Abstract:The primary objective of this study was to develop and evaluate new methods of analyzing laser-induced choroidal neovascularization (CNV), in order to make recommendations for improving the reporting of experimental CNV in the literature. Six laser burns of sufficient power to rupture Bruch's membrane were concentrically placed in each eye of 18 adult Norway rats. Eyes received intravitreal injections of either triamcinolone acetonide, ketorolac, or balanced salt solution (BSS). Fluorescein angiography (FA) wa… Show more
“…This indicates the potential of topically applied anti-VEGF DARPin to treat chorioretinal neovascularization.Fig. 6The effect of DARPin eye-drops on rat choroidal neovascularization (CNV) in comparison to Triamcinolon (positive control, ivt) [45] or PBS (buffer control, ivt). a Comparison of mean CNV lesion size of the different study groups.…”
Section: Resultsmentioning
confidence: 99%
“…The effect of DARPin eye-drops on rat choroidal neovascularization (CNV) in comparison to Triamcinolon (positive control, ivt) [45] or PBS (buffer control, ivt). a Comparison of mean CNV lesion size of the different study groups.…”
The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations. We show here the generation of a set of highly potent VEGF-A antagonistic DARPins (designed ankyrin repeat proteins) delivering these properties. DARPins with single-digit picomolar affinity to human VEGF-A were generated using ribosome display selections. Specific and potent human VEGF-A binding was confirmed by ELISA and endothelial cell sprouting assays. Cross-reactivity with VEGF-A of several species was confirmed by ELISA. Intravitreally injected DARPin penetrated into the retina and reduced fluorescein extravasation in a rabbit model of vascular leakage. In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model. Even at elevated doses, DARPins were safe to use. The fact that several DARPins are highly active in various assays illustrates the favorable class behavior of the selected binders. Anti-VEGF-A DARPins thus represent a novel class of highly potent and specific drug candidates for the treatment of neovascular eye diseases in both the posterior and the anterior eye chamber.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-012-9302-0) contains supplementary material, which is available to authorized users.
“…This indicates the potential of topically applied anti-VEGF DARPin to treat chorioretinal neovascularization.Fig. 6The effect of DARPin eye-drops on rat choroidal neovascularization (CNV) in comparison to Triamcinolon (positive control, ivt) [45] or PBS (buffer control, ivt). a Comparison of mean CNV lesion size of the different study groups.…”
Section: Resultsmentioning
confidence: 99%
“…The effect of DARPin eye-drops on rat choroidal neovascularization (CNV) in comparison to Triamcinolon (positive control, ivt) [45] or PBS (buffer control, ivt). a Comparison of mean CNV lesion size of the different study groups.…”
The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations. We show here the generation of a set of highly potent VEGF-A antagonistic DARPins (designed ankyrin repeat proteins) delivering these properties. DARPins with single-digit picomolar affinity to human VEGF-A were generated using ribosome display selections. Specific and potent human VEGF-A binding was confirmed by ELISA and endothelial cell sprouting assays. Cross-reactivity with VEGF-A of several species was confirmed by ELISA. Intravitreally injected DARPin penetrated into the retina and reduced fluorescein extravasation in a rabbit model of vascular leakage. In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model. Even at elevated doses, DARPins were safe to use. The fact that several DARPins are highly active in various assays illustrates the favorable class behavior of the selected binders. Anti-VEGF-A DARPins thus represent a novel class of highly potent and specific drug candidates for the treatment of neovascular eye diseases in both the posterior and the anterior eye chamber.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-012-9302-0) contains supplementary material, which is available to authorized users.
