Use of a Tacrolimus-Eluting Stent to Inhibit Neointimal Hyperplasia in a Porcine Coronary Model

Materials and Methods Stent and stent coating. Stainless steel balloon-expandable stents (Jostent,™ Germany) were used for these studies. The bare stents, 16 mm long, were dip-coated in a biological polymer (SAE coating) or in a polymer/tacrolimus solution (200 µg/stent) for in vivo studies. In addition, 18 mm long bare stents were dip-coated in a polymer/tacrolimus solution to load 750 µg/stent of tacrolimus for in vitro release analysis. The surface characteristics of the coated stents were examined by microscopy. The stents were sterilized using ethylene oxide before implantation in porcine coronary arteries. In vitro drug release studies. Three 750 µg tacrolimus-loaded, SAE-coated stents were placed in vials containing 1 ml 0.9% NaCl at 37°C. Ultraviolet (UV) absorbance (Cary 4 E spectrophotometer, Varian Inc., California) was measured at 205 nm for tacrolimus each day for the first 14 days, and after 3 and 4 weeks, to determine the tacrolimus release. After every time point, the stents were replaced in a new vial containing NaCl. One control SAE-coated stent underwent the same procedure and the UV absorbance values were subtracted from the values of the drug-eluting stents. Impact of tacrolimus on vascular smooth muscle cell (SMC) growth in vitro. SMCs were isolated from New Zealand White rabbit aorta, passaged and cultured (50,000 cells/well) in six-well plates (Corning).¹⁵ Every third day, 0, 10-8, 10-7, 10-6, and 10-5 M tacrolimus or paclitaxel (Bristol-Meyers-Squibb), both dissolved in 20 µl ethanol, was added in combination with medium changes. After 7 days, SMC growth was determined directly with cell counting (Coulter Counter), and indirectly with total cell mass using protein quantification (BCA Protein Assay Kit, Pierce). Before protein quantification in the latter assay, cell viability was determined using neutral red uptake.¹⁶ A viability index for each concentration was calculated as percentage neutral red from control/% total protein from control. In vivo studies. Domestic cross-bred pigs of both sexes, weighing 20–25 kg were used. They were fed with a standard natural grain diet without lipid or cholesterol supplementation throughout the study. All animals were treated and cared for in accordance with the Belgium National Institute of Health Guidelines for care and use of laboratory animals. Surgical procedures and stent implantation in coronary arteries were performed according to the method described by De Scheerder et al.^17,18 Biocompatibility of the Polymer Coating Acute study. Five SAE-coated stents and 5 bare stents were randomly implanted in the right and left anterior descending coronary arteries of 5 pigs. The arterial segments were selected to obtain a 1.1:1 stent-to-artery ratio. Pigs were sacrificed after 5 days to evaluate injury, acute inflammatory response, and thrombus formation. Chronic study. Ten SAE-coated stents and 10 bare stents were implanted randomly in the coronary artery of 10 pigs with the same oversizing as in the acute study. Pigs were sacrificed after 4 weeks to evaluate peri-strut inflammation and neointimal hyperplasia. Stent-based tacrolimus delivery. Seventeen SAE-coated stents and 17 SAE-coated stents loaded with 200 µg tacrolimus were randomly deployed in porcine coronary arteries. The arterial segments were selected to obtain a 1.2:1 stent-to-artery ratio. Pigs were sacrificed after 4 weeks to evaluate the efficacy of local tacrolimus delivery on neointimal hyperplasia. Tissue processing and histomorphometric analysis. At 5 days or 4-week follow-up, the pigs were sacrificed and the stented coronary artery was perfused with a 10% formalin solution at 80 mmHg. Artery segments were carefully dissected together with a minimum 1 cm vessel segment, both proximal and distal, to the stent. The segment was fixed in a 10% formalin solution. The entire stent was cut into 4 segments: 1 proximal, 2 middle, and 1 distal part. From these, the proximal, one middle, and the distal segment were embedded in a cold-polymerizing resin (Technovit 7100, Heraus Kulzer GmbH, and Wehrheim, Germany) for histomorphometric analysis. Sections that were 5 microns thick were cut with a rotary, heavy-duty microtome HM 360 (Microm, Walldorf, Germany), equipped with a hard metal knife, and stained with hematoxylin-eosin, elastic, lectin, Movat, and phosphotungstic acid hematoxylin stain. Light microscopic examination was performed blinded to the type of stent used. Injury of the arterial wall due to stent deployment was evaluated for each stent filament site and graded as described.