Administration of a Decoy Against the Activator Protein-1 Binding Site Suppresses Neointimal Thickening in Rabbit Balloon-Injured Arteries
Background— Transcription factor activator protein-1 (AP-1) is activated and upregulated in injured arterial smooth muscle cells in vivo, yet the exact role of the AP-1–related pathway in vascular disease in vivo has remained unclear. We examined the role of the transfer of synthetic double-stranded cis-element decoy oligodeoxynucleotides (ODNs) in balloon-injured rabbit carotid arteries and the effects of these ODNs on neointimal thickening.
Methods and Results— Transfection of fluorescein isothiocyanate–labeled ODNs using the hemagglutinating virus of Japan liposome method resulted in widespread distribution of fluorescent nuclear signals over the entire medial layer in injured arteries. Gel mobility shift assay revealed that AP-1 DNA binding was activated and that the AP-1 decoy reduced AP-1 DNA binding activity as a result of specific binding affinity to AP-1 in vivo. In morphometric analyses, AP-1 decoy led to a significant reduction in the neointimal area and a significant reduction in cell number and transforming growth factor-β1 production of human aortic smooth muscle cells under conditions of platelet-derived growth factor stimulation.
Conclusions— Because AP-1 decoy transfection in vivo dramatically prevented neointimal thickening in balloon-injured arteries, AP-1 may be a useful molecular target for gene therapy to reduce restenosis.
Received July 23, 2001; revision received December 21, 2001; accepted December 21, 2001.
Progressive neointimal thickening and restenosis remain major critical problems, limiting the long-term efficacy of percutaneous transluminal coronary angioplasty or bypass surgery to treat patients with vascular occlusive diseases.1 Common characteristics of vascular responses are proliferation and migration of vascular smooth muscle cells (VSMCs)2 and accumulation of extracellular matrix components.3 There are various factors implicated in this process, including proto-oncogenes (c-fos and c-jun) and growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β).4,5⇓ PDGF was found to induce activation of PDGF receptor mitogen-activated protein kinase (MAPK) activator protein-1 (AP-1) signaling pathways in vivo.6 AP-1 DNA binding activity is significantly increased in balloon-injured arteries.7
Administration of decoy oligodeoxynucleotides (ODNs) was found to block the binding of nuclear factors to promoter regions of targeted genes, the result being inhibition of gene transactivation in in vitro8 and in vivo9 assay systems. Our working hypothesis is that transfection of VSMCs with sufficient quantities of decoy ODNs containing the AP-1 binding site would effectively bind AP-1, thus preventing transactivation of essential cell-cycle regulatory genes and thereby inhibiting VSMC proliferation and vascular wall remodeling.
We administrated AP-1 decoy ODNs into balloon-injured rabbit carotid arteries using the hemagglutinating virus of Japan (HVJ) liposomes method to directly and efficiently reduce AP-1 activity. In our ongoing series of experiments, we want to determine the exact role of AP-1–related signal transduction pathways in proliferative responses and vascular remodeling after vascular injury. The feasibility of AP-1–targeted therapy for patients with a restenosis can thus be addressed.
Synthesis of Oligodeoxynucleotides
Following are the sequences of phosphorothioate double-stranded ODNs that we used against AP-1 binding sites and scrambled ODNs: AP-1 decoy (consensus sequences are underlined), 5′-AGCTTGTGAGTCAGAAGCT-3′, 3′-TCGAACACTCAGTCT-TCGA-5′, scrambled decoy, 5′-AATGGCATGGACTGTATCG-3′, and 3′-TTACCGTACCTGACATAGC-5′.
Preparation of HVJ Liposome Complexes
HVJ liposome complexes used were as previously described.9 Briefly, Phosphatidylserine (Funakoshi, Inc), phosphatidylcholine, and cholesterol (Sigma) were mixed in a weight ratio of 1:4.8:2. The lipid mixture was deposited on sides of a flask by removal of tetrahydrofuran. Dried lipid was hydrated in ODNs in 150 μL H2O containing 30 nmol/L ODNs. Liposomes were prepared by shaking and sonication. Purified HVJ was inactivated by ultraviolet irradiation for 3 minutes just before use. The liposome suspension was mixed with HVJ in a total volume of 2 mL balanced salt solution (BSS) (137 mmol/L NaCl, 5.4 mmol/L KCl, and 10 mmol/L Tris-HCl, pH 7.6). Free HVJ was removed from the HVJ liposome complexes by sucrose density gradient centrifugation. The top layer of sucrose gradient was collected.
