ABSTRACT: Oligonucleotides capturing CD31 endothelial cells (= aptamer) were used for coating of intracoronary stents to improve endothelialization and vascular healing. Methods. Three different coronary stents were implanted in 9 farm-raised swine: 1) cobalt-chromium stent (CC, control stent); 2) aminoparylene-coated stent (AP, polymer); and 3) aminoparylene- and aptamer-coated stent (AA). Stent length was 18 mm, stent diameter 3 mm. Animals were restudied after 6 weeks. Minimal lumen diameter (MLD) and late loss were determined by quantitative coronary angiography. Vessel lumen, intimal proliferation and restenosis were determined by histomorphometry. Disruption of the lamina elastica interna (LEI) and inflammatory reactions were assessed in all sections. Results. The average MLD at baseline was 2.98 ± 0.65 mm and at follow up 2.18 ± 0.53 mm (p Conclusions. Stents coated with endothelial-cell-capturing aptamers show similar late loss and angiographic restenosis rates as uncoated cobalt-chromium stents. Neointimal proliferation was similar in all three stents suggesting comparable proliferative potentials. Inflammatory reactions were observed in 1/5 of all histologic sections. In the present study, no advantage of aptamer-coating on neointimal proliferation of intracoronary stents was found.
J INVASIVE CARDIOL 2010;22:481–487
Key words: neointimal proliferation, endothelial-cell-binding aptamer, inflammation, cobalt-chromium stent, porcine overstretch model
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 excellent angiographic results.1,2 Long-term studies have shown that bare-metal stents are associated with a considerable number of 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 that are coated with a polymer as carrier substance containing an antiproliferative or immunosuppressive agent to prevent in-stent restenosis.4–6 Recent data suggest that drug-eluting stents do not show vascular healing and reendothelialization is lacking in up to 50% of all cases.7–9 The lack of reendothelialization has been associated with very late stent thrombosis,8,9 which may occur in the presence of a prothrombotic state such as pneumonia, non-cardiac surgical interventions or immobilization. Thus, controlled reendothelialization would protect against thrombosis. There may be differences in reendothelialization of the new third-generation stents coated with everolimus or biolimus, members of the limus family. To improve reendothelialization, a new concept was developed with endothelial progenitor cell (EPC) attracting messengers which increase vascular repair and reendothelialization by attracting EPCs which proliferate into an endothelial layer. Synthetic materials do not contain EPC-attracting properties and, thus, result in high blood cell adhesion (platelets, neutrophils, monocytes, etc.), leading to intimal proliferation and reocclusion.
Recently, an EPC-capturing stent was introduced containing CD34 binding antibodies.10 It was postulated that this stent shows rapid vascular healing and reendothelialization. As an alternative, oligonucleotides with high power for attracting proteins and specific binding for EPCs were developed.11 One of these coatings is tested in the present study using aminoparylene as carrier substance and aptamer (= oligonucleotide) as active substance for attracting CD31 expressing endothelial cells.
Aptamers as therapeutic agent. Aptamers are single-stranded oligonucleotides that fold into a specific three-dimensional structure that enables them to directly bind to proteins and to inhibit a protein target. Aptamers are generated by an iterative, in vitro selection process termed SELEX (Systemic Evolution of Ligands by EXponential enrichment).11,12 SELEX technology has enabled the isolation of aptamers to a myriad of target proteins.13 Aptamers possess several properties that make them potentially quite suitable for use as CD31 attractants. First, aptamers typically exhibit high affinity and specific binding to their target protein, with dissociation constants in the high picomolar to low nanomolar range, and specificity constants of 103 or greater when one compares aptamers with targeted versus non-targeted proteins.12 Aptamer technology is well suited for the detection of direct-acting, potent CD31 attractants. Moreover, the high affinity of most aptamers allows for therapeutic dosing at submicromolar levels, which should reduce potential non-specific effects. Second, aptamers are purported to be nonimmunogenic. Their small size and similarity to endogenous molecules theoretically makes them poor antigens, and the lack of antigenicity of aptamers has been supported by recent clinical studies.12 Third, the pharmacokinetic properties of aptamers are tunable and, to date, aptamers have exhibited predictable pharmacokinetics with well-behaved pharmacokinetic and pharmacodynamic relationships. In fact, aptamers can be formulated to possess very short half-lives (minutes) or can be easily conjugated to high-molecular-weight polyethylene glycol to provide compounds with much longer half-lives (9–12 hours following bolus intravenous or subcutaneous injection).14
The purpose of the present study was to test this new aptamer with aminoparylene-coated stent attracting endothelial cells and to compare this to a carrier substance-coated stent (aminoparylene) and a cobalt-chromium control stent. The study hypothesis was that reendothelialization is enhanced in the aptamer-coated stents and, therefore, intimal proliferation is reduced compared to the other two stents. Furthermore, inflammatory reactions are expected to be minimal in the aptamer-coated stents due to the rapid reendothelialization.
