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Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA
VPH-06247; No of Pages 7 Vascular Pharmacology xxx (2015) xxx–xxx
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Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph
Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA Kun Liu a, Wen Zhou a, Honglin Chen b, Haiyan Pan c, Xiaohui Sun d, Qingsheng You a,⁎ a
Department of Cardiothoracic Surgery, Affiliated Hospital of Nantong University, Nantong 226001, China Nantong University, Nantong 226001, China Department of Cardiology, Affiliated Hospital of Nantong University, Nantong 226001, China d Department of Cardiology, Nantong Geriatric Rehabilitation Hosptial, Nantong 226001, China b c
a r t i c l e
i n f o
Article history: Received 20 November 2014 Received in revised form 30 July 2015 Accepted 9 August 2015 Available online xxxx Keywords: Nur77 RNA interfering Vascular smooth muscle cell Proliferation Vein graft restenosis
a b s t r a c t Neointimal hyperplasia plays an important role in autologous vein graft stenosis, and orphan receptor TR3/nur77 (Nur77) might play an essential role, but the mechanisms are still unclear. Here, we investigated the function of Nur77 in autologous vein graft stenosis. Rat vascular smooth muscle cell A7r5 was used for evaluating the function of Nur77 and screen siRNAs. Meanwhile, rat vein graft models were constructed for investigating the stenosis inhibition effects of Nur77-targeted siRNAs. The mRNA and protein levels of Nur77 were highly expressed in A7r5 cell, and could be significantly inhibited by the pre-designed siRNAs; the proliferation of A7r5 cell was also inhibited by the siRNAs. Furthermore, the intimal thickening in rat vein graft models was inhibited when knocking down the expression of Nur77 by siRNA. The results suggest that Nur77-targeted siRNA can inhibit autologous vein graft stenosis, Nur77 may play an important role in autologous vein graft stenosis, and Nur77 targeted siRNAs may be a therapy method for anti-stenosis of autologous vein graft. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Autogenous vein is one of the most commonly used materials in coronary artery bypass grafting [4], however, the arterialized vein is subject to a spectrum of hemodynamic, inflammatory, and humoral mechanisms of injury that may induce pathologic changes [6]. Such changes are responsible for a significant incidence of vein graft stenosis or occlusion, which occurs in approximately 30 to 50% of lower extremity bypasses within 3 to 5 years [5]. As well-known, stenosis or failure due to intimal hyperplasia and vascular smooth muscle cell (VSMC) proliferation is important long-term constraints [7,17,22]. In response to various injurious stimuli, VSMCs can dedifferentiate, converting from a quiescent, contractile phenotype to a highly proliferative, synthetic phenotype [24], the alterations of VSMCs were regulated by various genes, and the migration and proliferation of cells were controlled dependently [1,11–13]. Recent studies suggest that orphan receptor TR3/nur77 (Nur77) influences vascular homeostasis and disease by controlling the physiology of macrophages, smooth muscle cells (SMCs), and vascular endothelium [9], and other researches indicate that Nur77 can be induced by proinflammatory stimuli in cultured macrophages [3] and it expressed by macrophages in human atherosclerotic lesions [2,18]. Furthermore, in vitro studies have revealed that correlated Nur77 expression with ⁎ Corresponding author. E-mail address: [email protected] (Q. You).
vascular disease by revealing that Nur77 expressed in neointimal SMCs in both human atherosclerotic lesions and in murine femoral artery lesions but it not expressed in medial SMCs in healthy vessels [20]. The function of Nur77 has been illuminated by a combination of cell culture experiments and studies of transgenic mice which indicated that Nur77 expression can reduce neointima formation in injured arteries by suppressing SMC proliferation [8,23]. In this study, we used RNA interfering (RNAi) technology to design the siRNAs targeting Nur77 and demonstrated that Nur77 is highly expressed in VSMCs, and could promote the proliferation of VSMCs. In addition, the in-vivo study used the rat vein graft models to identify that Nur77-targeted siRNA could inhibit the Nur77 expression and furthermore inhibit the graft stenosis. 2. Materials and methods 2.1. Cell culture Rat vascular smooth muscle cell A7r5 cell line was purchased from ATCC (USA). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) at 37 °C in a humidified incubator with 5% CO2. 2.2. siRNA sequences and transfection in vitro The sequence of Nur77 gene was obtained from GenBank (Accession No. NM_024388). Three siRNAs targeting Nur77 with a length of 19 nt
http://dx.doi.org/10.1016/j.vph.2015.08.008 1537-1891/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
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K. Liu et al. / Vascular Pharmacology xxx (2015) xxx–xxx
with dTdT 3′ overhang were designed. Meanwhile, negative control siRNA (NC_siR) that has no homology with human genome was designed as a negative control. The sequences used for the experiments are shown in Table 1. All chemically synthesized siRNAs were obtained from Biomics Biotech (China). The cells were transfected with siRNAs using a Lipofectamine® 2000 transfection reagent (Life Technologies, USA) according to the manufactures' instructions. After 6 h at 37 °C, the DMEM was replaced with a complete growth medium (DMEM with 10% FBS). The cells without siRNA transfection were used as an untreated control.
