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Cardiovascular Research 2002 54(3):640-648; doi:10.1016/S0008-6363(02)00335-8
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Copyright © 2002, European Society of Cardiology

Down-regulation of the ERK1 and ERK2 mitogen-activated protein kinases using antisense oligonucleotides inhibits intimal hyperplasia in a porcine model of coronary balloon angioplasty

Bo Liu1, Michael Fisher1 and Peter Groves*

Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF4 14XN, UK

peter.groves{at}cardiffandvale.nhs.wales.co.uk

* Corresponding author. Tel.: +44-2920-742-338; fax: +44-2920-744-473

Received 14 September 2001; accepted 1 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Neointimal hyperplasia is a central feature in the pathogenesis of a variety of vascular pathologies. Mitogen-activated protein kinases (MAPK) are involved in the downstream transduction of signals from receptors for many of the molecules known to be instrumental in this process and thus represent a potential target for the modification of the proliferative response. We examined the hypothesis that down-regulation of MAPK would inhibit neointima formation in a porcine coronary injury model. Methods: Balloon angioplasty was performed on 38 coronary arteries from 23 large white pigs. Antisense oligonucleotides to the p42 and p44 MAPK were locally delivered to the site of injury immediately after balloon injury. At 7 or 21 days, arteries were harvested for morphometry, determination of cell proliferation and assessment of MAPK protein levels. Results: At 7 days, neointima formation was significantly reduced compared to controls (corrected intima/media ratio (IMR) 1.01±0.13 vs. 1.61±0.07, P<0.01) and this was associated with a 58% and 23% down-regulation of p42 and p44 protein levels, respectively. Intimal and medial proliferation rates were also reduced by 32% and 26%, respectively. At 21 days however, the effect of the treatment on MAPK protein levels was no longer significant and this correlated with a loss of the effects on IMR and cell proliferation. Conclusions: Down-regulation of MAPK inhibits early smooth muscle cell (SMC) proliferation and neointimal thickening in response to arterial injury, implying that it plays an important role in determining the early vascular response to injury. Inhibitory effects were less apparent at 21 days after a single delivery of oligonucleotide, implying that more sustained local delivery may be required to achieve longer term therapeutic benefit.

KEYWORDS Restenosis; Protein kinases; Coronary circulation; Angioplasty


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The formation of an arterial neointima is a characteristic feature of the vascular response to diverse forms of injury [1]. It is implicated in such disparate processes as atherosclerosis [2], vein graft failure following coronary artery bypass grafting [3] and the restenotic response following percutaneous transluminal coronary angioplasty (PTCA). Limitation of neointima formation has become particularly relevant to the clinical practice of PTCA with the widespread employment of intracoronary stents, as intravascular ultrasound studies have demonstrated that this is the predominant mechanism of in-stent restenosis [1]. While many cell types are involved in the process of neointima formation, the vascular smooth muscle cell (VSMC) is the most prominent participant [2,4]. Following arterial injury, a variety of growth factors and cytokines are elaborated which result in VSMC migration [5,6] and proliferation [7] and also promote intercellular matrix production [8,9]. With the involvement of a wide array of growth factors and cytokines, preventing the activation of VSMCs is likely to require an influence on downstream intracellular signalling events responsible for transducing these multiple extracellular signals. Possible candidates for such manipulation are the mitogen-activated protein kinases (MAPK) which are a group of highly conserved and ubiquitously expressed proteins that have been shown to become activated by phosphorylation in response to numerous stimuli and therefore may serve to integrate input from a variety of different receptor types [10–12]. There are at least five subfamilies of mammalian MAPK: the p38 family, the p42 and p44 group (also known as ERK1 and ERK2), MAPKjnk, MAPKerk3/4 and MAPKerk5. Of these, ERK1 and 2 are the most extensively studied. Phosphorylation of the ERK proteins is followed by translocation to the nucleus where they activate a number of important mediators of cell proliferation including the proto-oncogenes c-fos, c-myc and c-jun. In addition there is also evidence that ERK1 and 2 are involved in the transduction of VSMC migration signals such as platelet-derived growth factor (PDGF) [13,14]. Since ERK1 and 2 have been implicated in mediating these processes, their down-regulation would appear to be a potentially useful means of reducing neointima formation following vascular injury. This hypothesis was tested in this study by attempting to down-regulate MAPK expression in a porcine coronary angioplasty model.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animal preparation
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Chemicals were purchased from Sigma Aldrich (Poole, Dorset, UK) unless otherwise stated. Female large white pigs, between 20 and 25 kg were used (PSS Animal Supplies, London). The animals were sedated with ketamine 15 mg/kg by intramuscular injection; anaesthesia was induced by inhalation of 0.5% halothane and then maintained by intravenous infusion of fentanyl (0.01 mg/ml), etomidate (0.04 mg/ml) and ketamine (1 mg/ml). Animals were then intubated and mechanically ventilated. Antibiotic prophylaxis (benzyl penicillin, 600 mg and gentamycin, 80 mg) was administered as an intramuscular bolus injection. Animals received an intravenous bolus of heparin (50 U/kg) followed by an intravenous infusion of 50U/kg/h throughout the procedure. The electrocardiogram and arterial blood pressure was monitored continuously and blood pressure was recorded for all animals as soon as anaesthesia was established.

