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Small Interference RNA Targeting vascular endothelial growth factor gene effectively attenuates retinal neovascularization in mice Model Kong Yi-chun, Sun Bei, Zhao Kan-xing, Han Mei and Wang Yu-chuan Key words:RNA inteference;VEGF;PEDF;retinal neovascularization Abstract: Background. The mechanism of retinal neovascularization is not understood completely.Many growth factor are involved in the process of retinal neovascularization, such as vascular endothelial growth factor(VEGF),pigment epithelium-deprived factor(PEDF), which are the represents of angiogenic and antiangiogenic molecules respectively.Oxygen induced retinopathy(OIR) is a useful model to investigate retinal neovascularization. The present study was to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy(OIR) by regulating VEGF to PEDF ratio(VEGF/PEDF). Methods. In vitro, cultured EOMA cells were transfected with VEGF-siRNA (psi-HITM/EGFP/VEGF siRNA)and Lipofectamine 2000 for 24 h, 48 h, and 72 h respectively,. Expression of VEGF mRNA was evaluated by real time PCR and the level of VEGF protein was analysed by Western blot. In vivo, OIR model mice were established, mice(C57BL/6J) received intravitreal injection of 1 μL of mixture of psi-HITM/EGFP/VEGF siRNA and Lipofectamine 2000. Expressions of retinal VEGF and PEDF protein were measured by Western blot, retinal neovascularization was observed by Fluorescein angiography and quantified. Results. In vitro psi-HITM/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression. In vivo, with decreased VEGF and VEGF-PEDF ratio, significant attenuation of neovascular tufts, avascular regions, tortuous and dilated blood vessels were observed in the interference animals. Conclusion. VEGF plays a important role in OIR, and transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo, accompanied by downregulation of VEGF-PEDF ratio, simultaneously attenuation of retinal neovascularization was observed. These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interfering targeting VEGF expression represents a possible therapeutic strategy. Tianjin Eye hospital, Tianjin Key Lab of Ophthalmology and Visual Science, Tianjin Eye Institute, Clinical college of ophthalmology Tianjin Medical University.TianJin,300020,China. (Kong Yi-Chun, Zhao Kan-Xing, Han Mei and Wang Yu-chuan) Key Laboratory of Hormones and Development(Ministry of Health)Metabolic Diseases Hospital & Tianjin Institute of Endocrinology Tianjin Medical University.TianJin,300070,China.( Sun Bei) Correspondence to:Dr.. Kong Yi-Chun, Tianjin Eye hospital, Tianjin Key Lab of Ophthalmology and Visual Science, Tianjin Eye Institute, Clinical college of ophthalmology University.TianJin,300020,China(Email:[email protected]) This study was supported by the scientific fund of TianJin Health Bureau(NO.2011KY31) Tianjin Medical Many ocular disease such as retinopathy of prematurity (ROP), age-related macular degeneration and Proliferative diabetic retinopathy are characterized by retinal neovascularization. Neovascularization can lead to retinal detachment and in final loss of vision. It is a major cause of blindness in industrialized countries. It has been a hot topic for ophthalmologist to inhibit retinal neovascularization effectively. One of the mechanisms of neovascularization is disorder of balance between angiogenic stimulators and inhibitors. Of these factors, vascular endothelial growth factor(VEGF) is a key factor. It might be a possible method to inhibit retinal neovascularization by block VEGF expression. In this study we show the suppression of retinal neovascularization via knockdown of VEGF protein expression using RNA interference in vitro and in vivo. Materials and methods siRNA design and transfection: Based on principle of siRNA by Elbashir et al [1], siRNA sequence of mouse VEGF cDNA (GenBank NM_009505) was designed and chemically synthesized by GeneCopoeia,MD. This siRNA consisted a sense strand: 5′TGCTGTGAAGATGTACTCTATCTCGTGTTTTGGCCACTGACTGACACGAGA TAGTACATCTTCA -3′ and an antisense strand: 5′CCTGTGAAGATGTACTATCTCGTGTCAGTCAGTGGCCAAAACC-3′. Sequences identified were BLASTed against the GenBank database. EOMA cells purchased from Amercian Type Cultured Collection(ATCC). On the day before transfection, Cells were seeded in a 24-well plate and placed in antibiotics deprived DMEM supplemented with 15% (v/v) FBS. When the cells showed 70% confluence, transfection were performed .LipofectamineTM 2000 (Invitrogen) and plasmid were diluted by 50ul serum-free