From the Swiss Cardiovascular Center/University Hospital, Bern and the †Institute of Pathology, University Hospital, Bern, Switzerland.
Disclosure. Research supported by an educational grant of DSI, Busan, South Korea and Biosteel AG, Aachen, Germany.
Manuscript submitted May 25, 2010, provisional acceptance given June 29, 2010, final version accepted August 9, 2010.
Address for correspondence: Prof. Otto M. Hess, MD, FESC, FAHA, Swiss Cardiovascular Center/University Hospital, Bern, Swiss Cardiovascular Center, University Hospital, Bern, CH-3010, Switzerland. Email: firstname.lastname@example.org
MethodsThree types of stent were evaluated in the present study in 9 pigs each. One stent was implanted in each of the large epicardial arteries (left anterior descending coronary artery, left circumflex coronary artery and right coronary artery) under general anesthesia with sodium pentobarbital 10 mg/kg intravenous which was maintained by halothan inhalation.15,16 Mean body weight was 36 kg. All pigs received 250 mg acetylsalicylic acid intravenous followed by 100 mg aspirin for three days. The left carotid artery was dissected and a 7 French (Fr) vascular sheath was introduced. Then a 6 Fr guiding catheter for coronary angiography and stent implantation was placed in the left and right coronary. 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 18 mm long. The structure of the stent was a stainless steel stent with 9 cells and minimal shortening in the long axis.16 Stents were randomly implanted in either one of the three coronary arteries to prevent implantation bias. Balloon-to-artery ratio was aimed to achieve a value of 1.1 to 1.3. After implantation, animals were transported back to the farm the same day and kept for 6 weeks for healing from the intervention. The protocol has been approved by the local animal ethics committee. 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 overdose of KCl intravenously. 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 polymethyl-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 a proximal and distal reference segment. All sections were stained with paragon (7.3 g toluidine blue with basic fuchsin dissolved in 1000 ml of ethanol 30%). For comparison purposes data from a previous study with bare-metal stents of identical design16 were used for comparison purposes. Angiography and histomorphometry were carried out by the same observers and pathologists.
