Biocompatibility of the vital dye Acid Violet-17 on retinal pigment epithelial cells

Purpose: To examine the viability and differentiation of retinal pigment epithelial (RPE) cells after exposure to the vital dye Acid Violet-17 (AV-17). Methods: Bovine RPE cells were incubated with AV-17 (0.0625-0.5 mg/mL) for 30 seconds or 5 minutes. Viability was determined by live/dead staining, cleaved CASP3 immunostainings, and MTT test. Actin cytoskeleton was visualized by Alexa 488-phalloidin. Immunocytochemistry was performed to determine the levels of ZO-1, CTNNB1, and KRT19. Results: Exposure to AV-17 at the concentrations of 0.25-0.5 mg/mL resulted in a dose-dependent decrease in viability, the loss of ZO-1 from tight junctions, translocation of CTNNB1 into the cytoplasm and nucleus, disarrangement of the actin cytoskeleton, and a slight increase in KRT19. Conclusion: AV-17 at a concentration. 0.125 mg/mL is likely to be well tolerated by the RPE cells, whereas the concentrations from 0.25 mg/mL onward can reduce viability and induce dedifferentiation particularly after long-term exposure

Zinc Oxide Tetrapods Modulate Wound Healing and Cytokine Release In Vitro -A New Antiproliferative Substance in Glaucoma Filtering Surgery

Glaucoma filtering surgery is applied to reduce intraocular pressure (IOP) in cases of uncontrolled glaucoma. However, postoperative fibrosis reduces the long-term success of both standard trabeculectomy and microstents. The aim of this study was to test the antiproliferative and anti-inflammatory potential of ZnO-tetrapods (ZnO-T) on human Tenon's fibroblasts (HTFs) for glaucoma surgery. The toxicity of ZnO-T on HTFs was determined using an MTT test. For analysis of fibroblast proliferation, migration, and transdifferentiation, cultures were stained for Ki67, alpha-smooth muscle actin (α-SMA), and p-SMAD. A fully quantitative multiplex ELISA was used to determine the concentrations of different cytokines, platelet-derived growth factor (PDGF), and hepatocyte growth factor (HGF) in culture supernatants with and without previous ZnO-T treatment. Treatment with higher concentrations (10 and 20 µg/mL) was associated with HTF toxicity, as shown in the wound healing assay. Furthermore, the number of Ki67, α-SMA-positive, and pSMAD-positive cells, as well as IL-6 and HGF in supernatants, were significantly reduced following incubation with ZnO-T. In conclusion, we were able to show the antiproliferative and anti-inflammatory potentials of ZnO-T. Therefore, the use of ZnO-T may provide a new approach to reducing postoperative fibrosis in glaucoma filtering surgery

Die Rho-Kinase Signalkette in Tenonfibroblasten und retinalen Ganglienzellen: ein neues pharmakologisches Zielprotein, um den Erfolg der filtrierenden Glaukomchirurgie zu erhöhen und das Überleben retinaler Ganglienzellen in Glaukompatienten zu verbessern.

