Inhibitory Effect Of Small Interfering RNA Targeting HIF-1α And VEGF On Retinal Neovascularization In The Mouse

Chapter one:The inhibition of expression of HIF-1αand VEGF by small interfering RNA targeting HIF-1αand VEGF in the human vascular endothelial cellsObjective:To investigate the effect of small interfering RNA (siRNA)targeting hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor(VEGF)on expression of HIF-1αand VEGF in the human umbilical vein endothelial cells.Methods:HIF-1αsiRNA recombinant plasmid was constructed. Human umbilical vein endothelial cells were cultured in vitro and divided into normoxia group(20%)and hypoxia group(1%).Hypoxia group was then divided into control hypoxia group,vector group,HIF-1αgroup, VEGF group and co-transfection group randomly.The cells were transiently transfected with vector plasmids(vector group),HIF-1αsiRNA recombinant plasmid(HIF-1αgroup),VEGF₁₆₅siRNA recombinant plasmid(VEGF group),HIF-1αsiRNA and VEGF₁₆₅siRNA recombinant plasmids(co-transfection group)by the Lipofectamine 2000 (LF2000)method.No transfection were performed in the normoxia group and control,hypoxia group.The expression of HIF-1αsiRNA and VEGF₁₆₅siRNA recombinant plasmids were identified by reverse transcriptase- polymerase chain reaction(RT-PCR).The expression of HIF-1αand VEGF were detected by RT-PCR and immunocytochemical methods.Results:The expression of HIF-1αsiRNA and VEGF₁₆₅siRNA recombinant plasmids were identified by RT-PCR after 24 hours of transfection to the cells.The exression of HIF-1αmRNA was observed in the normoxia group,whereas nearly no expression of HIF-1αprotein was observed.HIF-1αmRNA and protein levels of control hypoxia goup and vector group were increased compared to normoxia group.HIF-1αmRNA and protein levels of HIF-1αgroup decreased compared to control hypoxia group(P＜0.01),mRNA level was reduced by 59.8%.The expression of VEGF mRNA and protein were faint in the normoxia group, but increased obviously after hypoxia(P＜0.01).The expression of VEGF mRNA and protein in HIF-1αgroup,VEGF group and co-transfection group were decreased as compared with control hypoxia group(P＜0.01), mRNA levels were reduced by 39.7%,68.1%and 82.3%,respectively. Co-transfection group showed the highest inhibitory effect.Conclusions:HIF-1αand VEGF₁₆₅siRNAs effectively inhibit the expression of HIF-1αand VEGF in the human umbilical vein endothelial cells.Chapter two:Inhibitory effect of small interfering RNA targeting HIF-1αand VEGF on retinal neovascularization in the mouseObjective:To evaluate the inhibitory effects of small interfering RNA(siRNA)targeting hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor(VEGF)on retinal neovascularization in the mouse.Methods:Liposome mediated pEGFP-N1 complex was injected into the vitreous of C57BL/6J mice.The expression of GFP was observed in retinal flat-mounts one day after injection.There were 21 seven-day-old C57BL/6J mice in the normal group and 96 mice which were divided into five groups randomly including control model group,vector group and gene therapy group(HIF-1αgroup,VEGF group and co-transfection group)were induced for retinal neovascularization by hypoxia.The mice received an intravitreous injection of vector plasmids(vector group), HIF-1αsiRNA(HIF-1αgroup),VEGF₁₆₅siRNA(VEGF group),HIF-1αsiRNA and VEGF siRNA(co-transfection group)by the Lipofectamine 2000(LF2000)method 1 day before mice were moved out to room air from the oxygen chamber.The expression of HIF-1αsiRNA and VEGF₁₆₅ siRNA recombinant plasmids were identified by RT-PCR.Fluorescein angiography was used to assess the vascular pattern.The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from retinas into the vitreous in cross-sections.HIF-1αand VEGF levels in retinas were measured by RT-PCR,Western blot and immunohistochemical methods.Results:The expression of HIF-1αsiRNA and VEGF₁₆₅siRNA recombinant plasmids were identified by RT-PCR.The GFP expression in retinal cells was observed one day after injection of liposome mediated pEGFP-N1 complex.Neovascular tufts and fluorescein leakage were decreased in gene therapy group especially co-transfection group compared to control model group and vector group.The neovascular nuclei were decreased in gene therapy group compared to the other three groups(P＜0.01).The expression of HIF-1αmRNA and protein in retinas were increased in control model group and vector group as compared with normal group,while decreased 57.4%and 52.5%respectively in HIF-1αgroup as compared with control model group(P＜0.01).The expression of VEGF mRNA and protein in retinas were increased significantly in control model group and vector group as compared with normal group,while decreased significantly in gene therapy group especially co-transfection group(decreased 85.6%and 80.9% respectively)as compared with control model group(P＜0.01).Conclusions:HIF-1αand VEGF₁₆₅siRNAs can inhibit retinal neovascularization in the mouse effectively.Co-transfection of these two siRNAs shows the greatest inhibitory effect.