Quantitative Coronary AngiographyAll coronary angiograms were assessed quantitatively15,16 in a blinded fashion by a standard software program (Medis SA, Medical Imaging Systems, Leiden, The Netherlands). Minimal luminal diameter (MLD), in-stent diameter at the proximal, middle and distal end as well as the proximal and distal reference diameter were measured quantitatively (Figure 1). Late loss was calculated by subtracting MLD at follow up from MLD at baseline. In-stent restenosis was calculated from MLD divided by the mean value of the proximal and distal reference diameter multiplied by 100. This software has been carefully validated. Inter- and intraobserver variability ranged between 5 and 8%.15,16
HistomorphometryAfter staining the samples (Figure 2), quantitative evaluation of the stented vessel was carried out in a blinded fashion on a digital system (Image Pro Plus, Media Cybernetics). 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 measured from stent lumen minus vessel lumen and in-stent restenosis was calculated from intimal proliferation divided by stent lumen times 100 (Figure 2). 3. Reendothelialization was assessed under high magnification light microscopy by an experienced pathologist (T.S.). In all sections, inflammatory cells and granulomas were visually assessed (mild, moderate and severe). An inflammatory score was calculated from the severity of the inflammatory reaction: mild = 1 point, moderate = 2 points and severe = 3 points. 4. Disruption of the lamina elastica interna (LEI) was determined by visual examination of all histologic sections (at low and high magnification image, Figure 3). 5. Scanning electron microscopy (SEM) was not used for technical reasons. SEM requires fresh samples using glutaraldehyde fixation with gold staining, whereas histomorphometry requires formalin fixation and paragon staining. Either technique can be used but not both because the stents have to be embedded in polymethyl-methacrylate for cutting. 6. Control data were obtained from a similar study16 using uncoated bare-metal stents as comparators.Statistics. All data are given as mean or median ±1 standard deviation. Figures 4 through 6 show box plots with median and standard deviation (upper graphs) and bar graphs with mean and standard deviation (lower graphs). Statistical comparison was performed with a paired T-test for angiographic measurements of minimal lumen diameter (Figure 4). All other comparisons (Tables 1 and 4, Figures 5 and 6) were done with a one-way analysis of variance (ANOVA) for 3 groups because only one time point (follow-up examination) was available. When the test was significant the Scheffé method was used as post hoc analysis. A test was considered statistically significant when p (Tables 2 and 3).
ResultsAll animals survived the intervention and the follow-up interval of 6 weeks. No clinical signs of stent thrombosis were observed. Due to a randomization error one aminoparylene-coated stent was placed more in the right coronary artery and one aptamer-coated stent more in the circumflex artery. Balloon-to-artery ratio was 1.19 for cobalt-chromium, 1.15 for aminoparylene-coated and 1.17 for aminoparylene- and aptamer-coated stents, respectively.
Angiographic DataMLD decreased from baseline to follow up by 21% for CC, 28% for AP, and 31% for AA (all ns) (Figure 4). There were no significant differences. Late loss was similar for the three stents, however, smaller (ns) in the uncoated cobalt-chromium (0.6 mm) than in the other two coated stents (Figure 5). Late loss was slightly higher in aptamer- than aminoparylene-coated stents (0.96 mm vs. 0.85 mm, ns). Restenosis (Table 1) was 22% for the cobalt-chromium stent, 28% for the aminoparylene and 32% for the aptamer stent (all ns). Histologic data. Representative histologic samples for the three tested stents are shown in Figure 2. All stents showed inflammatory reactions regardless of vessel (arrows, Figure 3), but mostly in the aptamer-coated stent (Table 2). A disruption of the LEI (high magnification image, Figure 3) was found in all three stents but most commonly in the cobalt-chromium, respectively aptamer-coated stent (Table 2). Correlating the disruption and the inflammatory reaction (Table 3) a significant relationship between these two parameters was found. Vessel lumen was larger in the aminoparylene- than aptamer-coated or cobalt-chromium stent (4.60 mm2, 4.00 mm2, 3.91 mm2) (Table 4). Differences were, however, small. Intimal proliferation (Table 4 and Figure 6) was highest in the cobalt-chromium stent (2.39 mm2) followed by the aptamer-coated stent (2.13 mm2) and the aminoparylene-coated stent (1.88 mm2). Restenosis was similar in all three stents, namely 39% in the cobalt-chromium, 38% in the aptamer and 29% in the aminoparylene-coated stent, respectively. When these data are compared to bare-metal stent data from a previous study, no significant differences were observed (Table 4). The correlation between MLD (angiography) and vessel lumen (histology) showed a significant relationship with a correlation coefficient of 0.561.
Inflammatory ReactionsInflammatory reactions were seen in approximately 1/5 of all stents but more commonly in the cobalt-chromium and aptamer-coated stents (Table 2). The average inflammatory score was 2.0 in cobalt-chromium stents indicating that 1/4 of the stents have inflammatory reactions of moderate severity (Table 2). In 10% of the aminoparylene-coated stents there were inflammatory reactions with mild severity and in 1/3 of the aptamer-coated stents with moderate severity, respectively. A typical example is shown for a cobalt-chromium stent at low and high magnification (Figure 3). The granulomas were typically found around the stent struts.
