Author + information
- Received May 4, 2001
- Revision received November 7, 2001
- Accepted November 28, 2001
- Published online February 20, 2002.
- ↵*Reprint requests and correspondence:
Dr. Arnd B. Buchwald, Department of Cardiology and Pneumology, University of Goettingen, Robert-Koch-Str. 40, 37075 Goettingen, Germany.
Objectives We sought to demonstrate, in an appropriate animal model, that co-medication with a transcription factor-blocking agent limits restenosis after percutaneous transluminal coronary angioplasty (PTCA).
Background Enhanced synthesis in the vessel wall of endothelin-1 (ET-1), a powerful co-mitogen for vascular smooth muscle cells, appears to be one mechanism that promotes restenosis after PTCA. Deformation-induced expression of prepro-ET-1 is governed by the transcription factor, activator protein-1 (AP-1).
Methods An anti-AP-1 decoy oligodeoxynucleotide (dODN) strategy was devised in which the dODN-containing solution (20 nmol) was administered locally through a Dispatch catheter into the coronary arteries of hypercholesterolemic minipigs at the time of PTCA (AVE-GFX stent).
Results Treatment with an AP-1 dODN, mimicking the consensus binding site of the transcription factor, significantly reduced neointimal formation in the coronary arteries of hypercholesterolemic minipigs (n = 10 to 12), compared with vehicle-treated coronary arteries, after four weeks of follow-up (neointimal area 2.64 ± 0.33 vs. 4.81 ± 1.04 mm2[mean ± SEM]; p < 0.05). This effect was maintained after eight weeks (neointimal area 2.04 ± 0.22 mm2; n = 3) and correlated with a reduction in both nuclear translocation of AP-1 and ET-1 synthesis in the vessel wall 48 h after PTCA (n = 4). In contrast, an AP-1 mutant dODN, to which the transcription factor does not bind, showed no effect on neointimal formation at either time point (n = 3 to 7). Moreover, a consensus dODN directed against CCAAT/enhancer binding protein (C/EBP), another deformation-sensitive transcription factor, did not significantly affect neointimal formation after four weeks (n = 3).
Conclusions These findings demonstrate the feasibility, efficacy and specificity of the anti-AP-1 dODN approach to the treatment of restenosis, which principally but not exclusively targets deformation-induced ET-1 synthesis in the vessel wall. Provided that these findings can be extrapolated to the situation of patients with coronary artery disease, the observed extent of the inhibitory effect of the AP-1 dODN treatment suggests that this co-medication may greatly reduce the incidence of in-stent restenosis.
The implantation of stents has substantially reduced the rate of restenosis after percutaneous transluminal coronary angioplasty (PTCA) (1,2). However, in 10% to 40% of patients, restenosis still occurs, resulting in revascularization of the target lesion in up to 20% of vessels accommodating ≥3.0-mm stents, and even higher rates in smaller arteries and long lesions (3,4). Thus, restenosis contributes substantially to morbidity and the costs of treating coronary artery disease. Restenosis after coronary artery stent implantation is characterized by extensive neointimal proliferation of vascular smooth muscle cells (SMCs) and the formation of extracellular matrix (5–7). Possible mechanisms resulting in this change in SMC phenotype, from a contractile to a synthetic state, appear to be related to an increased synthesis of endothelin-1 (ET-1) in the vessel wall. Thus, enhanced synthesis of ET-1 has been demonstrated to occur after pressure trauma to the vessel wall in experimental models and in humans (8,9).
Previous work from our group has shown that this effect occurs at the level of transcription of the prepro-ET-1 gene (ppET-1) and that a decoy oligodeoxynucleotide (dODN) directed against the transcription factor activator protein-1 (AP-1) inhibits both ppET-1 expression and ET-1 synthesis in cultured endothelial cells exposed to mechanical deformation, as well as in isolated, perfused, endothelium-intact blood vessels in response to a supraphysiologic increase in perfusion pressure (10). Although there are many genes whose expression may be affected by this transcription factor, only a few, such as ppET-1, are sensitive to deformation, and there are only a few in which AP-1 functions as an essential component of the transcription initiation complex.
