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Arsenic trioxide eluting stent reduces neointima formation in a rabbit iliac artery injury model

Wei Yang, Junbo Ge, Hongling Liu, Kan Zhao, Xuebo Liu, Xiufen Qu, Weimin Li, Yonglin Huang, Aijun Sun, Yunzeng Zou
DOI: http://dx.doi.org/10.1016/j.cardiores.2006.08.010 483-493 First published online: 1 December 2006

Abstract

Objective: In-stent restenosis is caused by the neointimal hyperplasia, which involves abnormal growth of vascular smooth muscle cells (VSMC). Arsenic trioxide (As2O3) is known to be a potent inhibitor of cell proliferation. We therefore studied the role of an As2O3 eluting stent in the prevention of restenosis in a rabbit iliac artery model.

Methods and results: Bare stents, or stents coated with poly-l-lactic acid (PLLA) and either 40 μg of As2O3, 180 μg of paclitaxel or vehicle were implanted into the left proximal iliac arteries of New Zealand rabbits. The delivery of drugs from stents in vitro and in vivo was evaluated by atomic fluorescence spectrophotometry and high-performance liquid chromatography, respectively. Histomorphometric measurements at 7 or 28 days showed that, comparing to rabbits receiving the PLLA stent, in animals treated with As2O3 eluting or paclitaxel eluting stent neointima thickness was reduced by 50% and 46%, the absolute neointimal area was reduced by 53% and 44%, while the absolute luminal area was increased by 46% and 43%, respectively. There were no significant differences in injury or inflammation scores among PLLA, As2O3 eluting and paclitaxel eluting stents. As2O3 eluting stent induced more TUNEL-positive VSMC than the other stents. As2O3 levels measured in the arterial tissue were much higher than those in serum, which were nearly undetectable at 7 days after stent implantation. In in vitro studies, cultured rabbit arterial VSMC were stimulated with As2O3 or paclitaxel and analyzed for their cell cycle progression and apoptosis by flow cytometry and electron microscopy. As2O3 treatment resulted in a reduction of VSMC number in G1 phase with a concomitant increase in apoptosis of VSMC, whereas paclitaxel treatment led to blocking of VSMC in the G2/M phase.

Conclusion: In a rabbit iliac artery model PLLA coated As2O3 eluting stent significantly suppressed in-stent restenosis by reducing proliferation and inducing apoptosis of VSMC.

Keywords
  • Angioplasty
  • Restenosis
  • Stents
  • Arsenic trioxide (As2O3)

1. Introduction

Percutaneous coronary intervention (PCI) has become the most effective method for revascularization in the treatment of coronary artery diseases. However, the benefits of PCI have been principally limited due to a major problem, the renarrowing of the stented vessel after the procedure (restenosis), which is known to be associated with three main processes: elastic vascular recoil; neointimal hyperplasia and matrix deposition; and constrictive arterial remodeling [1–4]. Although implantation of a bare metal stent can prevent elastic vascular recoil and constrictive arterial remodeling by scaffolding the vessel wall, local deep vascular injury, partial denudation of endothelium and balloon-artery interaction during the procedure induce more neointimal hyperplasia than PCI alone, leading to an in-stent restenosis in approximately 20% of patients [5,6]. Thus, inhibiting the neointimal hyperplasia after deployment of the stent is critical to the efficacy of PCI treatment. Since systemic application of antiproliferative and antiplatelet agents is inefficient in the prevention of restenosis and induces severe side effects [7,8], the focus has recently been shifted to the local delivery of a drug by the coated stent.

Stent-based local delivery of a drug (drug eluting stent, DES) is a new technology aiming to prevent the development of neointimal hyperplasia after PCI. Application of the DES has enabled dramatic reductions of restenosis rates. Currently, a number of DES has been developed, including different carrier stents, coatings and drugs, which are under evaluation for their effectiveness and safety [9–11]. Studies of sirolimus- and paclitaxel-eluting stents showed impressive results, with less than 5% of target vessel revascularization after 9 months in the selected patients and lesions [12,13]. However, one area of uncertainty is whether these two agents alone are sufficient to treat the wide variety of lesions found in day-to-day practice, just as a single antibiotic cannot treat all infections. It may therefore be that a variety of agents beside sirolimus and paclitaxel are necessary to treat the restenosis after PCI. In addition, restenosis still remains a great challenge due to the increased number of procedures and more complicated diseases treated with PCI [14].

