Rapamycin-Eluting Stents in the Arterial Duct
Experimental Observations in the Pig Model
Background— Maintaining arterial duct patency by stent implantation may be advantageous in congenital heart disease management algorithms. Rapamycin, an immunosuppressant drug that demonstrates antiproliferative properties and inhibits smooth muscle cell migration, may deter the intimal hyperplasia that occurs during spontaneous closure and after-stent implantation of the arterial duct.
Methods and Results— Twenty-eight Yorkshire piglets (7 to 11 days old; weight, 2.2 to 4.9 kg) underwent stent implantation of the arterial duct (rapamycin-eluting (n=14) or bare metal (n=14) stents, 3.5-mm diameter) and were euthanized at 2, 4, and 6 weeks. Dissected arterial ducts were analyzed for lumen diameter, smooth muscle cell, and extracellular matrix components. Isolated arterial duct–derived smooth muscle cells were cultured in the presence or absence of rapamycin. Cellular proliferation rates were assessed by Ki-67 detection and [3H]-thymidine incorporation. No significant neointimal proliferation was present in either stent type at 2 weeks. At 4 weeks, the median luminal diameters of the bare metal stents were 87% (P=0.009), 54% (P=0.004), and 77% (P=0.004) that of the drug-eluting stents at the middle and aortic and pulmonary artery ends, respectively. At 6 weeks, the median luminal diameters of the bare metal stents were 0% (P=0.18), 5% (P=0.25), and 61% (P=0.13) that of the drug-eluting stents at the same respective levels. Complete histological occlusion was found in at least 1 level of the lumen in 9 pigs: 1 (17%) in the BMS group at 4 weeks, 5 (83%) in the BMS group at 6 weeks, and 3 (50%) in the DES group at 6 weeks. In vitro studies demonstrated 50%-lower proliferation rates in rapamycin-treated cultures of duct-derived smooth muscle cell cultures (P<0.001).
Conclusions— Rapamycin has antiproliferative actions on the arterial duct. Drug-eluting stents may be a more efficient tool than current palliative options for maintaining patency in critically duct-dependent states, but there may be a finite time-related benefit.
Received August 30, 2007; accepted February 13, 2009.
Patency of the arterial duct is critical for maintaining pulmonary blood flow in many neonatal congenital heart disease states; however, it is programmed for spontaneous anatomic closure. This complex process involves an interplay of modulations within the extracellular matrix and smooth muscle cells (SMCs), resulting in contraction and migration of the SMCs of the tunica media into the subendothelial space in a process described as neointimal proliferation.1–4
Clinical Perspective p 2085
Continued ductal patency relies on active intervention, and in its absence, alternative sources of pulmonary blood flow are necessary. Important shortcomings exist with each of the current palliative options.5–12 Continuous prostaglandin E1 infusion involves prolonged intravenous access and hospitalization, high drug cost, and medical complications. Balloon dilation of the arterial duct has a high rate of reocclusion.5,9 A surgical shunt may be complicated by luminal compromise, pulmonary artery distortion, phrenic nerve and thoracic duct injuries, and seroma formation.8,10,11 In a multicenter report of 1004 infants with shunt-dependent pulmonary blood flow, the incidence of shunt compromise was 12%, and mortality at 1 year was 26%.11 In another series, shunt narrowing >50% was noted in 23% examined at the time of elective takedown, with the predominant mechanism being neointimal proliferation.8
More effective than balloon dilatation, ductal stenting with a bare metal stent (BMS) has been reported in a variety of conditions with duct-dependent systemic and pulmonary blood flows.6,9,12 The initial technical challenges of ductal stenting have been overcome by the use of coronary stents with a low profile and easy deliverability.6 Although acutely effective, the main shortcoming of ductal stenting is in-stent stenosis secondary to neointimal proliferation, resulting in progressive obstruction and necessitating premature reintervention.6,7,12,13 Vessel stenting incites a robust reaction of excessive extracellular matrix production and intimal hyperplasia.14–16 In this respect, similarity exists between the mechanisms of ductal closure and in-stent stenosis.
Attenuation of the neointimal proliferative process has been demonstrated in the model of adult coronary artery disease, with the most effective agents to date being stents impregnated with drugs such as rapamycin and paclitaxel.17,18 Rapamycin is an immunosuppressive agent with antiinflammatory and antiproliferative effects.19 It induces cell-cycle arrest, ultimately resulting in inhibition of SMC proliferation.16,19,20
In this study, we examined whether the rapamycin drug-eluting coronary stent (DES) implanted in the neonatal porcine arterial duct demonstrated a reduction in neointimal proliferation translating to increased luminal diameter compared with the conventional BMS. To further assess the mechanism by which rapamycin may affect this unique vessel, we tested the influence of the drug in cultures of arterial duct–derived SMCs.
