Swaantje Peters

Antiproliferative and cytotoxic properties of bevacizumab on different ocular cells

Aim: To evaluate the antiproliferative and cytotoxic properties of bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5), and pig choroidal endothelial cells (CEC). Methods: Monolayer cultures of ARPE19, RGC5, and CEC were used. Bevacizumab (0.008–2.5 mg/ml), diluted in culture medium, was added to cells that were growing on cell culture dishes. Cellular proliferative activity was monitored by 5′-bromo-2′-deoxyuridine (BrdU) incorporation into cellular DNA and the morphology assessed microscopically. For cytotoxicity assays ARPE19, RGC5, and CEC cells were grown to confluence and then cultured in a serum depleted medium to ensure a static milieu. The MTT test was performed after 1 day. The “Live/Dead” viability/cytotoxicity assay was performed and analysed by fluorescence microscopy after 6, 12, 18, 24, 30, 36, and 48 hours of incubation. Expression of VEGF, VEGF receptors (VEGFR1 and VEGFR2) and von Willebrand factor was analysed by immunohistochemistry. Results: No cytotoxicity of bevacizumab on RGC5, CEC, and ARPE19 cells could be observed after 1 day. However, after 2 days at a bevacizumab concentration of 2.5 mg/ml a moderate decrease in ARPE19 cell numbers and cell viability was observed. Bevacizumab caused a dose dependent suppression of DNA synthesis in CEC as a result of a moderate antiproliferative activity (maximum reduction 36.8%). No relevant antiproliferative effect of bevacizumab on RGC5 and ARPE19 cells could be observed when used at a concentration of 0.8 mg/ml or lower. CEC and ARPE 19 cells stained positively for VEGF, VEGFR1, and VEGFR2. More than 95% of the CEC were positive for von Willebrand factor. Conclusions: These experimental findings support the safety of intravitreal bevacizumab when used at the currently applied concentration of about 0.25 mg/ml. Bevacizumab exerts a moderate growth inhibition on CEC when used in concentrations of at least 0.025 mg/ml. However, at higher doses (2.5 mg/ml) bevacizumab may be harmful to the retinal pigment epithelium.

Antipermeability and antiproliferative effects of standard and frozen bevacizumab on choroidal endothelial cells

Background: Bevacizumab is an antiangiogenic compound developed to target tumour vessels. Its off-label use in ophthalmology requires in vitro testing on ocular cells. Aim: To quantify the antipermeability and antiproliferative effects of bevacizumab on cultured choroidal endothelial cells (CECs). It was examined whether deep-freezing of bevacizumab attenuates its antiangiogenic activity. Methods: Porcine CECs were cultured in permeable insert systems. Permeability of the cell monolayers was quantified by a fluorescent isothiocyanate-dextran assay after treatment with vascular endothelial growth factor (VEGF; 20–100 ng/ml) alone and in combination with bevacizumab (0.1–1 mg/ml). Proliferation of the CECs was tested using a “wound scratch” assay. The experiments were repeated with bevacizumab after freezing at −20°C for 5 days. Results: Bevacizumab significantly reduced VEGF-induced permeability in a dose-dependant manner. A molar ratio of 2.6:1 of bevacizumab to VEGF was required for complete blocking of VEGF-induced rise in permeability. CEC proliferation was significantly blocked by bevacizumab (0.5 mg/ml). Thawed bevacizumab after deep freezing showed a moderate, but not statistically significant loss in activity. Conclusion: Bevacizumab significantly reduces VEGF-induced permeability and proliferation of CECs. Freezing and thawing of bevacizumab will affect its biological activity.

Inhibition of Lysosomal Degradation in Retinal Pigment Epithelium Cells Induces Exocytosis of Phagocytic Residual Material at the Basolateral Plasma Membrane

Purpose: To analyze chloroquine-induced morphological changes in the retinal pigment epithelium (RPE) and Bruch’s membrane (BM). Methods: Retina-choroid complexes of chloroquine-treated Long-Evans rats were analyzed by electron microscopy. Results: Intercellular spaces between the RPE cells and BM were enlarged. Residual material from phagosomes was released into these enlarged spaces. Debris accumulated within BM and encircled choriocapillaris endothelial cells. Conclusion: There is a release of undegraded phagocytic material (rod outer segments) into the extracellular space between BM and RPE cells, following inhibition of lysosomal degradation. Electron-dense deposits in BM and choriocapillaris may lead to reduced oxygen and nutrition flow.

