Postersitzungen, Donnerstag, 1.10.2015

 
Foyer Estrel 13:15 - 14:15 01.10.2015
Postersitzung PDo01
Retina: Grundlagen 1 Retina: Basics 1
Vorsitzende/r: Anselm Kampik (München), Olaf Strauß (Berlin)

Referent/in: Nicole Schäfer (Regensburg)
Purpose: The CFH-related proteins (CFHR) comprise a group of five plasma proteins structurally and functionally related to the main negative complement regulator, factor H (CFH). Mutations within the CFH/CFHR gene cluster are associated with various human diseases such as age-related macular degeneration (AMD), the leading cause of irreversible vision loss in the elderly. Deletion of the CFHR3/1 genes goes along with a decreased risk for AMD although the precise functions of the CFHR proteins still remain unclear. Antibody-dependent blocking of CFHR3 to elucidate the influence of complement activity in AMD has not been studied so far. The aim of this work comprised the extended elucidation of CFHR3 in complement regulation in degenerative eye diseases with the help of previously generated high-affinity monoclonal antibodies (mAbs) against that CFH-related protein. Methods: MAbs were tested for specificity and avidity against human CFHR3 in indirect enzyme-linked immunosorbent assay (ELISA) and Western blot. MAbs were used for immunoprecipitation from human serum and following mass spectrometry analysis. Complement modulation was analyzed in functional in vitro hemolysis assays and complement ELISA. Sandwich ELISA for detection of CFHR3 complexes from serum of AMD patients compared to healthy controls was established. Interactions of CFH and CFHR3 with the oxidative stress marker CEP (ω-(2-carboxyethyl)pyrrole) and the complement component 3b (C3b) were tested. Results: Generated mAbs were highly specific for human CFHR3. MAb 269-5 showed the highest avidity and immunoprecipitated CFHR3 in complex with alternative and terminal complement components from human serum. Anti-CFHR3 mAbs inhibited the complement pathways in in vitro analyses. Binding of CFH and CFHR3 to the AMD risk factor CEP and to C3b could be shown. Both proteins competed for binding to these molecules, and mAb 269-5 interfered with this competition by inhibiting CFHR3 interaction. MAbs 269-5 and 552-3 detected CFHR3 complexes from human serum of AMD patients and healthy controls. Conclusion: The mAb-based inhibition of CFHR3 implies an attenuation of complement reactivity. We showed, consistent with genetic analyses, that CFHR3 is involved in complement dysregulation in AMD patients. A competition of CFH and CFHR3 to oxidative stress epitopes and C3b impairs the local complement system homeostasis in the eye. We identified CFHR3 as a promising target for immune modulation in AMD patients.
Referent/in: Jana Koch (Freiburg)
Background: Myeloid cells (MC), such as macrophages, neutrophils and microglia (MG) have been implicated in the development of choroidal neovascularization (CNV) in patients with exudative age-related macular degeneration. The Interferon-Regulating Factor 8 (IRF8) is a central transcription factor in myeloid cell maturation, differentiation and lineage commitment. The aim of this study was to determine the role of IRF8 for retinal MG development, neurovascular homeostasis and the development of CNV. Methods: Irf8-Venus mice were studied to determine the basal expression levels of Irf8 in the retina. Next, adult Irf8-deficient mice (Irf8-/-) and age-matched controls (WT) were assessed for MG distribution and morphology by immunohistochemstry. The vascular phenotype and the retinal function of Irf8-/- mice were analysed by immunohistochemistry and electroretinography respectively. The expression of factors involved in homeostasis but also clearance of apoptotic cells, including MERTK, CD64, CX3CR1 and F4/80 was determined in MG of Irf8-/- and WT mice by flow cytometry. Finally, the role of Irf8 for pathological choroidal angiogenesis was assessed using the laser-induced CNV model. Results: Irf8 Venus reporter mice demonstrated that Irf8 is exclusively expressed in retinal MG in the steady state. Deficiency of Irf8 was associated with an overall reduction of retinal MG density and severely altered MG morphology in adult Irf8-/- compared to WT mice. Flow cytometry analysis of MG cells revealed a downregulation of CD64 and CX3CR1 as well as an upregulaton of MERTK and F4/80 in adult Irf8-/- mice indicating a disturbed cellular homeostasis. The retinal vasculature and neuroretinal function were similar in adult Irf8-/- and WT animals. In the laser-induced CNV model, however, Irf8-deficient mice exhibited reduced myeloid cell recruitment to sites of laser injury and increased size of CNV compared to WT controls. Conclusion: Our study demonstrates that Irf8 is vital for MG development and function but not for retinal neurovascular development. Our findings indicate that IRF8 is a critical factor for transforming microglia into a reactive phenotype thereby suppressing retinal inflammation and development of CNV.