The DES implanted was the CYPHER Select; the BMS was the BX Sonic (Cordis Inc, Miami, Fla). Both stents, 23 mm in length, were mounted on 3.5-mm-diameter balloon platforms. The DES was loaded with 140 μg rapamycin per 1 cm2 or a total of 245 μg. A drug-free polymer layer was applied on top of the drug-polymer matrix as a diffusion barrier to prolong the release of the drug, with 50% of the rapamycin eluted over the first week, 80% over 30 days, and 100% over 90 days.18
Twenty-eight Yorkshire piglets (7 to 11 days old; median weight, 3.6 kg; range, 2.2 to 4.9 kg) were anesthetized with isoflurane and nitrous gases, intubated, and mechanically ventilated. A 4F sheath was percutaneously placed in the femoral artery using the modified Seldinger technique. As documented by aortography, the arterial duct was tiny or probe-patent in all cases. A 0.014-in Wizdom wire (Cordis Corp) was placed across the arterial duct into the pulmonary artery. Premounted stents (14 DES, 14 BMS) were implanted in the arterial duct over the wire with balloon inflation to 18 atm to achieve a stent diameter of 3.7 mm. The stents were placed so that the implant protruded into the main pulmonary artery but was flush with the aorta, ensuring complete ductal coverage (Figure 1). Penicillin 17 000 IU/kg IM was administered. For both stent groups, 2 pigs were euthanized at 2 weeks and 6 pigs at 4 and 6 weeks after the implantation. Weight ranges at the time of death were 5.1 to 5.8 kg at 2 weeks, 6.7 to 12.6 kg at 4 weeks, and 13.6 to 22.4 kg at 6 weeks.
Two additional piglets served as controls for comparative analyses. One was euthanized immediately after BMS implantation; the other was euthanized without stent implantation. The latter provided tissue to assess the mechanism of spontaneous ductal closure. Immediately before their deaths, 2-dimensional echocardiography was performed on the piglets to determine ductal patency with the Vivid 7 echocardiographic system (GE Corp, Wauwatosa, Wis).
The animals were killed by use of a euthanasia solution (Euthanyl, MTC Pharmaceuticals, Cambridge, Ontario, Canada). Heparin sulfate 1000 U IM was administered to limit acute thrombus formation during harvesting. The arterial duct was excised and fixed in buffered 10% formalin.
Tissue Processing and Histomorphometric Analysis
The excised fixed arterial duct was embedded in Spur resin. Transverse slices, each 5 μm thick, were obtained at the proximal pulmonary artery and the middle and distal aortic ends of the stent with an Isomet slow-speed saw (Buehler, Lake Bluff, Ill) and tungsten carbide knife.
The histological sections were stained with Movat pentachrome to visualize the presence and distribution of SMCs, elastin, and other components of the extracellular matrix21; α-actin for activated SMCs; picrosirius red for collagen; and anti-human von Willebrand factor antibody for vascular endothelium. For each stent, 3 slices were examined (1 from each of the marked areas), and the lumen diameter was measured at 4 points (0° to 135° in 45° increments) and then averaged. These parameters were compared with the dimensions of the control animal stented with the BMS that was euthanized at day 0 and with the naturally closed (untreated) arterial duct.
SMCs were isolated from 3 intact (not stented) arterial ducts by collagenase digestion as previously described22 and cultured for 7 days in α-minimum essential medium supplemented with 10% FBS in the presence or absence of 100 nmol/L rapamycin. The drug was added twice, at time 0 and at day 4, together with fresh medium.
