Search

Fucoidans from brown algae are described as anti-inflammatory, antioxidative, and antiangiogenic. We tested two Saccharina latissima fucoidans (SL-FRO and SL-NOR) regarding their potential biological effects against age-related macular degeneration (AMD). Primary porcine retinal pigment epithelium (RPE), human RPE cell line ARPE-19, and human uveal melanoma cell line OMM-1 were used. Cell survival was assessed in tetrazolium assay (MTT). Oxidative stress assays were induced with erastin or H2O2. Supernatants were harvested to assess secreted vascular endothelial growth factor A (VEGF-A) in ELISA. Barrier function was assessed by measurement of trans-epithelial electrical resistance (TEER). Protectin (CD59) and retinal pigment epithelium-specific 65 kDa protein (RPE65) were evaluated in western blot. Polymorphonuclear elastase and complement inhibition assays were performed. Phagocytosis of photoreceptor outer segments was tested in a fluorescence assay. Secretion and expression of proinflammatory cytokines were assessed with ELISA and real-time PCR. Fucoidans were chemically analyzed. Neither toxic nor antioxidative effects were detected in ARPE-19 or OMM-1. Interleukin 8 gene expression was slightly reduced by SL-NOR but induced by SL-FRO in RPE. VEGF secretion was reduced in ARPE-19 by SL-FRO and in RPE by both fucoidans. Polyinosinic:polycytidylic acid induced interleukin 6 and interleukin 8 secretion was reduced by both fucoidans in RPE. CD59 expression was positively influenced by fucoidans, and they exhibited a complement and elastase inhibitory effect in cell-free assay. RPE65 expression was reduced by SL-NOR in RPE. Barrier function of RPE was transiently reduced. Phagocytosis ability was slightly reduced by both fucoidans in primary RPE but not in ARPE-19. Fucoidans from Saccharina latissima, especially SL-FRO, are promising agents against AMD, as they reduce angiogenic cytokines and show anti-inflammatory and complement inhibiting properties; however, potential effects on gene expression and RPE functions need to be considered for further research.

Purpose Systemic inflammation may be of importance in the development of AMD. RPE cells can recognize danger signals with toll-like receptors (TLR) and may react in a pro-inflammatory manner. In this study, we evaluated the basal and apical secretions of TNFα, IL-6, and IL-1β in primary RPE cells and RPE/choroid explant cells under basolateral stimulation of TLR2, 3, and 4; the effects on barrier function; and their influence on neuronal cell viability. Methods RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers; primary porcine RPE cells on transwell plates. Cells were basally stimulated with agonists Pam2CSK4 (Pam; TLR2), polyinosinic/polycytidylic acid (Poly I:C; TLR3), and lipopolysaccharide (LPS; TLR4) for 24 h. Supernatants were evaluated with ELISA for cytokines TNFα, IL-6, and IL-1β. Apical supernatants were applied to SHSY-5Y cells, and cell viability was evaluated in MTT assay. Barrier function was tested by measuring transepithelial electrical resistance (TER) and occludin immunostaining. Results None of the tested TLR agonists was toxic on RPE cells after 24 h of exposure. Unstimulated RPE cells secreted hardly any cytokines. Pam induced IL-6, IL-1ß, and TNFα on the basal and apical sides at all concentrations tested. Poly I:C induced IL-6 and TNFα primarily at the basal side at lower but on both sides at higher concentrations. LPS induced IL-6, IL-1ß, and TNFα apically and basally at all concentrations tested. In the RPE/choroid, a strong difference between apical and basal secretions could be found. IL-6 was constitutively secreted basally, but not apically, but was induced by all agonists on both sides. IL-1ß and TNFα alpha were strongly induced on the basal side by all agonists. TER was reduced by all agonists, with Pam and LPS being effective in all concentrations tested. Occludin expression was unaltered, but the distribution was influenced by the agonists, with a less distinct localization at the cell borders after treatment. None of the agonists or supernatants of treated RPE and RPE/choroid organ cultures exerted any effect on viability of SHSY-5Y cells. Conclusions Danger signals activating TLRs can induce polarized cytokine expression and contribute to the loss of barrier function in the RPE.

Background and objectives The thermal stimulation therapy of the retinal pigment epithelium (TSR) is a sublethal laser technique for thermal stimulation of the retinal pigment epithelium (RPE)-Bruch's membrane (BrM)-complex. The aim of this study was to investigate the influence of TSR on the release of age-related macular degeneration (AMD)-relevant cell mediators. Study design/materials and methods Porcine RPE-BrM-choroid explants were irradiated with a 532 nm continuous wave laser using different spot sizes (100-300 µm, duration 100 milliseconds, 15-100 mW). Cell death was investigated by calcein staining. Explants were treated with grids of sublethal spots and cultivated in modified Ussing chambers. The effect on matrix metalloproteinase-2 (MMP-2) and -9 was investigated by zymography and quantitative reverse transcription polymerase chain reaction. Secretion of vascular endothelial growth factor (VEGF), pigment epithelium derived factor (PEDF), and transforming growth factor-β (TGF-β) was analyzed by enzyme-linked immunosorbent assay and expression of HSP70 was examined by western blot. Integrity of the RPE/BrM-complex was analyzed by scanning electron microscopy. Results Laser powers of 15 mW (100 µm) and 45 mW (300 µm) did not induce RPE cell death. The integrity of the RPE/BrM-complex was not impaired after TSR. After TSR with 300 µm spot size, we observed a significant increase of active MMP-2 in the basal compartments. The content of PEDF significantly increased in treated explants in both compartments with 100 and 300 µm spot sizes. VEGF and TGF-β secretion was not triggered by TSR. Conclusions TSR represents a possible RPE stimulating treatment for dry AMD. TSR increases the basal release of active MMP-2, which might reverse age-related thickening of BrM. VEGF secretion was not triggered by TSR while anti-angiogenic PEDF was increased, indicating an induction of an anti-angiogenic and neuroprotective environment. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Purpose Silicone oil is used as endotamponade in combination with vitrectomy. Thinning of retinal layers and loss of retinal cells under silicone oil use have been found. Here, we investigate the influence of silicone oil on primary microglia cells. Methods Primary microglia cells were prepared from the porcine retina. Microglia identity was assessed with Iba1 staining. Silicone oil was emulsified by sonification. Cell morphology and silicone oil uptake were evaluated by light microscopy after Coomassie blue staining. Cytokine secretion was evaluated with ELISA. Toxicity of silicone oil on microglia and toxic effect of silicone oil–treated microglia on neuronal cell line PC12 were evaluated by MTT or WST assay, respectively. Results Microglia took up silicone oil droplets after 72 h of incubation. Silicone oil induced no toxicity but increased the metabolism in microglial cells. In addition, the secretion of IL-6 and IL-8, but not of IL-1ß or TNF-α, was induced. Silicone oil–treated microglia did not exert any neurotoxic effect on differentiated PC12 cells but induced an increase in metabolism. Conclusion Emulsified silicone oil changes the activity level of microglia and induces the secretion of IL-6 and IL-8. Neurotoxicity is not induced. Further experiments are required to investigate the long-term effect of silicone oil on microglia and their consequent effect on neuronal cells.

