5-Lipoxygenase Inhibition Protects Retinal Pigment Epithelium from Sodium Iodate-Induced Ferroptosis and Prevents Retinal Degeneration

Excessive reactive oxygen species (ROS) contribute to damage of retinal cells and the development of retinal diseases including age-related macular degeneration (AMD). ROS result in increased metabolites of lipoxygenases (LOXs), which react with ROS to induce lipid peroxidation and may lead to ferroptosis. In this study, the effect of 5-LOX inhibition on alleviating ROS-induced cell death was evaluated using sodium iodate (NaIO3) in the retinal pigment epithelium (RPE) cell line ARPE-19 and a mouse model investigating oxidative stress in AMD. We demonstrated that NaIO3 induced cell death in the RPE cells through mechanisms including ferroptosis. Inhibition of 5-LOX with specific inhibitor, Zileuton, or siRNA knockdown of ALXO5 mitigated NaIO3-induced lipid peroxidation, mitochondrial damage, DNA impairment, and cell death in ARPE-19 cells. Additionally, in the mouse model, pretreatment with Zileuton reduced the NaIO3-induced lipid peroxidation of RPE cells, cell death in the photoreceptor layer of the retina, inflammatory responses, and degeneration of both the neuroretina and RPE monolayer cells. Our results suggest that 5-LOX plays a crucial role in ROS-induced cell death in the RPE and that regulating 5-LOX activity could be a useful approach to control ROS and ferroptosis-induced damage, which promote degeneration in retinal diseases.


Introduction
Oxidative stress has been implicated in the pathogenesis of various retinal diseases. Excessive production of reactive oxygen species (ROS) may lead to impairment of retinal cells including retinal pigment epithelium (RPE) cells and vascular endothelial cells. It may also promote development and acceleration of retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), glaucoma, retinal vascular occlusion, and inherited retinal diseases [1].
Various signaling pathways have been proposed to clarify the mechanisms of ROS-induced retinal degeneration. Glutamate is an important neurotransmitter in the retina. However, excessive glutamate triggers calcium overload and subsequently increases mitochondrial ROS, which may prompt retinal ganglion cell death via a mitochondrialtargeting mechanism [2][3][4]. Treatments that alleviate ROS, such as nuclear factor (erythroid-derived 2)-like 2 gene therapy and spermidine, an endogenous ROS scavenger, successfully protect retinal cells from cell death in experimental models [5][6][7]. Mechanisms of cell death, including apoptosis, necroptosis, and recently, ferroptosis, have been reported in the research of retinal diseases [8][9][10][11][12]. Iron overload in the neuroretina and RPE may be a contributing factor in the progression of AMD or other diseases involving intraocular hemorrhage [13][14][15]. Ferroptosis is one of the nonapoptotic forms of programmed cell death, which is characterized by the development of iron-dependent lipid peroxidation [16]. It is regulated by the enzyme, glutathione peroxidase 4 (GPx4), with glutathione (GSH) synthesized from amino acids [17]. The membrane cystine transporter system xc-and GPx4 protect cells from ferroptosis [18]. Toxic substances may develop when superoxide interacts with membrane lipids and produce lipid hydroperoxides through Fenton reaction in the presence of excessive iron or deficiencies in antioxidant defense responses [19].
Both enzymatic and nonenzymatic metabolic pathways, such as cystine deprivation (CD), mevalonate, lipid metabolism, GPx4, iron metabolism, and ferritinophagy pathways, have been reported to be involved in the regulation of ferroptosis [20]. Lipoxygenases (LOXs), a group of enzymes that metabolize arachidonic acid as well as polyunsaturated fatty acids (PUFA), may contribute to lipid peroxidation and induce ferroptosis [21,22]. Inhibitors of LOX inhibitors, such as 5-LOX inhibitors Zileuton and BWB70C, 12/15-LOX inhibitor baicalin or baicalein, and pan-LOX inhibitor nordihydroguaiaretic (NDGA), have been reported to clear free radicals and therefore decrease the levels of lipid and protein oxidation [23]. Recombinant pigment epitheliumderived factor receptor (PEDF-R) proteins inhibit 5-LOX and protect RPE from ROS-induced cell death in vitro [24]. In ARPE-19 cells, docosahexaenoic acid (DHA) suppressed the H 2 O 2 -induced transcriptional upregulation of 5-LOX and proinflammatory hydroxyeicosatetraenoic acids (HETEs) following omega-6 PUFA oxidation [25]. These results imply the role of 5-LOX in lipid peroxidation in RPE cells. However, to the best of our knowledge, no studies have implicated the direct involvement of 5-LOX in RPE or retinal cell death under excessive oxidative stress in in vitro or in vivo models.
The role of LOXs in retinal or RPE diseases is still poorly understood. In this study, we investigated the role of 5-LOX in ROS-damaged RPE and retina with sodium iodate-(NaIO 3 -) induced cell death in RPE. We used ARPE-19 cells as an in vitro model, as well as a murine model of NaIO 3induced acute retinal damage, which has been widely used for investigating ROS-induced effects in AMD [26,27]. Both pharmacological and genetic approaches were used to verify the effect of LOX inhibition on protecting the retina or RPE from regulated cell death such as ferroptosis.

