[Inhibitory effect of interfering RNA targeting HIF-1alpha and VEGF on retinal neovascularization in the mouse].
OBJECTIVE
To evaluate the inhibitory effect of small interfering RNA (siRNA) on the expressions of hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in retinal neovascularization in the mouse.


METHODS
HIF-1alpha siRNA recombinant plasmid was constructed. Liposome mediated the expressive plasmid of enhanced green fluorescent protein (EGFP) 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. Randomized controlled trial was performed. There were totally ninety (seven-day-old) C57BL/6J mice in which seventeen mice were chosen as normal group and seventy-three mice were divided into five groups randomly including control model group, vector group and gene therapy group (HIF-1alpha siRNA group, VEGF siRNA group and co-transfection group), in which retinal neovascularization was induced by hypoxia. Liposome with vector plasmid, HIF-1alpha siRNA and VEGF165 siRNA were injected into the vitreous in the vector group, HIF-1alpha siRNA group and VEGF siRNA group respectively one day before mice were moved out to room air from the cabin. Liposome with HIF-1alpha siRNA and VEGF165 siRNA was injected in the co-transfection group at the same time point. Fluorescent angiography was used to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. HIF-1alpha and VEGF levels in retinas were measured by reverse transcriptase-polymerase chain reaction and Western blot. Significant differences between groups were evaluated by one-way analysis of variance, followed by a least-significant difference analysis.


RESULTS
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 in co-transfection group compared to the control model group. The neovascular nuclei were decreased in gene therapy group [HIF-1alpha siRNA group (27.73 +/- 2.33), VEGF siRNA group (15.43 +/- 3.23), co-transfection group (8.70 +/- 2.88)] compared to the other three groups (F = 3016.537, P < 0.01). The expression of HIF-1alpha mRNA and protein in retinas were increased in control model group (1.08 +/- 0.06, 0.383 +/- 0.009) and vector group (1.09 +/- 0.05, 0.386 +/- 0.010) as compared with normal group (0.81 +/- 0.07, 0.035 +/- 0.003), while decreased 57.4% and 52.5% respectively in the HIF-1alpha siRNA group (0.46 +/- 0.06, 0.182 +/- 0.008) as compared with control model group (F = 139.804, 2686.001; P < 0.01). The expression of VEGF mRNA and protein in retinas were increased significantly in control model group (1.53 +/- 0.07, 0.340 +/- 0.004) and vector group (1.59 +/- 0.06, 0.337 +/- 0.009) as compared with normal group (0.27 +/- 0.08, 0.051 +/- 0.008), while decreased significantly in gene therapy group especially co-transfection group (decreased 85.6% and 80.9% respectively) as compared with control model group (F = 421.423, 2513.583; P < 0.01).


CONCLUSIONS
HIF-1alpha siRNA and VEGF165 siRNA can inhibit retinal neovascularization in the mouse effectively. Co-transfection of these two siRNAs shows the greatest inhibitory effect.
Jiang
Jian
J
Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, China.
Xia
Xiao-bo
XB
Xu
Hui-zhuo
HZ
Xiong
Yu
Y
Song
Wei-tao
WT
Xiong
Si-qi
SQ
Li
Yan
Y
D000818:Animals; D005260:Female; D015316:Genetic Therapy; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D034741:RNA, Small Interfering; D015861:Retinal Neovascularization / Q000235:genetics / Q000517:prevention & control*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse