Endostatin Gene Transfer For Inhibition Of Retinal Angiogenesis In Mouse

Angiogenesis plays a role in several retinal diseases including diabetic retinopathy, retinal vein occlusion and retinopathy of prematurity. These diseases can lead to irreversible vision loss if there is progression from retinal neovasculrization to retinal detachment. The potent treatment is inhibition of retinal angiogenesis. Endostatin is a cleavage product of collagen X VI and a endogenous angiogensis inhibitors that inhibit tumor angiogenesis and growth. While inhibitors of tumor angiogensis does not necessarily inhibit ocular angiogenesis. On the other hand, antiangiogenesis therapy requires prolonged administration and high doses of recombinant protein. Gene therapy could provide an alternative approach to continuous local delivery of this antiangiogenesis inhibitor in vivo. So in this study human umbilical vein endothelial cells (ECV-304) was trasfected by liposomes mediated PCDNAa-ES to evaluate the effect of liposomes mediated plasmids encoding endostatin on endothelium cell proliferation in vitro. Then liposomes mediated plasmids encoding endostatin were injection into the vitreous to evaluate the effect of ES to inhibit experimental retinal neovascularization in mouse.MethodsEndostatin plasmidA plasmid containing an expression cassette for human endostatin wasconstructed and supplied by Professor Zhao Yueran.Immune dot blot hybridization methodCationic liposomes mediated endostatin (ES) expression plasmid PCDNAs-ESwas transferred to Cos-7 cells. ES protein expression was tested with immune dotblot hybridization method.Cell line and ES transfer in vitroHuman umbilical vein endothelial cells (ECV-304) was cultured in RPMI-1640medium and was trasfected by liposomes mediated PCDNAa-ES. The ES proteinexpression was tested with immunofluorescence.MTT for cell proliferationThe effect of cell proliferation was determined by methyl thiazolyl tetrazolium(MTT) method.Flow cytometric for cell cycleThe effect on cell cycle after ES transfection was examined by flow cytometricanalysis.Mouse model of Oxygen-induced RetinopathyOne-week-old C5yBL/6N mice were exposed to 75% oxygen for 5 days. Thenreturned to the room air to induce retinal neovascularization. Cationic liposomesmediated ES complex 2 u 1 were injected in the vitreous body in thetreatment group. Complex to PGDN AS or PBS 2 V- 1 were injected in the controlgroup oES protein expression in retinaES protein expression in the retina was tested with immunohistological methodsat 1, 3, 7,14 days after injection.Retinal fluorescein AngiographyRetinal neovascularization was evaluated by injection fluorescein-dextron andretinal agiography after 5 days in room air.Quantification of neovascular proliferative retinopathyAt P17 the eyes of mice were enucleated and fixed for 24 hours. And embedded inparaffin. Serial asfittal sections of whole eyes were stained with PeriodicAcid-Schiff. The endothelial cell nuclei that were located on the vitreal side if theinternal limiting membrane was count and compared.Toxicity of retina after ES transferTo examine the toxicity of plasmid PCDNAa-ES complex, the histological changein the retina was examined by light and electron microscopy.ResultsES protein expression in cos-7 cells and ECV-304 cellsES protein expression was detected in ES trasfected COS-7 cells andsupernatant .The ES expression in ECV-304 cells was observed and reached thehighest level on the second day.ES on proliferation of endotheliutn cellsThe proliferation of ECV-304 was inhibited by ES transfection with the ratio ofGO/GI phase increased and the ratio of the S phase decreased.ES protein expression in retinaThere is ES protein express in the retina at 24 hours after injection. Most of thempresent in the retinal gangling layer. The expression increased after 3 days, andsome of them can be seen in the inner nuclear layer. They are las...