One proposed mechanism of action of PS-targeting antibodies in tumors is by attracting macrophages and inducing ADCC.15More work is needed to determine if ADCC is in fact involved MK-2894 sodium salt in the inhibition of CNV growth. of flat mounts. == Results == We found that PS was exposed in CNV lesions and colocalized with vascular endothelial staining. Treatment with PS-targeting antibodies led to a 40% to 80% reduction in CNV lesion area when compared to treatment with a control antibody. The effect was the same as that seen using an equal dose of an anti-VEGF antibody. Results were confirmed using the choroid sprouting assay, an ex vivo model of choroidal angiogenesis. == Conclusions == We demonstrated that PS is exposed in choroidal neovascular endothelium. Furthermore, targeting this exposed PS with antibodies may be of therapeutic value in CNV. Keywords:choroidal neovascularization, phosphatidylserine, PS-targeting antibody, choroidal angiogenesis, choroidal sprouting, PS-exposure Age-related macular degeneration (AMD) is a prevalent blinding disease. An estimated 30% of Americans older than 75 years of age have some degree of macular degeneration.1,2Furthermore, advanced AMD afflicts MK-2894 sodium salt approximately 1.8 million individuals in the United States alone.1Approximately 90% of severe vision loss in advanced AMD can be attributed MK-2894 sodium salt to neovascular or wet AMD, characterized by the development of pathological angiogenesis originating in the choroid (choroidal neovascularization, CNV).2Recent therapeutic advances have CDC46 led to improvements in clinical outcomes for neovascular AMD,3but there remains a significant need for new therapeutic strategies.4,5Anti-VEGF therapy has provided significant therapeutic visual benefit, yet it does not decrease the size of the choroidal MK-2894 sodium salt neovascular complex (it only prevents a further increase in size).6Thus, frequent intravitreal injections are needed indefinitely to keep the disease at bay. Targeting different pathways related to the neovascular process may allow for combination therapies that improve clinical success, improve the quality of life, and decrease the treatment burden on patients with wet AMD. In normal cells, including vascular endothelium, the aminophospholipid phosphatidylserine (PS) is asymmetrically distributed across the plasma membrane lipid bilayer, such that it is exclusively localized to the membrane’s inner leaflet.7,8This asymmetry is maintained by enzymes known as aminophospholipid translocases that actively transport PS from the external to the internal leaflet of the plasma membrane.9,10On the other hand, the activation of phospholipid scramblases can lead to the loss of the asymmetric distribution of phospholipids in the plasma membrane of cells.11Virus-infected cells12and, importantly, tumor vascular endothelial cells13lose their capacity to maintain PS asymmetry. One potential trigger for this phenomenon is oxidative stress.13,14Phosphatidylserine-targeting antibodies can bind to exposed PS in the outer leaflet of the plasma membrane of tumor vascular endothelium, enabling antibody-dependent cell-mediated cytotoxicity (ADCC).15Antibody-dependent cell-mediated cytotoxicity is mediated by monocytes and macrophages and can result in the collapse of the tumor neovasculature.15 In this work we propose and test the hypotheses that (1) the abnormal MK-2894 sodium salt endothelium of choroidal neovascular membranes lacks the ability to maintain PS asymmetry in the plasma membrane lipid bilayer, and (2) PS-targeting antibodies may provide a new therapeutic approach for CNV. We were able to use an in vivo model (laser-induced CNV in mice) to demonstrate that PS is indeed exposed on the neovascular endothelium of CNV. Furthermore, in the laser CNV model and in an ex vivo model (choroidal sprouting assay), we were able to demonstrate that PS-targeting antibodies can inhibit choroidal angiogenesis. == Materials and Methods == == Animals == C57BL/6J (Jackson Laboratory, Bar Harbor, ME, USA) mice were used for laser-induced CNV experiments and for the choroidal sprouting assay. Mice were kept in a barrier animal facility at the University of Texas (UT) Southwestern Medical Center under normal lighting conditions with 12-hour-on/12-hour-off cycles. All applicable international, national, and institutional guidelines for the care and use of animals, including the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, were followed. All experiments were approved by the UT Southwestern Medical Center Institutional Animal Care and Use Committee (IACUC). Before all procedures, animals were anesthetized one at a time with a ketamine-xylazine cocktail (100 mg/kg ketamine, 5 mg/kg xylazine). == Antibodies == All antibodies were dissolved in PBS. The PS-targeting antibodies that were used in this study were 1N11 (a fully human antibody, also known as PGN635; used only for in vivo staining.
One proposed mechanism of action of PS-targeting antibodies in tumors is by attracting macrophages and inducing ADCC
