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.

Islet Co-Expression of CD133 and ABCB5 in Human Retinoblastoma Specimens.

BACKGROUND: The role of CD133 und ABCB5 is discussed in treatment resistance in several types of cancer. The objective of this study was to evaluate whether CD133+/ABCB5+ colocalization differs in untreated, in beam radiation treated, and in chemotherapy treated retinoblastoma specimens. Additionally, CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 gene expression was analyzed in WERI-RB1 (WERI RB1) and etoposide-resistant WERI RB1 subclones (WERI ETOR). METHODS: Active human untreated retinoblastoma specimens (n = 12), active human retinoblastoma specimens pretreated with beam radiation before enucleation (n = 8), and active human retinoblastoma specimens pretreated with chemotherapy before enucleation (n = 7) were investigated for localization and expression of CD133 and ABCB5 by immunohistochemistry. Only specimens with IIRC D, but not E, were included in this study. Furthermore, WERI RB1 and WERI ETOR cell lines were analyzed for CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 by the real-time polymerase chain reaction (RT-PCR). RESULTS: Immunohistochemical analysis revealed the same amount of CD133+/ABCB5+ colocalization islets in untreated and treated human retinoblastoma specimens. Quantitative RT-PCR analysis showed a statistically significant upregulation of CD133 in WERI ETOR (p = 0.002). No ABCB5 expression was detected in WERI RB1 and WERI ETOR. On the other hand, SPHK1 (p = 0.0027) and SPHK2 (p = 0.017) showed significant downregulation in WERI ETOR compared to WERI RB1. CONCLUSIONS: CD133+/ABCB5+ co-localization islets were noted in untreated and treated human retinoblastoma specimens. Therefore, we assume that CD133+/ABCB5+ islets might play a role in retinoblastoma genesis, but not in retinoblastoma treatment resistance.

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.

CXCR4, CXCR5 and CD44 May Be Involved in Homing of Lymphoma Cells into the Eye in a Patient Derived Xenograft Homing Mouse Model for Primary Vitreoretinal Lymphoma.

Background: Primary vitreoretinal lymphoma (PVRL), a rare malignancy of the eye, is strongly related to primary central nervous system lymphoma (PCNSL). We hypothesized that lymphoma cells disseminate to the CNS and eye tissue via distinct homing receptors. The objective of this study was to test expression of CXCR4, CXCR5, CXCR7 and CD44 homing receptors on CD20 positive B-lymphoma cells on enucleated eyes using a PCNSL xenograft mouse model. Methods: We used indirect immunofluorescence double staining for CD20/CXCR4, CD20/CXCR5, CD20/CXCR7 and CD20/CD44 on enucleated eyes of a PCNSL xenograft mouse model with PVRL phenotype (PCNSL group) in comparison to a secondary CNS lymphoma xenograft mouse model (SCNSL group). Lymphoma infiltration was evaluated with an immunoreactive score (IRS). Results: 11/13 paired eyes of the PCNSL but none of the SCNSL group were infiltrated by CD20-positive cells. Particularly the choroid and to a lesser extent the retina of the PCNSL group were infiltrated by CD20+/CXCR4+, CD20+/CXCR5+, few CD20+/CD44+ but no CD20+/CXCR7+ cells. Expression of CXCR4 (p = 0.0205), CXCR5 (p = 0.0004) and CD44 (p < 0.0001) was significantly increased in the PCNSL compared to the SCNSL group. Conclusions: CD20+ PCNSL lymphoma cells infiltrating the eye co-express distinct homing receptors such as CXCR4 and CXCR5 in a PVRL homing mouse model. These receptors may be involved in PVRL homing into the eye.

Monosomy-3 Alters the Expression Profile of the Glucose Transporters GLUT1-3 in Uveal Melanoma.

