- Email: [email protected]
Small Interfering RNA Targeting Nuclear Factor Kappa B to Prevent Vein Graft Stenosis in Rat Models
Small Interfering RNA Targeting Nuclear Factor Kappa B to Prevent Vein Graft Stenosis in Rat Models X.B. Meng, X.L. Bi, H.L. Zhao, J.B. Feng, J.P. Zhang, G.M. Song, W.Y. Sun, and Y.W. Bi ABSTRACT Background. Intimal hyperplasia plays an important role in vein graft stenosis. Inflammatory injury, especially nuclear factor kappaB (NF-kB) gene activation, is highly involved in stenosis progression. We examined whether neointimal hyperplasia and vein graft stenosis could be inhibited by silencing the NF-kB gene with small interference RNA (siRNA). Methods. Sixty adult male Sprague-Dawley rats were randomly divided into a normal vein group, a vein graft group, a scrambled siRNA group, and an NF-kB siRNA group. We performed reverse interpositional grafting of the autologous external jugular vein to the abdominal aorta. Vein grafts were treated with liposome and gel complexes containing NF-kB siRNA or scrambled siRNA. The levels of monocyte chemoattractant protein -1, tumor necrosis factor-a, and NF-kB p65 in vessel tissues were evaluated after surgery for content of proliferating cell nuclear antigen (PCNA) and vascular wall thickness. Results. NF-kB siRNA treated vein graft showed less neointimal formation and fewer positive PCNA cells (P < .05). In addition there were lower levels of, NF-kB p65 protein and of inflammatory mediators (P < .05) compared with the vein graft group. Conclusion. Our study suggested that siRNA transfection suppressed NF-kB expression, reduced inflammatory factors, lessened neointimal proliferation, and suppressed PCNA.
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ORONARY ARTERY BYPASS GRAFTING (CABG) is an important treatment for this serious disease. Autologous veins are the most commonly used graft material for CABG, but stenosis or failure due to intimal hyperplasia and vascular smooth muscle cell (VSMC) proliferation are important long-term constraints.1e3 In recent years, many studies have revealed that inflammatory injuries play crucial roles in the progression of vein graft stenosis. As a crucial transcription factor in inflammatory responses, nuclear factor kappa B (NF-kB) controls transcription of many genes. It has an established role in atherosclerosis with p65, the key active subunit in NF-kB transcription.4e7 To prevent inflammatory injury and inhibit vein graft stenosis, efficient methods are needed to interrupt NF-kB activation. Gene silencing by RNA interference (RNAi) can induce sequence-specific degradation of homologous mRNA, interfering with gene expression.8,9 Animal experiments have shown it to be a promising solution to prevent vein graft restenosis, as well as reduce neointimal formation and atherosclerosis.10 In our previous study, we have shown that NF-kB small interference RNA (siRNA) significantly inhibits cell
proliferation, reduces p65 expression, increases the ratio of G0/G1 stage cells, and decreases the proportion of S stage cells in vitro.11 In this in vivo study, we treated vein grafts at the time of surgery with siRNA, seeking to identify its inhibitory effects on cell proliferation or graft stenosis. MATERIALS AND METHODS Construction of NF-kB p65 siRNA Expression Vector The 21-nt siRNA sequences were designed and synthesized by Ambion Company according to the complete gene sequence of the rat NF-kB p65 subunit from GeneBank. We selected an siRNA sequence targeting the rat NF-kB p65 gene for synthesis as reported
From the Department of Cardiovascular Surgery (X.B.M., X.L.B., H.L.Z., G.M.S., W.Y.S., Y.W.B.), Central Laboratory (J.B.F.), and Department of Pathology, (J.P.Z.), QiLu Hospital, Shandong University, Jinan, Shandong Province, China. Address reprint requests to Yan Wen Bi, Department of Cardiovascular Surgery, Qilu Hospital, Shandong University, 107# Wenhua Xi Road, Jinan 250012, Shandong Province, People’s Republic of China. E-mail: [email protected]
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.03.045
Transplantation Proceedings, 45, 2553e2558 (2013)
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by He XP et al.11 The sequence 5 -AAGCACAGATACCACTAA GAC-30 was located between 211 bp and 231 bp. The specific sequence was BLAST (basic local alignment search tool) searched against the expressed sequence tags rat libraries to ensure that it was not homologous to others. Furthermore, we synthesized 2 complementary oligodeoxyribonucleotides of specific short hairpin RNA (shRNA), which encoded the 21-bp designed sequence, and annealed them to form a double-stranded RNA, which was ligated into the linearized plasmid pGenesil-1.2 with an mU6 promoter, in order to construct a recombinant plasmid expressing NF-kB p65 siRNA in eukaryotic cytoplasm after transfectiond“pGenesil1.2-p65siRNA.” Meanwhile, we constructed a negative control plasmid “scrambled siRNA.” The GC content of p65 sequences was 42.9%. The multiple cloning site of plasmid pGenesil-1.2 was EGFP (enhanced green fluorescence protein)-SacI-CMV (cytomegalovirus) promoter-MLUI-HINDIII-SACI-shRNAmU6 promoter. Enzyme analysis and DNA sequencing confirmed that the inserted sequences has been successfully cloned in the vector.
examinations. We detected NF-kB p65 protein levels in vein grafts that has been stored in liquid nitrogen.