“…Toma et al (2010) previously evaluated several edge detection techniques for measuring experimental CNV area from FA images using commercial software. The techniques had some level of automation; however, retinal specialists were still needed to outline the lesion or the user was required to manually adjust the threshold level for each image until the irrelevant microvasculature surrounding the lesion disappeared.…”
The purpose of this study was to develop a non-biased method of quantitatively measuring choroidal neovascularization (CNV) areas based on late-phase fluorescein angiography (FA) images. Experimental CNV was induced in Long Evans rats by laser disruption of the Bruch’s membrane. FA was performed weekly for 5 weeks. Multi-Otsu thresholding (MOT) was used to quantify CNV in late-phase FA images from both experimental rodent CNV and wet age-related macular degeneration patients (wAMD). Images were automatically thresholded into three levels based on the image histogram, with the highest level containing CNV. To determine the technique’s ability to quantify CNV areas, rats were given either triamcinolone acetonide or dexamethasone sodium phosphate to treat CNV and compared to untreated rats. The rat CNV lesion areas measured from 5-week histology sections from each treatment group were compared to areas measured from the corresponding FA images. MOT was able to detect statistical decreases in rodent CNV area in the treatment groups versus control from weeks 3 through 5. The ratio of CNV area measured from histology to area measured from FA images was not statistically different between groups. Finally, to determine the usefulness of MOT on pathological morphologies of CNV, MOT was performed on late-phase FA images from patients with classic and diffuse CNV. The technique was able to segment classical CNV in wAMD patients, but performed poorly with diffuse CNV. MOT provides a robust, objective, and quantifiable area measurement of CNV lesion area in both experimentally-induced and pathological CNV. The results indicate that MOT could be a useful research tool in helping evaluate the effects of therapeutics on CNV growth.
“…[12][13][14][15] Although studies of the pathogenesis and treatment of AMD have been delayed by lacking of ideal animal models, 16 laserinduced CNV rodent models are commonly used to study CNV in wet AMD. [17][18][19][20] Moreover, our previous study has shown that the Wnt signaling pathway is activated in very low-density lipoprotein receptor (VLDLR) knockout (KO) mice, a genetic animal model of subretinal neovascularization (NV). 21 The present study investigated the role of the Wnt signaling pathway in laser-induced CNV and explored therapeutic potential of a blocker of Wnt signaling in laser-induced CNV and VLDLR KO models.…”
PURPOSE. Choroidal neovascularization (CNV) is a severe complication of AMD. The Wnt signaling pathway has been shown to mediate angiogenesis. The purpose of this study was to investigate the pathogenic role of the Wnt pathway in CNV and explore the therapeutic potential of a novel Wnt signaling inhibitor in CNV.METHODS. Adult rats and mice were photocoagulated using diode laser to induce CNV. On the same day, the animals were intravitreally injected with a monoclonal antibody (Mab2F1) blocking LRP6 or nonspecific mouse IgG. The Wnt signaling activation and target gene expression in the eyecup were determined by Western blot analysis. Fundus angiography was used to examine leakage from the laser lesion. CNV areas were measured on choroidal flatmount using FITC-dextran. The canonical wingless-type MMTV integration site (Wnt) signaling pathway plays a critical role in the regulation of inflammation and angiogenesis. 5,6 Wnt ligands are secreted, cysteine-rich glycosylated proteins, 3 which bind to frizzled (Fz) receptors or to the coreceptor complex of Fz and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6).
RESULTS. Levels of
4-6Binding of Wnt ligands results in LRP6 phosphorylation and activation, 7,8 leading to dissociation of the kinase complex containing glycogen synthase kinase-3b (GSK3b), 9 axin and adenomatous polyposis.3 The GSK3b complex dissociation prevents transcription factor b-catenin from phosphorylation and degradation.10 Consequently, b-catenin is accumulated in the cytoplasm and translocated into the nucleus, complexes with TCF/LEF family transcription factors, 11 regulating expression of Wnt target genes including VEGF, which is the key pathogenic factor in CNV. [12][13][14][15] Although studies of the pathogenesis and treatment of AMD have been delayed by lacking of ideal animal models, 16 laserinduced CNV rodent models are commonly used to study CNV in wet AMD. [17][18][19][20] Moreover, our previous study has shown that the Wnt signaling pathway is activated in very low-density lipoprotein receptor (VLDLR) knockout (KO) mice, a genetic animal model of subretinal neovascularization (NV). 21 The present study investigated the role of the Wnt signaling pathway in laser-induced CNV and explored therapeutic potential of a blocker of Wnt signaling in laser-induced CNV and VLDLR KO models.
METHODS AnimalsCare, use, and treatment of experimental animals were in strict agreement with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male C57BL/6J mice and VLDLR KO mice (10 weeks old; The Jackson Laboratory, Bar Harbor, ME) and male Brown Norway rats (8-10 weeks old; Charles River, Wilmington, MA) were used in this study. In all procedures, animals were anesthetized by intramuscular injection of 50 mg/kg ketamine hydrochloride (Vedco, St. Joseph, MO) and 10 mg/kg xylazine (Vedco), and pupils were dilated with topical administration of 1% cyclopentolate (Wilson, Mustang, OK).
CNV InductionLaser photocoagulation (532 nm, 150-250 mW, 0.01 second, 50 lm; model di...
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