雷帕霉素电纺丝缓释药膜抑制大鼠自体移植静脉狭窄的实验研究
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摘要
研究背景：
冠状动脉旁路移植术(Coronary Arterial Bypass Grafting, CABG)是冠状动脉粥样硬化性心脏病的重要临床治疗措施,2012年国际权威杂志NEJM的冠心病观察性研究证实患者接受CABG治疗可获得比PCI治疗更长的生存期[1]。临床上作为CABG移植材料的主要有乳内动脉、大隐静脉和桡动脉,由于乳内动脉和桡动脉取材的限制性,大隐静脉一直作为CABG的重要取材血管。但移植后大隐静脉桥狭窄发生率较高,成为心血管外科亟需解决的临床难题[2]。目前有关大隐静脉桥狭窄防治的实验研究集中在血管内皮细胞及中层平滑肌细胞的干预治疗上,而对于大隐静脉主要管壁结构——血管外膜的研究却极少。近年来,血管外膜在移植血管狭窄中的作用开始受到重视,有研究提示血管外膜对于血管结构和功能有着直接或间接的重要调控作用[3]。外膜成纤维细胞是血管外膜的主要细胞类型,越来越多的研究证实血管外膜成纤维细胞在移植血管病变过程中并非是无辜的旁观者,而是积极的参与者[4]。最近我们参与的课题组发现静脉移植于动脉后因血管张力发生改变首先启动移植静脉外膜的成纤维细胞,分泌细胞外基质(ECM)、各种生长因子、细胞因子,如TGF-β1、MCP-1、MMPs等,诱导外膜成纤维细胞增殖并转化为成肌纤维细胞(myofibroblast,MF)[5],向中膜层及内膜迁移,参与新内膜的形成。而目前国内外尚无以外膜成纤维细胞为靶点,防治自体移植静脉狭窄的研究。
雷帕霉素(Rapamycin),又称西罗莫司,是一种亲脂性三烯含氮的大环内酯抗生素类免疫抑制剂,具有很好的抗排斥作用,被广泛应用于器官移植[6,7]。由于它具有极好的抑制血管再狭窄的作用,近年来受到心血管研究领域的极大关注,被广泛应用于药物支架涂层来预防血管成形术后再狭窄。同时,随着对血管外膜研究的深入,雷帕霉素对血管外膜的作用也开始受到越来越多的关注[8]。临床上大隐静脉桥的狭窄一般发生于移植后几周到几个月的时间内,对移植静脉长期的干预治疗才能有效地改善桥血管的狭窄[9]。因此我们建立大鼠自体静脉移植模型,在移植静脉局部包裹携带雷帕霉素的新型电纺丝缓释药膜,观察研究雷帕霉素缓释系统能否通过血管外周释放有效抑制自体移植静脉新内膜形成,从而改善移植静脉的中远期通畅率。
药物缓释载体是防治移植静脉的关键部分。目前用于制作药物缓释载体的材料多为人工合成的聚合物,如聚乙醇酸(PGA)及其共聚物PLGA,聚乳酸(PLA)等[10]。在实验过程中发现此类聚合物存在不少缺点,如亲水性差,细胞粘附力弱,产生酸性降解产物从而引起无菌性炎症等等。而可降解聚碳酸亚丙酯(PPC)是不释放酸性产物的新型聚合物,其最终降解产物是二氧化碳和水[11],最大程度地避免了药膜接触所引起的局部无菌炎症。并且PPC的亲水性和细胞粘附力明显优于PGA和PLA。电纺丝是一种制备高分子纳米／微米纤维的新兴方法,受到较多关注,由于电纺丝的直径为微米／纳米级[12],大大增加了药物释放面积,其微观结构多孔,方便能量和物质的跨膜交换。电纺丝可通过调整结晶性、共混比例、共聚组成及微观结构等来调节降解速度,从而有效控制药物缓释[13]。我们通过建立雷帕霉素电纺丝缓释药膜,以探讨其在防治自体移植静脉狭窄的可行性和中远期效果。
哺乳动物雷帕霉素靶蛋白(mTOR)作为雷帕霉素的主要作用靶点,是细胞生长和增殖的重要调节因子。mTOR信号及其表达产物的研究越来越受到人们的关注,大量研究显示mTOR信号途径的调控与细胞的生长与增殖密切相关[14].mTOR在TOR1和TOR2被发现后即被鉴定并克隆,其氨基酸序列高度保守,属于磷酯酰肌醇激酶相关蛋白家族(PI3K)家族的成员,这一家族中的成员与细胞的基本功能有关,如：细胞增殖、DNA损伤修复、细胞周期以及端粒长度维护等[15]。FAK和MMPs(尤其是MMP2和MMP9)与血管外膜成纤维细胞增殖、迁移、粘附功能密切相关,PI3K/Akt/mTOR细胞传导通路在成纤维细胞中的研究甚少[16]。研究雷帕霉素在此通路中的作用及能否调控FAK及MMPs的表达从而有效抑制成纤维细胞的增殖迁移,对移植静脉外膜炎症及桥血管狭窄的研究有重要意义。
目的：
建立大鼠颈静脉—腹主动脉自体血管移植模型,观察自体移植静脉狭窄病变,分析移植静脉狭窄与外膜成纤维细胞、外膜炎症的关联。利用新兴电纺丝技术制作雷帕霉素缓释药膜,将缓释药膜用于大鼠移植静脉外周,观察药膜对移植静脉狭窄及外膜炎症的抑制作用。以自体移植静脉的外膜成纤维细胞为研究靶点,从细胞水平分析外膜成纤维细胞增殖迁移对移植静脉狭窄的影响,并从分子水平探讨雷帕霉素通过外膜成纤维细胞的PI3K/Akt/mTOR细胞传导通路对FAK及MMPs表达的影响,从而防治移植静脉的狭窄。为临床冠状动脉旁路移植术中,在大隐静脉外周局部用药抑制术后静脉桥狭窄提供新的思路。
方法：
1、利用"Cuff"套管法制作Wistar大鼠颈静脉—腹主动脉自体血管移植模型,分别于术后1周、4周、8周用血管超声检测桥血管是否通畅,并测定桥血管搏动指数(PI)及阻力指数(RI),处死动物后移植静脉行HE染色,计算血管面积指数,血管外膜面积百分数及新生内膜面积百分比,并行免疫组化染色,分析移植静脉狭窄与外膜成纤维细胞、外膜炎症的关联。
2、利用新兴电纺丝技术,将PPC与雷帕霉素混匀后制成缓释药膜,共制三种药膜(低浓度药膜、高浓度药膜、空白对照药膜),用扫描电镜观察雷帕霉素缓释药膜的表面形态特征,将药膜完全溶解后用紫外分光光度法测定雷帕霉素的载药量和包封率。精确称取雷帕霉素缓释药膜,放入PBS中,置于37℃恒温水浴摇床中,在0.5d、1d、2d、4d、6d、8d、10d、12d、14d、16d、18d、20d、22d、24d、26d、28d后,检测残余药品中药物含量,得到雷帕霉素药物释放曲线。