To verify the therapeutic potential of this dODN approach, we have investigated whether a single administration of AP-1 consensus dODN at the time of PTCA can limit neointimal proliferation in a coronary stent angioplasty model in hypercholesterolemic minipigs.
Decoy ODN technique
Double-stranded dODN (0.4 mmol/l) was prepared from complementary single-stranded phosphorothioate-bonded ODN obtained from Eurogentec (Köln, Germany), by melting it at 95°C for 5 min, followed by a cool-down phase of 3 to 4 h at an ambient temperature. After that, the solutions were split into 50 μl aliquots and frozen at −80°C until further use. The efficiency of the hybridization reaction was verified with 2.5% agarose gel electrophoresis and usually exceeded 98%. The sequences of the dODN for AP-1, CCAAT/enhancer binding protein (C/EBP) and the corresponding mutant dODN were as follows (underlined letters denote phosphorothioate-bonded bases; bold letters denote the core binding sequence for the transcription factor; and italic letters denote the mutated bases; cons = consensus dODN; mut = mutant dODN):
Before infusion, an aliquot of the dODN solution (50 μl) was defrosted and diluted in 10 ml phosphate-buffered saline (PBS; 2 μmol/l final concentration). As buffer control (vehicle), a corresponding volume of TEN (Tris, EDTA and Nacl) buffer (10 mmol/l Tris-HCl, 1 mmol/l EDTA and 150 mmol/l NaCl, at pH 7.5) was diluted in 10 ml PBS.
Animal experiments were done in accordance with the guidelines of the local Committee on Animal Welfare and approved by the local authorities. The experimental model has been described in detail previously (11,12). Briefly, in 22 minipigs under general anesthesia, PTCA was performed through a carotid artery. Per animal, two or three coronary artery segments of a slightly smaller diameter (0.3 to 0.5 mm less) than the 3.0-mm stent-carrying balloon were chosen for treatment, as indicated subsequently. This resulted in placement of stents more distally in some vessels and more proximally in other vessels, depending on their size. Operators had no knowledge of the treatment given to a specific vessel segment (dODN or vehicle). Before PTCA, 0.2 mg of intracoronary nitroglycerin was administered. Stents (AVE-GFX, Medtronic, Düsseldorf, Germany) 12 mm in length were implanted at 16 atm of pressure for 20 s. The balloons were withdrawn, and a Dispatch catheter (Interventional Techniques, San Diego, California, through Cardiologic, Munich, Germany) was advanced into the stented segment and inflated to 6 atm, and infusion of the dODN solution or vehicle was started.
The Dispatch catheter has an inner lumen allowing for peripheral perfusion during inflation. Flow through this inner lumen was verified in 5-min intervals. On inflation, a helical-like–shaped balloon creates a space between the vessel wall and the catheter, into which there is drainage through multiple end holes of the infusion line. There is a slow, distal run-off into the artery, allowing for continuous drug infusion. After the desired volume (10 ml at 0.5 ml/min; corresponding to a total amount of 20 nmol dODN) was infused, the balloon was deflated and withdrawn, the carotid artery was ligated, and the neck wound was closed. The animals were returned to their cages until after the planned follow-up period (see subsequently), when they were euthanized.
After four or eight weeks of follow-up, a thoracotomy under deep anesthesia was performed, and the animals were euthanized by an overdose of barbiturate. The hearts were excised and immediately perfused with PBS at a pressure of 100 mm Hg, followed by perfusion-fixation with buffered 4% formaldehyde (1,000 ml). The angioplasty segments were then excised and embedded in methylmethacrylate. Three sections per 12-mm segment were subsequently analyzed morphometrically using a digital microscopic video camera and the Image Pro software (version 2.0, Media Cybernetics, Silver Spring, Maryland). Areas of the lumen, neointima, media and adventitia, as well as neointimal thickness over each stent strut, were measured. Penetration of each strut into the vessel wall was graded using a modified injury score, as originally described by Schwartz et al. (13), where injury ranged in increments from 1 (superficial) to 4 (into the adventitia). The investigator had no knowledge of the treatment of the segments during morphometry.
