纳米微孔人EIA激活基因阻遏子糖蛋白洗脱支架预防再狭窄研究
摘要
目的药物洗脱支架已被证实可以有效抑制新生内膜增殖和再狭窄的发生。然而,聚合物涂层药物洗脱支架的高分子聚合物长期存留体内,引起内皮愈合延迟以及持续炎症细胞浸润,而这些可能导致晚期支架内血栓形成以及晚期再狭窄等并发症。理论上,无聚合物载体药物洗脱支架在有效抑制新生内膜增殖的同时,可以在一定程度上避免上述晚期不良反应的发生。研究表明E1A激活基因阻遏子是成熟生物体内维持组织和细胞分化的重要分子,在肿瘤细胞诱导分化和抑制损伤血管VSMCs去分化、促进内皮功能恢复中均具有积极的生物学功能。本文选用本室自主研发的人E1A激活基因阻遏子糖蛋白为洗脱药物,纳米微孔支架为平台,利用被动吸附的方法制备纳米微孔人E1A激活基因阻遏子糖蛋白洗脱支架。在制备并检测了纳米微孔人E1A激活基因阻遏子糖蛋白洗脱支架体外特性的基础上,应用小型猪为动物模型评价了纳米微孔人E1A激活基因阻遏子糖蛋白洗脱支架的生物安全性和防治支架内再狭窄的有效性。
方法1:将人293F细胞株用Lipofectamine 2000转染,筛选稳定表达的细胞克隆,免疫应迹方法鉴定hCREG/myc-His融合蛋白的表达,糖苷酶法及Western blotting行hCREG/myc-His融合蛋白的糖基化分析。应用Ni-NTA柱纯化CREG糖蛋白,用HiTrap脱盐柱将纯化后的CREG蛋白脱盐。2:将清洗和消毒好的纳米微孔支架在37℃条件下浸泡于缓冲液稀释至0.5mg/ml,1.0mg/ml,1.5 mg/ml的CREG糖蛋白溶液中。采用125I放射性标记单抗CREG蛋白的方法对CREG蛋白在支架上的吸附以及CREGES在体外药代动力学进行了检测。应用免疫荧光的及扫描电镜的方法确定CREG糖蛋白在支架上的吸附。以培养的血管平滑肌细胞和内皮细胞为模型对CREG洗脱支架体外生物活性分析。3:将纳米微孔裸支架、聚合物载体雷帕霉素洗脱支架以及CREG洗脱支架随机植入21头小型猪的前降支、回旋支和右冠脉。在植入后7天、14天和1个月复查造影,并行组织形态学测量以及病理分析。
结果:裂解稳定表达pcDNA 3.1 myc-His/hCREG的293F细胞及未转染的对照细胞,行Western blot检测。hCREG抗体,myc抗体及His抗体均检测到大小约为30KD的融合蛋白表达。为证明带有myc和His标签的重组hCREG蛋白为糖基化蛋白,将蛋白用PNGaseF处理,结果发现hCREG融合蛋白分子量由30KD降低至25KD,电泳条带下移。将纯化后CREG糖蛋白以及收集的洗脱液经离心超滤管浓缩后,经BCA法测定并与蛋白标准曲线比较,发现重组hCREG蛋白浓度为1.6 mg/mL,测量纯度为93%。浓缩并脱盐后的纯化蛋白经PNGaseF处理后分子量由30KD降至25KD,证实纯化后的蛋白仍保留了糖基化形式。
125I放射标记单抗的方法对CREG白在支架上的吸附以及CREG蛋白在体外的释放进行了定量的检测。从吸附实验的结果来看, CREG蛋白在支架上的吸附量与蛋白溶液的浓度和吸附时间有关。实验检测了支架在三种不同的蛋白浓度中吸附48h的实验数据,最大的蛋白吸附量是在1.5mg/ml的蛋白溶液中吸附24h时得到的。支架上蛋白的释放时间可达到2周以上。为了研究CREGES体外生物学效应及蛋白稳定性,直接将支架置入内皮细胞及平滑肌细胞的培养皿中,观察其对内皮细胞和平滑肌细胞增殖的影响。结果显示在3天和4天时与BMS相比,CREGES组内皮细胞数明显增高(P﹤0.05),而PARNTER组内皮细胞数明显减少(P﹤0.05)。在平滑肌增殖试验研究表明CREG可以显著抑制平滑肌细胞增殖,在4天、6天及8天时与BMS相比CREGES及PARTENER支架可以明显抑制平滑肌细胞增殖(P﹤0.05),而CREGES及PARTENER支架对平滑肌细胞增殖影响无差别。但是在10天时PARTENER支架抑制平滑肌细胞增殖程度显著强于CREGES及BMS(P﹤0.05)。
在体研究结果表明CREGES具有良好的安全性,未发现支架内血栓和支架周围炎症。3组冠状动脉大小和血管损伤积分程度无显著差异。4周时,3组支架均观察到不同程度的内膜增生。4周时各组间管腔面积及中膜面积无显著差异,但是各组间新生内膜面积存在显著差异(p=0.01),其中PARNTER组及CREG组新生内膜面积较BMS组显著减少,而PARNTER组及CREG组间新生内膜面积无显著差异(p=0.87)。同时,各组间管腔狭窄程度存在显著差异(p<0.01),其中BMS组管腔狭窄程度比PARNTER组(48%±4% vs. 15%±4%, p<0.01)及CREG组(48%±4% vs.20%±3%, p<0.01)明显增大,PARNTER组与CREG组间管腔狭窄程度无显著差异(20%±4% vs. 15%±3%, p=0.06)。免疫组化显示a-SMC-actin含量及CD68阳性指数在3组之间无差别。但是,CREGES组及PARNTER组的细胞增殖标记物Ki67指数明显高于BMS组,而CREGES组与PARNTER组Ki67指数无显著差别。为了研究3种支架对内皮化程度的影响,本研究进行了在不同时间点进行CD31染色并且计算了内皮化积分。结果显示7天时3组血管表面粗糙,很少有内皮覆盖。14天时CREGES组内皮化程度较PARNTER组BMS组明显增高,而PARNTER组及BMS组内皮化程度无差别。28天时CREGES组及BMS组已经基本内皮化,内皮化程度,无差别,而PARNTER组内皮覆盖不完全,内皮化程度较PARNTER组及BMS组明显减低。
结论: CREGES在小型猪模型中,植入1个月可以有效抑制新生内膜形成,并且表现出良好的安全性。其长期有效性有待于进一步研究证实。
Object Multiple studies showed a remarkable reduction in the rate of restenosis and need for new revascularization procedures associated with DES compared to conventional bare metal (BM) stents. However, the long-term safety of these devices is far less certain. Recent reports pointed to the risk of late stent thrombosis and delayed arterial healing, which may be increased in DES using a polymer-based coating for retardation of drug release. There is evidence that application of polymers may lead to hypersensitivity reactions and, in few cases, late cardiac death. Furthermore, the issue of late-stent thrombosis in DES on the basis of delayed arterial healing, particularly after discontinuation of antiplatelet therapy, is currently subject to ongoing discussions. With the current technology, major hurdles to long-term success have not yet been taken. Thus, the development of novel stent designs with different anti-proliferative drugs, biodegradable polymers, or bioabsorbable stent matrices are being investigated. Cellular repressor of E1A-stimulated genes (CREG) is a recently identified secreted glycoprotein that antagonizes transcription activation and cellular transformation