Stent-Based Local Delivery of Nuclear Factor-κB Decoy Attenuates In-Stent Restenosis in Hypercholesterolemic Rabbits
Background— Nuclear factor-κB (NF-κB) plays a critical role in the vascular response to injury. However, the role of NF-κB in the mechanism of in-stent restenosis remains unclear. We therefore tested the hypothesis that blockade of NF-κB by stent-based delivery of a cis-element “decoy” of NF-κB reduces in-stent neointimal formation.
Methods and Results— Stents were coated with a polymer containing or not containing NF-κB decoy, which represented a fast-release formulation (<7 days). Bare, polymer-coated, and NF-κB decoy–eluting stents were implanted in iliac arteries of hypercholesterolemic rabbits. Increased NF-κB activity was noted at early stages after stenting, which was suppressed by stent-based delivery of NF-κB decoy. NF-κB decoy–eluting stents also reduced monocyte infiltration and monocyte chemoattractant protein-1 expression and suppressed CD14 activation on circulating leukocytes. Importantly, NF-κB decoy–eluting stents attenuated neointimal formation on day 28. There was no evidence of an incomplete healing process (persistent inflammation, hemorrhage, fibrin deposition, impaired endothelial regeneration) at the site of NF-κB decoy–eluting stents. Transfection of NF-κB decoy suppressed proliferation of human coronary artery smooth muscle cells in vitro. No systemic adverse effects of NF-κB decoy were detected.
Conclusions— Stent-based local delivery of NF-κB decoy reduced in-stent neointimal formation with no evidence of incomplete healing. These data suggest that this strategy may be a practical and promising means for prevention of in-stent restenosis in humans.
Received August 11, 2005; revision received October 10, 2006; accepted October 13, 2006.
Each year, >1.5 million patients worldwide undergo percutaneous coronary intervention for atherothrombotic lesions. Local drug delivery by drug-eluting stents is now becoming a useful strategy for prevention of restenosis because of promising results in animal studies and clinical trials.1,2 Currently marketed first-generation drug-eluting stents use antiproliferative drugs including rapamycin, its analogues, or paclitaxel. The current antiproliferative strategies are no longer a panacea, however, because this strategy involves potential problems such as impaired endothelial regeneration and an incomplete healing process (excessive inflammation and fibrin deposition) associated with increased risk of stent thrombosis.3–5 Lack of long-term effects of sirolimus-eluting stents due to delayed inflammation and proliferation has been reported in a porcine coronary model.6
Clinical Perspective p 2779
Recent experimental and clinical studies suggest that inhibition of stent-associated inflammation (monocyte recruitment and activation) can be a promising next-generation approach.7–9 Nuclear factor-κB (NF-κB) is a redox-sensitive transcription factor that regulates inflammation and thus plays a critical role in the vascular response to injury.10 Activated NF-κB is detected in human atherosclerotic and restenotic lesions of smooth muscle cells, monocytes, and endothelial cells.11 In contrast, activated NF-κB is rarely detected in normal uninjured arteries. After vascular injury, rapid activation of NF-κB in smooth muscle cells correlates with proliferation of smooth muscle cells and induced expression of NF-κB–dependent genes.12 Recently, blockade of NF-κB by transfection of adenoviral inhibitor-κB or NF-κB “decoy” oligodeoxynucleotides attenuated restenotic changes (neointimal formation) after balloon injury in animal models associated with reduced NF-κB–dependent genes like monocyte chemoattractant protein-1 (MCP-1).13,14 As a clinically feasible technique, however, these gene transfer approaches are usually hampered by prolonged arterial occlusions. It has not yet been directly determined whether blockade of NF-κB inhibits neointima formation after stenting. This is important because the mechanisms underlying neointima formation differ between balloon injury and stent-induced injury, and stenting is the most frequently performed vascular interventional technique.
