Summary
Background
The optimal therapy for in-stent restenosis (ISR) is controversial. We evaluated three different strategies for the treatment of in-stent restenosis: cutting balloon angioplasty (CBA), paclitaxel-eluting balloon angioplasty (PEBA) and cutting balloon followed by paclitaxel-eluting balloon angioplasty (CB + PEBA).
Methods
Forty-five coronary arteries in 45 mini-pigs underwent oversized bare-metal stent (stent-to-artery ratio, 1.2:1) implantation to induce in-stent restenosis. After 28 days, vessels with in-stent restenosis (≥ 50% diameter stenosis) were randomly divided into three groups: CBA, PEBA and CB + PEBA. In vivo angiography was performed before intervention, immediately after intervention and at 28-day follow-up. Stented arteries were harvested for pathological analyses. The proliferation and apoptosis of vascular smooth muscle cells were evaluated by immunohistochemical staining and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, respectively.
Results
Acute lumen gain was not different between the three groups. Late lumen loss and neointimal area at follow-up were lower for CB + PEBA compared with CBA but similar for CB + PEBA compared with PEBA. There were no significant differences in proliferating cell nuclear antigen-positive vascular smooth muscle cells and TUNEL-positive vascular smooth muscle cells between the CB + PEBA and PEBA groups.
Conclusions
PEBA with or without cutting balloon was superior to CBA alone for in-stent restenosis. The underlying mechanism was probably related to inhibition of smooth muscle cell proliferation and increased apoptosis. In this porcine coronary artery restenosis model, PEBA with or without cutting balloon was superior.
Résumé
Justification
Le traitement optimal de la resténose intra-stent reste controversé. Nous avons évalué trois stratégies différentes pour le traitement de la resténose intra-stent : angioplastie avec cutting balloon , angioplastie avec ballon au paclitaxel et cutting balloon suivi d’une angioplastie avec ballon au paclitaxel.
Méthode
Quarante-cinq artères coronaires chez 45 cochons ont bénéficié d’une angioplastie par un stent métallique surdimensionné (ratio stent/artère : 2.1) afin d’induire une resténose intra-stent. Après 28 jours, une resténose intra-stent définie comme un diamètre de sténose supérieur ou égal à 50 % de l’artère coronaire ont été randomisés dans trois groupes : angioplastie avec cutting balloon , angioplastie avec ballon au paclitaxel et cutting balloon suivi d’une angioplastie par ballon au paclitaxel. L’angiographie in vivo a été réalisée avant l’intervention, immédiatement au décours et à 28 jours. Les artères stentées ont été prélevées pour analyse anatomopathologique. La prolifération et l’apoptose des cellules musculaires lisses vasculaires ont été évaluées par immuno-histochimie et par un marquage terminal deoxynucléotidyle transferase d’UTD (marquage TUNEL), respectivement.
Résultats
La réduction immédiate de calibre coronaire n’était pas différente dans les trois groupes. La réduction tardive du diamètre luminal et la surface néo-intimale au suivi était moindre dans le groupe cutting balloon suivi d’une angioplastie avec ballon au paclitaxel, comparativement au groupe angioplastie par cutting balloon mais similaire au groupe angioplastie par ballon au paclitaxel. Il n’y avait pas de différence significative dans la détection de prolifération de cellules musculaires lisses vasculaires par reconnaissance antigénique ou par le test TUNEL, entre le groupe cutting balloon suivi d’une angioplastie par ballon au paclitaxel et le groupe angioplastie par ballon au paclitaxel.
Conclusion
L’angioplastie par ballon au paclitaxel avec ou sans cutting balloon s’avère supérieure à l’angioplastie par cutting balloon seule pour la prévention de la resténose intra-stent. Le mécanisme est probablement lié à l’inhibition de la prolifération des cellules musculaires lisses vasculaires, et une augmentation de l’apoptose. Dans ce modèle porcin de resténose coronaire, l’angioplastie par ballon au paclitaxel avec ou sans cutting balloon s’avère supérieure aux autres méthodes testées.
Introduction
Treatment of in-stent restenosis (ISR) remains challenging. Several studies have shown variable results using balloon angioplasty alone , repeat stenting , cutting balloon angioplasty (CBA) , intracoronary irradiation (brachytherapy) or excimer laser angioplasty . Other studies have compared these different techniques and it is still unclear which one, if any, will provide the most favourable outcomes .
Recently, paclitaxel-eluting balloon angioplasty (PEBA) has been developed as a novel approach, which combines the features of conventional balloon angioplasty with paclitaxel eluting for the treatment of ISR. Preclinical trials have demonstrated that the efficacy of PEBA in the treatment of ISR is superior to that of conventional balloon angioplasty and not inferior to that of a paclitaxel-eluting stent . However, given the structure of the paclitaxel-eluting balloon, it must have some of the shortcomings of conventional balloon angioplasty, such as balloon slippage and edge dissections post procedure. All these shortcomings have been associated with cumbersome procedures, suboptimal results and adverse clinical and angiographical outcomes . The use of CBA could potentially reduce the occurrence of these complications. However, the outcome of cutting balloon predilatation followed by PEBA for the treatment of ISR compared with PEBA or CBA alone is unknown.
The aims of this study are to compare 28-day imaging and pathology outcomes between PEBA, CBA and cutting balloon followed by paclitaxel-eluting balloon angioplasty (CB + PEBA) for ISR in pig models and to elucidate the possible mechanism.
Methods
All animal care and procedures conformed to 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) and were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Harbin Medical University (2009-X023).
