Abstract
BACKGROUND: Clinical trials have shown that oral administration of valsartan can decrease in-stent restenosis after stent implantation. But whether valsartan used locally also has the same effect and the possible mechanism should be validated.
OBJECTIVE: To observe the effect of valsartan-eluting stents on collagen deposition in neointima and AT2 receptor expression after implanting valsartan-eluting stents into rabbit abdominal aorta.
DESIGN: Randomized and controlled animal experiment.
SETTING: Beijing Friendship Hospital.
MATERIALS: The experiment was performed at the Laboratory of Beijing Friendship Hospital between October 2004 and March 2006. Fifteen New Zealand white rabbits, irrespective of gender, weighing 2.75-3.25 kg were selected (Animal Laboratory of Beijing Friendship Hospital). The rabbits were adaptively fed for one week. All the operations of rabbits during the experiment were accorded with animal ethical standards. Valsartan powder was presented as a gift by Novartis, China; Reagent of MASSON was provided by Department of Pathology of Beijing Friendship Hospital; 1% picrosirius solution was provided by the Department of Pathology of China-Japan Friendship Hospital; Mice-anti-rabbit monoclonal AT2 antibody was product of Santa Cruz Biotechnology (USA); Envision reagent was purphased from Dako; primers were synthesized by SBS Genetech (SBS).
METHODS: ①The animals were randomized into bare-metal stent group, carrier-eluting stent group and valsartan-eluting stent group with 5 animals in each group. All rabbits were implanted with corresponding types of above-mentioned stents into abdominal aortas down below renal artery. ②Quantitative angiography before, immediately after and 3 months after stent implantation were performed to compare vascular diameters of the aortas. ③Three months later, the rabbits were executed after anaesthesia. The vessels with stents were processed with HE staining. Indices of the vascular neointimal formation, i.e. inner and external elastic membrane luminal area, the maximal intimal thickness, neointimal area and stenosis area percent were measured. ④The collagen deposition in neointima was observed through MASSON staining, and the type of collagen was identified through picrosirius stain. ⑤The expressions of AT2R mRNA and proteins were also compared by RT-PCR and immunohistochemistry among three groups.
MAIN OUTCOME MEASURES: ①The diameters of aorta with stent at different time; ②Inner and external elastic membrane luminal area, the maximal intimal thickness, neointimal area and stenosis area percent; ③Collagen deposition and type of collagen of the aorta with stent; ④AT2R mRNA and protein expressions.
RESULTS: Of 15 rabbits selected in the experiment, 1 rabbit of the bare-metal stent group died during stent implanting, and 1 of the carrier-eluting stent group died during breeding after stenting. Finally, 13 rabbits were included in final analysis. ①There were no significant differences in the mean aortic diameters between any two of the three groups before, immediately after and 3 months after stent implantation (P > 0.05). ②A larger luminal area and a less neointimal hyperplasia in valsartan eluting-stents group were found compared with the other two groups (P < 0.01). ③MASSON staining showed that collagen deposition was rich in neointima of bare-metal stent group and carrier-eluting stent group while rare in neointima of valsartan eluting stent group. Picrosirius staining suggested that the deposited collagen was type Ⅲ collagen predominantly accompanied by typeⅠcollagen around stents struts; the type Ⅲ collagen deposition was obviously decreased in valsartan eluting stent group. ④AT2R protein only expressed in adventitia of bare-metal stent group and carrier-eluting stent group while expressed in all layers of valsartan eluting-stents group. The AT2R mRNA/β-Actin mRNA of valsartan eluting stent group was significantly higher than that in the other two groups (P < 0.01).
CONCLUSION: Valsartan eluting-stents inhibits neointimal hyperplasia after stenting by decreasing collagen deposition, especially collagen Ⅲ. The mechanism may be related with the upregulation of AT2R mRNA and protein expressions by valsartan-eluting stent.

