蛋白涂层支架携带一氧化氮合酶基因转染小型猪冠状动脉的可行性研究

目的 :探索蛋白涂层支架携带质粒介导人肝脏诱导型一氧化氮合酶 (i NOS)基因转染小型猪冠状动脉可行性。　　方法 :使用蛋白支架吸附去内毒素纯化质粒 ,以常规支架置入技术置入小型猪冠状动脉前降支中段。置入后第 7天取出前降支置入段 ,分别提取总核糖核酸 (RAN)并进行逆向多聚酶链反应 (RT- PCR) ,免疫组化染色检测导入人肝脏i NOS蛋白的表达。　　结果 :小型猪前降支置入支架处显示人 i NOS基因信使核糖核酸 (m RNA)转录 ,免疫组化染色显示中膜、内膜人i NOS基因表达人 i NOS蛋白的颗粒 ,以平滑肌细胞最明显。　　结论 :蛋白涂层支架吸附去内毒素携带人 i NOS基因质粒植入小型猪前降支冠状动脉 ,RT- PCR显示人 i NOS基因的 m RNA转录 ,免疫组化显示人 i NOS蛋白的表达。     

改进的明胶蛋白涂层支架用于小型猪冠状动脉转染一氧化氮合酶基因的可行性研究

目的为了判定明胶蛋白涂层支架携带质粒介导的一氧化氮合酶(iNOS)基因转染小型猪冠状动脉的可行性.方法将自行构建的pcDNA3.1/iNOS(L24553)质粒以浸泡(15～60min)及涂抹(60min)的方法携带在明胶蛋白涂层支架(由上海微创公司提供)上,采用标准球囊导管技术置入小型猪(n=5)冠状动脉前降支及右冠近中段,支架与置入处血管直径之比1.1～1.31.结果支架置入术后7天,在支架置入处血管段使用对人特异性引物完成的逆转录多聚酶链反应(RT-PCR)反应表明有iNOS基因转染,免疫组织化学检测表明有iNOS蛋白生成.结论明胶蛋白涂层支架携带质粒介导的iNOS基因可有效转染小型猪冠状动脉成形部位.     

蛋白涂层支架携带质粒介导的一氧化氮 合酶基因预防冠状动脉球囊扩张后 再狭窄的实验研究

目的　为评价蛋白涂层金属支架携带质粒介导的诱导性一氧化氮合酶 (iNOS)基因局部转染血管壁 ,预防冠状动脉内血管成形术后再狭窄的效果。方法　金属支架涂层为胶联明胶制成。载体为去内毒素纯化pcDNA3。采用标准球囊导管技术 ,将吸附有质粒介导的人肝脏的iNOS基因(pcDNA3hepiNOS)涂层支架置入小型猪 (n =9)冠状动脉前降支中段 ,以相同方法置入单纯蛋白涂层支架做为对照组 (n =9) ,支架与血管直径之比为 1.1～ 1.3:1。结果　在支架置入后 7d ,RT PCR检测和免疫组织化学染色 ,证实在pcDNA3hepiNOS转染的血管段有iNOSmRNA的表达和iNOS蛋白生成 ,而远离器官则无基因的表达。 3个月时冠状动脉造影显示 :转染pcDNA3hepiNOS组 (n =5 )无再狭窄发生 ,而对照组均发生显著的再狭窄。组织病理学形态分析结果显示 :pcDNA3hepiNOS组新生内膜面积 (1.7± 0 .8)mm2 、平均百分狭窄面积 (2 6 .5± 7.5 ) %、平均管腔狭窄百分数 (4 1.2± 16 .5 ) % ,均较对照组小 ,对照组分别为 (2 .8± 0 .8)mm2 ,P <0 .0 5 ;(94.2± 14.3) % ,P <0 .0 0 1;(88.0± 16 .6 ) % ,P <0 .0 0 1;比较内膜面积 /中膜面积比值 (I/M)治疗组较对照组减少了 5 9.8%。结论　在小型猪模型使用蛋白涂层支架携带纯化质粒介导的iNOS基因可直接导入血管     

