Anti-Inflammatory Effects of Arsenic Trioxide Eluting Stents in a Porcine Coronary Model

Previous research from our group has demonstrated arsenic trioxide eluting stents significantly reduced neointimal area and thickness compared with bare metal stents. In the present study, the anti-inflammatory effects of arsenic trioxide in vitro and arsenic trioxide eluting stents in a porcine coronary model have been explored. Sixty-five pigs underwent placement of 139 oversized stents in the coronary arteries with histologic analysis, endothelial function analysis, and immunohistochemical and western blot analyses. Arsenic trioxide eluting stents effectively inhibited local inflammatory reactions, while no significant difference in endothelialization and endothelial function between arsenic trioxide eluting stents and bare metal stents was observed. Arsenic trioxide eluting stents favorably modulate neointimal formation due to less augmentation of early inflammatory reactions, and quick endothelialization of the stent surface, which might contribute to long-term safety and efficacy of drug eluting stents.

DESs are designed to reduce in-stent neointimal growth through the elution of agents that arrest the cycle of cell proliferation. An ideal DES drug should suppress excessive neointimal growth while maintaining the proliferation of endothelial cells and functioning endothelium [15]. Arsenic trioxide (As 2 O 3 ) has been used in the treatment of patients with acute promyelocytic leukemia (APL) [16,17] and other types of malignant cancers [18][19][20][21][22]. As 2 O 3 acts on cells through a variety of mechanisms, in�uencing numerous signal transduction pathways and resulting in a vast range of cellular effects that include apoptosis induction, growth inhibition, promotion or inhibition of differentiation, and angiogenesis inhibition [23,24]. In a recent report [25], a heparin-immobilized copolymer of L-lactide (LA) and 5methyl-5-benzyloxycarbonate-1,3-dioxan-2-one (MBC) has been synthesized as a biodegradable coating material for local As 2 O 3 delivery. is copolymer was found to reduce thrombosis, localized hypersensitivity, and in�ammation, which belong to late-stage adverse events.
e pathological process of in-stent restenosis (ISR) is characterized by an in�ammatory healing response a�er stretch and damage of the vessel wall [26][27][28][29][30][31][32][33][34]. Stent restenotic reduction of DESs can be mainly ascribed to its antiproliferative activity, but anti-in�ammation may also be an important explanation for controlling in�ammation-triggered proliferative process [35,36]. As 2 O 3 eluting stents (AESs) effectively reduce neointimal thickening in rabbit and porcine models through inhibiting the cell cycle and inducing vascular smooth muscle cell (VSMC) apoptosis [25,37]. e present study was performed to evaluate the behavior of in�ammatory cells to As 2 O 3 and effect of AESs in a porcine coronary model, thus reveal the mechanism of AESs in reducing ISR.

Materials and Methods
2.1. Animals. Pigs (∼20 kg weight) were obtained from the Shanghai Animal Administration Center and received daily oral antiplatelet medication until termination. All animal experiments were approved by the Animal Care and Use Committee of Fudan University and were in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Academy Press (NIH Publication No. 85-23, revised in 1996).

Stent Implantation.
On the procedure day, thirty male pigs were anesthetized with ketamine (20 mg/kg intramuscularly) and xylazine (2 mg/kg intramuscularly). BMSs ( 21, 7 for one week, 5 for two weeks, and 9 for four weeks), PCSs ( 16, 5 for one week, 5 for two weeks, and 6 for four weeks), and AESs ( 32, 9 for one week, 11 for two weeks, and 12 for four weeks) were implanted in 2-3 coronary arteries per pig by random assignment to anatomic location. e resulting stent-to-artery ratio was about 1.2-1.3 : 1 by quantitative coronary angiography analysis. e animals were anesthetized with ketamine (20 mg/kg) and xylazine (2 mg/kg) for follow-up angiography in the same orthogonal views before death with 20 mL of potassium chloride intracoronary injection. e stented arteries were carefully dissected from the myocardium and cut into two pieces, each about 9 mm long for cross sections preparation and SEM imaging.