The following animal experiments were reviewed by the Committee on Ethics on Animal Experiments in the Faculty of Medicine, Kyushu University and were carried out under the Guidelines for Animal Experiments in the Faculty of Medicine, Kyushu University and the Law (No. 105) and Notification (No. 6) of the Government of Japan. The “Principles of Laboratory Animals” (publication No. NIH 80-23, revised 1985) were also followed. The animals were purchased from KBT Oriental Co (Fukuoka, Japan).
Japanese White rabbits weighing between 2.5 and 3.0 kg were used in the present study. All surgical procedures were performed under general anesthesia by injection of xylazine (15 mg/kg) and keteral (0.5 mL/kg) into subcutaneous tissues. Arterial injury was accomplished with a 2-F Fogarty balloon catheter as described.10
The effects of balloon injury on arterial AP-1 DNA binding activity were examined (n=35). Left common carotid artery at 1, 3, 6, and 24 hours after injury and noninjured right common carotid artery (control) were immediately excised and then placed in liquid nitrogen and stored at −80° until use.
Next, the effects of AP-1 decoy on AP-1 DNA binding activity and neointimal thickening in the balloon-injured artery (n=25) were examined. Rabbits were separated into the following 4 groups: (1) buffer-treated (control, incubated BSS), (2) vehicle-transfected (incubated HVJ liposome only), (3) scrambled decoy–transferred (incubated HVJ liposome complexes containing scrambled decoy), and (4) AP-1 decoy–transferred (incubated HVJ liposome complexes containing AP-1 decoy). The lumen of the artery was incubated with 15 nmol/L ODNs under the pressure of 150 mm Hg for 15 minutes at room temperature. The injured or transfected arteries were collected, as described above, at 3 hours after balloon injury for measurement of AP-1 DNA binding activity and at 4 weeks for measurement of the neointimal thickening.
FITC-labeled ODNs (10 μmol/L) were transferred into the injured carotid arteries, as described above and elsewhere.11 After balloon injury (n=12), the artery was incubated in buffer (control, n=2), FITC-labeled AP-1 decoy (naked FITC-ODNs) (n=5), or HVJ liposome complexes containing FITC-labeled AP-1 decoy (HVJ+FITC-ODNs, containing 10 μmol/L ODNs) (n=5). At 6 hours after transfection, the arteries were harvested, immediately embedded in OCT compound (Tissue Tek, Miles, Inc), and frozen in dry ice/acetone and the frozen specimens were cut into 4-μm sections on a cryostat.
Intimal thickening of the injured carotid arteries was determined by quantitative morphometry, as described.12 At 4 weeks after transfection, carotid arteries of each experimental group were harvested. Cross-section rings (5 μm) were stained with elastica van Gieson. The intimal and medial entire areas of all histopathological sections were determined with image analysis software (Mac SCOPE, Mitani Corporation). Then the intima to media ratio (the area of the intima divided by the area of the media) was calculated for each artery.