2.3. Vein graft model and experimental groups The study performed conforms with the NIH guidelines (guide for the care and use of laboratory). All rats were treated and all procedures were conducted in accordance with the guidelines for experimental animals approved by the Animal Care and Use Committee of Nantong University (China). Adult male Sprague–Dawley (SD) rats, 6–8 weeks old and weight 400–500 g obtained from the animal center of Nantong University (China) were initially housed in an air-conditioned environment. Briefly, the rats were treated with chloral hydrate at a dose of 400 mg/kg (IP) body weight and concentrations of 100 mg/mL, and the duration of anesthesia about 2 h (±0.5 h). And, additional chloral hydrate (200 mg/kg) was given to keep narcotism if the surgical protocol was more than 2 h. Respiration, heart rate and the color of the tongue were monitored to exclude overdose of anesthetic during the operation, and the legs of rat in spasm were observed after the anesthetic was added once. The hairs on the neck of the rats were cut off, and the rats were disinfected by Iodophor and alcohol, and then a 1-cm segment of the left external jugular vein was obtained through blunt separation of submandibular gland and sternocleidomastoid muscle, the veins were put in standby 20 U/mL concentration of heparin sodium solution. After that, the left sternocleidomastoid was pulled open by a hook, the carotid sheath was exposed, and 1-cm carotid artery was isolated. A microsurgical technique was used to ligate the jugular vein which was cut from the rats in the method of end to end anastomosis of common carotid artery in the proximal and distal ends using 11-0 vascular anastomosis sutures, and the incision was sutured. To alleviate the pain after operation, a small dose of chloral hydrate (50 mg/kg) was used on the next day. The siRNAs were mixed with 25% (m/v) Pluronic F-127 (PF-127, diluted in normal saline at 4 °C condition, Sigma, USA), and then applied to the vein graft surface evenly. Forty rats were randomly divided into 4 groups (n = 5 for 14 days and n = 5 for 28 days in each group): PF-127 containing Nur77_siR3, PF-127 containing NC_siR, blank control (PF-127) and untreated group. Five rats of each group were euthanized with chloral hydrate at a dose of 400 mg/kg before killed at each time point of 14 and 28 days, respectively. Then the rats were sacrificed by cervical vertebra dislocation for the isolation and analysis of the vein graft. The part of the vein grafts was harvested to be fixed in 4% paraformaldehyde for histologic examination, and others were stored at −80 °C for RT-qPCR and Western blot analysis.
2.4. Real-time quantity PCR (RT-qPCR) Total RNAs of A7r5 cells and vein grafts were extracted using RISO™ RNA isolation reagent (Biomics Biotech, China) according to the manufacturer's procedure and then were submitted to a 25 μL PCR reaction in the presence of 12.5 μL of 2 × Master Mix, 1 μL of each forward and reverse primer mix (10 μM each), 0.5 μL of 50 × SYBR Green I and 4 μL mRNA as template. The mRNA of Nur77 was detected by forward primer (5′-TGGAGAAGCGTGCCTCAG-3′) and reverse primer (5′-CCAG AGAGCAAGTCATAAAATTGC-3′). RNAs were reverse transcribed to first strand cDNA at 42 °C for 30 min, and then amplified for 2 min at 94 °C for pre-denature, followed by 45 cycles of denature at 94 °C for 20 s, annealing at 58 °C for 20 s and extension at 72 °C for 30 s. All reactions were run in triplicate. Rat β-actin served as the internal control (the primer was forward: 5′-GGGAAATCGTGCGTGACATT-3′ and reverse: 5′-GCGGCAGTGGCCATCTC-3′) for mRNA determination of Nur77. The experiment was performed in triplicate, and the results were analyzed by 2−ΔΔCt method [14].
Table 1 Sequences of siRNAs targeting Nur77 gene. siRNA names
Sequence (5′-3′)
Nur77_siR1
Sense: CCAAGUUGGACUAUUCCAAdTdT Antisense: UUGGAAUAGUCCAACUUGGdTdT Sense: GACAAGAUCUUUAUGGACAdTdT Antisense: UGUCCAUAAAGAUCUUGUCdTdT Sense: UGGCCCAGAGUUCCCUGAAdTdT Antisense: UUCAGGGAACUCUGGGCCAdTdT Sense: UUCUCCGAACGUGUCACGUdTdT Antisense: ACGUGACACGUUCGGAGAAdTdT
Nur77_siR2 Nur77_siR3 NC_siR
Fig. 1. The mRNA and protein relative level of Nur77 with pre-designed siRNA treatments in A7r5 cells. A. The mRNA levels of Nur77 in A7r5 cells detected by RT-qPCR. B. The protein levels of Nur77 in A7r5 cells detected by Western blot. Data are mean + SD from three independent experiments, each performed in triplicate. *P b 0.05 vs untreated group.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
K. Liu et al. / Vascular Pharmacology xxx (2015) xxx–xxx
Fig. 2. Cell proliferation was analyzed by CCK8 assay. Growth curve of A7r5 cells was shown for each treatment at 0, 24, 48 and 72 h. Data are mean + SD from three independent experiments, each performed in triplicate. *P b 0.05 Nur77_siR3 vs untreated group at 48 h and 72 h.
2.5. Western blotting A7r5 cells were plated at 1 × 106 cells per well in a 6-well plate, and were grown for 24 h until 70–80% confluence. The cells were divided into five groups with different treatments: Nur77_siR1, Nur77_siR2, Nur77_siR3 and NC_siR. Then, cells were harvested at 48 h post-transfection, and then lysed in ice-cold 1 × SDS buffer (50 mM Tris–HCl, pH 7.6; 1% SDS; 150 mM NaCl; 0.5% Triton-X 100; 5 mM EDTA; 5% βmercaptoethanol (BME); 1 mM PMSF) and then centrifuged at 10,000 rpm for 20 min at 4 °C. Protein concentrations were determined with a BCA protein assay kit (Pierce, USA). The supernatant was diluted
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in 5 × SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (50 mM Tris–HCl (pH 6.8), 10%(W/V) SDS, 0.5%(W/V) bromophenol blue, 50%(V/V) glycerol, 5%(W/V) β-mercaptoethanol) and boiled. 20 μg of total cell lysates per lane was separated with SDSPAGE and electroblotted onto polyvinylidene difluoride filter (PVDF) membranes (Millipore, USA), followed by blocking with 5% skim milk in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 2 h at room temperature. The membrane was incubated with a primary antibody of rabbit monoclonal to Nur77 (Abcam, USA, 1:500 dilution), and mouse-anti-human β-actin (Boster, China, 1:500 dilution) as an internal control. After being washed with TBST, the membrane was incubated with the secondary antibody conjugated to horseradish peroxidase (HRP) (goat anti-rabbit IgG-HRP with 1:1000 dilutions for Nur77; goat anti-mouse IgG-HRP with 1:2000 dilutions for β-actin) for about 1.5 h at room temperature, and then washed for three times in TBST for 5 min. The specific proteins were detected with BeyoECL Plus kit (Beyotime, China). At the 14-day and the 28-day assessment, vein graft tissues were subjected to Western blot analysis. The whole vein grafts (about 20 mg) with different treatments were homogenized in ice-cold 1 × SDS buffer, and the protein dissolved in SDS-PAGE, and the following steps were same as cell's operations. 2.6. Cell proliferation assay The proliferation of cells was measured using CCK8 kit (Dojindo, Japan). A7r5 cells (5 × 103 cells per well) were plated into a 96-well plate before transfection and were grown to 70–80% confluence for 24 h. After siRNA treatment for 0, 24, 48, 72 and 96 h, 10 μL of CCK8
