Ultrasound in Med. & Biol., Vol. 41, No. 5, pp. 1363–1371, 2015 Copyright Ó 2015 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter

http://dx.doi.org/10.1016/j.ultrasmedbio.2014.12.665

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Original Contribution EFFECTS OF LOW-INTENSITY ULTRASOUND ON OXIDATIVE DAMAGE IN RETINAL PIGMENT EPITHELIAL CELLS IN VITRO NA KYEONG KIM,* CHAN YUN KIM,y MIN JOO CHOI,z SO RA PARK,* and BYUNG HYUNE CHOIx * Department of Physiology, Inha University College of Medicine, Incheon, Republic of Korea; y Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Republic of Korea; z Department of Medicine, College of Medicine, Cheju National University, Cheju, Republic of Korea; and x Department of Biomedical Sciences, Inha University College of Medicine, Incheon, Republic of Korea (Received 12 February 2014; revised 19 December 2014; in final form 22 December 2014)

Abstract—Oxidative stress in retinal pigment epithelium (RPE) is one of the key causative factors of RPE injury in age-related macular degeneration (AMD). Low-intensity ultrasound (LIUS) less than 1 W/cm2 in intensity has been found to have cytoprotective and anti-inflammatory effects in many cell types and diseases. In this study, we investigated for the first time the feasibility of using LIUS to protect RPE cells from oxidative damage. ARPE-19 cells were treated with H2O2 (an exogenous source of reactive oxygen species) or L-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthase inhibitor, and exposed immediately to LIUS at intensities of 50, 100 and 200 mW/cm2 and a frequency of 1 MHz for 20 min. Both H2O2 and BSO increased the percentage of cells positive for mitochondrial reactive oxygen species at 1 h, but not at 24 h. Co-treatment with LIUS clearly repressed these cells similarly at all intensities by approximately 34%–43% for H2O2 and 24%–25% for BSO (p , 0.05). The percentage of cells with mitochondrial membrane depolarization also increased with H2O2 and BSO treatment, particularly at 1 h, and decreased by approximately 60% with LIUS at 100 mW/cm2 (p , 0.05). The amount of intracellular calcium ion ([Ca21]i) was elevated only by BSO at 24 h and was also significantly diminished, by approximately 45%, by LIUS at 100 mW/ cm2 (p , 0.05). Both H2O2 and BSO significantly hampered cell viability at 24 h, but LIUS at 100 mW/cm2 restored only BSO-induced cell viability by approximately 2.7-fold (p , 0.05). This study illustrated that LIUS has a protective effect on RPE cells against oxidative damage caused by BSO, an endogenous mitochondrial reactive oxygen species generator. We speculate that LIUS has the potential to treat oxidative damage and related pathologic changes in RPE. (E-mail: [email protected] or [email protected]) Ó 2015 World Federation for Ultrasound in Medicine & Biology. Key Words: Retinal pigment epithelium, Low-intensity ultrasound, Oxidative stress, Mitochondria, Cytoprotection.

changes in RPE functions (Kinnunen et al. 2012). RPE is composed of a monolayer of tightly connected pigmented cells and is important in normal vision function. Structural deformation and/or inflammation in RPE commonly accompany many cases of severely aggravated macular degeneration. Chronic oxidative stress is thought to be a main cause of RPE injury and to be a promising therapeutic target in the treatment of AMD (Beatty et al. 2000; Cai et al. 2000; Jarrett and Boulton 2012). However, current treatments of AMD, including laser treatment, laser photocoagulation, photodynamic therapy and vascular endothelial growth factor (VEGF) inhibitors, all aim at the reduction of macular edema and tension (Coleman et al. 2008). The oxidative damage of RPE results from an imbalance between the generation and elimination of reactive oxygen species (ROS). Mitochondria play a key role in

INTRODUCTION Age-related macular degeneration (AMD) is a degenerative disease that causes central vision loss in the elderly (Friedman et al. 2004). Common risk factors for AMD include increasing age, cigarette smoking, obesity and some genetic polymorphism (Coleman et al. 2008). The pathophysiology of AMD is characterized by degeneration of retinal photoreceptors, retinal pigment epithelium (RPE) and Bruch’s membrane or proliferation of choroidal neovascularization, both of which include