Purpose We examined the influence of retinal degeneration 8 (rd8) mutation of crumbs homolog 1 (CRB1) gene on age-related macular degeneration (AMD) phenotype in nuclear factor E2-related factor 2 knock out (NRF2−/−) mouse model. Methods CRB1rd8 mutation genotype was determined by polymerase chain reaction from tail clips in 73 NRF2−/− mice originating from C57BL/6J background on mixed C57BL/6J and C57BL/6N ancestry. The clinical grade of AMD-like fundus alterations was determined by funduscopy, optical coherence tomography (OCT) and fluorescein angiography (FLA) at the age of 9 or 12 months. Results Twelve NRF2−/− mice were wildtype CRB1+/+, 61 NRF2−/− were homozygous CRB1rd8/rd8. NRF2−/−CRB1rd8/rd8 mice had a significantly higher probability to show an advanced grade (grade 4 and 5) of AMD-like fundus alterations known to appear in NRF2−/− mice. Choroidal neovascularization (CNV) was only detected in NRF2−/−CRB1rd8/rd8 homozygous mice. Conclusions Homozygous CRB1rd8/rd8 mutation is common in commercial vendor mice strains of C57BL/6J origin if partly on C57BL/6N ancestry. The mutation has an influence on the extent of AMD-like retinal alterations in NRF2−/− mice and favors CNV formation.

We rely on vision more than on any other sense to obtain information about our environment. Hence, the loss or even impairment of vision profoundly affects our quality of life. Diet or food components have already demonstrated beneficial effects on the development of retinal diseases. Recently, there has been a growing interest in resources from marine animals and plants for the prevention of retinal diseases through nutrition. Especially fish intake and omega-3 fatty acids have already led to promising results, including associations with a reduced incidence of retinal diseases. However, the underlying molecular mechanisms are insufficiently explained. The aim of this review was to summarize the known mechanistic effects of marine resources on the pathophysiological processes in retinal diseases. We performed a systematic literature review following the PRISMA guidelines and identified 107 studies investigating marine resources in the context of retinal diseases. Of these, 46 studies described the underlying mechanisms including anti-inflammatory, antioxidant, antiangiogenic/vasoprotective, cytoprotective, metabolic, and retinal function effects, which we critically summarize. We further discuss perspectives on the use of marine resources for human nutrition to prevent retinal diseases with a particular focus on regulatory aspects, health claims, safety, and bioavailability.

Fucoidans are polysaccharides and constituents of cell walls of brown algae such as Laminaria hyperborea (LH). They exhibit promising effects regarding age-related macular degeneration (AMD). However, the safety of this compound needs to be assured. The focus of this study lies on influences of an LH fucoidan on the retinal pigment epithelium (RPE). The high-molecular weight LH fucoidan Fuc1 was applied to primary porcine RPE cells, and a tetrazolium (MTT) cell viability assay was conducted. Further tests included a scratch assay to measure wound healing, Western blotting to measure expression of retinal pigment epithelium-specific 65 kDa protein (RPE65), as well as immunofluorescence to measure uptake of opsonized fluorescence beads into RPE cells. Lipopolysaccharide was used to proinflammatorily activate the RPE, and interleukin 6 (IL-6) and interleukin 8 (IL-8) secretion was measured. RPE/choroid cultures were used to assess vascular endothelial growth factor (VEGF) secretion. Real-time polymerase chain reaction (real-time PCR) was performed to detect the gene expression of 91 different genes in a specific porcine RPE gene array. Fuc1 slightly reduced wound healing, but did not influence cell viability, phagocytosis or RPE65 expression. Fuc1 lowered IL-6, IL-8 and VEGF secretion. Furthermore, Fuc1 did not change tested RPE genes. In conclusion, Fuc1 does not impair RPE cellular functions and shows antiangiogenic and anti-inflammatory activities, which indicates its safety and strengthens its suitability concerning ocular diseases.

(1) Background: Inflammation is a major pathomechanism in the development and progression of age-related macular degeneration (AMD). The retinal pigment epithelium (RPE) may contribute to retinal inflammation via activation of its Toll-like receptors (TLR). TLR are pattern recognition receptors that detect the pathogen- or danger-associated molecular pattern. The involvement of TLR activation in AMD is so far not understood. (2) Methods: We performed a systematic literature research, consulting the National Library of Medicine (PubMed). (3) Results: We identified 106 studies, of which 54 were included in this review. Based on these studies, the current status of TLR in AMD, the effects of TLR in RPE activation and of the interaction of TLR activated RPE with monocytic cells are given, and the potential of TLR activation in RPE as part of the AMD development is discussed. (4) Conclusion: The activation of TLR2, -3, and -4 induces a profound pro-inflammatory response in the RPE that may contribute to (long-term) inflammation by induction of pro-inflammatory cytokines, reducing RPE function and causing RPE cell degeneration, thereby potentially constantly providing new TLR ligands, which could perpetuate and, in the long run, exacerbate the inflammatory response, which may contribute to AMD development. Furthermore, the combined activation of RPE and microglia may exacerbate neurotoxic effects.