Immunohistochemical localisation of intravitreally injected bevacizumab in the anterior chamber angle, iris and ciliary body of the primate eye

Aims: To locate bevacizumab in the tissues related to neovascularisation in the anterior segment within 1–14 days after intravitreal injection in the primate eye. Methods: Four cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. Control eyes remained untreated. The eyes were enucleated on day 1, 4 and 14 for immunohistochemistry, using donkey anti-human Cy3-IgG. Results: Immunoreactivity for bevacizumab was found in the blood vessels walls of the iris, anterior chamber angle and ciliary body. In the iris and chamber angle, immunoreactivity was most prominent on day 1 after injection and diminished until day 14. In the ciliary body, staining was most intense on day 4 and remained prominent until day 14. Immunoreactivity was also present in certain vessel lumens, especially in the ciliary body and the iris on day 4 and 14. Conclusion: Bevacizumab penetrates quickly into the iris, anterior chamber angle and ciliary body after intravitreal injection in the primate eye and accumulates particularly in blood-vessel walls. The highest concentration of bevacizumab in these tissues is present on day 1–4, the iris and anterior chamber angle being penetrated slightly earlier than the ciliary body. Our findings support the clinically observed rapid effect in the treatment of iris neovascularisation.

Subretinal Injection of Rod Outer Segments Leads to an Increase in the Number of Early-Stage Melanosomes in Retinal Pigment Epithelial Cells

Our study was performed to test the hypothesis that subretinally injected protein can induce melanogenesis in the retinal pigment epithelium (RPE). Rod outer segments (ROS) were isolated from cattle eyes and injected into the subretinal space of Long Evans rats. Five days after surgery, the injected eyes were investigated by electron microscopy. The number of early-stage melanosomes and small melanin granules was compared in injected and noninjected eyes. It was found that the injected ROS were phagocytized by the RPE cells, and the number of early-stage melanosomes in the RPE was significantly increased in injected eyes compared to eyes without injection. The ROS-containing endosomes fused with melanolysosomes in which melanogenesis took place. The increased number of early-stage melanosomes indicates new formation of melanin.

Safety Parameters for Indocyanine Green in Vitreoretinal Surgery

Since the early nineties removal of the internal limiting membrane (ILM) has been shown to be an effective and safe treatment option for conditions that involve the vitreoretinal interface. Peeling of the barely visible ILM, however, represents a challenge and complete removal is difficult and not always obvious. Damage at the vitreoretinal interface or unsatisfactory peeling may therefore be the result of the genuine procedure. Introduction of indocyanine green (ICG) for ILM staining led to better visibility of the ILM and greatly facilitated this surgical maneuver making ILM peeling more controllable, easier and faster. Consequently, enthusiastic acceptance resulted in an uncritical use not supported by preclinical safety data. Soon thereafter some clinical reports raised concerns about potential cytotoxic effects related to the intravitreal use of ICG. The following chapter summarizes the results of in vitro, ex vivo, in vivo and clinical studies related to the use of ICG in vitreoretinal surgery. Critical appraisal of the methodical procedures and results leads to the nonnegligible fact that ICG has a cytotoxic effect enhanced by photoactivation. The results of several studies as well as our experimental workup, however, showed that ICG toxicity to the retinal pigment epithelium is dependent on the dye concentration, the osmolarity of the solvent solutions, as well as on the lengths of dye exposure time and of the vitrectomy endolight illumination time. With respect to the safety margins and profile, ICG is therefore a useful surgical tool that is still widely applied, but that may be replaced by more inert and as efficient vital dyes.