Address correspondence to: Byung Hyune Choi, Department of Biomedical Sciences & So Ra Park, Department of Physiology, Inha University College of Medicine, B/3 Jeongsuk Building, Sinheungdong 3 ga, Jung-gu, Incheon, Republic of Korea. E-mail: srpark@ inha.ac.kr or [email protected] 1363

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the cellular response to oxidative stress and intracellular ROS generation (Park et al. 2011). RPE cells also produce intracellular ROS from the electron transport chain of mitochondrial NAD(P)H oxidases and lipid peroxidation from phagocytosed rod outer segments. Normally, mitochondrial ROS (mtROS) are eliminated immediately by antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase (Beatty et al. 2000; Jarrett and Boulton 2012). However, failure in the function of these antioxidant enzymes during aging causes accumulation of mtROS in RPE cells and may induce RPE injury and AMD. Mitochondria play a key role in the homeostasis of cytosolic Ca21 levels, and mitochondrial dysfunction results in Ca21 overload and eventually cell death (Rizzuto et al. 2012). In addition, the oxidative stress caused by t-butyl hydroperoxide includes Ca21 release (Zavodnik et al. 2013), and hydrogen peroxide (H2O2) activates directly a Ca21permeable melastatin-like transient receptor potential 2 (TRPM2) channel on the mitochondrial membrane (Naziroglu 2007). Intracellular Ca21 overload can also cause mitochondrial dysfunction and mtROS generation, thereby establishing mutual cross-talk between mitochondrial Ca21 and ROS (Feissner et al. 2009). Mechanical stimuli including ultrasound are associated with oxidative stress in cells in various ways depending on the physical properties of the stimuli and the surrounding environment. Shear stress, for example, induces antioxidant activity in endothelial cells (Chen et al. 2003), and cyclic mechanical strain increases production of ROS in pulmonary epithelial cells (Chapman et al. 2005). Ultrasound has cytotoxic effects on cells at high intensities generating thermal energy and/or large amounts of free radicals (Feril and Kondo 2004). Increases in the amounts of mitochondrial ROS and intracellular Ca21 appear to be involved in the ultrasound-induced apoptosis of U937 lymphoma cells (Honda et al. 2004). In contrast, ultrasound, with various parameters, also has a therapeutic effect on ischemia–reperfusion injury by reducing oxidative stress and increasing nitric oxide (NO) in endothelial cells (Bertuglia 2007). The mechanism of action appears to involve the physical effect of ultrasound in producing shear force of blood flow. Low-intensity ultrasound (LIUS) is generally defined as an ultrasonic wave less than 1 W/cm2 in intensity. Therapeutic ultrasound, including LIUS, can generate heat energy and destructive forces and is commonly used in thrombolysis, drug delivery and other therapies. In particular, LIUS is expected to regulate directly diverse cellular functions and control disease progression without any destructive effects (Hensel et al. 2011; Min et al. 2007). LIUS has been proven therapeutically effective in many diseases such as bone fractures, muscle laceration and osteoarthritis