¹⁹ Inflammatory reaction at every stent filament site was carefully examined searching for inflammatory cells, and scored as followed: 1 = sparsely located histiolymphocytic infiltrate around the stent filament; 2 = more densely located histiolymphocytic infiltrate covering the stent filament, but no foreign body guanuloma or giant cells; 3 = diffusely located inflammatory cells and/or giant cells, also invading the media. The mean score was calculated as the sum of scores for each filament/ number of filaments present. Morphometric analysis of the coronary segments harvested was performed on 3 slices (proximal, middle, and distal stent parts) by using a computerized morphometry program (Leitz CBA 8000). The areas of the arterial lumen, the area inside the internal elastic lamina (IEL), and the area inside the external elastic lamina (EEL), respectively, were measured. Furthermore, the area stenosis (1-lumen area/IEL area) and the area of neointimal hyperplasia (area of IEL- area of lumen) were calculated. Immunohistochemistry staining. The second middle segment was embedded in the cold-polymerizing resin Technovit 9100 (Heraus Kulzer GmbH, and Wehrheim, Germany), which allows immunohistochemistric analysis. Here, 5 µm sections were cut in a similar manner as desribed above. Immunohistochemistry was then performed with monoclonal antibodies against proliferation cell nuclear antigen (PCNA, Dako), macrophages (CD-68, mac-387, 1/300, Serotec), and endothelium [von Willebrand factor (vWf) The Birmingham Site]. The PCNA and mac-387 density (number of positive cells/mm²) at 4 stent struts (at 3, 6, 9, and 12 o’clock) were measured as described,²⁰ and the average for each segment was calculated. The vWf staining for each segment was graded as follows: I = 90% positivity of the lumen circumference. Statistics. Data are presented as mean values ± SD. The in vitro data (cell growth and viability assay) were evaluated by means of a one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison post hoc test. For comparison of histomorphometric data between different groups, a non-paired t-test was used. A p value Images of the coated stents. The surface analysis of SAE-coated stents is presented in Figure 1. Dipcoating resulted in a smooth ultrathin coating (10 µm). Impregnation of the coating with 750 µg tacrolimus did not alter the surface of the coating. In vitro drug release. Drug release curves showed that after 24 hours, only 28% of the loaded tacrolimus was released from the stents. After the first 24 hours, the release continuously rose, showing that after 1 week, 65% of the loaded tacrolimus was released. Finally, after 4 weeks of incubation in NaCl at 37°C, almost complete (92 %) release was obtained (Figure 2). Effect of tacrolimus on SMC growth and cell viability. The total protein quantification assay showed, after 7 days of incubation with tacrolimus, no reduction of SMC proliferation (control: 96.3 ± 2.1 versus 10-8 M: 88.7 ± 3.2; 10-7 M: 88.7 ± 2.5; 10-6 M: 86.3 ± 2.3; 10-5 M: 86.0 ± 5.6; all data are µg/ml; n = 3) as opposes to the dose-dependent reduction by paclitaxel (control: 87.3 ± 1.2 versus 10-8 M: 82.7 ± 3.8; 10-7 M: 69.3 ± 2.5, p In vivo biocompatibility of the SAE coating. At 5 days follow-up, the bare metallic and SAE coated stents showed a similar histopathological response. The arterial media was intact and mildly compressed. A few inflammatory cells were seen adjacent to the stent filaments (Figure 4A). Stent struts with moderate inflammatory reaction were rare. A thin thrombotic meshwork covering the stent filaments was observed. The inflammatory and thrombus score of the coated stents and the bare stents were not significantly different. Arterial injury caused by stent deployment was low and identical for the two groups. At 4 weeks follow-up, a well organized neointima with a few inflammatory cells was observed. The histopathological reaction of the vessel wall was comparable between the SAE-coated and the bare stent group. The mean lumen area, neointimal hyperplasia (bare stents 1.45 ± 0.81 versus coated stents 1.30 ± 0.68, p > 0.05) and area stenosis (22 ± 14 versus 19 ± 12, p > 0.05) were similar for the two groups. The peri-strut inflammation (1.12 ± 0.33 versus 1.03 ± 0.09, p > 0.05) and arterial injury (0.30 ± 0.43 versus 0.21 ± 0.21, p > 0.05) of the SAE-coated and the bare stents were identical. Furthermore, compared to the 5 days follow-up, the peri-strut inflammation of SAE-coated stents did not show an increased response. Stent-based tacrolimus delivery (Table 1). With an increased stent/artery ratio, the SAE-coated stents showed an increased arterial injury and inflammatory response. The stent struts showed a moderate compression of the arterial media. Internal elastic lamina disruption and medial laceration were observed. A few stent filaments showed a moderate inflammatory reaction, although inflammatory cells in the media and adventitia were rare (Figure 4B). In contrast, tacrolimus-loaded stents showed a limited inflammatory response. A few inflammatory cells around the stent filaments were