Western Blot Analysis At the 7-day assessment, vein graft tissues were subjected to Western blot analysis to detect NF-kB p65 protein levels. The whole graft was snap-frozen and ground into a powder. Portion of vein graft tissues (20 mg) were treated with lysis buffer. Equal amounts of protein dissolved in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) sample buffer and electrophoresed were transferred onto polyvinylidene fluoride membranes. The membranes were incubated with the respective primary antibodies at 4 C overnight before incubation with the secondary antibody for 60 minutes at room temperature with continuous shaking. In all cases, b-actin was used as the internal reference. Membranes were exposed to X-ray film to visualize the bands. We quantified the intensities of p65 protein and b-actin bands.
Histologic and Immunohistochemistry Analysis Vein Graft Model and Experimental Groups Adult male Sprague-Dawley rats (weight, 300e350 g) obtained from our Animal Research Center were initially housed in a temperature (21 Ce26 C) and 12-hour cycled, light-controlled rooms with free access to food and water. Briefly, animals anesthetized with pentobarbital sodium (60 mg/kg) by intraperitoneal injection were systemically heparinized (200 U/kg). A microsurgical technique was used to dissect a 15-mm segment of the right external jugular vein through a midline vertical neck incision. All branches were carefully divided and ligated with 8-0 polypropylene sutures. A 10-mm segment of the abdominal aorta was denuded and resected, before the divided ends were irrigated with heparinized saline solution. An autologous external jugular veinetoeabdominal aorta reverse interposition was performed in rats as previously described12 using a cuff technique. The arterial occlusion time ranged from 5 to 10 minutes. We confirmed immediate restoration of blood flow upon removal of the arterial occlusion clamps. Sixty rats were divided into 4 groups. Normal vein group (vein, n ¼ 15), vein grafted to abdominal aorta with no treatment group (vein graft, n ¼ 15), scrambled siRNA group (n ¼ 15), with grafted vein treated with Lipofectamine 2000 (Invitrogen) and scrambled siRNA complex for 10 minutes before the arteriovenous anastomosis. Poluronic F-127 gel (Sigma) and scrambled siRNA complex were applied to the vein graft surface immediately after the anastomosis. The final NF-kB siRNA group (n ¼ 15) had vein grafts treated with Lipofectamine 2000 and p65 siRNA complex for 10 minutes before the anastomosis with p65 siRNA and gel complex applied to the graft surface.
siRNA in vivo Transfection and Tissue Preparation To increase transfection efficiency, we use 2 methods to apply siRNA to the vein graft. First, liposome and siRNA complex were perfused into vein graft under pressure (20 mm Hg). Second, the siRNA-gel complex was applied to the graft surface, allowing longstanding suppression of the target gene. There delivery systems can provide sufficient suppressive effects on gene expression. Rats in each group were humanely killed under heavy anesthesia and vein grafts harvested after the surgery. The proximal portion of the vein grafts was harvested to be fixed in 4% paraformaldehyde or stored in liquid nitrogen for research. At the 7-day assessment, vein graft tissues were examined by polymerase chain reaction (PCR) and Western blot analysis. At the 14-day assessment, vein graft tissues were subjected to histologic and immunohistochemical
Tissue segment of vein grafts harvested at 14 days were fixed in formaldehyde (4%) and embedded in paraffin. To quantify graft thickness, we did not distinguish intima and media because there was no morphologic border. Neointimal thickness was measured at 6 randomly selected points for each section. Image analysis software was used to quantify vein graft thickness. Immunohistochemical examination was performed using the avidin-biotin peroxidase complex method to detect proliferating cell nuclear antigen (antiPCNA, 1:100, DAKO). The PCNA proliferation index was determined by dividing the number of positively stained nuclei by the total number of nuclei in the intima and media of each section. The average index was calculated for each vessel to determine the intimal thickness and proliferation index.
Analysis of Inflammatory Mediators Expressions of MCP-1 and tumor necrosis factor-a (TNF-a) inflammatory mediators in vein grafts were quantified by PCR (polymerase chain reaction). Specific primer sequences (MCP-1, TNF-a, and b-actin) obtained from Genbank were synthesized by the Jinan BoYa Biological Engineering Company (China). Total RNA was isolated with the RISOTM RNA Isolation Reagent (Biomics, USA) as described in the manufacture’s instructions. The RNA samples were normalized using the housekeeping gene b-actin. The primer sequences were: MCP-l (forward 50 -ATGCAGGTCTCTGTCACG CT-30 ; reverse 50 -GGTGCTGAAGTCCTTAGGGT-30 ). TNF-a (forward 50 -CTTATCTACTCCCAGGTTCTCTCAA-30 ; reverse 50 -GAG ACTCCTCCCAGGTACATGG-30 ). b-actin (forward 50 -AGACCTT CAACACCCCAG-30 ; reverse 50 -CACGATTTCCCTCTCAGC-30 ). RNA extracted from tissues with TRIzol (Invitrogen) was reverse transcribed to cDNA. Using specific primers, amplication was perfomed in the ABI Prism 7500 Real-Time PCR System (Applied Biosystems). The 40-cycle reaction consitions were 60 minutes at 42 C, 10 seconds at 95 C, 10 seconds at 55 C, and 35 seconds at 72 C. All experiments were performed in triplicate. The relative mRNA expressions of target genes were calculated and normalized by the comparative CT method.
Statistical Analysis Results are expressed as mean values standard deviations. Statistical significance was determined using one-way analysis of variance. The least significant difference test or Dunnett T3 test was used to compare 2 groups. SPSS 13.0 was used for statistical analyses. P value less than .05 were considered to be statistically significant.