For immunohistochemical analysis, the angioplasty segments were excised immediately after euthanasia 48 h after PTCA; the stents were removed; and a 3-mm portion was fixed in formaldehyde and embedded in paraffin. The remaining portion was deep-frozen in liquid nitrogen for subsequent electrophoretic mobility shift analysis. Of the paraffin blocks, 5-μm-thick sections were cut and mounted on siliconized slides. After deparaffinization and dehydration, the sections were first stained with hematoxylin, followed by overnight incubation at 4°C with a mouse monoclonal anti-ET-1 antibody (Dianova, Hamburg, Germany) at a dilution of 1:250. For detection of the bound primary antibody, the Histostain Plus kit (Zymed, San Francisco, California) was used, comprising a biotinylated goat–anti-mouse, streptavidin-peroxidase conjugate and the chromogenic substrate 3-amino-9-ethylcarbazol (AEC), yielding a red deposit.
Electrophoretic mobility shift analysis
Nuclear extracts from the coronary artery segments were prepared as described previously for rabbit blood vessels (14). The double-stranded gel shift oligonucleotides (Santa Cruz Biotechnology, Heidelberg, Germany) for AP-1 and C/EBP (sequences identical to AP-1cons and C/EBP dODN, as described earlier) were end-labeled with (γ-32P)adenosine triphosphate by using the 5" end-labeling kit from Amersham Pharmacia Biotech (Freiburg, Germany). Typically, the binding mixture contained 5 μg of nuclear extract, 20,000 cpm of the 32P-labeled oligonucleotide probe (0.5 ng), 1 μg poly(d[I-C]) and 1.33 mmol/l of dl-dithiothreitol in a total volume of 15 μl binding buffer.
Treatment groups and statistical analysis
Angioplasty of three coronary arteries per animal was performed in six minipigs, with a follow-up period of four weeks (comparisons of vehicle, AP-1 consensus dODN and AP-1 mutant dODN, n = 3; comparisons of vehicle, C/EBP consensus dODN and C/EBP mutant dODN, n = 3), and of two coronary arteries per animal in 16 minipigs, with a follow-up period of 48 h (comparison of vehicle and AP-1 consensus dODN, n = 4), four weeks (comparison of vehicle and AP-1 consensus dODN, n = 5; AP-1 consensus dODN vs. AP-1 mutant dODN, n = 4) or eight weeks (comparison of AP-1 consensus dODN and AP-1 mutant dODN, n = 3).
The results are expressed as the mean value ± SEM, with n referring to the number of coronary arteries analyzed per treatment group, regardless of comparisons made in individual animals. Statistical analysis using InStat software, version 3.0 (GraphPad, San Diego, California) was performed by Kruskal-Wallis one-way analysis of variance, followed by Dunn’s post test for selected groups or the unpaired ttest with the Welch correction, as appropriate (comparisons of three or two treatment groups, respectively), with p < 0.05 considered as statistically significant.
Serum cholesterol level
Animals were fed a standard diet supplemented with 2% cholesterol and 10 g/day of sodium cholate for at least eight weeks before PTCA, because in a previous study (11), cholesterol levels remained stable thereafter. This resulted in an increase in serum cholesterol from 69.4 ± 19.4 mg/dl at baseline to 211.3 ± 42.6 mg/dl at the time of PTCA (n = 22).
Effect of the AP-1 consensus dODN on ET-1 synthesis
In a separate series of experiments, immunohistochemical analysis of coronary arteries harvested at 2, 3, 4, 7, 14 and 28 days after stent placement (n = 3 per time point) revealed positive ET-1 staining, beginning at day 2 and reaching a maximum at day 4; after 28 days, all segments stained negative (A. B. Buchwald, unpublished observation). Hence, ET-1 synthesis in this model starts early after PTCA and subsides after approximately two weeks.
As shown in Figure 1, in AP-1 consensus dODN-treated segments, ET-1 was hardly detectable 48 h after PTCA. In contrast, there was a distinct ET-1 immunoreactivity in vehicle-treated segments, showing particularly intense staining in the periluminal cell layers.