observed. No foreign body granuloma or giant cells were present. The inflammatory score (1.05 ± 0.08 versus 1.27 ± 0.51, p = 0.088) and arterial injury (0.17 ± 0.10 versus 0.48 ± 0.44, p 2, p > 0.05, Figure 5, A, B). PCNA staining was more variable in the SAE-coated stent group, with one valued as 884 cells/mm2. Excluding this extremely value, however, the positive cells of PCNA staining was compared between the two groups (tacrolimus-loaded 92.4 ± 79.7 versus SAE-coated 85.5 ± 74.1 cells/mm,² p > 0.05, Figure 5, C, D). Furthermore, equal vWf staining surrounding the lumen was observed in both groups (tacrolimus-loaded: 3.4 ± 0.5 versus SAE-coated: 3.5 ± 0.6, p > 0.05, Figure 5, E , F). Discussion In the present study, a porcine coronary stenting model was used to evaluate the biocompatibility of a polymer-coated stent, and the efficacy of stent-based tacrolimus delivery to reduce neointimal hyperplasia. Stainless steel stents coated with a biological polymer showed a biocompatible response when implanted in porcine coronary arteries. No increased peri-strut inflammation and neointimal hyperplasia with coated stents were seen when compared to bare stents. Stent-based tacrolimus delivery significantly reduced the neointimal formation, which was associated with a decreased peri-strut inflammation. Since the injury score was also lower in the tacrolimus group, we must be cautious in claiming an antirestenotic therapeutic effect by tacrolimus due to the well known injury/hyperplasia relationship. Our interpretation of the results is, however, in favor of tacrolimus, since the balloon/artery oversizing was similar in both groups, which is a well known determinant of vascular injury after stent implantation. With a similar oversizing, the lower injury score can be explained by the potent anti-inflammatory effect of tacrolimus. Polymer coatings for local drug delivery. Polymers have been applied to the stent surface as a reservoir for sustained drug release at the stenting site. Drugs can elute from a polymer matrix or be released with the degradation of the polymer coating. Studies have shown that some synthetic polymers, biodegradable or nonbiodegradable, result in an important inflammatory and proliferative tissue response.^21,22 These reactions may counteract the biological effect of the locally released drug and can contribute to neointimal hyperplasia. Therefore, the polymer matrix is a crucial determinant for the optimal function of a drug-eluting stent. Biological polymers, such as fibrin and phosphorylcholine (PC), have the advantage of minimizing the inflammatory response. They also may be beneficial in limiting thrombus formation and neointimal hyperplasia.^23,24 It has been found that fibrin film-coated stents, compared to polyurethane-coated stents, showed significantly decreased neointimal hyperplasia and foreign-body tissue reaction.²⁴ PC-coated metallic stents did not increase neointimal formation in a porcine stent model.²⁵ In this study, we used a biological polymer to coat the metallic stent surface. At 5 days, a few inflammatory cells adjacent to the stent struts were observed, which was comparable to the bare metal stent. No increased fibrin deposition on the stent struts was observed. At 4-week follow-up, the peri-strut inflammatory response was low, and no significant difference was observed between 5 days and 4-week follow-up. These results suggest that this biological coating is biocompatible when implanted in coronary arteries. In addition, this coating material slowly dissolves within weeks without inducing an inflammatory reaction. The SAE-coated stent group did not induce an increased neointimal formation when compared to the bare stent group at 4 weeks. These data indicate that the SAE-coating can serve as a suitable coating for local drug delivery. Tacrolimus-eluting stent: Possible mechanism for anti-restenosis effects. Tacrolimus is a potent immunosuppressive agent with potent anti-inflammatory properties. It is a hydrophobic drug and can easily penetrate the plasma membrane to enter the cytoplasm. Binding to the intracellular FK-binding protein (FK-BP), tacrolimus forms a complex and then binds to calcineurin. This binding inhibits the activation of calcineurin and disrupts the dephosphorylation nuclear translocation of nuclear factor of activated T cells (NF-AT). Transcription proteins, required for the generation of interleukin (IL)-2 and other proinflammatory cytokines such as IL-3, IL-4, IL-5, IL-6, IL-8 and its receptor, tumor necrosis factor, are inhibited.