Fig. 3. Rat vein graft model. A. Common carotid artery indicated by white arrow was separated. B. External jugular vein indicated by white arrow was separated. C. Vein graft was indicated by white arrow. D. The vein graft with reperfusion performed by an arteriovenous anastomosis with the vein. E. The vein graft with applied with PF-127. F. The rat vein model with treatment for observation.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
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reagents was added to each well of a 96-well plate containing 100 μL culture medium and the plate was incubated for 4 h at 37 °C, 150 μL DMSO were added and incubated for 10 min at 37 °C. Then the optical density (OD) was measured at 490 nm using a Microplate Reader (Bio-Rad, USA). 2.7. Hematoxylin and eosin (HE) staining The proximal portion of the vein grafts was harvested to be formalin-fixed and paraffin-embedded, and sections (4 μm thick) were prepared for standard HE staining. The changes in histology were assessed under a light microscope. 2.8. Immunohistochemistry (IHC) staining The vein grafts were harvested to be formalin-fixed and paraffinembedded, and sections (4 μm thick) were immuno-stained for Ki67. IHC was performed by a standard Envision Plus/Horseradish Peroxidase system (DAKO Corp, USA). The sections were incubated with antiNur77 antibody (dilution 1:100, Abcam, USA), anti-Ki67 antibody (dilution 1:100, Abcam, USA) or anti-CD31 antibody (dilution 1:100, Abcam, USA) at 4 °C overnight, and rabbit IgG (dilution 1:100, Abcam, USA) used as an isotype control for CD31. After washing with phosphate buffered saline (PBS), the sections were incubated using the Envision Plus secondary antibody for 30 min, followed by diaminobenzidine (DAB) for 5 min. Positively stained cells were counted by an investigator in a blind fashion. 2.9. Statistical analysis All experiments were performed independently three times, the results were shown as mean ± standard deviation (SD), and statistical analyses were performed using SPSS19.0 software. The differences between groups were compared using Student's t-test to assess statistical significance. All P values were based on a two-sided statistical analysis and P b 0.05 was considered to indicate statistical significance.
at the mRNA level up to 78% (Nur77_siR3) in comparison to the untreated ones. And the silencing effects of Nur77 targeted siRNAs were observed at protein level up to 65% (Fig. 1B). 3.2. Inhibition effects of A7r5 cell proliferative activity by Nur77 targeted siRNAs A7r5 cell proliferation inhibition effects lead by Nur77-targeted siRNAs were detected by CCK8 assay. The absorbance values of A7r5 cells at 24, 48 and 72 h post-transfection with Nur77_siR3 were significantly lower than those of the untreated cells. There was no significant difference between the growth of cells treated with Nur77_siR2 and that of NC_siR. The cell proliferation inhibition rate treated with Nur77_siR3 showed a significant decrease (Fig. 2). 3.3. Construction of rat vein graft models A total of forty adult male SD rats were used to construct the vein graft models successfully according to the guidelines for experimental animals approved by the Animal Care and Use Committee of Nantong University (China). And a microsurgical technique was used for treating the vein grafts with different methods (Fig. 3). 3.4. Inhibition effects of Nur77-targeted siRNA at mRNA level in rat vein graft models The mRNA levels of Nur77 were determined by RT-qPCR after treated with siRNAs for 14 and 28 days in rat vein graft models. As shown in Fig. 4, Nur77 expression inhibited by Nur77_siR3 at the mRNA level up to 75% at 14 days post-treated with siRNA and PF-127 mixture in comparison to the untreated ones. And Nur77 expression inhibited by Nur77_siR3 at the mRNA level up to 58% at 28 days. The mRNA expression level of Nur77 in the grafts with no Nur77_siR3 treatment was all higher than that of ungraft veins (P b 0.05).
3. Results 3.1. Inhibition effects of pre-designed siRNAs that target Nur77 on mRNA and protein level The mRNA and protein levels of Nur77 were determined by RT-qPCR and Western blot after treated with siRNAs for 48 h in A7r5 cells. As shown in Fig. 1A, Nur77 targeted siRNAs inhibited the Nur77 expression
Fig. 4. The mRNA relative level of Nur77 with different treatments in rat vein graft models. Data are mean + SD from three independent experiments, each performed in triplicate. *P b 0.05 vs ungrafted veins. **P b 0.05 vs untreated and NC_siR at 14 days, #P b 0.05 vs ungrafted veins, ##P b 0.05 vs untreated and NC_siR at 28 days.
Fig. 5. The protein relative level of Nur77 with different treatments in rat vein graft models. Data are mean + SD from three independent experiments, each performed in triplicate. *P b 0.05 vs ungrafted veins. **P b 0.05 vs untreated and NC_siR at 14 days, # P b 0.05 vs ungrafted veins, ##P b 0.05 vs untreated and NC_siR at 28 days.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
K. Liu et al. / Vascular Pharmacology xxx (2015) xxx–xxx
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Fig. 6. HE staining (magnification 40× and 200×) and the thickness of the intimal and medial layers. A. HE staining at 14 days post-surgery. B. The thickness of the intimal and medial layers at 14 days post-surgery. C. HE staining at 28 days post-surgery. D. The thickness of the intimal and medial layers at 28 days post-surgery. Data showed as mean + SD, n = 5. *P b 0.05 vs untreated group.
