Introduction

The use of conventional bare-metal stents for the treatment of proliferative atherosclerotic lesions has resulted in a reduction of acute vessel closure with better long-term angiographic results after percutaneous coronary intervention as compared with angioplasty alone.¹ However, a high rate of in-stent restenosis (ISR) persists with the use of these stents.² The local delivery of antiproliferative drugs using drug-eluting stents (DESs) has assisted in achieving a reduction in the restenosis rate observed with bare metal stents.^2,3 Although there is a definite short to midterm benefit with a reduction in the revascularization procedures using DES, recent long-term meta-analysis of DES studies has raised questions about their long-term safety.^4,5 Poor long-term results with the use of DES may stem from the unselected inhibition of endothelial and smooth muscle cell (SMC) division by antiproliferative drugs resulting in incomplete re-endothelialization of stented vessels with an increased risk of late-stent thrombosis.⁶

As with DES, gene-eluting stents can deliver biologically active agents locally for prolonged duration to the site of vascular disease. We have previously shown that adenovirus and adeno-associated virus serotype 2 vectors can be used to deliver marker genes to the blood vessel wall for at least up to 28 days using phosphorylcholine (PC)-coated stents as delivery platform.⁷ The use of stents as scaffolds for localized and prolonged gene delivery of therapeutic genes into the diseased blood vessel wall is an appealing strategy for targeting the biological basis of restenosis. This process is a multifactorial pathological event with endothelial denudation and vascular SMC proliferation as its primary contributors. Accelerated re-endothelialization of stents is a desirable post-intervention event, which can result in a significant reduction in the risk of late stent thrombosis.⁸ In addition, endothelial regeneration can in turn reduce excessive neointimal formation by inhibiting SMC proliferation and migration.⁹ All major coronary risk factors are associated with endothelial dysfunction.¹⁰ Most of these risk factors cause endothelial dysfunction by increased production of oxygen-free radicals (predominantly super oxide anion) that combine with and inactivate nitric oxide (NO)¹¹ resulting in increased platelet aggregation, monocyte adhesion, vasoconstriction, and decreased fibrinolysis, all of which are important factors in the development of atherosclerosis, plaque rupture, and vessel thrombosis.

An ideal therapeutic approach to prevent ISR would allow endothelial regeneration while inhibiting SMC proliferation. Augmentation of vascular NO levels may achieve this outcome. In this study, we delivered an adenoviral vector encoding endothelial NO synthase (eNOS) to the blood vessel wall using PC-coated stents as a delivery platform. Local delivery of eNOS increases NO production, which is a pleiotropic agent acting as a potent vasodilator in many vascular beds with additional antithrombotic¹² and antiproliferative properties.¹³ Previous studies have suggested that eNOS gene delivery to the blood vessel wall after balloon injury can result in accelerated re-endothelialization,⁹ alter vascular reactivity,^14,15 and reduce neointimal formation.^9,16 However, gene-eluting stents have not been used to deliver eNOS to assess its therapeutic effect on the response to vascular injury in diseased animals.

Results

Detection of eNOS expression

Stented iliac arteries transduced with AdGal and AdeNOS were harvested 3 days after stent placement. Following qualitatively reverse transcriptase-PCR analysis, eNOS signal expression could be observed in all the AdeNOS-transduced arteries examined (n = 3). RNA obtained from these tissue samples demonstrated the presence of the appropriate-sized band for eNOS in arteries tested after reverse transcriptase-PCR analysis as shown in Figure 1.

Figure 1.

Detection of endothelial nitric oxide synthase (eNOS) expression by reverse transcriptase PCR of AdeNOS-stented vessel segments after 72 hours. eNOS expression was not visualized in the control phosphorylcholine (PC) or naive blood vessels or those treated with AdGal.

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Localization of eNOS gene expression

Stent-based delivery of AdeNOS to the rabbit iliac arteries resulted in localized overexpression of eNOS. This eNOS expression was localized to the areas around the stent struts in the intimal and medial layers as previously demonstrated with -galactosidase gene delivery using AdGal- and AAV2Gal-coated stents.⁷ The expression was localized at 6 days after eNOS delivery and in contrast there was no eNOS staining observed in the blood vessels treated with AdGal-coated stents. Moreover, control antibody–treated stents did not demonstrate any background staining (Figure 2).

Figure 2.