Referent/in: Jakob Siedlecki (München)
Fragestellung: Die anti-VEGF-Therapie ist der Therapiestandard bei neovaskulären Erkrankungen des Auges. Der klinische Erfolg beruht auf einer Verringerung der Gefäßleckage, ist aber durch eine unzureichende Zurückdrändung neovaskulärer Gefäße limitiert. Ein Grund dafür ist die Rekrutierung des Perizyten, der zusammen mit der Endothelzelle Kapillaren bildet und neue Gefäße vor einer Rückbildung schützt. Ein Schlüsselmolekül der Resistenz ist hierbei PDGF (platelet derived growth factor), das Perizyten zu den Orten der Angiogenese migrieren und dort proliferieren lässt. Axitinib ist ein Multi-Tyrosinkinaseinhibitor, der vorrangig VEGF-R und PDGF-R hemmt. Die vorliegende Studie untersucht potentielle inhibitorische Effekte von Axitinib auf den Perizyten anhand zellulärer Migration und Proliferation. Methodik: Humane primäre Perizyten (hPC-PL) wurden mit Axitinib-Konzentrationen von 0,1pg/ml bis 20µg/ml behandelt. Zur Messung antiproliferativer Eigenschaften wurde der kolorimetrische MTT-Assay genutzt. Zellmigration wurde mittels Boyden Chamber nach Stimulation mit PDGF sowie simultaner Zugabe von 0,5, 1 und 2,5µg/ml Axitinib evaluiert. Zum Ausschluß toxischer Effekte wurden ein MTT-Assay sowie eine fluoreszenzmikroskopische Färbung mittels Hoechst-Farbstoff und Propidiumiodid durchgeführt. Ergebnisse: Axtinib reduzierte die perizytäre Proliferation signifikant zwischen 1 und 20µg/ml (p< 0.05), die IC50 betrug 1.63µg/ml ± 0.01µg/ml (SD r2=0.95). Die perizytäre Migration wurde durch PDGF deutlich erhöht (p< 0.001), eine Konzentration von 0.5µg/ml Axitinib normalisierte diesen Effekt wieder (p< 0.001). 2.5µg/ml Axitinib senkten die Migration signifikant unter das Ausgangslevel ohne PDGF-Stimulation (p< 0.001). Alle gezeigten Effekte lagen außerhalb des toxischen Konzentrationsbereichs von Axitinib. Schlussfolgerung: In dieser in vitro-Studie konnte gezeigt werden, dass Axitinib ein potenter Inhibitor der perizytären Migration und Proliferation ist. Eine Ergänzung der anti-VEGF-Therapie um PDGF-Inhibition mittels Axitinib könnte somit auch im klinischen Einsatz vielversprechend sein und neben der Verringerung der Gefäßleckage eine Rückbildung neovaskulärer Gefäße ermöglichen.
