Search

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.

Oxidative stress is considered a major factor in the deterioration of retinal pigment epithelium (RPE) cells in dry age-related macular degeneration (AMD). The MAPK ERK1/2 can be activated by oxidative stress, may exert both pro- and anti-apoptotic functions, and has recently been proposed as a major factor in RPE degeneration in atrophic changes. Nrf2 is a master regulator of oxidative stress defense and ERK1/2 is an upstream activator of Nrf2. In this study, we investigate the participation of ERK1/2 in oxidative stress pathways in connection with Nrf2.Nrf2 knock-out and wild-type primary RPE cells were prepared from mouse eyes. Oxidative stress was induced by different concentrations of t-butylhydroperoxide. Mitogen-activated protein kinases (MAPKs) were blocked by commercially available inhibitors (SB203580, U0126, SP600125). Cell viability was determined by MTT assay. ERK1/2 expression and activation were assessed by Western blotting.Oxidative stress induced concentration dependent cell death, which occurred at lower concentrations in Nrf2 knock-out RPE. Western blot analysis displayed a biphasic activation of ERK1/2 in murine wild-type RPE and the inhibition of late, but not early activation of ERK1/2 exerted protection in wild-type murine RPE cells. The biphasic activation of ERK1/2 is lost in Nrf2 knock-out mice, and inhibition of ERK1/2 was generally protective. The inhibition of MAPK JNK or p38 exerted no protection, irrespective of Nrf2.RPE cells display a biphasic activation of ERK1/2 after oxidative insult, of which the late activation is pro-apoptotic. The biphasic activation is lost in Nrf2 knock-outs, suggesting that early ERK1/2 activation may be connected to Nrf2 signaling. In addition, ERK1/2 activation in Nrf2 knock-outs mediates oxidative stress-induced cell death.

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.

Purpose Microglia are the local cells of the innate immunity in the retina. Toll-like receptor (TLR) 3 is a receptor of the innate immune system, recognizing viral double-stranded RNA. Retinal pigment epithelium (RPE) cells express TLR-3 and react to TLR-3 stimulation. In this study, we investigated the effect of TLR-3-activated RPE on microglia. Methods Primary porcine RPE cells were prepared from freshly prepared pigs' eyes. Retinal microglia were prepared from porcine retina. Expression of the microglia marker Iba1 was evaluated using immunocytochemistry. RPE cells were treated with polyinosinic/polycytidylic acid (Poly I:C; 100 ng/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) for 24 hr. Either the supernatant was applied to microglia for 6 or 24 hr or microglia cells were directly treated with Poly I:C for 6 and 24 hr. Expression of interleukin (IL)-1s, IL-6, tumour necrosis factor (TNF)α, IL-10, Cox2 and iNOS was evaluated in quantitative PCR. Phagocytosis was evaluated with a microscope-based and a fluoroscan-based phagocytosis assay. Results Retinal pigment epithelium (RPE) cells induce the expression of IL-6, IL-1s and IL-10 in microglia cells. Microglia cells respond to Poly I:C stimulation in a concentration-dependent manner with the induction of IL-1s, IL-6, TNFα, Cox2, iNOS and, to a lesser degree, IL-10. Stimulation of microglia cells with supernatant of Poly I:C-treated RPE cells further elevated IL-6, IL-1s and Cox2 expression, while it reduced the expression of iNOS. No changes in phagocytosis could be detected. Conclusions TLR-3-activated RPE exacerbates inflammatory response of microglia in a differentiated manner. This indicates that viral infections in the RPE may have a proinflammatory influence on retinal microglia.

Age-related macular degeneration (AMD) is a complex, degenerative and progressive disease involving multiple genetic and environmental factors. It can result in severe visual loss e.g. AMD is the leading cause of blindness in the elderly in the western countries. Although age, genetics, diet, smoking, and many cardiovascular factors are known to be linked with this disease there is increasing evidence that long-term oxidative stress, impaired autophagy clearance and inflammasome mediated inflammation are involved in the pathogenesis. Under certain conditions these may trigger detrimental processes e.g. release of vascular endothelial growth factor (VEGF), causing choroidal neovascularization e.g. in wet AMD. This review ties together these crucial pathological threads in AMD.

Purpose: To compare the influence of aerobic and resistance exercise on intraocular pressure (IOP). Methods: Twenty-one healthy subjects participated. Aerobic exercise was performed using a cycle ergometer, and resistance exercise was performed with a leg curl and a butterfly machine. Intraocular pressure was measured at baseline, during exercise and 10 min after. During resistance exercise, a Valsalva manoeuvre was prevented. Results: Before aerobic exercise, the mean IOP was 18.8 ± 2.7 mmHg. It was 16.5 ± 2.8 after 10, 17.1 ± 2.6 after 20 and 16.7 ± 3.3 mmHg after 30 min of exercise. After 10 min, the IOP returned to baseline (18.8 ± 2.7 mmHg). The mean IOP before resistance exercise with the leg curl machine was 17.0 (15.6–18.4; 65%Wmax) and 16.8 (15.3–18.3) mmHg; 75%Wmax) and did not change significantly during the experiment. The mean IOP before resistance exercise with the butterfly machine (65%Wmax) was 16.4 (15.2–17.6) and increased to 17.2 (16.0–18.4) mmHg (p

Purpose The retinal pigment epithelium (RPE) is a major source of vascular endothelial growth factor (VEGF) in the eye. Despite the role of VEGF in ocular pathology, VEGF is an important factor in maintaining the choroid and the RPE. Accordingly, the VEGF is constitutively expressed in RPE. In this study, the regulation of constitutive VEGF expression was investigated in an RPE/choroid organ culture. Methods To investigate VEGF regulation, RPE/choroid of porcine origin were used. VEGF content was evaluated with enzyme-linked immunosorbent assay. The influence of several molecular factors was assessed with commercially available inhibitors (SU1498, bisindolylmaleimide, LY294002, nuclear factor kappaB [NFkB] activation inhibitor, mithramycin, YC-1, Stattic, SB203580). For toxicity measurements of inhibitors, primary RPE cells of porcine origin were used, and toxicity was evaluated with methyl thiazolyl tetrazolium assay. Results VEGF secretion as measured in the RPE/choroid organ culture was diminished after long-term (48 h) inhibition of vascular endothelial growth factor receptor-2 by VEGFR-2-antagonist SU1498. VEGF secretion was also diminished after phosphatidylinositol 3 kinase was inhibited by LY294002 for 48 h. Coapplication of the substances did not show an additive effect, suggesting that they use the same pathway in an autocrine-positive VEGF regulation loop. Inhibition of protein kinase C by bisindolylmaleimide, on the other hand, did not influence VEGF secretion in organ culture. Inhibition of the transcription factor SP-1 by mithramycin displayed effects after 24 h and 48 h. Inhibiting hypoxia-inducible factor-1 (HIF-1) and Stat3 did not show any influence on constitutive VEGF secretion. Inhibition of the transcription factor NFkB diminished VEGF secretion after 6 h (earliest measured time point) and remained diminished at all measured time points (24 h, 48 h). The same pattern was found when the inhibitor of mitogen-activated kinase p38 was applied. A combination of NFkB and p38 inhibitors displayed an additive effect, completely abolishing VEGF secretion. Conclusions Constitutive VEGF secretion in the RPE/choroid seems to be regulated by the transcription factor NFkB and the mitogen-activated kinase p38 in an independent manner. Constitutive VEGF secretion may be regulated to a lesser extent by the transcription factor SP-1, while Stat3 and hypoxia-inducible factor-1 do not seem to be involved. Additionally, VEGF secretion seems to be regulated long-term by an autocrine positive loop via vascular endothelial growth factor receptor-2 and phosphatidylinositol 3 kinase.

