Lentivirus-Mediated SFLT-1 Gene Fragments Transfer Represses Retinal Neovascularization

1. Construction of lentiviral expression plasmids carrying different fragments of sFlt-1 geneObjective: To construct the lentiviral expression plasmids carrying different fragments of sFlt-1 gene.Methods: The objective fragments, sFlt-1(2-3) and sFlt-1(2-4), were amplified by RT-PCR from fresh human placenta. Then the fragments were subcloned into the lentiviral vector,pRRL-GFP,to generate the lentiviral expression vector,pRRL/sFlt-1(2-3) and pRRL/sFlt-1(2-4). The corrected sFlt-1 gene fragments were confirmed by endoenzym digestion,sequencing. Recombinant lentiviruses were produced by HEK293T cells following the co-transfection of pRRL/sFlt-1, with the packaging plasmids pMDLg/pRRE, PRSV/REV and pMD2.G. The resulting recombinant lentiviruses Lenti.sFlt-1(2-3) and Lenti.sFlt-1(2-4) were then used to infect human retinal pigment epithelial cells. SFlt-1 expression in human retinal pigment epithelial cells was detected by RT-PCR and Western blot analysis.Results: SFlt-1 gene fragments, sFlt-1(2-3) and sFlt-1(2-4), were cloned from fresh human placenta by RT-PCR successfully. The recombinant lentiviruses carrying sFlt-1(2-3) or sFlt-1(2-4), which were produced by co-transfecting pRRL/sFlt-1 to 293T cells, could infect and deliver sFlt-1 gene to human retinal pigment epithelial cells.Conclusion: The recombinant lentiviruses can deliver target sFlt-1 gene fragments and have high infection efficiency, which bases further investigation of the role of sFlt-1 in the gene therapy of retinal neovascularization.2. The establishment and identification of a retinal neovascularization modelObjective: To set up a stable and reliable animal model of retinal neovascularization. Methods: Neonatal mice were exposed to an atmosphere of hyperoxia (75±2% O2) from postnatal day 7 (P7) to P12. On P12, they were returned to room air (21% O2) until P17 to induce retinal neovascularization. Controls were exposed in constant normoxia from P0 to P17. Retinal neovascularization was examined by fluorescence angiography and hematoxylin/eosin(H-E) staining.Results: Fluorescence angiography showed a pattern of pathological changes such as central non-perfused areas, tortuous and dilated blood vessels and vascular leakage in model mice. And the serial H-E staining sections also confirmed the results. It showed 48.65±6.24 neovascular nucleis/section in oxygen-exposed mice.Conclusion: The oxygen-induced ischemic retinopathy mouse model is a successful animal model of retinal neovascularization, which could be a reproducible model of angiogenesis in the research for retinal neovascularization.3. Lentivirus-mediated different fragments of sFlt-1 gene transfer represses retinal neovascularizationObjective: The vascular endothelial growth factor (VEGF) has been shown to be involved in retinal vasculogenesis. SFlt-1, a soluble extracellular domain of VEGF receptor-1, is an endogenous specific inhibitor for VEGF. The objective of our study was to determine the effect of Lenti.sFlt-1(2-3) and Lenti.sFlt-1(2-4) on blocking retinal neovascularization in a murine oxygen-induced retinopathy model in vivo.Methods: Retinal neovasuclarization model was induced by returning the neonatal mice to normoxia (21% O2) after exposure to hyperoxia (75% O2) from postnatal day 7 (P7) to P12. After sFlt-1(2-3) and sFlt-1(2-4) gene fragments transfer, their effects on blocking retinal neovascularization were identified by fluorescein angiography and hematoxylin/eosin staining. In addition, the expression changes of VEGF and its functional receptor KDR/Flk-1 were detected by Western blot and immunohistochemistry analysis.Results: Lenti.sFlt-1(2-3) and Lenti.sFlt-1(2-4) significantly inhibited neovascularization in the oxygen-induced retina neovascularization model. Retinas from mice treated with Lenti.sFlt-1(2-3) demonstrated a more obvious reduction in the number of neovascular cell nucleis compared with retinas treated with Lenti.sFlt-1(2-4). And fluorescence angiography confirmed the results. VEGF protein levels were nearly not affected in the two Lenti.sFlt-1 treated groups, while KDR/Flk-1 protein expression was obviously inhibited. Furthermore, KDR/Flk-1 protein expression showed a more significant reduction in retinas treated with Lenti.sFlt-1(2-3).Conclusion: SFlt-1 gene fragments mediated by lentivirus can significantly inhibit retinal neovascularization in the oxygen-induced ischemic retinopathy model, which indicates the potential therapeutic strategies for preventing retinal neovascularization by sFlt-1 gene transfer.