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(Chan et al. 2010; Loyola-Sanchez et al. 2012; Park et al. 2005; Pilla et al. 1990). The mechanism underlying the effect of LIUS is not clear, but possibly includes cytoprotective and anti-inflammatory actions as observed on chondrocytes and synoviocytes in vitro (Lee et al. 2007; Nakamura et al. 2011) and in animal models of osteoarthritis (Chung et al. 2012; Ito et al. 2012; Park et al. 2005). These results suggest the possibility that LIUS can also have a protective effect on other cell types and disease cases under different damaging conditions like AMD. In this study, we investigated the effect of LIUS on the mitochondrial activity and viability of RPE cells under oxidative stress in vitro. As mentioned above, ultrasound was previously found to produce ROS at high intensities and reduce oxidative damage in endothelial cells after ischemia/reperfusion injury via generation of shear stress in vivo. Therefore, this study is the first to address the therapeutic action of ultrasound, particularly at low intensities, in reducing oxidative stress at the cell level in vitro. METHODS Cell culture and induction of oxidative stress ARPE-19 cells were obtained from the American Type Culture Collection (CRL-2302, Manassas, VA, USA) and cultured with Dulbecco’s modified Eagle’s medium with F12 supplement (DMEM/F12) containing 10% fetal bovine serum (FBS), 10 mg/mL streptomycin and 100 IU/L penicillin at 37 C under 5% CO2. To induce oxidative stress, cells were plated at 0.3 3 105 cells/cm2 in 35-mm dishes 24 h before treatment with H2O2 (Junsei Chemical, Tokyo, Japan) or L-buthionine-(S,R)-sulfoximine (BSO, Sigma-Aldrich, St. Louis, MO, USA) at 1 mM unless otherwise indicated in the figures. H2O2 and BSO are commonly used to induce oxidative stress in RPE cells (Jin et al. 2005; Kaczara et al. 2010). H2O2 itself is a ROS and can damage cells directly at the cell membrane and cytoplasmic levels, which can occur very rapidly and independently of mtROS production and mitochondrial dysfunction (Fridovich 1995). In contrast, BSO is a glutathione (GHS) synthesis inhibitor and induces ROS generation in cells to exert its cytotoxic effect (Griffith and Meister 1979). Cells were assayed at 1 and 24 h. LIUS treatment A LIUS generator with six 30-mm-diameter plane disk transducers, resonating at a frequency of 1 MHz, was custom-made in cooperation with Korust (Anyang, Korea). The generator can produce ultrasound waves with an intensity range (spatial average temporal average) from 30 to 500 mW/cm2 (Fig. 1). Transducers were

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Fig. 1. Thirty-millimeter-diameter ultrasound transducers are housed in a water chamber equipped with a culture dish stand on top. The distance between the transducer and the bottom of the culture plate was approximately 50 mm when the 35-mm-diameter culture dish was placed.

housed in water chambers to easily disseminate heat energy and calibrated regularly using an ultrasound power meter (Ohmic Instruments, St. Easton, MD, USA). Culture dishes were placed in the near field in which the ultrasonic beam was confined within the effective radiating area of the transducer. In the present experiment, the bottom surface of culture dishes was located 5 cm from the surface of transducers (Fig. 1). Cells were treated with LIUS at 50, 100 or 200 mW/cm2 once for 20 min immediately after H2O2 or BSO treatment. Ultrasonic tone bursts were used at a duty factor of 50%. The upper limit of LIUS intensity was 200 mW/cm2, which was chosen on the basis of our pilot study on cell viability in human trabecular meshwork (HTM) cells and also confirmed to have no harmful effects on the viability of ARPE-19 cells (data not shown). The intensity of the six transducers could be adjusted separately. Each sample including the untreated control (0 mW/cm2) was randomly assigned to one of the six transducers. LIUS treatment was carried out at room temperature with the culture medium degassed. We found that the temperature in the culture medium increased by less than 1 C. This insignificant temperature rise is expected to have no effect on cell viability or other assays. It should be noted that the LIUS power actually delivered to cells is different from the power measured in water by the power meter, which results mainly from standing waves that are formed not only in the culture medium (Choi et al. 2012, 2013; Hensel et al. 2011), but also in the space between the transducer surface and the outer bottom of the culture dish. The pressure field in the culture medium is