INTRODUCTION

Recently, a series of intravascular studies demonstrate that elastic recoil and negative remodeling after percutaneous transluminal coronary angioplasty is counteracted by stents, so in-stent restenosis is mainly caused by intimal hyperplasia [1-3]. Drug-eluting stents (DES) coated with Rapamycin or Paclitaxel, strong antiproliferative agents, have been demonstrated to be potent anti-restenotic strategies. However, with the use of DES in the "real world", target vessel revascularization remains necessary in approximately 4%. Recently, there is an increasing interest in antirestenotic therapies by means of restoring physiological function of vessel wall. Many basic studies have proved that angiotonin Ⅱ (Ang Ⅱ) accelerates restenosis through acting with growth factors. AT1 receptor antagonists can inhibit restenosis by blocking the combination of Ang II and AT1 receptor to reduce the concentration of restenosis-related growth factors. Val-PREST and VALVACE trials have confirmed that oral administration of valsartan can reduce restenosis rate after stenting but the result and possible mechanism of local use of valsartan are unclear.
To explore the effect of valsartan eluting stent on intimal hyperplasia after stenting and the possible mechanism of valsartan eluting stent to prevent in-stent restenosis, we made valsartan eluting stents
by multi-layer coated technique and observed the effect of valsartan eluting stents on collagen deposition in neointima and the expression of AT2 receptor.

MATERIALS AND METHODS

Materials
The experiment was performed at the laboratory of Friendship Hospital in Beijing between October 2004 and March 2006. Fifteen clean-grade New Zealand white rabbits, irrespective of gender, weighing 2.75-3.25 kg were provided by Animal Laboratory of Beijing Friendship Hospital [No. SCXK(Jing) 2002-0003]. The rabbits were adaptively fed for one week. All the operations of rabbits during the experiment were accorded with animal ethical standards. The animals were randomly divided into bare-metal stent group, carrier-eluting stent group and valsartan-eluting stent group. Valsartan powder was presented as a gift by Novartis, China; Reagent of MASSON was provided by Department of Pathology of Beijing Friendship Hospital; 1% picrosirius solution was provided by the Department of Pathology of China-Japan Friendship Hospital; Mice-anti-rabbit monoclonal AT2 antibody was product of Santa Cruz Biotechnology (USA); Envision reagent was purchased from Dako; primers were synthesized by SBS Genetech (SBS).

Methods
Preparation of eluting stents: Thirty 316 L stainless steel stents of 10 mm in length and diameter of 3.0 mm were selected. Using multi-layers coated technique (patent: CN 02136332.3), 20 of the 30 stents were eluted, i.e. in 10 valsartan-eluting stents, valsartan was embedded in base coating with organic-inorganic compound, and covered by highly-compatible polymer full of phospholipid groups; using the same method, another 10 carrier-eluting stents were eluted by no drug was embed in base.
Experiment process:The animals were anesthetized celiacly with Ketamine (60 mg/Kg) and fixed on bench. The right common femoral artery (2-3 cm) was surgically exposed, and a 4-French arterial sheath was introduced through the incision up to aorta and fixed by thread. Heparin in a dosage of 100 IU/kg was injected through the sheath. An angioplasty balloon with stent was introduced by guiding wire (0.014''×190 cm) in superficial femoral artery and advanced into common femoral artery to a standardized location just distal to the bifurcation of renal artery. The balloon was inflated to 12-14 atm in 20-30 seconds before it was withdrawn and released the stent. The other stent was implanted in the same way and the distance between the stents was about 1-2 cm. The catheter was then removed, and the incision was closed after the implantation. Regular injection of 400 000 U Benzylpenicillin Sodium was administered for 3 days in order to prevent infection. All the rabbits were raised for 3 months.
Angiographic analysis: Abdominal aorta converse angiography was performed before, immediately and 3 months after stent implantation to detect vessel diameter.
Slice preparation and imaging analysis: Three months after repetitive angiography, the rabbits were executed. The vessels with stents were taken out and put into formalin (4%) for fixation, then were embedded by stiff plastic. Slices were made by polycut slice machine and were processed with HE, MASSON and picrosirius staining. Indices of the vascular neointimal formation, i.e. inner and external elastic membrane luminal area, the maximal intimal thickness, neointimal area and stenosis area percent were analyzed by Leica Q550CW image analysis system in HE staining. Collagen deposition was analyzed by MASSON staining under light microscope. Type Ⅰ and Ⅲ collagen percentage and distribution was analyzed quantitatively by picrosirius staining under polarimicroscope.
AT2 receptor detected by immunohistochemistry: The vessels with stents were taken out and put into formalin (4%) for fixation, then was embedded by paraffin to make slices. The slices were deparaffinaged and hydrated. Activity of endogenous peroxydase was eliminated using 3%H2O2. Complex digestive juice was added, then the slices were washed with buffer solution, and sealing fluid was added. Mice-anti-rabbit monoclonal AT2 antibody (1:50 dilution) was added, put in refrigerator at 4 ℃ overnight, then Envision reagent was added, and colored with 0.04%DAB+0.03% H2O2. Positive products showed brown.
AT2 receptor mRNA expression detected by RT-PCR: The vessels with stents were taken out, put into eppendorf tube and placed into refrigerator at -80 ℃. Total RNA was extracted from arterial sample according to Trizol Reagent introduction. The A260 : A280 of all total RNA measured by spectrophotometer was between 1.8 and 2.0. 1 μg total RNA was taken to synthesize cDNA using reverse transcriptase AMV (total 20 μL, 42 ℃ 15 minutes, 95 ℃ 5 minutes, 0-5 ℃ 5 minutes). Reverse transcript product was used for target gene and conserved gene synthesis by PCR method. Primers were designed with Primer5 software. AT2: 5'A G G T T T C C A G C A T T T A C A T C 3'; 3' C T A T C G C A A C C G A C C A C T G 5'. Reaction condition was pre-denaturalization at 95 ℃ for 3 minutes, denaturalization at 94 ℃ for 60 seconds, anneal at 58 ℃ for 45 seconds, extension at 72℃ for 40 seconds, after 35 circles delay 10 minutes at 72 ℃. The product was 404 bp.β-actin: 5' A G G A A G G A G G G C T G G A A C A 3'; 5' C C C A T C T A C G A G G G C T A C G C 3'. Reaction condition was pre-denaturalization at 94 ℃ for 2 minutes, denaturalization at 94 ℃ for 30 seconds, anneal at 60 ℃ for 30 seconds, extension at 72 ℃ for 90 seconds, after 28 circles delay 20 minutes at 72 ℃. The product was 311 bp. Agarose gel electrophoresis of PCR product was performed. AT2 mRNA was synthesized by PCR, and target gene/conserved gene was calculated.
Statistical analysis: All data were analyzed using SPSS 12.0 software by the first author. Descriptive data were expressed as Mean±SD. ANOVA was used in comparison between the groups. Statistic significance was assessed in a level of P < 0.05.

RESULTS

Quantitative analysis of animals
Fifteen rabbits were selected in the experiment. One rabbit of the bare-metal stent group died during stent implanting, and one rabbit of the carrier-eluting stent group died during breeding after stenting. Finally, 13 rabbits were included in final analysis.
Abdominal aortic diameter
There was no significant difference in the mean aortic diameters between any two of the three groups before operation (P > 0.05), and there was neither significant difference immediately after or 3 months post-implantation (P > 0.05). No in-stent restenosis (diameter stenosis > 50%) was found in any group according to the repeated angiography 3 months after implantation.

MASSON staining analysis and image analysis
A lot of collagen deposition was found in neointima in the bare-metal stent group and carrier-eluting stent group by MASSION staining but there was much less collagen in neointima of valsartan-eluting stent group (Figure 1).

Collagen deposition
Picrosirius staining suggested collagen III deposition was detected in neointima under polarimicroscope and collagen I was detected near the strut of stent occasionally. Collagen III deposition in valsartan-eluting stent group was decreased obviously compared with the other two groups (Figure 2).

Expression of AT2 receptor
AT2 receptor was found mainly in adventitia of the bare-metal stent group and carrier-eluting stent group. It was expressed significantly from adventitia to intima in valsartan-eluting stent group (Figure 3).

Image analysis of HE stained slices
The analysis showed that the luminal area (LA) of valsartan eluting stent group was significantly larger than the other two groups (P < 0.01). The maximal inner-membrane thickness (MIT) and neointimal area (NIA) in valsartan-eluting group were significantly smaller than the other two groups (P < 0.01, Table 1).

RT-PCR results
The expressions of AT2 mRNA were found in all three groups (Figure 4). The expression of AT2 mRNA/β-Actin in valsartan-eluting stent group was significantly higher than that in carrier-eluting stent group and bare-metal stent group (0.863±0.136, 0.441±0.082, 0.472±0.149, P < 0.01).

