Lentivirus Mediated Endostatin Gene Transfected Endothelial Progenitor Cells Inhibited Retinal Neovascularization

Part 1:Generating an Antiangiogenic Endothelial Progenitor Cell Line Using Endostatin Gene Transfer and Exploring The Effects of Endostatin Transfection on EPC Function.ObjectiveEndostatin(ES)is an endogenous inhibitor of angiogenesis.ES doesn’t affect the normal vascular tissue,and has no side effect.Increase of the expression of endogenous endostatin is urgently required for treating retinal neovascularization(NV).Simultaneously,the endothelial progenitor cells(EPCs)were investigated as a treatment option in retinal NV.The aims of present in vitro study were to generate EPCs with endostatin overexpression,and to explore the effects of expression of vascular endothelial growth factor(VEGF)on endostatin transfected EPCs and EPC function.MethodsEPCs were obtained from rat peripheral blood samples(collected from the right ventricle).The layer of peripheral blood mononuclear cells was isolated with density centrifugation,and was then re-suspended in an EGM-2MV medium of fibronectin-coated vessel.Flow cytometry and immunofluorescence assays were used to verify the EPCs,respectively.The endostatin fragment was cloned by polymerase chain reaction(PCR).The recombinant LV vector(LV5-EFla-GFP+PURO-Endostatin)was produced by co-transfection of 293T human embryonic kidney cells with 3 plasmids(pLV/helper-SL3,pLV/helper-SL4,and pLV/helper-SL5),and with Lipofectamine 2000 reagent and infected with rat EPCs,then these transfected cells were subjected to a puromycin selection.After ES transfected EPC line was obtained finally,the qRT-PCR and western blot assays were applied to determine the expression levels of endostatin mRNA and protein,respectively.Vascular endothelial growth factor(VEGF)expression levels were also detected to observe the anti-angiogenic effects of the endostatin-transfected EPCs.The transfection effects of endostatin overexpression on the proliferation,migratory,differentiation,apoptosis and the cell cycle of this cell line were also determined.ResultsAfter puromycin(lug/ml)selection for 4 days,a stable endostatin-transfected EPCline was generated.The qRT-PCR and western blot assays showed the high expression levels of endostatin mRNA and protein(P<0.001).While,the expression levels of VEGF decreased significantly(P<0.05).A cell counting kit-8 assay showed that endostatin overexpression inhibited significantly EPC proliferation(P<0.001).The transwell assay indicated that endostatin overexpression could significantly suppress EPC migration(P<0.01).Furthermore,endostatin overexpression enhanced significantly apoptosis(P<0.001),induced significantly the differentiation(P<0.001)and blocked significantly the cell cycle(P<0.001).As compared with negative control group,EPC viability significantly decreased in gene transfection group.ConclusionThe results of present study showed that EPCs can be genetically modified to over-express endostatin(transfected with LV-Endostatin-GFP),potentially giving them an anti-agiogenic effect through the increased secretion of endostatin and the decreased secretion of VEGF.The results of our study exhibited that the cellular viability(proliferation,migration,and differentiation abilities)was decreased,the cell cycle was inhibited,and apoptosis was induced in EPCs with endostatin transfection.On the other hand,there was a control group(viral transfection of no load gene)in our study,which could determine that the transfection procedure did not influence the cell viability.In conclusion,the present study determined the feasibility of lentivirus-mediated endostatin gene transfer,and indirectly proved the effect of endostatin secretion on EPCs(VEGF expression decreased).The experimental animal studies should be conducted in the next step.Part 2:Intravitreal Injection of Endostatin Gene Transfected Endothelial Progenitor Cells Inhibited Neovascularization in Oxygen-induced Rat ModelsObjectiveTransplantation of endothelial progenitor cells(EPCs)with transfected gene provides a novel gene therapy method for treatment of several neovascularization diseases.In this study,the recombinant LV vector(LV-ES)was produced and infected with EPCs,then endostatin gene transfected EPCs(ES-EPCs)were injected intravitreally into oxygen-induced rat models.The purpose of this study was to investigate the efficacy of endostatin transfected EPCs in preventing the retinal neovascularization diseases.MethodsNeonatal SD rats(P7)were randomly divided into experimental and control groups.Experimental group was exposed to hyperoxia(70%)for 5 days to induce oxygen-induced retinopathy(OIR),while the control group was put in normal air condition.Neovascular areas and retinal neovascularization were compared using fundus fluoresce in angiography(FFA)at P14,P15,P17,P19 to ensure the established OIR model.Then the OIR rats were randomly divided into four groups.Two days after returning to room air,on postnatal day 14(P14),OIR rats were injected intravitreal with the same volume of empty-LV,endostatin-LV,EPCs,and