DMEM. For the transfection, cells were incubated with 50nM plasmid, either psi-HITM/EGFP or psi-HITM/EGFP/VEGF siRNA, and 1ul of the LipofectamineTM for 6 h at 37°C. 3 groups were established in the experiment: A: blank control group(no interference);B: negative control group(transfected with psi-HITM/EGFP) and C: siRNA group(transfected with psi-HITM/EGFP/VEGF siRNA). Cells were collected 24,48,72 h after transfection and analyzed based on the intensity of EGFP monitored with a Nikon Eclipse TE2000-U immunofluorescent microscope and recorded with a CCD digital camera attached to the microscope. Real time RT-PCR for VEGF mRNA of cells: After 24,48,72h transfection respectively, cells were washed with PBS twice, total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. cDNA was synthesized using the cDNA Synthesis Kit (TaKaRa, Japan). Real-time PCR reactions with SYBR Green qPCR Kit (TaKaRa, Japan) were performed with VEGF-specific primers: Fw- CAGAAACACGACAAACCCATC and RVTAAGCCACTCACACACACAGCC.The cycling parameters were at 94°C for 30sec, then 35 cycles at 94°C for 10 sec, at 58°C for 10 sec, and at 72°C for 15 sec. The cycle threshold (Ct) indicates the fractional cycle number at which the PCR product was first detected above a fixed threshold. Changes in the expression of target genes were calculated with the 2−ΔΔCt method, where ΔΔCt = [Ct (target sample) − Ct (β-actin sample)] − [Ct (target calibrator) − Ct (β-actin calibrator)]. Western blot analysis for VEGF expression in cells: After 24,48,72h transfection respectively, cultured cell were collected. The protein expression of VEGF was determined by western blot assay as previously described [2]. In brief, total protein was extracted and 30ug protein from each sample was separated on a 10% SDS-PAGE gel(Bio-Rad) and electrophoretically transferred to nitrocellulose membrane(Amersham) at 120V, 2h.Membranes were blocked with 5% nonfat dry milk at 4℃ overnight and probed with primary antibody: anti-VEGF(1:500 Santa Cruz) or anti-β-actin(1:1000 Sigma) at 37℃ for 2h, followed by IgG-HRP conjugated secondary goat anti-rabbit antibody(BioLabs).The bands were visualized by chemiluminescent reagent(ECL, PIERCE).Density of bands was quantified by Bandscan 5 software and normalized to that of the β-actin band.. Animals and Oxygen Induced Retinopathy: Neonated CB57BL/6J mice were obtained from animal institute, Chinese Academy of Medical Sciences . Experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guideline for Care and Use of Laboratory Animals by Tianjin medical university, China. Neovascularization was induced as described by Smith et al.[3]. Briefly, 64 newborn CB57BL/6J mice were randomly assigned to experimental and normal groups(A).Normal group mice(16 mice) were raised in room air throughout the period. At postnatal day 7(p7), mice in the experimental group were exposed to hyperoxia (75% O2) for 5 days (p 7-12) and then returned to room air to induce retinal neovascularization. At p12, experimental group were equally divided into 3 subgroups: blank control(B); negative control: psi-HITM/EGFP treated (C)and VEGF siRNA group(D).No interference performed in normal and blank control; In other two groups:0.5ul psi-HITM/EGFP and 0.5ul lipofectomine were injected intravitreously in one eye of each mouse, contralateral eye was co-injected with 0.5ul psi-HITM/EGFP/ VEGF siRNA and 0.5ul lipofectamine. Intravitreal injections were performed at 11 o’clock,0.5 mm away from the corneal limbus of the eyes using a 10 ul Hamilton syringe fitted with a 33 G needle. Mice were chosen randomly in each group at P17:4 mice for retinal angiography, 4 mice for section and stained with hematoxylin eosin, 8 mice for Western Blot. Retinal angiography with high molecular weight fluorescein: Animals were anesthetized and perfused with fluorescein via retro-orbital injection of 2.5 mg/50ul of FITC-dextran (MW 2 × 106 Sigma-Aldrich, St. Louis, MO) as described previously.[4] The animals were immediately killed. The eyes were enucleated and fixed with 4% paraformaldehyde in PBS for 1h. The retina was then separated from the eyecup. 