Measurements of As 2 O 3 Levels in Tissues and Serum.
In additional animals at 0, 4, 24, 72 h, and 7 days aer deployment of As 2 O 3 eluting stents ( 6, 6 stents per time point) in the coronary arteries, the delivery of As 2 O 3 from stents to tissues and serum was evaluated using hydride generation reaction interfaced with atomic �uorescence spectrometry assay and HPLC, respectively [37]. In this experiment, �een pigs were used and each pig received two As 2 O 3 eluting stents.

Endothelialization of Stented Arteries and Evaluation of Endothelial
Function. e endothelialization of stented arteries was examined using SEM 1, 2, and 4 weeks aer stent implantation. Endothelial function aer stent implantation was estimated by measuring the coronary vasomotor reactivity in response to acetylcholine (Ach, 60 mg, performed at an infusion rate of 1 mL/min) infusion within 6-month followup. Ten pigs receiving two stents each and 20 stents (6 BMSs, 7 PCSs, and 7 AESs) were used in the experiment. Enddiastolic images for each segment were chosen and analyzed with the automated edge detection program (FD-10, Philips, Best, e Netherland). Two orthogonal views with less foreshortening or without overlapping of side branches were selected and averaged for biplane assessment by two experts blinded to stent type. About 5 mm distal to the site of stenting was chosen for analysis. Changes in coronary diameter in response to Ach coronary infusion were expressed as percent changes versus baseline angiograms.

Immunohistochemical and Western Blot Analyses.
For immunohistochemical and western blot analyses, ten pigs were used and each pig randomized received two stents (7 BMSs, 6 PCSs, and 7 AESs). Aer 4 weeks, the stented arteries were harvested and cut into two pieces. e stent struts were carefully removed from the coronary arteries. Serial sections from the stented arteries were �xed in phosphate buffered saline (PBS) containing 10% formalin, dehydrated by treatment with solutions of increasing alcohol content (70%, 85%, 95%, and 100%), followed by xylene and then embedded in paraffin. Immunohistochemical studies were performed using primary antibodies against interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), CD3, and S100. Brie�y, the sections were deparaffinized, heated at 95 ∘ C for 30 min in antigen retrieval solution, �xed in 0.3% hydrogen peroxide for 30 min, and then air dried. Nonspeci�c antibody binding sites were blocked by a 30 min incubation in normal horse serum (10%, 0.1% Triton X-100, 0.1 M PBS, pH 7.4). e slide was then incubated in the primary antibodies, respectively. (1 : 100, 0.1% Triton X-100, 0.1 M PBS) (R&D systems, Minneapolis, MN, USA) overnight at 4 ∘ C. Aer three washes in PBS, the slides were again blocked in normal horse serum as described above and exposed to the horseradish peroxidase-conjugated secondary antibody (1 : 400, 0.1% Triton X-100, 0.1 M PBS) (Beyotime, China), followed by the addition of 3,30-diaminobenzidine tetrahydrochloride (DAB) solution. Nuclei were also counterstained with hematoxylin. e total tissue area of the immunostained sections of each specimen was outlined manually and measured in mm 2 . e tissue areas of the immunostained sections occupied by positive cells were measured automatically using greyscale detection with a �xed threshold and the ratios of immunopositive areas were calculated as percentages of the total tissue area.
Vessel wall expression of in�ammatory-associated proteins (IL-6 and MCP-1) was determined by western blot analysis. Brie�y, protein extracts (50 g) were size fractionated on SDS-polyacrylamide gels, transferred to nitrocellulose membrane. Positive control for each target was run on the same gel. Membranes were incubated with an affinity puri�ed polyclonal antibody to IL-6 and MCP-1, respectively, washed, and incubated with secondary antibody. Signals were detected by the ECL chemiluminescence detection system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to ensure equal amount of protein extract in each sample.  Figure 1 shows the stereoscopic and SEM images of an As 2 O 3 eluting stent before and aer expansion. It can be clearly observed that As 2 O 3 crystals were dispersed between the basecoat and topcoat, while the coatings were still uniform. No delamination or destruction of the coatings on the stent can be observed, indicating the drug does not impair integrity of the coatings.