endostatin-LV-EPCs,respectively.Then the other group(the same age rats)was blank control group,which was put into the normal air condition.Neovascularization leakage areas were compared using FFA at 1 h、1 d、3 d and 5 d after intravitreal injections and then rats were killed.Hematoxylin-eosin(HE)staining method was used to observe and count the number of nuclear cells in endothelial cells out of the retinal inner limiting membrane of the retinal neovascularization.Retinal expression of endostatin,vascular endothelial growth factor(VEGF),CD31 and CD 133 were evaluated by immunohistochemistry method.ResultsFFA showed that neovascularization leakage was found in experimental group but was not seen in control group,which indicated that OIR Rat model had been established.Also FFA revealed that retinal neovascularization was significantly reduced in endostatin-LV and endostatin-LV-EPCs group,and the inhibiting effects on retinal neovascularization were almost the same.OIR+ES OE group(LV-ES injected):As compared with blank control group,the retinal NV leakage did not increase significantly in OIR+ES OE group at each time;as compared with NC(empty LV injected)group,the retinal NV leakage significantly reduced on day 3(P<0.01)and day 5(P<0.001).So it can be inferred that retinal NV leakage was inhibited in OIR+ES OE group.OIR+EPCs group(EPCs injected):As compared with blank control group,the retinal NV leakage significantly increased on each time(P<0.05);as compared with NC group,the significant difference between different time points was not found;as compared with OIR+ES OE group,the retinal NV leakage significantly increased on day 1(P<0.05),day 3(P<0.01)and day 5(P<0.001)-It can be inferred that EPC did nothave the inhibiting effect on retinal NV leakage,also did not have the promoting effect on retinal NV leakage.OIR+ES-EPCs group(LV-ES-EPC injected):As compared with blank control group,the retinal NV leakage in OIR+ES-EPCs group did not increase significantly at each time;as compared with NC group,the retinal NV leakage in OIR+ES-EPCs group significantly reduced on day 5(P<0.001);as compared with OIR+ES OE group,the significant difference between different time points was not found;as compared with OIR+EPCs group,the retinal NV leakage in OIR+ES-EPCs group significantly decreased on day 1(P<0.05),day 3(P<0.01)and day 5(P<0.001).So it can be inferred that the retinal NV leakage was inhibited in OIR+ES-EPCs group.The HE results showed that:There were few nucleus of vascular endothelium in blank control group;as compared with the blank control group,the nucleus of vascular endothelium increased significantly in OIR+NC group and OIR+EPCs group(P<0.01),so the retinal NV significantly increased;as compared with NC group,the nucleus of vascular endothelium decreased significantly in OIR+ES-OE group and OIR+ES-EPCs group(P<0.05),so the retinal NV decreased significantly;as compared with OIR+ES OE group,the nucleus of vascular endothelium increased significantly in OIR+EPCs group(P<0.05),so the retinal NV significantly increased;as compared with OIR+EPCs group,the nucleus of vascular endothelium decreased significantly in OIR+ES-EPCs group(P<0.01),so the retinal NV decreased significantly.Immunohistochemistry illustrated that the endostatin expression decreased significantly in OIR+NC group(P<0.05)as compared with blank control group;as compared with NC group,the endostatin expression increased significantly in OIR+ES-OE and OIR+ES-EPCs groups(P<0.01):as compared with OIR+ES OE group,the endostatin expression decreased significantly in OIR+EPCs group(P<0.05);as compared with OIR+EPCs group,the endostatin expression significantly increased in OIR+ES-EPCs group(P<0.05).As compared with blank control group,VEGF expression significantly increased in OIR+NC group(P<0.05);as compared with NC group,the significant difference in OIR+ES-OE,OIR+EPCs and OIR+ES-EPCs groups was not found;also no difference among OIR+ES-OE,OIR+EPCs and OIR+ES-EPCs was seen.As compared with blank control group,CD31 expression significantly increased in OIR+NC and OIR+EPCs group(P<0.05);as compared with NC group,CD31 expression significantly decreased in OIR+ES-OE group(P<0.05);as compared with OIR+ES-OE group,CD31 expression significantly increased in OIR+EPCs group(P<0.05);as compared with OIR+EPCs group,the significant difference in OIR+ES-EPCs group was found.As compared with blank control group,CD 133 expression significantly decreased in OIR+NC,OIR+EPCs and OIR+ES-EPCs groups(P<0.05);as compared with NC group,CD 133 expression significantly increased in OIR+ES-OE group(P<0.05);as compared with OIR+ES-OE group,CD 133 expression significantly decreased in OIR+EPCs and OIR+ES-EPCs groups(P<0.05);as compared with OIR+EPCs group,the significant difference in OIR+ES-EPCs group was seen.ConclusionFFA,HE and ICC methods determined that EPC with transferred gene EC could significantly inhibit the retinal neovascularization,also simple EPC could not improve the neovascularization,which provides the basis of new therapy direction for retinal NV diseases.