Referent/in: Jihong Lin (Mannheim)
Background: Retinal degeneration is accompanied by glia activation. Glial cells play an important role in the cross-talk between neurons and vascular cells. The impaired neurovascular coupling results in retinal neurodegeneration and vasoregression. Müller glia undergo reactive gliosis following acute injury or chronic neuronal stress. Inflammatory responses, in particular microglia, are involved in retinal degeneration. Recruitment and activation of microglia are regulated by Notch signaling and lipid metabolic products such as docosahexaenoic acid (DHA). Additionally, retinal vessel maturation and quiescence as well as mural cell recruitment are linked to Notch signaling. Recently, a critical role of sEH plays a critical role in regulation of vascular formation and inflammatory biology was described. Moreover, ɷ-3 polyunsaturated fatty acids (PUFA) such as docosahexenoic acid (DHA) can attenuate early retinal degeneration. However, the role of sEH in retinal vasoregression is still unclear. Aim: We want to know if sEH inhibition can prevent retinal vasoregression in rats. Methods: The transgenic polycystic kidney disease (PKD) rat is a model of ciliopathy, which expresses a truncated human polycystin-2 gene. Male homozygote PKD rats were daily treated with intraperitoneal injection of a sEH specific inhibitor (30mg/kg) for four weeks. Retinal digestion preparation was used for quantitative morphometry. sEH expression and microglial activation were quantified upon using cryosection and whole mount immunofluorecent staining, respectively. sEH activity was measured using a mass spectrometer. Inflammatory and immune-related cytokines were detected using real time PCR. Activation of the Notch signaling pathway was analyzed using western blotting. Results: sEH was upregulated in Müller cells of PKD rats. Retinal vasoregression was prevented by sEH inhibition. sEH inhibition was able to effectively reduce retinal pericyte loss and to repress pericyte migration. sEH inhibition decreased the activation of microglia and altered microglial distribution in PKD rats. The inflammatory cytokines were down-regulated. Moreover, Notch signaling pathway was involved in regulation of microglial recruitment.
Referent/in: Margrit Hollborn (Leipzig)
Purpose: Diabetic retinopathy is associated with osmotic stress resulting from hyperglycemia and intracellular sorbitol accumulation. Systemic hypertension is the main secondary risk factor of diabetic retinopathy. High intake of dietary salt increases extracellular osmolarity resulting in hypertension. We determined the effects of high extracellular osmolarity, chemical hypoxia, and oxidative stress on the gene expression of enzymes involved in sorbitol production and conversion in cultured human retinal pigment epithelial (RPE) cells. Methods: Alterations in the expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH) genes were examined with real-time RT-PCR. Protein levels were determined by Western blot analysis. Nuclear factor of activated T cell 5 (NFAT5) was knocked down with siRNA. Results: AR gene expression in RPE cells was increased by high (25 mM) glucose, CoCl2 (150 µM)-induced chemical hypoxia, H2O2 (20 µM)-induced oxidative stress, and high extracellular osmolarity induced by addition of NaCl (100 mM) or sucrose (100 mM). High extracellular osmolarity, but not chemical hypoxia, also induced increased cellular AR protein content. SDH gene expression was increased by chemical hypoxia and oxidative stress, but not NaCl- or sucrose-induced hyperosmolarity. Hyperosmolarity and hypoxia did not alter the SDH protein content of the cells. The hyperosmotic AR gene expression was dependent on activation of metalloproteinases, autocrine/paracrine TGF-β signaling, activation of p38 MAPK, ERK1/2, and PI3K signal transduction pathways, and the activity of NFAT5. Knockdown of NFAT5 or inhibition of AR activity decreased cell proliferation under hyperosmotic, but not hypoxic conditions. Conclusions: The data suggest that sorbitol accumulation in RPE cells occurs under hyperosmotic conditions, but not under hypoxic and oxidative stress conditions. It is suggested that high intake of dietary salt may aggravate diabetic retinopathy via NFAT5-mediated stimulation of retinal sorbitol accumulation.