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group, transfection with scrambled siRNA did not change p65 protein levels (P > .05), NF-kB siRNA significantly suppressed p65 protein expression (P < .05, Fig 1). The mRNA Expressions of TNF-a and MCP-1 in Vein Graft
QT-PCR was used to analyze TNF-a and MCP-1 mRNA expressions in grafts during thickening and accelerated atherosclerosis. At 7 days, the relative mRNA levels of TNF-a and MCP-1 expressions were higher and peaked in both the vein graft and the scrambled siRNA groups (P < .05, Fig 2). However, the mRNA levels of TNF-a and MCP-1 in the NF-kB siRNA group were less (P < .05, Fig 2). Vein Graft Proliferative Activity
Fig 1. The relative levels of nuclear factor kappa B (NF-kB) p65 protein expressions in each group *P < .05 vs vein group, **P < .05 vs vein group. siRNA, small interference RNA.
RESULTS NF-kB p65 Protein Expression of Vein Graft
Quantitative analysis showed that NF-kB p65 protein expression was significantly increased in the vein graft relative to the vein group (P < .05, Fig 1), indicating activation of the NF-kB pathway. Compared with the vein graft
PCNA reflects proliferative activity. The normal vein showed no evidence of PCNA-positive cells. Many of PCNA positive cells were evident in the intima and media of the vein grafts at 14 days after surgery group (P < .05). The scrambled siRNA group showed a similar result as the vein graft group. The ratio of PCNA-positive cells in the NF-kB siRNA group was lower than that in the vein graft group (P < .05, Fig 3). Therefore, NF-kB p65 siRNA treatment caused less proliferative activity vascular in the wall (Fig 3). Vein Graft Neointimal Hyperplasia
Histologic analysis evaluated the proliferation and thickness of the intima and media. At 14 days postsurgery, vein graft showed obvious neointima hyperplasia with significantly thicker intima and media both among the untreated and the scrambled siRNA compared with the in situ vein group
Fig 2. The relative levels of MCP-1 mRNA and tumor necrosis factor-a (TNF-a) mRNA expressions in different groups. *P< .05 vs vein graft group, **P <.05 vs vein group. siRNA, small interference RNA; NF-kB, nuclear factor kappa B.
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Fig 3. The ratio of proliferating cell nuclear antigen (PCNA)-positive cells in each group. Immunohistochemistry staining, magnification 400. *P < .05 vs vein graft group, **P< .05 vs vein group. siRNA, small interference RNA; NF-kB, nuclear factor kappa B.
(P < .05, Fig 4). There were no significant differences in thickness between the vein graft group and the scrambled siRNA group. However the intimal and media thickness in the NF-kB siRNA group were less than those in the vein graft group (P < .05, Fig 4). Therefore, NF-kB siRNA transfection significantly decreased vein graft thickness (Fig 4). DISCUSSION
Long-term patency of vein grafts is mainly limited by progressive neointimal hyperplasia, VSMC proliferation, thrombogenesis, and accelerated atherosclerosis. In animal models, neointimal hyperplasia is an early event lasting as
Fig 4. The thickness of intima and media in different group. Hematoxylin and eosin staining, magnification 200. *P < .05 vs vein graft group, **P < .05 vs vein group. siRNA, small interference RNA.
long as several weeks after vein grafting.l2e14 The progression of stenosis may represent an adaptive response of the vein graft to the arterialized environment. The injury stimulus for hyperplastic responses is assumed to result from VSMC and endothelial cell expression of cytokines and growth factor genes that stimulate cell migration, proliferation, and extracellular matrix production, inevitably leading to stenosis.15e17 Inflammation is a key mediator in the pathogenesis of atherosclerosis. Various approaches have attempted to control vein graft stenosis. Several recent reports have suggested that NF-kB inhibitition may be linked to improved patency of vein grafts. Indeed, NF-kB inhibitors,
NF-kB AND VEIN GRAFT STENOSIS
such as bortezomib12 or NF-kB decoy oligonucleotides, reduce macrophage recruitment and smooth muscle cell accumulation in experimental vein grafts.18e21 In particular, gene silencing by siRNA transfection holds promise for the control of vascular disease. Various animal experiments targeting inflammatory factors in vein graft can prevent the initiation of inflammation and the process of stenosis. Several studies have suggested that NF-kB influences vein graft disease. NF-kB signaling in vein grafts leads to inflammatory gene expressions, including ICAM-1 (intercellular adhesion molecule -1), VCAM-1 (vascular cell adhesion molecule -1), TNF-a, interleukin (IL)-1, IL-6, IL-8, and MCP1, that recruit inflammatory cells to the vessel wall.22e24 In atherosclerotic lesion of the human aorta. Inflammatory cytokines activated NF-kB (p65) have been detected NF-kB overlyies early lesions, of a vein graft. Nuclear p65 and p50 have been demonstrated in VSMC of human lesions, whereas NF-kB was inactive in VSMC of healthy tissues.25e29 In our study, jugular veins were grafted to the abdominal aorta. To increase siRNA transfection efficiency, vein grafts were treated with NF-kB siRNA liposome complex by soaking and by pressure perfusion before the anastomosis. siRNA plus gel complex was applied to the outer wall of the vein graft immediately after transplanation. At 7 days the treatment drastialy reduced the expression of NF-kB p65 protein as well as the mRNA levels of MCP-1 and TNF-a. In contrast the mRNA levels of MCP-1 and TNF-a in both the untreated vein graft and the scrambled siRNA groups were higher, suggesting that inflammatory mediators are involved in the early response, playing important roles in chronic stenosis. Up-regulation of NF-kB p65 protein accompanied by elevations of TNF-a and MCP-1 mRNAs were significantly inhibited by NF-kB siRNA, but not affected by the scrambled siRNA. NF-kB siRNA transfection not only suppressed NF-kB expression effectively, but also reduced levels of inflammatory mediators to some extent. At the 14-day assessment, the number of PCNA-positive cells in vein graft vessel walls were significantly increased, a process that was inhibited by NF-kB siRNA. Consistent with the PCNA results, the intima and media thickness were less in NF-kB siRNA than the vein graft group, suggesting inhibition of VSMC proliferation with reduced neointimal hyperplasia in vivo. The inhibitory effects of NF-kB siRNA on neointimal hyperplasia likely resulted from decreased production of NF-kB p65 protein, TNF-a, and MCP-1, known mediators of vein graft stenosis. Surgical damage to the vascular wall initiates inflammatory processess, activating the NF-kB pathway which was inhibited by siRNA. All our results indicated a possible therapeutic role of NF-kB siRNA to prevent stenosis of vein bypass grafts. The increase in inflammatory mediators in vein grafts occurs within the first 7 days, peaking thereafter (Figs 1 and 2), a time course that matches and first 1 to 4 weeks to shows the onset of stenosis well detect (Figs 3 and 4). However, vein graft durability is inevitably affected by many factors. Only one gene therapy cannot completely eliminate restenosis.