Arsenic compounds are natural substances that have been used in the treatment of patients with acute promyelocytic leukemia (APL) [15,16]. Recently many studies demonstrated that arsenic trioxide could treat not only patients with leukemia but also patients with other types of malignant cancers [17–20]. It has been reported that arsenic trioxide (As2O3) might inhibit the cell growth and angiogenesis, and induce partial cytodifferentiation and apoptosis in cancer. We therefore investigated whether an As2O3-coated stent might be a feasible, safe and efficient method to prevent the in-stent restenosis in a rabbit iliac artery model.

2. Materials and methods

2.1. Construction of the drug eluting stents

The As2O3 eluting stent was prepared by coating the stainless steel stent (Beijing Amsinomed Medical Company, China) with a monolithic matrix of a high molecular weight polymer of ∼320 KD poly-l-lactic acid (PLLA) and As2O3 base. As control, a paclitaxel eluting stent was prepared by similar coating. Using a proprietary technique, a 1% (w/w) solution of PLLA in chloroform containing either As2O3 or paclitaxel (Sigma) was sprayed onto the stent wires to form the eluting coating. Each coated stent contained ∼0.1 mg of PLLA plus 40 μg of As2O3 or 180 μg of paclitaxel. The thickness of the polymer layer on the steel wire ranged from 6 to 10 μm (mean 8 μm). This polymer coating was sufficiently flexible to allow the stent to expand without cracking or peeling from the wire (Supplementary Data 1). Stents were mounted on commercially available angioplasty balloons and sterilized using a conventional ethylene oxide gas technique.

The kinetic elution of As2O3 from the sterilized polymer-coated stent was assessed in an in vitro system. As2O3 eluting stents were placed in vials containing 10 ml of PBS at 37 °C. An AFS-810 model atomic fluorescence spectrophotometer equipped with an arsenic hollow cathode lamp (Beijing Titan-Instruments Co. Ltd, China) was employed for measurement of the atomic fluorescence intensity of arsenic. An automatic intermittent hydride generation device was used to generate the arsenic hydride. Argon gas was used as the carrier gas for transportation of arsenic hydride from the hydride generator to the atomizer. The desired trace element As3+ in the 5% (w/v) HCl as carrier solution, or in 2% KBH4–1% NaOH as reaction solution was transferred to the hydride-generating reaction cell using a peristaltic pump. Arsenic hydride produced was transferred to the atomizer using argon gas and the atomic fluorescence signal was measured at 193.7 nm. The concentration of As3+ in the unknown sample solutions was finally calculated by the standard curve method. As2O3 concentrations in PBS were measured at a series of time points in a period of 28 days.

Paclitaxel eluting stents were also checked for the uniformity of the drug application. Stents were randomly selected from each batch and the drug was extracted in 10 ml calf serum (Gibco BRL, Life Technologies) at 37 °C and measured by high-performance liquid chromatography (HPLC, Gilson). Paclitaxel was eluted from a 25 cm×34.6 mm pentafluorophenyl column (ES Industries) in a mobile phase consisting of 45% acetonitrile and 55% water flowing at 1.5 ml/min and monitored at 227 nm with a UV detector (Gilson model 116).

2.2. Procedure

Male New Zealand rabbits (3.0–3.5 kg) were obtained from the Shanghai Animal Administration Center. All animal experiments were approved by the Animal Care and Use Committee of Fudan University. Right carotid artery access was achieved under general anesthesia. Rabbits (n=100) were randomly divided into four equal groups of 25 animals per group: uncoated stents, PLLA control stents, As2O3 eluting stents, and paclitaxel eluting stents. Stents appeared identical and the operators were blind to the group assignment. Each stent was hand crimped on a 3.0 mm angioplasty balloon and deployed (8-atm balloon inflation for 45 s) in the proximal iliac artery, achieving an approximate balloon-to-artery ratio of 1.2:1. Angiographic arterial diameters were measured with digital clippers. The rabbits received 40 mg of aspirin orally 24 h before surgery and daily thereafter. Euthanasia was performed at 7 days (n=7 in every group) or 28 days (n=18 in every group) after stent deployment.

