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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 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.

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.

(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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Background. The polysaccharide fucoidan is widely investigated as an anti-cancer agent. Here, we tested the effect of fucoidan on uveal melanoma cell lines. Methods. The effect of 100 µM fucoidan was investigated on five cell lines (92.1, Mel270 OMM1, OMM2.3, OMM2.5) and of 1 µg/mL–1 mg/mL fucoidan in two cell lines (OMM1, OMM2.3). Cell proliferation and viability were investigated with a WST-1 assay, migration in a wound healing (scratch) assay. Vascular Endothelial Growth Factor (VEGF) was measured in ELISA. Angiogenesis was evaluated in co-cultures with endothelial cells. Cell toxicity was induced by hydrogen-peroxide. Protein expression (Akt, ERK1/2, Bcl-2, Bax) was investigated in Western blot. Results. Fucoidan increased proliferation in two and reduced it in one cell line. Migration was reduced in three cell lines. The effect of fucoidan on VEGF was cell type and concentration dependent. In endothelial co-culture with 92.1, fucoidan significantly increased tubular structures. Moreover, fucoidan significantly protected all tested uveal melanoma cell lines from hydrogen-peroxide induced cell death. Under oxidative stress, fucoidan did not alter the expression of Bcl-2, Bax or ERK1/2, while inducing Akt expression in 92.1 cells but not in any other cell line. Conclusion. Fucoidan did not show anti-tumorigenic effects but displayed protective and pro-angiogenic properties, rendering fucoidan unsuitable as a potential new drug for the treatment of uveal melanoma.

Fucoidans extracted from brown algae exert manifold biological activities paving the way for the development of numerous applications including treatments outside tumor therapy such as age-related macular degeneration or tissue engineering. In this study, we investigated the antiproliferative effects of fucoidans extracted from six different algae (Fucus vesiculosus, F. serratus, F. distichus subsp. evanescens, Dictyosiphon foeniculaceus, Laminaria digitata, Saccharina latissima) as well as three reference compounds (Sigma fucoidan, heparin, enoxaparin) on tumor (HL-60, Raji, HeLa, OMM-1, A-375, HCT-116, Hep G2) and non-tumor (ARPE-19, HaCaT) cell lines. All fucoidans were extracted according to a standardized procedure and tested in a commercially available MTS assay. Cell viability was measured after 24 h incubation with test compounds (1&ndash, 100 &micro, g/mL). Apart from few exceptions, fucoidans and heparins did not impair cell viability. In contrast, fucoidans significantly increased cell viability of suspension cell lines, but not of adherent cells. Fucoidans slightly increased viability of tumor cells and had no impact on the viability of non-tumor cells. The cell viability of HeLa and ARPE-19 cells negatively correlated with protein content and total phenolic content (TPC) of fucoidans, respectively. In summary, none of the tested fucoidans turned out to be anti-proliferative, rendering them interesting for future studies and applications.

To evaluate the effect of an intravitreally applied anti-IL-6 antibody for the treatment of experimental autoimmune uveitis (EAU).EAU was induced in female B10.RIII mice by Inter-Photoreceptor-Binding-Protein (IRBP) in complete Freund's adjuvant, boosted by Pertussis toxin. Single blinded intravitreal injections of anti-IL-6 antibody were applied 5-7days as well as 8-10days (3day interval) after EAU induction into the randomized treatment eye and phosphate buffered saline (PBS) into the fellow control eye. Clinical and fluorescein angiography scoring (6 EAU grades) was done at each injection day and at enucleation day 14. Enucleated eyes were either scored histologically (6 EAU grades) or examined by ELISA for levels of IL-6, IL-17 and IL-6 soluble Receptor (sIL-6R).Uveitis developed in all 12 mice. Clinical uveitis score was significantly reduced (p=0.035) in treated eyes (median 2.0, range 0-4.0, n=12) compared to the fellow control eyes (median 3.0, range 1.0-4.0, n=12). Angiography scores were reduced in 9/12 treated eyes and histological scores in 3/4 treated eyes compared to the fellow control eyes. Cytokine levels were determined in 8 mice, of which 4 responded to anti-IL-6 treatment and 4 did not respond. All mice responding to treatment had a significant reduction of IL-6 (p0.01) and IL-17 (p=0.01) levels in treated eyes compared to the fellow control eyes. This difference was not seen in non-responding mice.Intravitreal anti-IL-6 treatment significantly attenuates experimental autoimmune uveitis in mice. EAU activity correlates with ocular IL-6 and IL-17 levels.

