Intimal Proliferation and Restenosis in Paclitaxel-Eluting Stents with Aminoparylene as Carrier Substance in Swines

From the *Division of Cardiology, Swiss Cardiovascular Center, Bern, and the §Institute of Pathology, University Hospital Bern, Switzerland. Disclosures: This study was funded with an unrestricted educational grant from Fumedica AG, Switzerland. No author has a direct relationship or shares of Fumedica AG, Switzerland. Manuscript submitted August 12, 2008, provisional acceptance given October 16, 2008, final version accepted October 29, 2008. Address for correspondence: Otto M. Hess, MD, FAHA, FESC, Professor of Cardiology, Swiss Cardiovascular Center, University Hospital, CH-3010 Bern, Switzerland. E-mail: otto.hess@insel.ch

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ABSTRACT: Background. Polymer as carrier substance for drug-eluting stents (DES) has been accused of inducing inflammation and hypersensitivity reactions leading to restenosis and stent thrombosis. Thus, a new paclitaxel-eluting stent (PES) with aminoparylene as a carrier substance is tested in the present study. Methods. In 10 pigs, stents were implanted in the epicardial coronary arteries: 1) bare-metal stents (BMS, control stent); 2) cobalt-chromium stents (CCS); and 3) PES. Stent length was 15 mm, and diameter was 3 mm. The animals were restudied after 6 weeks. Quantitative coronary angiography was performed at baseline and follow up. Minimum luminal diameter (MLD) and late loss were calculated in all animals. Histologic vessel lumen, intimal proliferation and restenosis were determined by morphometry. Disruption of the lamina elastica interna (LEI) and inflammatory reactions were assessed by histology. Results. The MLD at baseline was 2.83 ± 0.28 mm, and at follow up it was 2.29 ± 0.44 (p Conclusions. PTs with aminoparylene as a carrier substance show similar late loss and angiographic restenosis to that of BM and CCS. The incidence of inflammatory reactions (35% of all histologic sections) is similar in all stents, but highest in PES. The mechanism of this reaction is unclear, but may be either due to the drug itself, the disruption of the LEI or to a hypersensitivity reaction.