3、重新建立大鼠自体血管移植模型,术中将雷帕霉素缓释药膜均匀紧密地缠绕于静脉桥外周,分为对照组、空白药膜组、低浓度药物药膜组、高浓度药物药膜组。于术后4周处死,处死后行HE染色,计算血管面积指数,新生内膜面积百分比及血管外膜面积百分比,观察雷帕霉素药膜对移植静脉狭窄及外膜炎症的抑制作用。并从细胞水平分析外膜成纤维细胞增殖迁移对移植静脉狭窄的影响。
4、取幼年大鼠腹主动脉外膜组织,剪碎后用培养瓶贴块法培养原代血管外膜成纤维细胞。传代成功后行细胞鉴定,MTT法检测雷帕霉素药膜对细胞活性的影响。细胞流式分析检测各组药膜对成纤维细胞周期的影响。用Transwell小室检测空白药膜及低、高浓度药膜对细胞迁移能力的影响。Western Blot检测药膜对外膜成纤维细胞FAK、pFAK、mTOR、pmTOR、MMP2、MMP9、ERK1/2、PCNA等表达含量的影响。分析雷帕霉素通过外膜成纤维细胞的PI3K/Akt/mTOR细胞传导通路对FAK及MMPs表达的影响。
结果：
1、静脉移植手术成功率90%,术后1周移植静脉通畅率100%,术后4周通畅率88.9%,术后8周通畅率77.8%。血管超声检查显示术后4周开始桥血管管壁明显增厚,术后4周及术后8周桥血管搏动指数PI及阻力指数RI均明显大于静脉对照组。大鼠处死后静脉桥HE染色显示,术后4周及术后8周静脉桥内膜增生明显。其管壁面积指数AI及外膜面积百分比均明显增高于静脉对照组。说明术后4周及8周桥血管阻力增加,血管外膜增厚伴新内膜形成,造成移植静脉的狭窄。免疫组化显示静脉桥形成的新内膜有大量vimentin和α-SMA表达阳性的细胞,说明在静脉移植术后,静脉外膜最先出现病理变化,外膜成纤维细胞增殖,转型为成肌纤维细胞,迁移至静脉内膜参与内膜增生,造成移植静脉狭窄的病理过程。
2、雷帕霉素/PPC缓释药膜的制作条件较为严格,必须是纺丝距离为15—20cnl,纺丝电压为10—15kV才能顺利流畅地进行电纺丝制作。通过扫描电镜观察,电纺丝纤维光滑均匀,交织成立体网格状,纤维平均直径为3μm。利用紫外分光光度法测得雷帕霉素在低浓度药膜和高浓度药膜中载药量分别为4.5%、10.3%,包封率分别为95.1%、90.6%。利用HPLC法测得雷帕霉素缓释药膜的释放曲线,5%雷帕霉素缓释药膜在前8天释放较快,释放41%,到28天释放91.5%。10%雷帕霉素缓释药膜在前6天释放较快,释放40%,到28天释放93%。是一种成功的雷帕霉素缓释系统。
3、将雷帕霉素缓释药膜用于动物模型,术后4周缓释药膜明显抑制了静脉桥新内膜的形成,内膜上成纤维细胞明显减少,移植静脉狭窄明显减轻,同时静脉外膜厚度亦明显变薄。免疫组化显示药膜明显降低血管外膜FAK、PCNA、mTOR、MMP2及MMP9的阳性表达百分比。
4、培养瓶组织贴块法行细胞培养时,于第7-9天开始组织块周围长出细胞,第10-12天细胞融合至80%左右,原代传代,细胞生长旺盛,2-4天即可再次传代。细胞免疫荧光检测vimentin为阳性表达,而α-SMA和desmin为阴性表达,可以鉴定为血管外膜成纤维细胞。扫描电镜观察发现在药膜上生长3天的VAF仍呈伸展状态,有的细胞进入电纺丝网格内,但在雷帕霉素药膜上的细胞伸展性明显差于空白PPC膜上的细胞。MTT检测提示PPC材料对成纤维细胞活性无影响,雷帕霉素药膜可以明显抑制成纤维细胞的活性。细胞流式分析显示雷帕霉素药膜明显抑制VAF由G1期进入S合成期。Transwell迁移分析显示低浓度及高浓度药膜均对成纤维细胞的迁移产生明显抑制作用。Western Blot分析示低浓度药膜和高浓度药膜均能明显抑制成纤维细胞mTOR、pmTOR、ERK1/2、pERK1/2的表达,说明雷帕霉素参与到成纤维细胞PI3K/Akt/mTOR细胞通路中,药膜同时抑制成纤维细胞FAK、pFAK、MMP2、MMP9的表达,减弱了细胞的粘附迁移功能。
结论：
1、利用“Cuff”套管法可成功制作大鼠颈静脉-腹主动脉自体血管移植模型。自体静脉移植术后最先出现移植静脉外膜的增厚及外膜成纤维细胞的增殖和向内膜迁移,成纤维细胞参与静脉桥新内膜的形成,从而造成移植静脉狭窄。
2、雷帕霉素缓释药膜可稳定释放药物,能持续释放28天甚或更长时间,对自体移植静脉狭窄及外膜炎症有明显抑制作用。
3、通过组织贴块法成功培养血管外膜成纤维细胞,雷帕霉素缓释药膜可明显抑制成纤维细胞的增殖和迁移,从而抑制移植静脉的狭窄。
4、雷帕霉素参与外膜成纤维细胞PI3K/Akt/mTOR细胞通路,且有效抑制成纤维细胞FAK及MMPs的表达,从而抑制移植静脉的狭窄。但是确切机制尚需进一步研究证实。
Background
Coronary Arterial Bypass Grafting is surgical therapeutic measurement for atherosclerosis of the coronary arteries, while graft stenosis is the main limiting factor for long-term survival of CAD patients. Observational studies on coronary heart diseases of the2012International authoritative magazine NEJM confirmed that patients who underwent CABG treatment had a longer survival than that who underwent PCI. In the clinical, transplantation materials mainly include internal mammary artery,great saphenous vein and radial arteries. Great saphenous vein has been the important materials of CABG because of the restrict of the internal mammary artery and radial artery. But high incidence of venous bridge stenosis