Transcription factor activation
Electrophoretic mobility shift analysis revealed that the AP-1 consensus dODN completely prevented nuclear translocation of AP-1 in endothelium-intact, in vitro porcine coronary artery segments that had been exposed to 1 μmol/l of phorbol dibutyrate to upregulate AP-1 activity through stimulation of protein kinase C (Fig. 2a). In contrast, the AP-1 mutant dODN did not affect AP-1 abundance in the nucleus under these conditions.
Treatment of the coronary arteries with AP-1 consensus dODN in vivo resulted in a markedly reduced AP-1 activity in the vessel wall 48 h after PTCA, compared with the vehicle-treated coronary artery from the same animal (Fig. 2b). Treatment with AP-1 dODN, in contrast, had no effect on the nuclear translocation of C/EBPβ and -δ, two deformation-sensitive members of the C/EBP family of transcription factors (10).
Proliferative vessel wall response with dODN treatment
Injury scores in AP-1 consensus and AP-1 mutant dODN-treated arteries (range 2.0 to 2.1) were comparable to those in the buffer control group (Table 1). Figure 3displays the morphometric analysis of a typical experiment with three different coronary arteries from the same animal treated with either vehicle, AP-1 consensus dODN or AP-1 mutant dODN. Of note, the AP-1 consensus dODN treatment had a pronounced effect on neointimal proliferation, despite comparable injury in all three arteries. After four weeks of follow-up, the minimal lumen area was clearly larger and the neointimal area was significantly smaller in AP-1 consensus dODN-treated segments than in vehicle-treated or AP-1 mutant dODN-treated segments (Fig. 4). The media and total vessel area were comparable between the treatment groups. There was no evidence of decreased cellularity in the media in any of the treatment groups, as an indicator of potential toxic induction of fibrosis or calcification (Table 1).
This effect of AP-1 consensus dODN, as well as its effects on the adventitial, luminal (Table 1) and neointimal area (Fig. 5a), were maintained at eight weeks after PTCA, compared with AP-1 mutant dODN. No comparison with the buffer control group was made at this point in time, as previous studies with the minipig model have shown that neointimal formation manifests at four weeks and does not progress or regress over the following eight weeks (11).
To confirm that AP-1 consensus dODN exerts a specific effect that cannot be mimicked by a dODN directed against another transcription factor that is also deformation-sensitive and involved in the regulation of ppET-1 expression in another species (rabbit carotid artery ), the effects of C/EBP consensus dODN on restenosis four weeks after PTCA were investigated as well. However, both the consensus and corresponding mutant dODN did not inhibit neointimal formation, compared with vehicle-treated segments (Table 1, Fig. 5b).
The main findings obtained in the minipig angioplasty model can be summarized as follows. Infusion of dODN against the transcription factor AP-1, using a local drug delivery catheter, results in a profound decrease in AP-1 activity and marked suppression of intravascular ET-1 synthesis 48 h after PTCA. This, in turn, contributes to a significant reduction of neointimal proliferation as the major determinant of in-stent restenosis, compared with buffer as the control, by >50% after four weeks. The corresponding mutant dODN, in contrast, has no effect. Moreover, inhibition of proliferation is maintained after eight weeks, indicating a definitive effect, not merely a delay of proliferation, occurring later than that in control arteries. The effect is achieved by a single application of the “naked” dODN at the time of PTCA, with no further treatment.
Role of ET-1 in in-stent restenosis
The action of ET-1 has been previously suggested as an important contributor to post-PTCA restenosis. Tissue ET-1 levels increase after endothelial denudation in a rabbit carotid artery model (8), and ET-1 is released from human atherosclerotic coronary arteries after injury (9). Selective antagonism of the endothelin A (ETA) receptor reduced the proliferative vessel wall response in a rat carotid artery (15)and a porcine femoral and carotid artery PTCA model (16), in accordance with the findings of this study. Similarly, the approach of selective ETAreceptor antagonism in a non-hypercholesterolemic porcine coronary stent angioplasty model reduced neointimal hyperplasia (17). However, a successful reduction in proliferation was observed, with modest proliferation in the control/placebo groups (e.g., average neointimal thickness of 0.45 mm in the study by McKenna et al. (17) vs. 1.03 mm in the present study). In contrast, in this study, inhibition of ET-1 synthesis by the AP-1 dODN limited neointimal proliferation, irrespective of the degree of arterial injury. Moreover, this effect was achieved with a single periprocedural local infusion of dODN, while antagonizing the receptor-required systemic administration of the drug over several days.