DiscussionCapture molecules have been recently introduced into stent technology for attracting endothelial cells which have been shown to stimulate vascular healing.11 Antibodies against CD34 expressing endothelial progenitor cells have been used in clinical practice for rapid vascular healing and prevention of adverse events such as in-stent restenosis and acute or subacute stent thrombosis.10 EPCs play an important role as precursors for angiogenesis and endothelial repair. Aptamers capturing CD31 expressing endothelial cells have been developed and bound to an aminoparylene-containing carrier substance (= polymer). This aptamer-coating was shown in a bioreactor to attract porcine CD31 expressing EPCs.11 In the present study, endothelial-cell-binding aptamer stents showed similar neointimal proliferation and restenosis rates to the uncoated cobalt-chromium (control) stent and the aminoparylene-coated carrier stent. In this experimental setup no effect on vascular healing was observed, but pigs have a high potential for rapid healing even in the presence of antiproliferative substances (sirolimus).17 An interesting observation was the occurrence of inflammatory reactions in both cobalt-chromium and aptamer-coated stents in approximately 25–30%. These inflammatory reactions were mainly seen in the presence of a ruptured lamina elastica interna. However, these inflammatory reactions were mainly seen around stent struts and could be explained by a hypersensitivity reaction to the coating of the aptamer stent as it was described in humans with sirolimus-eluting stents.8,18 The negative result of the present study could be either due to the lack of vascular healing by aptamers or due to the high potential of the pigs for rapid reendothelialization. No effect of this stent was found in human studies using CD-34-binding antibodies.10,19,20 Inflammatory reactions. Coronary inflammation was observed in 22% of all stents. The aptamer-coating stent showed the highest inflammatory reactions with 30%, followed by the uncoated cobalt-chromium stent with 24%. A typical inflammatory response is shown in Figure 3 for an uncoated cobalt-chromium stent. Typically, the inflammatory reaction is around the stent struts in the presence of the disruption of the lamina elastica interna. The nature of this inflammatory reaction is, however, not clear.21 It may be due either to the disruption of the lamina elastica interna or to a hypersensitivity reaction to the polymer as seen in patients with drug-eluting stents.8,18 Similar reactions were found in another study with bare-metal stents.16 Twenty-three percent of all sections showed inflammatory reactions in bare-metal stents similar as in the present study with either cobalt-chromium or aptamer-coated stents. There was also a correlation between the disruption of the lamina elastica interna and the inflammatory reactions (Table 3). These data suggest that the inflammatory reactions are probably due to procedural causes rather than to hypersensitivity reactions to cobalt-chromium or aptamer-coated stents. Limitations. Several potential limitations to the present study have to be mentioned:
1. Farm-raised swine show rapid vascular healing and even drug-eluting stents have been shown to be endothelialized within days or weeks.17 This may have limited the beneficial effect of the aptamer-coated stent because healing might have been as rapid in the uncoated as in the coated stent. 2. A potential cross-reaction between human and porcine endothelial cells may exist but since we have used porcine endothelial cells the effect on the aptamer may not be translated into humans. 3. Endothelialization was assessed 6 weeks after implantation by an experienced observer but no experiments were carried out to measure endothelialization by scanning electron microscopy (SEM) to show directly the binding of the endothelial cells to the stent surface. 4. An animal model with atherosclerotic coronary arteries would have been preferable, but such models exist only in small and medium-sized animals (rabbits, monkeys) receiving high cholesterol-rich diets. This model is artificial and does not represent the true clinical situation in patients with coronary artery disease. 5. A shorter follow up of 2 weeks may have shown some differences between the 3 stents17 which may have been lost due to the high healing potency during the follow up of 6 weeks. However, 6 to 8 weeks represent a gold standard for vascular healing in the porcine overstretch model. Shorter durations may underestimate the proliferative potency of the stent and longer observation periods may be associated with a late catch-up phenomenon.
