Elisabeth Richert

Fucoidan Reduces Secretion and Expression of Vascular Endothelial Growth Factor in the Retinal Pigment Epithelium and Reduces Angiogenesis In Vitro

Fucoidan is a polysaccharide isolated from brown algae which is of current interest for anti-tumor therapy. In this study, we investigated the effect of fucoidan on the retinal pigment epithelium (RPE), looking at physiology, vascular endothelial growth factor (VEGF) secretion, and angiogenesis, thus investigating a potential use of fucoidan for the treatment of exudative age-related macular degeneration. For this study, human RPE cell line ARPE-19 and primary porcine RPE cells were used, as well as RPE/choroid perfusion organ cultures. The effect of fucoidan on RPE cells was investigated with methyl thiazolyl tetrazolium – assay, trypan blue exclusion assay, phagocytosis assay and a wound healing assay. VEGF expression was evaluated in immunocytochemistry and Western blot, VEGF secretion was evaluated in ELISA. The effect of fucoidan on angiogenesis was tested in a Matrigel assay using calcein-AM vital staining, evaluated by confocal laser scanning microcopy and quantitative image analysis. Fucoidan displays no toxicity and does not diminish proliferation or phagocytosis, but reduces wound healing in RPE cells. Fucoidan decreases VEGF secretion in RPE/choroid explants and RPE cells. Furthermore, it diminishes VEGF expression in RPE cells even when co-applied with bevacizumab. Furthermore, fucoidan reduces RPE-supernatant- and VEGF-induced angiogenesis of peripheral endothelial cells. In conclusion, fucoidan is a non-toxic agent that reduces VEGF expression and angiogenesis in vitro and may be of interest for further studies as a potential therapy against exudative age-related macular degeneration.

Effects of aflibercept on primary RPE cells: toxicity, wound healing, uptake and phagocytosis

Background/aim Anti-VEGF treatment is the therapy of choice in age-related macular degeneration, and is also applied in diabetic macular oedema or retinal vein occlusion. Recently, the fusion protein, aflibercept, has been approved for therapeutic use. In this study, we investigate the effects of aflibercept on primary RPE cells. Methods Primary RPE cells were prepared from freshly slaughtered pigs’ eyes. The impact of aflibercept on cell viability was investigated with MTT and trypan blue exclusion assay. The influence of aflibercept on wound healing was assessed with a scratch assay. Intracellular uptake of aflibercept was investigated in immunohistochemistry and its influence on phagocytosis with a phagocytosis assay using opsonised latex beads. Results Aflibercept displays no cytotoxicity on RPE cells but impairs its wound healing ability. It is taken up into RPE cells and can be intracellularly detected for at least 7 days. Intracellular aflibercept impairs the phagocytic capacity of RPE cells. Conclusions Aflibercept interferes with the physiology of RPE cells, as it is taken up into RPE cells, which is accompanied by a reduction of the phagocytic ability. Additionally, it impairs the wound healing capacity of RPE cells. These effects on the physiology of RPE cells may indicate possible side effects.

Alpha synuclein and crystallin expression in human lens in Parkinson's disease.

