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S.L. Chen, T.J. McFarland, Y. Zhang, B. Appukuttan, J.T. Stout; Tenon's Fibroblast Expression of Antiangiogenic Factors via Lentiviral Transduction . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3794.
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Purpose: Vascular Endothelial Growth Factor (VEGF) plays an essential role in neovascularization of the eye, and many modalities involving the inhibition of VEGF are being explored as possible therapies. We wish to transduce tenon's cells, in vitro, with lentiviral vectors harboring the potentially antiangiogenic genes soluble Neuropilin-1 (sNRP1) and soluble Kinase domain receptor (sKDR). Transgenes have been designed to produce these secretable proteins able to act locally to inhibit ocular neovascularization. Methods: The cDNAs coding sNRP1 and sKDR were cloned into separate pHR' vectors under the control of the CMV promoter. To act as control and for verification of transduction, a PHR'-CMV-eGFP plasmid was also constructed. Replication-defective lentivirus was prepared by three-plasmid co-transfection into 293T cells. Tenon's tissue was harvested from one New Zealand White rabbit and cultured in a six well culture plate. Confluent tenon's fibroblasts were incubated with the lentiviral vectors containing eGFP, sNRP1 and sKDR. In vitro expression of sNRP1 and sKDR was verified by RT-PCR. Results: RT-PCR verified transduction of tenon's fibroblast cultures. Cells transduced with eGFP, demonstrated fluorescence by fluorescent microscopy. Conclusions: Our study demonstrated successful in-vitro tenon's fibroblast expression of antiangiogenic factors, sNRP1 and sVEGFR2 (KDR) via lentiviral transduction. Expression and secretion of antiangiogenic factors by tenon's fibroblasts is an attractive ex vivo approach to antiangiogenesis of the anterior segment.
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