Seven days after laser, the mice were given an intraperitoneal injection of 12 l/g body weight of 1% fluorescein sodium (Alcon, Fort Worth, Texas) and after 5 minutes they were euthanized. nonhuman primates, electroretinograms and fluorescein angiograms were normal, and light microscopy of ocular sections showed no evidence of structural damage. These data show for the first time that S1P stimulates both choroidal and retinal NV and. suggest that sonepcizumab could be considered for evaluation in patients with choroidal or retinal NV. Introduction Several proteins have been demonstrated to promote angiogenesis in various vascular beds, but lipid molecules such as sphingosine-1-phosphate (S1P) may also contribute. S1P acts through a family of five G protein-coupled receptors, S1P receptors 1C5 (Lee et al., 1998), first identified on vascular endothelial cells as the products of genes upregulated during differentiation of endothelial cells (endothelial differentiation genes, EDG) (Hla and Maciag, 1990). Acting through S1P receptors, S1P stimulates migration and survival of cultured vascular endothelial cells, and formation of cell-cell adherence junctions (Lee et al., 1998; Paik et al., 2001). Mice deficient in S1P1 receptor die between embryonic day 13.5 and 14.5 due to lack of pericytes recruitment around developing vessels resulting in lethal hemorrhages (Liu et al., 2000). In adult mice, injection of S1P1 receptor multiplex siRNAs into tumor xenografts or injection of a monoclonal antibody directed against S1P suppressed angiogenesis and tumor growth (Chae et al., 2004; Visentin et al., 2006) suggesting a role for S1P in tumor angiogenesis. However, bioactive lipids such as S1P have not previously been demonstrated to directly promote ocular angiogenesis. In ischemic retina, S1P2 receptor, but not S1P1 or S1P3 receptors, is strongly upregulated and compared to wild PF-06471553 type controls, mice deficient in S1P2 receptor develop significantly less ischemia-induced retinal neovascularization (NV) (Skoura et al., 2007). This suggests the hypothesis that an antagonist of S1P would inhibit ischemia-induced retinal NV and possibly other types of ocular NV. In this study, we used a humanized and optimized monoclonal antibody that binds S1P (sonepcizumab) to test that hypothesis. Materials and Methods Mice Pathogen-free C57BL/6 mice (Charles River, Wilmington, MA) were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Animal Care and Use Committee at the Johns Hopkins University Medical School. A humanized monoclonal antibody that specifically binds SP1 The generation and characterization of a monoclonal antibody directed against SP1 has been previously described (Visentin et al., 2006). A humanized version Rabbit polyclonal to F10 of the antibody (LT1009, sonepcizumab, Lpath Therapeutics, Inc., San Diego, CA) was utilized in this study. Intravitreous injections Intravitreous injections were done under a dissecting microscope with a Harvard Pump Microinjection System and PF-06471553 pulled glass micropipettes as previously described (Mori et al., 2001). Tracer studies with radiolabeled sonepcizumab [H3]-labeled sonepcizumab was produced at LPath Therapeutics, Inc by radiolabeling with tritium [propiony-3H] with specific activity of 2.0 mCi/mg[H3] by Vitrax. Three g of a 1:4 mixture of labeled to unlabeled sonepcizumab was PF-06471553 injected into the vitreous cavity of each eye of 8C10 week old female C57BL/6 mice. At 1, 7, and 14 days after injection, mice were euthanized and eyes were removed. Anterior segments were removed and lenses, retinas, and eyecups (retinal pigmented epithelium, choroid, and sclera) were weighed and briefly sonocated in lysis buffer (phosphate buffered saline (PBS) containing 20 mM EDTA and 1% Triton X-100) and briefly solublized in NaOH. Samples were added to vials containing scintillation fluid and radioactivity was counted. Dosage amount and frequency of sonepcizumab administration in mice Five to 6-week-old female C57BL/6 mice were randomized and treated in masked fashion. One group of mice received an intraocular injection of 1 1 l of PBS or PBS containing 0.05, 0.5, 1.0 or 3.0 g of sonepcizumab and the following day Bruchs membrane was ruptured at 3 locations in each eye. A second group of mice received an intraocular injection of PBS or 0.5 g of sonepcizumab one day prior to PF-06471553 and 6 days after laser-induced rupture of Bruchs membrane. Mouse model of choroidal NV Choroidal NV was induced by laser photocoagulation-induced rupture of Bruchs membrane as previously described (Tobe et al., 1998). Briefly, 5 to 6-week-old female C57BL/6 mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight) and pupils were dilated. Laser photocoagulation (75m spot size, 0.1 second duration, 120 mW) was performed in the 9, 12, and 3 oclock positions of the posterior pole of each eye with the slit lamp delivery system of an OcuLight GL diode laser (Iridex, Mountain View, CA) and a handheld cover slip as a contact lens to view the retina. Production of a bubble at the time of laser, which indicates rupture of Bruchs membrane, is an important factor in obtaining choroidal NV; therefore, only burns in which a bubble was produced.
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