2.2 Porcine coronary angioplasty procedure
Following surgical exposure, an 8F sheath was placed in the right femoral artery and a 0.035 inch guide wire introduced and used to guide an appropriate 8F guiding catheter into the left or right coronary ostium. A 0.014 inch hi-torque floppy coronary guide wire (ACS Inc., USA) was then manoeuvred distally down the target vessel and used to advance a standard 3x20 mm dilatation balloon into the artery. The vessel was then dilated three times at 8 atmospheres for 30 s, with an intervening period of 60 s of deflation. Immediately following injury the dilatation balloon was removed and a DispatchTM local delivery catheter (Boston Scientific Inc., USA) advanced to the site of balloon dilatation. Oligodeoxynucleotide (ODN) or normal saline (NS) control to a total volume of 2 ml was then slowly infused through the delivery catheter over 30 to 40 s. Local delivery as opposed to intravenous administration of ODN was performed at the site of injury in order to achieve as high a concentration as possible within the injured vessel and to minimize degradation of the ODN before it reached its target. The sheath was then removed, the femoral artery tied off and the wound sutured.

2.3 Study design
A total of 38 coronary arteries were studied from 23 animals. These were divided into a 7-day group comprising those treated with normal saline control (NS, n=8), sense oligonucleotide control (SMK, n=7) and antisense ODN (AMK, n=7) and a 21-day group comprising NS (n=6), SMK (n=5) and AMK (n=5)-treated vessels. These points were chosen to study times when intimal proliferation is known to be around its maximal point (7 days) and a later time-point when neointimal proliferation is virtually complete [15].

2.4 Harvesting of arteries
In order to harvest the arteries, animals were anaesthetised and an arterial sheath placed in the left femoral artery as described above. An 8F pig-tail catheter was advanced into the ascending aorta and Evan's Blue (0.5 mg/kg) dissolved in normal saline was infused in order to delineate the site of arterial injury. Thirty minutes later, pigs were killed by a lethal intravenous injection of pentabarbitone sodium, the chest was opened and the heart removed. The appropriate coronary arteries were carefully dissected out, rinsed in normal saline to remove blood and the angioplasty site (as demonstrated by the Evans's Blue staining; 20 mm long) was retrieved. Rings of tissue were taken for histological examination from the middle and the ends of the specimen, leaving two larger segments, which were snap frozen in liquid nitrogen for protein extraction and Western blot analysis (see below). Arterial segments were fixed in 10% formalin for 4 h prior to paraffin embedding and 6 µm sections were taken from each segment for histological and immunocytochemical examination.

2.5 Oligodeoxynucleotides
Porcine p42 and p44 MAPK have not been cloned and sequenced, however ERKs are highly conserved between species and a search of the GenBank database using the GCG package revealed that the region of the translation initiation site of the gene is identical in rat and human. ODN sequences were therefore based on these identical sequences and were synthesised by the King's College School of Medicine and Dentistry Oligo Synthesis Service, London, UK. The antisense ODN used was a 17-mer directed against a sequence starting at the initiation codon (AMK 5'-GCC GCC GCC GCC AT-3'). Sense ODN (SMK 5'-ATG GCG GCG GCG GC-3') was utilised as a control. All bases were protected by phosphorothioation. The antisense sequence used has been characterised in detail and demonstrated to selectively down-regulate the p42 and p44 isoforms of MAPK, without affecting upstream or downstream elements in the transduction cascade [16].