Immunohistochemical localization of endothelial nitric oxide synthase (eNOS) protein within the stented rabbit iliac artery 6 days after transduction and stent deployment. Expression was noted in (a) the endothelial/intimal layer and (b) medial layers. (c) eNOS expression was not detected in AdGal-treated vessels. Original magnification40. (d) AdeNOS-treated arteries probed using anti-green fluorescent protein mouse monoclonal antibody did not demonstrate any background staining. Original magnification10. Stent struts are indicated with S.

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Endothelial regeneration

Endothelial regeneration was determined using three independent methodologies: Evan's blue staining, scanning electron microscopy, and histomorphometry.

Two weeks after balloon injury and stent placement, endothelial regeneration was assessed in the normocholesterolemic-diet animals treated with AdeNOS- and AdGal-coated stents using Evans blue staining (n = 4 each). Luminal staining for Evans blue demonstrated that the balloon angioplasty procedure completely denuded the right iliac artery (Figure 3). Re-endothelialization was significantly greater in the AdeNOS-stented vessels (85.34% 7.4) in comparison with AdGal-treated control vessels (62.66% 10.5) (P < 0.05) (Figure 3).

Figure 3.

Re-endothelialisation of vessels following stent deployment as detected by Evans blue staining. (a) A control unstented artery demonstrates an intact endothelial layer, which does not take up Evans blue stain. Decreased uptake of Evans blue was noted for stents coated with (b) AdeNOS at 14 days after balloon injury and stent implantation compared with (c) AdGal-coated stents. (d) Similarly, high levels of Evans blue uptake denoting an injured vessel wall were noted in control arteries treated with angioplasty alone and stained within 30 minutes of injury. (e) Quantification of percentage re-endothelialization at 14 days after Evans blue staining using imaging software in the AdeNOS- and control AdGal-treated vessels. *P < 0.05.

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Furthermore, at 2 weeks after stent deployment, a cobblestone appearance was noted between the struts for AdeNOS-treated stents using scanning electron microscopy, indicating re-endothelialization (Figure 4a). Moreover, cells morphologically consistent with endothelial cells covering stent struts were noted (Figure 4c). In contrast, AdGal-treated vessels lacked endothelial cell morphology between struts (Figure 4b).

Figure 4.

Re-endothelialisation of vessels following stent deployment as detected by electron microscopy and histology. Early regeneration of the cobblestone appearance of endothelial cells in the (a) endothelial nitric oxide synthase (eNOS)-treated stents (original magnification350) as compared with (b) control AdGal stents (original magnification368). (c) Cells consistent with endothelial morphology were noted on the surface of AdeNOS-treated stent struts after deployment at day 14 (original magnification410). (d) Graphic assessment of re-endothelialization by histological analysis at day 28 after stent placement in the AdeNOS-treated vessels and control AdGal stents in both normal and high cholesterol–diet animals.*, **P < 0.05.

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Four weeks after the stent placement, histological assessment of endothelial regeneration demonstrated a persistence of significant difference in endothelial regeneration between the two groups in the normal-diet animals. The percentage of endothelial cells observed in the AdeNOS-stented vessels was significantly higher than in the control vessels (91.1% 10 versus 63.1% 22; P < 0.05). Endothelial regeneration was also significantly higher at 4 weeks in the AdeNOS group in the hypercholesterolemic animals versus AdGal group (96.97% 3.2 versus 28.33% 38.76; P < 0.05) (Figure 4d).

Histomorphometric analysis

The morphometric data of cross-sectional vessel areas and neointimal response of the normocholesterolemic animals treated with AdeNOS, AdGal, or PC stents are shown in Table 1 and Figure 5. A significant reduction in intimal area was observed in the AdeNOS-treated vessels in comparison with AdGal control (AdeNOS 1.17 1.13 mm² versus AdGal 1.46 0.76 mm²; P < 0.05). However, no significant differences were observed in any other parameters including the luminal area (P > 0.05), medial area (P > 0.05), and percentage stenosis (P > 0.05) between the three groups (Table 1). The injury scores after stent deployment were comparable in all the three groups in the normocholesterolemic animals (AdeNOS 1.03 0.22, AdGal 1.12 0.31, PC 1.19 0.26; P > 0.05). There was a nonsignificant trend toward a reduced inflammation score in the AdeNOS-treated vessels in comparison with controls (AdeNOS group 0.3 0.46, AdGal 0.7 0.32, PC 0.5 0.4; P = 0.06 and 0.3, respectively).

Figure 5.