Assessment of Cell Proliferation
Cellular proliferation rates of control and rapamycin-treated SMCs, derived from 3 separate arterial ducts, were compared by [3H]-thymidine incorporation and immunohistochemical detection of the proliferative antigen Ki-67 in quadruplicate cultures within each experimental group. Briefly, 1 μCi [3H]-thymidine per 1 mL media was added to the 4-day-old cultures at the time of the second rapamycin treatment, and the cultures were incubated for an additional 72 hours. The cultures were then washed twice with cold 5% trichloroacetic at 4°C and incubated with 0.5 mL of 0.3N NaOH for 30 minutes. Then, 200-μL aliquots from each well were added to 5 mL liquid scintillation cocktail and counted with a Win Spectral 1414 liquid scintillation counter (Wallac, Turku, Finland) as previously described.21
The parallel 7-day-old cultures were fixed in cold 100% methanol and exposed to antibody detecting the Ki-67 antigen in proliferating cells and then to peroxidase-labeled secondary antibody. The cultures were then counterstained with hematoxylin. For each of the quadruplicate cultures in each experimental group, the numbers of positively and negatively stained cells were counted under ×200 magnification in 30 separate fields. The percent of positively staining cells was determined within each field and averaged over the 30 fields examined.
Assessment of Elastin Production
The parallel 7-day-old cultures were probed with specific antibodies to elastin, collagen type I, and chondroitin sulfate–containing glycosaminoglycans, and the results were evaluated quantitatively by morphometry. The presence of elastic fibers was detected with polyclonal antibody to tropoelastin. The immunoreactions were visualized with fluorescein isothiocyanate–conjugated goat anti-rabbit secondary antibody. Nuclei were counterstained red with propidium iodide.21,22 The production of a new (metabolically labeled) insoluble elastin also was assessed in parallel 7-day-old cultures that were incubated for 72 hours with [3H]-valine.21 Deposition of insoluble elastin was reflected by levels of radioactive valine present in residues remaining after the cell layers of the same cultures were boiled in 0.1N NaOH for 45 minutes. This procedure removes all cellular and extracellular components except the cross-linked elastin. Quadruplicate cultures within each experimental group were analyzed. For each culture, 30 fields (magnification ×200) were randomly selected, and the area occupied by the particular immunodetectable component was quantified. The presence of each component was expressed as a percentage area of the entire analyzed field.
Descriptive statistics are reported as medians and median absolute deviation and first and third quartiles. Comparison of BMS and DES parameters was performed with the Wilcoxon rank-sum test. Data from the cultures of arterial duct–derived SMCs were analyzed by Student t test and are reported as means and SDs. Statistical significance was defined as P<0.05. All data analysis was performed with R 126.96.36.199
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Echocardiographic assessment of ductal patency corresponded with gross and histological examination findings in all piglets except 1 BMS piglet at 4 weeks, in which the lumen was very small on histological examination but appeared occluded on gross inspection and had no demonstrable flow by echocardiography. Effective luminal diameter throughout the length of the stent could not be measured accurately with echocardiography. For example, even in the widely patent stented arterial duct with minimal neointimal proliferation, the diameter of the color flow was not uniform throughout the stent length.
Insertion of both the DES and BMS prevented the rapid natural contraction and consequent closure of piglet arterial duct (Figure 2). At 2 weeks, no significant neointimal hyperplasia was found in either stent type resulting in a lumen diameter similar to that of the stented arterial duct harvested immediately after implantation (Figures 2 and 3⇓). At 4 weeks, the median luminal diameters of the BMS were 87% (P=0.009), 54% (P=0.004), and 77% (P=0.004) that of the DES at the middle and aortic and pulmonary artery ends, respectively (Figure 3). At 6 weeks, the median luminal diameters of the BMS were 0% (P=0.18), 5% (P=0.25), and 61% (P=0.13) that of the DES at the same respective levels (Figure 4). Despite the striking differences in median luminal diameter at 6 weeks, no statistical difference resulting from the scatter of the values was found. Complete histological occlusion occurred in at least 1 level of the lumen in 9 pigs: 1 (17%) in the BMS group at 4 weeks, 5 (83%) in the BMS group at 6 weeks, and 3 (50%) in the DES group at 6 weeks. Occlusion involved at least the middle level of the stent in all cases. Nine (50%) of the 18 segments at 6 weeks in the BMS group showed complete occlusion compared with 4 (22%) in the DES group.
Histological analysis revealed that neither stent type induced significant inflammatory infiltration of the arterial wall. Neither implant induced any progressive remodeling of tunica adventitia, and luminal compromise was a result of neointimal formation. Immunostaining with α-actin antibody showed relative abundance of activated SMCs in the neointima, particularly in the BMS group (Figure 5). Histochemical evaluation of transverse sections indicated that the milder and mostly focal intraluminal outgrowth found in the DES-treated arterial ducts, particularly in close proximity to the stent struts, contained more elastic fibers and less collagen than observed in BMS-treated ducts (Figure 6). At 2 weeks, evidence of endothelialization was found in the tissue between stent struts; however, the struts themselves were not covered because there neointimal proliferation was minimal. By 4 to 6 weeks, intimal coverage of stent struts had begun but was not complete in all sections (Figure 6).