The delivery of As2O3 or paclitaxel from stent to tissue and serum was evaluated using hydride generation reaction interfaced with atomic fluorescence spectrometry assay and HPLC, respectively, in additional animals at 2 h, 1 day, 3 days, 7 days, 14 days and 28 days after deployment of As2O3 eluting stents (n=7, 7 stents per time point) or paclitaxel eluting stents (n=3, 3 stents per time point) in the iliac arteries. Segments of the arteries were excised immediately distal and proximal to the stented segment. The stent was carefully removed from the artery and the artery was then blotted dry, weighed, snapped-frozen and stored at −70 °C for later analysis. Blood samples were also collected to measure serum concentration of As2O3 or paclitaxel.

2.3. Tissue preparation

Vessels with stents from every group at 7 days (n=7 for each group) or 28 days (n=15 for each group) after implantation were cut into 5 pieces, each 3 mm long, fixed in 10% buffered formalin, and embedded in glycol methacrylate or in paraffin. Cross sections (obtained by using a Leica section cutter, Germany) from proximal, distal and medial pieces were stained with hematoxylin and eosin (H–E) for the measurement of vessel area and histological analysis. Sections from the other pieces were used for immunohistochemistry and TUNEL analysis.

Three stented vessels collected at 28 days from every group were cut open lengthwise and fixed with 1% osmium tetroxide for scanning by an electron microscope (HITACHI S-520).

2.4. Histological analysis of neointimal hyperplasia

Vessel area was measured by tracing the external elastic lamina (EEL; mm2), the internal elastic lamina (IEL; mm2), and the lumen area (LA; mm2). Neointimal area (NA; mm2) was calculated as follows: NA=IEL−LA. The percentage of neointimal stenosis was calculated as follows: % stenosis=IEL−LA/IEL×100. Mean neointimal thickness, neointimal area, lumen area and injury and inflammatory scores were calculated using an imaging analysis system (Imagine Pro Plus). The depth of the vessel wall injury produced by the oversized stent (the stimulus for neointimal formation) at every wire site was determined and assigned a number in the injury score system [21]: 0=intact internal elastic lamina; 1=lacerated internal elastic lamina; 2=lacerated internal elastic lamina and media; 3=lacerated external elastic lamina. The inflammation of the vessel at every wire site was graded on a four-point scale [22]: 0=no neointimal inflammation; 1=inflammatory cells present in < 25% of neointima; 2=inflammatory cells present in ≥25%, but < 50% of neointima; 3=inflammatory cells present in > 50% of neointima.

2.5. Analysis of VSMC apoptosis

Apoptosis of VSMC was evaluated by the TUNEL method. VSMC in neointima and media were counted in three cross-sections for each arterial specimen. The number of TUNEL-positive VSMC was expressed as percentage of the total number of VSMC.

2.6. Cell culture

VSMC were isolated by digestion of New Zealand white rabbit aortas with collagenase and elastase. Cells were maintained in IDME containing 5% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 pg/ml) to confluence and then subcultured at a density of 5×l03 cells/cm2. The cultured cells exhibited typical morphology of VSMC including growth in a “hill and valley” pattern. The identity of VSMC was further confirmed by staining with a monoclonal anti HHF-35 antibody that selectively recognizes the smooth muscle form of α-actin. Cells were grown to confluence in 6-well or 24-well plates (Costar, Cambridge, MA) for further use.

2.7. Cell cycle analysis by flow cytometry

Cells were plated in 6-well plates at a density of 3×l04 cells/well and cultured for 2 days. As2O3 or Paclitaxel (both from Sigma) was added to the culture medium at concentrations of 0, 1, 3 or 5 μmol/L. Three days later the cells were collected, treated with 0.1% Triton X and 0.5% RNase A and then incubated with propidium iodide. All samples were analyzed on a flow cytometry system (FACS) (Becton Dickinson). 10,000 nuclei were noted in each analysis. The percentage of cells in each phase of the cell cycle was estimated using the program of Cell FIT cell cycle analysis, version 2.0 (Becton Dickinson). All the experiments were performed three times.