2.6 Preparation of the liposomal transfection medium
ODNs were mixed with antibiotic and serum-free medium (DMEM, GIBCO) to a concentration of 0.8 µM. This was then mixed with an equal volume of medium containing 80 µg/ml of lipofectin (Life Technologies), vortexed and allowed to stand for 15 min at room temperature. A volume of 1.25 ml of this mixture was then added to an equal volume of medium giving a final concentration of 0.2 µM for the ODNs and 20 µg/ml of lipofectin. The concentration of ODN chosen was based on previous studies [17,18] which revealed that 0.2 µM ODN effectively down-regulated MAPK in vitro and that a higher concentration of 0.4 µM showed evidence of non-specific toxicity. The final concentration within the vessel wall in vivo is likely to have been somewhat less than was achieved in the rather more controlled environment of the in vitro studies due to clearance and degradation of ODN, but we were concerned to avoid increasing the concentration to levels where non-specific toxicity may have become a factor.

2.7 Histology and morphometry
Sections were stained with Haematoxylin and Eosin for the assessment of morphology and with Van Gieson stain to visualise the internal elastic lamina (IEL). Slides were coded prior to analysis in order to blind the observer to the treatment group from which the specimen originated. Morphometry was performed using a video microscope (Olympus) linked to an IBAS 2 image analyser (Kontron, Germany) incorporating a computer graphics programme which allows the delineation and calculation of areas of interest. The intimal and medial areas were measured and the ratio of the two calculated for each section. The intima was defined as the area separating the IEL from the lumen of the vessel and the media as the region between the internal and external elastic laminae. In the regions of IEL rupture, the neointima was identified by the difference in histological appearance of the two layers. Cells are small and closely packed in the neointima whereas in the media they are larger and more widely separated with abundant interspersed elastin.

It is well established that in the porcine coronary injury model, the neointima is much more prominent in regions of IEL rupture than elsewhere and indeed there is an excellent correlation between the extent of damage to the IEL and the intimal/medial (I/M) area ratio [19]. It is therefore necessary to normalise the I/M ratio to the degree of IEL damage in order to be able to meaningfully compare vessels that have undergone different treatments. This is achieved by calculating a rupture index (RI) by dividing the length of the rupture in the IEL by the length of the non-ruptured segment (Fig. 1). The I/M area ratio is then normalised by dividing by the RI giving a corrected I/M ratio which may then be used to legitimately compare groups of vessels [19].


Figure 1
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  		Fig. 1 Calculation of the corrected I/M ratio. A rupture index (RI) is calculated by dividing the rupture length by the length of the residual IEL. The I/M ratio (IMR) is calculated as the area of the neointima divided by the area of the media measured by computerised planimetry. The corrected I/M ratio is then calculated as

Figure 1
.

 
2.8 Detection of proliferation
This was achieved by immunohistochemical staining for Ki67, a marker of cell proliferation believed to be more specific than the more commonly used PCNA, which has been shown to also label cells undergoing DNA repair [20]. Staining was performed using a slight modification of the method described by Shi et al. [21]. Briefly, arterial sections were mounted on poly-L-lysine-coated slides and dewaxed and rehydrated through xylene and an alcohol series. Sections were blocked with 0.5% hydrogen peroxide and then autoclaved for 10 min in 0.01 M citrate buffer. Slides were incubated with a monoclonal anti-Ki67 antibody overnight at 4 °C (clone MIB-1, 1:80 dilution, Immunotech, Marseilles, France) and then with a biotinylated anti-mouse secondary antibody for 30 min (Vector Laboratories, UK). Positive cells were then visualised using a standard ABC staining kit according to the manufacturer's instructions (DAKO, UK). All staining runs contained a section treated with non-immune rabbit serum instead of the primary antibody as a negative control and a section of tonsil as a positive control. Any runs in which the controls were not satisfactory were discarded and the staining repeated.