induced by the adenovirus E1A oncoprotein. CREG has a critical role in maintenance of smooth muscle cells in a mature, growth-quiescent state in the normal artery, and the downregulation of CREG contributes to neointimal hyperplasia after vascular injury. Our study also found that CREG facilitates migration and proliferation of ECs and contributes to the maintenance of vascular homeostasis. In this study we investigate the in vitro pharmacokinetic of nanoporous CREG eluting-stent.To evaluate the efficacy and safty of nanoporous CREG-eluting stent (CREGES) in inhibiting neointima proliferation in porcine coronary model.
Methods 1: The human 293F cells were transfected with pcDNA3.1 myc-His/hCREG using Lipofectamine 2000. The expression of recombinant secreted hCREG/myc-His protein was identified by Western blotting. The glycosylation of hCREG/myc-His protein was analyzed by PNGaseF digestion and Western blot. 2: For the absorption of the CREG protein by the nanoporous stent ,the stents were totally immersed and kept vertical in a 0.5 mg/mL ,1.0 mg/mL and 1.5 mg/ml solution of CREG protein in a phosphate buffer at 37℃in a 1.5 mL polypropylene (Eppendorf tube). These stent wires were further transferred in a 5% solution of BSA at 37℃for 1 h, followed by three rinsing cycles in PBS pH 7.4, 2 min each. Then, they were immersed in a 20μg/mL solution of fluorescein rabbit anti-huamn IgG in PBS, pH 7.4, at 378C for 30 min, followed by three rinsing cycles in PBS, pH 7.4, 2 min each. In vitro proliferation assays were performed using isolated endothelial and smooth-muscle-cells from to investigate the cellspecific pharmacokinetic effect of CREG protein and rapamycin. 3: The nanoporous bare metal stent(BMS), nanoporous CREG eluting-stent(CREGES) and sirolimus-eluting stent(PARNTER) were implanted in left anterior descending coronary,left circumflex coronary and right coronary artery of fourty porcines in random. And after 7, 14 and 28-day,animals were sacrificed for histomorphologic and pathologic score analysis.
Results: The lysates of 293F cells transfected pcDNA3.1 myc-His/hCREG plasmid were detected by Western blot with Anti-hCREG, Anti-myc and Anti-His respectively. The recombinanted fusion protein about 30KD was identified in transfected cells by Western blot using Anti-myc and Anti-His. The recombinant hCREG protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. After the elution was concentrated with Centriprep centrifugal filter devices, the concentration of recombinant protein was determined to be 1.6 mg/mL by BCA assay. The purity of recombinant protein reached 92% identified with image-J software analysis. Stents eluting the CREG protein were tested for their adsorption characteristics by radioisotope technique with 125I labeled CREG protein. The amount of CREG protein adsorbed onto the nanoporous bare metal stent was dependent on the concentration and duration of immersion in the solution.We have tested three different concentrations for 48h.Maximal CREG protein binding was therefore defined as the amount of agent bound to stent wires after 48 hours immersion in a 1.5 mg/ml solution. We implant the CREG eluting stents and 316L stainless steel stents the control in pig model to study the bio-security validity of prevention and cure ISR by sliding microtome,SEM,transmission electron microscope(TEM),immunohistochemi- stry,tissue stain and biochemistry methods. The results suggests the CREG eluting stents can inhibit the thrombosis and neointimal hyperplasia by accelerating the endothelialization of the stent surface and inhibit the smooth muscle cell proliferation.