3.5. Inhibition effects of Nur77-targeted siRNA at protein level in rat vein graft models The protein levels of Nur77 were determined by Western blot after treated with siRNAs for 14 and 28 days in rat vein graft models. As shown in Fig. 5, Nur77 expression inhibited by Nur77_siR3 at the protein level up to 55% at 14 days post-treated with siRNA and PF-127 mixture in comparison to the untreated ones. And Nur77 expression was inhibited by Nur77_siR3 at the mRNA level up to 37% at 28 days. The protein expression level of Nur77 in the grafts with no Nur77_siR3 treatment was all higher than that of ungraft veins (P b 0.05).
3.6. Inhibition effects of vein graft neointimal hyperplasia by Nur77targeted siRNA in rat vein graft models Histologic analysis evaluated the inhibition effects of vein graft neointimal hyperplasia in rat vein graft models, and the thickness of the intima and media was calculated. At 14 and 28 days post-surgery, the vein graft treated with Nur77_siR3 showed obvious neointima hyperplasia and significantly thicker intima in comparison to PF-127, NC_siR and untreated ones (P b 0.05, Fig. 6). There were no significant differences in thickness
among PF-127, NC_siR and untreated groups. The thickening of vein graft was inhibited significantly by Nur77 targeted siRNA (Fig. 6). The proliferative activity of smooth muscle cells at 14 days and 28 days on grafted veins with NC_siR and Nur77_siR3 treatment was monitored by Ki67 immuno-staining (Ki67 is a key marker of nuclear proliferation). The result showed that Ki67-positive cells appeared in the intima and media of vein graft with NC_siR treatment at 14 days and 28 days, and the ratio of Ki67-positive cells in Nur77_siR3 treated cells was lower than that of NC_siR treated ones (P b 0.05) (Fig. 7). The inhibition of proliferation in smooth muscle cells at 14 days and 28 days was induced by Nur77_siR3 successfully. Moreover, the expression of CD31 on grafted veins with NC_siR and Nur77_siR3 treatment at 14 and 28 days were no obvious differences, the result (Fig. 8) showed that Nur77_siR3 does not affect endothelial regeneration following engraftment, isotype control staining was used to demonstrate specificity. 4. Discussion VSMCs play an important role in vascular remodeling [21]. Heterogeneity and phenotypic changes in VSMCs are usually accompanied by a morphological difference, which directly regulates VSMC proliferation. Nur77 was found to be expressed in VSMCs; Arkenbout et al. [2] reported that TR3 could inhibit vascular lesion formation. Nur77 is a member
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
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Fig. 7. The expression of Ki67 was analyzed by immunohistochemistry staining (magnification 200×). A. Ki67-immunostained sections of vein grafts with NC_siR and Nur77_siR3 treatments at 14 and 28 days. B. The graphs showed the percentage of Ki67-positive cells in the vein grafts. The results were expressed as mean ± SD, n = 5. *P b 0.05 vs NC_siR treated group at 14 days, #P b 0.05 vs NC_siR treated group at 28 days.
of nuclear receptor superfamily, and it's a product of immediate early gene which is expressed rapidly after induced by several proliferation factors such as platelet-derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), phorbol ester
(PMA) [15], inflammatory factor, lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL), and fatty acid (FA) [3,16]. In the study, we determined the expression of Nur77 in rat VSMCs A7r5, meanwhile, the siRNAs were designed and used to knockdown the expression of Nur77, and we found that Nur77 was highly expressed in VSMCs, and pre-designed siRNAs could knockdown the expression of Nur77 effectively, especially Nur77_siR3 (Table 1, and Fig. 1). In addition, we demonstrated that Nur77_siR3 can also inhibit the proliferation of the VSMCs A7r5 (Fig. 2). But, there is still unclear mechanism about the relationship between Nur77 and the proliferation of VSMCs. Presently, we constructed the rat vein graft models (Fig. 3) and demonstrated that knockdown Nur77 by siRNA (Nur77_siR3) (Figs. 4 and 5) could suppress intimal layer thickening at 14 days (Fig. 6A and B) and 28 days (Fig. 6C and D). In the siRNA transfection in-vivo, we used Pluronic F-127 for siRNA delivery [10,14,19], and the results showed that Pluronic F-127 could help the siRNA transfection effectively, since how to deliver the siRNA to a target tissue is a difficult issue in the development of siRNA drugs, Pluronic F-127 might be a good delivery mediator in vein graft, especially in stenosis inhibition. On the other hand, RNAi has been proven recently to be a newly advanced and powerful tool for the development of therapeutic agents. And, recent years, there are over 30 siRNA drug successes in clinical trials; this is a novel and efficient tool to suppress the target gene in mRNA level [25], and the approach is better than the oligonucleotide decoy strategy, i.e. edifoligide that against E2F, which clinical trials failed to show efficacy [26]. In conclusion, our study has proved the role of Nur77 in the promoting of VSMC proliferation, and the down-regulation of Nur77 by siRNA can inhibit the proliferation of VSMCs. Furthermore, in rat vein graft models, the intimal thickening was also inhibited via knockdown of the Nur77 with siRNA. Therefore, Nur77 gene may potentially play a key role in the autologous vein graft stenosis and the results suggest that Nur77 may be a good target for inhibiting of autologous vein graft stenosis or intimal thickening post-surgery.
Disclosures We declare that none of the authors has any kind of conflict of interest related to the present work.
Fig. 8. The expression of CD31 was analyzed by immunohistochemistry (magnification 200×). A. CD31-immunostained sections of vein grafts with NC_siR and Nur77_siR3 treatments at 14 and 28 days. B. CD31 positive cells were counted and expressed as mean ± SD, n = 5, there was no difference in endothelial coverage between NC_siR and Nur77_siR3, *P N 0.05 vs NC_siR treated group at 14 days, #P N 0.05 vs NC_siR treated group at 28 days.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
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Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (no. 81100154), the Six Talent Peaks Project in Jiangsu Province of China (no. 2014-YY-006), the China Postdoctoral Science Foundation (no. 2013M541705), the Postdoctoral Research Foundation of Jiangsu Province, China (no. 1301072C) and the Science Foundation of Nantong City, Jiangsu Province, China (no. HS2012025).