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.

Vascular endothelial growth factor-signaling in human tenon fibroblasts (hTFs) has recently become a target for antifibrotic treatment in glaucoma filtration surgery. The anti-VEGF antibody bevacizumab (BVC) has been shown to increase filtration bleb size. Given the relatively high concentration of BVC needed to obtain an effect, we investigated whether BVC acts through VEGF inhibition or via non-antigen-dependent ways.Human tenon fibroblast primary cultures were obtained from strabismus surgery subjects. Under low (0.2%) and high (10%) serum conditions, cells were incubated with BVC, ranibizumab (RNB), aflibercept (AFB), or rituximab (RTX) at different concentrations. Total number of cells and number of dead or proliferating (5-bromo-2-deoxy-uridine-positive) cells were assessed after 24 hours. Concentrations of VEGF-A in cell culture media was measured with ELISA. Intracellular IgG was detected with immunostaining and Western blot analysis.In quiescent hTF culture (0.2% serum) the addition of 5 mg/mL BVC induced widespread cell death. Under proliferative conditions (10% serum), BVC reduced the number of proliferating cells. No such effect was observed with 2.5 mg/mL BVC or with 10 mg/mL AFB or 2.5 mg/mL RNB, although they were equally effective in binding free VEGF-A in the culture media. Instead, the CD20 antibody RTX, which did not bind VEGF, induced hTF death and inhibited proliferation in a BVC-comparable fashion. Bevacizumab, AFB, and RTX were detected intracellularly in a concentration-dependent manner.The cell death-inducing and antiproliferative effect of 5 mg/mL BVC appeared not to depend on VEGF inhibition. Our data question a direct role of VEGF for hTF survival and proliferation.

Exsudative altersbedingte Makuladegeneration (AMD) ist die haufigste Ursache akuter submakularer Blutungen (SMB). Unbehandelt fuhren SMB bei AMD praktisch immer zur Ausbildung einer Makulanarbe mit stark eingeschrankter Sehfahigkeit. Es gibt verschiedene chirurgische Therapieansatze, aber bisher noch kein allgemein anerkanntes einheitliches Therapieverfahren. Die subretinale Koapplikation von „recombinant tissue plasminogen activator“ (rtPA) und Bevacizumab im Rahmen einer Vitrektomie mit Gastamponade ist ein neues Verfahren, das in klinischen Studien gute funktionelle Ergebnissen gezeigt hat. Das Ziel der gleichzeitigen Gabe von rtPA und Bevacizumab ist es, zu einem moglichst fruhen Zeitpunkt erstens die Blutung aus der Fovea zu verdrangen und zweitens die der Blutung zugrunde liegende Neovaskularisationsmembran effektiv antiangiogenetisch zu behandeln. Experimentelle Untersuchungen haben gezeigt, dass rtPA und Bevacizumab bei gleichzeitiger Gabe kompatibel sind.

The purpose of this study was to investigate release of matrix metalloproteinases (MMP) 2 and 9 during retinal pigment epithelium (RPE) wound healing after Selective Retina Therapy (SRT) with laser energy levels below and above the threshold of RPE cell death. Following exposure to SRT using a prototype pulsed Nd:YLF laser with energies of 80-180 mJ/cm(2) fresh porcine RPE-monolayers with Bruch's membrane and choroid were cultured in modified Ussing chambers which separate the apical (RPE-facing) and basal (choroid facing) sides of the RPE monolayer. Threshold energy for RPE cell death and wound healing were determined with calcein-AM viability test. Inactive and active forms of MMP 2 and 9 were quantified within tissue samples and in the culture medium of the apical and basal compartments of the Ussing chamber using gelatine zymography. Laser energies of 160-180 mJ/cm(2) resulted in cell death within 1 h while 120-140 mJ/cm(2) resulted in delayed death of exposed RPE cells. All cells survived 80 and 100 mJ/cm(2). Laser spots healed within 6 days after SRT accompanied by a transient vectorial increase of MMPs. SRT with 180 mJ/cm(2) increased active MMP 2 by 1.9 (p < 0.05) and 1.6 (p < 0.05) fold in tissue and basal compartments, respectively, without alterations in the apical compartment. Pro-MMP 2 levels were also significantly increased in all compartments (p < 0.05). Release of MMP 9 was not altered. Laser energy below the threshold of RPE cell death did not alter the release of MMP 2 or 9. The findings suggest that the release of active MMP 2 on the basal side of the RPE during wound healing following SRT may address age-related pathological changes of Bruch's membrane with a potential to slow degenerative macular ageing processes before irreversible functional loss has occurred.

Acta Ophthalmol. 2010: 88: 885–890 Abstract. Purpose: Prior to phakic intraocular lens implantation, it is important to obtain precise knowledge of the anterior chamber depth (ACD). Accurate topographic evaluation of the iridocorneal angle is helpful in estimating risk for angle-closure glaucoma. This study investigated the use of the Orbscan II system to measure ACD and the iridocorneal angle in healthy subjects and assessed the influences of age, gender and spherical equivalent on these parameters. Methods: The Orbscan II system was used to determine the ACD and iridocorneal angle in eight different positions in 390 healthy White subjects with a mean age of 41 ± 16 years (range 10–80 years). The sample included 242 male and 148 female subjects. The influences of age, gender and spherical equivalent were assessed using multiple regression analysis. Results: Mean ACD was 2.87 ± 0.04 mm in male subjects and 2.81 ± 0.37 mm in female subjects. The explanatory variables relevant to the ACD were age (partial regression coefficient B = − 0.0115, p

Purpose The purpose of this study was to assess the light-induced cytotoxicity of indocyanine green (ICG) using different light sources commonly used in macular surgery and to assess the effect of optical filters. Methods Primary cultures of porcine retinal pigment epithelium cells were incubated with 0.5 mg/mL ICG solution dissolved in 5% glucose and illuminated with a surgical light fiber for 3 or 15 minutes. Halogen, mercury vapor, xenon, and metal halide light sources were used. Cell viability was assessed using the MTT assay. Retinal pigment epithelium cells without illumination served as controls. The decomposition of ICG after illumination was analyzed by high-performance liquid chromatography. Results Illumination of retinal pigment epithelium cells with all light sources with or without previous incubation with ICG did not affect cell viability compared with controls. Cell viability was significantly reduced when the cells were not rinsed immediately after incubation. The cytotoxic effect was abolished by a 475-nm long-pass filter. The high-performance liquid chromatography analysis of the illuminated ICG solution identified six cytotoxic ICG decomposition products. Conclusion Optical filters that narrow the emission spectrum of the light sources reduce the light-induced cytotoxicity of ICG. Optical filters applied in ICG-assisted macular surgery may reduce the risk of intraoperative cell damage.