J INVASIVE CARDIOL 2009;21:128–132

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Intracoronary stenting has become the standard procedure in patients with coronary artery disease undergoing percutaneous coronary interventions (PCI). Coronary stenting is currently used during most interventions to prevent abrupt vessel closure and to achieve an excellent angiographic result.^1,2 Long-term studies have shown that bare-metal stents (BMS) are associated with considerable in-stent restenosis (up to 20–25%) and an increased need for reintervention in 12–15% of all cases.³ More recently, drug-eluting stents (DES) have been introduced which are coated with a polymer as a carrier substance containing an antiproliferative or immunosuppressive agent to prevent neointimal proliferation.^4–7 Most DES have shown a significant reduction in angiographic restenosis and short-term outcomes have been excellent with regard to major adverse cardiac events (MACE). However, recent data suggest that DES may show delayed vascular healing and reduced reendothelialization in up to 50% of all stents.^8–10 Furthermore, a new phenomenon of late and very late stent thrombosis has been observed in patients ≥ 1 year after stent implantation.^11,12 This phenomenon has been attributed either to the lack of reendothelialization, a hypersensitivity reaction to the polymer or incomplete stent apposition late after implantation.¹³ Stent thrombosis has been found to increase linearly after implantation, with a yearly rate of 0.6% leading up to 3.5% 4 years after implantation.¹² For prevention of acute stent thrombosis, dual-antiplatelet therapy^7,14 was recommended with aspirin (lifelong) and clopidogrel (approximately 12 months). In the present study, a new carrier substance, aminoparylene, was tested to reduce inflammatory reactions and hypersensitivity. Study design. Three types of stents were tested in the present analysis in 10 pigs. One stent was implanted in each of the large epicardial arteries (left anterior descending coronary artery, left circumflex coronary artery and right coronary artery) during general anesthesia, which was induced with sodium pentobarbital 10 mg/kg intravenously (IV) and maintained by halothan inhalation^15,16 (mean body weight was 41 kg). The left carotid artery was dissected and a 6 Fr vascular sheath was introduced. Then a 6 Fr guiding catheter for the left coronary artery was placed into the left and right coronary arteries for selective angiography and stent placement. Stents were implanted with a pressure of 12 or 16 bar, depending on the size of the artery. Stents were 3 mm in diameter and 15 mm long and were randomly implanted in either one of the three coronary arteries to prevent implantation bias. The balloon-to-artery ratio was 1.27 for bare-metal stents (BMS), 1.21 for cobalt-chromium stents (CCS) and 1.22 for paclitaxel-eluting stents (PES), respectively (all ns). All three stents are commercially available (Eurocor GmbH, Bonn, Germany). Delivery systems were identical in the three groups, and durability of the coating and the drug were excellent, with good stability data. After implantation, the animals were transported on the same day back to the farm and kept for 6 weeks for stent healing. After this time span, the animals were brought from the farm to the hospital for a second coronary angiography. Immediately after angiography, the animals were euthanized with an IV overdose of KCl. Then the heart was removed and coronary arteries with the stents were dissected. All hearts were pressurized to prevent collapsing of the coronary arteries. After excision of the stents, the samples were fixed in formalin. Three-to-four weeks after fixation, the stents were embedded in poly(methyl methacrylate) and cut with a special microtome into 800 µm thin slices and polished to a thickness of 100 µm. Three segments of the stents were examined, a proximal, a middle and a distal segment, as well as proximal and distal reference segments. All sections were stained with paragon (7.3 g toluidine blue with basic fuchsin dissolved in 1,000 ml of ethanol 30%). Quantitative coronary angiography. All digital angiograms were assessed quantitatively^15,16 by a standard software program (Medis SA, Medical Imaging Systems, Leiden, The Netherlands). Minimal lumen diameter (MLD), vessel diameter at the proximal, middle and distal ends, as well as the proximal and distal reference diameters were measured quantitatively. Late loss was calculated by subtracting the MLD at follow up from the MLD at baseline. In-stent restenosis was calculated from the MLD divided by the mean of the proximal and distal reference diameters multiplied by 100. This software was carefully validated and inter- and intraobserver variability ranged from 5–8%.^15,16Histomorphometry. After staining the samples (Figure 1), quantitative evaluation of the stented vessel was carried out using the Image Pro Plus digital system (Media Cybernetics, Inc., Bethesda, Maryland). The following parameters were determined:^15,16

1. Vessel lumen, intimal proliferation and stent lumen (vessel lumen plus intimal proliferation). Mean and median values, as well as standard deviations were calculated for each stent section. 2. Intimal proliferation was calculated from the stent lumen minus the vessel lumen (mm2) and in-stent restenosis from intimal proliferation divided by the stent lumen multiplied by 100. 3. Reendothelialization was assessed under high-magnification light microscopy. In all sections, inflammatory cells and granulomas were visually assessed (mild, moderate, severe). 4. Disruption of the lamina elastica interna (LEI) was assessed by visual examination (Figure 1).

Statistics. All data are given as mean or median ± 1 standard deviation. Figures 2 through 5 show box plots with median and standard deviation (upper panel) and bar graphs with mean and standard deviation (lower panel). Statistical comparison was performed with a paired t-test for angiographic measurements of the MLD (Figure 2). All other comparisons (Tables 1 and 4, and Figures 3, 4 and 5) were done with a one-way analysis of variance (ANOVA) for three groups because only one time point (follow-up examination) was available (equal to multiple t-test). A test was considered statistically significant when p Results All animals survived the intervention and the follow-up interval of 6 weeks. No clinical signs of stent thrombosis were observed. Angiographic data. The MLD decreased in all 3 stents from baseline to follow up by approximately 15–25% (Figure 2). There were no significant differences between the three types of stents. Late loss was similar for all three stents, but was smaller (ns) in the uncoated CCS (0.37 mm) and slightly higher in PES (0.73 mm) compared to BMS (0.55 mm) (Figure 3). Angiographic restenosis (Table 1) was 26% for the PES, 20% for BMS and 16% for CCS (Figure 5). Thus, restenosis was highest in the PES, although the differences were not statistically significant. Histologic data. Representative histologic samples for the three evaluated stents are shown in Figure 1. Inflammatory granulomas can be seen around the stent struts of all three samples (arrows). Most stents showed inflammatory reactions, but mostly in the PES and the uncoated CCS (Table 2). A disruption of the LEI (Figure 1) was most frequently found in PES and CCS, respectively (Table 2). Correlating the disruption and inflammatory reaction (Table 3), there was a highly significant relationship between these two parameters. The vessel lumen was larger in CCS (4.06 mm2) than in BMS (3.82 mm2) or PES (3.44 mm2) (Table 4), however, the differences were small. Intimal proliferation (Table 4 and Figure 4) was highest in PES (1.64 mm²), followed by BMS (1.49 mm²) and CCS (1.38 mm²). Restenosis was greatest in PES (33%), followed by BMS (30%) and CCS (27%), respectively (Figure 5). The PES showed the highest restenosis rates compared to the other two stent types (Table 4).