Conclusion The nanoporous CREG-eluting stent in this study represents a novel promising device in preventing in-stent restenosis
方法1:将人293F细胞株用Lipofectamine 2000转染,筛选稳定表达的细胞克隆,免疫应迹方法鉴定hCREG/myc-His融合蛋白的表达,糖苷酶法及Western blotting行hCREG/myc-His融合蛋白的糖基化分析。应用Ni-NTA柱纯化CREG糖蛋白,用HiTrap脱盐柱将纯化后的CREG蛋白脱盐。2:将清洗和消毒好的纳米微孔支架在37℃条件下浸泡于缓冲液稀释至0.5mg/ml,1.0mg/ml,1.5 mg/ml的CREG糖蛋白溶液中。采用125I放射性标记单抗CREG蛋白的方法对CREG蛋白在支架上的吸附以及CREGES在体外药代动力学进行了检测。应用免疫荧光的及扫描电镜的方法确定CREG糖蛋白在支架上的吸附。以培养的血管平滑肌细胞和内皮细胞为模型对CREG洗脱支架体外生物活性分析。3:将纳米微孔裸支架、聚合物载体雷帕霉素洗脱支架以及CREG洗脱支架随机植入21头小型猪的前降支、回旋支和右冠脉。在植入后7天、14天和1个月复查造影,并行组织形态学测量以及病理分析。
结果:裂解稳定表达pcDNA 3.1 myc-His/hCREG的293F细胞及未转染的对照细胞,行Western blot检测。hCREG抗体,myc抗体及His抗体均检测到大小约为30KD的融合蛋白表达。为证明带有myc和His标签的重组hCREG蛋白为糖基化蛋白,将蛋白用PNGaseF处理,结果发现hCREG融合蛋白分子量由30KD降低至25KD,电泳条带下移。将纯化后CREG糖蛋白以及收集的洗脱液经离心超滤管浓缩后,经BCA法测定并与蛋白标准曲线比较,发现重组hCREG蛋白浓度为1.6 mg/mL,测量纯度为93%。浓缩并脱盐后的纯化蛋白经PNGaseF处理后分子量由30KD降至25KD,证实纯化后的蛋白仍保留了糖基化形式。
125I放射标记单抗的方法对CREG白在支架上的吸附以及CREG蛋白在体外的释放进行了定量的检测。从吸附实验的结果来看, CREG蛋白在支架上的吸附量与蛋白溶液的浓度和吸附时间有关。实验检测了支架在三种不同的蛋白浓度中吸附48h的实验数据,最大的蛋白吸附量是在1.5mg/ml的蛋白溶液中吸附24h时得到的。支架上蛋白的释放时间可达到2周以上。为了研究CREGES体外生物学效应及蛋白稳定性,直接将支架置入内皮细胞及平滑肌细胞的培养皿中,观察其对内皮细胞和平滑肌细胞增殖的影响。结果显示在3天和4天时与BMS相比,CREGES组内皮细胞数明显增高(P﹤0.05),而PARNTER组内皮细胞数明显减少(P﹤0.05)。在平滑肌增殖试验研究表明CREG可以显著抑制平滑肌细胞增殖,在4天、6天及8天时与BMS相比CREGES及PARTENER支架可以明显抑制平滑肌细胞增殖(P﹤0.05),而CREGES及PARTENER支架对平滑肌细胞增殖影响无差别。但是在10天时PARTENER支架抑制平滑肌细胞增殖程度显著强于CREGES及BMS(P﹤0.05)。
在体研究结果表明CREGES具有良好的安全性,未发现支架内血栓和支架周围炎症。3组冠状动脉大小和血管损伤积分程度无显著差异。4周时,3组支架均观察到不同程度的内膜增生。4周时各组间管腔面积及中膜面积无显著差异,但是各组间新生内膜面积存在显著差异(p=0.01),其中PARNTER组及CREG组新生内膜面积较BMS组显著减少,而PARNTER组及CREG组间新生内膜面积无显著差异(p=0.87)。同时,各组间管腔狭窄程度存在显著差异(p<0.01),其中BMS组管腔狭窄程度比PARNTER组(48%±4% vs. 15%±4%, p<0.01)及CREG组(48%±4% vs.20%±3%, p<0.01)明显增大,PARNTER组与CREG组间管腔狭窄程度无显著差异(20%±4% vs. 15%±3%, p=0.06)。免疫组化显示a-SMC-actin含量及CD68阳性指数在3组之间无差别。但是,CREGES组及PARNTER组的细胞增殖标记物Ki67指数明显高于BMS组,而CREGES组与PARNTER组Ki67指数无显著差别。为了研究3种支架对内皮化程度的影响,本研究进行了在不同时间点进行CD31染色并且计算了内皮化积分。结果显示7天时3组血管表面粗糙,很少有内皮覆盖。14天时CREGES组内皮化程度较PARNTER组BMS组明显增高,而PARNTER组及BMS组内皮化程度无差别。28天时CREGES组及BMS组已经基本内皮化,内皮化程度,无差别,而PARNTER组内皮覆盖不完全,内皮化程度较PARNTER组及BMS组明显减低。
结论: CREGES在小型猪模型中,植入1个月可以有效抑制新生内膜形成,并且表现出良好的安全性。其长期有效性有待于进一步研究证实。
Object Multiple studies showed a remarkable reduction in the rate of restenosis and need for new revascularization procedures associated with DES compared to conventional bare metal (BM) stents. However, the long-term safety of these devices is far less certain. Recent reports pointed to the risk of late stent thrombosis and delayed arterial healing, which may be increased in DES using a polymer-based coating for retardation of drug release. There is evidence that application of polymers may lead to hypersensitivity reactions and, in few cases, late cardiac death. Furthermore, the issue of late-stent thrombosis in DES on the basis of delayed arterial healing, particularly after discontinuation of antiplatelet therapy, is currently subject to ongoing discussions. With the current technology, major hurdles to long-term success have not yet been taken. Thus, the development of novel stent designs with different anti-proliferative drugs, biodegradable polymers, or bioabsorbable stent matrices are being investigated. Cellular repressor of E1A-stimulated genes (CREG) is a recently identified secreted glycoprotein that antagonizes transcription activation and cellular transformation induced by the adenovirus E1A oncoprotein. CREG has a critical role in maintenance of smooth muscle cells in a mature, growth-quiescent state in the normal artery, and the downregulation of CREG contributes to neointimal hyperplasia after vascular injury. Our study also found that CREG facilitates migration and proliferation of ECs and contributes to the maintenance of vascular homeostasis. In this study we investigate the in vitro pharmacokinetic of nanoporous CREG eluting-stent.To evaluate the efficacy and safty of nanoporous CREG-eluting stent (CREGES) in inhibiting neointima proliferation in porcine coronary model.
