Structure and function of the N-cadherin/catenin complex in retinoblastoma.

PURPOSE. To identify in human retinoblastoma and normal retinal tissue the type of cadherin, its relationship with cytoplasmic catenins, and its participation in invasion. METHODS. The cadherin/catenin complex was characterized in surgical retinoblastoma. specimens from five patients and human retinas from four donor eyes by immunocytochemistry, flow cytometry, and coimmunoprecipitation with antibodies against N-cadherin, alpha-catenin, and beta-catenin, followed by Western blot analysis or autoradiography. Y79 and WERI-Rb-1 retinoblastoma cell lines serve the evaluation of the cadherin/ catenin complex in aggregation and Collagen type I invasion in vitro. The association of the cadherin/catenin complex with the cytoskeleton was examined by an antibody-capping assay. RESULTS. In retinoblastoma and normal retina N-cadherin associated with a-catenin and beta-catenin but not E- or P-cadhcrin. The N-cadherin/catenin complex formed a regular, linear, and continuous honeycomb pattern in normal retina that was irregular, clustered, and interrupted in retinoblastoma. The Ncadherin/catenin complex was found also in the retinoblastoma cell lines WERI-Rb and Y79, in which it also showed an irregular pattern. Both cell lines were invasive in Collagen type I, and invasion was inhibited by the GC-4 antibody, which functionally neutralizes N-cadherin. Less GC-4 antibody was needed to inhibit invasion of Y79 cells, which expressed N-cadherin at a lower level, than to inhibit invasion of WERI-Rb-1 cells. In both cell lines, antibody capping of the N-cadherin/ catenin complex indicated that its linkage with the cytoskeleton were weak or absent. CONCLUSIONS. Retinoblastoma cells, in contrast with normal retina, express an N-cadherin/catenin complex that is irregularly distributed and weakly linked to the cytoskeleton. In retinoblastoma, this complex acts as an invasion promoter

Expression of the SST receptor 2 in uveal melanoma is not a prognostic marker

Introduction: Uveal melanoma (UM) cells and neurohormone-producing cells both originate from the neural crest. Somatostatin receptors subtype 2 (SSTR2) are over-expressed in several tumors, often from neuroendocrine origin, and synthetic antagonists like octreotide and octreotate are being used as diagnostic or therapeutic agents. We investigated the SSTR2 expression in UM, and determined whether this expression was related to prognosis of the disease. Materials and methods: UM cell lines and fresh primary UM samples were tested for SSTR2 expression by autoradiography (AR) using 125I-Tyr3-octreotate. Furthermore, UM cell lines were analyzed for SSTR2 mRNA expression with quantitative real-time RT-PCR. Results: Using AR, cell-surface SSTR2 expression was demonstrated in two UM metastatic cell lines, but no expression was detected in three cell lines derived from primary UM. However, all primary and metastatic UM cell lines showed mRNA expression levels for SSTR2 using quantitative real-time RT-PCR. Only three of 14 primary UM demonstrated moderate SSTR2 expression, and this expression was not significantly associated with tumor-free survival or any tested prognostic factor. Conclusions: Based on the rare and low expression of SSTR2 found in primary UM specimens and in UM cell lines, we conclude that SSTR2 is not widely expressed in UM. Furthermore, SSTR2 expression was not associated with tumor-free survival and prognostic factors. Therefore SSTR2 is not suited as prognostic marker or therapeutic target in UM

Real-World Weekly Efficacy Analysis of Faricimab in Patients with Age-Related Macular Degeneration

Objectives This study entailed a weekly analysis of real-world data (RWD) on the safety and efficacy of intravitreal (IVT) faricimab in neovascular age-related macular degeneration (nAMD). Methods A retrospective, single-centre clinical trial was conducted at the Department of Ophthalmology, University Hospital Zurich, University of Zurich, Switzerland, approved by the Cantonal Ethics Committee of Zurich, Switzerland. Patients with nAMD were included. Data from patient charts and imaging were analysed. The safety and efficacy of the first faricimab injection were evaluated weekly until 4 weeks after injection. Results Sixty-three eyes with a complete 4-week follow-up were enrolled. Six eyes were treatment-naïve; fifty-seven eyes were switched to faricimab from another treatment. Neither group showed signs of retinal vasculitis during the 4 weeks after injection. Central subfield thickness (CST) and volume (CSV) showed a statistically significant decrease compared to the baseline in the switched group (CST: p = 0.00383; CSV: p = 0.00702) after 4 weeks. The corrected visual acuity returned to the baseline level in both groups. The macular neovascularization area decreased in both groups, but this was not statistically significant. A complete resolution of sub- and intraretinal fluid after 4 weeks was found in 40% (switched) and 75% (naïve) of the treated patients. Conclusions The weekly follow-ups reflect the structure–function relationship beginning with a fast functional improvement within two weeks after injection followed by a return to near-baseline levels after week 3. The first faricimab injection in our cohort showed a high safety profile and a statistically significant reduction in macular oedema in switched nAMD patients