Cell
Culture. The RPE cell line ARPE-19 (ATCC, Manassas, VA, USA) was seeded at densities of 1:2 × 10 6 cells per well in 6-well plates, 6 × 10 5 cells per well in 12well plates, or 5 × 10 4 cells per well in 96-well transparent plates and grown to full confluence. The cells were cultured in DMEM/F-12 medium supplemented with HEPES (Thermo Fisher), 10% FBS, and 100 U/mL of penicillinstreptomycin at 37°C with 5% CO 2 . Cells used in experiments were within five passages after thawing from liquid nitrogen.
In vitro experiments were carried out 5 days after the start of the transfection procedure. Effectiveness of the ALOX5 knockdown was examined using western blot analysis for 5-LOX protein.
2.4. Cell Viability Assay. Cells were seeded in sterile transparent 96-well plates. The culture media was mixed with 10% (v/v) CCK-8 reagent (Dojindo) and incubated for 1 h at 37°C in an incubator. The absorption was then measured at 450 nm using the Hidex Sense Microplate Reader (Hidex, Turku, Finland). The cell viability was assessed with ARPE-19 seeded in 6 independently culture wells (n = 6) for each treatment condition. . Then, 100 μL of the sample was fortified with isotopically labeled analogues of arachidonic acid and 5-hydroxyeicosatetraenoic acid (5-HETE) that functioned as isotope internal standards. Next, the sample was acidified with 0.5 mL 1% formic acid aqueous solution and mixed with ethyl acetate solution to extract the method analytes, including arachidonic acid, 5-HETE, and their isotope-labeled analogues. The extract was concentrated to dryness with nitrogen and then reconstituted in 50 μL acetonitrile and 50 μL 0.1% formic acid aqueous solution. The analytes were separated on a Poroshell EC-18 column (Agilent Technologies, Santa Clara, CA, USA) with gradient elution using a mobile phase made of ultrapure water and acetonitrile. Detection was carried out using negative electrospray ionization and multiple reaction monitoring (MRM) in liquid chromatography-tandem mass spectrometry. The concentration of arachidonic acid and 5-HETE was calculated using the isotope internal standard. The data was acquired and processed using Agilent Mas-sHunter Workstation Version 10.1 software (Agilent Technologies). Detailed methodology is described in Supplemental Method S1.

Evaluation of Mitochondrial Function and Integrity.
Mitochondrial function and integrity of ARPE-19 cells were evaluated using ATP Detection Assay Kit-Luminescence (Cayman) and JC-1 staining (Cayman), respectively. In the ATP detection, the luciferin in assay reagent was converted to oxyluciferin with light emission that was quantitatively proportional to the amount of intracellular ATP (n = 6 per treatment condition). JC-1 staining (1 μM 30 min, n = 6 per treatment condition) was performed as per the manufacturer's protocol. Both luminescence and fluorescence were detected using the Hidex Sense Microplate Reader (Hidex). Signaling), and mouse xCT (NB300-318, Novus, Centennial, CO, USA), diluted in TBST with 5% (w/v) BSA overnight at 4°C. The membranes were then incubated with HRP-linked secondary antibodies for 1 h at room temperature (RT). The protein bands were visualized with HRP substrate reagent (Thermo Fisher) and detected with an ECL imaging system (Bio-Rad, Hercules, CA, USA). The relative expression of target protein was obtained after normalizing to β-actin (ab8227, Abcam) expression.