complex, as illustrated by Choi et al. (2012, 2013). It is highly sensitive to the height of the medium and also relies on the inner radius of the dish. In addition to the standing wave effect, ultrasound loses energy during propagation through the cell culture dish, as discussed in Min et al. (2006). The field characterization and measurement in the culture medium are out of the scope of the present study, and further details can be found in Choi et al. (2012, 2013) and Hensel et al. (2011). Nevertheless, the ultrasonic field inside the present culture medium was predicted using a finite-element method so as to estimate the actual ultrasonic intensity exerted on the cells. Figure 2a illustrates the geometry for the simulation performed with PzFlux (Weidlinger Associates, New York, NY, USA). The culture dish has an inner diameter of 34 mm, height (L) of 9 mm and bottom plate and side wall thickness (b) of 1 mm. The culture medium was considered to have the same acoustic property as distilled water. The height of the culture medium (h) was calculated to be 3.23 mm from the 2-mL volume injected into the dish. The peak-to-peak pressure on the transducer surface (Pi) was estimated, under the continuous wave condition, to be 38.83, 54.77 and 77.46 kPa for transducer output intensity settings of 50, 100 and 200 mW/cm2, respectively. An example of a prediction for the intensity setting of 100 mW/cm2 is illustrated in Figure 2b. At the top left of Figure 2b is the steady-state distribution of temporal peak pressure, P(z, r), in front of the transducer predicted for the intensity output setting of 100 mW/cm2 500 ms from the onset of transducer operation. At this time, the standing wave field is fully developed, as seen in the

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Fig. 2. Simulation of the ultrasonic field to which cells were exposed. (a) Geometry and medium for the finite-element method simulation. The cell culture dish is made of polystyrene, and the acoustic property of the culture medium was regarded to be the same as that of distilled water. (b) Temporal history of the pressure predicted in the culture medium at the center of the inner bottom surface of the dish, p(t, r 5 0, z 5 0) (top right); steady-state distribution of the temporal peak pressure, P(z, r), in front of the transducer predicted for the intensity output setting of 100 mW/cm2 500 ms from the onset of transducer operation when the standing wave field is fully developed (top left); temporal peak pressure profiles along the radial direction (r) in the culture medium on the inner bottom of the cell culture dish, P(r, z 5 0) (bottom left); and temporal peak pressure profiles along the height (z) of the dish, P(r 5 0, z) (bottom right). Note that the pressure at the transducer surface (Pi) was 54.8 kPa, but the temporal peak pressure in the culture medium on the bottom surface of the cell culture dish, P(z 5 0, r), which is regarded as the actual pressure to which the cells were exposed, varied greatly from 7.7 kPa (r 5 616.9 mm) to 79.1 kPa (r 5 60.8 mm). The mean temporal peak pressure on the cells was predicted to be 42.4 kPa, represents a decrease to 77.5% of Pi. (c) Predicted (spatial and temporal peak) ultrasonic intensities to which the cells were exposed versus the intensity settings of 50, 100, and 200 mW/cm2 considered in the present study. The predicted intensity varied from 2.0% to 208.3% of the setting intensity, with a spatial average of 60.0% of the setting intensity. The solid line represents the case in which the setting intensity is the same as the predicted intensity.

temporal history of the pressure predicted in the culture medium at the center of the inner bottom surface of the dish, p(t, r 5 0, z 5 0) (Fig. 2b, top right). The predicted ultrasonic field is characterized by the near-field structure mixed with the standing waves. The largest pressure, 215.2 kPa (3.9 3 Pi) is measured at the location z 5 20.94 mm, r 5 0 (inside bottom plate of the dish), as illustrated in the axial temporal peak pressure profile P(z, r 5 0) of (Fig. 2b, (bottom right). There are four local temporal peak pressures appeared inside the culture medium, and the largest is 171.3 kPa (3.1 3 Pi) at the location z 5 0.56 mm, r 5 0. Because the cells are cultured on the bottom of the dish and are about several tens of microns in size, the temporal peak pressure in the culture medium on the bottom surface of the cell culture dish is regarded to be the actual pressure to which the cells are exposed. At the bottom left of Figure 2b is the temporal

peak pressure profile along the radial direction, P(r, z 5 0). It has the peak value of 79.1 kPa (144.3% of Pi) near the center (r 5 60.8 mm) and decreases gradually, fluctuating, as jrj increases, to the minimum value of 7.7 kPa (14.1% of Pi) near the wall of the dish (r 5 616.9 mm). The mean temporal peak pressure is predicted to be 42.4 kPa, representing a decrease to 77.5% of Pi. In Figure 2c, the predicted (spatial and temporal peaks) ultrasonic intensity to which the cells are exposed is plotted against the three intensity settings of 50, 100 and 200 mW/cm2 considered in the present study. The figure indicates that the cells are expected to be exposed to widely varying ultrasonic intensity in the range 2.0% to 208.3% of the setting intensity, and the spatial average intensity is reduced to 60.0% of the setting intensity. The ratio of ultrasonic power delivered to the cells to that produced by the transducer is expected to remain unchanged

