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S. A. Morales, D. Telander, S. Mareninov, A. Nagy, J. Braun, L. K. Gordon; Novel Therapies to Reduce Proliferative Vitreoretinopathy, Evidence From an in vitro Model. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2713.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferative vitreoretinopathy (PVR) is an aberrant wound healing that occurs as a complication of severe ocular trauma including penetrating injuries or rhegmatagenous retinal detachments. Epithelial membrane protein 2 (EMP2) regulates collagen gel contraction by the retinal pigment epithelium cell line ARPE-19, an in vitro model for PVR, by modulating FAK activation. The purpose of this study is to investigate the efficacy of an EMP2 specific recombinant diabody in blocking collagen gel contraction by ARPE-19 cells.
EMP2 diabody was recombinantly constructed using a phage library to select for reactivity against a human EMP2 peptide. In all experiments ARPE-19 cells were pretreated with 20µg/ml of anti-EMP2 or control diabody for 2 hours. Toxicity, adhesion, and migration were assessed respectively through flow cytometry using Annexin V, binding to collagen type 1, and a wound healing assay. Collagen gel contraction was assessed using an in vitro assay.
No toxicity was observed using the anti-EMP2 diabody. The anti-EMP2 diabody did not significantly alter adhesion or migration. Exposure to anti-EMP2 diabody resulted in a 75% reduction in EMP2 protein levels at 4 hours. EMP2 recovery to pre-treatment levels was seen after 16 hours. Concordant decreases in basal levels of activated FAK were also observed. Collagen stimulated FAK activation was reduced by 25% in ARPE-19 cells treated with anti-EMP2 diabody. Anti-EMP2 diabody treatment significantly reduced collagen gel contraction by the ARPE-19 cells (p<0.001) and was dose dependent.
Anti-EMP2 diabody decreases contractile capacity through down regulation of EMP2 leading to decreased FAK activation and inhibition of collagen gel contraction. The anti-EMP2 diabody, if validated in in vivo studies, may provide a new therapeutic approach to PVR prevention.
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