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Further research must focus on the ways of NF-kB siRNA to these downstream inflammatory factors production and the signaling mechanisms that result in the stenosis. In conclusion, we demonstrated that suppression of NFkB gene expression with a specific siRNA effectively inhibited vein graft thickening through dampening neointimal hyperplasia and inflammation cascades, which may provide useful approach to prevent early stenosis and late occlusion. REFERENCES 1. Parang P, Arora R. Coronary vein graft disease: pathogenesis and prevention. Can J Cardiol. 2009;25:e57ee62. 2. Shuhaiber JH, Evans AN, Massad MG, et al. Mechanisms and future directions for prevention of vein graft failure in coronary bypass surgery. Eur J Cardiothorac Surg. 2002;22:387e396. 3. Toprak V, Sirin BH, Tok D, et al. The effect of cardiopulmonary bypass on the expression of inducible nitric oxide synthase, endothelial nitric oxide synthase, and vascular endothelial growth factor in the internal mammary artery. J Cardiothorac Vasc Anesth. 2006;20:63e67. 4. Collins T, Cybulsky MI. NF-kappaB: pivotal mediator or innocent bystander in atherogenesis? J Clin Invest. 2001;107: 255e264. 5. Corrado E, Rizzo M, Coppola G, et al. An update on the role of markers of inflammation in atherosclerosis. J Atheroscler Thromb. 2010;17:1e11. 6. Van der Heiden K, Cuhlmann S, Luong le A, et al. Role of nuclear factor kappaB in cardiovascular health and disease. Clin Sci (Lond). 2010;118:593e605. 7. de Winther MP, Kanters E, Kraal G, et al. Nuclear factor kappaB signaling in atherogenesis. Arterioscler Thromb Vasc Biol. 2005;25:904e914. 8. Shrey K, Suchit A, Nishant M, et al. RNA interference: emerging diagnostics and therapeutics tool. Biochem Biophys Res Commun. 2009;386:273e277. 9. Aagaard L, Rossi JJ. RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev. 2007;59:75e86. 10. Wang J, Liu K, Shen L, et al. Small interfering RNA to c-myc inhibits vein graft restenosis in a rat vein graft model. J Surg Res. 2011;169:e85ee91. 11. He XP, Li XX, Wang ZH, et al. Transfection of hairpin small interfering RNA expression vector targeting rat nuclear factor (NF) (kB) inhibits rat cell proliferation induced by NF-kB signal pathway activation. Transplant Proc. 2010;42:4633e4637. 12. He XP, Li XX, Bi YW, et al. The proteasome inhibitor bortezomib inhibits intimal hyperplasia of autologous vein grafting in rat model. Transplant Proc. 2008;40:1722e1726. 13. Wu J, Zhang C. Neointimal hyperplasia, vein graft remodeling, and long-term patency. Am J Physiol. 2009;297:H1194eH1195. 14. Hinokiyama K, Valen G, Tokuno S, et al. Vein graft harvesting induces inflammation and impairs vessel reactivity. Ann Thorac Surg. 2006;82:1458e1464. 15. Scott NA. Restenosis following implantation of bare metal coronary stents: pathophysiology and pathways involved in the vascular response to injury. Adv Drug Delivery Rev. 2006;58: 358e376. 16. Kwei S, Stavrakis G, Takahas M, et al. Early adaptive responses of the vascular wall during venous arterialization in mice. Am J Pathol. 2004;164:81e89. 17. Motwani JG, Topol EJ. Aortocoronary saphenous vein graft disease: pathogenesis, predisposition, and prevention. Circulation. 1998;97:916e931. 18. Shimizu N, Azuma N, Nishikawa T, et al. Effect on vein graft intimal hyperplasia of nuclear factor-kB decoy transfection using the second generation of HVJ vector. J Cardiovasc. 2007;48:463e470.