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Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
Contents lists available at ScienceDirect
Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph
Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA Kun Liu a, Wen Zhou a, Honglin Chen b, Haiyan Pan c, Xiaohui Sun d, Qingsheng You a,⁎ a
Department of Cardiothoracic Surgery, Affiliated Hospital of Nantong University, Nantong 226001, China Nantong University, Nantong 226001, China Department of Cardiology, Affiliated Hospital of Nantong University, Nantong 226001, China d Department of Cardiology, Nantong Geriatric Rehabilitation Hosptial, Nantong 226001, China b c
a r t i c l e
i n f o
Article history: Received 20 November 2014 Received in revised form 30 July 2015 Accepted 9 August 2015 Available online xxxx Keywords: Nur77 RNA interfering Vascular smooth muscle cell Proliferation Vein graft restenosis
a b s t r a c t Neointimal hyperplasia plays an important role in autologous vein graft stenosis, and orphan receptor TR3/nur77 (Nur77) might play an essential role, but the mechanisms are still unclear. Here, we investigated the function of Nur77 in autologous vein graft stenosis. Rat vascular smooth muscle cell A7r5 was used for evaluating the function of Nur77 and screen siRNAs. Meanwhile, rat vein graft models were constructed for investigating the stenosis inhibition effects of Nur77-targeted siRNAs. The mRNA and protein levels of Nur77 were highly expressed in A7r5 cell, and could be significantly inhibited by the pre-designed siRNAs; the proliferation of A7r5 cell was also inhibited by the siRNAs. Furthermore, the intimal thickening in rat vein graft models was inhibited when knocking down the expression of Nur77 by siRNA. The results suggest that Nur77-targeted siRNA can inhibit autologous vein graft stenosis, Nur77 may play an important role in autologous vein graft stenosis, and Nur77 targeted siRNAs may be a therapy method for anti-stenosis of autologous vein graft. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Autogenous vein is one of the most commonly used materials in coronary artery bypass grafting [4], however, the arterialized vein is subject to a spectrum of hemodynamic, inflammatory, and humoral mechanisms of injury that may induce pathologic changes [6]. Such changes are responsible for a significant incidence of vein graft stenosis or occlusion, which occurs in approximately 30 to 50% of lower extremity bypasses within 3 to 5 years [5]. As well-known, stenosis or failure due to intimal hyperplasia and vascular smooth muscle cell (VSMC) proliferation is important long-term constraints [7,17,22]. In response to various injurious stimuli, VSMCs can dedifferentiate, converting from a quiescent, contractile phenotype to a highly proliferative, synthetic phenotype [24], the alterations of VSMCs were regulated by various genes, and the migration and proliferation of cells were controlled dependently [1,11–13]. Recent studies suggest that orphan receptor TR3/nur77 (Nur77) influences vascular homeostasis and disease by controlling the physiology of macrophages, smooth muscle cells (SMCs), and vascular endothelium [9], and other researches indicate that Nur77 can be induced by proinflammatory stimuli in cultured macrophages [3] and it expressed by macrophages in human atherosclerotic lesions [2,18]. Furthermore, in vitro studies have revealed that correlated Nur77 expression with ⁎ Corresponding author. E-mail address: [email protected] (Q. You).
vascular disease by revealing that Nur77 expressed in neointimal SMCs in both human atherosclerotic lesions and in murine femoral artery lesions but it not expressed in medial SMCs in healthy vessels [20]. The function of Nur77 has been illuminated by a combination of cell culture experiments and studies of transgenic mice which indicated that Nur77 expression can reduce neointima formation in injured arteries by suppressing SMC proliferation [8,23]. In this study, we used RNA interfering (RNAi) technology to design the siRNAs targeting Nur77 and demonstrated that Nur77 is highly expressed in VSMCs, and could promote the proliferation of VSMCs. In addition, the in-vivo study used the rat vein graft models to identify that Nur77-targeted siRNA could inhibit the Nur77 expression and furthermore inhibit the graft stenosis. 2. Materials and methods 2.1. Cell culture Rat vascular smooth muscle cell A7r5 cell line was purchased from ATCC (USA). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) at 37 °C in a humidified incubator with 5% CO2. 2.2. siRNA sequences and transfection in vitro The sequence of Nur77 gene was obtained from GenBank (Accession No. NM_024388). Three siRNAs targeting Nur77 with a length of 19 nt
http://dx.doi.org/10.1016/j.vph.2015.08.008 1537-1891/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
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K. Liu et al. / Vascular Pharmacology xxx (2015) xxx–xxx
with dTdT 3′ overhang were designed. Meanwhile, negative control siRNA (NC_siR) that has no homology with human genome was designed as a negative control. The sequences used for the experiments are shown in Table 1. All chemically synthesized siRNAs were obtained from Biomics Biotech (China). The cells were transfected with siRNAs using a Lipofectamine® 2000 transfection reagent (Life Technologies, USA) according to the manufactures' instructions. After 6 h at 37 °C, the DMEM was replaced with a complete growth medium (DMEM with 10% FBS). The cells without siRNA transfection were used as an untreated control.