质粒介导下转染血管内皮生长因子基因不能防治小型猪冠状动脉再狭窄

目的 :评估质粒介导下蛋白涂层金属支架局部转染血管内皮生长因子 (VEGF)基因对小型猪冠状动脉再狭窄的作用。　　方法 :选用 1 4只正常小型猪作为实验动物 ,载体为携带有VEGF基因的PcDNA3质粒。用常规球囊导管技术 ,将携带有VEGF基因的蛋白涂层支架置入冠状动脉前降支中段作为VEGF转基因组 (n =7) ,以相同方法置入单纯蛋白涂层支架作为对照组 (n =7) ,球囊与血管直径之比为 1 1∶1。在基因转染后 7天 ,行反转录聚合酶链反应和免疫组化染色。在支架置入后 3个月重复冠状动脉造影 ,测定管腔狭窄直径 ;同时行组织病理学检查。　　结果 :支架被成功置入 1 4只动物冠状动脉前降支中段。支架置入后 7天 ,反转录聚合酶链反应结果证实 ,在转染部位的动脉壁内有VEGF信使核糖核酸的表达。免疫组化染色显示在中层平滑肌细胞浆内及外膜有VEGF蛋白质生成。在支架置入后 3个月重复冠状动脉造影显示 :转基因组和对照组均发生再狭窄 ,平均管腔狭窄直径分别是 ( 6 9±1 0 ) %和 ( 80± 1 4 ) % ,无显著性差异 (P =0 2 4 7)。组织病理学形态分析结果显示 :转基因组和对照组 ,在平均损伤积分、新生内膜厚度、平均新生内膜面积、百分狭窄面积和内膜 /中膜值等各项参数上均无显著性差别。　　结论 :在本实验的再狭窄动物模     

金属蛋白涂层支架用于小型猪冠状动脉质粒介导下转基因研究

目的 :评价在质粒介导下 ,金属蛋白涂层支架向血管内局部转基因的可行性、效率和选择性。　　方法 :金属支架由 316 L不锈钢丝编织而成 ,其涂层是通过把支架放入有交联剂的明胶溶液中浸泡而成。基因载体为Pc DNA2质粒 ,并携带有β-半乳糖苷酶标记基因 ,该基因编码核特异性β-半乳糖苷酶。首先将蛋白涂层支架分别固定在3.0 mm或 3.5 mm经皮冠状动脉腔内成形术球囊上并在浓度为 8μg/μl的基因原液中浸泡 3分钟 ,然后通过 8F大腔引导导管将支架送入小型猪冠状动脉前降支中段 (转基因组 ,n=3) ,另外把没有浸泡过基因的支架也送入小型猪冠状动脉前降支中段 (对照组 ,n=3)。在支架植入后 7天处死动物。β-半乳糖苷酶表达由 X- Gal染色评估。　　结果 :所有转基因动物均有基因表达。转基因表达出现在内膜、中层和外膜。中层平滑肌细胞转染率为 3.0 %。远处器官和对照组冠状动脉均未显示核特异性β-半乳糖苷酶阳性表达。　　结论 :蛋白涂层支架在质粒介导下向血管内转基因有效、可行 ,因此 ,它有可能成为冠状动脉腔内成形术后再狭窄基因治疗的有效转基因系统。     