2.8. Annexin-V staining

Cells were plated in the 6-well plates at a density of 5×l03 cells/well and cultured for 2 days. As2O3 or Paclitaxel was added to the culture medium at concentrations of 0, 1, 3 or 5 μmol/L and the cells were further cultured for 72 h. At the end of this period cells were incubated with a FITC-conjugated Annexin-V antibody according to the recommendations of the manufacturer (Immunotech). Nuclei were counterstained with propidium iodide (PI) and the cells were screened by FACS (approximately 10,000 events). Early apoptosis was estimated by the relative number of FITC+PI cells, while FITC+PI+ cells represented either secondary necrotic or postapoptotic populations. All the experiments were performed three times.

2.9. Electron microscopy

After 72 h treatment with either PBS or 3 μmol/L As2O3, VSMC were collected, washed twice with PBS, fixed with 1% osmium tetroxide, dehydrated with solutions containing increasing concentrations of ethanol and propylene oxide and embedded in araldite. Thin sections were collected on Quick-Coat (Electron Microscopy Sciences, Fort Washington, PA) and treated with copper grids. After staining with uranyl acetate and lead citrate, samples were viewed at 80 kV on a JEOL 1200 EX electron microscope.

2.10. Statistical analysis

Numerical data are presented as mean±SEM. Continuous variables were compared by ANOVA (t test with Bonferroni correction), and categorical variables were compared by χ2 test. A p value ≤0.05 was considered significant.

3. Results

3.1. Quantitative assessment of As2O3 and paclitaxel in vitro delivery

We first evaluated the in vitro delivery of As2O3 or paclitaxel from the As2O3 eluting stents (n=7) or the paclitaxel eluting stents (n=6), respectively (Fig. 1A and B). More than 50% of As2O3 elution occurred within the first 4–6 days, reaching a plateau after 8 days. On the other hand, more than 50% of paclitaxel elution occurred within the first 7–10 days, reaching a plateau after 14 days. These results indicated that in vitro the delivery of As2O3 occurred earlier than that of paclitaxel.

Fig. 1

Analysis of in vitro drug release from the As2O3 and paclitaxel eluting stents. The As2O3 (A) and the paclitaxel (B) eluting stents were placed in 10 ml of phosphate-buffered saline or calf serum, respectively, and their concentrations in the respective solutions were measured at the indicated times for a period of 28 days.

3.2. Stent implantation

In total 100 stents were placed in the iliac arteries of 100 rabbits, all stents being easily deployed at relatively low balloon inflation pressure. There was no systemic toxicity observed after the stent implantation. A complete blood cell count showed similar results for white blood cell counts, hematocrit and platelet counts among all groups (Supplementary Data 2). All stents were angiographically open at 7 and 28 days. Post-surgery mortality rate was zero.

3.3. Measurements of As2O3 and paclitaxel levels in tissue and serum

The levels of As2O3 or paclitaxel measured in arterial tissue (μg/g wet tissue weight) and serum (μg/ml serum) after stent implantation are presented in Tables 1 and 2. At 2 h after stent deployment, the mean concentration of As2O3 within the adjacent arterial tissue was 16.2±4.4 μg/g, whereas the mean serum concentration was 0.0087±0.0014 μg/ml, nearly five orders of magnitude lower than that in the tissue. Tissue concentration of As2O3 increased slightly at 1 day, but decreased thereafter, whereas serum concentration of As2O3 progressively declined after 1 day. Even at 7 days after stent implantation, As2O3 levels in the arterial wall were nearly four orders of magnitude higher than those in the serum. They remained high throughout the first 28 days, while serum As2O3 was not detected after 14 days. The changes in the concentration of paclitaxel in arterial tissue and serum after the stent implantation followed a pattern that was similar to that of As2O3.