Gel Mobility Shift Assay
For gel mobility shift assay, 8 carotid arteries were pooled so as to contain 1 sample. Arterial extract was prepared from the artery according to Gorski et al13 but with some modifications. In brief, arteries were homogenized in 0.8 mL of 10 mmol/L HEPES (pH 7.6) containing 25 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 2 mol/L sucrose, and 20% glycerol, incubated on ice for 15 minutes, and centrifuged at 75,000g at 4°C for 60 minutes. The pelleted nuclei were resuspended in 0.4 mL nuclear lysis buffer (10 mmol/L HEPES [pH 7.6] containing 100 mmol/L KCl, 1.5 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 20% glycerol, 0.2 mmol/L DTT, 0.5 mmol/L PMSF, 60 μg/mL aprotinin, and 2 μg/mL leupeptin), incubated on ice at ≈4°C for 15 minutes, and centrifuged at 10,000g at 4°C for 10 minutes. The resulting supernatant was assayed for protein concentrations and stored at −80°C until use. Detailed procedures regarding the gel mobility shift assay were according to Maniatis et al.14 In brief, 5 μg of nuclear protein extracts was used for each lane. Samples of nuclear protein extracts were incubated with 5 pmol of a 32P-labeled phosphorothioate double-stranded ODN probe containing the consensus AP-1 binding sequence underlined (5′-AGCTTGTGAGTCAGAAGCT-3′) at 22°C for 30 minutes in 20 μL of the binding buffer, consisting of 20 mmol/L Tris-HCl, 50 mmol/L NaCl, 10% glycerol, 0.1 mmol/L DTT, and 2 μg polydeoxyinosinicdeoxycytidylic acid (poly[dI-dC]; Pharmacia) as a nonspecific competitor. For competition experiments, the double-stranded ODNs containing the consensus nuclear factor-κB (NF-κB) binding sequences underlined (5′-CCTTGAAGGGATTTCCCTCC-3′) were also used as a cold competitor. The DNA protein complexes were electrophoresed on 4% nondenaturing polyacrylamide gels, and the gels were subjected to autoradiography. As a positive control sample of AP-1, we used nuclear extracts from raf-1–transformed rat fibroblasts stimulated with phorbol ester (3611-RF-phorbol) and known to be rich in AP-1 (Santa Cruz Biotechnology, Inc).
Supershift assays were done with rabbit polyclonal anti–c-Fos antibody and anti–c-Jun antibody (Oncogene Research Products). Each antibody (2 μg each) was added to the samples after the initial binding reaction between the positive control sample and 32P-labeled consensus AP-1 oligonucleotide; the reaction was allowed to occur at 22° for 30 minutes and subjected to electrophoresis, as described above.
A primary culture of human aortic smooth muscle cell (HASMC),15 originally derived from an explant of a 22-year-old man, was purchased from Kurabo Co (Osaka, Japan). These cells were maintained in a HuMedia-SG2 medium from Kurabo Co with 5% fetal calf serum, human epithelium growth factor (0.5 ng/mL), human fibroblast growth factor (2 ng/mL), insulin (5 μg/mL), gentamycin (50 μg/mL), and amphotericin B (50 ng/mL).15 This medium contained L-cysteine HCl H2O (110 μmol/L) and L-glutamic acid (72 μmol/L). Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2 with medium changes every 2 days.15 Cells from the 5th to 7th passages were used. Studies of HASMC proliferation were done on subconfluent cells made quiescent by incubation in low serum medium (LSM) (1%) containing insulin (5 μg/mL), gentamycin (50 μg/mL), and amphotericin B (50 ng/mL) for 24 hours.
AP-1 Binding Activity, Proliferation, and TGF-β1 Production Studies
In setting up experiments for determination of cell number and TGF-β1 production, cells were grown to 80% subconfluence in 96-well culture dishes. After reaching subconfluence, cells were washed 3 times with BSS. The medium was changed to fresh LSM with or without 10 ng/mL recombinant human platelet-derived growth factor-BB (PDGF-BB) (Pepro Tech Inc). Then 2 μL HVJ-liposome complexes (50 nmol/L encapsulated ODN) was added to the wells followed by incubation for 30 minutes at 37°C. Fresh LSM or LSM with PDGF-BB (10 ng/mL) was added and the cells were incubated for 3 hours or overnight. For AP-1 binding activity studies, we used cells incubated for 3 hours. Nuclear protein preparation was similar to that described elsewhere,16 and gel mobility shift assays were similar to those described as above. For cell proliferation and TGF-β1 production studies, we used cells that have been incubated overnight. On days 1 and 3, the medium was again changed to fresh LSM with or without PDGF. On day 4, cell numbers were counted, and measurement of TGF-β1 was done using ELISA (R&D Systems).
All values were expressed as mean±SEM. The data were statistically analyzed using one-way ANOVA followed by Schiff’s F test.
Transfection of FITC-Labeled AP-1 Decoy Into the Artery
We used fluorescence to trace the exact site of AP-1 decoy in the carotid balloon-injured arterial wall. Transfection of naked FITC-ODNs resulted in scattered fluorescence in the injured arterial wall (Figures 1C and 1D), whereas HVJ+FITC-ODN transfection showed widespread FITC signals in many nuclei in the injured arterial wall (Figures 1E and 1F). The control artery showed background fluorescence in elastic fibers (Figures 1A and 1B).