Methods 1: The human 293F cells were transfected with pcDNA3.1 myc-His/hCREG using Lipofectamine 2000. The expression of recombinant secreted hCREG/myc-His protein was identified by Western blotting. The glycosylation of hCREG/myc-His protein was analyzed by PNGaseF digestion and Western blot. 2: For the absorption of the CREG protein by the nanoporous stent ,the stents were totally immersed and kept vertical in a 0.5 mg/mL ,1.0 mg/mL and 1.5 mg/ml solution of CREG protein in a phosphate buffer at 37℃in a 1.5 mL polypropylene (Eppendorf tube). These stent wires were further transferred in a 5% solution of BSA at 37℃for 1 h, followed by three rinsing cycles in PBS pH 7.4, 2 min each. Then, they were immersed in a 20μg/mL solution of fluorescein rabbit anti-huamn IgG in PBS, pH 7.4, at 378C for 30 min, followed by three rinsing cycles in PBS, pH 7.4, 2 min each. In vitro proliferation assays were performed using isolated endothelial and smooth-muscle-cells from to investigate the cellspecific pharmacokinetic effect of CREG protein and rapamycin. 3: The nanoporous bare metal stent(BMS), nanoporous CREG eluting-stent(CREGES) and sirolimus-eluting stent(PARNTER) were implanted in left anterior descending coronary,left circumflex coronary and right coronary artery of fourty porcines in random. And after 7, 14 and 28-day,animals were sacrificed for histomorphologic and pathologic score analysis.
Results: The lysates of 293F cells transfected pcDNA3.1 myc-His/hCREG plasmid were detected by Western blot with Anti-hCREG, Anti-myc and Anti-His respectively. The recombinanted fusion protein about 30KD was identified in transfected cells by Western blot using Anti-myc and Anti-His. The recombinant hCREG protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. After the elution was concentrated with Centriprep centrifugal filter devices, the concentration of recombinant protein was determined to be 1.6 mg/mL by BCA assay. The purity of recombinant protein reached 92% identified with image-J software analysis. Stents eluting the CREG protein were tested for their adsorption characteristics by radioisotope technique with 125I labeled CREG protein. The amount of CREG protein adsorbed onto the nanoporous bare metal stent was dependent on the concentration and duration of immersion in the solution.We have tested three different concentrations for 48h.Maximal CREG protein binding was therefore defined as the amount of agent bound to stent wires after 48 hours immersion in a 1.5 mg/ml solution. We implant the CREG eluting stents and 316L stainless steel stents the control in pig model to study the bio-security validity of prevention and cure ISR by sliding microtome,SEM,transmission electron microscope(TEM),immunohistochemi- stry,tissue stain and biochemistry methods. The results suggests the CREG eluting stents can inhibit the thrombosis and neointimal hyperplasia by accelerating the endothelialization of the stent surface and inhibit the smooth muscle cell proliferation.
Conclusion The nanoporous CREG-eluting stent in this study represents a novel promising device in preventing in-stent restenosis
引文
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15. van der Giessen, W.J., et al., Marked inflammatory sequelae to implantation of biodegradable and nonbiodegradable polymers in porcine coronary arteries. Circulation, 1996. 94(7): p. 1690-7.
16. Kereiakes, D.J., New drug-eluting stent platforms to prevent stent thrombosis. Rev Cardiovasc Med, 2007. 8 Suppl 1: p. S34-43.
17. Sims, J.M., Update on drug-eluting stents. Dimens Crit Care Nurs, 2007. 26(6): p.237-40.
18. Kipshidze, N., et al., Update on sirolimus drug-eluting stents. Curr Pharm Des, 2004. 10(4): p. 337-48.
19. Sousa, J.E., et al., New frontiers in interventional cardiology. Circulation, 2005. 111(5): p. 671-81.
20. Morton, A.C., R.D. Walker, and J. Gunn, Current challenges in coronary stenting: from bench to bedside. Biochem Soc Trans, 2007. 35(Pt 5): p. 900-4.
21. McFadden, E.P., et al., Late thrombosis in drug-eluting coronary stents after discontinuation of antiplatelet therapy. Lancet, 2004. 364(9444): p. 1519-21.
22. Mehilli, J., et al., Randomized trial of a nonpolymer-based rapamycin-eluting stent versus a polymer-based paclitaxel-eluting stent for the reduction of late lumen loss. Circulation, 2006. 113(2): p. 273-9.
23. Serruys, P.W., et al., The effect of variable dose and release kinetics on neointimal hyperplasia using a novel paclitaxel-eluting stent platform: the Paclitaxel In-Stent Controlled Elution Study (PISCES). J Am Coll Cardiol, 2005. 46(2): p. 253-60.
24. Tsujino, I., et al., Drug delivery via nano-, micro and macroporous coronary stent surfaces. Expert Opin Drug Deliv, 2007. 4(3): p. 287-95.