DISCUSSION

Though stents could counteract aortic recoil and remodeling, the occurrence of in-stent restenosis, which is caused by intimal proliferation [4], is still 15%-20%. In-stent restenosis is a major drawback of percutaneous transluminal coronary angioplasty with stent placement. Recent advances in the development of drug-eluting stents have reduced these numbers tremendously. There are two kinds of drug-eluting stents widely used clinically to reduce restenosis effectively: rapamycin-eluting stent and paclitaxel-eluting stent. Both rapamycin and paclitaxel are strongly anti-proliferative agents. They can inhibit smooth muscle cell (SMC) proliferation and migration to inhibit neointimal formation and reduce restenosis. However, late thrombosis, inflammation, hypersensitivity reactions and endothelium delayed healing are of major concerns. Physiological endothelium and arterial recovering antirestenotic strategy for the prevention of in-stent restenosis are in focuses recently. In light of the recent interest in more physiological endothelium and arterial-recovering antirestenotic therapies, RAS intervention attracts people. RAS is not the sole factor for restenosis; however, it plays a role in pathophysiology. RAS intervention would be an attractive way to prevent in-stent restenosis in a more physiological manner. RAS intervention has endothelium-protective properties, even in the setting of vascular injury[5-6]. VAL-PREST[7] and VALVACE[8] trials have proved that systemically administered AT1 receptor blockade could reduce in-stent restenosis. To study the effect of valsartan eluting-stents on vascular neointimal formation and assess the feasibility to prevent restenosis, we coated stents with valsartan and carried out an animal experiment. The results show that valsartan eluting-stents inhibit neointimal hyperplasia and might play an important role in preventing restenosis.
Collagen and restenosis: Recently, extracellular matrix accumulation has been recognized as a very important component of in-stent restenosis in chronic phase after stent implantation. A substantial portion of the neointima consists of matrix rather than cells[9]. From 2 to 6 months, stented porcine coronary arteries show a reduction in neointimal hyaluronan associated with reduced neointimal type Ⅲ and increased type I collagen[10]. Observations to a more chronic phase of neointimal evolution have been extended to more than 18 months and found that the neointima in stents in place up to 18 months remains hypercellular (confluent SMCs) and rich in type Ⅲ collagen, versican, and hyaluronan with relatively little type I collagen and decorin. In contrast, stents ≥ 18 months demonstrated weaker staining for versican, hyaluronan, and type Ⅲ collagen and stronger staining for type I collagen and decorin. Neointimal cell density, area occupied by SMCs, and in-stent stenosis were smaller in stents ≥ 18 months old group compared with stents < 18 months old group. These data demonstrate that intimal lesions 18 months after stenting resemble wounds that are not fully healed and suggest that neointimal retraction occurs subsequently[11].
Renin-Angiotensin System (RAS) and Restenosis: It is known that there are 4 subtype angiotensin Ⅱ receptors: AT1, AT2, AT3 and AT4. RAS exerts its physiological effects through Angiotensin Ⅱ stimulating angiotensin Ⅱ receptors, especially AT1 and AT2. Activation of the AT1 receptor has detrimental effects on cardiovascular system, namely cell migration and proliferation, extracellular matrix deposition, inflammation, promotion of thrombosis, and production of reactive oxygen species. Activation of the AT2 receptor could counteract effects of the AT1 receptor activation. Activation of the AT2 receptor is associated with nitric oxide (NO) production, inhibition of proliferation, and induction of apoptosis. Selective AT1 receptor blockers inhibit stimulation of AT1 receptor by Angiotensin Ⅱ, the AT2 receptor can still be activated. So adverse effects of Angiotensin Ⅱ, such as cell migration and proliferation, extracellular matrix deposition, inflammation, promotion of thrombosis, and production of reactive oxygen species, are blockade, and the beneficial effects of AT2 activation still remain. AT1 receptor and AT2 receptor interacts each other. Wu et al [12] elucidated the function of the AT2 receptor while AT1 receptor antagonism after vascular injury. Neointimal formation is attenuated in wild mice by AT1 receptor blockade; this effect is less prominent in AT2 knockout mice. Moreover, anti-inflammatory effects of AT1 receptor blockade are decreased in AT2 knockout mice. The conclusion is that stimulation of the AT2 receptor during AT1 receptor antagonism is important in the decrease of neointimal formation after vascular injury. Furthermore, Some scholars demonstrated neointimal formation as well as DNA synthesis in vascular smooth cells after vascular injury was exaggerated in AT2 knockout mice, but they were both suppressed in AT1 knockout mice compared with those in wild-type mice. In contrast, the number of apoptosis cells in the injured artery in VSMC is significantly increased in AT1 knockout mice but decrease in AT2 knockout mice. The result suggests AT2 exerts antiproliferative effects and changes in VSMC by counteracting AT1 in the process of neointimal formation after vascular injury.
Our study shows that valsartan-eluting stents, local application of valsartan, could inhibit neointimal formation. It may be an attractive method to prevent in-stent restenosis.