Cell counting was performed using the same image analysis system used for the morphometry. A proliferation index was calculated separately for the intima and media using the following equation

Formula

2.9 Western blotting
Arterial segments were first ground to a powder under liquid nitrogen and the powder was then added to 500 µl of extraction buffer (20 mM Tris, pH 7.5, 5 mM EGTA, 1 mM dithiothreiotol (DTT), 20 mM β-glycerophosphate, 10 mM sodium fluoride, 1 µg/ml aprotinin and 1 mM each of PMF, TPCK and TLCK). Samples were then vortexed at 100,000 g for 5 min, placed on ice for 30 min and then vortexed again at the same speed for 10 min at 4 °C. Protein content was determined using the Bradford protein assay kit. Following addition of 3xLaemmli buffer, (0.33 mol/l Tris/HCL, 10% sodium dodecyl sulphate, 13% glycerol, 0.1 mol/l DTT, 0.13 mg/ml Bromophenol Blue), samples were denatured by boiling for 5 min and then resolved on 12% polyacrylamide gels and transferred to PVDF membranes. Equal quantities of protein were loaded in each lane, determined by the Bradford assay and this was further confirmed by Ponceau staining of the PVDF membranes. Any membranes in which there appeared to be unequal loading of the lanes were discarded. Non-specific binding to the membranes was blocked using 5% milk powder in PBS/0.05% Tween 20 (PBS/Tween) for 1 h and then anti-ERK antibody (Zymed, USA) was added to a concentration of 1:3000 and the membranes incubated overnight at 4 °C. Following three washes in PBS/Tween, the membranes were exposed to the secondary antibody, diluted 1 in 5000 in PBS/Tween/1% milk powder for 1 h at room temperature. After three further washes in PBS/Tween, the membranes were developed using ECL reagent (Amersham International, UK) according to the manufacturers instructions and autoradiograms recorded on Kodak Ektachrome film. Quantitation of the results was by scanning using a Bio-Rad GS3000 densitometer connected to an Apple Power Macintosh computer, followed by analysis using the Bio-Rad Molecular Analyst software. Initial studies had shown that this system produced a linear relationship between the quantity of ERK protein run on the gel and the determined density of the band (R2=0.98, data not shown). The densitometric values were normalised in each case to produce a value for the normal saline controls of 100% to account for exposure differences between different blots.

2.10 Statistical analysis
Data are presented as means±S.E.M. unless otherwise stated. Statistical comparisons between paired groups were performed using the paired t-test. Comparisons of multiple groups were done using one-way ANOVA, with subsequent testing for individual intergroup significant differences using the Student's Newman–Keuls post test. Linear regression analysis was used to test the relationship between the RI and the I/M ratio and ANOVA to detect differences in the gradient of the regression lines. A P-value of less than 0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Histological examination
At 7 days after injury, the degree of neointima formation, was significantly reduced by antisense ODN to MAPK as compared with sense (37%, P<0.01) and saline controls (43%, P<0.01) (Figs. 2 and 3Go). The degree of intimal response, reflected most accurately by the corrected I/M ratio, was shown to be directly and linearly related to the degree of injury in all three treatment groups (Fig. 4) in agreement with previous work [25]. The gradient of the regression lines in Fig. 4 represents the corrected I/M ratio for each of the treatment groups and revealed a significant reduction by AMK compared with sense and saline controls implying an abrogated response to injury (P<0.01). These early inhibitory effects of antisense ODN to MAPK were no longer seen at 21 days after injury when there was no significant difference between any of the treatment groups.


Figure 2
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  		Fig. 2 Photomicrographs illustrating the response to balloon angioplasty in pig coronary arteries at 7 days after injury. (A) Control uninjured vessel. (B) Vessel treated with saline control. (C) Vessel treated with sense oligonucleotide. (D) Vessel treated with antisense oligonucleotide. The results illustrate the significant reduction in neointima (NI) formation particularly evident at the site of internal elastic lamina (IEL) rupture and greatest medial (M) injury.

 

Figure 3
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  		Fig. 3 Injury response at 7 days in the different treatment groups. *P<0.01 vs. both SMK and NS groups.

 

Figure 4
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  		Fig. 4 Seven-day injury response data shown as a plot of the uncorrected I/M ratio against the rupture index. Note the strong linear relationship between the two variables and also the reduction in the corrected I/M ratio in the AMK group as indicated by the significant reduction in the gradient of the regression line. The equations of the regression lines and their R2 values are shown in the legend. *Gradient of line P<0.01 vs. NS and SMK groups.