View this table:
Table 2

Concentrations (μg/ml) of As2O3 and paclitaxel in the serum of animals with the drug eluting stent implantation

Time2 h1 day3 days7 days
As2O30.0087±0.00140.0097±0.00130.0053±0.00140.0016±0.0011
Paclitaxel0.0066±0.00220.0086±0.00260.0067±0.00220.0033±0.0012
  • Data are mean±SEM (n=7 for As2O3; n=3 for paclitaxel).

View this table:
Table 1

Concentrations (μg/g) of As2O3 and paclitaxel in the arterial tissues implanted with the drug eluting stents

Time2 h1 day3 days7 days14 days28 days
As2O316.2±4.418.6±6.18.1±4.23.2±1.30.8±0.30.06±0.01
Paclitaxel3.7±1.412.6±3.110.1±1.24.2±1.31.8±0.30.3±0.1
  • Data are mean±SEM (n=7 for As2O3; n=3 for paclitaxel).

3.4. Histological observations and morphometric measurements

No in-stent thrombosis was observed in any stenting groups. The arteries with As2O3 eluting or paclitaxel eluting stents had a larger lumen and reduced neointimal thickness versus those with uncoated stents or PLLA stents (Fig. 2). Comparing to the arteries implanted with uncoated or PLLA stents, there was a significant reduction in neointimal thickness and area in the arteries with As2O3 eluting or paclitaxel eluting stents, as follows: uncoated stent: 0.19±0.04 mm and 1.80±0.36 mm2; PLLA stent: 0.24±0.05 mm and 2.32±0.50 mm2; As2O3 eluting stent: 0.12±0.03 mm and 1.11±0.24 mm2; paclitaxel eluting stent: 0.13±0.04 mm and 1.21±0.34 mm2 (Fig. 3). The As2O3 eluting stent reduced neointimal thickness by 37% and 50% in comparison to the uncoated or PLLA stent, respectively, while paclitaxel eluting stent reduced neointimal thickness by 32% and 46% compared to uncoated and PLLA stent, respectively. There were no significant differences regarding any of the above parameters between As2O3 eluting stent and paclitaxel eluting stent groups.

Fig. 3

Histomorphometric analysis of the stented arteries. At 28 days after deployment, arteries with uncoated, PLLA, As2O3 eluting, or paclitaxel eluting stents were prepared, stained and analyzed as described in Materials and methods. (A) Neointimal thickness; (B) Neointimal area; (C) Lumen area; (D) % of neointimal stenosis. Results are presented as mean±SEM (n=15). *, p<0.01 vs. uncoated and PLLA stents. There is no significant difference between As2O3 eluting and paclitaxel eluting stents (p>0.05).

Fig. 2

Histological photographs of the stented arteries. Representative gross photographs of H–E stained sections of arteries deployed with either uncoated (A), PLLA (B), As2O3 eluting (C) or paclitaxel eluting stent (D) at 28 days (Bar 500 μm).

3.5. Injury scores and inflammatory responses

Injury scores did not significantly differ among the four groups (data not shown and Supplementary Data 3). Only mild inflammatory responses such as infiltration of mononuclear cells, lymphocytes and multinucleated giant cells at the polymer–tissue interface were observed in some arteries with PLLA coated stents (data not shown and Supplementary Data 3).

3.6. Endothelization of the stented arteries

We used scanning electron microscopy to analyze the endothelization of the stented arteries (n=3 for each group) at 28 days after the stent implantation. The arteries with uncoated or PLLA stents were fully endothelized, the luminal surface of the vessel wall and the stent struts being covered by confluent endothelial cells (Fig. 4A, B). However, endothelization of the arteries with As2O3 eluting or paclitaxel eluting stents was incomplete, similarly in both groups. The endothelized surface was 87.4±3.5% in As2O3 eluting stent and 85.8±4.6% in paclitaxel eluting stent (Fig. 4C, D).