We therefore created the NF-κB decoy–eluting stent using polymer technology that facilitates local delivery of the NF-κB decoy oligodeoxynucleotide during stent expansion by balloon dilatation. This decoy-eluting stent strategy is a clinically feasible approach. Although local gene delivery from a polymeric plasmid DNA-coated stent has been reported, local vascular transfer of decoy oligodeoxynucleotide by polymeric-coated stent has not been reported thus far. We herein report inhibition of in-stent neointimal formation by stent-based local transfer of NF-κB decoy in vivo.
The aims of this study were (1) to create a NF-κB decoy–eluting metallic stent by the use of water-soluble polymer; (2) to evaluate the in vivo blockade of NF-κB activation by NF-κB decoy–eluting stent implantations; and (3) to determine whether the NF-κB decoy–eluting stent attenuates stent-associated inflammation and neointimal formation in vivo.
The NF-κB decoy sequences are 5′-CCTTGAAGGGA-TTTCCCTCC-3′ and 3′-GGAACTTCCCTAAAGGGAGG-5′. GGGATTTCCC is the consensus sequence for the NF-κB binding site. The decoy is directed against the NF-κB binding site in the promoter region that corresponds to NF-κB–responsive genes.15,16 The decoy works to inhibit binding of this transcription factor to the promoter region.15,16 The NF-κB decoy oligodeoxynucleotides have been shown to bind to free NF-κB, preventing NF-κB transactivation of the cytokine genes. Because NF-κB is activated immediately after stenting, we designed the NF-κB decoy–eluting stent as an early-release formulation. The 15-mm-long stainless steel balloon-expandable stent (Kawasumi Co, Osaka, Japan) was dip-coated with multiple thin layers of polyurethane containing or not containing NF-κB decoy (500 to 600 μg per stent) under sterile conditions. Another layer of decoy-free polyurethane was applied on top of the decoy-polyurethane matrix. We selected polyurethane as a polymer matrix material for stent coating because (1) it is water soluble and therefore stably absorbs NF-κB decoy oligodeoxynucleotides and (2) metallic stents coated with polyurethane containing DNA are reported to be useful for transgene delivery to the iliac arterial wall of rabbits.17 In addition, of a number of polymer matrix materials evaluated for stent coating, polyurethane has been shown to prevent the thrombosis and inflammation that can occur with uncoated stents and some polymers used for stent coating.18
The coated stent was then mounted over a 3-mm balloon catheter. Uncoated bare stent mounted over the same balloon catheter was used as a control. Before implantation, all stents were sterilized with the use of ethylene oxide.
In Vitro Kinetics
In vitro kinetics studies were performed by placing a NF-κB decoy–coated stent in Tris-EDTA buffer at 37°C. The stent was periodically removed from the buffer, and the decoy eluted into the buffer was measured by a high-performance liquid chromatography system. The incremental quantities of the decoy released from the stent were plotted against time (n=8).
Stent Implantation in Animal Models
The experiments were reviewed and approved by the Committee on Ethics on Animal Experiments, Kyushu University Faculty of Medicine, and were performed according to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Male Japanese white rabbits (KBT Oriental, Tokyo, Japan) weighing 3.0 to 3.5 kg were fed a high-cholesterol diet containing 1% cholesterol and 3% peanut oil for 2 weeks before stent implantation. Animals were anesthetized and were divided randomly into 3 groups, which underwent deployment of either an uncoated bare metal stent (n=22); decoy-free polyurethane-coated stent (n=22); or NF-κB decoy–coated stent (n=22) in the right femoral artery as described previously.9 All animals received aspirin at 20 mg/d until euthanasia from 3 days before the stent implantation procedure. After venous blood samples were taken, animals were killed with a lethal dose of anesthesia at days 3 (n=6 each), 10 (n=8 each), and 28 (n=8 each). Stented arterial sites and contralateral unstented sites were excised for biochemical, immunohistochemical, and morphometric analyses.