 
3.2 MAPK protein levels
Both isoforms of MAPK were up-regulated by arterial injury compared with uninjured segments, in agreement with existing data from rodent models [22]. At 7 days after injury, treatment with antisense oligonucleotide significantly down-regulated ERK-1 by 54% (P<0.01) and ERK-2 by 23% (P<0.05) as compared with saline, while sense ODN produced no effect on MAPK protein expression (Fig. 5a). By 21 days after injury, the down-regulation of ERK-2 was still present (20% vs. saline control, P<0.05), although less marked, while ERK-1 protein levels were not significantly influenced by antisense ODN (Fig. 5b).


Figure 5
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  		Fig. 5 (a) Effect of treatment group on MAPK protein expression at 7 days post angioplasty. Control is uninjured artery. NS, normal saline treatment group. SMK and AMK are sense and antisense oligonucleotide groups, respectively. *P<0.05 vs. NS. **P<0.01 vs. NS. (b) Effect of treatment group on MAPK expression at 21 days.

 
3.3 Cell proliferation
SMC proliferation was assessed separately in the intima and media at early and late time-points. As has been previously demonstrated in porcine balloon injury models, levels of proliferation in the neointima and media were greatest at 7 days after injury [23]. Treatment with antisense, but not sense, ODN led to a significant inhibition of cell proliferation at 7 days after injury, which was apparent in both intima and media (Fig. 6). These inhibitory effects were lost by 21 days, a time-point at which levels of cell turnover were returning to baseline values.


Figure 6
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  		Fig. 6 Effects of treatment on Ki67 proliferation index at 7 and 21 days. Labelling is as described for Fig. 4.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The results of this study show that a single, per-lumenal treatment with locally delivered antisense ODN to MAPK resulted in down-regulation of ERK-1 and ERK-2, which was associated with a significant early reduction in neointima formation and inhibition of SMC proliferation following coronary arterial balloon injury. Our results corroborate the importance of ERK in mediating early post PTCA responses by demonstrating its up-regulation following injury [22] and, for the first time in a large animal model, the impact of its inhibition on both proliferation and neointimal volume following vascular injury. Previous studies have demonstrated that down-regulation of elements of the MAPK signalling pathway can reduce neointima formation in response to arterial injury in vivo [24,25] but these have been performed in rodent models. While it seems that the intracellular signalling cascades are similar in different species [12], there are known to be important inter-species differences in the mechanisms and histology of the arterial injury response [26]. The porcine coronary model of arterial balloon injury is generally considered to closely represent the human situation in that the structure of the vessels resembles those of the human and the mechanism of injury involves dissection and thrombus formation, both features of clinical angioplasty [27,28].

An early reduction in neointima was clearly apparent from the reduction in mean corrected I/M ratio in the antisense-treated group. Studying the extent of neointima formation in relation to the severity of internal elastic lamina rupture and injury demonstrated two additional important features of our results. Firstly, there was a consistent linear relationship between the degree of injury and the neointimal response in all treatment groups. This underpins the importance of controlling for degree of injury by the use of the corrected I/M ratio in similar future studies. Secondly, a reduction in ERK was associated with an abrogation of the intensity of intimal response at all degrees of injury, implying the ability of this intervention to down-regulate intimal proliferation across a full range of mechanical trauma. The mechanism by which this is achieved is speculative. Previous in vitro studies have demonstrated that the MAPK system is instrumental in transducing the epidermal growth factor-induced proliferative response in vascular smooth muscle cells [29] and also is involved in the intracellular signalling responses to platelet-derived growth factor and angiotensin II [30], all factors which have been implicated in neointima formation in vivo [2,5,31]. Binding of the appropriate ligand to the cell surface receptor leads to activation of the ras/raf system which in turn activates a kinase cascade culminating in the phosphorylation of ERK itself [11]. This active form of ERK translocates to the nucleus where it leads to progression through the cell cycle through the activation of a variety of nuclear proteins [30,32]. We have previously demonstrated that antisense oligonucleotides to the p42 and p44 MAPKinase down-regulate target protein and inhibit porcine VSMC proliferation in vitro [18]. The present results extend this study by demonstrating that delivery of the same ODN is associated with inhibition of proliferation in vivo. It cannot be assumed, however, that this was the only mechanism by which intima formation was reduced, since inhibition of ERK may also have the potential to inhibit VSMC migration, although these differential effects will need to be assessed in future studies.