Results from the in vitro studies demonstrated that rapamycin-treated cultures of arterial duct–derived SMCs demonstrated 50%-lower proliferation rates (P<0.001) than untreated counterparts as demonstrated by [3H]-thymidine incorporation and immunohistochemical detection of the proliferative antigen Ki-67 (Figure 7). The rapamycin-treated arterial duct SMCs also tripled their production of elastin (Figure 8).
The newborn arterial duct is a unique blood vessel programmed for spontaneous closure within the first few hours to days of postnatal life. Stent implantation of blood vessels also incites a reactive cellular response.14–16 Thus, the stented arterial duct provided a good model of a highly proliferative milieu of neointimal hyperplasia to compare the effects of the DES and BMS. This may have important clinical implications for the management of the neonate with duct-dependent blood flow.5–7,9,12,13
Rapamycin: Clinical Applications
Clinical experience with rapamycin has been predominantly as an immunosuppressive agent in posttransplant recipients and as the rapamycin-eluting stent implanted into adult coronary arteries. Only sporadic reports exist of successful rapamycin treatment in other settings of intimal hyperplasia. Pulmonary vein stenosis acquired from radiofrequency ablation to cure atrial fibrillation has been treated successfully with endovascular stenting and adjunctive oral rapamycin.24 Recently, a rapamycin DES was implanted in the arterial ductal component of an isolated left pulmonary artery in a 7-week-old infant with follow-up stent patency confirmed at 7 months.25
Clinical studies of coronary artery stents demonstrated substantially reduced in-stent intimal hyperplasia with the DES compared with the BMS, translating to a reduction in the need for repeat revascularization procedures.17,18 This initial enthusiasm and change in practice toward the DES have been tempered by safety concerns around emerging evidence of late thrombosis (1.3%) associated with the DES compared with BMS.26 The strongest independent predictors of stent thrombosis were premature discontinuation of and inadequate responsiveness to antiplatelet therapy.26 Delayed endothelialization after DES implantation is hypothesized as a causative factor.27 The issue of late thrombosis of the DES in an arterial duct is of limited concern because patency rates beyond 3 to 6 months are usually not necessary.
Local delivery via drug-impregnated stents allows localized therapeutic concentrations within the vessel walls with substantially reduced risk of systemic toxicity.20,28,29 In pig models with a DES containing 185 μg rapamycin, the whole-blood concentration of the drug peaks at 1 hour (mean, 2.63±0.74 ng/mL) and then declines below the lower limit of detection by 3 days while achieving therapeutic arterial tissue concentrations.20 In adults implanted with 150 and 178 μg/18 mm DES, peak drug concentration occurred between 3 and 4 hours at a level of 0.57±0.12 and 1.05±0.39 ng/mL with 1 or 2 stents, respectively, and minimal measurable blood levels were detectable at day 7.30
We aimed at estimating the serum concentrations of rapamycin that would be produced in a 3-kg infant on the basis of extrapolation of oral pharmacokinetics. The mean apparent clearance rate observed in pediatric renal and small bowel transplant recipients is 8.61±8.1 mL · min−1 · kg−1 or 517 mL · kg−1 · h−1.28,31 The systemic bioavailability of oral rapamycin is 14%.32 The CYPHER Select contained 245 μg rapamycin, half of which was released within 1 week (ie, 0.73 μg/h or 0.24 μg · kg−1 · h−1 for a 3-kg infant). Assuming 100% bioavailability of rapamycin from the DES, the mean clearance rate would be 72 mL · kg−1 · h−1. Hence, the steady-state concentration would be 3.3 ng/mL (dose rate divided by clearance rate) during the first week after implantation. In comparison, targeted steady-state trough levels range between 5 and 20 ng/mL for pediatric heart and renal transplant recipients.29,31 Thus, anticipated rapamycin serum levels with the DES would be in the subtherapeutic or therapeutic range during the first week after implantation and even lower thereafter; however, in vivo pharmacokinetics are required to confirm our hypothetical calculations, particularly given the known wide variability among patients.