^14,26,27 With its lipophilic characteristic, suppression of proinflammatory cytokines expression, and inhibition of T-cell proliferation, tacrolimus is a potentially interesting drug for stent-based delivery to decrease peri-strut inflammation and neointimal hyperplasia. The lipophilic nature of tacrolimus may enable it to pass easily through the cell membrane and minimize blood flow loss. The SAE coating material is able to contain a high dose of tacrolimus, keeping a gradient from the stent struts to arterial tissue and facilitate the tissue uptake. To analyze the release characteristics, we loaded a high dose of tacrolimus (750 µg) into the SAE matrix. In the first 24 hours, only 28% of the loaded tacrolimus was released, and the release period lasted for at least 4 weeks. The role of inflammation in the cascade of neointimal formation has been well documented. The arterial injury and vessel stretching during stenting, focal thrombus formation at the stent struts, and local atherosclerotic lesions, can activate and recruit leukocytes, monocytes, and macrophages from the circulating blood and adventitia at the stenting site. Clinical studies have shown that an inflammatory reaction is common in neointimal hyperplasia.^7,8 The macrophage number and inflammatory markers could predict the rate of restenosis in patients undergoing coronary angioplasty.²⁸ In addition, a positive relationship has been found between the extent of the inflammatory reaction and the amount of neointimal formation in an animal study.²⁹ By releasing chemostatic and growth factors, inflammatory cells can regulate the vascular repair and neointimal formation. Anti-inflammatory agents delivered using coated stents have shown to provide beneficial effects on in-stent restenosis in experimental and clinical studies.^30,31 It has been reported that coronary stent graft (CSG) loaded with a high dose of tacrolimus caused a moderate reduction of neointimal formation in a rabbit iliac artery.³² In this study, we used an overstretched coronary stent model to increase arterial injury and inflammatory response, and evaluated the effects of tacrolimus on neointimal formation. Our studies demonstrated that local delivery of tacrolimus using SAE-coated stents could significantly inhibit peri-strut inflammation. The peri-strut macrophage contents of the tacrolimus-coated stents were decreased by nearly 50% compared to the control stents. In rabbit carotid arteries, ceramic-coated stents loaded with tacrolimus, compared to the uncoated stents, also showed a decreased lymphocyte and macrophage score.³² Furthermore, compared to the SAE-coated stents, neointimal hyperplasia and area stenosis of tacrolimus-loaded stents were significantly reduced, although the PCNA-positive staining cells of the two stent groups were comparable. Locally released tacrolimus used to prevent macrophage and T-cell migration and proliferation inhibits the release of pro-inflammatory factors after stenting and may contribute to these reductions. Both cytostatic and cytotoxic agents have been used for stent-based delivery. With cytotoxic agents, a delayed healing process has been observed in some drug-eluting stents.^33,34 Using chondroitin sulfate and gelatin (CSG)-coated stents with paclitaxel, Farb et al. demonstrated an incomplete healing in the higher dose (42.0 and 20.2 µg) groups.³³ Intimal fibrin deposition and hemorrhage, medial necrosis, and adventitial inflammation were observed. Although the mechanism of the vascular toxicity of paclitaxel is uncertain, a reduction in cell numbers and extracellular protein mass were observed. As SMCs are the dominant cells in the arterial medial layer, we compared the effects of tacrolimus with paclitaxel on SMCs in vitro. No reduction of SMC growth by means of direct cell counting and indirect total cells mass quantification was found when SMCs were incubated with tacrolimus in concentrations ranging from 10-8 to 10-5 M. Moreover, these SMCs showed excellent viability at all concentrations. In contrast however, a dose-dependent reduction of SMC proliferation and a decreased viability were noted with paclitaxel. These findings suggest that tacrolimus has a higher safety range than paclitaxel. In another study, Lüscher et al. found that tacrolimus showed a toxic effect on SMC in vitro cell culture, however, the tacrolimus concentration to kill 50% cells was over a hundred times higher than paclitaxel (personal communication). In our in vivo study, the score of vWf staining of tacrolimus-loaded stents was comparable to the coated control stents, which suggests that locally released tacrolimus from the coated stents did not retard endothelial regrowth. Furthermore, no medial necrosis, increased adventitial inflammation, or other local arterial toxic effects with tacrolimus-loaded stents were observed. In conclusion, compared to bare stents, SAE-coated stents did not elicit an increased inflammatory response or proliferative tissue reaction. Stent-based tacrolimus delivery using the SAE coating could effectively reduce neointimal hyperplasia. This effect relates most probably to its anti-inflammatory properties.

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