Discussion

In the present study, a new carrier substance (aminoparylene coating^17,18) was tested in the experimental animal. Intimal proliferation was most pronounced in the PES with aminoparylene coating followed by the BMS and CCS. In a previous study, aminoparylene coating was associated with a mild reduction in intimal proliferation of 21% compared to the CCS.¹⁹ This reduction is not statistically significant. Intimal proliferation was in the range of 1.5 mm² in the present study, which is rather low compared to previous experimental studies^15,16,20 which showed a range of 2.3–2.6 mm², suggesting better stent designs or more biocompatible stent coatings. Implantation pressure was standardized and balloon-to-artery ratio (stent oversizing) was similar in the three stent groups. Thus, mechanical injury to the vessel is compatible for the three stent groups and does not explain a higher rate of intimal proliferation. Overall, the proliferative response was mild in all three stents, with no statistical significance. Inflammatory reactions. Coronary inflammation was observed in 35% of all stents. The PES had the highest inflammation probability with 46%, followed by CCS with 36%, and BMS with 23%. In Figure 1, examples of the three stents are shown with granulomas around the stent struts (arrows) with severe intimal proliferation. Similar granulomas were found in many, but not all stents (Table 2). The nature of this inflammatory reaction is not clear, but may be due to disruption of the LEI by the stent struts,¹⁹ with an enhanced mechanical injury (Table 3). By histologic examination, disruption of the LEI was found in 32 out of a total of 80 samples (40%). Disruption of the LEI was found in all coronary arteries (Table 2). Those stents with disruption of the LEI typically showed more inflammatory reactions (Table 3), suggesting that the pathophysiologic mechanism is the disruption of the inner elastic membrane. Another explanation for the inflammatory reactions could be a hypersensitivity reaction either due to the drug or the aminoparylene coating. Virmani et al⁹ and Nebeker et al²¹ described a hypersensitivity reaction due to the polymer coating found in patients who died from late stent thrombosis. The higher inflammatory reactions in the PES compared to the BMS and CCS could be attributed — at least in part — to the aminoparylene coating and not simply to the disruption of the LEI. However, as shown previously,¹⁹ we found no increased inflammatory reaction with the aminoparylene coating. In the present study, the type of inflammation did not differ between the three stents.

Conclusions

PES with aminoparylene coating show a similar late loss and angiographic restenosis as BMS and CCS, and therefore PES have no better antiproliferative effect than the uncoated control stents. This could be explained by a “too-low” paclitaxel dose or an enhanced inflammatory reaction to the stent coating. The mechanism responsible for this phenomenon, however, remains unclear. Thirty-five percent of all stents showed inflammatory reactions after stent implantation. Similar data have been found in a previous experimental study.¹⁹ The incidence of inflammatory reactions was similar in all three stent types, but was highest in the PES. This could be attributed, as discussed earlier, either to the drug itself, the disruption of the LEI or the hypersensitivity reaction to the aminoparylene coating. The exact nature of this inflammatory reaction is not clear, but may enhance the proliferative reaction of BMS and DES as well.

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