25. Hausleiter, J., et al., Prevention of restenosis by a novel drug-eluting stent system with a dose-adjustable, polymer-free, on-site stent coating. Eur Heart J, 2005. 26(15): p. 1475-81.
26. Wieneke, H., et al., Synergistic effects of a novel nanoporous stent coating and tacrolimus on intima proliferation in rabbits. Catheter Cardiovasc Interv, 2003. 60(3): p. 399-407.
27. Moses, J.W., et al., Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med, 2003. 349(14): p. 1315-23.
28. Schofer, J., et al., Sirolimus-eluting stents for treatment of patients with long atherosclerotic lesions in small coronary arteries: double-blind, randomised controlled trial (E-SIRIUS). Lancet, 2003. 362(9390): p. 1093-9.
29. Schampaert, E., et al., The Canadian study of the sirolimus-eluting stent in the treatment of patients with long de novo lesions in small native coronary arteries (C-SIRIUS). J Am Coll Cardiol, 2004. 43(6): p. 1110-5.
30. Lorcy, J.M., et al., Coaxial nickel/poly(p-phenylene vinylene) nanowires as luminescent building blocks manipulated magnetically. Nanotechnology, 2009. 20(40): p. 405601.
31. Bartorelli, A.L., et al., Synergy of passive coating and targeted drug delivery: the tacrolimus-eluting Janus CarboStent. J Interv Cardiol, 2003. 16(6): p. 499-505.
32. Han, Y.L., et al., Midterm outcomes of prospective, randomized, single-center study of the Janus tacrolimus-eluting stent for treatment of native coronary artery lesions. Chin Med J (Engl), 2007. 120(7): p. 552-6.
33. Finkelstein, A., et al., Local drug delivery via a coronary stent with programmablerelease pharmacokinetics. Circulation, 2003. 107(5): p. 777-84.
34. Wiemer, M., et al., Scanning electron microscopic analysis of different drug eluting stents after failed implantation: From nearly undamaged to major damaged polymers. Catheter Cardiovasc Interv, 2009.
35. Scott, N.A., Restenosis following implantation of bare metal coronary stents: pathophysiology and pathways involved in the vascular response to injury. Adv Drug Deliv Rev, 2006. 58(3): p. 358-76.
36. Kavanagh, C.A., et al., Local drug delivery in restenosis injury: thermoresponsive co-polymers as potential drug delivery systems. Pharmacol Ther, 2004. 102(1): p. 1-15.
37. Jovinge, S., et al., Tumor necrosis factor-alpha activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol, 1997. 17(3): p. 490-7.
38. Clausell, N., et al., In vivo blockade of tumor necrosis factor-alpha in cholesterol-fed rabbits after cardiac transplant inhibits acute coronary artery neointimal formation. Circulation, 1994. 89(6): p. 2768-79.
39. Foo, R.S., et al., Inhibition of platelet thrombosis using an activated protein C-loaded stent: in vitro and in vivo results. Thromb Haemost, 2000. 83(3): p. 496-502.
40. Javed, Q., et al., Tumor necrosis factor-alpha antibody eluting stents reduce vascular smooth muscle cell proliferation in saphenous vein organ culture. Exp Mol Pathol, 2002. 73(2): p. 104-11.
41. Lefkovits, J., E.F. Plow, and E.J. Topol, Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med, 1995. 332(23): p. 1553-9.
42. Azrin, M.A., et al., Preparation, characterization, and evaluation of a monoclonal antibody against the rabbit platelet glycoprotein IIb/IIIa in an experimental angioplasty model. Circ Res, 1994. 75(2): p. 268-77.
43. Aggarwal, R.K., et al., Antithrombotic potential of polymer-coated stents eluting platelet glycoprotein IIb/IIIa receptor antibody. Circulation, 1996. 94(12): p. 3311-7.
44. Matsuno, H., et al., Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation. Circulation, 1994. 90(5): p. 2203-6.
45. Valgimigli, M., et al., Tirofiban and sirolimus-eluting stent vs abciximab and bare-metal stent for acute myocardial infarction: a randomized trial. JAMA, 2005. 293(17): p. 2109-17.
46. Baron, J.H., et al., In vitro evaluation of c7E3-Fab (ReoPro) eluting polymer-coated coronary stents. Cardiovasc Res, 2000. 46(3): p. 585-94.
47. Hong, Y.J., et al., Effect of abciximab-coated stent on in-stent intimal hyperplasia in human coronary arteries. Am J Cardiol, 2004. 94(8): p. 1050-4.
48. Kim, W., et al., The clinical results of a platelet glycoprotein IIb/IIIa receptor blocker (abciximab: ReoPro)-coated stent in acute myocardial infarction. J Am Coll Cardiol, 2006. 47(5): p. 933-8.
49. Grainger, D.W., Controlled-release and local delivery of therapeutic antibodies. Expert Opin Biol Ther, 2004. 4(7): p. 1029-44.
50. Sousa, J.E., P.W. Serruys, and M.A. Costa, New frontiers in cardiology: drug-eluting stents: Part I. Circulation, 2003. 107(17): p. 2274-9.
51. Melo, L.G., et al., Endothelium-targeted gene and cell-based therapies for cardiovascular disease. Arterioscler Thromb Vasc Biol, 2004. 24(10): p. 1761-74.
52. Kong, D., et al., Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells. Circulation, 2004. 109(14): p. 1769-75.
53. Werner, N. and G. Nickenig, Clinical and therapeutical implications of EPC biology in atherosclerosis. J Cell Mol Med, 2006. 10(2): p. 318-32.
54. Wissink, M.J., et al., Endothelial cell seeding of (heparinized) collagen matrices: effects of bFGF pre-loading on proliferation (after low density seeding) and pro-coagulant factors. J Control Release, 2000. 67(2-3): p. 141-55.