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regardless of the setting intensity, because the ultrasound is of low intensity. Measurement of reactive oxygen species Mitochondrial superoxide levels of ARPE-19 cells were measured using MitoSOX Red (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The dye is permeant to live cells and rapidly and selectively targets the mitochondria to be oxidized by superoxide to produce red fluorescence. Cells were washed twice with wash buffer and then incubated with 1 mL of 5 mM dye reagent for 20 min at 37 C. After thorough washing, the proportion of cells with positive signals was determined by flow cytometry using a BD FACScalibur (BD Biosciences, San Jose, CA, USA) at 590 nm. JC-1 assay for mitochondrial membrane potential The mitochondrial membrane potential of ARPE-19 cells was measured using the probe 5,50 ,6,60 -tetrachloro1,10 ,3,30 -tetraethylbenzimidazolyl carbocyanine iodide (JC-1, Invitrogen). Cells were suspended in 1 mL of warm medium at 1 3 106 cells/mL, mixed with 10 mL of 200 mM JC-1 (2 mM final concentration) and incubated at 37 C under 5% CO2 for 15 to 30 min. The samples were analyzed by flow cytometry at 488 nm for green fluorescence and at 590 nm for red fluorescence. Cells with both red and green fluorescence (double positive) were regarded as having normal mitochondrial membrane potential, and a decrease in the double-positive population was regarded as loss of the potential. Data are expressed as the portion of double-positive cells calculated and normalized relative to that of the untreated control. Intracellular calcium ion measurement Intracellular Ca21 levels ([Ca21]i) in ARPE-19 cells were measured using the FluoForte Calcium Assay Kit (Enzo Life Science, Farmingdale, NY, USA) according to the manufacturer’s instructions. ARPE-19 cells in a 96-well plate were washed several times with culture medium and incubated with 100 mL FluoForte Dyeloading solution for 1 h at room temperature. The amount of intracellular Ca21 in cells was determined using a fluorometric plate reader (LS55 luminescence spectrometer, Perkins Elmer Instruments, Shelton, CT, USA) and expressed in relative fluorescence units (RFU). An emission wavelength of 490 nm and excitation wavelength of 525 nm were used for analysis. Wst-1 assay for cell viability ARPE-19 cells were plated at 1 3 104 cells per well in 96-well culture plates. After 24 h, cells were treated with oxidative stress (H2O2 or BSO) at increasing concentrations of 0, 0.1, 0.5, 1 and 2 mM. LIUS was applied