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.

Aim To test the microablation of excess graft choroidal tissue with the pulsed electron avalanche knife (PEAK-fc) in an in-vitro model of autologous retinal pigment epithelium (RPE)–choroid translocation. Methods Choroidal tissue of porcine RPE–choroid explants was ablated with the PEAK-fc. Tissue morphology was assessed by light microscopy (LM) and scanning electron microscopy (SEM). The amount of ablated choroidal tissue was analysed as a function of three PEAK-fc parameters: (1) amplitude of biphasic voltage (70–100%); (2) distance between choroidal tissue and tip of the PEAK-fc (0–300 μm); and (3) exposure time (2–8 s). Results LM and SEM showed a smooth plain within the ablation area with well defined cutting edges and preserved adjacent tissue structure. The mean amount of ablated tissue correlated linearly with applied voltage (range 79–120 μm, r=0.34) and distance between choroidal tissue and PEAK-fc tip (range 10–100 μm, r=0.74). The mean amount of ablated tissue increased with exposure time between 2 and 4 s (36–88 μm, r=0.4) and remained constant between 4 and 8 s. Conclusion The PEAK-fc accurately microablates choroidal tissue in-vitro. The adjacent choroidal tissue structure and Bruch9s membrane are preserved. Patient studies are required to test the PEAK-fc in RPE–choroid translocation surgery.

PURPOSE. To investigate the influence of VEGF antagonists on the barrier function of the retinal pigment epithelium and underlying mechanisms. METHODS. Porcine RPE cells were cultured on six-well membrane inserts. The cells were exposed to bevacizumab (62.5 g/mL) or ranibizumab (25 g/mL) for 24 hours (short term) or 9 days (long term). Transepithelial flux of FITC-dextran and intracellular levels of reduced glutathione (GSH) at normal and low-glucose conditions were investigated at different points in time. The influence of the addition of triamcinolone acetonide (TA) was investigated. The effect of GSH depletion on RPE permeability was examined using L-buthionine sulfoximine (BSO), a -glutamylcysteine synthethase inhibitor. RESULTS. After short-term exposure, VEGF antagonists increased the transepithelial flux of FITC-dextran significantly on day 2. Bevacizumab, but not ranibizumab, increased permeability up to 9 days. Under long-term exposure, both drugs enhanced permeability for 7 days; bevacizumab had the stronger effect. The addition of TA inhibited this increase. At the ninth day of short- and long-term exposure, bevacizumab-exposed cells, but not ranibizumab-exposed cells, exhibited a significantly lower GSH level. In the low-glucose condition, both drugs accelerated the decrease of intracellular GSH for the first 48 hours. GSH depletion increased the permeability of retinal pigment epithelium. TA had no effect on BSO-induced GSH depletion. CONCLUSIONS. The results suggest that bevacizumab and ranibizumab may decrease RPE barrier function, with bevacizumab exhibiting a prolonged and more profound effect. Combination with TA is thought to be beneficial because of its protective effect on stabilizing RPE junctional integrity. (Invest Ophthalmol Vis Sci. 2010;51:4848‐4855) DOI:10.1167/iovs.09-4699

Deferoxamine mesylate is clinically used as a chelating agent but might induce retinopathy. To evaluate its effect on the retinal pigment epithelium (RPE), porcine RPE cells were stimulated with deferoxamine. Cell death was assessed with trypan blue exclusion assay. To investigate the pathway of cell death, the mitogen-activated protein kinases (MAPKs) Erk, JNK, and p38 were inhibited with U0126, SP600125, and SB203580, respectively. Their activity was determined by Western blot. Deferoxamine induces significant cell death in RPE cells, accompanied by phosphorylation of p38 and Erk. Inhibition of p38 attenuates cell death. In conclusion, deferoxamine is directly toxic on RPE cells, its toxicity depending on p38.

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.

We have previously shown that bevacizumab, but not ranibizumab, is taken up by porcine RPE cells. In this study, the effects of bevacizumab and ranibizumab on proliferation, wound healing and phagocytosis of the RPE were investigated.Primary porcine RPE cell culture were prepared from fresh eyes, cultivated and treated with clinically relevant concentrations of bevacizumab or ranibizumab respectively. Proliferation was investigated in a proliferation assay, wound healing in a wound scratch assay and phagocytosis was investigated by feeding RPE cells photoreceptor outer segment-opsonized FITC-labeled latex beads.Bevacizumab, and to a lesser extend ranibizumab, impair the proliferation of RPE cells but do not affect wound healing. Bevacizumab, but not ranibizumab, reduces the phagocytotic function of RPE cells.The uptake of bevacizumab reduces phagocytosis in RPE cells, which indicates possible long-term effects of repeated bevacizumab treatment.

Hintergrund und Ziel Daten zur Expression von Matrixmetalloproteinase-19 (MMP-19) in der humanen Kornea liegen in der Literatur nicht vor. Ziel dieser Arbeit ist es, die Expression von MMP-19 in der humanen Kornea zu untersuchen. Im MMP-19-knock-out-Mausmodell soll eine mogliche Bedeutung dieser Protease bei der kornealen Wundheilung aufgezeigt werden.

Purpose Vascular endothelial growth factor (VEGF) is a fundamental factor for angiogenesis. It plays important roles in pathological conditions (e.g. the development of wet AMD), but also in the healthy organism) e.g. in maintaining the vasculature and supporting the retina). Recent therapies to treat the wet AMD focus on neutralizing VEGF indiscriminately. VEGF is constitutively expressed in the retina, but its expression is upregulated by various (noxious) stimuli, e.g. oxidative stress or hypoxia. Discrimination between constitutive expression of VEGF and its pathological upregulation might provide the possibility of focusing on inhibiting the pathological expression only. Here, we focused on the influence of different mitogen-activated protein kinase (MAPK) (p38, Erk, JNK) on the secretion and expression of VEGF, with or without being challenged by oxidative stress. Methods VEGF secretion was measured using a perfusion organ culture model; expression was examined in primary RPE culture and Western blotting. Results Constitutive VEGF expression and secretion can be diminished by inhibiting p38, while inhibiting Erk or JNK does not show a significant effect. When challenged with oxidative stress (250 µM t-butylhydroperoxide), VEGF expression and secretion increases and the influence of the MAPK changes: While p38 still accounts for about 30% of the secretion, Erk shows a similar influence. Inhibiting JNK presents conflicting results. In organ culture, inhibiting JNK significantly increases VEGF secretion after stimulation with 250 µM tBH, while with regard to VEGF expression in RPE cell culture, this effect could not be seen. Conclusion Constitutive and oxidative stress induced VEGF secretion, and expression is differently regulated, which might offer an opportunity to selectively inhibit pathological VEGF expression only.

Introduction For ongoing progress in refractive surgery, exact knowledge about the anatomical properties of the cornea is useful. Thus, the aim of the study was to characterize the thinnest point of the cornea compared with the central corneal thickness in normal subjects and to investigate with regard to influencing factors such as sex, age, refraction, and intraocular pressure. Materials and methods The central corneal thickness and the thinnest point of the cornea were determined with the Orbscan II in 390 white normal subjects. Difference between the 2 eyes, influence of sex, and measuring repetition accuracy were tested for statistical significance with t tests, and the influence of age was tested with nonparametrical test methods. Results In the right eyes, the mean central corneal thickness was 548 +/- 37 microm and the thinnest point 537 +/- 37 microm. In the left eyes, the mean central corneal thickness was 547 +/- 37 microm and the thinnest point 535 +/- 39 microm. The difference between the central corneal thickness and the thinnest point was found to be significant in both eyes in paired t test (P > 0.001). No influence of sex, refraction, and intraocular pressure on the thickness of the thinnest point of the cornea could be observed. The difference between central corneal thickness and thickness at the thinnest point was not subject to a statistically significant influence of age. Conclusions In the calculation of the residual corneal layer thickness in laser refractive surgery, the thinnest point of the cornea should form the basis.

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.

Age-related macular degeneration (AMD) is the main cause for legal blindness of the elderly in the industrialized world and due to the demographic changes of the Western societies the prevalence is expected to increase strongly. The late forms of this disease are differentiated into geographic atrophy and exsudative (wet) AMD, where vessels grow from the choroid into the retina. On a biological level, the most important structure ofAMD pathogenesis in the photoreceptor/retinal pigment epithelium (RPE)/choroid complex. The interaction of the components of this structure enables the photoreceptors to function. Age-related alterations of this complex play an important role in the development of AMD. The exact triggers for AMD onset, however, are not known. There are no available therapies for the early forms of AMD or for geographic atrophy. However, the exsudative form can be treated by inhibiting vascular endothelial growth factor (VEGF). The currently available therapeutics (ranibizumab, aflibercept, bevacizumab) show good results in the clinic, however, in order to uphold the therapeutic effect they have to be regularly injected into the vitreous body. The inhibitors differ on a molecular level as well as on a biological level concerning their interaction with retinal pigment epithelial cells.

Monocytes are important mediators of inflammatory processes and are in the focus of immunological studies. While the preparation of human monocytes is widely established, little is published on the isolation of porcine monocytes for experimental studies. The aim of this study is to establish a cost efficient method of preparing and culturing porcine monocytes of considerable purity and reasonable yield. In our method, we combined and modified different protocols of human monocyte preparation. The blood of a single pig is harvested and treated with EDTA to prevent coagulation. Peripheral blood mononuclear cells are obtained by a density gradient centrifugation using a Bicoll gradient and monocytes are harvested by culturing on serum-treated culture flasks, rinsing and tapping. A high yield is obtained by constant cooling of flasks and tubes. The purity of the culture is evaluated by the expression of CD14, using flow cytometry. Using this method, we reached a purity of 92.6 % (+/− 3.06 %). With this procedure, we established a reliable method to prepare and cultivate porcine monocytes which can be performed cost effectively and does not require special equipment.

Immunophilin ligands such as FK506 and Cyclosporin A, used in immunosuppression, are well-characterized drugs. In the past, they had been the center of attention as a putative therapeutic strategy for neuroregeneration and neuroprotection. In contrast to Cyclosporin A, FK506 readily crosses the brain-blood-barrier and, thus together with its derivatives, may represent a novel approach to the treatment of neurological disorders. FK506 exerts profound neuroprotective and neuroregenerative effects in vivo and in vitro. The mechanism underlying neuroregeneration is fairly well understood. It is independent of the inhibition of calcineurin, which is responsible for the immunosuppression, but operates via the binding of FKBP52 and the heat shock protein (Hsp) 90. In contrast, the underlying pathways of neuroprotection are far less understood. Protection is apparently independent of calcineurin, as shown by non-calcineurin inhibiting derivatives, such as V-10,367 and GPI-1046, but the intracellular actions remain to be defined. FK506 has been shown to interfere with the apoptotic pathway of neuronal cells, including inhibiting JNK activity, cytochrome c release, caspase 3 activation, and CD95 ligand expression. These effects are in part mediated by the inhibition of calcineurin and may not contribute to protection. Our recent studies suggest that the protective properties of FK506 and its non-calcineurin inhibiting derivatives are realized by a fast induction of heat shock proteins. The induction of the heat shock response by immunophilin ligands might prove to be an interesting target for neuroregeneration and neuroprotection.

Purpose Members of the vascular endothelial growth factor (VEGF) family are strongly involved in pathological processes in the retina, such as age-related macular degeneration and diabetic retinopathy. Cells of the retinal pigment epithelium (RPE) constitutively secrete VEGF-A, and the secretion of placental growth factor (PlGF) has also been described. RPE cells are strongly polarized cells with different secretome at the apical and basal side. In this study, we evaluated the basal and apical regulation of VEGF-A and PlGF secretion in RPE/choroid explants and primary RPE cells. Methods RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers, separating apical (RPE) and basal (choroid) supernatant. Primary RPE cells were also prepared from porcine eyes and cultivated on Transwell plates. Explants and cells were treated with inhibitors for VEGFR-2 (SU1498), p38 (SB203580), and the transcription factors nuclear factor-kappa B (NF-κB) and SP-1 (mithramycin), respectively. VEGF-A and PlGF content was evaluated with enzyme-linked immunosorbent assay (ELISA). In addition, western blots were performed. Results In the RPE/choroid, VEGF-A can initially be found on the apical and basal sides with significantly more pronounced secretion on the basal side. VEGF-A secretion is differentially regulated on the apical and basal sides, with the inhibition of SP-1 and NF-κB showing strong effects apically and basally after 24 h and 48 h, the inhibition of p38 displaying its effect mainly on the basal side with some effect apically after 48 h, and the inhibition of VEGFR-2 reducing the secretion of VEGF only on the apical side at 24 h and 48 h. In the RPE cell culture, similar effects were found, with inhibition of NF-κB or SP-1 displaying a strong decrease in VEGF-A on both sides, and p38 inhibition displaying only an inhibitory effect on the basal side. In contrast, an apical effect of VEGFR-2 inhibition was not found. However, the western blot experiments exhibited a significant decrease in the VEGF-A protein under SU1498 treatment. In the RPE/choroid organ cultures, PlGF was initially found mainly on the basal site with only minute amounts of PlGF found apically. NF-κB and SP-1 were strongly involved in PlGF regulation apically and basally, while VEGFR2 and to a lesser degree p38 displayed some regulation at the basal site. In the primary RPE cell culture, PlGF was not found on the apical or basal side. Conclusions VEGF-A and PlGF were constitutively secreted and regulated by the RPE/choroid complex, with PlGF secreted mainly by the choroid. Although the transcription factors NF-κB and SP-1 were involved in apical and basal regulation of both growth factors, VEGFR-2 displayed a strong polarity, with regulation of apical VEGF-A and basal PlGF secretion.

Background/aims Subretinal coapplication of recombinant tissue plasminogen activator (rtPA) and vascular endothelial growth factor (VEGF)-antagonists is a new treatment option for age-related macular degeneration complicated by submacular haemorrhage. Here, we investigate the compatibility of rtPA and aflibercept or ranibizumab in vitro because intraoperatively, rtPA or rtPA-induced plasmin may cleave aflibercept or ranibizumab. Methods Aflibercept and ranibizumab, respectively, were incubated with rtPA or plasmin, separated in gel electrophoresis and stained with Coomassie or silver. The antiangiogenic activity of the VEGF-antagonists was quantified by VEGF-ELISA after incubation with the supernatant of primary porcine retinal pigment epithelium cell cultures. Results In electrophoresis, ranibizumab displayed no additional fragments when it was coapplied with rtPA or plasmin. Its VEGF-inhibiting efficacy remained unchanged in coapplication with rtPA with or without blood, or plasmin. rtPA did not cleave or functionally compromise aflibercept. When aflibercept was coapplied with plasmin, electrophoresis displayed additional bands in Coomassie (30 kDa, 27 kDa, 19 kDa, 15 kDa) and silver staining (31 kDa, 26 kDa, 21 kDa, 19 kDa, 15 kDa). While at a clinical dosage (800 µg/mL) VEGF was inhibited by aflibercept when coapplied with plasmin, at borderline concentrations (400 ng/mL) VEGF-binding ability of aflibercept was abolished. Conclusions Ranibizumab is not cleaved or functionally compromised by rtPA or plasmin. Aflibercept is cleaved and its VEGF-binding ability is reduced when coapplied with plasmin. In clinical practice, rtPA and ranibizumab can be coapplied as a treatment for neovascular age-related macular degeneration with submacular haemorrhage while the antiangiogenic activity of aflibercept may be compromised when coapplied with rtPA in the presence of plasmin.

Purpose To describe long-term functional and morphologic retinal changes by deferoxamine, a chelating agent used to treat systemic iron overload. Methods Ophthalmologic examination of acute deferoxamine maculopathy and follow-up of 5 years, using ophthalmologic examination, fluorescence angiography, electrophysiology, color vision testing, fundus image-correlated microperimetry, time-domain and spectral-domain optical coherence tomographies. The patient is a 53-year old woman who presented with acute visual deterioration to 20/50 in the right eye, 20/60 in the left eye, and reduced color vision. She had been treated with deferoxamine for 6 months. The diagnosis of deferoxamine maculopathy was made, and the drug discontinued immediately. Results After 5 months, visual acuity was 20/25 bilaterally, but mottling of the retinal pigment epithelium (RPE) had developed. Five years later, visual acuity was 20/20 in both eyes. Fundus autofluorescence showed progressively clumped hyper- and hypofluorescence with larger areas of total RPE atrophy in the left eye. Spectral-domain optical coherence tomography visualized areas of RPE thickening, photoreceptor outer segment elongation, outer retinal tubulation, and outer retinal and RPE atrophy. Fundus image-correlated microperimetry showed relative central scotomas at the beginning, some of which recovered to normal light sensitivity, while others deteriorated to absolute scotomas. Conclusion Although visual acuity had recovered after cessation of deferoxamine, functional and morphologic residues remained, which included disturbed color vision, central microscotomas, and progressive RPE and outer retinal degeneration.

The anti-VEGF antibody bevacizumab is widely used off-label for the treatment of various ocular diseases, most commonly in age-related macular degeneration and diabetic macular edema. Bevacizumab is able to penetrate the retina and is found in the choroid after intravitreal injection in a time dependent manner. It has previously been shown to be taken up by the retinal pigment epithelium (RPE). In this study, we have investigated the intracellular pathway following uptake of bevacizumab in RPE cells, tested both in primary porcine RPE cells and in the human cell line ARPE19. Bevacizumab displays a characteristic, time-dependent pattern of intracellular distribution, as detected by immunofluorescence and pulse chase experiments. In both primary cells and the cell line, intracellular bevacizumab can be found after seven days, as detected by immunofluorescence and Western blotting. Immediately after application, bevacizumab partially colocalizes with Rab5, indicating some uptake in early endosomes. Intracellularly, bevacizumab is detected in the cytoskeletal fraction, aligning with actin filaments, as revealed by subcellular fractioning and immunofluorescence. Bevacizumab seems to travel along actin filaments by myosin7a, as determined by triple staining immunofluorescence. Interestingly, over a period of seven days, bevacizumab seems to accumulate in certain storage areas, as observed by immunofluorescence. Furthermore, results obtained with immunocytochemistry, Western blotting and flow cytometry indicate that bevacizumab may be released from the RPE cells via exosomes. In conclusion, bevacizumab is taken up by and transported in the retinal pigment epithelial cells in a characteristic, time-dependent manner, where it seems to move along actin filaments by myosin7a and seem to be partially released from the cells via exosomes.

Localized application of hyperthermia is a potential treatment for retinal diseases. Vascular endothelial growth factor (VEGF) derived from the retinal pigment epithelium (RPE) is implicated in a variety of retinal pathologies. As it has been recently shown that hyperthermia may induce VEGF in the RPE, the aim of this study was to investigate hyperthermia-induced VEGF secretion and the pathways of hyperthermal VEGF upregulation in the RPE. The human RPE cell line (Arpe-19) was exposed to 40°, 42°, 45° and 50 °C for one, five and 15 min. Cell viability was evaluated using a trypan blue exclusion assay, VEGF secretion was evaluated by an enzyme-linked immunosorbent assay ELISA) and VEGF expression was investigated using a Western blot. Involvement of mitogen-activated protein kinase (MAPK) pathways (ERK1/2, JNK, p38) and transient receptor potential vanilloid (TRPV) channels on VEGF induction was investigated using commercially available inhibitors (U0126, SB203580, SP600125, ruthenium red). Expression and phosphorylation of MAPKs was investigated using a Western blot. Hyperthermia induces time- and temperature-dependent cell death in human RPE cells. VEGF expression and secretion is induced by hyperthermia in a time- and temperature-dependent manner mediated by p38 and to a lesser degree by JNK. TRPV channels seem to play a minor role in regulation of hyperthermia-induced VEGF secretion. Hyperthermia induces temperature-dependent secretion of VEGF in the RPE, which is mediated by p38 and, to a lesser extent, JNK. This may lead to undesired effects from hyperthermal treatment of retinal diseases.

Retinal pigment epithelium (RPE) cells may alter the phenotype of monocytes by soluble factors that may be influenced by stimulation of the RPE. Since RPE cells carry the toll-like receptor-3 (TLR3) that detects and reacts to viral infection through binding of dsRNA we investigated the effects of RPE cells with or without TLR3 stimulation on blood-derived monocytes with respect to regulation of pro-/anti-inflammatory cytokines, anti-angiogenic factors and migratory properties. Primary RPE cells were prepared from porcine eyes; monocytes were prepared from porcine blood. TLR3 activation was induced by polyinosinic:polycytidylic acid (Poly I:C). RPE cells were stimulated with Poly I:C in different concentrations for 24 hours and a cell culture supernatant was applied to the monocytes. Expression of CD14 and Fas ligand (FasL) was determined via flow cytometry. The expression of IL-6, IL-1s, TNFα, Cox2, iNOS and IL-10 was determined via quantitative RT-PCR. Migration was determined using Boyden chamber experiments. The supernatant of RPE cells, irrespective of TLR3 activation, induced FasL expression in the monocytes. Expression of iNOS and Cox2 was reduced by RPE cells and the reduction of Cox2 but not if iNOS was lost under TLR3 activation. No induction of IL-6, IL-1s, IL-10 or TNFα by the RPE was seen. TLR3-activated RPE cells induced monocyte migration. RPE cells induce an upregulation of FasL and a downregulation of iNOS and Cox2 without upregulating inflammatory cytokines, possibly inducing an anti-angiogenic phenotype in the monocytes. This phenotype is still upheld after challenging RPE cells with dsRNA, mimicking a viral infection.