血管内皮生长因子基因治疗小型猪冠状动脉闭塞的实验研究

目的探讨应用血管内皮生长因子(VEGF)基因治疗动物实验性冠状动脉闭塞的疗效.方法中国实验用小型猪19只,结扎左冠状动脉前降支中远段,心肌内多点注射自行构建的pcD2/hVEGF121真核表达质粒.应用逆转录聚合酶链反应、VEGF免疫组化染色、Ⅷ因子相关抗原免疫组化染色等方法检测VEGF基因在心肌中的表达及其生物学作用,用常规冠状动脉造影观察VEGF基因治疗对闭塞冠脉侧支循环建立的作用.结果转移VEGF基因后,小型猪心肌内VEGF mRNA高表达,VEGF免疫组化染色提示VEGF蛋白表达水平升高;Ⅷ因子相关抗原免疫组化染色显示心肌毛细血管增加;冠状动脉造影证明VEGF基因治疗能够促进闭塞冠脉侧支循环的建立.结论心肌内注射pcD2/hVEGF121真核表达质粒能够获得VEGF mRNA和蛋白的有效表达,促进心肌毛细血管增生,促进侧支循环建立.     

在小型猪模型由蛋白涂层金属支架局部转染尿激酶前体基因 …

目的为了评价蛋白涂层金属支架局部转染尿激酶前体基因对冠状动脉内血小板沉积、早期血栓形成和平滑肌细胞增生的影响。方法 金属支架涂层为交联明胶制成。载体为复制缺陷的、携载Ｐｒｏ＝ＵＫ基因的重组腺病毒。采用标准球囊导管技术，将携带有Ｐｒｏ－ＵＫ基因的涂层支架置入小型猪冠状动脉前降支中段，以相同方法置入单纯蛋白涂层支架或裸露支架做为对照。结果 支架置入后３天，Ｐｒｏ－ＵＫ基因转染血管段（ｎ＝６）＾１１１Ｉ     

血管支架固定化抗体携带和靶向投递基因研究

目的将基因通过化学偶联和特异性免疫结合在血管支架上,评价基因递送、局部转染及预防再狭窄的效果。方法以增强型绿色荧光蛋白基因为报告基因,通过化学偶联和特异性免疫结合在血管支架上进行细胞转染实验,评价其基因转染效率;以人肝脏来源的诱导型一氧化氮合酶基因为治疗基因,将携带治疗基因的蛋白涂层支架植入猪冠状动脉进行动物在体实验研究,评价其进行局部转染的效果。结果细胞转染实验发现,实验组支架胶原涂层的表面有大量绿色荧光蛋白基因转染的细胞浸润生长,与支架接触的培养皿表面生长的细胞转染效率约为21.8%,明显高于单纯物理吸附携带基因的支架,而未与支架直接接触的周边细胞几乎没有被转染。猪冠状动脉支架植入实验中,支架植入28天后逆转录聚合酶链反应表明支架段血管内有诱导型一氧化氮合酶基因的表达,肺、肝、肾等远离组织内没有该基因的表达。结论通过化学和免疫双重偶联将基因固定在血管支架上的新型基因递送体系具有局部靶向和高效基因转运的特征,猪冠状动脉实验初步验证通过该方法携带治疗基因进行局部转染、靶向投递基因的有效性。     

携带血管内皮生长因子的蛋白涂层支架预防冠状动脉球囊扩张后再狭窄的预防

目的 :评价蛋白涂层支架携带血管内皮生长因子 (VEGF)预防冠状动脉成形术后再狭窄的效果。方法 :金属支架涂层为胶联明胶制成。应用标准球囊导管技术 ,将包被有VEGF的涂层支架置入小型猪 (n =10 )冠状动脉前降支中段 ,以相同方法置入单纯蛋白涂层支架作为对照组 (n =10 ) ,支架与血管直径之比为 1.1～ 1.3∶1。结果 :在支架置入后 3个月时冠状动脉造影显示 :VEGF组无再狭窄发生 ,而对照组均发生显著的再狭窄。组织病理学形态分析结果显示 :VEGF组新生内膜面积 (1.8± 0 .6 )mm2 ,平均百分狭窄面积 (2 5 .9± 6 .5 ) % ,平均管腔狭窄百分数 (40 .4± 13.7) % ,均较对照组〔分别为 (2 .6± 0 .7)mm2 ,P <0 .0 5 ;(93.1± 11.5 ) % ,P <0 .0 1;(88.2± 14 .4 ) % ,P <0 .0 1〕小 ;内膜面积 /中膜面积比值 ,治疗组较对照组减少了 5 5 .3%。结论 :在小型猪模型使用蛋白涂层包被VEGF支架 ,能预防内膜过度增殖 ,从而预防再狭窄的发生。     

