Endothelialization of Sirolimus-Eluting Stents with Slow and Extended Drug Release in the Porcine Overstretch Model

ABSTRACT: Background. Vascular healing of intracoronary stents has been shown to be delayed in drug-eluting stents (DES) due to the cytotoxic compounds on the stent surface that prevent stent ingrowth and endothelialization. The lack of endothelialization explains the occurrence of late and very late stent thrombosis in DES. Materials and Methods. In 11 house swines (body weight 38–45 kg), 3 stents were implanted randomly into the 3 large epicardial coronary arteries, namely a bare-metal stent (BMS), a sirolimus-eluting stent with slow-release (SES) and a SES with extended-release (SESXR). Stent length was 18 mm, and stent diameter 3 mm. All stents were of identical design. Animals were followed for 3 (n = 3), 7 (n = 4) and 14 (n = 4) days, respectively. One animal died before implantation due to hyperthermia. On the day of explantation, the animals were euthanized and endothelialization was tested by scanning electron microscopy after drying and sputtering the samples. Endothelial coverage was determined semiquantitatively by 2 observers. Results. Endothelialization was more rapid with BMS and SESXR than SES at 3 and 14 days. At 7 days there were no significant differences between the 2 SES. Conclusions. Endothelialization of intracoronary stents is faster with BMS and SESXR at 3 days than with SES. These differences persist at 14 days, suggesting delayed vascular healing with the slow-release SES. J INVASIVE CARDIOL 2008;20:631–634 Vascular healing after stent implantation has been shown to be delayed in drug-eluting stents (DES) due to the fact that drugs released from the stent surface are cytotoxic and prevent stent ingrowth and endothelialization.1–3 DES are used to reduce intimal proliferation and to prevent restenosis after implantation.4,5 In some cases, drug concentrations have been in the cytotoxic range and induced vascular necrosis with aneurysmal formation.6 This phenomenon has been associated with stent thrombosis7,8 and has been called incomplete stent apposition.9 In contrast, bare-metal stents (BMS) show rapid ingrowth within 4 to 6 weeks, but have a high potential for neointimal proliferation causing in-stent restenosis.10,11 The delayed reendothelialization and vascular ingrowth of the DES plays an important role in late complications and clinical outcomes in patients with DES. New attempts to overcome these problems have resulted in the development of slow- and extended-release antiproliferative agents such as sirolimus to improve endothelialization and to accelerate vascular healing. The purpose of the present study was to evaluate the endothelialization of three different stents (BMS, sirolimus-eluting stent [SES], extended-release SES [SESXR]) in the porcine overstretch model. Endothelialization was studied early, at mid-term and 2 weeks after implantation. Material and Methods Three different stents were tested in 11 house swines with a body weight of 38–45 kg. The animals were brought directly from the animal farm to the experimental surgical laboratory. During general anesthesia with intravenous (IV) sodium pentobarbital 10 mg/kg, which was maintained by halothan inhalation, the left carotid artery was dissected free and a vascular sheath was introduced.12,13 Next, the animals were brought to the catheterization laboratory and a 6 Fr guiding catheter for the left coronary artery was introduced under fluoroscopic guidance. Selective coronary angiography was performed and a guidewire was placed in the left anterior descending coronary artery, then in the left circumflex and the right coronary arteries for stent implantation. Stents were randomly selected and implanted with a pressure of 12–16 bar depending on the size of the artery. Stents were 3 mm in diameter and 18 mm in length. In the slow-release group, the drug was formulated in a biostable polymer matrix with a total concentration of 1.4 g/mm2, while in the extended-release group, the drug was formulated with a pharmaceutical excipient acting as a top coat, which provides the release, while the drug is not mixed with any polymers at all. The slow-release stent is currently used in India and several European countries; the extended-release stent has not yet been marketed. After successful implantation, the animals were brought back to the farm on the same day and kept for 3, 7 and 14 days, respectively, for healing of the stents. On the day of restudy, the animals were brought back from the farm to the hospital and were then euthanized with an IV overdose of KCl. At the next step, the heart was removed and the coronary arteries with the stents were dissected free. All hearts were pressure perfused to prevent collapsing of the coronary arteries. After excision of the stents, samples were fixed in 2.5% glutaraldehyde in 0.03 M potassium phosphate buffer. Tissue processing. For scanning electron microscopy (SEM) analysis, selected tissue samples were dehydrated in ascending concentrations of ethanol, critical-point dried in liquid carbon dioxide, and then sputter-coated with gold. The specimens were mounted on aluminum stubs and viewed on a Philips XL30 FEG SEM.14 For each sample, 9 images were taken for further analysis. Two observers who were blinded to the experimental groups evaluated all samples and determined endothelial coverage of the stent struts. The same design was used for all 3 stents. Intraobserver