Referent/in: Rebecca Dollinger (Tübingen)
Hintergrund: In den vergangenen Jahrzehnten wurde das Gebiet der Neuroprotektion in der ophthalmologischen Wissenschaft intensiv erforscht. Neuroprotektion ist u.a. wichtig bei Erkrankungen wie dem Glaukom, retinalen Gefäßverschlüssen, Netzhautablösung und der diabetischen Retinopathie. Ziel dieser Studie war ein standardisiertes Stressmodell für zukünftige vorklinische Therapietestungen zu etablieren. Methodik: Netzhäute von Rindern wurden präpariert, mit Sauerstoff angereicherter Standardlösung umspült und ERG-Messungen durchgeführt. Nach Erhalt stabiler b-Wellen wurde durch Hypoxie (reines N2) oder Glutamat (250µm) für 45 Minuten ein Stress auf die Netzhäute ausgeübt. Um gezielt die alleinigen Effekte auf die Funktion der Photorezeptoren zu testen, wurde zum Erhalt von a-Wellen 1mM Aspartat hinzugefügt. Anschließend wurde eine 75minütige Auswaschphase anhand ERG-Messungen dokumentiert. Ergebnisse: Für die Hypoxie wurde nach Exposition von 45 Minuten eine Abnahme der a-Wellen Amplitude von 87,0% verzeichnet (p< 0,01) (eine Verringerung um 36,5% am Ende der Auswaschphase p=0,03). Zusätzlich wurde anfangs eine statistisch signifikante Abnahme der b-Wellen Amplitude von 87,23% festgehalten (p< 0,01) (Abnahme von 25,5% am Ende der Auswaschphase p=0,03). Mit 250µm Glutamat wurde initial eine Reduktion der a-Wellen Amplitude um 7,8% (p>0,05) festgehalten, gefolgt von einer Reduktion um 1,9% (p>0,05). Bei der b-Wellen Amplitude wurde eine Verminderung von 83,7% (p< 0,01) und nach 75 Minuten Auswaschen eine Verminderung von 2,3% (p=0,62) verzeichnet. Schlussfolgerung: In dieser Studie präsentieren wir ein standardisiertes Stressmodell, welches in Zukunft hilfreich beim Erkennen von neuroprotektiven Effekten sein kann.
Referent/in: Harutyun Melkonyan (Münster)
Introduction: The systemical administration of NaIO3 has been used in different animal models to induce retinal degeneration secondary to a selective damage of the retinal pigment epithelium (RPE) in-vivo. The exact mechanisms remain unclear. The aim of this work was to explore exact mechanisms and selectivity of NaIO3 within the RPE. Methods: ARPE-19 cells were exposed to NaIO3 in different concentrations (from 0,2 mM to 2 mM) and time frames (1d-4d). Morphological changes were studied by time-lapse microscopical imaging. The viability was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. Changes in protein expression and mRNA levels involved in oxidative stress, e.g., haemoxingenase-1 (hmox-1), glutamate-cysteine ligase catalytic subunit (GCLC), and toll-like receptors (e. g., TLR-2) as well as apoptotis-related factors (e. g., Bax, Bcl-2) were quantified by immunofluorescence studies, western blotting, and quantitative real-time PCR. Primary RPE-cells of Callithrix jacchus and humans as wells as non-ocular cells (e.g., HeLa cells) was used to confirm recent findings. Results: Alterations in cell morphology, proliferation-rates and viability was found in used RPE-cells in relation to NaIO3 administration. A dramatically increase of hmox, GCLC, and TLR-2 confirm an underlying oxidative stress. A switch of Bax/Bcl-2 ratio indicates increased apoptotic-processes. Discussion: The presented data indicate that NaIO3-associated RPE damage is induced by elevated oxidative stress in-vitro. More over, the in-vitro results do not support the reported selectivity of a RPE damage by NaIO3 administration. However, further studies are mandatory, in order to state whether RPE in-vivo responds in a similar way or not.