三氧化二砷洗脱支架抑制猪损伤冠状动脉局部炎性因子的表达

目的 探讨三氧化二砷(As2O3)多聚左旋乳糖酸(PLLA)涂层支架对猪损伤冠状动脉局部炎性因子表达及炎性细胞浸润的影响,了解As2O3洗脱支架对局部炎性反应的作用. 方法 在8只小型家猪的前降支、回旋支和右冠状动脉随机双盲植入裸金属支架(裸支架组)、西罗莫司洗脱支架和As2O3洗脱支架,7 d处死,检测支架段血管单核细胞趋化蛋白(MCP)-1、白细胞介素(IL)-6蛋白和mRNA表达,HE和免疫组化检测炎性细胞浸润,体外观察As2O3对人T淋巴细胞的凋亡诱导作用. 结果裸支架组MCP-1蛋白和mRNA表达分别为0.857±0.053和0.724±0.027,IL-6蛋白和mRNA表达分别为0.551±0.032和1.015±0.041,As2O3洗脱支架和西罗莫司洗脱支架均降低支架段血管MCP-1(分别为0.421±0.055和0.406±0.042)和IL-6(分别为0.151±0.032和0.146±0.051)蛋白的表达(P＜0.01),并同时降低MCP-1(分别为0.338±0.047和0.327±0.051)和IL-6(分别为0.531±0.052和0.523±0.061)mRNA的表达(P＜0.01),As2O3洗脱支架和西罗莫司洗脱支架段血管局部炎性细胞浸润较裸支架组明显减少.体外细胞培养显示,As2O3具有诱导入T淋巴细胞凋亡作用. 结论 As2O3洗脱支架具有减少猪损伤冠状动脉炎性细胞浸润和抑制炎性因子MCP-1、IL-6表达的作用,诱导炎性细胞凋亡可能是其抗炎机制之一.     

Examination of Stent Deformation and Gap Formation after Complex Stenting of Left Main Coronary Artery Bifurcations Using Microfocus Computed Tomography

Background: Fluoroscopy and intravascular ultrasound (IVUS) lack sufficient resolution for assessing the results of complex stenting in true bifurcation lesions.
Objectives: After diverse bifurcation stenting at the left main coronary artery (LM) bifurcation model, the results were examined using microfocus computed tomography (MFCT).
Methods: The strut distribution of three kinds of stents deployed on a straight vessel segment was investigated. Classical crush, double kissing (DK)–double crush, and culotte stenting were performed on a three-dimensional (3D) LM model. The results were assessed using cross-sectional, longitudinal, and 3D reconstruction views of MFCT.
Results: Nonuniform strut distribution was observed in a corrugated stent design deployed on a straight vessel segment. Following classical crush stenting, a relatively large gap at the nonmyocardial site was observed in the corrugated stents. When the guidewire recrossed outside the ostium of the crushed side branch stent, kissing balloon inflation caused further crushing of the stent at the more distal segment. The dilated strut rose up from the main vessel bed after the first kissing balloon inflation in DK crush stenting; the advantage of DK would be cancelled after main vessel stenting due to recrushing the raised strut. The culotte stenting with closed-cell stents showed the restriction of the expansion at the branch ostium when it was dilated with a 3.5-mm balloon. The culotte stenting with open-cell-based stents showed a good stent apposition except for a tiny gap and small metallic carina at the distal bifurcation.
Conclusion: MFCT analysis in the 3D phantom model is useful to assess the structural deformation of the stents and gap on vessel wall coverage after complex stenting at the LM bifurcation.     