Fig. 4

Scanning microscopy photographs of the rabbit iliac arteries 28 days after stent implantation. (A) Uncoated stent, appearing fully coated with endothelial cells covering the smooth muscle layer. (B) PLLA stent, showing an apparently complete endothelization. (C) As2O3 eluting stent, partly endothelized; the arrow indicates the focal area without endothelization. (D) Paclitaxel eluting stent, showing partial endothelization; the arrow indicates an area lacking endothelization, a denuded strut not covered by endothelial cells. (15 kV, ×30, bar 100 μm).

3.7. Apoptosis of VSMC in the stented arteries

Many TUNEL-positive cells scattered throughout the neointima and media of the arteries with As2O3 eluting stents were observed at 7 days post implantation (Fig. 5A, B). However, at 28 days, the number of TUNEL-positive cells significantly decreased (Fig. 5C, D).

Fig. 5

TUNEL staining of the stented iliac artery. At 1 (A and B) and 4 weeks (C and D) after implantation with an As2O3 eluting stent (A and C) or a PLLA stent (B and D), vessels were stained by the TUNEL method. Apoptotic cells are indicated by their nuclear brown staining (arrows). The sections were counterstained with hematoxylin. * Strut region. ×400, bar 50 μm.

A quantification of the TUNEL staining in the intima of the stented arteries at 7 days post implantation showed that in the case of the As2O3 eluting stent TUNEL-positive cells represented 64.6±11.7% of the total number of VSMC. By comparison, only 39.6±6.4%, 37.7±4.9% and 41.3±4.9% of VSMC were TUNEL-positive in the arteries with uncoated, PLLA and paclitaxel eluting stents, respectively. At 28 days after stenting, TUNEL-positive VSMC number decreased to 39.1%±6.3% in As2O3 eluting stent arteries, and to 19.4±5.9%, 18.7±5.5%, and 23.3±6.6% in uncoated, PLLA and paclitaxel eluting stent arteries, respectively (Fig. 6A).

Fig. 6

Quantification of the TUNEL-positive cells in the neointima and media at 1 and 4 weeks after uncoated, PLLA, As2O3 eluting, or paclitaxel eluting stent implantation. Results are presented as mean±SEM (n=7). (A) In the neointima; * p<0.01 vs. uncoated, PLLA and paclitaxel eluting stents, p>0.05 vs. uncoated and PLLA stents. (B) In the media; * p<0.01 vs. uncoated, PLLA and paclitaxel eluting stents, p>0.05 vs. uncoated and PLLA stents.

In a similar way, quantification of the TUNEL staining in the media of the stented arteries at 7 days showed 66.8±9.4% positive VSMC in the As2O3 eluting stent, and lower numbers for the uncoated stent (41.1±8.3%), PLLA stent (40.8±6.9%) or paclitaxel eluting stent (44.0±6.7%). At 28 days after stenting, the number of TUNEL-positive VSMC decreased to 33.4±6.8% in As2O3 eluting stent arteries, and to 16.7±5.0%, 17.0±5.5% and 19.1±3.3% in uncoated stent, PLLA stent and paclitaxel eluting stent arteries, respectively (Fig. 6B).

3.8. As2O3 Effect on the cell cycle and apoptosis of cultured VSMC

To further confirm the in vivo data showing an increased percentage of TUNEL-positive VSMC in stents eluting As2O3, we tested its effects on cultured VSMC. As2O3 treatment resulted in a dose-dependent reduction of VSMC number in the G1 phase (Fig. 7), the percentage of cells in G1 phase being as follows: 66.9±6.5 in the absence of As2O3, 54.9±3.8 at 1 μmol/L, 45.7±5.5 at 3μmol/L and 23.6±5.4 at 5 μmol/L. At the same time, As2O3 treatment demonstrated a dose-dependent increase in apoptosis of VSMC. In response to 0 μmol/L, 1 μmol/L, 3 μmol/L or 5 μmol/L, the apoptotic VSMC represented the following percentages of the total number of cells: 4.9±1.3%, 17.9±3.3%, 36.2±3.8% and 51.3±4.3%, respectively. In contrast, paclitaxel dose-dependently increased the number of VSMC blocked in G2/M phase: 15.9±4.3% at 0 μmol/L, 25.9±4.3% at 1 μmol/L, 35.7±5.3% at 3 μmol/L and 43.6±4.9% at 5 μmol/L. Unlike As2O3, paclitaxel did not induce significant apoptosis of cultured VSMC (data not shown).