Histopathological and Immunohistochemical Analysis
The stented artery segments were processed as described previously.9 The segment was divided into 2 parts at the center of the stent. The proximal part was embedded in methyl methacrylate mixed with n-butyl methacrylate to allow for sectioning through metal stent struts. Serial sections were stained with elastica van Gieson and hematoxylin-eosin. Neointimal area, the area within the internal elastic lamina and external elastic lamina, and the lumen area were measured by computerized morphometry. A single observer who was blinded to the experiment protocol performed morphometry. All images were captured by an Olympus microscope equipped with a digital camera (HC-2500) and were analyzed with the use of Adobe Photoshop 6.0 and Scion Image 1.62 Software.
The injury and inflammatory scores were determined at each strut site, and mean values were calculated for each stented segment as previously described19,20 (see Table I in the online-only Data Supplement for details).
The distal part was used for immunohistochemical analysis. After stent struts were removed gently with microforceps, the tissue was dehydrated, embedded in paraffin, and cut into 5-μm-thick slices. They were subjected to immunostaining with antibodies against rabbit monocytes/macrophages (RAM-11; Dako, Glostrup, Denmark), endothelial cells (CD31; Dako), an epitope (α-p65) on the p65 subunit of NF-κB (α-p65; Boehringer Mannheim, Roche Diagnostics, Basel, Switzerland), MCP-1 (a gift from Dr Matsukawa, Kumamoto University), or nonimmune mouse IgG (Dako). The α-p65 monoclonal antibody recognizes an epitope on the p65 subunit that is masked by bound I-κB.11 Therefore, this antibody exclusively detects activated NF-κB. For quantification of immunohistochemical images, care was taken to select stented sites with minor injury in the neointima induced by detachment of stent strut. Because this process of selecting sections with the least injury may introduce bias, at least 5 representative images were selected, and the percentage of immunopositive cells per total cells in each image was calculated. The average of the 5 images was reported for each animal.
Electrophoretic Mobility Shift Assay
The electrophoretic mobility gel shift assay was performed on nuclear extracts prepared immediately from rabbit femoral arteries after stent implantation with the method described previously.21 For competition studies, a 50-fold molar excess of unlabeled probe for NF-κB was added. For supershift assays, 1 μg anti-p50 or anti-p65 (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) antibodies was added and incubated for 20 minutes. Nuclear extracts of HeLa cells were used as positive control.
Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction
Real-time polymerase chain reaction amplification was performed with the rabbit cDNA with the use of the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, Calif) as described previously.22 The respective polymerase chain reaction primers and TaqMan probes were designed from GenBank databases with a software program (Applied Biosystems; Table II in the online-only Data Supplement).
Fluorescence-Activated Cell Sorting
Peripheral blood was obtained at day 10 after stent implantation (n=7 each). Flow cytometry for CD14+ cells was performed with the use of R-phycoerythrin–conjugated anti-CD14 (Dako). Data were analyzed by a flow cytometer and software (Becton, Dickinson and Co, Franklin Lakes, NJ).
Blood Cholesterol Measurements
Plasma total cholesterol levels were determined with commercially available kits (Wako Pure Chemical Industries, Ltd, Osaka, Japan).
Human Coronary Artery Smooth Muscle Cell Culture
This section is available in the online-only Data Supplement.
Potential Systemic Adverse Effects or Toxicity
To examine systemic adverse effects, biochemical markers were measured before and after implantation of the NF-κB decoy–eluting stent in rabbits (n=7). Five 5-year-old male cynomolgus monkeys weighing 4.2 to 5.0 kg were purchased and fed a normal diet (n=5). Biochemical markers were measured before and after intravenous injection of NF-κB decoy at 1 mg.
Data are expressed as mean±SD. Statistical analysis of differences between the 2 groups was performed by unpaired t test. Statistical analysis of differences among the 3 groups was performed with the use of ANOVA and Bonferroni multiple comparison tests. A level of P<0.05 was considered statistically significant.