Should the left main be covered entirely with drug-eluting stents in percutaneous intervention

Restenosis after percutaneous intervention in the left main coronary artery may present as sudden cardiac death. Although drug-eluting stents have demonstrated promising results, there remains the question about appropriate length of the left main artery to be covered with the stent. We describe a patient who received two drug-eluting stents with the balloon crushing technique in the distal left main coronary artery. Three months later, this patient presented with a new lesion in the segment of the left main artery not covered with stent, but instead at the site where the balloon was inflated in the initial procedure.     

Unintentional extraction of a coronary stent deployed 4 months earlier during cutting-balloon angioplasty for in-stent restenosis

In general, coronary stents, when deployed in a coronary artery by conventional balloon expansion, appear to be tightly forced into the vessel wall, virtually precluding intentional or unintentional removal of the stents. Here, we present a case of unintended coronary stent extraction during cutting balloon angioplasty for high-grade in-stent restenosis of a stent successfully deployed 4 months earlier. The blades of the cutting balloon became stuck in the stent struts. Retrieval of the cutting balloon was only possible in conjunction with the stent using increased traction. Subsequent vessel closure was recanalized and a paclitaxel-eluting stent was implanted, covering the entire region of the previous stent and rendering an excellent angiographic result. Careful inspection of the extracted stent demonstrated complete removal. Retrospective analysis of the stent implantation procedure 4 months prior revealed complete stent expansion and closely matched stent and vessel dimensions as assessed by angiographic criteria.     

Stents covered by an autologous arterial graft in porcine coronary arteries: feasibility, vascular injury and effect on neointimal hyperplasia.

OBJECTIVE: The use of stents has improved results after balloon coronary angioplasty. Several materials have been proposed for covering the metallic surface of the stent to reduce the rate of subacute thrombosis and restenosis. In our institution, an autologous arterial graft was used for covering the external surface of a conventional stent. The angiographic and histological response in a porcine coronary artery model was investigated. METHODS: An autologous arterial graft was removed from the femoral artery and carefully prepared. Subsequently, a conventional stent was covered externally by the arterial graft. Twenty-two covered stents and 22 uncovered regular stents were implanted alternatively in the coronary arteries of 22 pigs. One animal died immediately after the procedure, due to thrombus formation in the uncovered stent. Six animals were sacrificed at seven days and the remaining animals were sacrificed at two months. Before the sacrifice, coronary angiography was performed in all animals. RESULTS: Thrombosis was detected in two control segments and in one covered stented segment. After seven days, the luminal surface of the covered stents was covered by a new endothelial layer in contrast to partial endothelial cell appearance in the control group. The angiographic parameters were similar between the two groups. Histologically, the covered stents were associated with less vascular injury compared to uncovered stents. In covered stents a trend towards reduction of maximal intimal hyperplasia was detected (covered: 116.6 +/- 47.75 vs uncovered: 150.25 +/- 46.81 microns, p = 0.08); also the thickness of the arterial media was reduced (covered: 21.34 +/- 10.28 vs uncovered: 102.63 +/- 18.71 microns, p = 0.02). The luminal and vessel areas were similar in the two groups. CONCLUSIONS: The preparation and implantation of the autologous arterial graft-covered stent is technically safe and feasible. This type of covered stent results in accelerated endothelialization, less vascular injury, thinning of the arterial media and a trend to reduce the intimal hyperplasia in normal coronary arteries.     

Simple and complex stent strategies for bifurcated coronary arterial stenosis involving the side branch origin