Referent/in: Marina Renner (Bochum)
Purpose: Several studies demonstrated that ischemia-reperfusion (I/R) leads in morphological and functional changes of different retinal cell types. Additionally, axonal or myelin damage of the optic nerve (ON) can observed after I/R. However, little is known about the ischemic effects on the ON. Therefore, we analyzed ONs after ischemia-reperfusion injury. Methods: I/R was induced by raising the intraocular pressure (IOP) in one eye of rats to 140 mmHg for 1 h (n=5/group). The other eye served as control (Co). 21 days after ischemia the ONs were removed and longitudinal optic nerve sections were prepared. They were stained with H&E (structure) and LFB (myelin). Additionally, the microglial immigration (Iba1), activation of macrophages (ED1), and the macroglial response (GFAP and nestin) were analyzed using immunohistochemistry. Alteration and demyelination grade was evaluated using a scoring system. ImageJ software was used for microglia cell count and to measure GFAP+ and nestin+ area. Statistical analysis was performed using student’s t-test. Results: Histology showed a significant damage of the ONs of I/R eyes. Significantly more inflammatory cells infiltrated the ONs of the I/R group (p˂0.001). Additionally, large areas of demyelination could be observed after I/R (p˂0.001). Microglial staining confirmed these observations. Significantly more Iba1+ cells were detected in I/R ONs (p˂0.01). Furthermore, the ED1+ cell counts revealed a significant activation of macrophages (p˂0.01). Regarding macroglial staining, no differences could be noted in GFAP+ and nestin+ area between both groups (p>0.05). Conclusions: The global retinal ischemic event also has an intense effect on the optic nerve. 21 days after ischemia-reperfusion injury inflammation and demyelination right up to tissue dissolution of the optic nerves could be revealed. Furthermore we noted a distinct activation of microglial cells. On this basis we conclude, that the retinal damage after I/R is the reason for the axonal degeneration. No group differences could be shown in the macroglial staining area, probably due to the grave degeneration of the optic nerve at this late point of time.
Referent/in: Andreas Bringmann (Leipzig)
Purpose: Osmotic swelling of neurons and glial cells contributes to retinal edema and neurodegeneration. Brain-derived neurotrophic factor (BDNF), a major neuroprotectant in the retina, was shown to inhibit osmotic swelling of glial (Müller) and bipolar cells in the rat retina; the effect of BDNF on bipolar cell swelling is mediated by inducing a release of neuroprotective cytokines from Müller cells (Berk et al., 2015, Neuroscience, in press). Here, we determined whether BDNF-mediated cell volume regulation is altered after transient retinal ischemia. Methods: Freshly isolated retinal slices of rats which undergone a 1-h pressure-induced retinal ischemia and from control eyes were superfused with a hypoosmotic solution (60% osmolarity). BDNF and TrkB were immunocytochemically stained in freshly isolated Müller and bipolar cells. Retinal expression of BDNF and TrkB genes was determined by real-time RT-PCR. Results: BDNF prevented osmotic swelling of Müller cells in both control and postischemic retinal slices. BDNF also prevented osmotic swelling of bipolar cells in the control, but not in the ischemic retina. Freshly isolated Müller cells of control retinas displayed immunoreactivity of truncated TrkB, but not of full-length TrkB. In contrast, Müller cells of postischemic retinas displayed immunoeractivities of both TrkB isoforms. Bipolar cells isolated from control and postischemic retinas were immunolabeled for both TrkB isoforms. The expression levels of BDNF und TrkB isoforms genes were decreased in postischemic retinas, while p75NTR gene expression was increased compared to control. Conclusions: It is suggested that the ischemic abrogation of the BDNF effect in bipolar cells is related to altered BDNF receptor expression in Müller cells. Glial upregulation of full-length TrkB may support the survival of Müller cells in the ischemic retina, but may impair the BDNF-induced release of neuroprotective cytokines from Müller cells.