Histomorphometric analysis of stented vessels from normocholesterolemic animals. Representative cross-sectional histology of AdeNOS stents (a,b) compared with AdGal stents (c,d) and phosphorylcholine (PC) stents (e,f) at 28 days after stent deployment. (g) Percent restenosis in AdeNOS-, AdGal-, and PC-treated vessels at 28 day after stenting (AdGal, AdeNOS n = 9 each, PC n = 8).

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Table 1 - Histomorphometric data comparing stents with and without viral coatings.

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The serum low-density lipoprotein and triglyceride levels in the high cholesterol animals were seven- and threefold higher than normal chow animals, respectively (P > 0.05). There was no difference in the levels of low-density lipoprotein and TG serum concentration between high cholesterol therapeutic animals and control animals (P < 0.05). In contrast to normal-diet animals, morphometric analysis in hypercholesterolemic animals showed a significantly larger luminal area in the AdeNOS-treated blood vessels (n = 6) in contrast to AdGal- and PC-treated vessels (n = 6 and 9, respectively) (Table 1, AdeNOS 2.73 mm² 1.18, AdGal 0.98 mm² 0.98, PC 1.87 mm² 1.18; P < 0.05), whereas the percentage restenosis (AdeNOS 45.23 20.81, AdGal 79.6 20.31, PC 70.16 22.2; P < 0.05) and neointimal area (AdeNOS 2.32 mm² 1.13, AdGal 3.73 mm² 0.95, PC 3.2 mm² 0.94; P < 0.05) was significantly greater in the control stents at 4-week follow-up (Figure 6).Two vessels in the AdGal-treated group in high cholesterol diet resulted in 100% ISR by excessive neointimal formation. There was no significant difference in the injury score between the AdeNOS- and the AdGal-treated vessels (AdeNOS 1.38 0.21, AdGal 0.98 0.21, PC 1.21 0.22; P > 0.05).

Figure 6.

Histomorphometric analysis of stented vessels from hypercholesterolemic animals. Representative cross-sectional histology of AdeNOS-stented vessels (a,b) and AdGal-stented vessels (c,d) and phosphorylcholine (PC)-stented vessels (e,f) at 28 days after stent deployment. (g) Percent restenosis in AdeNos-, AdGal-, and PC-treated vessels at 28 day after stenting. (AdGal, AdeNOS n = 6 each, PC n = 9) *P < 0.05.

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Quantitative angiography

Iliac artery angiography was performed at 4 weeks after balloon injury and stenting in the AdeNOS, AdGal, and PC groups in normal diet (n = 9, 9, and 8, respectively) and in the hypercholesterolemic animals (AdeNOS and AdGal n = 6 each and PC n = 9) In the normocholesterolemic group, there was no significant difference observed regarding the extent of ISR between the PC, AdGal, and AdeNOS stents (P > 0.05) (Figure 7a). This data was consistent with the morphometric data concerning luminal area stated above. However, in the hypercholesterolemic animals, there was a significant difference in the percentage of ISR between the AdeNOS (n = 6), AdGal (n = 6), and PC groups (n = 9) (AdeNOS 15.95% 7.63, AdGal 56.9% 38.6, PC 58 34.6; P < 0.05). Similar to morphometric results there was 100% restenosis in two control vessels on follow-up angiography. Results of angiographic assessment of ISR in the normal diet and hypercholesterolemic animals are summarized in Figure 7a and representative angiographic images of bilateral stenting in hypercholesterolemic animals with AdeNOS and AdGal stents are shown in Figure 7b and c.

Figure 7.

The effect of AdeNOS delivery on in-stent restenosis assessed using quantitative angiography. (a) Comparison of quantitative angiography at 28 days after stent deployment in normocholesterolemic and hypercholesterolemic rabbits. Stent restenosis in the AdGal and phosphorylcholine (PC) stents was significantly higher with comparison to the AdeNOS stents in hypercholesterolemic animals *P < 0.05. (b,c) Representative angiographic images of iliac arteries with bilateral stents coated with AdeNOS compared with AdGal in hypercholesterolemic animals at 28 days after stent deployment, respectively. Significant in-stent restenosis can be observed in AdGal-treated stents in both iliac arteries and is highlighted by arrows.

