Aysegül Tura

Safety, penetration and efficacy of topically applied bevacizumab: evaluation of eyedrops in corneal neovascularization after chemical burn

Purpose:  That vascular endothelial growth factor (VEGF) plays a major role in inflammatory angiogenesis has been well established. This pilot study was designed to evaluate experimental treatment with bevacizumab eyedrops in corneal neovascularization induced by alkali burn. The feasibility of topical administration, corneal cell viability and corneal penetration were investigated in an animal model.

Melanin Precursor 5,6-Dihydroxyindol: Protective Effects and Cytotoxicity on Retinal Cells in vitro and in vivo

5,6-Dihydroxyindole (DHI) is a melanin pigment precursor with antioxidant properties. In the light of a report about cytotoxicity of DHI, the aim of this study was to assess possible toxic effects of DHI on cells related to the eye, such as human ARPE-19 cells and mouse retinal explants. Moreover, DHI was tested on its effects on retinal function in vivo using electroretinography. We found cytotoxicity of DHI against ARPE-19 cells at 100 μM, but not at 10 μM. 10 μM DHI exhibited a slight, though not significant protective activity against UV-A damage in ARPE-19 cells. We found cytoprotection in cultured mouse retinas by 50 μM DHI or its diacetylated derivative 5,6-diacetoxyindole (DAI), respectively. In ERG measurements in vivo, amplitudes were decreased only slightly by 100 μM DHI compared to saline, whereas a better preservation of amplitudes was visible at 10 μM DHI, in particular with respect to cones. In histological sections, more cones were found at 10 μM DHI than at 100 μM DHI. As a conclusion, DHI shows a slight protective effect at 10 μM both in vitro and in vivo. At 100 μM, it shows a strong cytotoxicity in vitro, which is strongly reduced in vivo.

Identification of Circulating Melanoma Cells in Uveal Melanoma Patients by Dual-Marker Immunoenrichment

PURPOSE Despite successful local tumor control, uveal melanoma (UM) patients may develop lethal metastases. To reliably identify circulating melanoma cells (CMC) in UM patients, we set out to test a new immunomagnetic enrichment assay and screened UM patients for the presence of CMC. We also determined whether we could find CMC in culture; for example, for future drug testing. METHODS A dual-immunomagnetic enrichment assay using antibodies against two melanoma markers (NKI/C3 and NKI/beteb) was used to determine the presence of UM cells in blood. The sensitivity of the assay was determined by spiking normal blood with 92.1 cells (concentration range, 1-10(4) cells/mL). Isolated cells were characterized by immunocytochemistry directly after immunoenrichment and after a 2-week culture. The presence of CMC was determined in the peripheral blood of 31 patients with UM, and results were compared to clinical prognostic factors at the time of presentation. RESULTS The CMC were detected in 93.5% (n = 29 of 31) of the patients with primary nonmetastatic UM at a median density of 3.5 cells/10 mL blood (range, 0-10.2 cells), as well as in blood cultures. No significant association was observed between the presence or number of CMC and any clinical prognostic factors. CONCLUSIONS The improved dual-immunoenrichment assay enabled the detection of intact and viable CMC in the majority of UM patients. We also were able to identify CMC after short-term culturing. Molecular characterization of the CMC rather than the prevalence of these cells is expected to provide relevant information on the individual risk of metastasis.

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.

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

This study evaluates the use of the TGF-β receptor 1 inhibitor LY-364947 (LY) to prevent proliferative vitreoretinopathy (PVR). For the in vitro experiments Human Tenons 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-β receptor 1 inhibitor LY reduces HTF proliferation and RPE transdifferentiation in vitro and prevents proliferative vitreoretinopathy and subsequent tractional retinal detachment in vivo.

ApoA-I Mimetic Peptide 4F Reduces Age-Related Lipid Deposition in Murine Bruch’s Membrane and Causes Its Structural Remodeling

ABSTRACT Purpose: Accumulation of lipoprotein-derived lipids including esterified and unesterified cholesterol in Bruch’s membrane of human eyes is a major age-related change involved in initiating and sustaining soft drusen in age-related macular degeneration (AMD). The apolipoprotein (apo) A-I mimetic peptide 4F is a small anti-inflammatory and anti-atherogenic agent, and potent modifier of plasma membranes. We evaluated the effect of intravitreally-injected 4F on murine Bruch’s membrane. Methods: We tested single intravitreal injections of 4F doses (0.6 µg, 1.2 µg, 2.4 µg, and placebo scrambled peptide) in ApoEnull mice ≥10 months of age. After 30 days, mice were euthanized. Eyes were processed for either direct immunofluorescence detection of esterified cholesterol (EC) in Bruch’s membrane whole mounts via a perfringolysin O-based marker linked to green fluorescent protein or by transmission electron microscopic visualization of Bruch’s membrane integrity. Fluorescein isothiocyanate-conjugated 4F was traced after injection. Results: All injected eyes showed a dose-dependent reduction of Bruch’s membrane EC with a concomitant ultrastructural improvement compared to placebo treated eyes. At a 2.4 µg dose of 4F, EC was reduced on average by ~60% and Bruch’s membrane returned to a regular pentalaminar structure and thickness. Tracer studies confirmed that injected 4F reached intraocular targets. Conclusion: We demonstrated a highly effective pharmacological reduction of EC and restoration of Bruch’s membrane ultrastructure. The apoA-I mimetic peptide 4F is a novel way to treat a critical AMD disease process and thus represents a new candidate for treating the underlying cause of AMD.

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.

Analysis of monosomy-3 in immunomagnetically isolated circulating melanoma cells in uveal melanoma patients.

Monosomy‐3 in primary uveal melanoma (UM) is associated with a high risk of metastasis and mortality. Although circulating melanoma cells (CMC) can be found in most UM patients, only approximately 50% of the patients develop metastases. We utilized a novel immuno‐FISH assay to detect chromosome‐3 in intact CMC isolated by dual immunomagnetic enrichment. Circulating melanoma cells were detected in 91% of the patients (n = 44) with primary non‐metastatic UM, of which 58% were positive for monosomy‐3. The monosomy‐3 status of CMC corresponded to the monosomy‐3 status of the primary tumor in 10 of the 11 patients where this could be tested. Monosomy‐3 in the CMC was associated with an advanced tumor stage (P = 0.046) and was detected in all four patients who developed metastasis within the follow‐up period of 4 yr. This non‐invasive technique may enable the identification of UM patients at risk for metastasis particularly when a primary tumor specimen is unavailable.

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.

Analysis of Caveolin‐1 and Phosphoinositol‐3 Kinase expression in primary uveal melanomas

To evaluate the regulation of blood supply in primary uveal melanomas through caveolin‐1 (Cav‐1)/phosphoinositol‐3 kinase (PI3K).