Monosomy-3 in uveal melanoma (UM) cells increases the risk of fatal metastases. The gene encoding the low-affinity glucose transporter GLUT2 resides on chromosome 3q26.2. Here, we analyzed the expression of the glucose transporters GLUT1, GLUT2, and GLUT3 with regard to the histological and clinical factors by performing immunohistochemistry on the primary tumors of n = 33 UM patients. UMs with monosomy-3 exhibited a 57% lower immunoreactivity for GLUT2 and a 1.8×-fold higher ratio of GLUT1 to total GLUT1-3. The combined levels of GLUT1-3 proteins were reduced in the irradiated but not the non-irradiated tumors with monosomy-3. GLUT3 expression was stronger in the irradiated samples with disomy-3 versus monosomy-3, but the ratio of the GLUT3 isoform to total GLUT1-3 did not differ with regard to the monosomy-3 status in the irradiated or non-irradiated subgroups. Systemic metastases were associated with the presence of monosomy-3 in the primary and circulating tumor cells as well as a higher GLUT1 ratio. Upregulation of the high-affinity glucose transporter GLUT1 possibly as a compensation for the low-affinity isoform GLUT2 may be enhancing the basal glucose uptake in the UM cells with monosomy-3. Prevention of hyperglycemia might, therefore, be a valuable approach to delay the lethal UM metastases.

Ranibizumab interacts with the VEGF-A/VEGFR-2 signaling pathway in human RPE cells at different levels.

Vascular endothelial growth factor (VEGF) secreted by the retinal pigment epithelium (RPE) plays an important role in ocular homeostasis, but also in diseases, most notably age-related macular degeneration (AMD). To date, anti-VEGF drugs like ranibizumab have been shown to be most effective in treating these pathologic conditions. However, clinical trials suggest that the RPE could degenerate and perish through anti-VEGF treatment. Herein, we evaluated possible pathways and outcomes of the interaction between ranibizumab and human RPE cells (ARPE-19). Results indicate that ranibizumab affects the VEGF-A metabolism in RPE cells from an extra- as well as intracellular site. The drug is taken up into the cells, with the VEGF receptor 2 (VEGFR-2) being involved, and decreases VEGF-A protein levels within the cells as well as extracellularly. Oxidative stress plays a key role in various inflammatory disorders of the eye. Our results suggest that oxidative stress inhibits RPE cell proliferation. This anti-proliferative effect on RPE cells is significantly enhanced through ranibizumab, which does not inhibit RPE cell proliferation substantially in absence of relevant oxidative stress. Therefore, we emphasize that anti-VEGF treatment should be selected carefully in AMD patients with preexistent extensive RPE atrophy.

CD147 and matrix-metalloproteinase-2 expression in metastatic and non-metastatic uveal melanomas.

BACKGROUND: Extracellular matrix remodelling regulated by matrix-metalloproteinase (MMP) inducer (CD147) is a crucial process during tumor cell invasion and regulation of blood supply. In this study, we evaluated the correlation of CD147 and MMP-2 expression with major prognostic factors for uveal melanoma and the development of metastasis. METHODS: The expression of CD147 and MMP-2 was analyzed in 49 samples of uveal melanomas. Triple immunofluorescence stainings using markers against glial cells (GFAP), endothelial cells (CD34) and macrophages (CD68) were performed to further analyse the exact localisation of CD147 and MMP-2 positivity. In 28 cases clinical metastatic disease were found. The remaining 21 cases showed no signs of metastatic disease for an average follow-up of 10 years. Correlation analysis (Pearson correlation) was performed to analyse the association of CD147 and MMP-2 expression with known prognostic factors, vasculogenic mimicry (VM), the mature vasculature (von Willebrand Factor) and tumor induced angiogenesis (by means of Endoglin expression). RESULTS: CD147 and MMP-2 were expressed in 47 (96.0 %) of the uveal melanomas. CD147 up-regulation was significantly correlated with a higher MMP-2 expression. The overall expression analysis revealed no significant difference in the metastatic (p = 0.777) and non-metastatic subgroup (p = 0.585). No correlation of CD147 expression and any system of blood supply was evident. In the non-metastatic sub-group a significant correlation of clustered CD147 positive cells with largest basal diameter (p = 0.039), height (p = 0.047) and TNM-stage (p = 0.013) was evident. CONCLUSIONS: These data may indicate that CD147 regulates MMP-2 expression in uveal melanoma cells.

Analysis of caveolin-1 and phosphoinositol-3 kinase expression in primary uveal melanomas.