Coronary lesions located in major bifurcations constitute a challenge for the use of stents. Although the occlusion of a side branch covered by a stent is infrequent, the maintenance of a patent, stenosis-free bifurcation may result in a complex procedure. Between September 1994 and April 1998, 70 patients were treated by stent implantation for coronary bifurcation stenosis. The side branch always had a diameter >2 mm. The pairs of treated arteries were: left anterior descending (LAD)/diagonal artery in 32 patients, circumflex/obtuse marginal in 26, right coronary/posterior descending artery in 5, and LAD/circumflex in 7. We applied 2 different techniques of stent implantation: (1) deployment of 1 stent in the parent vessel covering the takeoff of the side branch and subsequent angioplasty of the side branch across the metallic structure (group A, n = 47 patients), and (2) implantation of 1 stent at the ostium of the side branch and complete reconstruction of the entire bifurcation with additional implantation of 1 or 2 stents at the parent vessel (group B, n = 23 patients). There were no significant differences between groups at baseline variables. Procedural success was similar in both groups: 42 (89%) in group A versus 21 (91%) in group B. However, major cardiac events at 18 months follow-up were higher in group B (event-free probability 44% vs 75%, p <0.05). Selected patients with coronary stenosis at major bifurcations can be treated with an acceptable rate of primary and late success. Complex techniques providing radical stent reconstruction of the bifurcation seems to provide no advantages over the simpler stent jail followed by ostial side branch balloon dilation.     

Management of coronary artery rupture: Covered stent or microcoil embolization

The management of three cases of coronary artery rupture is described: (1) after high-pressure balloon angioplasty following uneventful placement of three Gianturco-Roubin stents, (2) following balloon angioplasty of an occluded diagonal branch, and (3) subsequent to rotational ablation of a left main and proximal circumflex arteries. Placement of an autologous veincovered Palmaz stent or microcoil embolic vessel occlusion solved each problem. In each case, emergency surgery was avoided; subsequent management, including anticoagulation (when indicated), was performed without incident. This is the first communication detailing correction of a coronary vessel rupture with an autologous vein-covered stent or by microcoil embolic vessel occlusion.     

Neoatherosclerosis: Coronary stents seal atherosclerotic lesions but result in making a new problem of atherosclerosis

Chronic inflammation of the native vessel wall with infiltration of lipid-laden foamy macrophages through impaired endothelium results in atherosclerosis. Percutaneous coronary intervention, including metallic stent implantation, is now widely utilized for the treatment of atherosclerotic lesions of the coronary artery. Bare-metal stents and the subsequently developed drug-eluting stents seal the atherosclerosis and resolve lumen stenosis or obstruction of the epicardial coronary artery and myocardial ischemia. After stent implantation, neointima proliferates within the stented segment. Chronic inflammation caused by a foreign body reaction to the implanted stent and subsequent neovascularization, which is characterized by the continuous recruitment of macrophages into the vessel, result in the transformation of the usual neointima into an atheromatous neointima. Neointima with an atherosclerotic appearance, such as that caused by thin-cap fibroatheromas, is now recognized as neoatherosclerosis, which can sometimes cause in-stent restenosis and acute thrombotic occlusion originating from the stent segment following disruption of the atheroma. Neoatherosclerosis is emerging as a new coronary stent-associated problem that has not yet been resolved. In this review article, we will discuss possible mechanisms, clinical challenges, and the future outlook of neoatherosclerosis.     

Vascular endoscopic and macroscopic observations after crush stenting of coronary artery bifurcations in pigs.

The crush stent technique has recently been proposed to limit the development of restenosis between drug-eluting stents implanted at coronary artery bifurcations. We studied the stent expansion, apposition to the vessel, and aspect of the overlapping stents after in vivo crush stent implantation. Crush stent implantation was performed at coronary bifurcations in anesthetized swines. The treated sites were examined using intravascular ultrasound and a vascular endoscope. The stents removed from the vessel were analyzed macroscopically. After final kissing balloon inflation, an adequate apposition of the stent to the vessel wall was confirmed by vascular endoscopy and visual inspection. However, the side-branch stent was narrowed at the site of stent overlap, and the overlapping stents in the main branch created a metal mass, which could promote the development of thrombosis. The technique of crush stent implantation with additional kissing balloon inflation is feasible and promising. However, it may be limited by thrombosis and restenosis at the carina because of stent overlapping and potential incomplete apposition. Additional studies are needed to confirm the safety and long-term clinical results of this technique.