Animal
2.13. Immunofluorescence Imaging for In Vitro Studies. For immunofluorescence imaging of intracellular structure, ARPE-19 cells were seeded at a density of 0:8 × 10 5 cells per well in 8-well chamber slides and fixed with 4% PFA and permeabilized with 0.2% Triton X-100 (in PBS). Following blocking with 5% normal goat serum, the slide was incubated with primary antibody for cytochrome c oxidase subunit 4 (COX-4, ab16056, Abcam), succinate dehydrogenase [ubiquinone] iron-sulfur subunit (SDHB, 10620-1-AP, Proteintech, Rosemont, IL, USA) or 8-hydroxy-2 ′ -deoxyguanosine (8-OHdG) conjugated with Alexa Fluor® 647 (sc-393871 AF647, Santa Cruz) at 4°C overnight. These slides were then incubated with Alexa-conjugated secondary antibody for 60 min at RT for unconjugated primary antibodies. Nuclei were counterstained with DAPI. Images were captured using an immunofluorescence microscope Axio Imager M2 (Carl Zeiss, Oberkochen, Germany). 8-OHdGstained cells were counted in three 400x power fields (more than 50 cells in each field) per slide. The mean percentages of 8-OHdG-positive cells in each slide were compared among the groups with four independent experiments.
2.14. Immunofluorescence Imaging for In Vivo Studies. The primary antibodies used include anti-Iba1 (019-19741, Fujifilm Wako, Osaka, Japan), anti-4-hydroxynonenal (HNE) (HNEJ-2, JaICA, Shizuoka, Japan), and anti-zonula occludens-1 (ZO-1, 61-7300, Thermo Fisher). For flat mount imaging of the RPE monolayer, the eyeball was cut along the equator. After removal of the anterior segment and neuroretina, the eyecup was fixed with 4% PFA for 3 h, blocked with mouse-on-mouse blocking reagent (ab269452, Abcam) for mouse-derived primary antibodies or 5% normal goat serum for the rabbit-derived primary antibodies with 0.3% Triton X-100 in PBS for 1 h at RT. Next, the eyecup was incubated with primary antibodies 4-HNE (n = 4, 24 h after NaIO 3 injection), Iba1 (n = 4, 3 days after NaIO 3 injection), or ZO-1 (n = 12 animals, 14 days after NaIO 3 ) overnight at 4°C and then with Alexa floursecondary antibody for 1 h. The eyecup was flat-mounted on a glass slide after eight radial cuts and prepared in mounting medium for immunofluorescence imaging. Fluorescence intensity of 4-HNE was measured in the total surface area of the RPE monolayer under low-power 50x images. The number of Iba1-positive cells was counted in four independent 100 μm × 100 μm square areas, which were chosen about 400 μm away from the optic disc under 100x images. In each 10000 μm 2 area, the mean counts of Iba1positive cells were compared between the four retinas in each treatment condition. The integrity of RPE monolayer, hexagonal structure of intercellular junction between RPE cells, in the mouse retina was evaluated with ZO-1 staining. RPE monolayer without clear and regularly aligned hexagonal ZO-1 staining in more than 25% area of the retina (under 200x magnification) was considered destructed. Cryosections of eyeballs were used for TUNEL staining and retinal thickness assessment. TUNEL staining of mouse retinal cryosections was performed with DAPI counterstained nuclei using the manufacturer's protocol (Roche, Basel, Switzerland). TUNEL-positive cells were counted in the region of the retina 200 to 800 μm away from the optic disc. The mean number of cells in three sections from each eyeball was compared among groups (n = 8). Retinal thickness was evaluated with DAPI-stained retinal cryosections obtained after 14 days of treatment with NaIO 3 . The average retinal thickness of the three sections from each eyeball 200, 400, 600, and 800 μm away from the optic disc was compared (n = 6 in each group). Both flat-mount and retinalsectional images were obtained using the immunofluorescence microscope Axio Imager M2 (Carl Zeiss). Fluorescence intensities in the images were analyzed for quantitative comparison between groups with ImageJ.