Dynamics of Treatment Response to Faricimab for Diabetic Macular Edema

This study analyzes the dynamics of short-term treatment response to the first intravitreal faricimab injection in eyes with diabetic macular edema (DME). This retrospective, single-center, clinical trial was conducted at the Department of Ophthalmology, University Hospital Zurich. Patients with treatment-naïve and pretreated DME were included. Patient chart data and imaging were analyzed. Safety and efficacy (corrected visual acuity (CVA), central subfield thickness (CST), and signs of intraocular inflammation (IOI)) of the first faricimab intravitreal therapy (IVT) were evaluated weekly until 4 weeks after injection. Forty-three eyes (81% pretreated) of 31 patients were included. Four weeks after the first faricimab IVT, CVA remained stable and median CST (µm) decreased significantly (p < 0.001) from 325.0 (293.5–399.0) at baseline to 304.0 (286.5–358.0). CVA at week 4 was only associated with baseline CVA (p < 0.001). CST was the only predictive variable (p = 0.002) between baseline and week 4 CST. Weekly safety assessments did not show any sign of clinically significant IOI. This study suggests faricimab is an effective treatment for (pretreated) DME, showing structural benefit 1 month following the first injection without short-term safety signals

Efficacy of fatty acids dietary supplement in polyethylene glycol-induced mouse model of retinal degeneration

Current knowledge of the benefits of nutrition supplements for eye pathologies is based largely on the use of appropriate animal models, together with defined dietary supplementation. Here, C57BL6 mice were subretinally injected with polyethylene glycol (PEG)-400, an established model of retinal degeneration with a dry age-related macular degeneration (AMD)-like phenotype, an eye pathology that lacks treatment. In response to PEG-400, markers of the complement system, angiogenesis,inflammation,gliosis,andmacrophageinfiltrationwereupregulatedinbothretinasand retinal pigment epithelium (RPE)/choroids, whereas dietary supplementation with a mixture based on fatty acids counteracted their upregulation. Major effects include a reduction of inflammation, in both retinas and RPE/choroids, and an inhibition of macrophage infiltration in the choroid, yet not in the retina, suggesting a targeted action through the choroidal vasculature. Histological analysis revealed a thinning of the outer nuclear layer (ONL), together with dysregulation of the epithelium layer in response to PEG-400. In addition, immunohistofluorescence demonstrated Müller cell gliosis and macrophage infiltration into subretinal tissues supporting the molecular findings. Reduced ONL thickness,gliosis,andmacrophageinfiltrationwerecounteractedbythedietsupplement. The present data suggest that fatty acids may represent a useful form of diet supplementation to prevent or limit the progression of dry AMD

Echinomycin mitigates ocular angiogenesis by transcriptional inhibition of the hypoxia-inducible factor-1

Background: Echinomycin (EKN), an inhibitor of hypoxia-inducible factor (HIF)-1 DNA-binding activity, has been implied as a possible therapeutic agent in ischemic diseases. Here, we assess EKN in hypoxia-driven responses in vitro using human primary adult retinal pigment epithelium cells (aRPE) and retinal endothelial cells (hREC), and in vivo using the laser-induced mouse choroidal neovascularization (CNV) model. Methods: Effects of EKN on hypoxia-mediated pathways in aRPE were analyzed by Western blotting for HIF-1α protein, quantitative PCR of HIF-target genes, and proteome array for soluble angiogenic factors. In vitro inhibition of angiogenesis by EKN was determined in hREC. In vivo inhibition of angiogenesis by EKN was determined in the mouse laser-induced CNV, as a model of HIF-associated ocular neovascularization. CNV lesion area was determined by fundus fluorescein angiography. Results: aRPE treated with EKN showed hypoxia-dependent significantly decreased cell recovery in the wound healing assay. These results were supported by lower levels of HIF-mediated transcripts detected in hypoxic aRPE cells treated with EKN compared with non-treated controls, and confirmed by proteome profiler for angiogenic factors. hREC exposed to aRPE EKN-conditioned medium displayed reduced sprouting angiogenesis. Mice with laser-induced CNV treated with intravitreally injected EKN showed significantly decreased vascular lesion area when compared with a mouse equivalent of aflibercept, or vehicle-treated controls. Conclusions: Our data proposes EKN as a potent inhibitor of HIF-mediated angiogenesis in retinal cells and in the mouse model of CNV, which could have future implications in the treatment of patients with neovascular age-related macular degeneration.Ministerio de Ciencia, Innovación y Universidades de España. FPU17/0346

Gaining insight on mitigation of rubeosis iridis by UPARANT in a mouse model associated with proliferative retinopathy