BACKGROUND: To evaluate the regulation of blood supply in primary uveal melanomas through caveolin-1 (Cav-1)/phosphoinositol-3 kinase (PI3K). METHODS: The expression of Cav-1 and PI3K was analysed in 51 paraffin sections of metastatic (n = 30) and non-metastastic uveal melanomas (n = 21). Two trained observers quantified Cav-1 and PI3K immunofluorescensce expression by determining intensity of staining and percentage of positive cells. The expression was correlated with known prognostic factors. Besides angiogenesis by means of endoglin expression, the normal vasculature (von Willebrand Factor expression) was evaluated semi-quantitatively. Vasculogenic mimicry (VM) was analysed by CD31/PAS staining. RESULTS: All examined specimens expressed Cav-1 with a mean of 90.34% Cav-1 positive cells (range, 3.23-100%). Metastatic disease was associated with a higher Cav-1 expression. The correlation of Cav-1 with well-established prognostic factors showed a significant association between Cav-1 expression and largest tumour diameter (P = 0.022), tumour node metastasis classification (P = 0.008) and invasion of optic nerve head (P = 0.048). PI3K was expressed by all uveal melanomas with a mean of 87.28% cells showing PI3K expression. A higher level of PI3K was significantly associated with larger height (P = 0.042) and progressed tumour node metastasis stage (P = 0.016). The percentage of PI3K and Cav-1 positive cells were significantly associated (P = 0.034). For PI3K and Cav-1 expression a non-significant association with VM was shown (P = 0.064 and P = 0.072, respectively). No correlation of PI3K or Cav-1 with angiogenesis or mature vasculature was seen (P > 0.05). CONCLUSIONS: Cav-1 expression may be especially up-regulated in larger uveal melanomas. As it was correlated with PI3K expression and VM in this series of uveal melanoma, Cav-1 might induce the formation of VM via the PI3K-signalling cascade.

Differential expression of vascular endothelial growth factor-a isoforms in neovascular age-related macular degeneration.

PURPOSE: To investigate the role of vascular endothelial growth factor-A (VEGF-A) isoforms in neovascular age-related macular degeneration. METHODS: Choroidal neovascular membranes (CNV) were excised in 24 patients, 8 of them underwent previous photodynamic therapy. All procedures were performed before anti-VEGF therapies were implemented in Germany. Normal human donor eyes served as controls. Messenger RNA expression of total VEGF-A and VEGF-A isoforms was measured. RESULTS: Vascular endothelial growth factor-A121 is the most abundant isoform in CNV and control tissues. In controls, VEGF-A121 is lowest in neural retina and highest in choroids. For total VEGF-A and VEGF-A165, this is vice versa. VEGF-A165 and VEGF-A189 are significantly higher in CNV than in control choroids, the opposite is found for VEGF-A121. After photodynamic therapy, total VEGF-A and VEGF-A121 are increased, VEGF-A165 and VEGF-A189 are decreased. Age-dependently, there is an increase in VEGF-A165 and a decrease in VEGF-A121. CONCLUSION: Vascular endothelial growth factor-A isoforms are differentially distributed, suggesting that tissue-specific regulation of various isoforms is physiologically important. The disruption of this homeostasis in CNV membranes may be significant in the onset and progression of neovascular age-related macular degeneration. Our findings support the dominant role of VEGF-A121 in neovascular age-related macular degeneration but hint that VEGF-A165 may have an equivalent role in other neovascular retinal pathology.

A TGF-ß receptor 1 inhibitor for prevention of proliferative vitreoretinopathy.

This study evaluates the use of the TGF-ß receptor 1 inhibitor LY-364947 (LY) to prevent proliferative vitreoretinopathy (PVR). For the in vitro experiments Human Tenon's Fibroblasts (HTFs) and retinal pigment epithelial (RPE) cells were treated with different concentrations of LY to determine HTF proliferation and RPE transdifferentiation. For in vivo testing 30 rabbits underwent a PVR trauma model. The animals received different concentrations of intravitreally injected LY, with or without vitrectomy. LY treatment reduced HTF proliferation and RPE transdifferentiation in vitro. In vivo intravitreal injection of LY prevented PVR development significantly. This positive effect was also present when LY injection was combined with vitrectomy. Intravitreal injection of LY prevented tractional retinal detachment in 14 out of 15 animals. In conclusion, treatment with the TGF-ß receptor 1 inhibitor LY reduces HTF proliferation and RPE transdifferentiation in vitro and prevents proliferative vitreoretinopathy and subsequent tractional retinal detachment in vivo.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Impact of Nintedanib and Anti-Angiogenic Agents on Uveal Melanoma Cell Behavior.