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at 100 mW/cm2 for 20 min immediately after the oxidative stress, as described above. Concentrations of viable cells were determined at 1 and 24 h with the Wst-1 assay (Roche Diagnostics, Indianapolis, IN, ISA). Optical densities (ODs) at 420 nm are presented. Statistical analysis All experiments were performed at least three times; each trial comprised triplicate samples per group. Data are expressed as means 6 standard deviations of the mean. Statistical analysis was performed using SPSS Version 12.0K (SPSS, Chicago, IL, USA). Intergroup differences were analyzed using a non-paired Student t-test. Null hypotheses of no difference were rejected at p-values , 0.05. RESULTS LIUS suppressed H2O2- and BSO-induced increases in mtROS-positive cells The proportion of mtROS-positive ARPE-19 cells increased approximately from 11.1 6 1.7% to 66.1 6 6.3% after treatment with 1 mM H2O2 for 1 h (Fig. 3a) and to 50.5 6 6.5% after treatment with BSO (Fig. 3b). When monitored at 1, 3, 6, 12 and 24 h after exposure to the chemicals, the percentage of mtROS-positive cells peaked at 1 h and then decreased until 24 h, particularly rapidly in the H2O2-treated samples (data not shown). When the treated cells were also exposed to LIUS at 50, 100 and 200 mW/cm2 for 20 min, the proportion of mtROS-positive cells decreased significantly to 37.8%– 43.4% for H2O2 and 36.8%–38.4% for BSO (p , 0.05). The LIUS effect exhibited no clear dose dependency, but was similar for 50, 100 and 200 mW/cm2. It is not clear why there was no significant difference among the different LIUS intensities. We speculate that 50 mW/cm2 was high enough to cause the positive effect of the nearplateau level. When measured after a longer time of 24 h, mtROS-positive cells decreased even in the H2O2 and BSO groups, and exposure to LIUS at 100 mW/cm2 did not have a significant inhibitory effect (Fig. 3c, d). Treatment with LIUS alone at 50, 100 and 200 mW/cm2 resulted in a marginal increase ,10% in the proportion of mtROSpositive RPE cells (data not shown). LIUS suppressed H2O2- and BSO-induced increases in mitochondrial membrane potential Changes in mtROS levels in cells can depolarize mitochondrial membrane potential and subsequent cellular events. We first examined changes in mitochondrial membrane potential of ARPE-19 cells after treatment with 1 mM H2O2 or BSO, with or without exposure to LIUS at 100 mW/cm2 as described above. As illustrated in Figure 3a, the proportion of depolarized cells was 13.0 6 3.2% at rest and increased significantly in response to

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Fig. 3. Effect of LIUS on mtROS-positive cells. (a, b) ARPE-19 cells were treated with 1 mM H2O2 (a) or L-buthionine(S,R)-sulfoximine (BSO) (b), and immediately mock-exposed or exposed to LIUS at 50, 100 and 200 mW/cm2 for 20 min. The percentage of mtROS-positive cells was calculated by flow cytometry using the MitoSOX Red kit after 1 h. (c, d) ARPE-19 cells were treated with 1 mM H2 O2 (c) or BSO (d) as described above and exposed to LIUS at 100 mW/ cm2 for 20 min. The percentage of mtROS-positive cells was calculated as described above after 1 and 24 h. In histograms, data presented are the means 6 standard deviations from three independent experiments (n 5 3). *p , 0.05. LIUS 5 lowintensity ultrasound; mtROS 5 mitochondrial reactive oxygen species.

H2O2 treatment at 1 h (approximately 48.8 6 7.5%) and to BSO at 1 and 24 h (56.2 6 2.1% and 32.1 6 2.0%, respectively). Co-exposure of cells to H2O2 or BSO and LIUS significantly reduced the 1-h values to levels similar to the initial level (19.2 6 2.3% for H2O2 and 25 6 6.9% for BSO) (p , 0.05). This result suggests that LIUS can efficiently modulate the change in mitochondrial activity in response to oxidative stress, even when mtROS levels are not completely decreased by LIUS. LIUS suppressed the BSO-induced increase in [Ca21]i Changes in [Ca21]i can be indicative of a disease state and are closely related to cellular ROS levels and mitochondrial activity in RPE cells (Rizzuto et al. 2012). When examined in ARPE-19 cells at 1 and 24 h, [Ca21]i was elevated approximately threefold by BSO only at 24 h, and significantly suppressed by approximately 45% by LIUS at 100 mW/cm2 (Fig. 4d) (p , 0.05). Treatment of cells with H2O2 did not increase [Ca21]i at any time point. This result suggests that LIUS can inhibit BSO-induced cellular events like calcium ion release. LIUS suppressed BSO-induced death of ARPE-19 cells Viability of ARPE-19 cells did not change after 1 h, but decreased significantly 24 h after treatment with 1 mM H2O2 or BSO treatment (Fig. 5a, b). When treated