The authors had full access to the data and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Eluting Stent and In Vitro Release Kinetics
Scanning electron microscopy analysis revealed that the polymer coating formed a uniform film over the outer surface of the stent (Figure 1A). After balloon expansion, stretching of the polymer with no fragmentation was observed (Figure 1A). In vitro release kinetics showed an early burst release of NF-κB decoy as designed (Figure 1B).
Early Activation of NF-κB After Stenting and Effects of NF-κB Decoy–Eluting Stents
Time course and localization of NF-κB activation were examined by immunohistochemical studies with the antibody against α-p65. This antibody recognizes the I-κB binding region on the p65 component of NF-κB.11 In the unstented artery, no positive cells for α-p65 were noted in the media and adventitia, whereas there were some positive cells in the endothelial layer (Figure 2A). On day 3, activation of NF-κB was noted in the smooth muscle cells in the media. On day 10, activation of NF-κB decreased markedly in the media, but it was noted in neointimal cells of the luminal side. On day 28, NF-κB activation was rarely noted in the media and neointima, but it was noted in cells around the stent strut.
The effects of NF-κB decoy–eluting stent on NF-κB activation were examined on day 3 (Figure 2B and 2C). As expected, compared with the uncoated stent site, the number of α-p65–positive cells in the media was less (P<0.01) in the NF-κB decoy–eluting stent site.
To confirm immunohistochemical data, an electrophoretic mobility shift assay was performed (Figure 3). No DNA binding activity of NF-κB was noted in samples from unstented arteries. In contrast, the binding activity increased strikingly in samples from the uncoated stent site, which peaked on day 1 and gradually decreased on days 3 and 7. This NF-κB binding activity was attenuated in samples from the NF-κB–eluting stent site. Competition for increased binding of NF-κB was observed by an excess amount of NF-κB. A diminution of main band with supershifted band was observed in samples from uncoated stent sites treated with the p65 antibody but not with the p50 antibody.
Inhibitory Effects of NF-κB Decoy–Eluting Stent on Neointimal Formation
The in-stent neointima was formed equally in the uncoated stent and polyurethane-coated stent sites. Quantitative analysis demonstrated a significant reduction (P<0.01) of neointimal formation (neointimal area and thickness) and percent stenosis in the NF-κB decoy–eluting stent site compared with the other 2 sites (Figure 4). In contrast, there were no significant differences in internal elastic lamina area, external elastic lamina area, and medial area among the 3 groups.
A semiquantitative histological scoring system demonstrated that there was no significant difference in the injury score and inflammation score among the 3 groups (Table I in the online-only Data Supplement). Endothelial cell linings, monitored by CD31 immunoreactivity, were observed equally in the 3 groups (Table I in the online-only Data Supplement). There was no significant treatment effect on serum cholesterol levels and body weight among the groups (data not shown).
Inhibitory Effects of NF-κB Decoy–Eluting Stents on Local and Systemic Inflammatory Changes
As we previously reported,9 infiltration of RAM-11–positive macrophages around stent strut was observed at 10 days after stent implantation (Figure 5A). NF-κB decoy–eluting stents reduced such inflammatory changes (Figure 5B).
CD14 expression on circulating monocytes was examined by flow cytometry for CD14+ cells as a systemic inflammation marker. Maximum fluorescence intensity of CD14 on circulating monocytes increased (P<0.01) 10 days after uncoated stent implantation compared with unstented controls. No increase in CD14 expression on monocytes was observed in animals implanted with NF-κB decoy–eluting stent (Figure 5C).
Inhibitory Effects of NF-κB Decoy–Eluting Stents on Expression of Proinflammatory Factors
NF-κB decoy–eluting stents reduced the increased (P<0.01) gene expression of MCP-1, interleukin-6, tumor necrosis factor-α, and tissue factor (Figure 6A). NF-κB decoy–eluting stents did not affect increased gene expression of interleukin-1β and vascular cell adhesion molecule-1. Immunohistochemical staining performed 10 days after stenting revealed increased immunoreactive MCP-1 in cells in the neointima and smooth muscle cells in the media, which was attenuated (P<0.01) in the NF-κB decoy–eluting stent group (Figure 6B).