2.3. Vein graft model and experimental groups The study performed conforms with the NIH guidelines (guide for the care and use of laboratory). All rats were treated and all procedures were conducted in accordance with the guidelines for experimental animals approved by the Animal Care and Use Committee of Nantong University (China). Adult male Sprague–Dawley (SD) rats, 6–8 weeks old and weight 400–500 g obtained from the animal center of Nantong University (China) were initially housed in an air-conditioned environment. Briefly, the rats were treated with chloral hydrate at a dose of 400 mg/kg (IP) body weight and concentrations of 100 mg/mL, and the duration of anesthesia about 2 h (±0.5 h). And, additional chloral hydrate (200 mg/kg) was given to keep narcotism if the surgical protocol was more than 2 h. Respiration, heart rate and the color of the tongue were monitored to exclude overdose of anesthetic during the operation, and the legs of rat in spasm were observed after the anesthetic was added once. The hairs on the neck of the rats were cut off, and the rats were disinfected by Iodophor and alcohol, and then a 1-cm segment of the left external jugular vein was obtained through blunt separation of submandibular gland and sternocleidomastoid muscle, the veins were put in standby 20 U/mL concentration of heparin sodium solution. After that, the left sternocleidomastoid was pulled open by a hook, the carotid sheath was exposed, and 1-cm carotid artery was isolated. A microsurgical technique was used to ligate the jugular vein which was cut from the rats in the method of end to end anastomosis of common carotid artery in the proximal and distal ends using 11-0 vascular anastomosis sutures, and the incision was sutured. To alleviate the pain after operation, a small dose of chloral hydrate (50 mg/kg) was used on the next day. The siRNAs were mixed with 25% (m/v) Pluronic F-127 (PF-127, diluted in normal saline at 4 °C condition, Sigma, USA), and then applied to the vein graft surface evenly. Forty rats were randomly divided into 4 groups (n = 5 for 14 days and n = 5 for 28 days in each group): PF-127 containing Nur77_siR3, PF-127 containing NC_siR, blank control (PF-127) and untreated group. Five rats of each group were euthanized with chloral hydrate at a dose of 400 mg/kg before killed at each time point of 14 and 28 days, respectively. Then the rats were sacrificed by cervical vertebra dislocation for the isolation and analysis of the vein graft. The part of the vein grafts was harvested to be fixed in 4% paraformaldehyde for histologic examination, and others were stored at −80 °C for RT-qPCR and Western blot analysis.
2.4. Real-time quantity PCR (RT-qPCR) Total RNAs of A7r5 cells and vein grafts were extracted using RISO™ RNA isolation reagent (Biomics Biotech, China) according to the manufacturer's procedure and then were submitted to a 25 μL PCR reaction in the presence of 12.5 μL of 2 × Master Mix, 1 μL of each forward and reverse primer mix (10 μM each), 0.5 μL of 50 × SYBR Green I and 4 μL mRNA as template. The mRNA of Nur77 was detected by forward primer (5′-TGGAGAAGCGTGCCTCAG-3′) and reverse primer (5′-CCAG AGAGCAAGTCATAAAATTGC-3′). RNAs were reverse transcribed to first strand cDNA at 42 °C for 30 min, and then amplified for 2 min at 94 °C for pre-denature, followed by 45 cycles of denature at 94 °C for 20 s, annealing at 58 °C for 20 s and extension at 72 °C for 30 s. All reactions were run in triplicate. Rat β-actin served as the internal control (the primer was forward: 5′-GGGAAATCGTGCGTGACATT-3′ and reverse: 5′-GCGGCAGTGGCCATCTC-3′) for mRNA determination of Nur77. The experiment was performed in triplicate, and the results were analyzed by 2−ΔΔCt method [14].
Table 1 Sequences of siRNAs targeting Nur77 gene. siRNA names
Sequence (5′-3′)
Nur77_siR1
Sense: CCAAGUUGGACUAUUCCAAdTdT Antisense: UUGGAAUAGUCCAACUUGGdTdT Sense: GACAAGAUCUUUAUGGACAdTdT Antisense: UGUCCAUAAAGAUCUUGUCdTdT Sense: UGGCCCAGAGUUCCCUGAAdTdT Antisense: UUCAGGGAACUCUGGGCCAdTdT Sense: UUCUCCGAACGUGUCACGUdTdT Antisense: ACGUGACACGUUCGGAGAAdTdT
Nur77_siR2 Nur77_siR3 NC_siR
Fig. 1. The mRNA and protein relative level of Nur77 with pre-designed siRNA treatments in A7r5 cells. A. The mRNA levels of Nur77 in A7r5 cells detected by RT-qPCR. B. The protein levels of Nur77 in A7r5 cells detected by Western blot. Data are mean + SD from three independent experiments, each performed in triplicate. *P b 0.05 vs untreated group.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
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Fig. 2. Cell proliferation was analyzed by CCK8 assay. Growth curve of A7r5 cells was shown for each treatment at 0, 24, 48 and 72 h. Data are mean + SD from three independent experiments, each performed in triplicate. *P b 0.05 Nur77_siR3 vs untreated group at 48 h and 72 h.
2.5. Western blotting A7r5 cells were plated at 1 × 106 cells per well in a 6-well plate, and were grown for 24 h until 70–80% confluence. The cells were divided into five groups with different treatments: Nur77_siR1, Nur77_siR2, Nur77_siR3 and NC_siR. Then, cells were harvested at 48 h post-transfection, and then lysed in ice-cold 1 × SDS buffer (50 mM Tris–HCl, pH 7.6; 1% SDS; 150 mM NaCl; 0.5% Triton-X 100; 5 mM EDTA; 5% βmercaptoethanol (BME); 1 mM PMSF) and then centrifuged at 10,000 rpm for 20 min at 4 °C. Protein concentrations were determined with a BCA protein assay kit (Pierce, USA). The supernatant was diluted
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in 5 × SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (50 mM Tris–HCl (pH 6.8), 10%(W/V) SDS, 0.5%(W/V) bromophenol blue, 50%(V/V) glycerol, 5%(W/V) β-mercaptoethanol) and boiled. 20 μg of total cell lysates per lane was separated with SDSPAGE and electroblotted onto polyvinylidene difluoride filter (PVDF) membranes (Millipore, USA), followed by blocking with 5% skim milk in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 2 h at room temperature. The membrane was incubated with a primary antibody of rabbit monoclonal to Nur77 (Abcam, USA, 1:500 dilution), and mouse-anti-human β-actin (Boster, China, 1:500 dilution) as an internal control. After being washed with TBST, the membrane was incubated with the secondary antibody conjugated to horseradish peroxidase (HRP) (goat anti-rabbit IgG-HRP with 1:1000 dilutions for Nur77; goat anti-mouse IgG-HRP with 1:2000 dilutions for β-actin) for about 1.5 h at room temperature, and then washed for three times in TBST for 5 min. The specific proteins were detected with BeyoECL Plus kit (Beyotime, China). At the 14-day and the 28-day assessment, vein graft tissues were subjected to Western blot analysis. The whole vein grafts (about 20 mg) with different treatments were homogenized in ice-cold 1 × SDS buffer, and the protein dissolved in SDS-PAGE, and the following steps were same as cell's operations. 2.6. Cell proliferation assay The proliferation of cells was measured using CCK8 kit (Dojindo, Japan). A7r5 cells (5 × 103 cells per well) were plated into a 96-well plate before transfection and were grown to 70–80% confluence for 24 h. After siRNA treatment for 0, 24, 48, 72 and 96 h, 10 μL of CCK8