55. Conklin, B.S., et al., Basic fibroblast growth factor coating and endothelial cell seeding of a decellularized heparin-coated vascular graft. Artif Organs, 2004. 28(7): p. 668-75.
56. Asahara, T., et al., Local delivery of vascular endothelial growth factor accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Circulation, 1995. 91(11): p. 2793-801.
57. Di Pasquale, P., et al., Safety and tolerability of abciximab in patients with acute myocardial infarction and failed thrombolysis. Int J Cardiol, 2003. 92(2-3): p. 265-70.
58. Zhu, G., S.R. Mallery, and S.P. Schwendeman, Stabilization of proteins encapsulated in injectable poly (lactide- co-glycolide). Nat Biotechnol, 2000. 18(1): p. 52-7.
59. Jiang, W. and S.P. Schwendeman, Stabilization and controlled release of bovine serum albumin encapsulated in poly(D, L-lactide) and poly(ethylene glycol) microsphere blends. Pharm Res, 2001. 18(6): p. 878-85.
60. Kavanagh, C.A., et al., Poly(N-isopropylacrylamide) copolymer films as vehicles for the sustained delivery of proteins to vascular endothelial cells. J Biomed Mater Res A, 2005. 72(1): p. 25-35.
61. Ehrbar, M., et al., Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth. Circ Res, 2004. 94(8): p. 1124-32.
62. Van Belle, E., et al., Accelerated endothelialization by local delivery of recombinant human vascular endothelial growth factor reduces in-stent intimal formation. Biochem Biophys Res Commun, 1997. 235(2): p. 311-6.
63. Arnljots, B. and B. Dahlback, Antithrombotic effects of activated protein C and protein S in a rabbit model of microarterial thrombosis. Arterioscler Thromb Vasc Biol, 1995. 15(7): p. 937-41.
64. Veal, E., et al., A cellular repressor of E1A-stimulated genes that inhibits activation by E2F. Mol Cell Biol, 1998. 18(9): p. 5032-41.
65. Veal, E., et al., The secreted glycoprotein CREG enhances differentiation of NTERA-2human embryonal carcinoma cells. Oncogene, 2000. 19(17): p. 2120-8.
66. Sacher, M., et al., The crystal structure of CREG, a secreted glycoprotein involved in cellular growth and differentiation. Proc Natl Acad Sci U S A, 2005. 102(51): p. 18326-31.
67. Di Bacco, A. and G. Gill, The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-like growth factor II receptor. Oncogene, 2003. 22(35): p. 5436-45.
68. Deng, J., et al., Overexpressing cellular repressor of E1A-stimulated genes protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis by activation of PI3K/Akt. Apoptosis, 2010. 15(4): p. 463-73.
69. Han, Y.L., et al., [Expression of cellular repressor of E1A-stimulated genes in vascular smooth muscle cells of different phenotypes]. Zhonghua Yi Xue Za Zhi, 2005. 85(1): p. 49-53.
70. Han, Y., et al., CREG promotes a mature smooth muscle cell phenotype and reduces neointimal formation in balloon-injured rat carotid artery. Cardiovasc Res, 2008. 78(3): p. 597-604.
71. Han, Y., et al., Cellular repressor of E1A-stimulated genes inhibits human vascular smooth muscle cell apoptosis via blocking P38/JNK MAP kinase activation. J Mol Cell Cardiol, 2010.
72. Han, Y., et al., CREG inhibits migration of human vascular smooth muscle cells by mediating IGF-II endocytosis. Exp Cell Res, 2009. 315(19): p. 3301-11.
73. Han, Y., et al., Adenovirus-mediated intra-arterial delivery of cellular repressor of E1A-stimulated genes inhibits neointima formation in rabbits after balloon injury. J Vasc Surg, 2008. 48(1): p. 201-9.
74. Schahs, P., et al., Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis. Exp Cell Res, 2008. 314(16): p. 3036-47.
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2. Zargham, R., Preventing restenosis after angioplasty: a multistage approach. Clin Sci (Lond), 2008. 114(4): p. 257-64.
3. Subbotin, V.M., Analysis of arterial intimal hyperplasia: review and hypothesis. Theor Biol Med Model, 2007. 4: p. 41.
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6. Versari, D., L.O. Lerman, and A. Lerman, The importance of reendothelialization after arterial injury. Curr Pharm Des, 2007. 13(17): p. 1811-24.
7. Hao, H., G. Gabbiani, and M.L. Bochaton-Piallat, Arterial smooth muscle cell heterogeneity: implications for atherosclerosis and restenosis development. Arterioscler Thromb Vasc Biol, 2003. 23(9): p. 1510-20.
8. Muto, A., et al., Smooth muscle cell signal transduction: implications of vascular biology for vascular surgeons. J Vasc Surg, 2007. 45 Suppl A: p. A15-24.
9. Babapulle, M.N. and M.J. Eisenberg, Coated stents for the prevention of restenosis: Part I. Circulation, 2002. 106(21): p. 2734-40.
10. Babapulle, M.N. and M.J. Eisenberg, Coated stents for the prevention of restenosis: Part II. Circulation, 2002. 106(22): p. 2859-66.
11. Kukreja, N., et al., The future of drug-eluting stents. Pharmacol Res, 2008.
12. Nebeker, J.R., et al., Hypersensitivity cases associated with drug-eluting coronary stents: a review of available cases from the Research on Adverse Drug Events and Reports (RADAR) project. J Am Coll Cardiol, 2006. 47(1): p. 175-81.
