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Ann M Chan, Meagan Kiyohara, Christen M Dillard, Daniel Diaz-Aguilar, Saad Shah, Teevit Dunnsiri, Madhuri Wadehra, Lynn K Gordon; Blockade of Epithelial Membrane Protein-2 Decreases Corneal Neovascularization in a Burn Model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5651. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal neovascularization (NV) can result from infection, prolonged use of contact lens, direct or chemical trauma, or immunologic disease. Vision loss may result from corneal NV. Epithelial membrane protein 2 (EMP2), a member of GAS3/PMP22 tetraspan protein family, is expressed in the corneal epithelium, and controls VEGF expression in ocular epithelial cell lines. The goal of this study is to examine the relationship between EMP2 and neovascularization of the cornea in a burn model.
Human corneal limbal epithelial (HCLE) cell line was exposed to nutrient deprivation, and used to examine the expression of pro-angiogenic proteins. Small hairpin-RNA was used to knockdown EMP2 expression in HCLE cells. Corneal NV was induced in 6-8 week old female Balb/c mice using a NaOH burn method. Mice were treated with a recombinant anti-EMP2 or control antibody via subconjunctival injection immediately after the corneal burn. Animals were clinically evaluated to determine the extent of NV. Seven days post treatment, the eyes were enucleated and immunohistochemical studies were performed to identify the vasculature using antibodies against CD34 and VEGF.
In vitro analysis on HCLE cells via Western blot showed a decrease in phospho-STAT3, upon EMP2 knockdown (p< 0.01). Subconjunctival injections of anti-EMP2 antibody, but not the control antibody, significantly decreased EMP2 expression in the cornea epithelium at day 7. Clinically, less corneal NV was observed in the eyes that received the subconjunctival injection of anti-EMP2 antibody than saline controls or with a control antibody (p< 0.03). In the anti- EMP2 treated animals, as compared to the controls, histological measurement of the central region of the cornea showed reduced thickness, and anti-CD34 and VEGF immunohistochemistry indicated a decrease in expression.
Knockdown of EMP2 expression in HCLE cells decreases STAT3 phosphorylation. Subconjunctival injections of anti-EMP2 antibody reduces the corneal epithelial expression of CD34 and VEGF as well as decreases clinical evidence of corneal NV following ocular burn using NaOH. Modulation of EMP2 expression may provide a new method of preventing corneal neovascularization following injury.
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