4 incisions were made to the retina, which was flat-mounted on a gelatin-coated slide. The vasculature was then examined under a fluorescent microscope (Nikon Eclipse TE20000-U, Nikon, Japan). Quantification of neovascularization: The eyes of mice from each group at p17 were enucleated, fixed with 10% formaldehyde, 6-μm-thick sections were made through the cornea parallel to the optic disc, and then stained with hematoxylin and eosin. The nuclei of vascular cells on the vitreal side of the retina were counted under a light microscope in a masked study. Ten sagittal sections from each eye were examined, and cell numbers were averaged in each group of animals. The average number of preretinal vascular nuclei was compared. Western Blot analysis for VEGF and PEDF expression in retina: Retinas of mice each group were isolated, lysed, and centrifuged. The protein expression of VEGF,PEDF was determined by western blot assay as experiment in vitro. 50ug protein measured by BCA assay (BCATM kit,PIERCE) from each sample was separated on a 10% SDS-PAGE gel(Bio-Rad). After protein transferred to nitrocellulose membrane(Amersham) ,membranes were probed with primary antibody: anti-VEGF(1:500 Santa Cruz),anti-PEDF(1:500 Santa Cruz) , anti-β-actin(1:1000 Sigma) at 37℃ for 2h, followed by IgG-HRP conjugated secondary goat anti-rabbit antibody(BioLabs). Density of bands was normalized to that of the β-actin band. Statistical Analyses: Results are expressed as mean ± SD. Statistical analysis of the data was performed by SPSS11.5 .To compare multiple sets of data, one-way ANOVA test was used. For paired data sets, LSD-t test was used. P < 0.05 was considered statistically significant. Results In vitro efficacy of VEGF siRNA: GFP fluorescence showed plasmid psi-HITM/EGFP/VEGF siRNA were successfully transfected into EOMA cells (Fig.1A).To demonstrate the levels of inhibition after transfection, cells were collected and RT-PCR analysis was performed. Real time PCR analysis clearly demonstrated that VEGF expression was significantly reduced by VEGF-specific siRNA but was not affected by treatment without siRNA and there was no difference between blank control and negative control (P>0.05) VEGF mRNA 24h post-transfection in siRNA treated cells and blank control was0.211±0.002 and 0.312±0.004, respectively, mRNA was inhibited by 21.1%.After 48 h and 72h transfection, VEGF mRNA was inhibited by69.8 % and80.4 % respectively.( Fig.1B) As shown in Fig.1C, 24,48,72h post-siRNA transfection, VEGF protein level of cells was0.717±0.033 , 0.633±0.014 , and 0.266±0.022,respectively.At the same time point, VEGF expression in blank control was 0.868±0.025, 0.884±0.022 and 0.906±0.028,respectively.VEGF level was significantly lower in cells treated with VEGF-specific siRNA(P=0.01,0.01,0.00). The results demonstrated that VEGF siRNA was an effective sequence to inhibit target gene and the capability of interference gradually increased in 72h after transfection. Retinal neovascularization in mice As shown by fluorescein angiography in flat-mounted retinas(Fig.2 upper panel), the mice raised in room air formed superficial and deep vascular layers in the retina. A mature capillary network that extended from the optic disc to the periphery filled in the entire retina. The typical retinal neovascularization was observed in blank control and negative control group (only psi-HITM/EGFP treatment), including neovascular tufts, avascular regions, tortuous and dilated blood vessels. The mice treated with siRNA developed light retinal neovascularization: tortuous blood vessels and central non perfused retina area decreased, normal retinal vasculature reappeared. The results showed that retinal neovascularization was inhibited by VEGF siRNA. Quantification of retinal neovascularization in mice There was no vascular cell nuclei broke through ILM in normal group, average vascular cell was not significantly different between blank control group and negative control group:60.37±6.78 and 59.89±6.56 respectively. siRNA group showed less neovascularization, with 10.85±1.26 preretinal cells per section.(Fig.2 lower panel) Changes in retinal VEGF,PEDF: At P17,in relative hypoxia condition(blank control), retinal VEGF reached the level that was 5.74 fold more than normal mice, but retinal PEDF level was decreased by 75%.After siRNA treatment, retinal VEGF level reduced by 68% compared with blank control, but retinal PEDF level changed reversed, which elevated by 2.29 fold(Fig.3).After siRNA application, an apparently decreased VEGF-to-PEDF ratio(ratio=0.58) was observed, which almost returned to normal(ratio=0.18). Nevertheless VEGF-to-PEDF ratio was markedly increased (ratio=4.19) without siRNA treated, meanwhile more severe retinal neovascularization occurred . Discussion RNA interference(RNAi) is a nature mechanism exist widely in mammal. It belongs to post-transcriptional gene silence that has been used to suppress gene expression thus reduce their protein