Fig. 7

Dose-dependent effect of As2O3 on the cell cycle of cultured VSMC. VSMC were incubated for 72 h with increasing concentrations of As2O3 as follows: 0 μmol/L (A), 1 μmol/L (B), 3 μmol/L (C), and 5 μmol/L (D). Cell cycle distribution was determined by a flow cytometry after staining of the cells with propidium iodide and analyzed using the ModFit software. 10,000 cells were analyzed in each group. A representative experiment from 3 independent ones is shown. The regions in black color represent cells arrested in the G1 phase.

Finally, we used electron microscopy to confirm the characteristics of apoptotic VSMC. Comparing to cells in the absence of treatment, the cells exposed for 72 h to 3.0 μmol/L As2O3 demonstrated reduced microvilli, a decreased endoplasmic reticulum, parts of which expanded into blebs and vesicles, undetectable nucleoli and condensed chromatin at the periphery of the nuclear envelope (Fig. 8).

Fig. 8

Electron microscopy photographs of cultured VSMC. VSMC were viewed on a JEOL 1200 EX electron microscope at 80 kV (×1000, bar 2 μm). A representative cell is shown from each group. (A) In the absence of As2O3 the cell displayed a regular structure. (B) After 72 h treatment with 3 μmol/L As2O3 the cell showed condensed chromatin at the periphery of the nuclear envelope.

4. Discussion

In the present study done in a rabbit iliac artery model we have demonstrated that local delivery of As2O3 via a PLLA coated stent inhibited in-stent neointima formation and preserved lumen area without significant systemic toxicity, suggesting that As2O3 eluting stent is a feasible, safe and efficient DES. The stents coated with either 40 μg As2O3 or with 180 μg paclitaxel had similar effects of inhibiting neointima proliferation and mildly delaying endothelization without an increase in inflammation up to 28 days after stenting. The mechanism by which As2O3 reduced neointima formation seems to be a complex one, involving not only inhibition of VSMC cycle progression but also induction of VSMC apoptosis.

The polymer PLLA has been utilized for orthopedic application in humans and found biocompatible for at least the first few months after implantation [23]. Lincoff et al. demonstrated that low molecular weight PLLA rather than high molecular weight one produced an inflammatory reaction within the vascular wall over 28 days [24]. In our present study we noticed only a mild inflammatory response in the arteries implanted with high molecular weight PLLA coated stents for 28 days. The degradation of PLLA products may play a causative role in the tissue inflammation [25].

Although further studies are necessary to determine whether products of degradation of the biodegradable polymer might trigger cellular responses with long-term effects, effective uneventful dissolution of such polymers might represent an important advantage over the potential long-term consequences of disruption of the integrity of non biodegradable stent coatings.

Recent success in early clinical trials with DES using the antiproliferative agents sirolimus and paclitaxel have been quite promising [26–28]. Indeed, this has contributed directly to the rapid expansion of effective DES in the world market. Rapamycin inhibits smooth muscle cell proliferation by blocking the cell cycle in the G1 (growth) phase when RNA is produced and proteins are synthesized. In doing so, it prevents the cell from entering the S (DNA synthesis) phase, thus preventing replication. The specific target of rapamycin (TOR) is considered to be the central protein kinase element of the downstream signaling pathway controlling mRNA translation and cell growth [29–31]. Paclitaxel is the potent, natural prototype of the taxane family of anticancer agents. It works by binding to polymerized tubulin, thereby stabilizing it against disassembly and inhibiting cell mitosis (G2/M phase of cell cycle).

Accumulating evidence suggests the possibility of development of drug resistance to rapamycin through genetic mutations or compensatory changes in mTOR-regulated proteins [32]. Likewise, overexpression of the multidrug resistance gene (MDR-1), molecular changes in the target protein β-tubulin, changes in mitosis checkpoint proteins, changes in apoptotic regulatory proteins, and overexpression of cytokines such as IL-6, have been suggested as possible mediators of resistance to Taxol [33,34]. Thus, as with antimicrobial therapy, it is likely that effective DES treatment will require a varied armamentarium in order to cope with developing changes in devices and materials, untoward tissue responses and drug resistances.