Establishment of the in-stent restenosis model
Forty-five mini-pigs (20–25 kg) were pretreated with aspirin (300 mg) and clopidogrel (75 mg) once a day starting 3 days prior to the procedure. Animals were intubated after sedation with ketamine (20 mg/kg, intramuscularly) and diazepam (0.4 mg/kg, intramuscularly), followed by 3% sodium pentobarbital through the marginal ear vein (25 mg/kg, intravenously). A 6-F guiding catheter was used. Continuous haemodynamic and surface electrocardiographic monitoring was maintained throughout the procedure. After intravenous heparin (150 U/kg) and intracoronary injection of nitroglycerin (100 μg), baseline angiography of the target vessel was performed. The methods of stent implantation have been published previously . One bare-metal stent (BMS) (3.0–3.5 × 18 mm; Lepu Medical Company, Beijing, China) was placed at 12–14 atm for 30 s in the left anterior descending artery of each pig. The stent-to-artery ratio was maintained at 1.2:1. After the equipment was removed, all mini-pigs were sent back to the animal house, where they were fed a normal diet, and received aspirin (300 mg, orally) and clopidogrel (75 mg, orally) daily.
Interventional procedure for in-stent restenosis
At 28 days after BMS implantation, a repeat angiography was performed. The segments with ISR (≥ 50% diameter stenosis) by quantitative coronary angiography were randomly assigned to one of the three treatment groups: CBA, PEBA or CB + PEBA.
Cutting balloon angioplasty
The length of the cutting balloon (Boston Scientific, Natick, MA, USA) was 10–15 mm and the diameter was chosen according to the size of the stent (cutting balloon-to-stent ratio, 1.1:1). The cutting balloon was positioned at the lesion site and inflated to the recommended maximal pressure of 10 atm once.
Paclitaxel-eluting balloon angioplasty
Predilation of the target lesion was usually required before placement of the study device. The diameter of the conventional non-study balloon catheter was 0.5 mm smaller than that of the paclitaxel-eluting balloon. The paclitaxel-eluting balloon (3.0–3.5 × 20 mm; SeQuent Please, BRAUN, Germany) was inflated in the same fashion as a conventional balloon catheter for 60 s and the pressure was 10 atm.
Cutting balloon followed by paclitaxel-eluting balloon angioplasty
Before the paclitaxel-eluting balloon was inflated, predilation of the target lesion was carried out with a cutting balloon catheter, which was similar in diameter to a paclitaxel-eluting balloon.
Quantitative coronary analyses
Angiograms were performed during the initial procedure, on day 28 after BMS implantation and 28 days after three different strategies for the treatment of ISR. A computerized coronary angiography analysis system (GE Company, Germany) was used for quantitative coronary analyses by two experienced cardiologists blinded to the treatment protocol. Discrepancies were resolved by mutual consensus. The immediate lumen diameter (LD) gain (minimum LD immediately after the interventional procedure minus minimum LD before the interventional procedure) and late lumen loss (minimum LD immediately after the interventional procedure minus minimum LD at follow-up) were calculated.
Pathological evaluation
For the morphometric analysis, stented arteries were harvested and fixed in 10% buffered formalin and embedded in glycol methacrylate. Stented segments were cut into three parts (proximal, mid and distal). Thin sections from each artery block were stained with haematoxylin and eosin and Verhoeff van Giesen for measurement of the external elastic lamina area (EELA), lumen area (LA) and internal elastic lamina area (IELA). The neointimal area (NA) was calculated using the equation NA = IELA − LA. The percentage of lumen area stenosis (AS%) was calculated using the equation AS% = (NA/IELA) × 100. Injury score at each strut site was assessed as described by Schwartz et al. , where: 0 = no injury; 1 = break in the internal elastic membrane; 2 = perforation of the media; and 3 = perforation of the external elastic membrane to the adventitia. The mean injury score for each segment was calculated by dividing the sum of injury scores by the total number of struts at the examined section.
Stent wires were carefully removed and the tissue was embedded in paraffin. Vessel-wall expression of proliferating cell nuclear antigen (PCNA) was evaluated by immunohistochemical analyses (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Apoptosis of vascular smooth muscle cells (VSMCs) was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. There were three cross-sections of each arterial specimen to analyse. The samples were incubated, developed, and counterstained with haematoxylin. The total number of VSMCs in each randomly chosen neointimal cross-section was measured with computer assistance at × 400 magnification (Pro Plus 5.0 software). The percentages of proliferating or apoptotic VSMCs were obtained by dividing the number of PCNA- or TUNEL-positive VSMCs by the total number of VSMCs in each cross-section; the results were then averaged.
Statistical analyses
Data are expressed as means ± standard deviations except where noted. The mean angiographical, histological and morphological data for each stent were compared by one-way analysis of variance with post hoc analysis for multiple comparisons. A value of P < 0.05 was considered significant. All statistics were calculated with SPSS version 18.0 software.
Results
Forty-five mini-pigs underwent successful implantation of 45 BMSs in the left anterior descending artery. All mini-pigs survived until the end of experiments. Thirty-one out of 45 lesions (68.9%) had greater or equal to 50% diameter stenosis and the mean angiographical stenosis was 62.2 ± 10.3%. At the end of the experiments, there were 11 lesions in the CBA group, 10 lesions in the PEBA group and 10 lesions in CB + PEBA group.
Angiographical findings
Quantitative coronary angiography results are summarized in Table 1 . There were no significant differences in baseline and immediate angiographical measurements among the three groups. At follow-up, late lumen loss in the CB + PEBA group was significantly less than that in the CBA group, but similar to that in the PEBA group ( P > 0.05).