NaIO 3 Induces Mitochondrial Damage in ARPE-19 Cells.
Mitochondria contain membranous structures and iron and therefore may be a target of oxidative damage in ferroptosis [20,30,31]. NaIO 3 treatment caused an increase in mitochondrial ROS, detected with MitoSOX staining (Figure 5(a)). Additionally, a decrease in mitochondrial membrane potential (ΔΨm) was detected with JC-1 staining

NaIO 3 Caused RPE Loss, Inflammatory Responses, and
Damage of Mouse Retina. To verify the effects of NaIO 3 and the role of 5-LOX in RPE in vivo, we used a murine model of NaIO 3 (intraperitoneal injection 40 mg/kg body weight)-induced acute retinal degeneration using 6-weekold male wild-type C57BL/6JNarl mice. To detect ferroptosis after NaIO 3 treatment in vivo, we examined genes associated with ferroptosis using real-time PCR. Changes in the mRNA expression of ferroptosis-associated genes ALOX5, GPX4, SLC7A11, CHAC1, CISD1, and HSPB1 were observed in RPE/choroid samples collected 1 day after NaIO 3 treatment ( Figure 6(a)). Unexpectedly, we observed a decrease in mRNA expressions of GPX4 and CIS1D, previously reported to protect mitochondria from lipid peroxidation [32], following the NaIO 3 treatment. NaIO 3 -induced change in the protein level of xCT (SLC7A11) was consistent with change in mRNA in RPE/choroid (Figure 6(b)). Pretreatment with Zileuton (20 mg/kg intraperitoneal, 24 h and 15min before NaIO 3 injection) modulated NaIO 3 -induced changes in mRNA expression including ALOX5. These results suggest the potential participation of 5-LOX in cell death such as ferroptosis induced by NaIO 3 in vivo. NaIO 3 induced lipid peroxidation as evidenced by positive staining for 4-HNE, particularly in the membrane of cells in the RPE monolayer ( Figure 6(c)). Meanwhile, cell death, demonstrated by positive TUNEL staining, was observed in the retinal photoreceptor layer 24 h after NaIO 3 treatment (Figure 7(a)). Pretreatment with Zileuton effectively reduced NaIO 3 -induced cell death in the retina and 4-HNE staining of RPE (Figures 7(a) and 6(c)). Induction of ferroptosis was supported by the protective effect of DFO, which reduced NaIO 3 -induced cell death in mouse retina (Figure 7(b)). Infiltration of inflammatory cells in the retina and/or RPE layers can be related to cell death. An increasing number of active microglia or monocytic cells with amoebic, round, or oval-shaped and positive Iba1-staining were observed in the RPE monolayers 3 days following NaIO 3 treatment (Figures 8(a) and 8(b)). Pretreatment of mice with Zileuton reduced the number of Iba1-positive cells on the RPE monolayer surface and downregulated the expression of proinflammatory genes, including CD80 and TNF in the cells derived from the RPE and eyecup (Figure 8(c)). Destruction of RPE monolayer integrity was observed 14 days following NaIO 3 treatment in mice. Pretreatment with Zileuton protected the RPE monolayer from NaIO 3 -induced destruction, which was demonstrated with ZO-1 staining. Pretreatment with ferroptosis inhibitors, such as DFO and ferrostatin-1, showed similar protective effects in the RPE monolayer (Figure 9(a)). This finding also supported the involvement of cell death mechanisms related to ferroptosis in NaIO 3 -induced destruction of RPE in vivo. Additionally, NaIO 3 induced reduction in the thickness of neuroretina, which was also mitigated with Zileuton (Figure 9(b)).

Discussion
In this study, NaIO 3 -induced lipid peroxidation and cell death were mitigated with the 5-LOX inhibitor Zileuton in ARPE-19 cells (Figures 2 and 4). Furthermore, the potential benefit of 5-LOX inhibition was validated for the first time in an in vivo model of NaIO 3 -induced acute retinal degeneration. Inhibition of 5-LOX decreased lipid peroxidation of RPE cells (Figure 6), cell death in the photoreceptor layer of mouse retina (Figure 7), activation of inflammatory cells (Figure 8), and degeneration of the neuroretina and RPE monolayer following NaIO 3 treatment (Figure 9). Pretreatment with ferroptosis inhibitors including DFO and ferrostatin-1 exerted similar effects in protecting RPE cells from NaIO 3 -induced damage both in in vitro and in vivo models (Figures 1, 2, and 9). Additionally, the role of 5-LOX in cell death caused by conventional ferroptosis inducers, such as Erastin was demonstrated in vitro ( Figure 3). Our results suggest that ferroptosis was one of the major mechanisms involved in oxidative stress-induced cell death and that 5-LOX was a crucial enzyme that participated in the ROS-induced response and induction of ferroptosis in RPE cells. Therefore, modulating 5-LOX activity could be a promising strategy to alleviate damages induced by accumulation of ROS and disturbance of iron homeostasis in retinal diseases, such as AMD. The graphic summary of the proposed mechanisms is presented in Figure 10.
Research on ferroptosis in the retina is still in its early stages. Ferroptosis has been implicated to be one of the mechanisms associated with stress-induced cell death and senescence in RPE [10-12, 33, 34]. The importance of antioxidative enzyme systems in the protection of retina degeneration has been emphasized previously. Increased expression of GPx4 provided strong functional and structural protection from oxidative damage in a transgenic mouse model [35]. Iron is essential for energy generation and metabolism in most tissues involved in energy homeostasis [36]. However, it has been reported that genetic ironoverload in diabetic Hfe-knockout mice escalated neuronal