In contrast to the results at 7 days, no significant effects were observed on either cell proliferation or I/M ratio at 21 days. Two explanations for this observation seem possible: the oligonucleotide may simply not have persisted long enough in the tissue and ‘catch up’ growth obliterated the early inhibition of neointima production; alternatively different mechanisms may operate to produce neointimal thickening at later time-points which are not dependent on MAPK-transduced signals. The fact that the down-regulation of MAPK protein levels was much less marked at 21 days provides some support for the former hypothesis. To our knowledge, little is known regarding the relative contribution of proliferation, migration, matrix production and apoptosis to neointima formation at different time-points in the porcine model. Future work should therefore aim to produce sufficiently sustained delivery of oligonucleotide to significantly down-regulate MAPK levels at late time-points as well as assessing the individual contribution of these mechanisms to neo-intimal volume.

A potential criticism of the current study concerns the fact that porcine ERK has not yet been cloned and sequenced and thus it is not known that the AMK sequence is truly complementary to the 5' end of the cDNA. In response to this it should be pointed out that the translation initiation site of both ERK-1 and ERK-2, against which AMK was directed, is completely conserved in all mammalian species in which the gene has been sequenced. More importantly the object of using an antisense strategy was to down-regulate the expression of ERK protein and we have demonstrated that this was achieved via the immunoblotting experiments. Non-specific effects of antisense ODNs have been previously noted, both in vitro [33] and in vivo in the porcine arterial injury model [34]. Therefore, possible different mechanisms of action need to be considered (see Bennett [33] for a full review). Studies investigating the antiproliferative mechanism of action of oligonucleotides directed against the proto-oncogene c-myb [35] have demonstrated that this effect was mediated via a non-antisense mechanism, being dependent on the presence of four successive guanine residues in the antisense ODN (the 4G effect) [36]. This is not applicable in the current study however, as the oligonucleotides used here do not contain a 4G sequence. Similarly, unmethylated GC nucleotide pairs have been demonstrated to have non-specific, inflammatory effects [37]. However, these do not appear to have played a significant part in these studies as the sense control sequence (SMK) contained an identical number of GC pairs, yet did not exhibit antiproliferative effects. Direct interaction of oligonucleotides with cellular proteins, so modifying their function, has also been described [38], but this so-called aptameric effect has only been noted to occur at ODN concentrations at least 50-fold higher than those used in the current experiments. It is notable that in the experiments of Gunn et al. [34], which noted non-specific effects of locally delivered ODNs, comparatively high concentrations of oligonucleotide, in excess of 10 µM, were used. In addition, down-regulation of the target proteins was not confirmed in the in vivo experiments. In contrast our experiments were performed using 0.2 µM oligonucleotide, a concentration which has been previously demonstrated to specifically down-regulate ERK [17,18] and in addition, down-regulation of the protein levels by the antisense sequence, but not by the sense control, was demonstrated by the immunoblotting experiments making non-specific effects unlikely.

In summary, we have demonstrated for the first time that down-regulation of MAPK protein is associated with a significant, early reduction in cellular proliferation and neointima formation in response to balloon injury in a large animal coronary model. Nevertheless, the loss of these effects at 3 weeks suggests that further work is required to obtain a greater duration of suppression of MAPK in order to determine whether this strategy is sufficient to affect the long-term arterial injury response. These data corroborate the importance of the p42 and p44 MAPK proteins in mediating the vascular injury response and identify a promising target for the modulation of this in humans and therefore the treatment of such clinical problems as restenosis following PTCA and vein graft failure following coronary bypass surgery.

Time for primary review 32 days.


   Acknowledgements
 
Dr Fisher is supported by the British Heart Foundation and the Medical Research Council, UK. Dr Liu is supported by a research grant from Boston Scientific Inc. This work benefited from the use of the SEQNET computing facility at Daresbury, UK.