variability was 5.2% for assessing endothelialization by SEM. Statistical analysis. All data are provided as mean and standard deviation in the case of continuous variables. Because Gaussian distribution of the data cannot be assumed, only nonparametric tests were used for data analysis. First, an overall test considering all time points simultaneously was performed by using a nonparametric analysis of variance (ANOVA) for longitudinal data (two-factorial design with the factors being stent type and time; SAS macro F1_LD_F1). For the particular time points, a one-factorial design was used (with stent type as the factor; SAS macro LD_F1).1 Pair-wise post hoc analyses between the three stent types were only followed at the particular time points where a significant difference was detected in the overall testing. For this pair-wise analysis, the Wilcoxon signed rank test was used, taking into account the correlated nature of the measurements (three stent types tested in the same animal). For all analyses, p-values were two-sided and differences were considered to be statistically significant with p Results Representative SEM images of endothelialized stents at 3, 7 and 14 days are shown in Figures 1–3. The best endothelialization was observed after 14 days and the least endothelialization after 3 days. Differences between the three stents were seen at all three time points. On Day 3 (Figure 4), endothelialization was good for the BMS, but significantly less so for the slow-release SES. Coverage of the SESXR was greater than with the SES, but less than what was observed with the BMS. At 7 days there were no significant differences between the three stents, but endothelialization was greater for the BMS than for the other two stents. At 14 days these differences remained, but endothelial coverage was significantly lower for the slow-release SES than for the BMS, and was slightly better for the extended-release SES (p = 0.061). Plotting endothelialization versus time (Figure 5), stent coverage was greatest at all three time points for the BMS and least for the slow-release SES. The extended-release SES lies between the two curves, being slightly better than the slow-release SES, but slightly worse than the BMS. Discussion Vascular healing is delayed in DES depending on the concentration of the drug and the pharmacokinetic profile of the substance.6 New drugs and carrier polymers have been developed15 to improve vascular healing. In the present study, an extended-release SES was tested for its healing properties using SEM to assess endothelialization at 3, 7 and 14 days. Endothelialization was fastest in the present study with BMS, and slowest with the slow-release SES, and the extended-release SES was between the two (Figures 4 and 5), suggesting slower vascular healing than with the BMS, but better healing than with the slow-release SES. Endothelialization of intracoronary stents. Joner et al3 and Virmani et al11 showed that endothelialization was close to 100% with BMS after 6 months, and was approximately 40–50% with DES up to 3 years after implantation. These data were obtained from human autopsy studies showing stent thrombosis as a potential complication after delayed or absent endothelialization. The risk of major adverse cardiac events (MACE)8 during late follow-up depends largely on vascular healing with full coverage of the implanted stents. Differences between slow and extended release of sirolimus. The extended-release SES clearly shows better vascular healing than slow-release SES, suggesting that vascular healing is dependent on the pharmacokinetic profile of the DES, i.e., the lower the drug concentration, the better the endothelialization. It is possible that the extended-release SES is not as efficacious as the slow-release SES for inhibition of restenosis. Restenosis with DES is usually below 10%, but with slow-release SES, this figure may be even lower. Since there are no studies addressing these issues, it is not clear whether the extended-release SES has less stent thrombosis due to more rapid endothelialization, but more restenosis due to a lower antiproliferative effect. It must be noted that the extended-release SES has a large initial burst of drug release followed by a slow release over several weeks. Lowering peak concentration of sirolimus in the prolonged-release group may improve endothelialization, but might enhance the restenosis rate. Therefore, a simple reduction in sirolimus concentration does not mimic the extended-release stent. The issue of restenosis has not been addressed in the present animal experiment because SEM was performed to determine endothelialization of the stent surface at 3, 7 and 14 days. Restenosis is usually observed after 4- to 6-week follow up using histologic sections for determination of intimal proliferation. Endothelialization and intimal proliferation cannot be assessed in the same animal model without losing either the early or the late effects. Conclusions Endothelialization of intracoronary stents was best in BMS followed by the extended-release SES in the porcine overstretch model. The slowest rate of endothelialization was observed with the slow-release SES, suggesting that the pharmacokinetic profile of drug release is important for endothelialization of the stent. At 14 days, endothelialization in the extended-release stent reached values similar to those of the BMS, suggesting improved vascular healing with this type of stent.