Porcine Model of DES
Despite the inherent differences between the pig coronary system and that of humans, it provides the most pragmatic animal model to study vessel response to injury and intervention. The time-dependent progression in cellular response to stenting of coronary vessels observed in the porcine model is mirrored in human postmortem studies.15,33 Pigs grow considerably faster than humans and appear to have accelerated rates of neointimal hyperplasia. Neointimal proliferation in stented pig coronaries is maximal at 1 month, with ≈25% regression in neointimal growth occurring over the subsequent 3 to 6 months.33 In humans, peak neointimal thickness occurs between 6 months and 1 year, with ≈22% regression after 1 year.15,33 In this study, a clear reduction in neointimal hyperplasia and increased lumen diameter was found in the DES group at 4 weeks, but this benefit was not sustained convincingly thereafter. Explanations include the possibility that the arterial duct receives ongoing signaling for continued neointimal proliferation until vessel closure, or it is feasible that the local drug concentration at 6 weeks is below levels needed to sustain SMC inhibitory effects because >80% of the drug has been eluted by 30 days.18 Of note, porcine coronary artery studies of BMS and DES have follow-up periods up to 28 to 30 days,29,34 and it is unknown whether differences would have appeared with longer follow-up in this particular model. Such comparative observations do not exist for the arterial duct, but the mechanism of spontaneous ductal closure appears to be similar in pigs and humans. In our study, pigs were euthanized at up to 42 days when they had obtained a mean weight of 19.2 kg, anticipating that they had reached the maximal period of neointimal hyperplasia. It remains to be determined whether the observed reduction in neointimal proliferation with the DES in the pig model will translate to important sufficient clinical difference in ductal patency in infants awaiting further cardiac intervention.
The shift of the extracellular matrix balance, toward elastin and away from proteoglycan and collagen production, observed in the DES is consistent with the previously established paradigm that higher elastin deposition coincides with lower SMC migration and proliferation and that proteoglycans interfere with normal assembly of elastin fiber.21,22
The arterial duct was either very small or probe-patent at the time of stent implantation. In infants, the arterial duct is generally larger, particularly with prostaglandin therapy. It is possible that the mechanical injury of balloon dilation was magnified in the smaller animal vessels, inciting a more hyperplastic response. However, such effects would be similar in both the DES and BMS groups and would reinforce the antiproliferative benefits of rapamycin.
The number of animals in our study was small but larger than in published animal studies of coronary stents.20,34 Further studies in animals are needed before this technology can be applied to infants.
No antiplatelet or anticoagulation treatment was administered during this study other than at time of euthanasia. This study does not draw any conclusions regarding the presence or role of thrombosis.
The contribution of thrombus to subsequent neointima formation is not thought to be significant, an observation supported by the lack of reduction in in-stent restenosis by antithrombotic agents.35 Luminal thrombosis was a rare finding on gross and histological assessments; however, its potential importance is underscored by our observation of incomplete endothelialization of stent struts at all time periods. In the clinical setting, antithrombotic therapy is strongly recommended after ductal stenting.
BMS implantation counteracts the immediate contraction-driven closure of the newborn arterial duct. Its effectiveness appears to be enhanced when impregnated with rapamycin, which inhibits the neointimal proliferation that is intrinsic to ductal closure and BMS implantation, proposing it as a potentially more efficient tool for maintaining patency in critically duct-dependent states. However, there may be a finite time-related benefit.
We are grateful for the assistance of Marvin Estrada in the animal laboratory and Yanting Wang from Dr Hinek’s research laboratory.
Source of Funding
CYPHER Select stents were donated by Johnson & Johnson Co (New Brunswick, NJ), which had no role in the concept, design, or results analysis of this study.
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Maintaining arterial duct patency by stent implantation may be advantageous in congenital heart disease management algorithms. Rapamycin has antiproliferative properties and inhibits smooth muscle cell migration. In this study, bare metal and rapamycin-eluting stents were implanted into the arterial duct of 28 newborn pigs. At 2 weeks after stent implantation, minimal neointimal proliferation was observed in both the bare metal and rapamycin-eluting groups. At 4 weeks, luminal diameters were significantly increased in the rapamycin-eluting stent group. The difference at 6 weeks was not statistically significant. In vitro studies examining the cultures of duct-derived smooth muscle cells in the absence or presence of rapamycin demonstrated 50%-lower proliferation rates in the rapamycin group. This study demonstrates that rapamycin has antiproliferative actions on the arterial duct. Drug-eluting stents may be a more efficient tool than current palliative options for maintaining patency in critically duct-dependent states, but there may be a finite time-related benefit.