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Oxidative Medicine and Cellular Longevity cell death, vascular damages, and defects in retinal barrier integrity [37]. Apart from DR, iron may also be involved in a broad range of retinal disorders. It has been reported that the iron levels in the human macula increase with age. Postmortem retinal tissues derived from patients with AMD had higher iron and iron-carrying transferrin protein (TF) than age-matched controls [15]. The increase in both iron and TF has been shown to be correlated with poor visual acuity in murine models of retinal detachment. Exogenous transferrin can thus help in iron clearance to decrease cell death induced by retinal detachment [38]. Ferrous ions have also been found to interact with bis-retinoids in the RPE and promote cell damage [39]. In another study, ironchelating reagent metallocomplex zinc-desferrioxamine lowered lipid peroxidation and oxidative DNA damage in a murine model of retinitis pigmentosa [40]. These findings suggest that iron metabolism potentially participates in cell death observed in retinal diseases. In addition to abnormal iron metabolism, research on ferroptosis-associated molecules and signaling pathways has also increased. Metabolism of cysteine, iron and lipid, mevalonate pathway, GPx4 activity, and autophagy have been reported to participate in the regulation of ferroptosis [20,31,41]. However, simple blockage of these metabolic and signaling pathways can cause disturbance and unexpected impairment in retinal functions; therefore, identification of a feasible treatment to control ferroptosis is challenging. In this study, NaIO 3 induced changes in the mRNA expression of ferroptosis-associated genes including ALXO5, GPX4, SLC7A11, CHAC1, and HSPB1 in the mouse RPE/eyecup tissue ( Figure 6) [32,42,43]. The understand-ing of 5-LOX activity in the RPE or retina is limited. We found that the protective effect of 5-LOX inhibition was mostly associated with NaIO 3 -induced oxidative stress, especially lipid peroxidation, and less likely associated with modulation of intracellular iron levels in ARPE-19 cells (Figure 4). It has previously been reported that DHA suppressed hydrogen peroxide-induced transcriptional upregulation of 5-LOX and associated proinflammatory HETEs, following omega-6 PUFA oxidation observed in ARPE-19 cells [25]. Some recombinant PEDF-R proteins, which can inhibit 5-LOX activity, have been shown to protect RPE cells from ROS-induced cell death in vitro via the inhibition of leukotriene production [24]. LOXs including 5-12-and 15-LOX can metabolize arachidonic acid and PUFA into inflammatory chemokines including leukotrienes and prostaglandins to counteract with the damages caused in diseases [44,45]. Nevertheless, it has been reported that downstream metabolites of LOXs, such as HPETEs, can react with ROS and contribute to the lipid peroxidation, which induces ferroptosis. Mobilization of arachidonic acid under oxidative stress has also been reported [28]. In this study, we found that NaIO 3 upregulated both arachidonic acid and 5-HETE in ARPE-19 cells (Figure 2). This could indicate the possibility of transient increase of unstable hydroperoxide derivatives, such as 5-HPETE, which may react with ROS to cause lipid peroxidation of cellular membrane structures through Fenton reaction inducing ferroptosis [21,22]. In addition to cell death, NaIO 3 caused a significant increase in the density of inflammatory cells in the RPE monolayer of the eyecup (Figure 8). ROS-damaged RPE cells potentially release proinflammatory signals, such as leukotriene B4,  Oxidative Medicine and Cellular Longevity which can lead to infiltration of inflammatory cells in the retina [24]. However, the exact role of 5-LOX inhibition in the differentiation of inflammatory cells was not explored in this study and requires further investigation.
Upregulation of antioxidant proteins including xCT (SLC7A11) in vitro ( Figure 4) and SCL7A11 and CHAC1 mRNA in vivo ( Figure 6) suggested the activation of the intracellular antioxidant system, which utilizes glutathione to counteract NaIO 3 -induced increase in ROS. Upregulation of xCT was observed in both in vitro and in vivo experiments, suggesting that xCT may be a sensitive marker of ROS in RPE cells. This can also indicate shortage of cysteine following oxidative stress, and induction of ferroptosis in cysteine deprivation condition [41]. In addition, we found that NaIO 3 induced significant loss of ATP production, mitochondrial transmembrane potential, and expression of mitochondrial proteins essential for citric acid cycle and the electron transport chain ( Figure 5). Mitochondria are iron-rich organelles, in which iron is incorporated to form iron-sulfur clusters in the electron transport chain. However, abnormality in iron metabolism, particularly ferrous ions or excessive ROS, can induce lipid peroxidation