Ultrastructural localization of lipid peroxides as benzidine-reactive substances in the albino mouse eye

Abstract · Background: Lipid peroxidation is considered to be a prominent feature of retinal degeneration and has also been proposed to be involved in the pathogenesis of age-related macular degeneration. Melanin protects against lipid peroxidation and takes part in the detoxification of lipid peroxides (LP). LP can be ultrastructurally detected as benzidine-reactive substances (BRS) using tetramethylbenzidine (TMB). Albino mice lack melanin. In the present study, LP were localized as BRS in the eyes of albino and pigmented mice. · Methods: Eye cups of an albino mouse lineage and of wild-type mice were fixed with 2% glutaraldehyde, incubated with 0.5 mg/ml TMB and embedded for electron microscopy. · Results: BRS were detected in the eyes of albino mice, but no reaction product was seen in pigmented eyes. BRS located in the retinal pigment epithelium (RPE) and in the choroid of the albino mouse; no BRS were found in intact rod outer segments (ROS). · Conclusion: The lack of melanin in albino mice is associated with a higher level of lipid peroxidation in RPE and choroid. Melanin seems to protect against LP in RPE and choroid. A lack of melanin is not associated with lipid peroxidation in intact ROS. The present investigation demonstrates the significance of melanin in protection against LP in RPE and choroid.

Cellular transport of subretinal material into choroidal and scleral blood vessels: an electron microscopic study

Abstract · Background: The fate of indigestible material injected into the subretinal space of rats was investigated. · Methods: The non-toxic dye Monastral Blue (MB), which cannot be digested within the lysosomal compartment, was injected transsclerally into the subretinal space of Long Evans and Wistar rats. After 5 and 12 days respectively the eyes were enucleated and examined by light and electron microscopy. Cryo sections were made of eyes 5 days after MB injection for the application of immunohistochemical techniques using markers for epithelial cells (cytokeratin) and macrophages (ED 1). · Results: Retina, choroid and sclera were not altered in their morphology in the circumference of the MB-containing bubble generated by subretinal injection. After both 5 and 12 days no injected material was found extracellularly in the subretinal space. Especially high amounts of MB were found, in particular 5 days after injection, in lysosomes and melanosomes of RPE cells as well as in cells between choroidal melanocytes. Cells containing MB were seen in contact with choroidal and scleral blood vessels. These MB-containing cells in the choroid and in the sclera were positive for macrophage antibodies. · Conclusion: Sub-retinal injection was confirmed as a suitable method for placing fluids into the subretinal space without affecting the morphology of the retina. Subretinal injected material was shown to be incorporated into lysosomes and melanosomes of RPE cells. The injected material was subsequently transported through Bruch’s membrane to be finally removed from the eye via choroidal and scleral veins, the process involving macrophages.

Anti-VEGF-Therapie: Grundlagen und Substanzen

Die Angiogenese ist ein physiologischer und lebensnotwendiger Prozess. Unter bestimmten Umstanden kann dieser aber auch Ausdruck krankhafter Mechanismen sein. Man spricht dann von pathologischen Neovaskularisationen. Diese konnen im Rahmen neoplastischer Prozesse auftreten. In den meisten Fallen handelt es sich aber um ein reaktives Ereignis auf ein sich veranderndes Milieu. Hierzu zahlen insbesondere hypoxische und entzundliche Vorgange. Neovaskularisationen am Auge wirken sich durch die besondere Anatomie und Funktion des Organs meistens funktionsbeeintrachtigend aus und konnen letztendlich zur Erblindung oder Verlust des Auges fuhren. Zahlreiche Studien weisen den Faktor VEGF (»vascular endothelial growth factor«) als potenten Stimulator okularer Neovaskularisationen aus.

Current and future strategies for nonexudative age-related macular degeneration

Background: Age-related macular degeneration (AMD) is the leading cause of blindness in the industrialized world. Recently, the anti-VEGF strategy revolutionized the treatment of the exudative ‘wet’ form of the disease and improved therapeutic outcomes. However, the current strategy does not treat the causative and even more common neurodegenerative ‘dry’ process of the disease. Objectives: In this article we review the current approach and rationale for the present and developing therapeutic strategies that target the nonexudative dry form of AMD. Methods: A review of the literature and ongoing studies was undertaken. Results: Currently, there is no proven drug for the treatment of dry AMD. Cessation of smoking, and nutritional recommendations and supplements are supposed to slow disease progression. Different strategies targeting perfusion, inflammation, oxidative stress and degeneration, or interfering early in – or even prior to – the formation of subretinal pigmented epithelium deposits, may finally succeed in treating the causative processes of the disease. Conclusion: For the nonexudative form of AMD, no established treatment exists, except for the use of oral supplements with high-dose antioxidants that may delay progression. Therefore, a fundamental treatment approach for the neurodegenerative process is needed. Potential future drugs are under development.