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.

Purpose: Toll-like receptor 3 (TLR3) is a receptor of the innate immune system, recognizing double-stranded RNA. TLR3 can lead to cytokine release or apoptosis and has recently been associated with the development of geographical atrophy via cytotoxic effects on the retinal pigment epithelium (RPE). The current study was conducted to elucidate the underlying pathways of TLR3 effects in the RPE. Methods: TLR3 activation via polyinosinic acid/polycytidylic acid (Poly I:C) was investigated in primary porcine RPE cells, focussing on cell death and vascular endothelial growth factor (VEGF) secretion. Primary cells were stimulated with different concentrations of Poly I:C. Cell death was investigated in trypan blue exclusion assay and cell death detection ELISA. VEGF and IFN-s secretion were also detected in ELISA. As Mitogen-activated protein kinases (MAPK) play an important part in TLR3-mediated signal transduction, we investigated the influence of JNK, ERK1/2 and p38 on cell death and VEGF secretion, using commercially available inhibitors. Results: Activation of TLR3 by Poly I:C induced concentration-dependent cell death, partly mediated by JNK. ERK1/2 was activated and exerted some protection. Furthermore, higher concentrations of Poly I:C increased VEGF secretion after 4 and 24 hr, which was independent of MAPK. Conclusion: The induction of cell death in RPE cells by TLR3 activation confirms possible involvement of TLR3 activation in GA. As cell death is partly mediated by JNK, more studies should be conducted investigating the role of JNK in RPE cell death to evaluate whether its inhibition might be a new therapeutic opportunity for the treatment of geographical atrophy. Additionally, effects on VEGF secretion can be found.

Purpose Transpupillary thermotherapy is a hyperthermia treatment for small uveal melanomas but its use is controversially discussed. In uveal melanomas, vascular endothelial growth factor (VEGF) increase is correlated with metastases, and VEGF increase can be found in treated melanomas. In this study, the effect of hyperthermia on the VEGF secretion in human RPE cells was studied. Methods Immortal human retinal pigment epithelium (RPE) cell line Arpe-19 was exposed to 40°, 42°, 45° and 50°C for 1 min, 5 min and 15 min. Toxicity was evaluated using trypan blue exclusion assay and VEGF secretion was evaluated by ELISA. Involvement of Mitogen activated protein kinase (MAPK) pathways and Transient Receptor Potential Vanilloid (TRPV) channels on VEGF induction was investigated using commercially available inhibitors. Results Hyperthermia induces cell death in a time and temperature dependent manner. VEGF expression and secretion is strongly induced by hyperthermia in a time and temperature dependent manner. VEGF induction is mediated by p38 and to a lesser degree by JNK. TRPV channels are only involved in VEGF secretion at lower temperatures. Conclusion Hyperthermia induces a temperature dependent secretion of VEGF in human RPE, which is mediated by p38. As VEGF may be involved in the development of micrometastases, these findings indicate that thermotherapy for the treatment of uveal melanomas should be regarded with caution.

Objective To investigate the compatibility of recombinant tissue plasminogen activator (rtPA) and bevacizumab in vitro because during surgery, rtPA or rtPA-induced plasmin may cleave and inactivate bevacizumab. Methods To simulate the intraoperative range of mixing ratios of rtPA, bevacizumab, and subretinal blood, we calculated the volumes of 12 submacular hemorrhages (SHs) with a spherical cap formula using measurements derived from fundus photographs and spectral-domain optical coherence tomographic images. Bevacizumab was incubated with rtPA or plasmin before gel electrophoresis with Coomassie blue and silver staining. The anti-angiogenetic activity of bevacizumab in the presence of rtPA with or without clotted human blood or of plasmin was quantified by vascular endothelial growth factor–enzyme-linked immunosorbent assay after incubation with the supernatant of porcine retinal pigment epithelium cell cultures. Results The mean (SD) volume of SH was 28.6 (24.7) mm 3 (range, 6.2-94.6 mm 3 ). In sodium dodecyl sulfate–polyacrylamid electrophoresis with Coomassie blue or silver staining, bevacizumab displayed characteristic patterns of protein bands. No additional fragments were detected in co-application of bevacizumab with either rtPA or plasmin. The anti-angiogenetic activity of bevacizumab remained unchanged in co-application with rtPA with or without blood or plasmin. Conclusions We demonstrated the absence of cleavage or functional inactivation of bevacizumab by rtPA in an in-vitro model of their intraoperative co-application as a treatment of SH. Clinical relevance In clinical practice, rtPA and bevacizumab can be co-applied as a treatment for neovascular age-related macular degeneration with SH to simultaneously clear SH and reduce choroidal new vessel activity.

The production of Vascular Endothelial Growth Factor (VEGF) in the retina is important to maintain the vasculature in the choroid and has protective function on the retinal pigment epithelium and the neuroretina. The expression of VEGF is mainly regulated by the presence of oxygen, a decline of oxygen partial pressure resulting in an activation of Hypoxia Inducible Factor 1α (HIF-1α), inducing the expression of VEGF. A plethora of other factors are also involved, including oxidative stress, hyperglycemia, or inflammatory cytokines. An increase in VEGF secretion can lead to pathological vascularization in the retina, as seen in exudative age-related macular degeneration (AMD), retinopathy of prematurity or in diabetic retinopathy. In order to treat pathological neovascularizations in the retina, VEGF antagonists have been introduced into the clinic and approved for the treatment of wet AMD. Recently, VEGF-antagonists have also been approved for the treatment of diabetic macular edema. New products are developed, e.g., VEGF-Trap Eye or VEGF receptor antagonists which are currently being tested in clinical trials. VEGF siRNAs are also being tested. VEGF-antagonists neutralize secreted VEGF by inhibiting the binding of VEGF to its receptor. Additional pathways are possible, e.g., interfering with autoregulatory pathways. VEGF-receptor antagonists inhibit the signal transduction induced by VEGF binding. Anti-VEGF-siRNA intracellularly inhibits the expression of VEGF and might also exert an RNA specific, VEGF-independent effect.