Proliferative retinopathies (PR) lead to an increase in neovascularization and inflammation factors, at times culminating in pathologic rubeosis iridis (RI). In mice, uveal puncture combined with injection of hypoxia-conditioned media mimics RI associated with proliferative retinopathies. Here, we investigated the effects of the urokinase plasminogen activator receptor (uPAR) antagonist-UPARANT-on the angiogenic and inflammatory processes that are dysregulated in this model. In addition, the effects of UPARANT were compared with those of anti-vascular endothelial growth factor (VEGF) therapies. Administration of UPARANT promptly decreased iris vasculature, while anti-VEGF effects were slower and less pronounced. Immunoblot and qPCR analysis suggested that UPARANT acts predominantly by reducing the upregulated inflammatory and extracellular matrix degradation responses. UPARANT appears to be more effective in comparison to anti-VEGF in the treatment of RI associated with PR in the murine model, by modulating multiple uPAR-associated signaling pathways. Furthermore, UPARANT effectiveness was maintained when systemically administered, which could open to novel improved therapies for proliferative ocular diseases, particularly those associated with PR. KEY MESSAGES: • Further evidence of UPARANT effectiveness in normalizing pathological iris neovascularization. • Both systemic and local administration of UPARANT reduce iris neovascularization in a model associated with proliferative retinopathies. • In the mouse models of rubeosis iridis associated with proliferative retinopathy, UPARANT displays stronger effects when compared with anti-vascular endothelial growth factor regimen

Systemic 7-methylxanthine in retarding axial eye growth and myopia progression: a 36-month pilot study

The adenosine antagonist 7-methylxanthine (7-mx) works against myopia in animal models. In a clinical trial, 68 myopic children (mean age 11.3 years) received either placebo or 7-mx tablets for 12 months. All participants subsequently received 7-mx for another 12 months, after which treatment was stopped. Axial length was measured with Zeiss IOL-Master and cycloplegic refraction with Nikon Retinomax at −6, 0, 12, 24, and 36 months. Axial growth was reduced among children treated with 7-mx for 24 months compared with those only treated for the last 12 months. Myopia progression and axial eye growth slowed down in periods with 7-mx treatment, but when the treatment was stopped, both myopia progression and axial eye growth continued with invariable speed. The results indicate that 7-mx reduces eye elongation and myopia progression in childhood myopia. The treatment is safe and without side effects and may be continued until 18–20 years of age when myopia progression normally stops

Suppression and Regression of Choroidal Neovascularization in Mice by a Novel CCR2 Antagonist, INCB3344

PURPOSE: To investigate the effect of an intravitreally administered CCR2 antagonist, INCB3344, on a mouse model of choroidal neovascularization (CNV). METHODS: CNV was induced by laser photocoagulation on Day 0 in wild type mice. INCB3344 or vehicle was administered intravitreally immediately after laser application. On Day 14, CNV areas were measured on retinal pigment epithelium (RPE)-choroid flat mounts and histopathologic examination was performed on 7 µm-thick sections. Macrophage infiltration was evaluated by immunohistochemistry on RPE-choroid flat mounts and quantified by flow cytometry on Day 3. Expression of vascular endothelial growth factor (VEGF) protein in RPE-choroid tissue was examined by immunohistochemistry and ELISA, VEGF mRNA in sorted macrophages in RPE-choroid tissue was examine by real-time PCR and expression of phosphorylated extracellular signal-regulated kinase (p-ERK 1/2) in RPE-choroid tissue was measured by Western blot analysis on Day 3. We also evaluated the efficacy of intravitreal INCB3344 to spontaneous CNV detected in Cu, Zn-superoxide dismutase (SOD1) deficient mice. Changes in CNV size were assessed between pre- and 1week post-INCB3344 or vehicle administration in fundus photography and fluorescence angiography (FA). RESULTS: The mean CNV area in INCB3344-treated mice decreased by 42.4% compared with the vehicle-treated control mice (p<0.001). INCB3344 treatment significantly inhibited macrophage infiltration into the laser-irradiated area (p<0.001), and suppressed the expression of VEGF protein (p = 0.012), VEGF mRNA in infiltrating macrophages (p<0.001) and the phosphorylation of ERK1/2 (p<0.001). The area of spontaneous CNV in Sod1⁻/⁻ mice regressed by 70.35% in INCB3344-treated animals while no change was detected in vehicle-treated control mice (p<0.001). CONCLUSIONS: INCB3344 both inhibits newly forming CNV and regresses established CNV. Controlling inflammation by suppressing macrophage infiltration and angiogenic ability via the CCR-2/MCP-1 signal may be a useful therapeutic strategy for treating CNV associated with age-related macular degeneration

Cigarette Smoke-Related Hydroquinone Dysregulates MCP-1, VEGF and PEDF Expression in Retinal Pigment Epithelium in Vitro and in Vivo

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice.MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD