which may lead to specific treatment algorithms. Apoptosis has both a commitment phase, which may be reversible, and an execution phase, at which time reversal is not possible.36 By elucidating and defining the specific mechanisms underlying the secondary degeneration after laser injury, it may be possible to identify specific compounds that will target key molecules in the process and possibly reverse it. Identifying the mechanisms of laser eye injury is becoming increasingly important due to the expanding use of lasers in industry, medicine, and the military. Indeed, laser weapons are being developed to damage battlefield electro-optical sensors and visually incapacitate soldiers.37 It is known that U.S. forces have developed several laser weapons, named "Cobra" and "Dazer,"38 and there are reports of other countries developing laser weapons.37 During the Persian Gulf War, the allied forces used laser eye protection, and at least one American soldier was injured by a probable Iraqi laser source.39 The battlefield eye injuries are especially devastating due to the young age of the victims and the tendency of the injury to be bilateral.40 By understanding the mechanisms involved in laser injuries, we may be able to minimize the damage caused to the retina and save the vision of these victims.

Current medical treatment for laser injury of the retina consists of high-dose corticosteroids to reduce cellular inflammation in a yet-undetermined mechanism. Morphologically, treated retinas showed rapid reestablishment of retinal and choroidal vasculature, proliferation, and organization of the RPE, less macrophage activity, and reduction of photoreceptor damage.41 42 Other studies have questioned the efficiency of corticosteroids in laser eye injuries. Systemic corticosteroids had an only short-term effect on argon laser-induced lesions in the retinas of pigmented rats.43 The glutamate receptor blocker MK-801was another compound used to limit the retinal lesion caused by laser injury,44 but it was found to be too toxic for human use.

In the present study, we have begun to unravel the identity of the underlying mechanism of cell death in the secondary degeneration after laser-induced apoptosis. By doing so, we have come closer to the time when apoptosis-preventing therapeutic agents can be introduced to alter the progression of cell death. Modulation of the expression of key molecular components of the cell death mechanism (e.g., the survival gene product Bcl-2) is an attractive strategy. An alternative strategy is to interfere with the signal transduction pathways activated during the apoptosis cascade, such as the ceramide pathway. We found a prolonged and persistent (a few hours) elevation in ceramide levels in hRPE cells after laser injury. In a previous study, we showed that synthetic ceramide induces apoptosis and that reactive oxygen intermediates production in hRPE cells.29 Our current findings are consistent with previous reports that documented a complex and variable response of ceramide formation after stress induction.14 Our finding that the ceramide elevation occurred several hours after the laser irradiation is somewhat puzzling, and may argue for a direct formation of ceramide that was not caused by the laser irradiation. In many other examined cells, however, the most pronounced changes in the amount of ceramide occurred hours after the stress induction.18 45 The kinetics of ceramide formation in response to these inducers are complex and variable. Reported responses range from seconds to hours, and the same inducer has generated very different ceramide responses in different studies. This range of findings has become a major source of confusion as to the possible relevant roles of ceramide in signal transduction and cell regulation. Acute changes in ceramide concentration within seconds or 1 to 2 minutes have been described, primarily with TNF-{alpha}, ionizing radiation, and engagement of the Fas receptor.25 46 47 These changes may be indicative of a role for ceramide in mediating some of the very early responses to TNF-{alpha}, such as activation of nuclear factor (NF)-{kappa}B.

Several extracellular stimuli and agents induce prolonged and persistent accumulation of ceramide that occurs over a period of several hours. Serum withdrawal in leukemia cells results in a 15-fold accumulation of ceramide over 24 to 48 hours, and TNF-{alpha} and Fas activation also induce a three- to eightfold increase in intracellular concentrations of ceramide over 12 to 24 hours.19 21 48 These findings have led to the concept of ceramide function as a "biostat." According to this concept, ceramide may function as a component of a "biostat" that measures and initiates responses to cellular stress, much like a thermostat that measures the temperature over a long period. The cell then responds to the changes in the ceramide levels by undergoing apoptosis or cell arrest, which occurs via multiple enzymatic pathways. Thus, ceramide levels act as a general measurement for the "stress level" of the cell. By manipulation of the ceramide levels, less apoptosis will occur, and so less tissue damage can be expected. We believe that the model we present herein is suitable for testing the effects of ceramide inhibitors (or various anti-apoptotic agents) on hRPE cells.

In the present study, we used the DGK assay to determine the ceramide levels. This assay is the most frequently used method for total endogenous ceramide mass measurements and is highly sensitive; thus, the result of a threefold increase in ceramide levels seems sufficient to conclude that the ceramide peak we measured is not a laboratory artifact.49 Nevertheless, a new method of ceramide determination, such as mass spectrometric analysis, may provide more confidence in the validity of the results.50

Our model has several drawbacks. During the laser ablation, we did not control the thermal conduction of the plastic in which the cells were grown. Thus, part of the effect may be due to thermal injury that may be different from in vivo effects due to differences in thermal relaxation properties of plastic and ocular tissues. A control experiment showing how much apoptosis is generated by the same thermal energy resulting from laser exposure will help solve this problem. Moreover, in the present study, we did not directly assess the amount of ceramide that was generated by the laser irradiation and that which was generated by the thermal injury. Measurements using controlled temperatures that will clarify the reason for ceramide generation are warranted. Another drawback is the lack of regulation of the direct effect of laser irradiation or oxidation on ceramide formation. It has been shown that the choline head group of sphingomyelin is cleaved with mild oxidation.51 Although the increase in ceramide levels takes effect immediately after irradiation, and the ceramide formation discovered in our study appeared several hours after laser irradiation, suggesting enzymatic generation, at least part of the ceramide formation may be attributed to a direct laser effect and not to enzymatic generation. In further studies, a slurry of pure sphingomyelin should be added to a plastic dish, and irradiation of the sphingomyelin should be performed to see whether ceramide generation is obtained.

Another weakness of our model is that we used cell cultures, and so it is difficult to estimate long-term changes and the possible mechanisms leading to laser scar expansion because of the rapid cellular division that occurs in cells surrounding the zone of cellular necrosis after laser treatment. Experiments that substitute cell cultures by organ cultures or live animals may help to confirm our observations.

In conclusion, in the present study, the apoptosis cascade was activated after laser irradiation of hRPE cells. The activation of the apoptosis cascade was accompanied by the production of ceramide, a key molecule in the sphingomyelin pathway. The identification of a signal transduction agent, ceramide, which is involved in the induction of apoptosis in hRPE cells may have clinical implications. Ceramide antagonists may be experimentally used to reduce secondary degeneration that accompanies laser-induced injury, thus reducing the amount of hRPE cell death associated with such injury.


   Footnotes
 
Supported by the Mills and Margaret Cox Foundation, Macula Society Research Grant (LSM), an unrestricted grant from Iridex, Ltd. (LSM), and a grant from Research to Prevent Blindness to the Department of Ophthalmology, University of California.

Submitted for publication August 1, 2004; revised January 2, 2005; accepted January 21, 2005.

Disclosure: A. Barak, None; T. Goldkorn, None; L.S. Morse, Iridex, Ltd. (F)

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Adiel Barak, Department of Ophthalmology, Tel Aviv Sourasky Medical Center, 6 Waitzmann Street, Tel Aviv 64239, Israel; omadb{at}post.tau.ac.il.


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 Materials and Methods
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 Discussion
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