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T.J. McFarland, B. Appukuttan, Y. Zhang, J.T. Stout; Assessment of Lentiviral Delivered sKDR and sNRP1 in a Rat Aortic Ring Assay, Potential Therapeutic Genes for Proliferative Retinal Disease . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4879.
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Purpose: To examine the effects of Lentiviral transduction using soluble Kinase Domain Receptor (sKDR) and soluble Neuropilin 1 (sNRP1) in a rat aortic ring assay. Methods: : Lentiviral vectors, which expressed the soluble form of KDR and NRP1 under the control of a CMV promoter, were produced by cotransfection into 293T cells. Rat aortas were dissected from Norwegian Brown rats and cut into 1mm rings. The rings were incubated in triplicate with PBS, eGFP virus, sKDR virus, sNRP1 virus, or co infected with sKDR and sNRP1. The rings were embedded in 600ul of Matrigel, covered with MCDB 131 media and incubated at 37°C 5% CO2. Rings were observed for 14 days by light microscopy and analyzed with Image-Pro Plus 4.0 digital software. RT-PCR was performed to confirm the presence of gene transcripts. Results: RT-PCR verified eGFP, sKDR, and sNRP1 mRNA presence in aortas incubated with the Lentiviral vectors. Peak vessel sprouting was seen at day 10. The aortas transduced with the eGFP vector displayed fluorescent sprouting as viewed by fluorescent microscopy. Preliminary data suggests an inhibitory effect in aortas transduced with sKDR and sNRP1 virus, both singly and in the co infected treatment. Vessel sprouts were longer in length and more frequent in control aortas as compared with test samples. Conclusions: KDR and NRP1 have both been shown to bind VEGF165, a potent mitogen of angiogenesis. The use of the soluble isoforms is an attractive therapeutic approach to competitively inhibit VEGF induced neovascularization of the retina. The vectors produced are efficient at transducing blood vessels and this assay provides a useful method to study the relationship between these genes in a biological manner.
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