Fig. 3. Rat vein graft model. A. Common carotid artery indicated by white arrow was separated. B. External jugular vein indicated by white arrow was separated. C. Vein graft was indicated by white arrow. D. The vein graft with reperfusion performed by an arteriovenous anastomosis with the vein. E. The vein graft with applied with PF-127. F. The rat vein model with treatment for observation.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
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reagents was added to each well of a 96-well plate containing 100 μL culture medium and the plate was incubated for 4 h at 37 °C, 150 μL DMSO were added and incubated for 10 min at 37 °C. Then the optical density (OD) was measured at 490 nm using a Microplate Reader (Bio-Rad, USA). 2.7. Hematoxylin and eosin (HE) staining The proximal portion of the vein grafts was harvested to be formalin-fixed and paraffin-embedded, and sections (4 μm thick) were prepared for standard HE staining. The changes in histology were assessed under a light microscope. 2.8. Immunohistochemistry (IHC) staining The vein grafts were harvested to be formalin-fixed and paraffinembedded, and sections (4 μm thick) were immuno-stained for Ki67. IHC was performed by a standard Envision Plus/Horseradish Peroxidase system (DAKO Corp, USA). The sections were incubated with antiNur77 antibody (dilution 1:100, Abcam, USA), anti-Ki67 antibody (dilution 1:100, Abcam, USA) or anti-CD31 antibody (dilution 1:100, Abcam, USA) at 4 °C overnight, and rabbit IgG (dilution 1:100, Abcam, USA) used as an isotype control for CD31. After washing with phosphate buffered saline (PBS), the sections were incubated using the Envision Plus secondary antibody for 30 min, followed by diaminobenzidine (DAB) for 5 min. Positively stained cells were counted by an investigator in a blind fashion. 2.9. Statistical analysis All experiments were performed independently three times, the results were shown as mean ± standard deviation (SD), and statistical analyses were performed using SPSS19.0 software. The differences between groups were compared using Student's t-test to assess statistical significance. All P values were based on a two-sided statistical analysis and P b 0.05 was considered to indicate statistical significance.
at the mRNA level up to 78% (Nur77_siR3) in comparison to the untreated ones. And the silencing effects of Nur77 targeted siRNAs were observed at protein level up to 65% (Fig. 1B). 3.2. Inhibition effects of A7r5 cell proliferative activity by Nur77 targeted siRNAs A7r5 cell proliferation inhibition effects lead by Nur77-targeted siRNAs were detected by CCK8 assay. The absorbance values of A7r5 cells at 24, 48 and 72 h post-transfection with Nur77_siR3 were significantly lower than those of the untreated cells. There was no significant difference between the growth of cells treated with Nur77_siR2 and that of NC_siR. The cell proliferation inhibition rate treated with Nur77_siR3 showed a significant decrease (Fig. 2). 3.3. Construction of rat vein graft models A total of forty adult male SD rats were used to construct the vein graft models successfully according to the guidelines for experimental animals approved by the Animal Care and Use Committee of Nantong University (China). And a microsurgical technique was used for treating the vein grafts with different methods (Fig. 3). 3.4. Inhibition effects of Nur77-targeted siRNA at mRNA level in rat vein graft models The mRNA levels of Nur77 were determined by RT-qPCR after treated with siRNAs for 14 and 28 days in rat vein graft models. As shown in Fig. 4, Nur77 expression inhibited by Nur77_siR3 at the mRNA level up to 75% at 14 days post-treated with siRNA and PF-127 mixture in comparison to the untreated ones. And Nur77 expression inhibited by Nur77_siR3 at the mRNA level up to 58% at 28 days. The mRNA expression level of Nur77 in the grafts with no Nur77_siR3 treatment was all higher than that of ungraft veins (P b 0.05).
3. Results 3.1. Inhibition effects of pre-designed siRNAs that target Nur77 on mRNA and protein level The mRNA and protein levels of Nur77 were determined by RT-qPCR and Western blot after treated with siRNAs for 48 h in A7r5 cells. As shown in Fig. 1A, Nur77 targeted siRNAs inhibited the Nur77 expression
Fig. 4. The mRNA relative level of Nur77 with different treatments in rat vein graft models. Data are mean + SD from three independent experiments, each performed in triplicate. *P b 0.05 vs ungrafted veins. **P b 0.05 vs untreated and NC_siR at 14 days, #P b 0.05 vs ungrafted veins, ##P b 0.05 vs untreated and NC_siR at 28 days.
Fig. 5. The protein relative level of Nur77 with different treatments in rat vein graft models. Data are mean + SD from three independent experiments, each performed in triplicate. *P b 0.05 vs ungrafted veins. **P b 0.05 vs untreated and NC_siR at 14 days, # P b 0.05 vs ungrafted veins, ##P b 0.05 vs untreated and NC_siR at 28 days.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
K. Liu et al. / Vascular Pharmacology xxx (2015) xxx–xxx
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Fig. 6. HE staining (magnification 40× and 200×) and the thickness of the intimal and medial layers. A. HE staining at 14 days post-surgery. B. The thickness of the intimal and medial layers at 14 days post-surgery. C. HE staining at 28 days post-surgery. D. The thickness of the intimal and medial layers at 28 days post-surgery. Data showed as mean + SD, n = 5. *P b 0.05 vs untreated group.