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Discussion

We investigated the possibility of using an eNOS gene–eluting stent as an alternative treatment strategy to reduce neointimal formation while accelerating endothelial regeneration. The rationale for this study was to develop a therapeutic strategy which would reduce ISR without increasing the risk of thrombosis. We used a PC-coated stent for adenovirus-mediated gene delivery to the vessel wall, a method which we have recently described.⁷ The data show that there was a significant acceleration of re-endothelialization in the eNOS-stented blood vessels as early as 14 days which persisted for at least up to 28 days after the procedure with stent-based eNOS gene delivery in comparison with the control stents. This was accompanied by a significant reduction in the formation of neointima in the eNOS-stented vessels in hypercholesterolemic animals, as observed by histomorphometric analysis and quantitative coronary analysis. Thus, we have demonstrated for the first time that stent-based eNOS gene delivery results in a reduction of ISR while promoting endothelial regeneration.

The use of DES has markedly reduced the incidence of ISR but is associated with a small but definite risk of late stent thrombosis. A recent published report has shown that DES may in fact increase long-term mortality and Q-wave infarction after discontinuation of clopidogrel in contrast to bare- metal stents.^4,5 Combination treatment with aspirin and clopidogrel has dramatically reduced the incidence of stent thrombosis. However, approaches to reduce ISR which would promote endothelial regeneration may provide an alternative strategy which would reduce the risk of in-stent thrombosis. A further advantage of this approach would be the avoidance of the deleterious consequences of discontinuation of antiplatelet drugs. Like DES, gene-eluting stents have the advantage of combining revascularization and delivery of a therapeutic agent to the local plaque site.

An optimal stent/polymer/gene combination will be required for the successful implementation of this therapeutic strategy. We have recently shown that PC-coated stents can be used to deliver genes successfully to the stented blood vessel wall. In particular, we have shown the superiority of adenoviral vectors to deliver reporter genes compared with adeno-associated virus serotype 2 for prolonged gene delivery.⁷ In this study, placement of an eNOS gene–eluting stent resulted in localized gene delivery at the site of stent implantation, and gene overexpression was observed around the stent struts. This would allow localized delivery of NO to the site of injury. eNOS expression was observed as early as 72 hours after stent deployment by real-time PCR and by immunohistochemical analysis at 6 days after stent placement. The viral dose used for this study was 5 10⁹ plaque-forming units which is consistent with previously used doses of this vector. We have previously shown that PC-coated stents result in transgene expression for at least 28 days and that this is located predominantly in the neointima at these later time points.⁷

Having previously determined the characteristics of stent-based gene delivery, in this study we sought to determine whether the resulting increase in vascular NO bioavailability would have beneficial therapeutic effects. We were particularly interested to determine whether augmenting vascular wall NO bioavailability through eNOS gene delivery would inhibit intimal hyperplasia while increasing endothelial regeneration. NO has pleiotropic beneficial effects in the blood vessel wall including vasodilatation, inhibition of platelet adhesion, matrix metalloproteinase inhibition, anti-inflammatory effects, and inhibition of SMC proliferation.^{11,12,13,14,15,16} We have previously demonstrated in vitro and in vivo,^9,17 the inhibitory effects of NO on SMC proliferation which has been consistently reported.^17,18 We have also shown that adenovirus-mediated gene delivery of eNOS to the blood vessel wall has beneficial effects in animal models of a large number of vascular disease states.^15,19,20 The current report adds to these observations by demonstrating inhibition of in-stent restenosis in a clinically relevant model after adenovirus-mediated gene delivery of eNOS from a PC-coated stent. We chose eNOS as the isoform to study because of our previous observation that iNOS overexpression in the injured blood vessel wall reduces intimal hyperplasia and also inhibits endothelial regeneration.⁹

The major new finding in this study is the effect of eNOS gene delivery on endothelial regeneration in this clinically relevant model of vascular injury. The effect of NO on endothelial cell proliferation has produced variable results in the literature.^21,22,23 There is less information available on the effect of adenovirus-mediated gene delivery of eNOS on endothelial cell proliferation but again results are inconsistent. We have demonstrated that adenovirus-mediated gene delivery of eNOS to endothelial cells in vitro inhibits cellular proliferation.¹⁷ In contrast, overexpression of eNOS in an injured artery in vivo improved re-endothelialization at 2 weeks.⁹ This finding in vivo has been confirmed in the current report in another model of vascular injury with more relevance to the clinical problem being addressed. The difference in the observed effect of NO on endothelial cell proliferation in vitro and in vivo is unclear but may be due to the relative amounts of the cofactor tetrahydrobiopterin present in different systems. Endothelial cells in culture have been shown to have low levels of expression of the enzyme guanosine triphosphate cyclohydrolase.²⁴ Further evidence in support of this hypothesis is the finding that increasing tetrahydrobiopterin levels by administration of sepiapterin improved the nitrite production from eNOS in vitro.¹⁷ Thus, we hypothesize that the in vitro inhibitory effect of eNOS overexpression on endothelial cell proliferation may be due to enzyme uncoupling related to reduced bioavailability of tetrahydrobiopterin. The uncoupled enzyme would produce superoxide anion which may inhibit endothelial cell proliferation. In contrast, our previous data from a carotid injury model and the results presented in the current report suggest that overexpression of eNOS in vivo is capable of selectively inhibiting SMC proliferation while promoting endothelial cell proliferation.⁹ One caveat to be acknowledged is the fact that while we have shown that the eNOS improves endothelial regeneration, an inhibition of in-stent thrombosis has not been demonstrated.