ConclusionsUncoated cobalt-chromium stents show similar neointimal proliferation with coated aptamer and uncoated bare-metal stents 6 weeks after implantation in the porcine overstretch model. Histologic data showed a restenosis rate of 39% for the cobalt-chromium, 38% for the aptamer, 29% for the aminoparylene-coated and 30% for the uncoated bare-metal stent, respectively. Reendothelialization was good in all 6 stents at 6-week follow up. However, inflammatory reactions were seen in some of the stents but somewhat more in the aptamer-coated and cobalt-chromium stents. The nature of this reaction is not clear but may be due to the disruption of the lamina elastica interna or due to a hypersensitivity reaction to the aptamer-coated stent as described in humans with drug-eluting stents.8
1. Serruys PW, Kutryk MJ, Ong AT. Coronary-artery stents. N Engl J Med 2006;354:483–495. 2. 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. 3. 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. 4. 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. 5. 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. 6. Serruys PW, Sianos G, Abizaid A, et al. 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. Farb A, Burke AP, Kolodgie FD, Virmani R. Pathological mechanisms of fatal late coronary stent thrombosis in humans. Circulation 2003;108:1701–1706. 8. 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. 9. 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. 10. Aoki J, Serruys PW, van Beusekom H, et al. Endothelial progenitor cell capture by stents coated with antibody against CD34: The HEALING-FIM (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth-First In Man) Registry. J Am Coll Cardiol 2005;45:1574–1579. 11. Hoffmann J, Paul A, Harwardt M, et al. Immobilized DNA aptamers used as potent attractors for porcine endothelial precursor cells. J Biomed Mater Res A 2008;84:614–621. 12. Nimjee SM, Rusconi CP, Harrington RA, Sullenger BA. The potential of aptamers as anticoagulants. Trends Cardiovasc Med 2005;15:41–45. 13. Gold L, Polisky B, Uhlenbeck O, Yarus M. Diversity of oligonucleotide functions. Annu Rev Biochem 1995;64:763–797. 14. Tucker CE, Chen LS, Judkins MB, et al. Detection and plasma pharmacokinetics of an antivascular endothelial growth factor oligonucleotide-aptamer (NX1838) in rhesus monkeys. J Chromatogr B Biomed Sci Appl 1999;732:203–212. 15. Windecker S, Mayer I, De Pasquale G, et al. Working Group on Novel Surface Coating of Biomedical Devices (SCOL). Stent coating with titanium-nitride-oxide for reduction of neointimal hyperplasia. Circulation 2001;104:928–933. 16. Flueckiger A, Strahm Y, Billinger M, et al. Intimal proliferation and restenosis in paclitaxel-eluting stents with aminoparylene as carrier substance in swines. J Invasive Cardiol 2009;21:128–132. 17. Frey D, Billinger M, Meier P, et al. Endothelialization of sirolimus-eluting stents with slow and extended drug release in the porcine overstretch model. J Invasive Cardiol 2008;20:631–634. 18. Nebeker JR, Virmani R, Bennet CL, et al. Hypersensitivity cases associated with drug-eluting coronary stents: A review of available cases from the Research on Adverse Drug Events and Reports (RADAR) project. J Am Coll Cardiol 2006;47:175–181. 19. Szmitko PE, Kutryk MJ, Stewart DJ, et al. Endothelial progenitor cell-coated stents under scrutiny. Can J Cardiology 2006;22:1117–1119. 20. Taljaard M, Ward MR, Kutryk MJ, et al. Rationale and design of Enhanced Angiogenic Cell Therapy in Acute Myocardial Infarction (ENACT-AMI): The first randomized placebo-controlled trial of enhanced progenitor cell therapy for acute myocardial infarction. Am Heart J 2010;159:354–360. 21. Van der Giessen WJ, Lincoff AM, Schwartz RS, et al. Marked inflammatory sequelae to implantation of biodegradable and non-biodegradable polymers in porcine coronary arteries. Circulation 1996;94:1690–1697.