Zusammenfassung Hintergrund Ziel der Studie war es den Einfluss der Selektiven Retinatherapie (SRT) auf die Ausschüttung inflammatorischer Zellmediatoren, wie dem Komplementfaktor-3 (CC3), Tumor Growth Factor-beta2 (TGF-β2), Tumor Necrosis Factor-alpha (TNF-α) und Interferon-gamma (IFN-γ) in einem porcinen Organkulturmodell zu untersuchen. Material und Methoden Porcine Organkulturexplantate aus retinalem Pigmentepithel (RPE), Bruch-Membran und Choroidea wurden mit 2 gepulsten Lasersystemen (SRTYLF und SRTYAG) behandelt (Nd : YLF, λ = 527 nm, Pulsdauer 1,7 µs und Nd : YAG, Wellenlänge 532 nm, Pulsdauer 2,4 – 3 µs). Es wurden 30 Pulse bei einer Repetitionsrate von 100 Hz und einer Spotgröße von 200 × 200 µm appliziert. Es wurde mit einer Energiedichte von 140 mJ/cm² pro Puls (auf der RPE-Zelltodschwelle) und 180 mJ/cm² pro Puls (über der RPE-Zelltodschwelle) behandelt. Die Explantate wurden in modifizierten Ussing-Kammern kultiviert und die Zellvitalität mittels Calcein-AM-Färbung untersucht. Die Sekretion und Expression der Zellmediatoren wurde mittels ELISA bzw. im Western Blot analysiert. Ergebnisse Vier Tage nach SRT wurde die Regeneration der RPE-Zellen im Bereich der Läsion beobachtet. Ein Tag nach SRT mit 140 mJ/cm² pro Puls zeigte sich eine Reduktion der basolateralen CC3-Sekretion. Nach der Behandlung mit 180 mJ/cm² pro Puls wurde nach 4 Tagen eine verminderte Sekretion von IFN-γ beobachtet. Schlussfolgerung Die SRT führt zu keiner Induktion der untersuchten proinflammatorischen Zytokine in vitro.

Purpose/Aim of the study: Vascular endothelial growth factor (VEGF)-antagonists are given over long time periods in the clinic, but the long term effects on retinal pigment epithelium (RPE) cells are not fully investigated. This study aims to investigate these effects with two clinical relevant VEGF antagonists, bevacizumab and aflibercept, on the function on primary RPE cells. Materials and methods All tests were conducted with primary porcine RPE. Cells were stimulated with bevacizumab or aflibercept (both 250 µg/ml) for 1 day, 7 days or 4 weeks. Cell viability was tested in MTT Assay. Secretion of TGF-s was tested in ELISA, phagocytosis in a microscopic assay, migration in a scratch assay, and expression of RPE65 in Western blot. Barrier function was tested for bevacizumab in transwell-cultured cells by measuring transepithelial electrical resistance for up to 3 days. Results Viability was reduced by both antagonists at all time points tested. TGF-s secretion was not altered by any treatment. Phagocytosis was not significantly reduced by any treatment. Wound healing ability was not significantly altered by any treatment. The expression of RPE65 was reduced by bevacizumab but not aflibercept after 4 weeks. Transepithelial electrical resistance was not altered. Conclusions Long term treatment with anti VEGF may affect viability of RPE cells, and treatment with bevacizumab may have effects on RPE function in long term treatment.

Properties of retinal pigment epithelium (RPE) are relevant for the development of cell culture models concerning an exact reproduction of the ocular cell biology. Here, we want to investigate how different carrier materials and coatings influence proliferation, differentiation and functions of RPE in regard to development of a three-dimensional cell culture model based on primary porcine RPE. Human RPE cell line ARPE-19 and primary porcine RPE were used. Cells were cultivated on plates which were coated with collagen I, collagen IV, laminin or fibronectin, respectively, and cell numbers were assessed after different time periods via trypan blue staining. Also, the ARPE-19 were cultivated on polydimethylsiloxane (PDMS), alginate, gelatin methacrylate (GelMA), poly-N-isopropylacrylamide (PNIPAM) and cells number were assessed. Primary RPE were cultured on PDMS material. Supernatants were collected and analyzed via ELISA for their vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) content. After day 14 cells were lysed and retinal pigment epithelium-specific 65 kDa protein (RPE65) and bestrophin-1 (BEST1) expression was investigated via Western blot. Cellular functions were tested on collagen I, collagen IV, laminin and fibronectin with and without PDMS. Scratch assay was performed to detect wound healing 24 and 48 h after scratch application. Immunolabeling was used to highlight tight junctions in concert with Hoechst staining and phalloidin to label cell nuclei and actin filaments, respectively. Phagocytosis of fluorescently labeled latex beads opsonized with photoreceptor outer segments (POS) was assessed via fluorescence microscopy. Transepithelial electrical resistance was measured for detection of cellular barrier. Gene expression of RDH11 (retinol dehydrogenase 11), BEST1 (bestrophin 1) and TGFB1 (transforming growth factor beta 1) was investigated via real-time PCR. Only PDMS carrier material was appropriate for primary RPE and ARPE-19 cell cultivation. Coating of PDMS with laminin led to increased proliferation. In primary RPE, VEGF secretion was increased if PDMS was coated with laminin or fibronectin compared to uncoated PDMS. No significant changes in phagocytic ability and generation of tight junctions were detected between different coatings, but RPE65 expression was reduced on fibronectin coated PDMS. Laminin coating decreased TGF-β and increased BEST1 protein expression. Also, RPE on collagen IV showed highest TEER on transwell plates. The genes RDH11 and TGFB1 were decreased when coated with collagen IV without PDMS as well as coated PDMS. Laminin and collagen IV coating led to an increased wound healing. Cultivation of RPE and ARPE-1 on PDMS is a possible alternative for cell culture models whereas alginate, GelMA and PNIPAM were not suitable. Coating with laminin increased the proliferation, wound healing and VEGF secretion of the cells. The results suggest that laminin coated PDMS as carrier material is suitable for the development of 3D culture model systems.

(1) Background: Retinal pigment epithelial cells (RPE) cells constitutively secrete vascular endothelial growth factor (VEGF) in the retina, protecting the neuronal cells and the choroid. Increased VEGF secretion, however, can result in neovascularization and edema. Many factors regulate VEGF secretion. In this study, we investigated the effect of external stimuli in relation to diurnal rhythm on constitutive VEGF secretion. (2) Methods: Single-eye RPE cell culture was prepared from porcine eyes. RPE cells were cultured in darkness, treated with daylight or room light, and treated with melatonin at different time frames, either respectively or in combination. Supernatants were collected and VEGF content evaluated using ELISA. Expression of the clock protein BMAL1 was evaluated with Western blot. (3) Results: VEGF secretion of the RPE shows a diurnal rhythm. While the rhythm is not influenced by either light or melatonin, the amount of secreted VEGF can be increased by nocturnal melatonin, especially in combination with morning daylight. These findings disclose another layer of VEGF regulation in the retina.

