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.

1Cardiovascular Center of Chuiyangliu Hospital, Beijing 100022, China; 2Cardiovascular Center of Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China

Li Gui-hua☆, Doctor, Attending physician, Cardiovascular Center of Chuiyangliu Hospital, Beijing 100022, China
lghua1971@163.
com

Received:2007-10-27
Accepted:2007-12-19
(07-50-10-5872/YWY)

Li GH, Wang L, Jia SQ, Zhao L, Yao DK, Ding RJ, Ren WL.Influence of valsartan-eluting stent implantation into rabbit abdominal aorta on collagen deposition and AT2 receptor expression. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(9):
1761-1765(China)

[www.zglckf.com/
zglckf/ejournal/
upfiles/08-9/
9k-1761(ps).pdf]

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

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。

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）