Glaucoma is characterized by the progressive loss of retinal ganglion cells (RGC) and their axons, giving rise to irreversible visual field defects leading to blindness when left untreated. The current methods of glaucoma treatment aim at reducing the intraocular pressure (IOP), the major risk factor arising from an imbalance between the production and outflow of aqueous humor. Glaucoma filtration surgery, which entails the creation of a scleral fistula that enables the drainage of aqueous humor, remains to be the most effective method for lowering the IOP. However, the wound healing activities of the Tenon’s capsule fibroblasts (TCF) result in the occlusion of the drainage channel and account for the surgical failure. The Rho-kinase/ROCK signaling pathway is an intracellular convergence point for signals initiating the cytoskeletal alterations underlying the dynamic events involved in wound healing. The first part of this study therefore focused on the functions of the ROCK-pathway in human TCFs in vitro. With this purpose, the fibroblasts stimulated with serum were treated with H-1152P, a specific ROCK-inhibitor. Incubation of the TCFs with 10 µM H-1152P resulted in an 80-90% reduction in cell proliferation. H-1152P also caused the disassembly of stress fibers in a dose-dependent manner without exerting toxicity and without interfering with the protein kinase A (PKA) pathway. Migration of the fibroblasts and their contractility were also suppressed by H-1152P together with the impairment of fibronectin network assembly. These findings provide further evidence for the role of the ROCK-pathway in the wound healing activities of human TCFs and underline the potential of H-1152P as a safe and specific means to suppress these events. Numerous extracellular signals that induce apoptosis also converge on the activation of ROCK. This places H-1152P mediated ROCK-inhibition as a novel approach for promoting the survival of RGCs in glaucoma, the efficacy of which was analyzed initially on isolated mouse retinae incubated without serum. The survival of the cells in the ganglion cell layer was severely impaired in this stringent in vitro model after 1 day. In contrast, 1 µM H-1152P considerably reduced the extent of apoptosis in all the retinal layers without interfering with PKA. The reactivity of astrocytes, Müller cells, and microglia were also significantly suppressed in retinae treated with H-1152P. In addition, a significant reduction was observed in the levels of various proinflammatory cytokines such as interleukin IL-3, IL-6, interferon-gamma, and tumor necrosis factor alpha released into the culture medium in response to H-1152P, which probably underlied the decrease in the toxicity of the conditioned media obtained from retinae incubated with this inhibitor. These results demonstrate that the neuroprotective effect of H-1152P mediated ROCK inhibition on retinal cells may be due to the suppression of glial cell reactivity to a certain extent, leading to the diminishing of cytokine release and to the suppression of secondary damage. To analyze the direct effects of H-1152P on RGCs, the rat retinal ganglion cell line RGC-5 cultivated without serum was also utilised in this study. Despite the stability of this cell line, H-1152P at 1 µM exerted direct anti-apoptotic effects by specifically interfering with ROCK-dependent phosphorylation events. However, increasing the concentration of the inhibitor to 10-20 µM resulted in a dramatic increase in the amount of cells undergoing an oncotic cell death characterized by a marked cellular swelling. These highly contrasting effects of H-1152P are likely to have arisen from the molecular differences of the transformed cell line RGC-5 from primary RGCs, the further characterization of which are believed to be useful in providing more insight into the properties of both cell types as well as the mechanisms of oncosis. The neuroprotective potential of H-1152P on RGCs after optic nerve crush (ONC) was also studied in collaboration with Dr. Frank Schüttauf from the University Eye Hospital in Tübingen. H-1152P administered at 1 µM led to a 1.6 fold increase in the number of surviving RGCs together with a decrease in the extent of glial cell reactivity 7 days after the ONC. These findings provide further support for the hypothesis suggesting that the suppression of reactive gliosis to a certain degree after axonal injury may indeed be beneficial in reducing the spread of secondary damage and rescuing the remaining ganglion cells. These data demonstrating the central role of the ROCK-signaling pathway in various cells implicated in the course of glaucoma also highlight the potential of the H-1152P-mediated targeting of this pathway as