Purpose Bevacizumab is taken up and transported through the retinal pigment epithelium. Inflammatory signaling may influence this interaction. In the present study, we have investigated the effect of pro-inflammatory stimuli on the uptake, intracellular localization, and transepithelial transport of bevacizumab. Methods ARPE-19 cell line or primary porcine RPE cells were treated with clinical relevant concentrations of bevacizumab (250 µg/ml). Pro-inflammatory signaling was induced by TLR-3 agonist polyinosinic:polycytidylic acid (Poly I:C). Viability was investigated with MTT and trypan-blue exclusion assay, and cell number, uptake, and intracellular localization were investigated with immunofluorescence, investigating also actin filaments, the motor protein myosin 7a and lysosomes. Immunofluorescence signals were quantified. Intracellular bevacizumab was additionally detected in Western blot. Barrier function was investigated with transepithelial resistant measurements (TER). The transepithelial transport of bevacizumab and its influence on cytokine (IL-6, IL-8, IL-1β, TNFα) secretion was investigated with ELISA. Results Poly I:C in combination with bevacizumab reduced the viability of the cells. Treatment with Poly I:C reduced the uptake of bevacizumab, changed the intensity of the actin filaments, and reduced the colocalization with myosin 7a. In addition, Poly I:C reduced the capacity of RPE cells to transport bevacizumab over the barrier. In addition, bevacizumab reduced the secretion of IL-8 and TNFα after Poly I:C stimulation at selected time points. Conclusions Pro-inflammatory activation of RPE cells with TLR-3 agonist Poly I:C changes the interaction of RPE cells with the anti-VEGF compound bevacizumab, reducing its uptake and transport. On the other hand, bevacizumab might influence pro-inflammatory cytokine release. Our data indicate that inflammation may influence the pharmacokinetic of bevacizumab in the retina.

Ingredients of brown seaweed like fucoidans are often described for their beneficial biological effects, that might be interesting for a medical application. In this study, we tested an extract from Dictyosiphon foeniculaceus (DF) to evaluate the effects in glioblastoma and uveal melanoma, looking for a possible anti-cancer treatment. We investigated toxicity, VEGF (vascular endothelial growth factor) secretion and gene expression of tumor and non-tumor cells. SVGA (human fetal astrocytes), the human RPE (retinal pigment epithelium) cell line ARPE-19, the tumor cell line OMM-1 (human uveal melanoma), and two different human primary glioblastoma cultures (116-14 and 118-14) were used. Tests for cell viability were conducted with MTS-Assay (3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), and the proliferation rate was determined with cell counting. VEGF secretion was assessed with ELISA (enzyme-linked immunosorbent assay). The gene expression of VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2) and VEGF-A was determined with real-time qPCR (quantitative polymerase chain reaction). DF lowered the cell viability of OMM-1. Proliferation rates of ARPE-19 and OMM-1 were decreased. The VEGF secretion was inhibited in ARPE-19 and OMM-1, whereas it was increased in SVGA and 116-14. The expression of VEGFR1 was absent and not influenced in OMM-1 and ARPE-19. VEGFR2 expression was lowered in 116-14 after 24 h, whereas VEGF-A was increased in 118-14 after 72 h. The extract lowered cell viability slightly and was anti-proliferative depending on the cell type investigated. VEGF was heterogeneously affected. The results in glioblastoma were not promising, but the anti-tumor properties in OMM-1 could make them interesting for further research concerning cancer diseases in the human eye.

Age-related macular degeneration (AMD) is the major reason for blindness in the industrialized world with limited treatment options. Important pathogenic pathways in AMD include oxidative stress and vascular endothelial growth factor (VEGF) secretion. Due to their bioactivities, fucoidans have recently been suggested as potential therapeutics. This review gives an overview of the recent developments in this field. Recent studies have characterized several fucoidans from different species, with different molecular characteristics and different extraction methods, in regard to their ability to reduce oxidative stress and inhibit VEGF in AMD-relevant in vitro systems. As shown in these studies, fucoidans exhibit a species dependency in their bioactivity. Additionally, molecular properties such as molecular weight and fucose content are important issues. Fucoidans from Saccharina latissima and Laminaria hyperborea were identified as the most promising candidates for further development. Further research is warranted to establish fucoidans as potential therapeutics for AMD.

Purpose Inflammatory processes play a major role within the multifactorial pathogenesis of age-related macular degeneration (AMD). Neuroretina sparing laser therapies, thermal stimulation of the retina (TSR) and selective retina therapy (SRT), are known to reduce AMD-like pathology in vitro and in vivo. We investigated the effect of TSR and SRT on inflammatory processes in AMD mouse models. Methods One randomized eye of 8 months old apolipoprotein (Apo)E and 9 months old nuclear factor (erythroid-derived 2) -like 2 (NRF2) knock out mice were treated by TSR (10 ms, 532 nm, 50 µm2 spot size, mean 4.5 W, ~200 spots) or SRT (~1.4 µs pulses, 532 nm, 50 µm spot size, 100 Hz over 300 ms, mean 2.5 µJ per pulse, ~200 spots). Fellow eyes, untreated knock out mice and wild-type BL/6J mice acted as controls. All mice were examined funduscopically and by optical coherence tomography (OCT) at the day of laser treatment. Mice were euthanized and enucleated either 1 day or 7 days after laser treatment and examined by gene expression analysis of 84 inflammatory genes. Results The inflammatory gene expression profile of both knock out models compared to healthy BL/6J mice suggests a regulation of pro- and anti-inflammatory processes especially concerning T-cell activity and immune cell recruitment. TSR resulted in downregulation of several pro-inflammatory cell-mediators both in ApoE -/- and NRF2-/- mice compared to treatment naive litter mates one day after treatment. In contrast, SRT induced pro-inflammatory cell-mediators connected with necrosis one day after treatment as expected following laser-induced selective RPE cell death. Seven days after laser treatment, both findings were reversed. Conclusions Both TSR and SRT influence inflammatory processes in AMD mouse models. However, they act conversely. TSR leads to anti-inflammatory processes shortly after laser therapy and induces immune-cell recruitment one week after treatment. SRT leads to a quick inflammatory response to laser induced RPE necrotic processes. One week after SRT inflammation is inhibited. It remains unclear, if and to what extent this might play a role in a therapeutic or preventive approach of both laser modalities on AMD pathology.