In the present study implantation of As2O3 eluting stents for 28 days significantly suppressed neointimal formation and reduced arterial stenosis, effects much more pronounced than in the case of the stents without drugs. These changes may be associated with a stronger inhibition of VSMC growth and cell cycle, as well as with induction of apoptosis in VSMC. As2O3 has been reported to inhibit the cell growth and cycle, and induce apoptosis in cancer cells. This study further indicated that As2O3 not only inhibits the cell cycle but also induces apoptosis in cultured VSMC. Drugs that interfere early in the cell cycle (early G1 or pre-G1) are considered to be cytostatic and elicit less cellular necrosis and inflammation than agents that affect the cell cycle at a later stage [35]. Thus, inhibition of VSMC proliferation by decreasing the cells in G1 stage and induction of VSMC apoptosis by As2O3 may provide an alternative with potentially lower toxicity.

In comparison to the uncoated and PLLA stents, As2O3 eluting stent increased the rate of VSMC apoptosis in the rabbit arteries both at 1 week and at 4 weeks after implantation. These effects were observed not only in the neointima but also in the media. Several authors suggested that VSMC proliferation is one of the major reasons for neointimal hyperplasia after balloon angioplasty, as well as stent implantation [36,37]. As shown by Zeymer et al. [38], a peak of proliferation of neointimal cells occurred 1 week after balloon denudation of the rat aorta. Schwartz and coworkers [39] observed that stent implantation increases the amount of neointima formation in comparison with balloon angioplasty. It is therefore critical to inhibit the proliferation of neointimal cells at 1 week after stent implantation. Moreover, it has been previously indicated that balloon angioplasty induces apoptosis [40], and stent implantation can cause more apoptosis in the media and neointima than balloon angioplasty [41]. Our data showed that in the rabbit model As2O3 eluting stent induced more VSMC apoptosis than uncoated, PLLA or paclitaxel eluting stents, and that most of VSMC apoptosis was observed at first week, decreasing then within 4 weeks after stent implantation.

In 2000 the Food and Drug Administration approved As2O3 for treatment of APL. The safe amount of As2O3 administered systemically is thought to be no more than 0.15 mg/kg−1/day [42]. Soignet et al. did a multicenter study of arsenic trioxide in APL. When patients were given arsenic trioxide with a median daily dose of approximately 0.16 mg/kg (range of 0.06–0.20 mg/kg) and a median cumulative dose of 360 mg (range of 160–515 mg), the safety profile of the drug was favorable. Adverse events were uncommon, generally self-limiting and reversible [43]. In our study, the highest level of As2O3 in the serum of rabbits implanted with As2O3 eluting stents was 0.0097±0.0013 μg/ml. Animals implanted with As2O3 eluting stents showed similar white blood cell counts, hematocrit and platelet counts comparing to rabbits with paclitaxel eluting stents, PLLA stents and uncoated stents, suggesting that 40 μg As2O3 eluting stent may be safe to use.

In conclusion, in a rabbit iliac artery model PLLA coated stents with As2O3 reduced the neointimal hyperplastic response to injury through inhibition of cell cycle and induction of apoptosis of VSMC. Although further studies are necessary to confirm the long-term effects and the effects in large animals, and to define the full pharmacokinetic profile, including dose–response relationships as well as efficacy in the presence of other treatment regimens, the present study suggests that As2O3 eluting stent may represent a novel device useful in preventing in-stent restenosis.

Acknowledgments

This study was supported by a grant from the National Natural Science Foundation of China (NSFC) (30170368). We thank Han FU, Jianguo JIA, Ruiming YAO for their assistance with the animal and cell experiments. We also thank Dr. S Goergescu at Tufts New England Medical Center, U.S.A. for English revision.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.08.010.

Footnotes

  • 1 These authors contribute equally to this study.

  • Time for primary review 25 days

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View Abstract