product. RNAi is now being exploited as a powerful tool for inhibit genetics and shows great promise for therapeutic applications[5][6]. This method has been successfully used to silence VEGF gene expression in many models [7][8][9] . Development of retinal neovascularization depends on the interaction of several signaling events influenced by a fine balance of angiogenic agonists and antagonists. Of many factors, two important factors are VEGF and PEDF. VEGF has long been considered as a key angiogenic factor [10][11] and PEDF is a major angiogenic inhibitor in the eye[12][13][14]. Thus, we used the VEGF-to-PEDF ratio as representative of the balance between angiogenic stimulators and inhibitors to understand the mechanism of retinal neovascularization. In this study, siRNA were injected intravitreally during the development of neovascularization. The results clearly indicated that, using this experimental approach, siRNA which shown to exert specificity and potency in vitro, VEGF expression as measured by Western blot was reduced. More importantly, treatment with VEGF-specific siRNA greatly attenuated the neovascular response suggesting that VEGF play a key role in the regulation of retinal neovascularization. After siRNA treatment, VEGF-PEDF ratio ranged from 4.19 to 0.58 which approximated normal level(ratio=0.18) .It is likely that the return the balance VEGF-PEDF ratio contributed to the reduced neovascular response. Retinal neovascularization is mediated, at least in part by VEGF-PEDF ratio[15][16]. It is supported by the experimental results that:1) overexpression of VEGF/PEDF in vivo led to occurrence of [2] neovascularization ;and2)inhibition of VEGF-PEDF ratio significantly decreased the extent of retinal neovascularization in this study.Although it is a effective method to decrease active vascular features by anti-VEGF agents[17][18],some drawback also was observed[19][20]. But there is no exact idea why VEGF siRNA lead to increased expression of PEDF protein. It is supposed that decreased expression of VEGF caused by siRNA attenuate retinal neovascularization; with avascular regions disappear, PEDF expression increased; rebalance of VEGF-PEDF ratio progressively reduce retinal neovascularization. In summary, although the mechanism of retinal neovascularization is not completely understood, The results of this study demonstrate that restoring the balance of VEGF/PEDF by inhibition of VEGF expression with siRNA can attenuate retinal neovascularization. Thus it is clear that a delicate balance between angiogenic stimulator and inhibitor plays a key role in regulating retinal angiogenesis. Our study reveals that siRNA to silencing VEGF may be a promising avenue for the treatment of retinal neovascularization. Acknowledgements: This work was finished in Tianjin Institute of Endocrinology (Tianjin Medical University) and Tianjin Key Lab of Ophthalmology and Visual Science (Tianjin eye hospital). The author acknowledges the assistance of all teachers worked in labs. REFERENCE 1.Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411:494–498. 2. Kong YC, Han M, Zhao KX. Expression of vascular endothelial growth factor and pigment epithelium derived factor in mouse oxygen-induced retinopathy and its significance. 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A VEGF mRNA level BB Figure 1. Inhibition of VEGF production by VEGF-specific siRNA in EOMA cells 24h,48h,72h after transfection respectively. A) EGFP fluorescence in EOMA cells after transfection (X40). B) relative VEGF mRNA expression measured by real time PCR(β-actin as control). C) relative VEGF expression measured by Western Blot(β-actin as control) 1.control 2.24h after transfection 3.48h after transfection 4.72h after transfection Normal siRNA treatment blank control(OIR) Figure.2 Quantification of retinal neovascularization in C57BL/6J mice. Upper panel : representative retinal angiographs in mice(×40): compared to normal retina, neovascular tufts, avascular regions, tortuous and dilated blood vessels were observed in OIR(blank control).(red arrow), after siRNA were injected,abnormal vessels diminished.(yellow arrow). lower panel: representative retinal sections of mice(×40):compared to retina in OIR(blank control), vascular cell nuclei broke through ILM reduced.(blue arrow) relative VEGF expression ① A B C D relative PEDF expression ② Figure.3 Retinal VEGF ,PEDF levels in retina. ①:relative VEGF expression ②: relative PEDF expression A: normal , B: blank control,C: negative control,D: VEGF siRNA treatment