Introduction: Anti-VEGF therapy is repeatedly given for an extended period of time to patients when treated for age-related macular degeneration. While short-term effects of anti-VEGF agents on retinal pigment epithelial cells have been investigated, the effects of long-term and repeated treatment on these cells are scarce. In this study, we have investigated the effects of anti-VEGF treatment (bevacizumab and aflibercept) after long-term, repeated treatment on uptake, storage, and subcellular localization. Methods: Experiments were conducted in primary porcine retinal pigment epithelium (RPE) cells in first passage and in ARPE-19 cell line. Cells were treated with 250 µg/mL bevacizumab, aflibercept, or, as a non-VEGF inhibiting antibody, rituximab once a week for 1 day, 7 days, 4, and 12 weeks. Cell survival was evaluated with methyl thiazolyl tetrazolium assay. Uptake and localization of compounds were investigated with immunofluorescence microscopy. Selective intracellular proteins were stained with specific respective primary antibodies; actin cytoskeleton was stained with phalloidin. For quantitative analysis, intracellular signals were normalized to light intensity and exposure time. Intracellular association with lysosomes (Lamp2) and exosomes (CD63) was also quantified. In addition, subcellular fractions (nucleus, plasma, membrane, and cytoskeleton) were generated and analyzed in Western blot. Results: Weekly treatment up to 12 weeks displayed no toxic effects on RPE cells in any substance tested. Intracellular signal of bevacizumab and aflibercept was strongest after 1 day, decreased after 1 and 4 weeks but increased again after 12 weeks. The signal of intracellular bevacizumab was significantly stronger than of aflibercept. In addition, in primary RPE, aflibercept was significantly more associated with Lamp2, indicating degradation of aflibercept. At all time points, the respective therapeutics could be detected at the cytoskeleton. In primary RPE cells, co-localization with exosome marker CD63 showed a maximum after 1 day for bevacizumab and after 12 weeks for aflibercept. Actin-encapsulated therapeutics can be found at any time point tested. Conclusion: Both bevacizumab and aflibercept display a distinctive time-dependent uptake in the RPE cells and are stored in actin-covered accumulations for extended periods of time. When normalized and quantified, less aflibercept can be found in RPE cells, while more aflibercept is co-localized with Lamp2. Our data suggest that bevacizumab is differently processed by RPE cells than aflibercept.

Zusammenfassung Hintergrund Die Rolle des oxidativen Stresses bei Krebserkrankungen ist komplex. Während die durch ihn ausgelösten pathologischen Veränderungen an der Krebsentstehung beteiligt sind, kann er in späteren Stadien das Absterben von Tumorzellen fördern und sogar Metastasierungen eindämmen. Krebszellen zeigen durch ihren veränderten Metabolismus häufig eine verstärkte Bildung von radikalen Sauerstoffspezies, was sie anfällig für zusätzlichen oxidativen Stress macht. Dies ist ein wichtiger Wirkmechanismus von verschiedenen Chemotherapeutika und von therapeutisch eingesetzter ionisierender Strahlung. Das uveale Melanom ist der häufigste primäre Tumor im adulten Auge. Für die bei etwa 50 % der Patienten auftretenden Metastasen sind zurzeit leider keine wirksamen Behandlungsoptionen verfügbar. Der Primärtumor kann aber mit gutem Erfolg mit ionisierender Strahlung behandelt werden. Eine wichtige Nebenwirkung ist hierbei die Strahlenretinopathie, die wiederum mit VEGF-Antagonisten (VEGF: Vascular endothelial Growth Factor) behandelt wird. Eine Therapie des Primärtumors mit Anti-VEGF-Substanzen wird ebenfalls in der Literatur diskutiert. Über eine solche Anwendung liegen bisher nur wenige Daten vor, es ist aber eine paradoxe Verschlechterung der Situation bei einem uvealen Melanommodell in der Maus unter Anti-VEGF-Therapie berichtet worden. Methoden In einer Studie haben wir den Einfluss von VEGF und dem VEGF-Antagonisten Bevacizumab auf das Überleben von 5 unterschiedlichen uvealen Melanomzelllinien, die mit oxidativem Stress (Wasserstoffperoxid) behandelt worden sind, untersucht. Im Weiteren haben wir uns die Expression relevanter Proteine sowie den Einfluss von Bevacizumab auf das Proliferationsverhalten der Zellen und auf das Angiogeneseverhalten von Endothelzellen in einer Kokultur mit uvealen Melanomzellen angesehen. Ergebnisse Wir konnten in dieser Studie zeigen, dass nicht nur VEGF, sondern paradoxerweise auch der VEGF-Antagonist Bevacizumab uveale Melanomzellen vor einem Zelltod durch oxidativen Stress schützen kann. Bevacizumab zeigt darüber hinaus keinen Einfluss auf die Proliferation der Zellen und ist nur bedingt in der Lage, angiogene Strukturen in diesem System zu verhindern. Schlussfolgerungen Die Protektion der uvealen Melanomzellen vor oxidativem Stress durch Bevacizumab ist, gerade in Anbetracht des Wirkprinzips der ionisierenden Strahlung, durch oxidativen Stress in den Tumorzellen einen Zelltod auszulösen, ein besorgniserregender Befund, der gegen eine Verwendung von Anti-VEGF bei uvealem Melanom spricht.

Rotating wire brushes are used, e.g., for rust removal. Detaching fragments can cause severe eye injuries. The purpose of this study was to investigate mechanism, severity, clinical outcome, validity of Ocular Trauma Score (OTS) and to assess the likelihood of final visual acuity. Twenty patients with traumatic ocular injuries by rotating wire brushes were included. Location and type of injury, grade of injury according to OTS, surgical procedure, and development of visual acuity were evaluated. Eleven accidents (55%) happened at work, nine at home (45%). Eighteen injuries (90%) were penetrating, one (5%) was perforating. In one case (5%), an intraocular foreign body was present. One case each was classified OTS 1 and 2 (5%), six cases OTS 3 (30%), four cases OTS 4 (20%), and eight cases OTS 5 (40%). None of the patients was wearing safety goggles. Fourteen patients (70%) were surgically treated. Of these, five were treated at the anterior segment only, nine additionally underwent pars-plana vitrectomy. Nine patients received phacoemulsification. Mean best corrected visual acuity was logMAR 1.0 (2/20) at admission and 0.3 (10/20) at last follow-up. Our results were similar to those in the OTS study, except for OTS 1 (p = 0.046). Comparing the categorical distribution of final visual acuity in all OTS categories, no statistically significant difference was found (p = 0.119) between our results and the OTS study group. OTS may provide prognostic information in traumatic ocular injuries by rotating wire brushes. The injuries could have been avoided by wearing safety goggles.

Vascular endothelial growth factor (VEGF) is a major pro-angiogenic cytokine, but also exerts many additional physiological functions in the retina. The retinal pigment epithelium (RPE) is a major source for VEGF in the retina, especially affecting the endothelium of choriocapillaris but also photoreceptors and the RPE itself. VEGF is regulated on many levels and the pathway of induction differs between the many stimuli. In this chapter, the role of RPE-derived VEGF will be discussed, as well as the physiological and induced pathways of VEGF regulation, especially induced by hypoxia, oxidative stress and hyperglycemia, inflammation and hyperthermia.

Cell culture of retinal pigment epithelium cells (RPE) has been in practice since the 1920s, using different species as donors. Besides primary cell cultures, of which the human is the most important origin, cell lines have been generated and are widely used for their convenience. In this chapter we discuss the challenges of RPE cell culture, and compare the advantages and limitation of cell lines, especially ARPE-19, and primary RPE cells, focusing mainly on fetal and adult cells of human origin as well as on adult cells of porcine origin. Finally, new developments concerning 3D- and microfluidic systems are discussed.

Purpose Degenerative ocular disorders like age-related macular degeneration (AMD) are associated with long-term pro-inflammatory signals on retinal pigment epithelial (RPE) cells. In this study, we investigated the effect of long term treatment of RPE cells with agonists of toll-like receptor (TLR) −3 (Polyinosinic:polycytidylic acid, Poly I:C), TLR-4 (lipopolysaccharide, LPS) and the pro-inflammatory cytokine TNFα. Methods All tests were conducted with primary porcine RPE. Cells were stimulated with Poly I:C (1, 10, 100 μg/ml), LPS (0.1, 1, 10 μg/ml) or TNFα (12.5, 25 or 50 ng/ml) for 1 day, 7 days or 4 weeks. Cell viability tests (MTT) were additionally tested in ARPE-19 cells. Cytokine secretion (IL-6, IL-1β, IL-8, TNFα, TGF-β) was tested in ELISA, phagocytosis in a microscopic assay, and expression of RPE65 in Western blot. Barrier function was tested in transwell-cultured cells by measuring transepithelial resistance for up to 3 days. Results LPS and TNFα significantly reduce cell viability after 1 day and 7 days, Poly I:C after 7 days and 4 weeks. LPS, Poly I:C and TNFα significantly induce the secretion of IL-6 and IL-8 at all tested time points. IL-1β is increased by LPS and Poly I:C after 1 day, but not by TNFα. TNFα secretion is increased by Poly I:C and LPS after 1 day but not at later time points. TGF-β secretion is not influenced by any stimulus. Concerning RPE function, LPS decreased phagocytosis after 7 days, while Poly I:C and TNFα showed no effect. RPE65 expression was strongly reduced by TNFα and LPS after 4 weeks. Wound healing capacity was reduced by Poly I:C but induced by LPS after 7 d and 4 w. Barrier function was not affected by Poly I:C or LPS, while TNFα reduced barrier function after 1 h, 4 h and 3 days. Conclusion Long term pro-inflammatory stimuli reduce RPE viability, barrier properties and cellular function and induce pro-inflammatory cytokines and therefore may contribute directly to atrophic changes in AMD.

For many retinal diseases, including age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy (DR), the exact pathogenesis is still unclear. Moreover, the currently available therapeutic options are often unsatisfactory. Research designed to remedy this situation heavily relies on experimental animals. However, animal models often do not faithfully reproduce human disease and, currently, there is strong pressure from society to reduce animal research. Overall, this creates a need for improved disease models to understand pathologies and develop treatment options that, at the same time, require fewer or no experimental animals. Here, we review recent advances in the field of in vitro and ex vivo models for AMD, glaucoma, and DR. We highlight the difficulties associated with studies on complex diseases, in which both the initial trigger and the ensuing pathomechanisms are unclear, and then delineate which model systems are optimal for disease modelling. To this end, we present a variety of model systems, ranging from primary cell cultures, over organotypic cultures and whole eye cultures, to animal models. Specific advantages and disadvantages of such models are discussed, with a special focus on their relevance to putative in vivo disease mechanisms. In many cases, a replacement of in vivo research will mean that several different in vitro models are used in conjunction, for instance to analyze and validate causative molecular pathways. Finally, we argue that the analytical decomposition into appropriate cell and tissue model systems will allow making significant progress in our understanding of complex retinal diseases and may furthermore advance the treatment testing.

Background: Sulfated fucans show interesting effects in the treatment of ocular diseases (e.g., age-related macular degeneration), depending on their chemical structure. Here, we compared three purified sulfated fucans from Laminaria hyperborea (LH) regarding cell viability, oxidative stress protection, and vascular endothelial growth factor (VEGF) secretion in ocular cells. Methods: High-molecular-weight sulfated fucan (Mw = 1548.6 kDa, Fuc1) was extracted with warm water and purified through ultrafiltration. Lower-molecular-weight samples (Mw = 499 kDa, Fuc2, 26.9 kDa, Fuc3) were obtained by mild acid hydrolysis of ultrapurified sulfated fucan and analyzed (SEC-MALS (Size-exclusion chromatography-Multi-Angle Light Scattering), ICP-MS, and GC). Concentrations between 1 and 100 &micro, g/mL were tested. Cell viability was measured after 24 h (uveal melanoma cell line (OMM-1), retinal pigment epithelium (RPE) cell line ARPE-19, primary RPE cells) via MTT/MTS (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide/3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Oxidative stress protection was determined after 24 h (OMM-1, ARPE-19). VEGF secretion was analyzed via ELISA after three days (ARPE-19, RPE). Results: Fuc2 and Fuc3 were antiproliferative for OMM-1, but not for ARPE. Fuc1 protected OMM-1. VEGF secretion was lowered with all fucans except Fuc3 in ARPE-19 and RPE. The results suggest a correlation between molecular weight and biological activity, with efficiency increasing with size. Conclusion: The LH sulfated fucan Fuc1 showed promising results regarding VEGF inhibition and protection, encouraging further medical research.