Time Course of AP-1-DNA Binding Activity in a Balloon-Injured Artery and Effects of AP-1 Decoy
The results obtained by the gel mobility shift assay of a positive control for AP-1 revealed that the specific AP-1 DNA binding complex could be detected using the present method. As shown in Figure 2A, the band (designated by a half bracket) was efficiently competed for by increasing concentrations of cold AP-1 but not by NF-κB ODNs. Furthermore, as shown in Figure 2B, the addition of anti–c-Fos or anti–c-Jun antibody to the binding reaction produced supershifted complexes.
To assess the time course of in vivo activation of AP-1 DNA binding and effect of decoy transfection in balloon-injured arteries, gel mobility shift assay was done using arterial extracts from rabbit carotid arteries. As shown in Figure 3A, arterial AP-1 DNA binding activity (corresponding to band AP-1 in Figure 2A) was increased at 3 hours after injury and gradually decreased thereafter. As shown in Figure 3B, compared with a very low level of AP-1 DNA binding activity in the uninjured control artery, increased AP-1 DNA binding activity was evident in the injured artery at 3 hours after balloon injury, and this activity was reduced in the AP-1 decoy–transferred artery but not competed for in the scrambled decoy–transferred artery. These results confirmed that treatment with AP-1 decoy prevented the increase in arterial AP-1 DNA binding activity after balloon injury. These experiments were repeated 5 times, all with representative results.
Inhibitory Effect of AP-1 Decoy Transfer on Neointimal Thickening in Balloon-Injured Arteries
At 4 weeks after decoy transfection, buffer-treated, vehicle-transfected, and scrambled decoy–transferred arteries after balloon injury all formed a similarly thickened neointima (Figures 4A, 4B and 4C). In contrast, AP-1 decoy–transferred arteries had a reduced intimal area (Figure 4D). The area of the thickened intima (mm2) was 0.55±0.06 in buffer-treated arteries after balloon injury, 0.38±0.04 in vehicle-transfected arteries, 0.36±0.05 in scrambled decoy-transferred arteries, and 0.17±0.01 in AP-1 decoy–transferred arteries. Quantitative analysis demonstrated a significant reduction in AP-1 decoy–transferred arteries compared with the other 3 groups (Figure 5A). In addition, the intimal/media ratio was 1.87±0.16 in buffer-treated arteries after balloon injury, 1.16±0.07 in vehicle-transfected arteries, 1.08±0.08 in scrambled decoy–transferred arteries, and 0.43±0.03 in AP-1 decoy–transferred arteries. Quantitative analysis demonstrated a significant reduction in AP-1 decoy–transferred arteries compared with findings in the other 3 groups (Figure 5B).
Effects of AP-1 Decoy on PDGF-Mediated AP-1 DNA Binding Activity, Cell Proliferation, and TGF-β1 Production in HASMCs
To assess PDGF-mediated AP-1 DNA binding activity and effects of AP-1 decoy, gel mobility shift assay was done using nuclear extracts of PDGF-stimulated HASMCs. As shown in Figure 6, compared with a very low level of AP-1 DNA binding activity in the control cells, increased AP-1 DNA binding activity was evident in the untreated cells. This AP-1 DNA binding activity was reduced in the AP-1 decoy–transferred cells but not competed for in the scrambled decoy–transferred cells. These results confirmed that PDGF stimulation increased AP-1 DNA binding activity in HASMCs and treatment with AP-1decoy prevented the increase in AP-1 DNA binding activity of HASMCs after PDGF stimulation. These experiments were repeated 5 times, all with representative results.