Vascular endothelial growth factor (VEGF) has emerged as a major player in retinal diseases and is strongly involved in choroidal neovascularisation in exudative age-related macular degeneration (AMD) or in the development of macular edema in diabetic retinopathy. An important source of VEGF in the retina is the Retinal Pigment Epithelium (RPE). The RPE is a highly polarized cell layer situated between the choroid and the photoreceptors and among its many functions is the secretion of cytokines. RPE-derived VEGF is important for the development of the vasculature of the retina in the premature eye. In the adult eye, the RPE constitutively secrets low amounts of VEGF, mainly on the basolateral side, in order to maintain the choroid and protect endothelial cells. Also, neuroprotective properties of (apical) VEGF have been described. Main isoforms of RPE-secreted VEGF are VEGF165 and, to a lesser extent, VEGF121. Also, minor amounts of VEGF189 are can be found. VEGF165b, an anti-angiogenic isoform, might also be secreted by the RPE. Under different noxious stimulations, RPE cells increase their VEGF secretion, via several, stimulus specific, pathways. Among these stimuli, hypoxia, oxidative stress, hyperglycemia, several cytokines and endoplasmic reticulum stress might be closely connected to the pathogenesis of exudative AMD and other neovascular alterations of the retina. Loss of polarity might also contribute to inappropriate VEGF secretion and subsequent neovascular changes. The dichotomy of protective physiological and vision-threatening pathological VEGF secretion renders the differences of physiological and pathological VEGF expression regulation a highly interesting target for the development of long-term VEGF inhibiting drugs.

To determine reference values for the HMC anomaloscope (Heidelberg multi-color anomaloscope) of healthy subjects.One hundred and thirteen healthy subjects were divided into four age groups:20 years of age (ten female, five male), 20-39 years of age (23 female, 15 male), 40-59 years of age (23 female, ten male) and60 years of age (nine female, 18 male). Match midpoint, matching range (MR) and anomaly quotient (AQ), according to the Moreland equation [blue (436 nm) + blue-green (490 nm) = cyan (480 nm) + yellow (589 nm)] and according to the Rayleigh equation [green (546 nm) + red (671 nm) = yellow (589 nm)] were determined. The neutral adaptation was done showing white light every 5 seconds in absolute mode and every 15 seconds in relative mode.The mean match midpoint according to the Rayleigh equation was 43.9 ± 2.6 scale units in absolute mode. It was highest between 20-39 years (45.2 ± 2.2) and lowest in subjects60 years of age (42.2 ± 2.2). The mean MR in absolute mode was 3.1 ± 3.5 scale units with a maximum60 years (4.4 ± 4.4). The MR in relative mode was between 1.6 ± 1.9 (20-39 years) and 4.4 ± 3.8 (60 years). The resulting mean AQ was 1.01 ± 0.15 in both modes. The mean match midpoint of the Moreland equation was 51.0 ± 5.2 scale units in absolute mode. It was highest between 20-39 years (52.5 ± 5.7), and lowest in subjects60 years of age (48.7 ± 3.6). The mean MR according to the Moreland equation was lower in absolute mode (13.4 ± 15.6) than in relative mode (16.2 ± 15.2). The mean resulting AQ was 1.02 ± 0.21 in both modes.The values of this study can be used as references for the diagnosis of red-green and blue perception impairment with the HMC anomaloscope.

Smoking is a strong environmental factor for the development of age-related macula degeneration. In this study, we investigated the effects of nicotine on RPE cell function in porcine in vitro models, focussing on cell death, VEGF secretion and phagocytotic ability. For these experiments, perfusion organ culture and primary RPE cell culture were used and exposed to nicotine up to 7 days. Survival was investigated in primary porcine RPE cells in an MTT and trypan blue exclusion assay. VEGF secretion was investigated in a porcine perfusion organ culture model using ELISA. A phagocytosis assay using FITC-labelled latex beads in primary RPE cells was used to assess the phagocytotic ability of the cells. Nicotine does not induce cell death in the RPE at any time point up to 7 days of stimulation at any tested concentration. VEGF secretion, however, is diminished compared to untreated control already after 1 day of nicotine treatment and even more profoundly up to 7 days. Furthermore, phagocytotic ability of the RPE is diminished by nicotine in the highest concentrations tested (100 μM). Nicotine impedes RPE function (VEGF secretion, phagocytosis), which could be directly involved in the development of dry AMD and geographic atrophy.

To investigate the influence of alcohol consumption on the occurrence of open-globe injuries in adults.A retrospective study was made of 100 consecutive patients (81 male, 19 female) with open-globe injuries. Of these patients, 18 exhibited alcohol intoxication (group Ai), and 82 exhibited no alcohol intoxication (group nAi). Investigated parameters were best-corrected visual acuity at day of admission and last examination (logMAR), type of injury according to BETT-classification, extraocular injuries, cause of injury, time and setting of injury, in relation to alcohol consumption and tested for statistical significance with Fisher's exact test or the Mann-Whitney U test, respectively.In group Ai, 83.3% of the patients were male, and in group nAi, 80.5%. Mean logMAR at day of admission was 1.06 ± 0.63 (20/250) in group Ai and 1.08 ± 0.59 (20/250) in group nAi. At last examination, mean logMAR in group Ai was 1.11 ± 0.59 (20/250), in group nAi 0.75 ± 0.60 (20/125). This difference was statistically significant (p = 0.02). In group Ai, significantly more ruptures according to BETT classification occurred (p = 0.05). In group Ai, significantly more additional extraocular injuries occurred compared to group nAi (38.9% versus 6.1%; p = 0.0009). In group Ai, the cause of injury was significantly more often glass (44.4% versus 2.4%; p = 0.0000), in group nAi the injury was more often directly or indirectly caused by tools (74.4% versus 33.3%; p = 0.001). In group Ai, the injury was significantly more often inflicted by others (50.0% versus 9.8%; p = 0.0003). The settings in which the injuries occurred were significantly more often the street in group Ai (44.4% versus 6.1%; p = 0.0002), in group nAi the garden or tool shed (31.7% versus 5.6%; p = 0.02) or the workplace (34.2 % versus 11.1 %; p = 0.04). In group Ai, the injuries occurred significantly more often at night (p = 0.0001) and on weekends (p = 0.0000).Open-globe eye injuries under alcohol intoxication are more often caused by a third party and have a worse prognosis. Open-globe injuries under alcohol intoxication occur in a different spatio-temporal setting and exhibit a more severe type of injury. Risk behavior combined with alcohol consumption therefore seems to be an independent factor for the incidence of open-globe eye injuries.

The retinal pigment epithelium (RPE) is the most active phagocyte in the human body, and this function is indispensable for vision. In this study, we introduce an easy and inexpensive way to quantify RPE phagocytosis by opsonizing FITC-labeled latex beads of a defined size with a crude mixture of photoreceptor outer segment fragments, using cells of porcine origin. After performance of the desired experiment, phagocytosis can easily be quantified by counting the ingested particles. With this protocol, we combine the advantage of a natural target with the advantages of the defined properties of latex beads. Moreover, in order to conduct these phagocytosis experiments, as laboratory equipment, in addition to cell culture equipment, only a centrifuge and a simple fluorescence microscope are needed.