   Notes
 
1 Contributed equally to the study. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Schwartz S.M, deBlois D, O'Brien E.R.M. The intima-soil for atherosclerosis and restenosis. Circ Res (1995) 77:445–465.[Free Full Text]
  2. Ross R. Cell biology of atherosclerosis. Annu Rev Physiol (1995) 57:791–804.[CrossRef][Web of Science][Medline]
  3. Bryan A.J, Angelini G.D. The biology of saphenous vein graft occlusion: etiology and strategies for prevention. Curr Opin Cardiol (1994) 9:641–649.[Web of Science][Medline]
  4. Clowes A.W, Clowes M.M. Kinetics of cellular proliferation after arterial injury. Circ Res (1986) 58:839–845.[Abstract/Free Full Text]
  5. Jackson C.L, Raines E.W, Ross R, Reidy M.A. Role of endogenous platelet-derived growth factor in arterial smooth muscle migration after balloon catheter injury. Arterioscler Thromb (1993) 13:1218–1226.[Abstract/Free Full Text]
  6. George S.J, Williams A, Newby A.C. An essential role for platelet-derived growth factor in neointima formation in human saphenous vein in vitro. Atherosclerosis (1996) 120:227–240.[CrossRef][Web of Science][Medline]
  7. Burgess W.H, Maciag T. The heparin binding (fibroblast) growth factor family of proteins. Annu Rev Biochem (1989) 58:575–606.[CrossRef][Web of Science][Medline]
  8. Post M.J, Borst C, Pasterkamp G, Haudenschild C.C. Arterial remodeling in atherosclerosis and restenosis: a vague concept of a distinct phenomenon. Atherosclerosis (1995) 118(Suppl.):S115–S123.[Web of Science][Medline]
  9. Schwartz R.S, Holmes D.R Jr., Topol E.J. The restenosis paradigm revisited: an alternative proposal for cellular mechanisms [editorial]. J Am Coll Cardiol (1992) 20:1284–1293.[Abstract]
  10. Pelech S.L, Sanghera J.S. Mitogen-activated protein kinases: versatile transducers for cell signalling. Trends Biochem Sci (1992) 17:223–238.[CrossRef][Web of Science][Medline]
  11. Davis R.J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem (1993) 268:14553–14556.[Free Full Text]
  12. Widmarm C, Gibson S, Jarpe M.B, Johnson G.L. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev (1999) 79:143–180.[Abstract/Free Full Text]
  13. Lundberg M.S, Curto K.A, Bilato C, Monticone R.E, Crow M.T. Regulation of vascular smooth muscle migration by mitogen-activated protein kinase and calcium/calmodulin-dependent protein kinase II signaling pathways. J Mol Cell Cardiol (1998) 30:2377–2389.[CrossRef][Web of Science][Medline]
  14. Graf K, Xi X.P, Yang D, Fleck E, Hsueh W.A, Law R.E. Mitogen-activated protein kinase activation is involved in platelet-derived growth factor-directed migration by vascular smooth muscle cells. Hypertension (1997) 29:334–339.[Abstract/Free Full Text]
  15. Strauss B, Robinson R, Batchelor W.B, Chisholm R.J, Ravi G, Natarajan R, et al. In vivo collagen turnover following experimental balloon angioplasty injury and the role of matrix metalloproteinases. Circ Res (1996) 79:541–550.[Abstract/Free Full Text]
  16. Robinson C.J.M, Scott P.H, Allan A.B, Jess T, Gould G.W, Plevin R. Treatment of vascular smooth muscle cells with antisense phosphorothioate oligodeoxynucleotides directed against p42 and p44 mitogen-activated protein kinases abolishes DNA synthesis in response to platelet-derived growth factor. Biochem J (1996) 320:123–127.[Web of Science][Medline]
  17. Glennon P.E, Kaddoura D, Sale E.M, Sale G.I, Fuller S.J, Sugden P.H. Depletion of mitogen-activated protein kinase using an antisense oligodeoxynucleotide approach downregulates the phenylephrine-induced hypertrophic response in rat cardiac myocytes. Circ Res (1996) 78:954–961.[Abstract/Free Full Text]
  18. Fisher N.I, Liu B, Glennon P.E, et al. Downregulation of the ERK 1 and ERK 2 mitogen activated protein kinases using antisense oligonucleotides inhibits proliferation of porcine vascular smooth muscle cells. Atherosclerosis (2001) 156:289–295.[CrossRef][Web of Science][Medline]