Purpose: The purpose of this study was to investigate the direct impact of the combined angiokinase inhibitor nintedanib as well as the anti-angiogenic agents ranibizumab, bevacizumab, and aflibercept on the primary uveal melanoma (UM) cell line Mel270 and liver metastasis UM cell line OMM2.5. Methods: The metabolic activity, viability, and oxidative stress levels were analyzed by the Thiazolyl Blue Tetrazolium Bromide (MTT), LIVE/DEAD, and reactive oxygen species (ROS) assays. Expression of intracellular VEGF-A165 and VEGF receptor-2 was detected by immunofluorescent staining. The secretion of VEGF-A165 into the cell culture supernatants was evaluated by VEGF-A165 ELISA. Results: Nintedanib, at a concentration of 1 µg/mL, resulted in a median reduction of metabolic activity (for Mel270 of approximately 38% and for OMM2.5 of 46% compared to the untreated control) without exerting toxicity in either cell line, whereas the other 3 substances did not result in any changes (which also means that none of the 4 substances led to an increased cell death). Moreover, nintedanib (1 µg/mL) induced oxidative stress in the Mel270 by approximately 1.2 to 1.5-fold compared to the untreated control, but not the OMM2.5 cells. Conclusions: Nintedanib could suppress the growth of UM cells in a concentration-dependent manner. The metastatic UM cell line OMM2.5 was not sensitive to the pro-oxidant activity of nintedanib. This study was the first to investigate nintedanib in the context of UM. We propose further investigation of this substance to elucidate its effects on this tumor entity with the hope of identifying advantageous therapeutic options for future adjuvant tumor therapies.

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

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.

Radiation-Induced DNA Damage in Uveal Melanoma Is Influenced by Dose Delivery and Chromosome 3 Status.

Purpose: The purpose of this study was to analyze the extent of DNA breaks in primary uveal melanoma (UM) with regard to radiotherapy dose delivery (single-dose versus fractionated) and monosomy 3 status. Methods: A total of 54 patients with UM were included. Stereotactic radiotherapy (SRT) was performed in 23 patients, with 8 undergoing single-dose SRT (sdSRT) treatment and 15 receiving fractionated SRT (fSRT). DNA breaks in the enucleated or endoresected tumors were visualized by a TUNEL assay and quantified by measuring the TUNEL-positive area. Protein expression was analyzed by immunohistochemistry. Co-detection of chromosome 3 with proteins was performed by immuno-fluorescent in situ hybridization. Results: The amount of DNA breaks in the total irradiated group was increased by 2.7-fold (P < 0.001) compared to non-irradiated tissue. Tumors treated with fSRT were affected more severely, showing 2.1-fold more DNA damage (P = 0.007) compared to the cases after single (high) dose irradiation (sdSRT). Monosomy 3 tumors showed less DNA breaks compared to disomy 3 samples (P = 0.004). The presence of metastases after radiotherapy correlated with monosomy 3 and less DNA breaks compared to patients with non-metastatic cancer in the combined group with fSRT and sdSRT (P < 0.05). Conclusions: Fractionated irradiation led to more DNA damage than single-dose treatment in primary UM. As tumors with monosomy 3 showed less DNA breaks than those with disomy 3, this may indicate that they are less radiosensitive, which may influence the efficacy of irradiation.

The neuroprotective potential of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in response to hypoxia in isolated bovine retina.