after transplantation is a clinical problem needed to be resolved in Cardiovascular surgery. Lots of experiment research emphasizes on intervention treatment of transplant endothelial cells and middle smooth muscle cells,while there is only few researches to vascular adventitia which is the main wall structure of great saphenous vein. In recent years, the important function of vascular adventitia in graft stenosis has been taken more and more attention to. A study suggests that vascular adventitia has directly or indirectly important regulations on vascular structure and function. Fibroblasts are the main cell type of vascular adventitia, and more and more research confirmed that adventitia fibroblasts were not innocent bystanders but active participants in the process of graft lesions. Recently domestic research group found when veins was transplanted to arteries adventitia fibroblasts were firstly triggered by changes of vascular tension, then secreted the extracellular matrix (ECM), kinds of growth factors and cytokines, such as TGF β-1, MCP-1, MMPs, etc and induced adventitia fibroblasts to proliferate and transform to myofibroblast(MF), which migrated to media and intima,then participated in the formation of neointima. Rapamycin,also known as sirolimus,is a macrocyclic lactone antibiotic produced by Streptomyces hygroscopicus,ofen used as a potent immunosuppressive agent.Recent years rapamycin is paid much attention to in cardiovascular reserach and is commonly used in drug-coated stents to delay coronary arterial restenosis after PTCA for its known effects for graft stenosis.With more and more research on adventitial inflammation,the effects of rapamycin on adventitia are of ardent concern.In our study we established Wistar jugular vein-abdomainal artery allograft model,position rapamycin slow-releasing electrospin layer around allograft veins tightly to verify its efficiency for inhibition of allograft restenosis.
Most attention on electrospinning biodegradable polymers has focused on synthetic materials,such as PGA,PLGA,and PLA.However,these materials has some disadvantages,such as low hydrophilicity,low cellular adhesion and aseptic inflammation caused by their acid degradation products.Poly-propylene carbonate(PPC) is a degradable material formed by copolymerization of propylene oxide and carbon dioxide. Electrospinning is an attractive approach for the fabrication of fibrous biomaterials.Electrospun mats have larger specific surface areas and smaller pore size for polymer degradation and drug diffusion compared to other drug delivery systems.