3.5. Inhibition effects of Nur77-targeted siRNA at protein level in rat vein graft models The protein levels of Nur77 were determined by Western blot after treated with siRNAs for 14 and 28 days in rat vein graft models. As shown in Fig. 5, Nur77 expression inhibited by Nur77_siR3 at the protein level up to 55% at 14 days post-treated with siRNA and PF-127 mixture in comparison to the untreated ones. And Nur77 expression was inhibited by Nur77_siR3 at the mRNA level up to 37% at 28 days. The protein expression level of Nur77 in the grafts with no Nur77_siR3 treatment was all higher than that of ungraft veins (P b 0.05).
3.6. Inhibition effects of vein graft neointimal hyperplasia by Nur77targeted siRNA in rat vein graft models Histologic analysis evaluated the inhibition effects of vein graft neointimal hyperplasia in rat vein graft models, and the thickness of the intima and media was calculated. At 14 and 28 days post-surgery, the vein graft treated with Nur77_siR3 showed obvious neointima hyperplasia and significantly thicker intima in comparison to PF-127, NC_siR and untreated ones (P b 0.05, Fig. 6). There were no significant differences in thickness
among PF-127, NC_siR and untreated groups. The thickening of vein graft was inhibited significantly by Nur77 targeted siRNA (Fig. 6). The proliferative activity of smooth muscle cells at 14 days and 28 days on grafted veins with NC_siR and Nur77_siR3 treatment was monitored by Ki67 immuno-staining (Ki67 is a key marker of nuclear proliferation). The result showed that Ki67-positive cells appeared in the intima and media of vein graft with NC_siR treatment at 14 days and 28 days, and the ratio of Ki67-positive cells in Nur77_siR3 treated cells was lower than that of NC_siR treated ones (P b 0.05) (Fig. 7). The inhibition of proliferation in smooth muscle cells at 14 days and 28 days was induced by Nur77_siR3 successfully. Moreover, the expression of CD31 on grafted veins with NC_siR and Nur77_siR3 treatment at 14 and 28 days were no obvious differences, the result (Fig. 8) showed that Nur77_siR3 does not affect endothelial regeneration following engraftment, isotype control staining was used to demonstrate specificity. 4. Discussion VSMCs play an important role in vascular remodeling [21]. Heterogeneity and phenotypic changes in VSMCs are usually accompanied by a morphological difference, which directly regulates VSMC proliferation. Nur77 was found to be expressed in VSMCs; Arkenbout et al. [2] reported that TR3 could inhibit vascular lesion formation. Nur77 is a member
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
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Fig. 7. The expression of Ki67 was analyzed by immunohistochemistry staining (magnification 200×). A. Ki67-immunostained sections of vein grafts with NC_siR and Nur77_siR3 treatments at 14 and 28 days. B. The graphs showed the percentage of Ki67-positive cells in the vein grafts. The results were expressed as mean ± SD, n = 5. *P b 0.05 vs NC_siR treated group at 14 days, #P b 0.05 vs NC_siR treated group at 28 days.
of nuclear receptor superfamily, and it's a product of immediate early gene which is expressed rapidly after induced by several proliferation factors such as platelet-derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), phorbol ester
(PMA) [15], inflammatory factor, lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL), and fatty acid (FA) [3,16]. In the study, we determined the expression of Nur77 in rat VSMCs A7r5, meanwhile, the siRNAs were designed and used to knockdown the expression of Nur77, and we found that Nur77 was highly expressed in VSMCs, and pre-designed siRNAs could knockdown the expression of Nur77 effectively, especially Nur77_siR3 (Table 1, and Fig. 1). In addition, we demonstrated that Nur77_siR3 can also inhibit the proliferation of the VSMCs A7r5 (Fig. 2). But, there is still unclear mechanism about the relationship between Nur77 and the proliferation of VSMCs. Presently, we constructed the rat vein graft models (Fig. 3) and demonstrated that knockdown Nur77 by siRNA (Nur77_siR3) (Figs. 4 and 5) could suppress intimal layer thickening at 14 days (Fig. 6A and B) and 28 days (Fig. 6C and D). In the siRNA transfection in-vivo, we used Pluronic F-127 for siRNA delivery [10,14,19], and the results showed that Pluronic F-127 could help the siRNA transfection effectively, since how to deliver the siRNA to a target tissue is a difficult issue in the development of siRNA drugs, Pluronic F-127 might be a good delivery mediator in vein graft, especially in stenosis inhibition. On the other hand, RNAi has been proven recently to be a newly advanced and powerful tool for the development of therapeutic agents. And, recent years, there are over 30 siRNA drug successes in clinical trials; this is a novel and efficient tool to suppress the target gene in mRNA level [25], and the approach is better than the oligonucleotide decoy strategy, i.e. edifoligide that against E2F, which clinical trials failed to show efficacy [26]. In conclusion, our study has proved the role of Nur77 in the promoting of VSMC proliferation, and the down-regulation of Nur77 by siRNA can inhibit the proliferation of VSMCs. Furthermore, in rat vein graft models, the intimal thickening was also inhibited via knockdown of the Nur77 with siRNA. Therefore, Nur77 gene may potentially play a key role in the autologous vein graft stenosis and the results suggest that Nur77 may be a good target for inhibiting of autologous vein graft stenosis or intimal thickening post-surgery.
Disclosures We declare that none of the authors has any kind of conflict of interest related to the present work.
Fig. 8. The expression of CD31 was analyzed by immunohistochemistry (magnification 200×). A. CD31-immunostained sections of vein grafts with NC_siR and Nur77_siR3 treatments at 14 and 28 days. B. CD31 positive cells were counted and expressed as mean ± SD, n = 5, there was no difference in endothelial coverage between NC_siR and Nur77_siR3, *P N 0.05 vs NC_siR treated group at 14 days, #P N 0.05 vs NC_siR treated group at 28 days.
Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008
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Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (no. 81100154), the Six Talent Peaks Project in Jiangsu Province of China (no. 2014-YY-006), the China Postdoctoral Science Foundation (no. 2013M541705), the Postdoctoral Research Foundation of Jiangsu Province, China (no. 1301072C) and the Science Foundation of Nantong City, Jiangsu Province, China (no. HS2012025).
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Please cite this article as: K. Liu, et al., Autologous vein graft stenosis inhibited by orphan nuclear receptor Nur77-targeted siRNA, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.08.008