Although vascular gene therapy has been shown to be feasible in animal models, no convincing evidence of benefit in controlled human studies is available to date. There are many potential reasons for this observation, one of which may be the means of the delivery of therapeutic gene to the blood vessel wall. Stent-based adenoviral gene delivery has been rarely studied.^25,26 The application of endovascular stents as carriers of therapeutic genes for prolonged and site-specific delivery is appealing and promising. This is the second report of a therapeutic benefit from stent-based gene delivery to the blood vessel wall resulting in reduced neointimal hyperplasia and enhanced endothelial regeneration²⁶ and the first showing this effect after eNOS gene delivery.

In summary, we have demonstrated that overexpression of eNOS in the context of stent deployment in a diseased vessel inhibits in-stent restenosis while promoting endothelial generation. This may represent an ideal strategy to address the problems of in-stent restenosis and late-stent thrombosis and could provide an alternative approach to the current use of DESs.

Materials and Methods

Construction, propagation and purification of adenoviral vectors. A recombinant replication-defective adenoviral vector encoding the Escherichia coli -galactosidase gene driven by the cytomegalovirus promoter was used as previously described.⁷ A recombinant adenovirus encoding the bovine eNOS gene driven by the cytomegalovirus promoter was generated as previously described.²⁷

Animals. Studies were performed in accordance with national regulations after an animal use license was obtained from the Department of Health and Children. A total of 66 male New Zealand White rabbits (Harlan, Bicester, UK) weighing 2.5–3.5 kg were used. Animals were fed a standard chow diet and water ad libitum. A subset of animals (n = 11) was fed 1% cholesterol with 3% peanut oil for 4 weeks before and after intervention.

In vivo catheter procedures. In vivo balloon injury and percutaneous stent placement in the right iliac arteries have been previously described in detail.⁷ Briefly, a 5-French (Fr) introducer sheath (Radifocus, Terumo, Japan) was surgically introduced into the right carotid artery and advanced to the lower abdominal aorta. A balloon injury was performed three times with a 2.5 15 mm balloon in the right external iliac artery. After balloon injury, a 3.0 15 mm BiodivYsio HI matrix-coated stent was deployed at the injury site (6 atmospheres for 30 second). Post-stent deployment angiography was carried out in all animals to exclude any acute thrombus formation at the site of stent deployment. Bilateral iliac arteries were stented in a subset of animals (n = 10).

Follow-up angiography was carried out through the contralateral left carotid artery using an introducer sheath (4 Fr Terumo).

BiodivYsio phosphorylcholine polymer stents. A total of seventy six BiodivYsio HI matrix PC-coated premounted stents (3.5 15 mm) were used for this study. All stents were supplied by Abbott Vascular (Galway, Ireland). Under sterile conditions, the PC stents were coated with a 50-l bolus of AdGal (n = 28) or AdeNOS (n = 28) at a concentration of 5 10⁹ plaque-forming units per stent. The stents were coated manually with a micro pipette and air dried for 45 minutes before deployment. A total of 20 stents were used as PC controls.

Analysis of transgene expression

Reverse transcriptase-PCR: RNA was extracted from the rabbit arteries 3 days after deployment of AdeNOS, AdGal, and PC stents (n = 3 each group) using the RNeasy kit (Qiagen, Crawley, UK). Extracted and purified RNA (200 ng) was then subjected to reverse transcription-PCR using the Quantitect SYBR Green RT-PCR kit (Qiagen) in conjunction with the ABI 7000 Sequence detection system (ABI). (50 °C, 30 minutes; 94 °C, 15 minutes; 35 cycles of 94 °C, 15 seconds; 60 °C 1 minute). PCR products were visualized on agarose gels. PCR primers were designed to amplify bovine eNOS. Bovine eNOS (NM_181037) primers were forward 5'-GAGAGGCTGCATGACATTGAGA-3'and reverse 5'-GGTA GAGATGGTCGAGTT GGGA-3' with an expected product size of 94 base pairs.