REFERENCES

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缬沙坦涂层支架置入兔腹主动脉对血管内膜胶原沉积及AT2受体表达的影响☆

李贵华1，王 雷2，贾三庆2，赵 林2，姚道阔2，丁荣晶2，任文林1
1北京市垂杨柳医院心脏中心，北京市 100022;2首都医科大学附属北京友谊医院心脏中心，北京市 100050
李贵华☆，男， 1971年生，山东省微山县人，汉族，2006年首都医科大学毕业，医学博士，主治医师，主要从事冠心病药物及介入治疗。
摘要
背景：有实验证实口服缬沙坦能降低血管支架置入后血管再狭窄的发生, 局部应用缬沙坦能否产生相同的效应且其可能的作用机制需验证。
目的：采用缬沙坦涂层支架置入实验兔腹主动脉，观察支架对血管狭窄情况、新生内膜中胶原沉积及AT2受体表达的影响。
设计：随机对照动物实验。
单位：北京市友谊医院。
材料：实验于2004-10/2006-03在北京市友谊医院实验室完成，选用15只新西兰大白兔，雌雄不拘，体质量2.75~3.25 kg，由北京友谊医院动物实验室提供。动物实验室喂养适应1周。实验过程中对动物的处置符合动物伦理学标准。缬沙坦粉剂由诺华公司馈赠， MASSON染色试剂为北京友谊医院病理科提供，1%天狼星红苦味酸溶液为中日友好医院病理科提供，鼠抗兔AT2单克隆抗体为美国Santa Cruz Biotechnology公司产品，Envision 试剂购自Dako公司，引物由北京赛百盛公司合成。
方法：①随机数字表法将实验兔分为裸支架组、载体涂层支架组及药物涂层支架组，每组5只，分别在实验兔腹主动脉的肾动脉开口下方植入裸支架、载体涂层支架及药物涂层支架。②支架置入前后即刻及置入后3个月分别行腹主动脉造影测量腹主动脉直径。③3个月后麻醉处死实验兔，取支架血管段切片并作苏木精-伊红染色，对管腔面积、内外弹力膜围绕面积、新生内膜面积及最大内膜厚度进行测定。④将支架血管段进行MASSON染色，观察胶原沉积情况，天狼星红苦味酸染色进一步观察胶原的类型。⑤免疫组化方法检测AT2受体蛋白质表达，并采用RT-PCR方法测定AT2受体 mRNA的表达。
主要观察指标：①不同时间腹主动脉直径测定结果。②支架血管段管腔面积、内外弹力膜围绕面积、新生内膜面积及最大内膜厚度。③支架血管段胶原类型及沉积情况。④AT2受体蛋白质及mRNA表达。
结果：纳入实验兔15只，裸支架组1只在支架置入过程中死亡，载体涂层支架组1只在饲养过程中死亡，其余13只均进入结果分析。①各组实验兔支架置入前后即刻及术后3个月组间比较结果显示血管直径差异无统计学意义（P > 0.05）。②缬沙坦组实验兔管腔面积大于裸支架组及载体支架组，差异有显著性意义（P < 0.01），新生内膜厚度及新生内膜面积均小于裸支架组及载体支架组，差异有显著性意义（P < 0.01）。③MASSON染色显示裸支架组及载体涂层支架组新生内膜中胶原大量沉积，缬沙坦洗脱支架新生内膜中胶原沉积较少；天狼星红-苦味酸染色经偏光显微镜下可见新生内膜中主要是Ⅲ型胶原沉积，在支架脚附近间或有Ⅰ型胶原的沉积，缬沙坦涂层支架组Ⅲ型胶原沉积明显减少。④裸支架组与载体涂层支架组AT2受体蛋白仅在外膜表达，缬沙坦药物涂层支架AT2受体蛋白自外膜至内膜均有表达，药物涂层支架组AT2受体/β-Actin表达高于载体支架组及裸支架组（P < 0.01)。
结论：缬沙坦涂层支架能通过减少胶原尤其是Ⅲ型胶原的沉积抑制支架术后血管内膜增生，其机制可能与缬沙坦涂层支架上调AT2受体表达有关。
关键词：涂层支架；缬沙坦；AT2受体；再狭窄； 胶原
中图分类号: R318 文献标识码: B 文章编号: 1673-8225(2008)09-01761-05
李贵华，王雷，贾三庆，赵林，姚道阔，丁荣晶，任文林.缬沙坦涂层支架置入兔腹主动脉对血管内膜胶原沉积及AT2受体表达的影响[J].中国组织工程研究与临床康复，2008，12(9):1761-1765
[www.zglckf.com/zglckf/ejournal/upfiles/08-9/9k-1761(ps).pdf]
（Edited by Salah A.M. Said/Su LL/Wang L）