Fucoidan extracts may have beneficial effects in age-related macular degeneration (AMD). Over-the-counter fucoidan preparations are generally undefined, crude extracts. In this study, we investigated the effect of a crude fucoidan extract from Fucus distichus subspecies evanescens (Fe) on the retinal pigment epithelium (RPE). Fe extract was investigated for chemical composition and molar mass. It was tested in primary RPE and RPE cell line ARPE19. Oxidative stress was induced with tert-butyl hydroperoxide, cell viability evaluated with MTT assay, VEGF secretion assessed in ELISA. Phagocytosis was evaluated in a fluorescence microscopic assay. Wound healing ability was tested in a scratch assay. Additionally, the inhibition of elastase and complement system by Fe extract was studied. The Fe extract contained about 61.9% fucose and high amounts of uronic acids (26.2%). The sulfate content was not as high as expected (6.9%). It was not toxic and not protective against oxidative stress. However, Fe extract was able to reduce VEGF secretion in ARPE19. Phagocytosis was also reduced. Concerning wound healing, a delay could be observed in higher concentrations. While some beneficial effects could be found, it seems to interfere with RPE function, which may reduce its beneficial effects in AMD treatment.

In diabetic patients, high blood glucose induces alterations in retinal function and can lead to visual impairment due to diabetic retinopathy. In immortalized retinal pigment epithelial (RPE) cultures, high glucose concentrations are shown to lead to impairment in epithelial barrier properties. For the first time, the induced pluripotent stem-cell-derived retinal pigment epithelium (hiPSC-RPE) cell lines derived from type 2 diabetics and healthy control patients were utilized to assess the effects of glucose concentration on the cellular functionality. We show that both type 2 diabetic and healthy control hiPSC-RPE lines differentiate and mature well, both in high and normal glucose concentrations, express RPE specific genes, secrete pigment epithelium derived factor, and form a polarized cell layer. Here, type 2 diabetic hiPSC-RPE cells had a decreased barrier function compared to controls. Added insulin increased the epithelial cell layer tightness in normal glucose concentrations, and the effect was more evident in type 2 diabetics than in healthy control hiPSC-RPE cells. In addition, the preliminary functionality assessments showed that type 2 diabetic hiPSC-RPE cells had attenuated autophagy detected via ubiquitin-binding protein p62/Sequestosome-1 (p62/SQSTM1) accumulation, and lowered pro- matrix metalloproteinase 2 (proMMP2) as well as increased pro-MMP9 secretion. These results suggest that the cellular ability to tolerate stress is possibly decreased in type 2 diabetic RPE cells. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:20 issue:15 ispartof: location:Switzerland status: published

Background: Fucoidans are interesting for potential usage in ophthalmology, and especially age-related macular degeneration. However, fucoidans from different species may vary in their effects. Here, we compare fucoidans from five algal species in terms of oxidative stress protection and vascular endothelial growth factor (VEGF) interference in ocular cells. Methods: Brown algae (Fucus vesiculosus, Fucus distichus subsp. evanescens, Fucus serratus, Laminaria digitata, Saccharina latissima) were harvested and fucoidans isolated by hot-water extraction. Fucoidans were tested in several concentrations (1, 10, 50, and 100 &micro, g/mL). Effects were measured on a uveal melanoma cell line (OMM-1) (oxidative stress), retinal pigment epithelium (RPE) cell line ARPE19 (oxidative stress and VEGF), and primary RPE cells (VEGF). Oxidative stress was induced by H2O2 or tert-Butyl hydroperoxide (TBHP). Cell viability was investigated with methyl thiazolyl tetrazolium (MTT or MTS) assay, and VEGF secretion with ELISA. Affinity to VEGF was determined by a competitive binding assay. Results: All fucoidans protected OMM-1 from oxidative stress. However, in ARPE19, only fucoidan from Saccharina latissima was protective. The affinity to VEGF of all fucoidans was stronger than that of heparin, and all reduced VEGF secretion in ARPE19. In primary RPE, only the fucoidan from Saccharina latissima was effective. Conclusion: Among the fucoidans from five different species, Saccharina latissima displayed the most promising results concerning oxidative stress protection and reduction of VEGF secretion.

Purpose To investigate the effect of selective retina therapy (SRT) on age-related macular degeneration (AMD)-like alterations of retinal pigment epithelium (RPE) and Bruch's membrane (BrM) in AMD mouse models as therapeutic approach for the treatment of dry AMD. Methods In B6.129P2-Apoetm1Unc /J (ApoE-/-) and B6.129X1-Nfe2I2 tm1Ywk /J (NRF2-/-), one randomized eye of each mouse in groups of 15 mice was treated by SRT (532 nm, 300 ms, ∼1.4-μs pulse, 100 Hz, 50-μm spot), the fellow eye and healthy C57BL/6J mice served as controls. Clinical examinations were obtained at treatment day and 1 month later, followed by enucleation to analyze BrM thickness and ultrastructural RPE morphology. Results Nearly all ApoE-/- and NRF2-/- mice showed AMD-like retinal alterations. BrM thickness was increased in both mouse models, RPE had vacuoles within the cell body and shortened apical microvilli. SRT neither affected neuroretinal anatomy nor function. BrM thickness as well as AMD-like ultrastructural alterations of the RPE were significantly reduced in laser-treated eyes compared with fellow control and untreated control eyes. Conclusions SRT reduces BrM thickness and AMD-like RPE alterations in AMD mouse models without damage to structural or functional properties of neuroretina. It may be a prophylactic or therapeutic option for dry AMD. Translational relevance SRT shows therapeutic effectivity in murine AMD models and might therefore become an option for the treatment of dry AMD.