2558 19. Miyake T, Aoki M, Shiraya S, et al. Inhibitory effects of NF kappaB decoy oligodeoxynucleotides on neointimal hyperplasia in a rabbit vein graft model. J Mol Cellur Cardiol. 2006;41: 431e440. 20. Gareus R, Kotsaki E, Xanthoulea S, et al. Endothelial cellspecific NF-kappaB inhibition protects mice from atherosclerosis. Cell Metab. 2008;8:372e383. 21. Yoshimura S, Morishita R, Hayashi K, et al. Inhibition of intimal hyperplasia after balloon injury in rat carotid artery model using cis-element “decoy” of nuclear factor-kappaB binding site as a novel molecular strategy. Gene ther. 2001;8:1635e1642. 22. Monaco C, Andreakos E, Kiriakidis S, et al. Canonical pathway of nuclear factor kappa B activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis. Proc Nat Acad Sci U S A. 2004;101:5634e5639. 23. Libby P, Okamoto Y, Rocha VZ, et al. Inflammation in atherosclerosis: transition from theory to practice. Circ J. 2010;74:213e220.
MENG, BI, ZHAO ET AL 24. Pesarini G, Amoruso A, Ferrero V, et al. Cytokines release inhibition from activated monocytes, and reduction of in-stent neointimal growth in humans. Atherosclerosis. 2010;211:242e248. 25. Wilcox JN, Nelken NA, Coughlin SR, et al. Local expression of inflammatory cytokines in human atherosclerotic plaques. J Atheroscler Thromb. 1994;1(Suppl 1):S10eS13. 26. Eslami MH, Gangadharan SP, Belkin M, et al. Monocyte adhesion to human vein grafts: a marker for occult intraoperative injury? J Vasc Surg. 2001;34:923e929. 27. Libby P. Inflammation in atherosclerosis. Nature. 2002;420: 868e874. 28. Xanthoulea S, Curfs DM, Hofker MH, et al. Nuclear factor kappa B signaling in macrophage function and atherogenesis. Curr Opin Lipidol. 2005;16:536e542. 29. Wang T, Zhang X, Li JJ. The role of NF-kappaB in the regulation of cell stress responses. Int Immunopharmacol. 2002;2: 1509e1520.
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ORONARY ARTERY BYPASS GRAFTING (CABG) is an important treatment for this serious disease. Autologous veins are the most commonly used graft material for CABG, but stenosis or failure due to intimal hyperplasia and vascular smooth muscle cell (VSMC) proliferation are important long-term constraints.1e3 In recent years, many studies have revealed that inflammatory injuries play crucial roles in the progression of vein graft stenosis. As a crucial transcription factor in inflammatory responses, nuclear factor kappa B (NF-kB) controls transcription of many genes. It has an established role in atherosclerosis with p65, the key active subunit in NF-kB transcription.4e7 To prevent inflammatory injury and inhibit vein graft stenosis, efficient methods are needed to interrupt NF-kB activation. Gene silencing by RNA interference (RNAi) can induce sequence-specific degradation of homologous mRNA, interfering with gene expression.8,9 Animal experiments have shown it to be a promising solution to prevent vein graft restenosis, as well as reduce neointimal formation and atherosclerosis.10 In our previous study, we have shown that NF-kB small interference RNA (siRNA) significantly inhibits cell
proliferation, reduces p65 expression, increases the ratio of G0/G1 stage cells, and decreases the proportion of S stage cells in vitro.11 In this in vivo study, we treated vein grafts at the time of surgery with siRNA, seeking to identify its inhibitory effects on cell proliferation or graft stenosis. MATERIALS AND METHODS Construction of NF-kB p65 siRNA Expression Vector The 21-nt siRNA sequences were designed and synthesized by Ambion Company according to the complete gene sequence of the rat NF-kB p65 subunit from GeneBank. We selected an siRNA sequence targeting the rat NF-kB p65 gene for synthesis as reported
From the Department of Cardiovascular Surgery (X.B.M., X.L.B., H.L.Z., G.M.S., W.Y.S., Y.W.B.), Central Laboratory (J.B.F.), and Department of Pathology, (J.P.Z.), QiLu Hospital, Shandong University, Jinan, Shandong Province, China. Address reprint requests to Yan Wen Bi, Department of Cardiovascular Surgery, Qilu Hospital, Shandong University, 107# Wenhua Xi Road, Jinan 250012, Shandong Province, People’s Republic of China. E-mail: [email protected]
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.03.045
Transplantation Proceedings, 45, 2553e2558 (2013)
2553
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MENG, BI, ZHAO ET AL 0
by He XP et al.11 The sequence 5 -AAGCACAGATACCACTAA GAC-30 was located between 211 bp and 231 bp. The specific sequence was BLAST (basic local alignment search tool) searched against the expressed sequence tags rat libraries to ensure that it was not homologous to others. Furthermore, we synthesized 2 complementary oligodeoxyribonucleotides of specific short hairpin RNA (shRNA), which encoded the 21-bp designed sequence, and annealed them to form a double-stranded RNA, which was ligated into the linearized plasmid pGenesil-1.2 with an mU6 promoter, in order to construct a recombinant plasmid expressing NF-kB p65 siRNA in eukaryotic cytoplasm after transfectiond“pGenesil1.2-p65siRNA.” Meanwhile, we constructed a negative control plasmid “scrambled siRNA.” The GC content of p65 sequences was 42.9%. The multiple cloning site of plasmid pGenesil-1.2 was EGFP (enhanced green fluorescence protein)-SacI-CMV (cytomegalovirus) promoter-MLUI-HINDIII-SACI-shRNAmU6 promoter. Enzyme analysis and DNA sequencing confirmed that the inserted sequences has been successfully cloned in the vector.
examinations. We detected NF-kB p65 protein levels in vein grafts that has been stored in liquid nitrogen.