Blockade of NF-κB Inhibits Proliferation of Human Coronary Artery Smooth Muscle Cells
The serum-induced proliferation of human coronary artery smooth muscle cells was nearly prevented (P<0.01) by the adenovirus-mediated gene transfer of dominant-active I-κB or by transfection of NF-κB decoy (Figure in the online-only Data Supplement).
No Adverse Systemic Effects of NF-κB Decoy
Biochemical markers were measured as described in the online-only Data Supplement. These data show that no systemic adverse effects of NF-κB decoy were noted in rabbits or monkeys.
The present study reports, for the first time, the formulation of a stent-based delivery system of the NF-κB decoy oligodeoxynucleotide. A water-soluble polymer (polyurethane) was used to create a rapid-release type because NF-κB was found to be activated only at early stages but not at later stages after stenting. The present study clearly showed early activation of NF-κB after uncoated bare stenting and its inhibition by stent-based local delivery of NF-κB decoy in 2 approaches (immunostaining of a specific marker of NF-κB activation and DNA-finding assay). The inhibition of NF-κB activation was associated with reduced inflammatory changes such as reduced CD14 expression on circulating leukocytes as well as monocyte recruitment into stent sites. Although multiple factors are involved in the mechanism of decoy transfection, the mechanical force during stent expansion by the balloon dilatation procedure is likely to be a major contributing factor. The decoy might be transfected into medial and neointimal smooth muscle cells, which in turn reduced expression of various NF-κB–dependent inflammation-promoting factors. This polymeric technology-driven delivery system could be used for delivery of any other potential candidates of decoy oligodeoxynucleotides.
It has been reported that prolonged inflammatory changes were detected in arteries exposed to polymeric stent-coating materials in experimental animals18,23 and humans.3–5 However, no such adverse reaction was noted in this study. In addition, there was no evidence of an impaired healing process and endothelial regeneration at sites of stents coated with polyurethane alone and polyurethane plus decoy. These data suggest that the polymers used in this study may not cause an adverse reaction during a 4-week observation period.
The most important finding of the present study was inhibition of neointimal formation by stent-based delivery of NF-κB decoy. The beneficial effects of NF-κB decoy–eluting stents were associated with reduced gene expression of NF-κB–dependent genes (eg, MCP-1, interleukin-1β, interleukin-6) and with no change in NF-κB–independent genes (platelet-derived growth factor) (Figure 6). Immunoreactive MCP-1 expression was also reduced at sites of NF-κB decoy–eluting stent. These data indicate a specific function of the NF-κB decoy–eluting stent on local NF-κB activation. It is known that injury-induced inflammatory and proliferative changes are critical in restenotic changes after vascular injury.8,24,25 We and others have reported that (1) increased monocyte-mediated inflammation correlates positively with in-stent neointimal formation7,26 and (2) blockade of MCP-1 reduces neointimal formation after vascular injury.9,27,28 Because the NF-κB–eluting stent reduced inflammation and MCP-1 expression in this study, the beneficial effects of NF-κB decoy–eluting stents can be attributable at least in part to inhibition of MCP-1–related inflammation resulting from reduced NF-κB activation. Otherwise, emerging evidence suggests that NF-κB regulates proliferation of vascular smooth muscle cells.29,30 In this regard, we found that blockade of NF-κB activation by transfection of NF-κB decoy or dominant-active I-κB suppressed proliferation of human coronary artery smooth muscle cells in vitro. Therefore, it is also likely that NF-κB decoy–eluting stents might inhibit proliferation of vascular smooth muscle cells induced by NF-κB activation.