3.2.
In Vivo As 2 O 3 Levels aer Stent Implantation. e levels of As 2 O 3 measured in arterial tissue, serum, and main organs aer AESs implantation are presented in Table 1. At 4 h aer stent deployment, the mean concentration of As 2 O 3 within the adjacent arterial tissue was 1.264 ± 0.056 g/g, whereas the value in serum was 0.012±0.004 g/g, nearly one hundred times higher than that in the serum. e concentration of As 2 O 3 in stented artery tissue reached a maximum level at 24 h, and decreased thereaer, whereas the values in serum, heart, liver, and kidney progressively declined aer 4 h. Even at day 7 aer stent implantation, the As 2 O 3 concentration in the arterial wall was 0.036 ± 0.002 g/g, while no As 2 O 3 can be detected in the serum, heart, liver, and kidney. Insert: Digital camera photographs of the inner wall of the stented arteries. At the �rst week, a thick layer of matrix consists of a lot of in�ammatory cells and �brin associated with foreign body reactions is present over the stent struts for BMSs. A much thinner layer of such matrix can be observed for AESs. At 2 weeks, the neointima consisting of amorphous material and in�ammatory cells formed. is amorphous material adjacent to the stent strut was never observed 2 weeks aer AESs implantation. At 4 weeks, the neointima consists of smooth muscle cells and matrix proteoglycans. e density of smooth muscle cells in neointima is much lower for AESs than BMSs.

Morphological Evaluation of the Stented Arteries.
e typical morphologies of the stented arteries 1, 2, and 4 weeks aer stent implantation are shown in Figure 2. In the �rst two weeks, BMSs and AESs had different appearance with �brin-platelet deposition and acute in�ammatory cells. At one week, a relatively thick layer of matrix composed of many in�ammatory cells and �brin-associated foreign body reactions was present over the stent struts for BMSs, while a much thinner layer of such matrix was observed in the AES group. e percent area stenosis for BMSs and AESs at this time point was 7.6 ± 2.7% and 4.3 ± 1.6%, respectively. Aer two weeks, neointima consisting of amorphous material and in�ammatory cells formed. e amorphous material adjacent to the stent strut was also less for AESs as compared with the controlled BMSs. Aer 4 weeks, neointima consists of SMCs and matrix proteoglycans formed. e density of SMCs in neointima was much lower in the AES group than in the BMS group. ere was a trend that the percent area stenosis at four weeks was lower in the AES group compared to BMS, although the difference was not signi�cant ( . 7 ).

Endothelialization of the Stented Arteries and Evaluation
of Endothelial Function. e progress of endothelialization of stented arteries was examined using SEM at 1, 2, and 4 weeks aer stent implantation as shown in Figure 3(a). At the �rst week, both AES and BMS had �uite low endothelial cells attachment. AES showed much less �brin-platelet deposition than BMS, which was consistent with the morphological results in Figure 2. For the second week of development, endothelial cell attachment could be found to occur on the BMS surface with a relative low density. Although AESs were reported to mildly delay endothelialization in a rabbit iliac artery injury model [25], no difference was observed in a porcine coronary model. Aer 4 weeks, there was no difference in endothelialization for AES and BMS. e arteries treated with BMSs or AESs were fully endothelialized; the lumen surface of the vessel wall and the stent struts were covered by con�uent endothelial cells.