Western Blot Analysis At the 7-day assessment, vein graft tissues were subjected to Western blot analysis to detect NF-kB p65 protein levels. The whole graft was snap-frozen and ground into a powder. Portion of vein graft tissues (20 mg) were treated with lysis buffer. Equal amounts of protein dissolved in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) sample buffer and electrophoresed were transferred onto polyvinylidene fluoride membranes. The membranes were incubated with the respective primary antibodies at 4 C overnight before incubation with the secondary antibody for 60 minutes at room temperature with continuous shaking. In all cases, b-actin was used as the internal reference. Membranes were exposed to X-ray film to visualize the bands. We quantified the intensities of p65 protein and b-actin bands.
Histologic and Immunohistochemistry Analysis Vein Graft Model and Experimental Groups Adult male Sprague-Dawley rats (weight, 300e350 g) obtained from our Animal Research Center were initially housed in a temperature (21 Ce26 C) and 12-hour cycled, light-controlled rooms with free access to food and water. Briefly, animals anesthetized with pentobarbital sodium (60 mg/kg) by intraperitoneal injection were systemically heparinized (200 U/kg). A microsurgical technique was used to dissect a 15-mm segment of the right external jugular vein through a midline vertical neck incision. All branches were carefully divided and ligated with 8-0 polypropylene sutures. A 10-mm segment of the abdominal aorta was denuded and resected, before the divided ends were irrigated with heparinized saline solution. An autologous external jugular veinetoeabdominal aorta reverse interposition was performed in rats as previously described12 using a cuff technique. The arterial occlusion time ranged from 5 to 10 minutes. We confirmed immediate restoration of blood flow upon removal of the arterial occlusion clamps. Sixty rats were divided into 4 groups. Normal vein group (vein, n ¼ 15), vein grafted to abdominal aorta with no treatment group (vein graft, n ¼ 15), scrambled siRNA group (n ¼ 15), with grafted vein treated with Lipofectamine 2000 (Invitrogen) and scrambled siRNA complex for 10 minutes before the arteriovenous anastomosis. Poluronic F-127 gel (Sigma) and scrambled siRNA complex were applied to the vein graft surface immediately after the anastomosis. The final NF-kB siRNA group (n ¼ 15) had vein grafts treated with Lipofectamine 2000 and p65 siRNA complex for 10 minutes before the anastomosis with p65 siRNA and gel complex applied to the graft surface.
siRNA in vivo Transfection and Tissue Preparation To increase transfection efficiency, we use 2 methods to apply siRNA to the vein graft. First, liposome and siRNA complex were perfused into vein graft under pressure (20 mm Hg). Second, the siRNA-gel complex was applied to the graft surface, allowing longstanding suppression of the target gene. There delivery systems can provide sufficient suppressive effects on gene expression. Rats in each group were humanely killed under heavy anesthesia and vein grafts harvested after the surgery. The proximal portion of the vein grafts was harvested to be fixed in 4% paraformaldehyde or stored in liquid nitrogen for research. At the 7-day assessment, vein graft tissues were examined by polymerase chain reaction (PCR) and Western blot analysis. At the 14-day assessment, vein graft tissues were subjected to histologic and immunohistochemical
Tissue segment of vein grafts harvested at 14 days were fixed in formaldehyde (4%) and embedded in paraffin. To quantify graft thickness, we did not distinguish intima and media because there was no morphologic border. Neointimal thickness was measured at 6 randomly selected points for each section. Image analysis software was used to quantify vein graft thickness. Immunohistochemical examination was performed using the avidin-biotin peroxidase complex method to detect proliferating cell nuclear antigen (antiPCNA, 1:100, DAKO). The PCNA proliferation index was determined by dividing the number of positively stained nuclei by the total number of nuclei in the intima and media of each section. The average index was calculated for each vessel to determine the intimal thickness and proliferation index.
Analysis of Inflammatory Mediators Expressions of MCP-1 and tumor necrosis factor-a (TNF-a) inflammatory mediators in vein grafts were quantified by PCR (polymerase chain reaction). Specific primer sequences (MCP-1, TNF-a, and b-actin) obtained from Genbank were synthesized by the Jinan BoYa Biological Engineering Company (China). Total RNA was isolated with the RISOTM RNA Isolation Reagent (Biomics, USA) as described in the manufacture’s instructions. The RNA samples were normalized using the housekeeping gene b-actin. The primer sequences were: MCP-l (forward 50 -ATGCAGGTCTCTGTCACG CT-30 ; reverse 50 -GGTGCTGAAGTCCTTAGGGT-30 ). TNF-a (forward 50 -CTTATCTACTCCCAGGTTCTCTCAA-30 ; reverse 50 -GAG ACTCCTCCCAGGTACATGG-30 ). b-actin (forward 50 -AGACCTT CAACACCCCAG-30 ; reverse 50 -CACGATTTCCCTCTCAGC-30 ). RNA extracted from tissues with TRIzol (Invitrogen) was reverse transcribed to cDNA. Using specific primers, amplication was perfomed in the ABI Prism 7500 Real-Time PCR System (Applied Biosystems). The 40-cycle reaction consitions were 60 minutes at 42 C, 10 seconds at 95 C, 10 seconds at 55 C, and 35 seconds at 72 C. All experiments were performed in triplicate. The relative mRNA expressions of target genes were calculated and normalized by the comparative CT method.