cells were also exposed to LIUS at 100 mW/cm2, cell viability at 24 h was significantly increased only in the BSO-treated cells (2.7-fold), not in the H2O2-treated cells. To address dose-dependent changes in cell viability, cells were treated with 0.1, 0.5, 1 and 2 mM H2O2 or BSO for 24 h in the presence of LIUS. H2O2 did not significantly affect cell viability at 0.1 mM, but abruptly decreased it from 0.5 mM (Fig. 5c), whereas BSO decreased cell viability gradually from 0.1 mM and had a moderate effect even at high dosages (Fig. 5d). Exposure of treated cells to 100 mW/cm2 LIUS increased the viability of BSO-treated cells only, but not of H2O2treated cells, at all dosages (p , 0.05). Cell viability was decreased slightly further by co-treatment with 0.5 mM H2O2 and LIUS. This result suggests that LIUS can reduce the cellular toxicity of BSO and increase the viability of ARPE-19 cells. DISCUSSION In this study, we found for the first time that LIUS at around 100 mW/cm2 efficiently inhibits BSO-induced oxidative stress and decreases in cell viability in ARPE19 cells, possibly by modulating mitochondrial activity and/or intracellular calcium ion levels. It is not clear if LIUS at 100 mW/cm2 also generated oxidative stress in ARPE-19 cells. The amount of ROS generated by 100

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Fig. 4. Effect of LIUS on mitochondrial membrane potential and [Ca21]i in ARPE-19 cells. Cells were treated with 1 mM H2O2 (a, c) or BSO (b, d) and immediately exposed to 100 mW/cm2 LIUS for 20 min. After 1 and 24 h, cells were subjected to flow cytometry with JC-1 staining to measure mitochondrial membrane potential (a, b) or fluorometric analysis using the FluoForte Calcium Assay Kit (Enzo Life Science) to measure [Ca21]i (c, d). Data presented are the means 6 standard deviations from three independent experiments (n 5 3). *p , 0.05. BSO 5 L-buthionine-(S,R)-sulfoximine.

mW/cm2 LIUS might be very small, if any, and not detrimental to cells, because we did not observe any decrease in cell viability in response to LIUS at 100 mW/cm2 (data not shown). Chronic oxidative stress and mitochondrial dysfunction are highly associated with RPE injury and cell senescence and development of AMD (Beatty et al. 2000; Jarrett and Boulton 2012). Therefore, they have been therapeutic targets in the treatment of AMD for a long time (Cai et al. 2000). Some endogenous antioxidants, including melatonin, glutathione-S-transferase and ascorbic acid, have been found to have a protective effect on the RPE against oxidative stress in vitro and in vivo (reviewed in Jarrett and Boulton 2012). Recently, OT-551 (1-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride), a disubstituted hydroxylamine with antioxidant properties, was well tolerated and therapeutic possibility in a phase II trial in patients with advanced geographic atrophy (Wong et al. 2010). SkQ1 (plastoquinonyl-decyl-triphenylphosphonium), a mitochondria-targeted antioxidant, was also found to regress retinal damage in a rodent model of AMD (Markovets et al. 2011). We therefore speculate that LIUS could be also evaluated for its therapeutic effect on RPE injury and vision loss in AMD patients. Ultrasonography, long used in clinics for ophthalmic diagnosis, uses high-frequency waves .8 MHz. Ultrasound of relatively low intensity, from 190 to 560 mW/cm2, and 880

kHz in frequency has been used to facilitate drug delivery through the cornea and revealed no structural changes in corneal epithelium (Zderic et al. 2004). We expect that LIUS parameters used in this study will have no significant adverse effects on human eyes and can be easily applied. However, careful investigation is required before long-term and/or repeated LIUS treatment, particularly in a disease environment. Therefore, further studies are needed to verify the therapeutic efficacy and optimize the utility of LIUS in animal models and patients. In this study, we used two sources of oxidative stress: H2O2 and BSO. Both H2O2 and BSO increased the percentages of mtROS-positive cells and mitochondrial membrane depolarization in ARPE-19 cells. In contrast, only BSO increased [Ca21]i, and the decrease in cell viability induced by BSO, but not that induced by H2O2, was efficiently blocked on LIUS treatment. These differences between H2O2 and BSO effects might be due to differences in their cellular action mechanisms. H2O2 is itself a ROS and damages cells directly at the cell membrane and cytoplasmic levels, which can occur very rapidly and independently of mtROS production and mitochondrial dysfunction (Fridovich 1995). H2O2 can also cause mtROS generation through the well-known ROS-induced ROS release (RIRR) process (Zorov et al. 2000). In contrast, BSO is a glutathione (GHS) synthesis inhibitor and induces ROS generation in cells to exert its cytotoxic effect (Griffith and Meister 1979).