The mammalian target of rapamycin(mTOR),known as target point of rapamycin, is a significant regulatory factor for cellular growth and proliferation.Thus mTOR and its expression products are of more and more concerns.The mTOR,which belongs to PIKK family and its amino acid sequence is highly conservative, owns PIKK's basic functions,such as proliferation,DNA repair,cellular cycle regulation and telomere preservation. mTOR was identified and cloned after that TOR1and TOR2was found. FAK and MMPs(especially MMP2and MMP9) were associated with proliferation,migration, and adhesion of vascular adventitial fibroblasts. The pathway of PI3K/Akt/mTOR was seldomly studied in adventitial fibroblasts.The study of rapamycin in this pathway which could regulate expression of FAK and MMPs was significant in graft adventitial inflammation and arteriovenous graft stenosis.
Purpose We establish Wistar jugular vein-abdomainal artery allograft model to observe adventitial inflammation and degree of arteriovenous graft stenosis.We make rapamycin-eluting electrospun film.observe the effects on adventitial fibroblasts,and position electrospun film around allograft veins tightly to verify its efficiency for inhibition of arteriovenous graft stenosis.To provide a new thought for clinical drug usage around arteriovenous grafts.
Methods
1. Wistar jugular vein-abdomainal artery allograft model was performed by the "Cuff" cannulation style.Echocardiography was performed to assess graft vein patency and to measure pulsatility index (PI) and resistance index(RI)1week,4weeks,8weeks posttransplantation respectively.HE staining was performed to graft veins harvested postoperation to measure vascular wall area index(AI) and adventitia area percentage,then the special cytokines were measured by immunohistochemisty(IHC).
2. We manufactured rapamycin slow-release layers(high dosage ones,low dosage ones and blank ones) with PPC and rapamycin mixed together by using newly electrospinning technology.The size, surface morphology and distribution of the fibers were measured by a scanning electron microscope after silver coating.Then we calculated encapsulation efficiency(EE) and drug loading rate by ultraviolet spectrophotometry.The drug layers were immersed into PBS buffer in tube incubated in37℃shaking water bath and rapamycin residued in layers was measured by HPLC analysis at each specified time intervals,0.5d,1d,2d,4d,6d,8d,10d,12d,14d,16d,18d,20d,22d,24d,26d,28dto get the time-cumulative drug releasing curve.
3. We cultivated primary adventitial fibroblasts with patched adventitia from abdominal arteries of young Wistar rats sticked to culture flask. When the cells were got successfully in the culture, we qualificated the cells by ICC using vimentin, a-SMA and desmin antibody. We detected the effects of rapamycin layers on cytoactivation of adventitial fibroblasts by MTT technology, while measured the effects on fibroblast migration by transwell method. Finally we measured the expression level of FAK、pFAK、mTOR、pmTOR、MMP2、MMP9、 PCNA in fibroblasts with rapamycin film in DMEM.
4. We positioned the rapamycin layers around graft veins tightly during operation and divided graft models into four groups (control group, blank layer group, low dosage layer group and high dosage layer group).The graft veins harvested4weeks postoperatively were performed HE staining and we measured vascular wall area index(AI) and adventitia area percentage to observe the therapeutical effects on graft stenosis. Finally the expression levels of FAK、ERK1/2、PCNA、mTOR、 pmTOR、MMP2、MMP9were calculated by IHC.
Results
1. The mortality of graft models was10%(6/60),while patency rates were100%1week,88.9%(16/18)4weeks,and77.8%(14/18)8weeks postoperatively respectively. Under echocardiography sonde, the obvious thickness of graft vascular wall were observed8weeks postoperatively. The PI (1.80±0.04)8weeks was obviously higher than PI(1.44±0.03)4weeks (P<0.05),PI(1.17±0.08)1week (P<0.05) and PI(0.24±0.02) control ones (P<0.05),while the PI(1.44±0.03)4weeks was obviously higher than PI(1.17±0.08)1week (P<1.05) and PI(0.24±0.02) control ones (P<0.05),the PI(1.17±0.08)1week was obviously higher than PI(0.24±0.02) control ones (P<0.05).Also, the RI (0.76±0.03)8weeks was obviously higher than RI(0.41±0.03)1week (P<0.05) and RI(0.28±0.02) control ones (P<0.05),while the RI(0.63±0.03)4weeks was obviously higher than RI(0.41±0.03)1week (P<0.05) and PI(0.28±0.02) control ones (P<0.05),but the RI(0.76±0.03)8weeks had no obvious contrast with the PI(0.63±0.03)4weeks. The graft veins harvested postoperatively were performed HE staining and we measured vascular wall area index(AI) and adventitia area percentage.The AI(30.15±9.85%) and adventitia area percentage (18.25±5.23%) in8weeks were both obviously higher than the AI(11.23±3.12%) and adventitia area percentage (8.12±2.05%) in control group(P<0.05), while the AI(23.35±7.12%) and adventitia area percentage (15.23±4.02%) in4weeks were both obviously higher than the AI(11.23±3.12%) and adventitia area percentage (8.12±2.05%) in control group(P<0.05). It indicated that the graft vessel wall, especially the adventitia, was thickened after4and8weeks postoperatively,and the vascular resistance was higher. The expression levels of FAK、vimentin、MMP2、MMP9in adventitia were all higher than those in control group.