Purpose To investigate the effect of thermal stimulation of the retina (TS-R) on Bruch's membrane (BrM) thickness in age-related macular degeneration (AMD) mouse models as a novel concept for the prophylaxis and treatment of dry AMD. Methods Two knockout AMD mouse models, B6.129P2-Apoetm1Unc/J (ApoE-/-) and B6.129X1-Nfe2I2tm1Ywk/J (NRF2-/-), were chosen. One randomized eye of each mouse in four different groups (two of different age, two of different genotype) of five mice was treated by TS-R (532 nm, 10-ms duration, 50-μm spot size), the fellow eye served as control. Laser power was titrated to barely visible laser burns, then reduced by 70% to guarantee for thermal elevation without damage to the neuroretina, then applied uniformly to the murine retina. Fundus, optical coherence tomography (OCT), and fluorescein angiography (FLA) images were obtained at the day of treatment and 1 month after treatment. Eyes were enucleated thereafter to analyze BrM thickness by transmission electron microscopy (TEM) in a standardized blinded manner. Results Fundus images revealed that all ApoE-/- and NRF2-/- mice had AMD associated retinal alterations. BrM thickness was increased in untreated controls of both mouse models. Subvisible TS-R laser spots were not detectable by fundus imaging, OCT, or FLA 2 hours or 1 month after laser treatment. TEM revealed a significant reduction of BrM thickness in laser-treated eyes of all four groups compared to their fellow control eyes. Conclusions TS-R reduces BrM thickness in AMD mouse models ApoE-/- and NRF2-/- without damage to the neuroretina. It may become a prophylactic or even therapeutic treatment option for dry AMD. Translational relevance TS-R may become a prophylactic or even therapeutic treatment option for dry AMD.

PURPOSE. To investigate the effect of selective retina therapy (SRT) on the release of AMD-relevant cell mediators, such as matrix metalloproteinases (MMPs), VEGF, and pigment epithelium derived factor (PEDF) using different laser spot sizes and densities. METHODS. Porcine RPE-choroid explants were treated with a pulsed 532 nm Nd:YAG laser using (1) large spot sizes, (2) small spot sizes with a high-density (hd) treatment, and (3) small spot sizes with a low-density (1d) treatment. Explains were cultivated in modified Ussing chambers. RPE regeneration and RPE cell death were investigated by calcein-AM staining and immunofluorescence. The MMP release was examined via zymography and immunofluorescence. VEGF and PEDF secretion was analyzed by ELISA. RESULTS. During pigment epithelium regeneration (PER), mitosis and RPE cell migration were observed. Four days after SRT (large spot size) the content of active MMP2 increased significantly (P < 0.01). Hd treatment with small spot sizes resulted also in an increase of active MMP2 (P < 0.05). In immunofluorescence explants showed a localized expression of MMP2 within the healing lesions after irradiation. The PEDF level increased significantly (P = 0.01) after SRT with large spot sizes. VEGF secretion decreased significantly (P < 0.05) following SRT with large spot sizes and with hd treatment of small spot sizes. CONCLUSIONS. SRT induces a cytokine profile, which may improve the flux across Brach's membrane, slows down progression of early AMD by RPE regeneration, and inhibits the formation of choroidal neovascularization. The cytokine release depends on the size and density of applied laser spots.

Fucoidan is a heterogeneous group of sulfated polysaccharide with a high content of l-fucose, which can be extracted from brown algae and marine invertebrates. It has many beneficial biological activities that make fucoidan an interesting candidate for therapeutic application in a variety of diseases. Age-related macular degeneration and diabetic retinopathy are major causes for vision loss and blindness in the industrialized countries and increasingly in the developing world. Some of the characteristics found in certain fucoidans, such as its anti-oxidant activity, complement inhibition or interaction with the Vascular Endothelial Growth factor, which would be of high interest for a potential application of fucoidan in age-related macular degeneration or diabetic retinopathy. However, the possible usage of fucoidan in ophthalmological diseases has received little attention so far. In this review, biological activities of fucoidan that could be of interest regarding these diseases will be discussed.

BACKGROUND:Vascular endothelial growth factor-A (VEGF-A) is intensively investigated in various medical fields. However, comparing VEGF-A measurements is difficult because sample acquisition and pre-analytic procedures differ between studies. We therefore investigated which variables act as confounders of VEGF-A measurements. METHODS:Following a standardized protocol, blood was taken at three clinical sites from six healthy participants (one male and one female participant at each center) twice one week apart. The following pre-analytical parameters were varied in order to analyze their impact on VEGF-A measurements: analyzing center, anticoagulant (EDTA vs. PECT / CTAD), cannula (butterfly vs. neonatal), type of centrifuge (swing-out vs. fixed-angle), time before and after centrifugation, filling level (completely filled vs. half-filled tubes) and analyzing method (ELISA vs. multiplex bead array). Additionally, intrapersonal variations over time and sex differences were explored. Statistical analysis was performed using a linear regression model. RESULTS:The following parameters were identified as statistically significant independent confounders of VEGF-A measurements: analyzing center, anticoagulant, centrifuge, analyzing method and sex of the proband. The following parameters were no significant confounders in our data set: intrapersonal variation over one week, cannula, time before and after centrifugation and filling level of collection tubes. CONCLUSION:VEGF-A measurement results can be affected significantly by the identified pre-analytical parameters. We recommend the use of CTAD anticoagulant, a standardized type of centrifuge and one central laboratory using the same analyzing method for all samples.