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Fig. 5. Changes in ARPE-19 cell viability. (a, b) ARPE-19 cells were treated with 1 mM H2O2 (a) or BSO (b) and immediately exposed to 100 mW/cm2 LIUS for 20 min. Viability of cells were measured using the Wst-1 assay (Invitrogen) at 1 and 24 h. (c, d) Cells were treated with 0.1, 0.5, 1 and 2 mM H2O2 (c) or BSO (d) and immediately exposed to 100 mW/cm2 LIUS for 20 min. Viability of cells was measured using the Wst-1 assay (Invitrogen) at 24 h. The amount of viable cells was expressed as mean OD 6 standard deviation from three independent experiments (n 5 3). *p , 0.05. BSO 5 L-buthionine-(S,R)-sulfoximine; OD 5 optical density.

The cellular target of the LIUS effect is not clear, but its mechanism must involve the mitochondria and Ca21 signaling, at least in BSO-treated ARPE-19 cells. Mitochondria regulate cytosolic Ca21 levels and also take up Ca21 into the mitochondrial matrix, both important in mitochondrial function and modulation of diverse cellular activities (Rizzuto et al. 2012). In reverse, accumulation of intracellular Ca21 at more than physiologic levels can also cause mitochondrial dysfunction and mtROS generation (Feissner et al. 2009). In our study, however, the increase in [Ca21]i was observed only at 24 h after BSO treatment and, therefore, might not be the cause of the mtROS increase and mitochondrial membrane depolarization observed 1 h after BSO treatment in ARPE-19 cells. We speculate that the increase in [Ca21]i was instead caused by ROSinduced mitochondrial dysfunction and could be the cause of the decrease in cell viability (Zavodnik et al. 2013). Local mechanical stimulation using a micropipette was also found to induce Ca21 movement in RPE cells (Stalmans and Himpens 1999). In submandibular gland cells, micropipette stimulation caused changes in Ca21 movement and mitochondrial function probably via the stretch-activated ion channels and ATP receptors (Ryu et al. 2010). Therefore, it may not be generalized, but the cellular effect of mechanical stimuli appears to commonly involve changes in the Ca21 signal and mitochondrial function. It is possible that LIUS regulated intracellular Ca21 signaling induced by BSO independently of its effect on ROS reduction, thereby

exerting its protective effect on cell viability. H2O2 was previously found to activate the Ca21-permeable TRPM2 channel on mitochondrial membranes in Chinese hamster ovary (CHO) cells (Naziroglu 2007). It is not clear why it did not induce [Ca21]i in ARPE-19 cells in our study. The inability of LIUS to increase cell viability in H2O2-treated cells might be correlated with its failure to inhibit the H2O2-induced increase in mtROS. We speculate that H2O2 has a direct cytotoxic effect and induces cell death independently of mitochondria and the Ca21 signal; therefore, its negative effect on cell viability was not inhibited by LIUS. Further studies are needed to identify the cellular target and precise mechanism of LIUS. CONCLUSIONS This study illustrated that low-intensity ultrasound (LIUS) of 100 mW/cm2 reduces BSO-induced oxidative stress in ARPE-19 cells and protects cells from cytotoxic cell death. The effect of LIUS appeared to involve modulation of mitochondrial activity and intracellular calcium ion levels. These results suggest that LIUS could be a potential treatment to inhibit oxidative damage and related pathologic changes in RPE cells. Acknowledgments—This study was supported by an Inha University grant and a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A101727).

LIUS effects on oxidative damage in retina d N. K. KIM et al.

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