Immunohistochemistry: Six days after stent placement, AdeNOS- and AdGal-treated blood vessels (n = 3 from each group) were retrieved and opened longitudinally and the stents were removed. Immunohistochemical staining for eNOS was performed as previously described.¹³ Briefly, paraffin-embedded 5-m sections were fixed, dried, and then blocked using 5% goat serum/phosphate-buffered saline–Tween 20. eNOS monoclonal antibodies (1:50 dilution; Transduction Laboratory) were applied for 60 minutes to appropriate slides for 2 hours at room temperature, followed by incubations with biotinylated rabbit anti-mouse F(ab')₂ secondary antibody (1:200, incubation time 1 hour; DAKO, Dublin, Ireland) and peroxidase-conjugated streptavidin (1:200, incubation time 1 hour; DAKO). eNOS immunoreactivity was visualized with diaminobenzidine and hematoxylin counterstaining. Control sections were treated in the same way with anti-GFP mouse monoclonal antibody (Chemicon) substituted as a control antibody.

Assessment of stent endothelialization

Evans blue: Fourteen days after stent deployment, a total of eight animals were anesthetized as above (n = 4 AdGal and AdeNOS each). Five ml of 1% Evans blue (Sigma-Aldrich, Dublin, Ireland) was injected into the left ear veins and the stents were retrieved 30 minutes after injection. The animals were killed before the retrieval. The stented blood vessels were fixed and examined longitudinally using Java image software.

Scanning electron microscopy: Re-endothelialization was also assessed in six animals at day 14 after stent placement (n = 3 AdGal and AdeNOS each). Retrieved stents were washed with saline and fixed in 4% paraformaldehyde and longitudinally incised as above and examined using scanning electron microscopy.

Histological assessment: Stents were retrieved at day 28 and embedded in resin after perfusion fixation. Three serial sections were taken per stent at proximal, mid, and distal points in the vessel and re-endothelialization was assessed directly under the microscope by a histologist blinded to treatment. Endothelial coverage was expressed as the percentage of the lumen circumference covered by the endothelial cells.

Morphometric analysis. Morphometric analysis was performed by CV Path Institute (Gaithersburg, MD). The effects of AdeNOS-, AdGal-, and PC-coated stents on the vessel morphology were assessed at 28 days in normal-diet animals (n = 9, 9, and 8, respectively) and also in hypercholesterolemic animals which included AdeNOS, AdGal (n = 6 each), and PC (n = 9) groups. Retrieved vessels were embedded in methylmethacrylate plastic. After polymerization, 3-mm sections were taken from the proximal, mid, and distal portions of each single stent. Four- and five-micron sections from the stents were cut on a rotary microtome, mounted and stained with hematoxylin, eosin, and elastic Van Gieson stains. All sections were examined by light microscopy for the presence of inflammation, thrombus and neointimal formation, and vessel wall injury (Table 2).

Table 2 - Injury and inflammation score method.

Full table (28K)

A vessel injury score was calculated according to the Schwartz method.²⁸ The cross-sectional areas (external elastic lamina, internal elastic lamina, and Lumen) were measured with digital morphometry. Neointimal thickness was measured as the distance from the inner surface of each stent strut to the luminal border.

Assessment of ISR by angiogram. Follow-up angiograms were performed through the left carotid artery, at 28 days after initial intervention in the AdeNOS-, AdGal-, and PC-coated stents in normal-diet animals (n = 9, 9, and 8, respectively) and in hypercholesterolemic animals (AdeNOS, AdGal (n = 6 each), PC n = 9). All the angiograms were analyzed for quantitative assessment of ISR using standard quantitative coronary analysis software by two operators blinded to the treatment groups.

Statistical analysis. Values are expressed as mean SD. Mean variables between the groups were compared with the one-way analysis of variance. Score data, including injury score and inflammation score were compared using the Wilcoxon/Kruskal–Wallis (Rank Sums) test. If a statistical difference was determined, treatment groups were then separately compared with the test article and a Bonferroni correction. A value of P < 0.05 was considered statistically significant.

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Acknowledgments

This work is funded by a grant from Enterprise Ireland (Dublin, Ireland). Stents were provided by Abbott Vascular Devices Ireland (Galway, Ireland). We also thank the Department of Cardiology at University college hospital Galway for their support in completion of study. Timothy O'Brien is supported by a Centre for Science Engineering and Technology award from Science Foundation Ireland.