AIMS: To investigate the outcomes of Rho-kinase inhibition in the electrophysiological ex vivo model of the isolated perfused vertebrate retina under hypoxia. METHODS: Bovine retinas were perfused with an oxygen saturated nutrient solution with or without the Rho-kinase inhibitor H-1152P. The retinas were stimulated repeatedly until stable amplitudes were reached and the electroretinogram was recorded at five minute intervals. Hypoxia was induced for 15, 30, and 45 minutes, after which the oxygen saturation was restored. The extent of the cell damage and glial reactivity was determined by Ethidium homodimer-1 staining, immunohistochemistry, and Western blot. RESULTS: Hypoxia caused a time-dependent reduction of the b-wave amplitudes, which could not be prevented by the H-1152P. Although the Rho-kinase inhibitor maintained higher b-wave amplitudes, these effects did not reach statistical significance. Hypoxia also resulted in an increase in cell damage and the activation of the glial cells in the untreated retinas whereas the administration of H-1152P significantly reduced the extent of these events. CONCLUSION: H-1152P exerted a neuroprotective effect against necrosis on the isolated bovine retina under hypoxia together with a reduction in glial cell reactivity. However, the inhibitor could not prevent the hypoxia induced retinal dysfunction possibly due to the interference with synaptic modulation.

Tetrapodal ZnO-Based Composite Stents for Minimally Invasive Glaucoma Surgery.

The glaucoma burden increases continuously and is estimated to affect more than 100 million people by 2040. As there is currently no cure to restore the optic nerve damage caused by glaucoma, the only controllable parameter is the intraocular pressure (IOP). In recent years, minimally invasive glaucoma surgery (MIGS) has emerged as an alternative to traditional treatments. It uses micro-sized drainage stents that are inserted through a small incision, minimizing the trauma to the tissue and reducing surgical and postoperative recovery time. However, a major challenge for MIGS devices is foreign body reaction and fibrosis, which can lead to a complete failure of the device. In this work, the antifibrotic potential of tetrapodal ZnO (t-ZnO) microparticles used as an additive is elucidated by using rat embryonic fibroblasts as a model. A simple, direct solvent-free process for the fabrication of stents with an outer diameter of 200-400 µm is presented, in which a high amount of t-ZnO particles (45-75 wt %) is mixed into polydimethylsiloxane (PDMS) and a highly viscous polymer/particle mixture is extruded. The fabricated stents possess increased elastic modulus compared to pure PDMS while remaining flexible to adapt to the curvature of an eye. In vitro experiments showed that the fibroblast cell viability was inhibited to 43 ± 3% when stents with 75 wt % t-ZnO were used. The results indicate that cell inhibiting properties can be attributed to an increased amount of protruding t-ZnO particles on the stent surface, leading to an increase in local contacts with cells and a disruption of the cell membrane. As a secondary mechanism, the released Zn ions could also contribute to the cell-inhibiting properties in the close vicinity of the stent surface. Overall, the fabrication method and the antifibrotic and mechanical properties of developed stents make them promising for application in MIGS.

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.

In Vitro Evaluation of Zinc Oxide Tetrapods as a New Material Component for Glaucoma Implants.

In our previous study we were able to show that zinc oxide (ZnO) tetrapods inhibit wound healing processes. Therefore, the aim of this study was to test the antiproliferative effect of two types of porous polydimethylsiloxane (PDMS)/ tetrapodal zinc oxide (ZnO-T) materials, as well as their usability for glaucoma implants. To find the best implant material, two different porous PDMS/ZnO-T materials were examined. One consisted of 3D interconnected PDMS coarse-pored foams with protruding ZnO-T particles; the other consisted of fine-pored 3D interconnected ZnO-T networks homogeneously coated by a thin PDMS film in the nanometer range. Fibroblast cell viability was investigated for both materials via MTT dye, and some implant material samples were further processed for electron microscopy. Both PDMS/ZnO-T materials showed reduced cell viability in the MTT staining. Furthermore, the electron microscopy revealed barely any fibroblasts growing on the implant materials. At the surface of the fine-pored implant material, however, fibroblasts could not be observed in the etched control samples without ZnO-T. It was found that post-processing of the material to the final stent diameter was highly challenging and that the fabrication method, therefore, had to be adapted. In conclusion, we were able to demonstrate the antiproliferative potential of the two different PDMS/ZnO-T materials. Furthermore, smaller pore size (in the range of tens of micrometers) in the implant material seems to be preferable.