Visual features including cataract (lenticular opacity) are common in PD. On the other hand, cataract disease is associated with a higher risk of developing PD. Cataract surgery provides in vivo access to lens tissue, which permits a premortem PD biomarker search. Aggregated a-synuclein (aSYN) is the hallmark of PD, and cataract disease is caused by protein (crystallin) accumulation. In a physiological state, a-crystallins prevent protein aggregation, but this ability is lost in cataract disease. In Lewy bodies, a-crystallins colocalize with aSYN. Forced lenticular aSYN expression has induced cataract disease in mice. However, little is known about proteins in human PD cataract disease. During standard cataract surgery using phacoemulsification, we collected the rinsing fluid and lenses of 6 eyes from 5 PD patients (4 men, mean age 71.8 [67-77] years and mean disease duration 9.6 [2-18] years) and 9 eyes from 7 age-matched controls (2 men, mean age 72.9 [66-78] years). Ethical approval and written consent were obtained. The rinsing fluid (0.9% NaCl, substituted with gentamicin and Suprarenin (Infectopharm, Heppenheim, Germany)) with lens remnants was centrifuged (15 minutes, 2500 rpm) and followed by denaturating protein lysis. The supernatant was centrifuged with Amicon Ultra filters (Millipore, Billerica, Massachusetts), enriching the proteins in 1.5 ml of fluid (3 3 50 minutes, 4000g). The filtrate was centrifuged (15 minutes, 13,000 rpm), the supernatant was harvested, and the resulting pellet of residual lens fragments was subjected to lysis. Samples were separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie (BioRad, M€ unchen, Germany) or silver. Western blots were conducted for 3 samples of 2 PD patients and 4 controls for aSYN (abcam: Cambridge, UK, ab51252), aB-crystallin (Santa Cruz: Heidelberg, Germany, sc-22744), and ß-actin (Cell Signaling Technology: Danvers, USA, #4967) followed by horseradish peroxidase–tagged secondary antibody (Cell Signaling Technology). Each specimen was loaded into 3 lanes: supernatant (S), pellet (P), and residual lens fragments in the supernatant (RLFS). We always applied 40 lg for a-synuclein blots and 5 lg for the ß-crystallin blots for PD patients and controls. The molecular weight and densitometric volume of the bands (data not shown; available on request) were assessed. PD patients showed no definite uniform band pattern in the proteins retrieved from the supernatant or pellet, but differences were seen in the RLFS. In Coomassie stains, a band at 30 to 34 kDa, which was present in all controls, was absent or very lowly expressed in 5 of the 6 PD samples (2 of them were from the same patient) (Figure 1A). On the other hand, in silver stains, 2 of the PD samples (from different patients) displayed an additional band at 76 kDa, which was absent in controls (Figure 1B). aSYN appeared as single or double bands (mean 23.3 and 18.8 kDa), and aB-crystallin appeared as a single band (mean 18.9 kDa). aSYN was prominent in the supernatant, but scarce in the pellet. In PD patients, aSYN in the RLFS tended to be more prominent when compared with controls. To our knowledge, this is the first in vivo study investigating protein profiles of PD cataract lenses. However, not all soluble oligomeric forms are detected by the sulfate polyacrylamide gel electrophoresis used here. Therefore, chromatography, proteomic analysis and differentiation of native versus phosphorylated aSYN should be performed in future studies to further entangle the role of aSYN in cataract formation. In conclusion, the possibility of detecting PD-associated proteins in living patients would benefit the diagnosis and treatment of these patients. Although our data did not reveal a robust biomarker, there are indications of differences between the lenses of PD patients and controls. We encourage further research of lenses in PD patients.

Reduction of GAPDH in lenses of Parkinson's disease patients: A possible new biomarker.

We previously showed that the protein pattern of lenses removed in cataract surgery differs between patients with Parkinsons disease and age‐matched controls. In this study, we identified the protein reduced in abundance in the 34‐ to 37‐kDa gel band.

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.

Intravitreal injection of anti-Interleukin (IL)-6 antibody attenuates experimental autoimmune uveitis in mice

Purpose To evaluate the effect of an intravitreally applied anti‐IL‐6 antibody for the treatment of experimental autoimmune uveitis (EAU). Methods 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 5–7 days as well as 8–10 days (3 day 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). Results 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 (p < 0.01) 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. Conclusions Intravitreal anti‐IL‐6 treatment significantly attenuates experimental autoimmune uveitis in mice. EAU activity correlates with ocular IL‐6 and IL‐17 levels. HighlightsNew treatment possibility for Experimental Autoimmune Uveitis (EAU).Intravitreal anti‐IL‐6 injections attenuate EAU.EAU activity is correlated with ocular IL‐6 and IL‐17 levels.

Thermal Stimulation of the Retina Reduces Bruch's Membrane Thickness in Age Related Macular Degeneration Mouse Models

Purpose To investigate the effect of thermal stimulation of the retina (TS-R) on Bruchs 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.

Release of Different Cell Mediators During Retinal Pigment Epithelium Regeneration Following Selective Retina Therapy

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 (ld) treatment. Explants 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 Bruchs 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.