2. Electrospun could be performed normally only when the distance was15-20cm and the voltage was10-15kV. SEM images showed that the surface of fibers was smooth and uniform, and, the diameter of the fiber was about3p.m.The loading rate and encapsulation efficiency was4.5%,95.1%in low dosage layer and10.3%,90.6%in high dosage layer. The time-cumulative drug releasing curve showed that rapamycin released41%at8th day and91%at28th day in5%RAPA/PPC film, while40%at6th day and93%at28th day in5%RAPA/PPC film.
3. We observed sporadic cells at7-91day and numerous cells at10-12th day which could transfer into new generations. Then we qualified the cells by ICC using vimentin,α-SMA and desmin antibody. The vimentin was positive while the α-SMA was negative, which indicated the cells cultured were adventitial fibroblasts but not smooth muscle cells. We detected the obvious inhibition of rapamycin layers on cytoactivation and migration of adventitial fibroblasts. Finally we measured the expression levels of FAK、pFAK、mTOR、pmTOR、 MMP2、MMP9、ERK1/2、PCNA in fibroblasts were all depressed by rapamycin layers in DMEM.
4. We positioned the rapamycin-eluting electrospun film around graft veins tightly during operation and the graft veins harvested4weeks postoperatively were performed HE staining. The AI(15.25±2.86%) and adventitia area percentage (9.21±2.03%) in low dosage layer group were both obviously lower than the AI(24.26±7.05%)(P<0.05)and adventitia area percentage (16.08±4.15%) in control group(P<0.05),while the same applied to high dosage layer group(AI13.44±2.55%, adventitia area percentage8.02±1.89%)(P<0.05). However, the high dosage group had no dominant advantage compared with low dosage group. Finally the expression levels of FAK、ERK1/2、PCNA、mTOR、 pmTOR、MMP2、MMP9were all depressed in high and low dosage layer groups.
Conclusions
1.Wistar jugular vein-abdominal artery allograft model performed by the "Cuff" annulation style was successful in researching graft stenosis. The graft vascular wall, especially the adventitia, was thickened after4and8weeks postoperatively, and the vascular resistance was higher, which was in accordance with pathological change of graft stenosis.
2.The electrospun rapamycin layer can release rapamycin steadily, and, can release rapamycin for28days or more. It has evident inhibition on adventitial inflammation and graft stenosis.
3.Adventitial fibroblasts were cultured in vitro successfully, and its proliferation and immigration were obviously depressed by rapamycin-eluting electrospun film, which then inhibited graft stenosis.
4.Rapamycin participated in PI3K/Akt/mTOR signaling pathway in adventitial fibroblasts. Moreover, rapamycin/PPC film cutdawn the expression of MMPs and FAK,the reason of which was unknown.
冠状动脉旁路移植术(Coronary Arterial Bypass Grafting, CABG)是冠状动脉粥样硬化性心脏病的重要临床治疗措施,2012年国际权威杂志NEJM的冠心病观察性研究证实患者接受CABG治疗可获得比PCI治疗更长的生存期[1]。临床上作为CABG移植材料的主要有乳内动脉、大隐静脉和桡动脉,由于乳内动脉和桡动脉取材的限制性,大隐静脉一直作为CABG的重要取材血管。但移植后大隐静脉桥狭窄发生率较高,成为心血管外科亟需解决的临床难题[2]。目前有关大隐静脉桥狭窄防治的实验研究集中在血管内皮细胞及中层平滑肌细胞的干预治疗上,而对于大隐静脉主要管壁结构——血管外膜的研究却极少。近年来,血管外膜在移植血管狭窄中的作用开始受到重视,有研究提示血管外膜对于血管结构和功能有着直接或间接的重要调控作用[3]。外膜成纤维细胞是血管外膜的主要细胞类型,越来越多的研究证实血管外膜成纤维细胞在移植血管病变过程中并非是无辜的旁观者,而是积极的参与者[4]。最近我们参与的课题组发现静脉移植于动脉后因血管张力发生改变首先启动移植静脉外膜的成纤维细胞,分泌细胞外基质(ECM)、各种生长因子、细胞因子,如TGF-β1、MCP-1、MMPs等,诱导外膜成纤维细胞增殖并转化为成肌纤维细胞(myofibroblast,MF)[5],向中膜层及内膜迁移,参与新内膜的形成。而目前国内外尚无以外膜成纤维细胞为靶点,防治自体移植静脉狭窄的研究。