There are several caveats in our present findings in regard to potential clinical applicability. First, application of the present findings to treatment of restenosis in humans could be limited because the ideal animal model for drug-eluting stent evaluation is uncertain according to the recommendation from the consensus group.31 They stated that the coronary arteries in pigs and iliac-femoral arteries of rabbits are suitable in that their size, access, and injury response are similar to those of human vessels and therefore they allow examination of devices that might be used in clinical evaluation. Thus, the rabbit peripheral artery model is considered an acceptable model of choice. Second, the observed efficacy and safety of NF-κB decoy and polymer at 28 days may be too short. Third, potential adverse effects or toxicity of NF-κB decoy may be important. In histopathological analysis, no adverse reactions such as incomplete healing or impaired endothelial regeneration were noted. Measurements of serum blood markers (glucose, aspartate aminotransferase, alanine aminotransferase, creatine kinase, γ-GTP, and C-reactive protein in Tables III and IV in the online-only Data Supplement) showed no systemic adverse effects. Because the dose of NF-κB decoy (500 to 600 μg per body) coated on the stent was very low from a toxicological point of view, our decoy-coated stent may not cause any toxicity in vivo. It has been reported that repeated bolus administration of high doses (eg, 10 mg/kg every other day for 28 days in monkeys, 20 mg/kg every other day for 28 days in mice) causes kidney damage.32 In addition, we recently completed a clinical trial to test the feasibility and safety of NF-κB decoy in which NF-κB decoy at doses of 1000, 2000, or 4000 μg per body was transfected into the stented coronary artery sites via a channel balloon catheter immediately after successful percutaneous coronary intervention in 16 patients with flow-limiting coronary stenosis. The initial 2 cases have been reported,33 and they showed no evidence of restenosis or systemic adverse effects during the 6-month observation period. Overall, these data support the notion that this NF-κB decoy–eluting stent system can be applied to the clinical setting.
In conclusion, the present study supports the experimental evidence that stent-based local delivery of NF-κB decoy reduces in-stent neointimal formation by inhibiting NF-κB–dependent gene expression and inflammation and perhaps by inhibiting proliferation of vascular smooth muscle cells. Inhibition of stent-associated inflammation by the NF-κB decoy–eluting stent may be a promising next-generation approach for the prevention of restenosis. Further preclinical studies and clinical trials are needed to prove this hypothesis.
Sources of Funding
This study was supported by grants-in-aid for scientific research (14657172, 14207036) from the Ministry of Education, Science, and Culture, Tokyo, Japan; by health science research grants (Research on Translational Research) from the Ministry of Health, Labor, and Welfare, Tokyo, Japan; and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, Tokyo, Japan.
Drs Egashira and Morishita hold a patent on the results reported in the present study. The remaining authors report no conflicts.
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Although first-generation drug-eluting stents are effective in reducing the rate of restenosis, the drug-eluting stent has no effect on the incidence of cardiovascular events compared with the bare-metal stent. In addition, recent clinical studies have demonstrated that drug-eluting stents increase the incidence of late stent thrombosis, leading to acute myocardial infarction and death after the discontinuation of clopidogrel. These serious late thrombotic events are thought to result from impaired endothelial regeneration and an incomplete healing process because of the drugs or polymers used in the construction of drug-eluting stents. Therefore, the formulation of a novel drug-eluting stent system with fewer adverse effects is warranted. In the present study, we formulate a nuclear factor-κB (NF-κB) decoy–eluting stent with biocompatible polymer technology and report inhibition of neointimal formation by stent-based delivery of NF-κB decoy. Importantly, no histopathological evidence of impaired endothelial regeneration and healing process was noted at sites of stents coated with polymer alone and polymer plus decoy. These data support the experimental evidence that the NF-κB decoy–eluting stent is effective in reducing in-stent neointimal formation and thrombosis. Our previous clinical trial testing the feasibility and safety of NF-κB decoy supports the notion that this NF-κB decoy–eluting stent system can be applied to the clinical setting. Ultimately, we propose that this system be used to treat vulnerable plaques leading to acute coronary syndrome and stroke.
The online-only Data Supplement, consisting of expanded Methods, tables, and a figure, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.582254/DC1.