Statistical Analysis Results are expressed as mean values standard deviations. Statistical significance was determined using one-way analysis of variance. The least significant difference test or Dunnett T3 test was used to compare 2 groups. SPSS 13.0 was used for statistical analyses. P value less than .05 were considered to be statistically significant.
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group, transfection with scrambled siRNA did not change p65 protein levels (P > .05), NF-kB siRNA significantly suppressed p65 protein expression (P < .05, Fig 1). The mRNA Expressions of TNF-a and MCP-1 in Vein Graft
QT-PCR was used to analyze TNF-a and MCP-1 mRNA expressions in grafts during thickening and accelerated atherosclerosis. At 7 days, the relative mRNA levels of TNF-a and MCP-1 expressions were higher and peaked in both the vein graft and the scrambled siRNA groups (P < .05, Fig 2). However, the mRNA levels of TNF-a and MCP-1 in the NF-kB siRNA group were less (P < .05, Fig 2). Vein Graft Proliferative Activity
Fig 1. The relative levels of nuclear factor kappa B (NF-kB) p65 protein expressions in each group *P < .05 vs vein group, **P < .05 vs vein group. siRNA, small interference RNA.
RESULTS NF-kB p65 Protein Expression of Vein Graft
Quantitative analysis showed that NF-kB p65 protein expression was significantly increased in the vein graft relative to the vein group (P < .05, Fig 1), indicating activation of the NF-kB pathway. Compared with the vein graft
PCNA reflects proliferative activity. The normal vein showed no evidence of PCNA-positive cells. Many of PCNA positive cells were evident in the intima and media of the vein grafts at 14 days after surgery group (P < .05). The scrambled siRNA group showed a similar result as the vein graft group. The ratio of PCNA-positive cells in the NF-kB siRNA group was lower than that in the vein graft group (P < .05, Fig 3). Therefore, NF-kB p65 siRNA treatment caused less proliferative activity vascular in the wall (Fig 3). Vein Graft Neointimal Hyperplasia
Histologic analysis evaluated the proliferation and thickness of the intima and media. At 14 days postsurgery, vein graft showed obvious neointima hyperplasia with significantly thicker intima and media both among the untreated and the scrambled siRNA compared with the in situ vein group
Fig 2. The relative levels of MCP-1 mRNA and tumor necrosis factor-a (TNF-a) mRNA expressions in different groups. *P< .05 vs vein graft group, **P <.05 vs vein group. siRNA, small interference RNA; NF-kB, nuclear factor kappa B.
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Fig 3. The ratio of proliferating cell nuclear antigen (PCNA)-positive cells in each group. Immunohistochemistry staining, magnification 400. *P < .05 vs vein graft group, **P< .05 vs vein group. siRNA, small interference RNA; NF-kB, nuclear factor kappa B.
(P < .05, Fig 4). There were no significant differences in thickness between the vein graft group and the scrambled siRNA group. However the intimal and media thickness in the NF-kB siRNA group were less than those in the vein graft group (P < .05, Fig 4). Therefore, NF-kB siRNA transfection significantly decreased vein graft thickness (Fig 4). DISCUSSION
Long-term patency of vein grafts is mainly limited by progressive neointimal hyperplasia, VSMC proliferation, thrombogenesis, and accelerated atherosclerosis. In animal models, neointimal hyperplasia is an early event lasting as
Fig 4. The thickness of intima and media in different group. Hematoxylin and eosin staining, magnification 200. *P < .05 vs vein graft group, **P < .05 vs vein group. siRNA, small interference RNA.
long as several weeks after vein grafting.l2e14 The progression of stenosis may represent an adaptive response of the vein graft to the arterialized environment. The injury stimulus for hyperplastic responses is assumed to result from VSMC and endothelial cell expression of cytokines and growth factor genes that stimulate cell migration, proliferation, and extracellular matrix production, inevitably leading to stenosis.15e17 Inflammation is a key mediator in the pathogenesis of atherosclerosis. Various approaches have attempted to control vein graft stenosis. Several recent reports have suggested that NF-kB inhibitition may be linked to improved patency of vein grafts. Indeed, NF-kB inhibitors,
NF-kB AND VEIN GRAFT STENOSIS
such as bortezomib12 or NF-kB decoy oligonucleotides, reduce macrophage recruitment and smooth muscle cell accumulation in experimental vein grafts.18e21 In particular, gene silencing by siRNA transfection holds promise for the control of vascular disease. Various animal experiments targeting inflammatory factors in vein graft can prevent the initiation of inflammation and the process of stenosis. Several studies have suggested that NF-kB influences vein graft disease. NF-kB signaling in vein grafts leads to inflammatory gene expressions, including ICAM-1 (intercellular adhesion molecule -1), VCAM-1 (vascular cell adhesion molecule -1), TNF-a, interleukin (IL)-1, IL-6, IL-8, and MCP1, that recruit inflammatory cells to the vessel wall.22e24 In atherosclerotic lesion of the human aorta. Inflammatory cytokines activated NF-kB (p65) have been detected NF-kB overlyies early lesions, of a vein graft. Nuclear p65 and p50 have been demonstrated in VSMC of human lesions, whereas NF-kB was inactive in VSMC of healthy tissues.25e29 In our study, jugular veins were grafted to the abdominal aorta. To increase siRNA transfection efficiency, vein grafts were