雷帕霉素(Rapamycin),又称西罗莫司,是一种亲脂性三烯含氮的大环内酯抗生素类免疫抑制剂,具有很好的抗排斥作用,被广泛应用于器官移植[6,7]。由于它具有极好的抑制血管再狭窄的作用,近年来受到心血管研究领域的极大关注,被广泛应用于药物支架涂层来预防血管成形术后再狭窄。同时,随着对血管外膜研究的深入,雷帕霉素对血管外膜的作用也开始受到越来越多的关注[8]。临床上大隐静脉桥的狭窄一般发生于移植后几周到几个月的时间内,对移植静脉长期的干预治疗才能有效地改善桥血管的狭窄[9]。因此我们建立大鼠自体静脉移植模型,在移植静脉局部包裹携带雷帕霉素的新型电纺丝缓释药膜,观察研究雷帕霉素缓释系统能否通过血管外周释放有效抑制自体移植静脉新内膜形成,从而改善移植静脉的中远期通畅率。
药物缓释载体是防治移植静脉的关键部分。目前用于制作药物缓释载体的材料多为人工合成的聚合物,如聚乙醇酸(PGA)及其共聚物PLGA,聚乳酸(PLA)等[10]。在实验过程中发现此类聚合物存在不少缺点,如亲水性差,细胞粘附力弱,产生酸性降解产物从而引起无菌性炎症等等。而可降解聚碳酸亚丙酯(PPC)是不释放酸性产物的新型聚合物,其最终降解产物是二氧化碳和水[11],最大程度地避免了药膜接触所引起的局部无菌炎症。并且PPC的亲水性和细胞粘附力明显优于PGA和PLA。电纺丝是一种制备高分子纳米／微米纤维的新兴方法,受到较多关注,由于电纺丝的直径为微米／纳米级[12],大大增加了药物释放面积,其微观结构多孔,方便能量和物质的跨膜交换。电纺丝可通过调整结晶性、共混比例、共聚组成及微观结构等来调节降解速度,从而有效控制药物缓释[13]。我们通过建立雷帕霉素电纺丝缓释药膜,以探讨其在防治自体移植静脉狭窄的可行性和中远期效果。
哺乳动物雷帕霉素靶蛋白(mTOR)作为雷帕霉素的主要作用靶点,是细胞生长和增殖的重要调节因子。mTOR信号及其表达产物的研究越来越受到人们的关注,大量研究显示mTOR信号途径的调控与细胞的生长与增殖密切相关[14].mTOR在TOR1和TOR2被发现后即被鉴定并克隆,其氨基酸序列高度保守,属于磷酯酰肌醇激酶相关蛋白家族(PI3K)家族的成员,这一家族中的成员与细胞的基本功能有关,如：细胞增殖、DNA损伤修复、细胞周期以及端粒长度维护等[15]。FAK和MMPs(尤其是MMP2和MMP9)与血管外膜成纤维细胞增殖、迁移、粘附功能密切相关,PI3K/Akt/mTOR细胞传导通路在成纤维细胞中的研究甚少[16]。研究雷帕霉素在此通路中的作用及能否调控FAK及MMPs的表达从而有效抑制成纤维细胞的增殖迁移,对移植静脉外膜炎症及桥血管狭窄的研究有重要意义。
目的：
建立大鼠颈静脉—腹主动脉自体血管移植模型,观察自体移植静脉狭窄病变,分析移植静脉狭窄与外膜成纤维细胞、外膜炎症的关联。利用新兴电纺丝技术制作雷帕霉素缓释药膜,将缓释药膜用于大鼠移植静脉外周,观察药膜对移植静脉狭窄及外膜炎症的抑制作用。以自体移植静脉的外膜成纤维细胞为研究靶点,从细胞水平分析外膜成纤维细胞增殖迁移对移植静脉狭窄的影响,并从分子水平探讨雷帕霉素通过外膜成纤维细胞的PI3K/Akt/mTOR细胞传导通路对FAK及MMPs表达的影响,从而防治移植静脉的狭窄。为临床冠状动脉旁路移植术中,在大隐静脉外周局部用药抑制术后静脉桥狭窄提供新的思路。
方法：
1、利用"Cuff"套管法制作Wistar大鼠颈静脉—腹主动脉自体血管移植模型,分别于术后1周、4周、8周用血管超声检测桥血管是否通畅,并测定桥血管搏动指数(PI)及阻力指数(RI),处死动物后移植静脉行HE染色,计算血管面积指数,血管外膜面积百分数及新生内膜面积百分比,并行免疫组化染色,分析移植静脉狭窄与外膜成纤维细胞、外膜炎症的关联。
2、利用新兴电纺丝技术,将PPC与雷帕霉素混匀后制成缓释药膜,共制三种药膜(低浓度药膜、高浓度药膜、空白对照药膜),用扫描电镜观察雷帕霉素缓释药膜的表面形态特征,将药膜完全溶解后用紫外分光光度法测定雷帕霉素的载药量和包封率。精确称取雷帕霉素缓释药膜,放入PBS中,置于37℃恒温水浴摇床中,在0.5d、1d、2d、4d、6d、8d、10d、12d、14d、16d、18d、20d、22d、24d、26d、28d后,检测残余药品中药物含量,得到雷帕霉素药物释放曲线。
3、重新建立大鼠自体血管移植模型,术中将雷帕霉素缓释药膜均匀紧密地缠绕于静脉桥外周,分为对照组、空白药膜组、低浓度药物药膜组、高浓度药物药膜组。于术后4周处死,处死后行HE染色,计算血管面积指数,新生内膜面积百分比及血管外膜面积百分比,观察雷帕霉素药膜对移植静脉狭窄及外膜炎症的抑制作用。并从细胞水平分析外膜成纤维细胞增殖迁移对移植静脉狭窄的影响。
4、取幼年大鼠腹主动脉外膜组织,剪碎后用培养瓶贴块法培养原代血管外膜成纤维细胞。传代成功后行细胞鉴定,MTT法检测雷帕霉素药膜对细胞活性的影响。细胞流式分析检测各组药膜对成纤维细胞周期的影响。用Transwell小室检测空白药膜及低、高浓度药膜对细胞迁移能力的影响。Western Blot检测药膜对外膜成纤维细胞FAK、pFAK、mTOR、pmTOR、MMP2、MMP9、ERK1/2、PCNA等表达含量的影响。分析雷帕霉素通过外膜成纤维细胞的PI3K/Akt/mTOR细胞传导通路对FAK及MMPs表达的影响。
结果：
1、静脉移植手术成功率90%,术后1周移植静脉通畅率100%,术后4周通畅率88.9%,术后8周通畅率77.8%。血管超声检查显示术后4周开始桥血管管壁明显增厚,术后4周及术后8周桥血管搏动指数PI及阻力指数RI均明显大于静脉对照组。大鼠处死后静脉桥HE染色显示,术后4周及术后8周静脉桥内膜增生明显。其管壁面积指数AI及外膜面积百分比均明显增高于静脉对照组。说明术后4周及8周桥血管阻力增加,血管外膜增厚伴新内膜形成,造成移植静脉的狭窄。免疫组化显示静脉桥形成的新内膜有大量vimentin和α-SMA表达阳性的细胞,说明在静脉移植术后,静脉外膜最先出现病理变化,外膜成纤维细胞增殖,转型为成肌纤维细胞,迁移至静脉内膜参与内膜增生,造成移植静脉狭窄的病理过程。
2、雷帕霉素/PPC缓释药膜的制作条件较为严格,必须是纺丝距离为15—20cnl,纺丝电压为10—15kV才能顺利流畅地进行电纺丝制作。通过扫描电镜观察,电纺丝纤维光滑均匀,交织成立体网格状,纤维平均直径为3μm。利用紫外分光光度法测得雷帕霉素在低浓度药膜和高浓度药膜中载药量分别为4.5%、10.3%,包封率分别为95.1%、90.6%。利用HPLC法测得雷帕霉素缓释药膜的释放曲线,5%雷帕霉素缓释药膜在前8天释放较快,释放41%,到28天释放91.5%。10%雷帕霉素缓释药膜在前6天释放较快,释放40%,到28天释放93%。是一种成功的雷帕霉素缓释系统。
3、将雷帕霉素缓释药膜用于动物模型,术后4周缓释药膜明显抑制了静脉桥新内膜的形成,内膜上成纤维细胞明显减少,移植静脉狭窄明显减轻,同时静脉外膜厚度亦明显变薄。免疫组化显示药膜明显降低血管外膜FAK、PCNA、mTOR、MMP2及MMP9的阳性表达百分比。