13. Virmani, R., et al., Localized hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent: should we be cautious? Circulation, 2004. 109(6): p. 701-5.
14. Commandeur, S., H.M. van Beusekom, and W.J. van der Giessen, Polymers, drug release, and drug-eluting stents. J Interv Cardiol, 2006. 19(6): p. 500-6.
15. van der Giessen, W.J., et al., Marked inflammatory sequelae to implantation of biodegradable and nonbiodegradable polymers in porcine coronary arteries. Circulation, 1996. 94(7): p. 1690-7.
16. Kereiakes, D.J., New drug-eluting stent platforms to prevent stent thrombosis. Rev Cardiovasc Med, 2007. 8 Suppl 1: p. S34-43.
17. Sims, J.M., Update on drug-eluting stents. Dimens Crit Care Nurs, 2007. 26(6): p.237-40.
18. Kipshidze, N., et al., Update on sirolimus drug-eluting stents. Curr Pharm Des, 2004. 10(4): p. 337-48.
19. Sousa, J.E., et al., New frontiers in interventional cardiology. Circulation, 2005. 111(5): p. 671-81.
20. Morton, A.C., R.D. Walker, and J. Gunn, Current challenges in coronary stenting: from bench to bedside. Biochem Soc Trans, 2007. 35(Pt 5): p. 900-4.
21. McFadden, E.P., et al., Late thrombosis in drug-eluting coronary stents after discontinuation of antiplatelet therapy. Lancet, 2004. 364(9444): p. 1519-21.
22. Mehilli, J., et al., Randomized trial of a nonpolymer-based rapamycin-eluting stent versus a polymer-based paclitaxel-eluting stent for the reduction of late lumen loss. Circulation, 2006. 113(2): p. 273-9.
23. Serruys, P.W., et al., The effect of variable dose and release kinetics on neointimal hyperplasia using a novel paclitaxel-eluting stent platform: the Paclitaxel In-Stent Controlled Elution Study (PISCES). J Am Coll Cardiol, 2005. 46(2): p. 253-60.
24. Tsujino, I., et al., Drug delivery via nano-, micro and macroporous coronary stent surfaces. Expert Opin Drug Deliv, 2007. 4(3): p. 287-95.
25. Hausleiter, J., et al., Prevention of restenosis by a novel drug-eluting stent system with a dose-adjustable, polymer-free, on-site stent coating. Eur Heart J, 2005. 26(15): p. 1475-81.
26. Wieneke, H., et al., Synergistic effects of a novel nanoporous stent coating and tacrolimus on intima proliferation in rabbits. Catheter Cardiovasc Interv, 2003. 60(3): p. 399-407.
27. Moses, J.W., et al., Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med, 2003. 349(14): p. 1315-23.
28. Schofer, J., et al., Sirolimus-eluting stents for treatment of patients with long atherosclerotic lesions in small coronary arteries: double-blind, randomised controlled trial (E-SIRIUS). Lancet, 2003. 362(9390): p. 1093-9.
29. Schampaert, E., et al., The Canadian study of the sirolimus-eluting stent in the treatment of patients with long de novo lesions in small native coronary arteries (C-SIRIUS). J Am Coll Cardiol, 2004. 43(6): p. 1110-5.
30. Lorcy, J.M., et al., Coaxial nickel/poly(p-phenylene vinylene) nanowires as luminescent building blocks manipulated magnetically. Nanotechnology, 2009. 20(40): p. 405601.
31. Bartorelli, A.L., et al., Synergy of passive coating and targeted drug delivery: the tacrolimus-eluting Janus CarboStent. J Interv Cardiol, 2003. 16(6): p. 499-505.
32. Han, Y.L., et al., Midterm outcomes of prospective, randomized, single-center study of the Janus tacrolimus-eluting stent for treatment of native coronary artery lesions. Chin Med J (Engl), 2007. 120(7): p. 552-6.
33. Finkelstein, A., et al., Local drug delivery via a coronary stent with programmablerelease pharmacokinetics. Circulation, 2003. 107(5): p. 777-84.
34. Wiemer, M., et al., Scanning electron microscopic analysis of different drug eluting stents after failed implantation: From nearly undamaged to major damaged polymers. Catheter Cardiovasc Interv, 2009.
35. Scott, N.A., Restenosis following implantation of bare metal coronary stents: pathophysiology and pathways involved in the vascular response to injury. Adv Drug Deliv Rev, 2006. 58(3): p. 358-76.
36. Kavanagh, C.A., et al., Local drug delivery in restenosis injury: thermoresponsive co-polymers as potential drug delivery systems. Pharmacol Ther, 2004. 102(1): p. 1-15.
37. Jovinge, S., et al., Tumor necrosis factor-alpha activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol, 1997. 17(3): p. 490-7.
38. Clausell, N., et al., In vivo blockade of tumor necrosis factor-alpha in cholesterol-fed rabbits after cardiac transplant inhibits acute coronary artery neointimal formation. Circulation, 1994. 89(6): p. 2768-79.
39. Foo, R.S., et al., Inhibition of platelet thrombosis using an activated protein C-loaded stent: in vitro and in vivo results. Thromb Haemost, 2000. 83(3): p. 496-502.
40. Javed, Q., et al., Tumor necrosis factor-alpha antibody eluting stents reduce vascular smooth muscle cell proliferation in saphenous vein organ culture. Exp Mol Pathol, 2002. 73(2): p. 104-11.
41. Lefkovits, J., E.F. Plow, and E.J. Topol, Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med, 1995. 332(23): p. 1553-9.
42. Azrin, M.A., et al., Preparation, characterization, and evaluation of a monoclonal antibody against the rabbit platelet glycoprotein IIb/IIIa in an experimental angioplasty model. Circ Res, 1994. 75(2): p. 268-77.