Referent/in: Lisa Strudel (Tübingen)
Purpose: Last year we presented a group of lipid-modified DNA based nanoparticles as a new drug delivery system for topical applied drugs. This year we will present the most important issue in development of a new drug delivery platform: safety and toxicity. Furthermore, we also quantified the amount of NP present on the eye after dropping them to the eye. Methods: To exclude toxicity of the NPs, three ocular cell lines (661W, ARPE-19 & RGC-5) were incubated with the NP for 24 h and cell viability via MTS-assay, cell amount via crystal violet staining and apoptosis via caspase 3/7 assay was measured. Additionally, the same tests were performed using primary corneal epithelial cells. In order to further determine the toxicity profile, the NP were dropped on rat eyes at different time-points and dosing regimes. Sections of the eyes were then analyzed via TUNEL-assay for apoptosis. As a last toxicity evaluation the upregulation of inflammatory pathways was screened. Through a PCR assay the influence of the NPs on expression of toll-like receptors (TLR), interleukins (IL) and tumor necrosis factor alpha (TNF-α) was determined. Finally, the residence time of the NPs was evaluated using an ocular fluorophotometer (Fluorotron™) Results: Neither in-vitro cell toxicity screening nor the in-vivo experiments showed any toxicity. Even more, higher concentration than the effective dose or an increased dropping interval could not cause a negative effect. Also the evaluation of TLR, IL and TNF-α regulation showed normal expression levels. Additionally, evaluation of the residence time by ocular fluorophotometry showed that the NP gets washed out slowly, whereas a small molecule control is already removed after 5 minutes. Conclusions: Thorough and extensive in-vivo and in-vitro testing confirmed the local safety of our NP-carriers system. The previous shown better efficacy could now be confirmed with quantitative numbers. These important findings allow the further safe utilization of the NP platform for ocular drug delivery. Nevertheless, further studies on systemic toxicity and bio distribution need to be performed before clinical trials can be performed.
Referent/in: Anna Dobias (Aachen)
Purpose: Neovascular age-related macular degeneration (AMD), which leads to vision loss within several months, is characterized by the growth of choroidal blood vessels (CNV) into the subretinal space. CNV results from an imbalance of the pro-angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic pigment epithelium-derived factor (PEDF). Current treatment, which consists of the intravitreal injection of anti-VEGF antibodies, improves vision in about 30% of patients, but it may be accompanied by severe side effects and non-compliance. We have theorized that transplantation of pigment epithelial cells that were genetically modified to overexpress human PEDF would be a safer and improved treatment. Stable transgene expression was ensured by the Sleeping Beauty (SB100X) transposon system delivered in pFAR4-plasmids, an expression vector free of antibiotic resistance markers. Here, we report that PEDF-transfected cells do not exhibit tumorigenic potential. Methods: Pre-cultivated (P0) and freshly isolated retinal pigment epithelial (RPE) cells from human donor eyes were transfected by means of electroporation using a pFAR4-plasmid encoding the enhanced SB100X transposase and a pFAR4-plasmid encoding the PEDF gene at a ratio of 1:16. Stability of recombinant PEDF secretion was analyzed by immunoblotting. To assay for tumorigenic potential, approximately 35,000 cells/cm2 were applied to a two-layer soft agar system. Colony development was analyzed after 1, 5, 7, and 14 days by phase contrast microscopy and compared to colony development of HeLa cells as the positive control. Results: The transfection of as few as 10,000 human RPE cells with the PEDF gene resulted in a stable increase of PEDF secretion. Pre-cultivated (P0) and freshly isolated PEDF-transfected RPE cells did not form colonies in soft agar at any time, whereas HeLa cells formed 89.4 ± 26.4 soft agar colonies per microscopic field at day 5. Conclusions: The results of this study show clearly indication that PEDF-transfected pre-cultivated (P0) and freshly isolated RPE cells have no tumorigenic potential. Additional safety studies will analyze the integration loci of the PEDF transgene as well as the biodistribution of PEDF-transfected RPE cells transplanted subretinally. This study is just one of many aspects demonstrating the feasibility of our gene/cell therapy strategy to treat neovascular AMD.