4、培养瓶组织贴块法行细胞培养时,于第7-9天开始组织块周围长出细胞,第10-12天细胞融合至80%左右,原代传代,细胞生长旺盛,2-4天即可再次传代。细胞免疫荧光检测vimentin为阳性表达,而α-SMA和desmin为阴性表达,可以鉴定为血管外膜成纤维细胞。扫描电镜观察发现在药膜上生长3天的VAF仍呈伸展状态,有的细胞进入电纺丝网格内,但在雷帕霉素药膜上的细胞伸展性明显差于空白PPC膜上的细胞。MTT检测提示PPC材料对成纤维细胞活性无影响,雷帕霉素药膜可以明显抑制成纤维细胞的活性。细胞流式分析显示雷帕霉素药膜明显抑制VAF由G1期进入S合成期。Transwell迁移分析显示低浓度及高浓度药膜均对成纤维细胞的迁移产生明显抑制作用。Western Blot分析示低浓度药膜和高浓度药膜均能明显抑制成纤维细胞mTOR、pmTOR、ERK1/2、pERK1/2的表达,说明雷帕霉素参与到成纤维细胞PI3K/Akt/mTOR细胞通路中,药膜同时抑制成纤维细胞FAK、pFAK、MMP2、MMP9的表达,减弱了细胞的粘附迁移功能。
结论：
1、利用“Cuff”套管法可成功制作大鼠颈静脉-腹主动脉自体血管移植模型。自体静脉移植术后最先出现移植静脉外膜的增厚及外膜成纤维细胞的增殖和向内膜迁移,成纤维细胞参与静脉桥新内膜的形成,从而造成移植静脉狭窄。
2、雷帕霉素缓释药膜可稳定释放药物,能持续释放28天甚或更长时间,对自体移植静脉狭窄及外膜炎症有明显抑制作用。
3、通过组织贴块法成功培养血管外膜成纤维细胞,雷帕霉素缓释药膜可明显抑制成纤维细胞的增殖和迁移,从而抑制移植静脉的狭窄。
4、雷帕霉素参与外膜成纤维细胞PI3K/Akt/mTOR细胞通路,且有效抑制成纤维细胞FAK及MMPs的表达,从而抑制移植静脉的狭窄。但是确切机制尚需进一步研究证实。
Background
Coronary Arterial Bypass Grafting is surgical therapeutic measurement for atherosclerosis of the coronary arteries, while graft stenosis is the main limiting factor for long-term survival of CAD patients. Observational studies on coronary heart diseases of the2012International authoritative magazine NEJM confirmed that patients who underwent CABG treatment had a longer survival than that who underwent PCI. In the clinical, transplantation materials mainly include internal mammary artery,great saphenous vein and radial arteries. Great saphenous vein has been the important materials of CABG because of the restrict of the internal mammary artery and radial artery. But high incidence of venous bridge stenosis after transplantation is a clinical problem needed to be resolved in Cardiovascular surgery. Lots of experiment research emphasizes on intervention treatment of transplant endothelial cells and middle smooth muscle cells,while there is only few researches to vascular adventitia which is the main wall structure of great saphenous vein. In recent years, the important function of vascular adventitia in graft stenosis has been taken more and more attention to. A study suggests that vascular adventitia has directly or indirectly important regulations on vascular structure and function. Fibroblasts are the main cell type of vascular adventitia, and more and more research confirmed that adventitia fibroblasts were not innocent bystanders but active participants in the process of graft lesions. Recently domestic research group found when veins was transplanted to arteries adventitia fibroblasts were firstly triggered by changes of vascular tension, then secreted the extracellular matrix (ECM), kinds of growth factors and cytokines, such as TGF β-1, MCP-1, MMPs, etc and induced adventitia fibroblasts to proliferate and transform to myofibroblast(MF), which migrated to media and intima,then participated in the formation of neointima. Rapamycin,also known as sirolimus,is a macrocyclic lactone antibiotic produced by Streptomyces hygroscopicus,ofen used as a potent immunosuppressive agent.Recent years rapamycin is paid much attention to in cardiovascular reserach and is commonly used in drug-coated stents to delay coronary arterial restenosis after PTCA for its known effects for graft stenosis.With more and more research on adventitial inflammation,the effects of rapamycin on adventitia are of ardent concern.In our study we established Wistar jugular vein-abdomainal artery allograft model,position rapamycin slow-releasing electrospin layer around allograft veins tightly to verify its efficiency for inhibition of allograft restenosis.