1. Serruys PW, Kutryk MJ, Ong AT. Coronary-artery stents. N Engl J Med 2006;354:483–495.
2. Farb A, Burke AP, Kolodgie FD, Virmani R. Pathological mechanisms of fatal late coronary stent thrombosis in humans. Circulation 2003;108:1701–1706.
3. Joner M, Finn AV, Farb A, et al. Pathology of drug-eluting stents in humans: Delayed healing and late thrombotic risk. J Am Coll Cardiol 2006;48:193–202.
4. Kastrati A, Mehilli J, Pache J, et al. Analysis of 14 trials comparing sirolimus-eluting stents with bare-metal stents. N Engl J Med 2007;356:1030–1039.
5. Kastrati A, Dibra A, Eberle S, et al. Sirolimus-eluting stents vs. paclitaxel-eluting stents in patients with coronary artery disease: Meta-analysis of randomized trials. JAMA 2005;294:819–825.
6. Serruys PW, Sianos G, Abizaid A, et el. The effect of variable dose and release kinetics on neointimal hyperplasia using a novel paclitaxel-eluting stent platform: The Paclitaxel In-Stent Controlled Elution Study (PISCES). J Am Coll Cardiol 2005;46:253–260.
7. Stone GW, Moses JW, Ellis SG, et al. Safety and efficacy of sirolimus- and paclitaxel-eluting coronary stents. N Engl J Med 2007;356:998–1008.
8. Schuehlen H, Kastrati A, Dirschinger J, et al. Intracoronary stenting and risk for major adverse cardiac events during the first month. Circulation 1998;98:104–111.
9. Cook S, Wenaweser P, Togni M, et al. Incomplete stent apposition and very late stent thrombosis after drug-eluting stent implantation. Circulation 2007;115:2426–2434.
10. Silber S, Borggrefe M, Böhm M, et al. Positionspapier der DGK zur Wirksamkeit und Sicherheit von Medikamente freisetzenden Koronarstents (DES). Eine evidenzbasierte Analyse von 71 randomisierten Studien mit 28 984 Patienten. Der Kardiologe 2007;1:84–111.
11. Virmani R, Guagliumi G, Farb A, et al. Localized hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent: Should we be cautious? Circulation 2004;109:701–705.
12. Billinger M, Buddeberg F, Hubbell JA, et al. Polymer stent coating for prevention of neointimal hyperplasia. J Invasive Cardiol 2006;18:423–426; Discussion 427.
13. Windecker S, Mayer I, De Pasquale G et al. Stent coating with titanium-nitrideoxide for reduction of neointimal hyperplasia. Circulation 2001;104:928–933.
14. Makanya AN, Sparrow MP, Warui CN, et al. Morphological analysis of the postnatally developing marsupial lung: The quokka wallaby. Anat Rec 2001;262:253–265.
15. Van der Giessen WJ, Lincoff M, Schwartz R, et al. Marked inflammatory sequelae to implantation of biodegradable and non-biodegradable polymers in porcine coronary arteries. Circulation 1996;94:1690–1697.