a promising anti-glaucoma therapy approach not only for suppressing the undesirable wound healing activities of Tenon’s capsule fibroblasts after glaucoma filtration surgery, but also for preventing further RGC loss.Die glaukomatöse Opticusneuropathie ist charakterisiert durch eine Degeneration der Ganglienzellaxone und den progressiven Verlust retinaler Ganglienzellen (RGZ), der eine irreversible Schädigung des visuellen Systems nach sich zieht. Die derzeitigen Behandlungsmethoden zielen auf die Senkung des Augeninnendrucks (IOD) ab, dem Hauptrisikofaktor für das Glaukom, resultierend aus einem Ungleichgewicht zwischen Produktion und Abfluss des Kammerwassers. Die filtrierende Glaukomchirurgie stellt die wirkungsvollste Methode für die Senkung des IOD dar. Postoperative Wundheilungsaktivitäten der humanen Tenonfibroblasten (HTF) wie erhöhte Zellproliferation, Zellmigration, Zellkontraktion und Matrixproduktion führen jedoch zu einem Verschluss des für den Kammerwasserabfluss präparierten Filterkissens und tragen zu einem Misserfolg der Drucksenkung bei. Eine zentrale Rolle bei der Organisation des Zytoskeletts während dynamischer Vorgänge wird dem RhoA-Protein und dessen Effektorprotein Rho-Kinase/ROCK zugeschrieben. In der vorliegenden Arbeit wurde die Rolle der ROCK-Signalkette bei den Vernarbungsaktivitäten der HTFs in vitro mittels H-1152P untersucht, eines wirksamen und spezifischen Inhibitors der ROCK. Es zeigte sich, dass sich die Proliferation um 80-90% in den Zellen, die mit 10 µM des Inhibitors für 4 Tage inkubiert wurden, reduzieren ließ. H-1152P führte zu einem dosis-abhängigen Abbau der Stressfasern ohne toxische Wirkungen auszuüben und wies keine deutliche Interferenz mit der Proteinkinase A (PKA)-Signalkette auf. H-1152P bewirkte weiterhin eine 3,5-fache Verminderung der Zellmigration, eine 5-fache Senkung der Kontraktion einer Kollagenmatrix und eine dosis-abhängige Beeinträchtigung im Aufbau des Fibronektinnetzwerks. Diese Befunde weisen auf die Bedeutung der ROCK für die Vernarbung hin und betonen das Potenzial von H-1152P als nicht-toxischer Hemmstoff bei unerwünschten Wundheilungsaktivitäten. Zahlreiche pro-apoptotische Signale aktivieren die ROCK-Signalkette. Eine H-1152P-vermittelte Hemmung der ROCK bietet sich daher als neue Strategie an, um das Überleben der RGZs beim Glaukom zu fördern. Ihre Wirksamkeit wurde zunächst in Organkulturen der Mausretina untersucht, in denen durch Serumentzug Zelltod insbesondere in der Ganglienzellebene induziert wurde. Inkubation mit 1 µM H-1152P verringerte das Ausmaß des Zelltods in allen retinalen Schichten deutlich ohne die PKA-Signalkette zu beeinflussen. Die Aktivierung der Astrozyten, Müllerzellen und Mikroglia wurde ebenfalls durch die Behandlung mit H-1152P stark abgeschwächt. Weiterhin wurde die Freisetzung pro-inflammatorischer Cytokine wie Interleukin (IL)-3, IL-6, Interferon-gamma, und TNFalpha signifikant reduziert, worauf die zu beobachtende verminderte Toxizität der Kulturüberstände zurückzuführen sein könnte. Diese Ergebnisse zeigen, dass die neuroprotektive Wirkung der ROCK-Hemmung auf retinale Zellen durch eine verminderte Gliazellreaktivität und den daraus resultierenden Sekundärschäden bedingt sein könnte. Eine direkte Wirkung der H-1152P-Behandlung auf RGZ wurde an einer transformierten Ganglienzelllinie der Ratte (RGC-5) analysiert. Unter Serumentzug übte 1 µM H-1152P eine direkte anti-apoptotische Wirkung aus. Jedoch führten höhere Konzentrationen (10-20 µM) zu einer drastischen Zunahme in der Anzahl onkotischer Zellen, die aufgrund einer beachtlichen Volumenzunahme im Zelltod endeten. Diese stark kontrastierenden Wirkungen von H-1152P beruhen vermutlich auf Unterschieden zwischen transformierten RGC-5 Zellen und den primären RGZ. Weitere Untersuchungen könnten nützliche Informationen bezüglich der Mechanismen der Onkose erbringen. Um die neuroprotektive Wirkung von H-1152P auf RGZ in vivo zu untersuchen wurde eine Sehnervquetschung (SNQ) in Ratten durchgeführt. Die intravitreale Injektion von 1 µM H-1152P führte zu einer 1,6-fachen Zunahme in der Anzahl lebender RGZ verbunden mit einer verminderten Reaktivität der Gliazellen 7 Tage nach SNQ. Die Befunde unterstützen die Hypothese, dass die Abschwächung der reaktiven Gliose in gewissem Maße Sekundärschaden verhindert und das Überleben der RGZ nach Verletzung des Axons fördert. Diese Daten zeigen die Rolle der ROCK-Kaskade in verschiedenen am Glaukom beteiligten Zellen auf und betonen das therapeutische Potenzial der H-1152P-abhängigen ROCK-Hemmung als eine aussichtsreiche Maßnahme nicht nur um die unerwünschten Wundheilungs-aktivitäten der Tenonfibroblasten zu unterdrücken, sondern auch um den weiteren Verlust der RGZ zu verhindern