Vessel wall trauma and therapeutic efficacy
The proliferative vessel wall response increases with increasing stretch, and thus injury to the vessel wall with PTCA (12,13). In accordance with the available evidence suggesting stretch-induced activation of ET-1 synthesis as a major determinant of the proliferative vessel wall response to injury, exceeding the adaptive proliferation secondary to mild stretch, the results of the present study support the hypothesis that inhibition of ppET-1 gene expression and, consequently, the synthesis of mature ET-1 results in a limited proliferative response, even in areas of deep vessel wall injury.
Specificity of the dODN approach
It could be argued that any mechanosensitive transcription factor can cause neointimal proliferation after PTCA, especially if this transcription factor is potentially associated with the expression of the target gene, too (10). However, the negative results obtained in the animals treated with the C/EBP dODN indicate that the proliferative response in this model is specifically mediated by AP-1. This does not mean, however, that the inhibitory effect of AP-1 dODN on in-stent restenosis is solely due to the blockade of deformation-induced ppET-1 expression. Other gene products involved in neointimal formation, the expression of which is influenced by AP-1, may be likewise affected, although from a therapeutic point of view, this does not necessarily pose a problem.
Given that the aforementioned results can be extrapolated to the situation in patients with coronary artery disease, the observed extent of the inhibitory action of dODN treatment suggests that this co-medication may greatly reduce the incidence of in-stent restenosis, which at present ranges from 10% to 40% after six months.
A potential limitation of this study is that it was performed in minipigs, and although these animals were fed an atherogenic diet, PTCA was performed in near-healthy coronary arteries. In the clinical situation, severe atherosclerosis and calcification, replacing parts of the normal vessel wall, may influence the role of the endothelin system in the reparative response after PTCA (9). Although the release of ET-1 from human coronary arteries after PTCA suggests a role for the peptide in human atherosclerosis, as well, the dODN approach must be tested in a clinical setting.
We are grateful for the expert technical assistance of Astrid Steen in preparing the histologic specimens.
☆ This study was supported by the Deutsche Forschungsgemeinschaft (He 1587/7-1) and by Cardion AG, Erkrath, Germany.
- activator protein-1
- CCAAT/enhancer binding protein
- decoy oligodeoxynucleotide
- endothelin A
- phosphate-buffered saline
- percutaneous transluminal coronary angioplasty
- smooth muscle cell
- Received May 4, 2001.
- Revision received November 7, 2001.
- Accepted November 28, 2001.
- American College of Cardiology
- Williams D.O,
- Holubkov R,
- Yeh W,
- et al.
- the STent REStenosis Study (STRESS) Investigators,
- Savage M.P,
- Fischman D.L,
- Rake R,
- et al.
- Karas S.P,
- Gravanis M.B,
- Santoian E.C,
- Robinson K.A,
- Anderberg K.A,
- King S.B 3d.
- Hoffmann R,
- Mintz G.S,
- Dussaillant G.R,
- et al.
- Kearney M,
- Pieczek A,
- Haley L,
- et al.
- Azuma H,
- Hamasaki H,
- Niimi Y,
- Terada T,
- Matsubara O
- Hasdai D,
- Holmes D.R,
- Garrat K.N,
- Edwards W.D,
- Lerman A
- Buchwald A.B,
- Unterberg C,
- Nebendahl K,
- Gröne H.J,
- Wiegand V
- Unterberg C,
- Sandrock D,
- Nebendahl K,
- Buchwald A.B
- Schwartz R.S,
- Huber K.C,
- Murphy J.G,
- et al.
- Douglas S.A,
- Louden C,
- Vickery-Clark L.M,
- et al.
- McKenna C.J,
- Burke S.E,
- Opgenorth T.J,
- et al.