Background We previously showed that the protein pattern of lenses removed in cataract surgery differs between patients with Parkinson's disease and age-matched controls. In this study, we identified the protein reduced in abundance in the 34- to 37-kDa gel band. Methods During cataract surgery (phacoemulsification), we collected the rinsing fluid and lens particles from the eyes of PD patients and controls. Residual lens fragments in the supernatant of 3 PD patients and aged-matched controls were studied for protein profiles using liquid chromatography-mass spectrometry and Western blots. Results We identified glyceraldehyde-3-phosphate dehydrogenase by mass spectrometry as the protein reduced in abundance and verified this finding in Western blots. Conclusions Glyceraldehyde-3-phosphate dehydrogenase has been implicated in PD development. The reduction of glyceraldehyde-3-phosphate dehydrogenase in the lenses of PD patients may be a new biomarker for PD and might also indicate an important role for this protein in PD development. © 2016 International Parkinson and Movement Disorder Society

In the ophthalmological clinic, intravitreally applied antibodies or Fc-containing fusion proteins are frequently used, but the biology and pharmacokinetics of these therapeutics in the retina are not well understood. We have previously shown intracellular uptake of Fc-containing molecules in RPE cells. In this study, we investigated the involvement of Fc-receptors, both Fcγ-receptors and the neonatal Fc-receptor (FcRn) in the uptake and intracellular trafficking of the VEGF-antagonists bevacizumab, aflibercept and the anti-CD20 antibody rituximab in three different model systems, primary porcine RPE cells, ARPE-19 cells and porcine RPE/choroid explants. The expression of Fcγ-receptors was tested in primary porcine RPE cells, and the expression of Fcγ-receptors I and II could be shown in RT-PCR and qRT-PCR, while the expression of FcRn was additionally confirmed in Western blot and immunocytochemistry. All three compounds, bevacizumab, rituximab and aflibercept, were taken up into the cells and displayed a characteristic time-dependent pattern, as shown in Western blot and immunohistochemistry. The uptake was not altered by the inhibition of Fcγ-receptors using different inhibitors (TruStain FcX, genistein, R406). However, the inhibition of FcRn with an antagonistic antibody reduced intracellular IgG in porcine RPE cells (rituximab) and ARPE-19 cells (bevacizumab, rituximab). Colocalisations between the tested compounds and myosin7a could be found. In addition, limited colocalization with FcRn and the tested compounds, as well as triple localization between compound, FcRn and myosin7a could be detected, indicating a role of myosin7a in FcRn mediated transport. However, the colocalizations are restricted to small fractions of the Fc-containing compounds. Furthermore, the FcRn is mainly found in the membrane section, where only minute amounts of the Fc-containing compounds are seen, suggesting a limited interaction. An apical to choroidal transport of IgG through the RPE/choroid can be found in RPE/choroid explants. Inhibition of FcRn increases the amount of bevacizumab found on the choroidal side, suggesting a role of FcRn in the recycling of bevacizumab. In conclusion, our data indicate a role for FcRn, but not Fcγ-receptors, in the uptake and transport of Fc-containing molecules in the RPE and indicate a recycling function of FcRn in the retina.

In age-related macular degeneration, inflammatory events are presumed to contribute to disease development. A primary suspect of this contribution is the microglia, the innate immune cell of the retina. In addition, retinal pigment epithelium (RPE) cells can be inflammatorily activated. In this study, we investigate the effect of activated RPE cells on retinal microglia and on neuronal cells. RPE cells and microglia were harvested from porcine eyes. In addition, a neuronal cell line (SHSY-5Y) of human origin was used. For inflammatory activation, agonists of toll-like receptors in different concentrations were used: Pam2CSK4 (Pam; TLR-2), Polyinosinic:polycytidylic acid (Poly I:C; TLR-3) and lipopolysaccharid (LPS; TLR-4). Cell viability was investigated with an MTT assay. The secretion of cytokines was assessed in an ELISA and their expression in real-time PCR. There was no effect of the agonists on cell viability in RPE cells. All agonists induced the secretion of IL-6 and IL-8 in RPE cells with the strongest effect induced by LPS. In microglia, pro-inflammatory stimulation increased the metabolic activity. All agonists induced the secretion of IL-1ß, IL-8, and TNFα in microglia cells while in real-time PCR, LPS and Pam induced the expression of IL-6, IL-1ß and iNOS. Direct stimulation of SHSY-5Y with the agonists induced only minor alterations of viability. Stimulated RPE cell supernatant reduced the secretion of TNFα and IL-8 irrespective of the inducing agent in microglia cells. Additionally a slight induction of IL-1ß was found in microglia treated with supernatant of RPE cells treated with Pam. In real time PCR, the supernatant of RPE cells stimulated with LPS significantly reduced the expression of iNOS and IL-6, but not of IL-1ß. Of note, the expression of iNOS was also reduced by naive RPE cells. The treatment of the SHSY-5Y with supernatant of microglia previously treated with RPE conditioned medium significantly decreased SHSY-5Y viability with and without pro-inflammatory treatment. In conclusion, inflammatory activated RPE cells have a regulatory effect on the pro-inflammatory activation of microglia, stressing the importance of the interaction between these two retinal cell types. Microglia treated with RPE supernatant reduced viability of a neuronal cell line, indicating a neurotoxic effect.

The cultivation of retinal pigment epithelium (RPE)-choroid explants gives the opportunity to study the RPE and Bruch’s membrane in its natural environment. Porcine eyes are easily available and an excellent model for human RPE. Explants are prepared less than 4 h postmortem from cooled eyes and are transferred in fixation rings. The tissues held between rings are cultured in a perfusion organ culture system for up to a week. Viability of the explants can be investigated by calcein staining.

The cultivation of retinal pigment epithelium (RPE)-choroid explants gives the opportunity to study the RPE and Bruch's membrane in its natural environment. Porcine eyes are easily available and an excellent model for human RPE. Explants are prepared less than 4 h postmortem from cooled eyes and are transferred in fixation rings. The tissues held between rings are cultured in a perfusion organ culture system for up to a week. Viability of the explants can be investigated by calcein staining.

We examined the influence of retinal degeneration 8 (rd8) mutation of crumbs homolog 1 (CRB1) gene on age-related macular degeneration (AMD) phenotype in nuclear factor E2-related factor 2 knock out (NRF2CRB1Twelve NRF2Homozygous CRB1