Influence of Retinal Microsecond Pulse Laser Treatment in Central Serous Chorioretinopathy: A Short-Term Optical Coherence Tomography Angiography Study

Background: Central serous chorioretinopathy (CSC) is a common macular condition characterized by detachment of the neuroretina and is a frequent cause of central vision loss in adults. Among the various therapeutic strategies, subthreshold microsecond pulsed laser (SML) treatment has become a useful option. Despite the suggested involvement of choroidal circulatory disturbances in CSC, the effects of this treatment on macular microperfusion have not been fully evaluated yet. Herein, we report the impact of SML on retinal and choroidal microvascular flow using non-invasive optical coherence tomography (OCT) angiography (OCTA). Methods: In this study, CSC patients with persistent subretinal fluid (SRF) with or without secondary choroidal neovascularization (CNV) were included (referred to as the pachychoroid neovasculopathy (PNV) group and the CSC group, respectively). SML was conducted using a yellow (577 nm) laser with a duty cycle of 10%, spot size of 200 µm and duration of 200 ms. Best corrected visual acuity (BCVA) as well as OCT and OCTA images were evaluated at baseline and 4 weeks after SML. OCTA parameters of interest included full retinal perfusion (FRP), choriocapillaris perfusion (CCP), Sattler’s layer perfusion (SLP), and Haller’s layer perfusion (HLP), which were evaluated longitudinally and compared to unaffected fellow eyes. Results: 27 affected eyes and 17 fellow eyes from 27 patients were included. Before treatment, central retinal thickness (CRT) and subfoveal choroidal thickness (SFCT) of affected eyes were significantly larger than in fellow eyes. Four weeks after SML, CRT decreased significantly, whereas perfusion parameters did not change. In subgroup analyses, the CSC group showed a significant decrease in SFCT, whereas the PNV group did not despite the decrease in CRT. Conclusion: Our results suggest that the SML may affect the SFCT of the CSC, but not the PNV patients at least within four weeks following treatment. This effect seems to be independent of the change in choroidal perfusion measured with OCTA

Uptake of Ranibizumab but Not Bevacizumab into Uveal Melanoma Cells Correlates with a Sustained Decline in VEGF-A Levels and Metastatic Activities

Despite the implication of vascular endothelial growth factor-A (VEGF-A) in the pathophysiology of uveal melanoma (UM), the anti-VEGF-A antibody bevacizumab yielded conflicting results on UM growth. Here, we evaluated whether bevacizumab and ranibizumab, a humanized Fab-fragment against VEGF-A, can enter UM cells and induce a sustained physiological response. The primary and metastatic UM cell lines Mel-270 and OMM-2.5 were exposed to bevacizumab or ranibizumab for one day and were maintained further in untreated medium for a total of three days. Both antibodies significantly reduced the levels of extracellular VEGF-A and the angiogenic potential of the conditioned medium after one day. These inhibitory effects of bevacizumab diminished by day three. Ranibizumab suppressed the metabolic activity, proliferation, and intracellular VEGF-A levels in a cell-type and concentration-dependent manner, whereas bevacizumab exerted no effect. Both drugs were detected inside early endosomes within the UM cells, with the stronger and sustained colocalization of ranibizumab. Our results therefore demonstrated the more potent and persistent suppressive activity of ranibizumab on the UM cells, possibly due to its higher level of uptake and prolonged intracellular retention. Further research on the endosome dynamics in UM cells might provide valuable insight into the response of these heterogenous tumors to therapeutic antibodies

Quercetin Impairs the Growth of Uveal Melanoma Cells by Interfering with Glucose Uptake and Metabolism

Monosomy 3 in uveal melanoma (UM) increases the risk of lethal metastases, mainly in the liver, which serves as the major site for the storage of excessive glucose and the metabolization of the dietary flavonoid quercetin. Although primary UMs with monosomy 3 exhibit a higher potential for basal glucose uptake, it remains unknown as to whether glycolytic capacity is altered in such tumors. Herein, we initially analyzed the expression of n = 151 genes involved in glycolysis and its interconnected branch, the “pentose phosphate pathway (PPP)”, in the UM cohort of The Cancer Genome Atlas Study and validated the differentially expressed genes in two independent cohorts. We also evaluated the effects of quercetin on the growth, survival, and glucose metabolism of the UM cell line 92.1. The rate-limiting glycolytic enzyme PFKP was overexpressed whereas the ZBTB20 gene (locus: 3q13.31) was downregulated in the patients with metastases in all cohorts. Quercetin was able to impair proliferation, viability, glucose uptake, glycolysis, ATP synthesis, and PPP rate-limiting enzyme activity while increasing oxidative stress. UMs with monosomy 3 display a stronger potential to utilize glucose for the generation of energy and biomass. Quercetin can prevent the growth of UM cells by interfering with glucose metabolism

Testing the effects of the dye Acid violet-17 on retinal function for an intraocular application in vitreo-retinal surgery