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Oxidative Medicine and Cellular Longevity through Fenton reaction, which may compromise mitochondrial membrane integrity and subsequently induce bioenergetic crisis and cell death [20]. This is of particular significance in the retina, which is a tissue with high metabolic demand and rich in mitochondria. In this study, Zileuton prevented an increase in ROS and mitigated the loss of mitochondrial proteins including SDHA, SDHB, and COX4, thereby preserving ATP production in ARPE-19 cells ( Figure 5). Consistent with our findings, 5-LOX was previously shown to mediate mitochondrial damage and cell death in mononuclear cells in end-stage retinal disease patients and PC-12 neuronal cells [46]. NaIO 3 -induced drop in CIS1D mRNA, which regulates iron homeostasis in mitochondria, may indicate the involvement of mitochondria in ferroptosis in RPE cells in vivo. Overall, our results support the hypothesis that mitochondria are implicated in ferroptosis, and inhibition of 5-LOX can partially preserve mitochondrial integrity and function in RPE cells.
Our results suggested that NaIO 3 -induced oxidative damage, although a fast model to investigate the effects of ROS in the retina and RPE, should be interpreted cautiously. NaIO 3 may induce cell death through multiple mechanisms besides ferroptosis in the RPE in vivo, and it is not easy to exclude the possibility that 5-LOX is activated by mechanisms other than NaIO 3 -induced ferroptosis in the retina. Nevertheless, NaIO 3 -induced ferroptosis was confirmed through the protective effects of the iron chelator, DFO, which inhibited cell death in the outer retinal layer and destruction of the RPE monolayer. Additionally, cell death induced by conventional ferroptosis inducers Erastin and RSL3 was successfully blocked via 5-LOX inhibition with Zileuton in ARPE-19 cells, providing evidence for the role of 5-LOX in ferroptosis in RPE cells in vitro. Moreover, combination of Zileuton with DFO did not exert additive effects when compared with Zileuton alone. Taken together, these results suggest that 5-LOX inhibition can effectively block ferroptosis in NaIO 3 -injured RPE.

Conclusions
In summary, we demonstrated the involvement of 5-LOX in oxidative stress in RPE cells, and that 5-LOX inhibition protected RPE cells from NaIO 3 -induced lipid peroxidation, mitochondrial damage, DNA damage, and cell death. The observed increase in 5-HETE provided evidence for the activation of the 5-LOX pathway and increase in the upstream unstable 5-HPETE. These eicosanoids can react with ROS to cause lipid peroxidation of cellular membrane structures ultimately leading to cell death through mechanisms including ferroptosis. A novel finding of our study was the protective effect of 5-LOX inhibition on RPE and retina from ROSinduced inflammation and cell death in vivo. Our results highlight the potential of 5-LOX inhibition as a feasible strategy for control of ROS damage in RPE and retina in retinal diseases, such as AMD. Future studies with genetic and pharmaceutical approaches are required to confirm the role of 5-LOX activity in managing retinal diseases associated with abnormal iron metabolism and oxidative stress.

Data Availability
Data is contained within the article or the supplemental material.

Conflicts of Interest
The authors declare no conflict of interest.