Background Vascular endothelial growth factor (VEGF) antagonists are currently the therapy of choice for agerelated macular degeneration. Here we compared the effects of FDA-approved Ranibizumab and off-label used Bevacizumab on RPE cells, investigating their respective uptake by RPE cells over time. Methods Primary porcine RPE cells were treated with Bevacizumab or Ranibizumab, respectively. Uptake of the respective VEGF-antagonists was assessed with confocal laser scanning microscopy and flow cytometry. Cell death was assessed with MTT assay and VEGF secretion was measured with ELISA. Results When clinical doses were applied for 1 h, Bevacizumab was taken up by RPE cells as assessed by confocal laser scanning microscopy and flow cytometry. After 24 h of incubation, and further assessed after 1d, 5d, and 7d, Bevacizumab was detected in RPE cells where it accumulated over time. The presence of Bevacizumab within RPE cells after 7d was confirmed by flow cytometry. While some Ranibizumab was found in RPE cells after 1 h of incubation when assessed with confocal laser microscopy but not by flow cytometry, no signal above control was detected after 1d, 5d, or 7d. Neither substance induced significant cell death after 7 days and no inhibitory effect on VEGF secretion was observed after day 3 of culture. Conclusions Bevacizumab, but not Ranibizumab, accumulates in RPE cells over time, implying substantial differences between these two drugs.

Aims: To evaluate the changes of morphological and functional characteristics of the retinal pigment epithelium (RPE)-choroid perfusion culture during cultivation. Methods: PorcineRPE-choroid tissue was cultivated in a perfusion tissue culture system. After the indicated times, histology, immunolocalization of collagen IV and von Willebrand factor, RPE cell viability with calcein-AM, TUNEL assay and occludin immunolocalization of RPE cells were examined. The tissue was treated with selective RPE treatment laser after different time periods and the wound healing response was characterized. Vascular endothelial growth factor secretion was measured by enzyme-linked immunosorbent assay. Results: On day 8, prominent morphological degenerative changes of RPE cells were observed in histology. According to the immunohistochemistry for collagen IV, the Bruch’s membrane did not display any obvious decomposition until day 8. Von Willebrand factor staining decreased during cultivation, especially at the choriocapillaris. Calcein-AM staining and TUNEL assay displayed the increase of apoptotic changes in only a minority of the cells on day 4, but in many cells on day 8. Occludin delocalization was observed on day 8. Selective RPE treatment laser-produced wounds were completely closed by monolayer RPE when wounded on fresh and 3-day-old cultures, but not when wounded on 6-day-old cultures. Vascular endothelial growth factor secretion was stable between days 2 and 5, but increased after that. Conclusion: Under the stated culture perfusion conditions, porcine RPE-choroid tissue was suitable for experimentation up to 5 days of maintenance.

Purpose Vascular endothelial growth factor (VEGF) antagonists are the therapy of choice for age-related macular degeneration. Ranibizumab and pegaptanib have been approved by the United States Food and Drug Administration, whereas bevacizumab is used off label. In this study, the authors compare these VEGF inhibitors directly regarding their efficiency to neutralize VEGF in a quantifiable in vitro system. Methods Porcine retina-retinal pigment epithelium-choroid organ culture and RPE cell culture were prepared from fresh eyes, cultivated in a perfusion chamber, and treated with clinically relevant concentrations of bevacizumab, ranibizumab and pegaptanib. VEGF content of the supernatant was analyzed with ELISA. Additionally, the influence of bevacizumab and ranibizumab on intracellular VEGF was analyzed with Western blot. Results At clinically significant doses, bevacizumab (0.25 mg/mL) and ranibizumab (0.125 mg/mL) neutralized VEGF completely for 6 hours, whereas pegaptanib (0.08 mg/mL) showed no effect. Bevacizumab and ranibizumab neutralized VEGF significantly up to 16 hours. When diluted, bevacizumab lost its inhibiting properties at a concentration of 975 ng/mL, and ranibizumab neutralized VEGF up to a concentration of 120 ng/mL. Both substances significantly diminished VEGF expression in Western blot. Conclusions At clinical doses, bevacizumab and ranibizumab are equally potent in neutralizing VEGF. To neutralize VEGF completely in this system, a fraction of the clinical dose is needed. Ranibizumab is more efficient at neutralizing VEGF when diluted. Pegaptanib showed no effect in this system, which might help explain the clinical experience regarding this drug. A direct effect of ranibizumab and bevacizumab on VEGF protein expression indicates additional pathways of VEGF inhibitors.

PURPOSE In several retinal complications, such as age-dependent macular degeneration (AMD), oxidative stress is increased and cytokine level is elevated. These are shown to alter the activation and expression of matrix metalloproteinase (MMP) both in human primary and immortalized retinal pigment epithelial (RPE) cells. However, the effects on human embryonic stem cell (hESC)-derived RPE cells remain to be elucidated. METHODS The mature hESC-RPE cells were exposed to inflammatory cytokines (IFN-γ or TNF-α) for 24 hours or oxidative stress (H2O2) for 1 hour. Effects on barrier properties were analyzed with transepithelial electrical resistance (TEER), the expression of MMP-1, MMP-2, MMP-3, MMP-9, collagen I, and collagen IV genes with quantitative RT-PCR, and the expression of MMP-1 and MMP-3 proteins with Western blot or ELISA, respectively. Also, activation and secretion of MMP-2 and -9 proteins were analyzed with zymography. RESULTS In normal state, mature hESC-RPE cells expressed MMP-1, -2, -3, and -9 genes in low levels, respectively. Tumor necrosis factor-α increased MMP-1 and -2 gene expression, and H2O2 increased MMP-3 and -9 gene expression. Zymography revealed IFN-γ- and TNF-α-induced secretion of MMP-2 and high-molecular-weight species of MMP (HMW MMP), but H2O2 decreased their secretion. Furthermore, TNF-α and H2O2 significantly decreased barrier properties. CONCLUSIONS Here, cytokines induced the MMP-1 and -2 gene and protein expression. Also, H2O2 induced MMP-3 and -9 gene expression, but not their protein secretion. These data propose that under oxidative stress and cytokine stimuli, mature hESC-RPE cells resemble their native counterpart in the human eye in regard to MMP secretion and expression and could be used to model retinal disorders involving alterations in MMP activity such as AMD, diabetic retinopathy, or proliferative vitreoretinopathy in vitro.

6-Hydroxydopamine (6-OHDA) causes death of dopaminergic neurons by mitochondrial dysfunction with JNKs as central mediators. Here we provide novel insights into specific actions of JNK isoforms in 6-OHDA-induced death of PC12 cells. Twenty five μm 6-OHDA enhanced total JNK activity in the cytoplasm, nucleus, and at the mitochondria. Inhibition of JNKs by 2 μm SP600125 or transfection with dominant-negative JNK2 (dnJNK2) rescued more than 60% of the otherwise dying PC12 cells after 24 h, whereas transfection with dnJNK1 had no protective effects. In contrast to constitutively present JNK1, JNK2 amounts increased in the nucleus and at the mitochondria after 6-OHDA stimulation. JNK inhibition by SP600125 or transfection of dnJNK2 reduced the pool of active JNKs in the nucleus, the release of cytochrome c, as well as the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase-1. Transfection with dnJNK1, however, had no effects on the translocation of JNKs to the mitochondria or the release of cytochrome c. Our data provide novel functional insights into the pathological role of individual JNK isoforms, the signalosome at the mitochondria, and the mode of JNK-induced release of cytochrome c.