Discussion
It is well known that ISR is a consequence of in�ammation, SMC proliferation, and migration [38]. e effects of As 2 O 3 as an antiproliferative agent for drug eluting stents at clinically achievable concentrations were investigated without severe side effects. In the studies done in rabbit iliac artery model and porcine coronary model, AESs effectively reduce neointimal thickening through inhibiting the cell cycle and inducing VSMC apoptosis. No in-stent thrombosis can be observed within six-month followup in porcine coronary arteries [25,37], suggesting that AESs are a feasible, safe, and e�cient DES. In the present study, anti-in�ammatory effect of As 2 O 3 in vitro and effect of AESs in a porcine coronary model have been evaluated. AESs were also found to signi�cantly decrease the protein expression of MCP-1, IL-6, and CD3 compared with BMSs and PCSs. But unfortunately, we failed to detect any decrease of S100 expression, a marker of DCs 4 weeks aer AESs implantation. Indeed, the expression of S100 in BMS, PCS, and AES treated arteries was low and the difference was not signi�cant at this time point. MCP-1 is the prototype of the C-C chemokine-subfamily and exhibits its most potent chemotactic activity toward monocytes and T lymphocytes. In addition to promoting the transmigration of circulating monocytes into tissue, MCP-1 exerts various other effects on monocytes, including superoxide anion induction, cytokines production, and adhesion molecule expression [39]. IL-6 is a cytokine that has been proven to be crucially involved in ischemic heart disease. is substance possesses pleiotropic functions that are mostly in favor of in�ammation. Patients with a higher release of IL-6 in the coronary sinus aer stent deployment have a higher risk of restenosis. Suzuki and coworkers [40] showed in an experimental model that sirolimus-and dexamethasone-covered stents induce a reduction in release of MCP-1 and IL-6 compared with BMSs, thus decreasing local in�ammation.
Endothelium plays an integral role in maintaining vascular homeostasis. is role is not limited to the modulation of vascular tone but also for regulation of in�ammation, platelet activation, and thrombosis [41,42]. Drug eluting stents substantially reduce in-stent restenosis in patients with coronary artery disease and also lead to disruption of the endothelial layer and leaves a thrombogenic metallic surface exposed to the blood stream. Early DES devices have been associated with late stent thrombosis (LST) in patients, a rare though life-threatening event that has become an increasingly controversial issue for interventional cardiology. Delayed or incomplete re-endothelialization is an important predictor of LST, which is increasingly apparent for DES in real-world applications. e early establishment of a functional endothelial layer aer vascular injury has been shown to assist in prevention of neointimal proliferation and thrombus formation [43,44]. In our previous report, stent thrombosis did not occur within 6-month followup for the AESs in a porcine coronary artery model [37]. Stent-based local delivery of sirolimus (Cypher) and paclitaxel (Taxus) profoundly inhibited neointima formation but caused vasomotor dysfunction in distal conduit vessel segments [44]. As revealed in previous reports, the segments proximal and distal to the stents were both more strongly constricted to Ach infusion in the SES group compared with the BMS group [45][46][47]. ere was no signi�cant difference in extensive endothelialization and endothelial function between AESs and BMSs aer 4 weeks, suggesting improved safety of AES than SES and PES. e early restoration of endothelial function aer AES implantation might be interpreted as the result of rapid elution of the loaded drug, which was completely released within 7 days, and thus minimized local toxicity [37]. In contrast, the loaded drug for sirolimus and paclitaxel eluting stents is released up to 60 days aer stent implantation. Another reasonable explanation is related to the biodegradable coating material that can induce mild foreign body reactions and quick endothelialization [37].

Conclusions
In a porcine coronary artery model AES reduced the neointimal hyperplastic response to injury through inhibition of cell cycle and induction of apoptosis of VSMC [27,48], less augmentation of early in�ammatory reactions, and quick endothelialization of the stent surface. Although further studies are necessary to con�rm the long-term e�cacy and safety of this novel medical device, our data highlight the necessity to improve our understanding regarding the performance of AESs in reducing ISR.

Con�ict of �nterests
e authors declare no con�ict of interests.