Retinal Pigment Epithelium Expressed Toll-like Receptors and Their Potential Role in Age-Related Macular Degeneration

(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

Differences in Uptake and Intracellular Fate between Bevacizumab and Aflibercept after Repetitive Long-Term Treatment in the Retinal Pigment Epithelium

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. 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

Interaction of High-Molecular Weight Fucoidan from Laminaria hyperborea with Natural Functions of the Retinal Pigment Epithelium

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

The Influence of Melatonin and Light on VEGF Secretion in Primary RPE Cells

(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

a clinical observation of vascular complications after ocular surgery

Background. Ocular vascular occlusions following intraocular procedures are a rare complication. We report a case series of patients with retinal vascular occlusions or anterior ischemic optic neuropathy (AION) after anterior and posterior segment surgery and demonstrate possible risk factors. Methods. Observational case series. Results. In ten patients, vascular occlusions were observed within ten weeks after intraocular surgery: branch retinal arterial occlusion (BRAO) (n = 2), central retinal artery occlusion (CRAO) (n = 2), central retinal vein occlusion (CRVO) (n = 1), branch retinal vein occlusion (BRVO) (n = 1), anterior ischemic optic neuropathy (AION) (n = 3), and combined central artery and vein occlusion (n = 1). AION occurred later (27–69 d) than arterial occlusions (14–60 d) or venous occlusions (1-2 d). In all cases, either specific surgical manipulations or general vascular disorders were identified as risk factors. In addition to general cardiovascular risk factors (arterial hypertension n = 6, diabetes mellitus n = 4), internal workup disclosed bilateral stenosis of the carotid arteries (n = 1) and myeloproliferative syndrome (n = 1). Conclusion. Vascular occlusions after surgical ocular procedures seem to be more frequent when cardiovascular diseases coexist. Surgical maneuvers and intra- or postoperative pressure changes may act as a triggering mechanism in patients with underlying systemic cardiovascular disorders. Affected patients should undergo thorough internal examination to identify possible underlying diseases

Patients' perspective on emergency treatment of ophthalmologic diseases during the first phase of SARS-CoV2 pandemic in a tertiary referral center in Germany - the COVID-DETOUR questionnaire study

Background During the first wave of the COVID-19 pandemic, the need of treatment of urgent ophthalmological diseases and the possible risk of a SARS-CoV-2 infection had to be weighed against each other. In this questionnaire study, we aimed to analyze potential barriers and patients' health beliefs during and after the lockdown early 2020 in a tertiary referral center in Kiel, Germany. Results Ninety-three patients were included, 43 in subgroup A (before April 20th) and 50 in subgroup B (April 20th or later). Retinal disorders were the most common causes for admission (approximately 60%).. Only 8 patients (8.6%) experienced a delay between their decision to visit a doctor until the actual examination. Every fourth patient was afraid of a COVID-19 infection, and expected a higher likelihood for an infection at the hospital. Patients with comorbidities tended to be more likely to be afraid of an infection (correlation coefficient 0.183, p = 0.0785) and were significantly more likely to be concerned about problems with organizing follow-up care (corr. Coefficient 0.222, p = 0.0328). Higher age was negatively correlated with fear of infection (corr. Coefficient - 0.218, p-value 0.034). Conclusion In this questionnaire study, only a minority of patients indicated a delay in treatment, regardless of whether symptoms occurred before or after the lockdown before April 20th, 2020. While patients with comorbidities were more concerned about infection and problems during follow-up care, patients of higher age - who have a higher mortality - were less afraid. Protection of high-risk groups should be prioritized during the SARS-CoV-2 pandemic. Trial registration The study was registered as DRKS00021630 at the DRKS (Deutsches Register Klinischer Studien) before the conduction of the study on May 5th, 2020

Effects of a Newly Developed Enzyme-Assisted Extraction Method on the Biological Activities of Fucoidans in Ocular Cells

Fucoidans from brown seaweeds are promising substances as potential drugs against age-related macular degeneration (AMD). The heterogeneity of fucoidans requires intensive research in order to find suitable species and extraction methods. Ten different fucoidan samples extracted enzymatically from Laminaria digitata (LD), Saccharina latissima (SL) and Fucus distichus subsp. evanescens (FE) were tested for toxicity, oxidative stress protection and VEGF (vascular endothelial growth factor) inhibition. For this study crude fucoidans were extracted from seaweeds using different enzymes and SL fucoidans were further separated into three fractions (SL_F1-F3) by ion-exchange chromatography (IEX). Fucoidan composition was analyzed by high performance anion exchange chromatography (HPAEC) after acid hydrolysis. The crude extracts contained alginate, while two of the fractionated SL fucoidans SL_F2 and SL_F3 were highly pure. Cell viability was assessed with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay in OMM-1 and ARPE-19. Protective effects were investigated after 24 h of stress insult in OMM-1 and ARPE-19. Secreted VEGF was analyzed via ELISA (enzyme-linked immunosorbent assay) in ARPE-19 cells. Fucoidans showed no toxic effects. In OMM-1 SL_F2 and several FE fucoidans were protective. LD_SiAT2 (Cellic®CTec2 + Sigma-Aldrich alginate lyase), FE_SiAT3 (Cellic® CTec3 + Sigma-Aldrich alginate lyase), SL_F2 and SL_F3 inhibited VEGF with the latter two as the most effective. We could show that enzyme treated fucoidans in general and the fractionated SL fucoidans SL_F2 and SL_F3 are very promising for beneficial AMD relevant biological activities

Fucoidan Does Not Exert Anti-Tumorigenic Effects on Uveal Melanoma Cell Lines

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

Evaluation of a Brown Seaweed Extract from Dictyosiphon foeniculaceus as a Potential Therapeutic Agent for the Treatment of Glioblastoma and Uveal Melanoma

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