treated with NF-kB siRNA liposome complex by soaking and by pressure perfusion before the anastomosis. siRNA plus gel complex was applied to the outer wall of the vein graft immediately after transplanation. At 7 days the treatment drastialy reduced the expression of NF-kB p65 protein as well as the mRNA levels of MCP-1 and TNF-a. In contrast the mRNA levels of MCP-1 and TNF-a in both the untreated vein graft and the scrambled siRNA groups were higher, suggesting that inflammatory mediators are involved in the early response, playing important roles in chronic stenosis. Up-regulation of NF-kB p65 protein accompanied by elevations of TNF-a and MCP-1 mRNAs were significantly inhibited by NF-kB siRNA, but not affected by the scrambled siRNA. NF-kB siRNA transfection not only suppressed NF-kB expression effectively, but also reduced levels of inflammatory mediators to some extent. At the 14-day assessment, the number of PCNA-positive cells in vein graft vessel walls were significantly increased, a process that was inhibited by NF-kB siRNA. Consistent with the PCNA results, the intima and media thickness were less in NF-kB siRNA than the vein graft group, suggesting inhibition of VSMC proliferation with reduced neointimal hyperplasia in vivo. The inhibitory effects of NF-kB siRNA on neointimal hyperplasia likely resulted from decreased production of NF-kB p65 protein, TNF-a, and MCP-1, known mediators of vein graft stenosis. Surgical damage to the vascular wall initiates inflammatory processess, activating the NF-kB pathway which was inhibited by siRNA. All our results indicated a possible therapeutic role of NF-kB siRNA to prevent stenosis of vein bypass grafts. The increase in inflammatory mediators in vein grafts occurs within the first 7 days, peaking thereafter (Figs 1 and 2), a time course that matches and first 1 to 4 weeks to shows the onset of stenosis well detect (Figs 3 and 4). However, vein graft durability is inevitably affected by many factors. Only one gene therapy cannot completely eliminate restenosis.
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Further research must focus on the ways of NF-kB siRNA to these downstream inflammatory factors production and the signaling mechanisms that result in the stenosis. In conclusion, we demonstrated that suppression of NFkB gene expression with a specific siRNA effectively inhibited vein graft thickening through dampening neointimal hyperplasia and inflammation cascades, which may provide useful approach to prevent early stenosis and late occlusion. REFERENCES 1. Parang P, Arora R. Coronary vein graft disease: pathogenesis and prevention. Can J Cardiol. 2009;25:e57ee62. 2. Shuhaiber JH, Evans AN, Massad MG, et al. Mechanisms and future directions for prevention of vein graft failure in coronary bypass surgery. Eur J Cardiothorac Surg. 2002;22:387e396. 3. Toprak V, Sirin BH, Tok D, et al. The effect of cardiopulmonary bypass on the expression of inducible nitric oxide synthase, endothelial nitric oxide synthase, and vascular endothelial growth factor in the internal mammary artery. J Cardiothorac Vasc Anesth. 2006;20:63e67. 4. Collins T, Cybulsky MI. NF-kappaB: pivotal mediator or innocent bystander in atherogenesis? J Clin Invest. 2001;107: 255e264. 5. Corrado E, Rizzo M, Coppola G, et al. An update on the role of markers of inflammation in atherosclerosis. J Atheroscler Thromb. 2010;17:1e11. 6. Van der Heiden K, Cuhlmann S, Luong le A, et al. Role of nuclear factor kappaB in cardiovascular health and disease. Clin Sci (Lond). 2010;118:593e605. 7. de Winther MP, Kanters E, Kraal G, et al. Nuclear factor kappaB signaling in atherogenesis. Arterioscler Thromb Vasc Biol. 2005;25:904e914. 8. Shrey K, Suchit A, Nishant M, et al. RNA interference: emerging diagnostics and therapeutics tool. Biochem Biophys Res Commun. 2009;386:273e277. 9. Aagaard L, Rossi JJ. RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev. 2007;59:75e86. 10. Wang J, Liu K, Shen L, et al. Small interfering RNA to c-myc inhibits vein graft restenosis in a rat vein graft model. J Surg Res. 2011;169:e85ee91. 11. He XP, Li XX, Wang ZH, et al. Transfection of hairpin small interfering RNA expression vector targeting rat nuclear factor (NF) (kB) inhibits rat cell proliferation induced by NF-kB signal pathway activation. Transplant Proc. 2010;42:4633e4637. 12. He XP, Li XX, Bi YW, et al. The proteasome inhibitor bortezomib inhibits intimal hyperplasia of autologous vein grafting in rat model. Transplant Proc. 2008;40:1722e1726. 13. Wu J, Zhang C. Neointimal hyperplasia, vein graft remodeling, and long-term patency. Am J Physiol. 2009;297:H1194eH1195. 14. Hinokiyama K, Valen G, Tokuno S, et al. Vein graft harvesting induces inflammation and impairs vessel reactivity. Ann Thorac Surg. 2006;82:1458e1464. 15. Scott NA. Restenosis following implantation of bare metal coronary stents: pathophysiology and pathways involved in the vascular response to injury. Adv Drug Delivery Rev. 2006;58: 358e376. 16. Kwei S, Stavrakis G, Takahas M, et al. Early adaptive responses of the vascular wall during venous arterialization in mice. Am J Pathol. 2004;164:81e89. 17. Motwani JG, Topol EJ. Aortocoronary saphenous vein graft disease: pathogenesis, predisposition, and prevention. Circulation. 1998;97:916e931. 18. Shimizu N, Azuma N, Nishikawa T, et al. Effect on vein graft intimal hyperplasia of nuclear factor-kB decoy transfection using the second generation of HVJ vector. J Cardiovasc. 2007;48:463e470.
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