  19. Bonan R, Paiement P, Scortichini D, Cloutier M.J, Leung T.K. Coronary restenosis: evaluation of a restenosis injury index in a swine model. Am Heart J (1993) 126:1334–1340.[CrossRef][Web of Science][Medline]
  20. Kanoh M, Takemura G, Misao I, Hayakawa Y, Aoyama T, Nishigaki K, et al. Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy: not apoptosis but DNA repair. Circulation (1999) 99:2757–2764.[Abstract/Free Full Text]
  21. Shi Y, Fard A, Galeo A, Hutchinson H.G, Vermani P, Dodge G.R, et al. Transcatheter delivery of c-myc antisense oligomers reduces neointimal formation in a porcine model of coronary artery balloon injury. Circulation (1994) 90:944–951.[Abstract/Free Full Text]
  22. Lai K, Wang H, Lee W.S, Jain M.K, Lee M.E, Haber E. Mitogen-activated protein kinase phosphatase-1 in rat arterial smooth muscle cell proliferation. J Clin Invest (1996) 98:1560–1567.[Web of Science][Medline]
  23. Malik N, Francis S.E, Holt C.M, Gunn I, Thomas G.L, Shepherd L, et al. Apoptosis and cell proliferation after porcine coronary angioplasty. Circulation (1998) 98:1657–1665.[Abstract/Free Full Text]
  24. Indolfi C, Avvedimento E.V, Rapacciuolo A, Di Lorenzo E, Esposito G, Feliciello A, et al. Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in vivo [see comments]. Nature Med (1995) 1:541–545.[CrossRef][Web of Science][Medline]
  25. Indolfi C, Avvedimento E.V, Rapacciuolo A, Esposito G, Di Lorenzo E, Leccia A, et al. In vivo gene transfer: prevention of neointima formation by inhibition of mitogen-activated protein kinase kinase. Basic Res Cardiol (1997) 92:378–384.[Web of Science][Medline]
  26. Muller D.M.W, Ellis S.G, Topol E.G. Experimental models of coronary artery restenosis. J Am Coll Cardiol (1992) 19:418–432.[Abstract]
  27. Bauters C, Labanche J, McFadden E, Hamon N.I, Bertrand M.E. Relation of coronary angioscopic findings at coronary angioplasty to angiographic restenosis. Circulation (1995) 92:2473–2479.[Abstract/Free Full Text]
  28. Peters M.G, Kok W.E.M, Di Mario C, Serruys P.W, Bar F.W.H.M, Pasterkamp G, et al. Prediction of restenosis after coronary balloon angioplasty. Circulation (1997) 95:2254–2261.[Abstract/Free Full Text]
  29. Yu S, Hung L, Lin C. cGMP-elevating agents suppress proliferation of vascular smooth muscle cells by inhibiting the activation of epidermal growth factor signalling pathway. Circulation (1997) 95:1269–1277.[Abstract/Free Full Text]
  30. Liao D.F, Duff I.L, Daum G, Pelech S.L, Berk B.C. Angiotensin II stimulates MAP kinase kinase kinase activity in vascular smooth muscle cells. Role of Raf. Circ Res (1996) 79:1007–1014.[Abstract/Free Full Text]
  31. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (1993) 362:801–809.[CrossRef][Medline]
  32. Gille H, Sharrocks A.D, Shaw P.E. Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at the c-fos promoter. Nature (1992) 358:414–417.[CrossRef][Medline]
  33. Bennett N.I.R, Schwartz S.M. Antisense therapy for angioplasty restenosis. Circulation (1995) 92:1981–1993.[Free Full Text]
  34. Gunn J, Holt C.M, Francis S.E, Shepherd L, Grohmann M, Newman C.M.H, et al. The effect of oligonucleotides to c-myb on vascular smooth muscle cell proliferation and neointima formation after porcine coronary angioplasty. Circ Res (1997) 80:520–531.[Abstract/Free Full Text]
  35. Simons M, Edelman E.R, DeKeyser J, Langer R, Rosenberg R. Antisense c-myb oligonucleotides inhibit arterial smooth muscle cell accumulation in vivo. Nature (1992) 359:67–70.[CrossRef][Medline]
  36. Castier Y, Chemia E, Nierat I, Heudes D, Vasseur M.A, Rajnoch C, et al. The activity of c-myb antisense oligonucleotide to prevent intimal hyperplasia is nonspecific. J Cardiovasc Surg (1998) 39:1–7.[Medline]
  37. Varga L.V, Toth S, Novak I, Falus A. Antisense strategies: functions and applications in immunology. Immunol Lett (1999) 69:217–224.[CrossRef][Web of Science][Medline]
  38. Bock L.C, Griffin L.C, Latham J.A, Vermaas E.H, Toole J.J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature (1992) 355:564–566.[CrossRef][Medline]

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