To facilitate epiretinal or inner limiting membrane peeling, dyes like Indocyanine Green (ICG) as well as Trypan Blue (TB) were used so far. However, toxic effects on the retina were described for both dyes. The aim of our study was to investigate the effects of a novel vital dye Acid violet-17 (AV-17) on retinal histology and function to assess a possible application in vitreo-retinal surgery. AV-17 was dissolved in a solvent with heavy water. An electroretinogram was recorded on perfused bovine retina. After reaching stable b-wave amplitudes, AV-17 (0.125-0.5 mg/ml) or the solvent was applied epiretinally for 30-300 seconds. The b-wave amplitudes were recorded before, during, and after treatment. Cultures of bovine retina were incubated for 30 or 300 seconds with the dye or solvent and processed for live/dead staining, immunohistochemistry, and immunoblotting. Reductions of the b-wave amplitudes were observed directly after the exposure to AV-17, which were rapidly and completely reversible within the recovery period for all exposure times at the concentrations of 0.125 and 0.25 mg/ml as opposed to the partial recovery after exposure to 0.5 mg/ml. A high degree of damage in the ganglion cell layer (GCL) and glial reactivity were detected at the concentrations of 0.25 and 0.5 mg/ml but not after exposure to lower concentrations or the solvent. Application of AV-17 at a concentration of up to 0.125 mg/ml was well tolerated in terms of retinal function, survival in the GCL, and glial reactivity whereas higher concentrations are not recommended

In vivo evaluation of a nanotechnology-based microshunt for filtering glaucoma surgery

Abstract To carry out the preclinical and histological evaluation of a novel nanotechnology-based microshunt for drainage glaucoma surgery. Twelve New Zealand White rabbits were implanted with a novel microshunt and followed up for 6 weeks. The new material composite consists of the silicone polydimethylsiloxane (PDMS) and tetrapodal Zinc Oxide (ZnO-T) nano-/microparticles. The microshunts were inserted ab externo to connect the subconjunctival space with the anterior chamber. Animals were euthanized after 2 and 6 weeks for histological evaluation. Ocular health and implant position were assessed at postoperative days 1, 3, 7 and twice a week thereafter by slit lamp biomicroscopy. Intraocular pressure (IOP) was measured using rebound tonometry. A good tolerability was observed in both short- and medium-term follow-up. Intraocular pressure was reduced following surgery but increased to preoperative levels after 2 weeks. No clinical or histological signs of inflammatory or toxic reactions were seen; the fibrotic encapsulation was barely noticeable after two weeks and very mild after six weeks. The new material composite PDMS/ZnO-T is well tolerated and the associated foreign body fibrotic reaction quite mild. The new microshunt reduces the IOP for 2 weeks. Further research will elucidate a tube-like shape to improve and prolong outflow performance and longer follow-up to exclude medium-term adverse effects

Protein Profiling of WERI-RB1 and Etoposide-Resistant WERI-ETOR Reveals New Insights into Topoisomerase Inhibitor Resistance in Retinoblastoma

Chemotherapy resistance is one of the reasons for eye loss in patients with retinoblastoma (RB). RB chemotherapy resistance has been studied in different cell culture models, such as WERI-RB1. In addition, chemotherapy-resistant RB subclones, such as the etoposide-resistant WERI-ETOR cell line have been established to improve the understanding of chemotherapy resistance in RB. The objective of this study was to characterize cell line models of an etoposide-sensitive WERI-RB1 and its etoposide-resistant subclone, WERI-ETOR, by proteomic analysis. Subsequently, quantitative proteomics data served for correlation analysis with known drug perturbation profiles. Methodically, WERI-RB1 and WERI-ETOR were cultured, and prepared for quantitative mass spectrometry (MS). This was carried out in a data-independent acquisition (DIA) mode. The raw SWATH (sequential window acquisition of all theoretical mass spectra) files were processed using neural networks in a library-free mode along with machine-learning algorithms. Pathway-enrichment analysis was performed using the REACTOME-pathway resource, and correlated to the molecular signature database (MSigDB) hallmark gene set collections for functional annotation. Furthermore, a drug-connectivity analysis using the L1000 database was carried out to associate the mechanism of action (MOA) for different anticancer reagents to WERI-RB1/WERI-ETOR signatures. A total of 4756 proteins were identified across all samples, showing a distinct clustering between the groups. Of these proteins, 64 were significantly altered (q < 0.05 & log2FC |>2|, 22 higher in WERI-ETOR). Pathway analysis revealed the “retinoid metabolism and transport” pathway as an enriched metabolic pathway in WERI-ETOR cells, while the “sphingolipid de novo biosynthesis” pathway was identified in the WERI-RB1 cell line. In addition, this study revealed similar protein signatures of topoisomerase inhibitors in WERI-ETOR cells as well as ATPase inhibitors, acetylcholine receptor antagonists, and vascular endothelial growth factor receptor (VEGFR) inhibitors in the WERI-RB1 cell line. In this study, WERI-RB1 and WERI-ETOR were analyzed as a cell line model for chemotherapy resistance in RB using data-independent MS. Analysis of the global proteome identified activation of “sphingolipid de novo biosynthesis” in WERI-RB1, and revealed future potential treatment options for etoposide resistance in RB