43. Aggarwal, R.K., et al., Antithrombotic potential of polymer-coated stents eluting platelet glycoprotein IIb/IIIa receptor antibody. Circulation, 1996. 94(12): p. 3311-7.
44. Matsuno, H., et al., Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation. Circulation, 1994. 90(5): p. 2203-6.
45. Valgimigli, M., et al., Tirofiban and sirolimus-eluting stent vs abciximab and bare-metal stent for acute myocardial infarction: a randomized trial. JAMA, 2005. 293(17): p. 2109-17.
46. Baron, J.H., et al., In vitro evaluation of c7E3-Fab (ReoPro) eluting polymer-coated coronary stents. Cardiovasc Res, 2000. 46(3): p. 585-94.
47. Hong, Y.J., et al., Effect of abciximab-coated stent on in-stent intimal hyperplasia in human coronary arteries. Am J Cardiol, 2004. 94(8): p. 1050-4.
48. Kim, W., et al., The clinical results of a platelet glycoprotein IIb/IIIa receptor blocker (abciximab: ReoPro)-coated stent in acute myocardial infarction. J Am Coll Cardiol, 2006. 47(5): p. 933-8.
49. Grainger, D.W., Controlled-release and local delivery of therapeutic antibodies. Expert Opin Biol Ther, 2004. 4(7): p. 1029-44.
50. Sousa, J.E., P.W. Serruys, and M.A. Costa, New frontiers in cardiology: drug-eluting stents: Part I. Circulation, 2003. 107(17): p. 2274-9.
51. Melo, L.G., et al., Endothelium-targeted gene and cell-based therapies for cardiovascular disease. Arterioscler Thromb Vasc Biol, 2004. 24(10): p. 1761-74.
52. Kong, D., et al., Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells. Circulation, 2004. 109(14): p. 1769-75.
53. Werner, N. and G. Nickenig, Clinical and therapeutical implications of EPC biology in atherosclerosis. J Cell Mol Med, 2006. 10(2): p. 318-32.
54. Wissink, M.J., et al., Endothelial cell seeding of (heparinized) collagen matrices: effects of bFGF pre-loading on proliferation (after low density seeding) and pro-coagulant factors. J Control Release, 2000. 67(2-3): p. 141-55.
55. Conklin, B.S., et al., Basic fibroblast growth factor coating and endothelial cell seeding of a decellularized heparin-coated vascular graft. Artif Organs, 2004. 28(7): p. 668-75.
56. Asahara, T., et al., Local delivery of vascular endothelial growth factor accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Circulation, 1995. 91(11): p. 2793-801.
57. Di Pasquale, P., et al., Safety and tolerability of abciximab in patients with acute myocardial infarction and failed thrombolysis. Int J Cardiol, 2003. 92(2-3): p. 265-70.
58. Zhu, G., S.R. Mallery, and S.P. Schwendeman, Stabilization of proteins encapsulated in injectable poly (lactide- co-glycolide). Nat Biotechnol, 2000. 18(1): p. 52-7.
59. Jiang, W. and S.P. Schwendeman, Stabilization and controlled release of bovine serum albumin encapsulated in poly(D, L-lactide) and poly(ethylene glycol) microsphere blends. Pharm Res, 2001. 18(6): p. 878-85.
60. Kavanagh, C.A., et al., Poly(N-isopropylacrylamide) copolymer films as vehicles for the sustained delivery of proteins to vascular endothelial cells. J Biomed Mater Res A, 2005. 72(1): p. 25-35.
61. Ehrbar, M., et al., Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth. Circ Res, 2004. 94(8): p. 1124-32.
62. Van Belle, E., et al., Accelerated endothelialization by local delivery of recombinant human vascular endothelial growth factor reduces in-stent intimal formation. Biochem Biophys Res Commun, 1997. 235(2): p. 311-6.
63. Arnljots, B. and B. Dahlback, Antithrombotic effects of activated protein C and protein S in a rabbit model of microarterial thrombosis. Arterioscler Thromb Vasc Biol, 1995. 15(7): p. 937-41.
64. Veal, E., et al., A cellular repressor of E1A-stimulated genes that inhibits activation by E2F. Mol Cell Biol, 1998. 18(9): p. 5032-41.
65. Veal, E., et al., The secreted glycoprotein CREG enhances differentiation of NTERA-2human embryonal carcinoma cells. Oncogene, 2000. 19(17): p. 2120-8.
66. Sacher, M., et al., The crystal structure of CREG, a secreted glycoprotein involved in cellular growth and differentiation. Proc Natl Acad Sci U S A, 2005. 102(51): p. 18326-31.
67. Di Bacco, A. and G. Gill, The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-like growth factor II receptor. Oncogene, 2003. 22(35): p. 5436-45.
68. Deng, J., et al., Overexpressing cellular repressor of E1A-stimulated genes protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis by activation of PI3K/Akt. Apoptosis, 2010. 15(4): p. 463-73.
69. Han, Y.L., et al., [Expression of cellular repressor of E1A-stimulated genes in vascular smooth muscle cells of different phenotypes]. Zhonghua Yi Xue Za Zhi, 2005. 85(1): p. 49-53.
70. Han, Y., et al., CREG promotes a mature smooth muscle cell phenotype and reduces neointimal formation in balloon-injured rat carotid artery. Cardiovasc Res, 2008. 78(3): p. 597-604.
71. Han, Y., et al., Cellular repressor of E1A-stimulated genes inhibits human vascular smooth muscle cell apoptosis via blocking P38/JNK MAP kinase activation. J Mol Cell Cardiol, 2010.
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