PDGF stimulated increases in cell number and TGF-β1, whereas transfection of AP-1 decoy showed a decrease in the presence of PDGF (Figures 7 and 8⇓). The number (×104) was 0.8±0.1 in control cells, 18.6±0.7 in untreated cells under PDGF-stimulated conditions, 16.9±0.7 in vehicle-transfected cells, 17.7±0.3 in scrambled decoy–transferred cells, and 12.8±0.7 in AP-1 decoy–transferred cells. The concentration (pg/mL) of TGF-β1 was 57.3±6.1 in control cells, 213.5±12.5 in untreated cells under PDGF-stimulated conditions, 211.9±15.9 in vehicle-transfected cells, 218.8±24.8 in scrambled decoy–transferred cells, and 147.0±9.3 in AP-1 decoy–transferred cells. Quantitative analysis demonstrated a significant reduction in AP-1 decoy–transferred cells compared with findings in untreated and scrambled decoy–transferred cells.
AP-1 is a collective term referring to dimeric transcription factors composed of Jun, Fos, or activating transcription factor subunits that bind to a common DNA site, the AP-1 DNA binding site. In vitro studies revealed significant roles of AP-1 activity in VSMC functions.17 Even if AP-1 is activated and upregulated in injured arterial smooth muscle cells in vivo,18 the contribution of AP-1 in vascular lesion formation in vivo remains to be determined.
VSMCs are the predominant cell type in atherosclerotic lesions and in the neointima after balloon angioplasty.19 We found definite and direct in vivo evidence for the activated AP-1 involvement in VSMCs and synthetic decoy ODNs with the AP-1 consensus sequence inhibiting AP-1 DNA binding activity in balloon-injured arteries, the result being a significant suppression of neointimal thickening. These findings strongly suggested that AP-1 activation in the injured vessel wall may play a substantial role in vivo during vascular injury remodeling processes, hence AP-1 is an important molecular target for restenosis therapy.
Our present study using gel mobility shift analysis showed that AP-1 DNA binding activity is increased in arteries 3 hours after balloon injury and then gradually decreased to normal levels within 24 hours and is reduced in arteries treated with AP-1 decoy ODNs. It was reported that in vivo transfer of ODNs using the HVJ liposome method resulted in widespread distribution of fluorescence in medial vascular cells, such as VSMCs, at 10 minutes after transfection.11 The fluorescence was localized primarily in cell nuclei and persisted for up to 2 weeks after transfection into the vessel wall.11 These observations suggested that transfer of AP-1 decoy by the HVJ liposome method inhibits arterial AP-1 DNA binding activity after vascular injury. Therefore, the AP-1 decoy strategy may prove to be an effective approach to treat subjects with vascular-related diseases.
We found that transfection of AP-1 decoy inhibited AP-1 DNA binding activity, cell proliferation, and production of TGF-β1 in response to PDGF-BB in HASMCs. PDGF is likely to stimulate PDGF-initiated PDGF receptor MAPK AP-1 signaling and AP-1 DNA binding activity.6 Therefore, given the specific competition of the AP-1 binding site, our studies using transfection of AP-1 decoy on proliferative responses of HASMCs under PDGF-stimulated conditions showed a significant reduction in numbers of HASMCs.
TGF-β1 has been implicated in the development of human restenosis after angioplasty20 as well as in neointimal lesions in balloon-injured arteries of experimental animals.3,20⇓ Transfection of TGF-β1 cDNA into the injured vessel wall indicates that TGF-β1 can promote both the formation and growth of neointima in laboratory animals.21 TGF-β1 mRNA levels were found to be significantly increased in restenotic lesions.20 It is worth noting that the TGF-β1 gene has an AP-1 consensus sequence in its promoter region.22 These results suggest that one of the mechanisms of neointimal thickening suppression by AP-1 decoy may involve suppression of VSMC proliferation by blocking PDGF-initiated PDGF receptor MAPK AP-1 signaling pathways and TGF-β1 production.
In summary, we found that in vivo delivery of synthetic decoy ODNs against the AP-1 binding site significantly suppressed neointimal thickening in rabbit carotid injured arteries. In addition, the in vitro delivery of synthetic decoy ODNs against the AP-1 binding site significantly suppressed AP-1 DNA binding activity, numbers of cells, and TGF-β1 production in HASMCs subjected to PDGF stimulation. These findings provide evidence for involvement of the AP-1–related pathway in the process of neointimal thickening after arterial injury. Ongoing experiments are expected to clarify the role of the AP-1–related pathway and detailed molecular mechanisms related to VSMCs.
This study was supported in part by a grant-in-aid from the Ministry of Education, Science, Technology, Sports, and Culture of Japan.
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