Inhibition of a small interfering dsRNA targeting hypoxia inducible factor-1α on retinal neovascularization in mice

Abstract

Background Hypoxia-inducible factor-1 α (HIF-1α) specific double-stranded RNA ( dsRNA ) mediated by liposome inhibit reinal neovascularization in mice at dose-dependent manner. Objective The present study was to investigate the inhibitory effect of dsRNA targeting HIF-1α on retinal neovascularization in mice.Methods Models of oxygen-induced retinal neovascularization were set up in C57BL/6J mouse through exposure of postnatal day 7 ( P7 ) to (75±3) ％ oxygen for 5 days.Fluorescein conjugated Dextran angiography of retinal vascular was performed to identify the retinal neovascularization.The 8 mice of the normal group were raised in the room air.Fifty-one P7 mice exposed to(75±3)％ oxygen for 5 days and then returned to the room air and assigned to control group ( 3 mice),empty vector group( 3 mice) and gene therapy group (45 mice),and the latter were medially divided to 9 groups randomly according to dose-ratio ( liposomes ∶ plasmid).The pSilencer 2.1-U6 hygro was injected in the model mice of empty vector group,and different dose-ratios of pSilencer2.1-U6 hygro-HIF-1α dsRNA were injected respectively in the model mice of various gene therapy groups.Fluorescein conjugated Dextran angiography of retinal vascular was performed to observe the morphology of new blood vessels,and retinal slides were prepared to score the numbers of nuclei extending beyond the inner limiting membrane( ILM ),and expression of vascular endothelial growth factor(VEGF) was detected in the retina by immunohistochemistry. Results The retinal blood vessels of the normal group formed a fined radial branching pattern.The retinal vascular patterns in the control group and the empty vector group were characterized by decreased central perfusion in both the superficial and the deep layers.The abundant vessels were distorted and irregular in the control group and empty vector group,and the obstructed capillary and lots of neovascular tufts were seen.The retinal neovascularization and non-perfusion distraction in the every gene therapy group were reduced markedly with the most severe appearance in 1 ∶ 1 ( liposomes ∶ plasmid) dose-ratio group.Few vascular endothelial cell nucleus extending beyond the ILM were found in the normal group;while a large number of vascular endothelial cell nucleus were showed in the control group and empty vector group with the occurring rate 100％.Statistically,no significant difference was seen in the number of nuclei extending beyond the ILM between the control group and the empty vector group(11.57±5.85 vs 11.53±6.15),however,that in 1∶1 (liposomes∶plasmid) group was reduced markedly ( 2.17 ± 4.23 ) ( P ＜ 0.01 ).Immunohistochemistry revealed that VEGF was faintly expressed in the normal group but strongly expressed in the control group and the gene therapy group.VEGF expressions of various gene therapy groups were weaker than ones of the control group and the empty vector group.Conclusions Retinal neovascularization can be efficiently inhibited by intravitreal injection of the pSilencer2.1-U6 hygro-HIF-1α dsRNA mediated by liposome.Proportion of 1 ∶ 1 (liposomes ∶ plasmid)has a maximized efficiency. Key words: Retinal neovascularization/therapy; Endothelium; Blood vessel; Growth substance; Gene therapy; Animal model