Most attention on electrospinning biodegradable polymers has focused on synthetic materials,such as PGA,PLGA,and PLA.However,these materials has some disadvantages,such as low hydrophilicity,low cellular adhesion and aseptic inflammation caused by their acid degradation products.Poly-propylene carbonate(PPC) is a degradable material formed by copolymerization of propylene oxide and carbon dioxide. Electrospinning is an attractive approach for the fabrication of fibrous biomaterials.Electrospun mats have larger specific surface areas and smaller pore size for polymer degradation and drug diffusion compared to other drug delivery systems.
The mammalian target of rapamycin(mTOR),known as target point of rapamycin, is a significant regulatory factor for cellular growth and proliferation.Thus mTOR and its expression products are of more and more concerns.The mTOR,which belongs to PIKK family and its amino acid sequence is highly conservative, owns PIKK's basic functions,such as proliferation,DNA repair,cellular cycle regulation and telomere preservation. mTOR was identified and cloned after that TOR1and TOR2was found. FAK and MMPs(especially MMP2and MMP9) were associated with proliferation,migration, and adhesion of vascular adventitial fibroblasts. The pathway of PI3K/Akt/mTOR was seldomly studied in adventitial fibroblasts.The study of rapamycin in this pathway which could regulate expression of FAK and MMPs was significant in graft adventitial inflammation and arteriovenous graft stenosis.
Purpose We establish Wistar jugular vein-abdomainal artery allograft model to observe adventitial inflammation and degree of arteriovenous graft stenosis.We make rapamycin-eluting electrospun film.observe the effects on adventitial fibroblasts,and position electrospun film around allograft veins tightly to verify its efficiency for inhibition of arteriovenous graft stenosis.To provide a new thought for clinical drug usage around arteriovenous grafts.
Methods
1. Wistar jugular vein-abdomainal artery allograft model was performed by the "Cuff" cannulation style.Echocardiography was performed to assess graft vein patency and to measure pulsatility index (PI) and resistance index(RI)1week,4weeks,8weeks posttransplantation respectively.HE staining was performed to graft veins harvested postoperation to measure vascular wall area index(AI) and adventitia area percentage,then the special cytokines were measured by immunohistochemisty(IHC).
2. We manufactured rapamycin slow-release layers(high dosage ones,low dosage ones and blank ones) with PPC and rapamycin mixed together by using newly electrospinning technology.The size, surface morphology and distribution of the fibers were measured by a scanning electron microscope after silver coating.Then we calculated encapsulation efficiency(EE) and drug loading rate by ultraviolet spectrophotometry.The drug layers were immersed into PBS buffer in tube incubated in37℃shaking water bath and rapamycin residued in layers was measured by HPLC analysis at each specified time intervals,0.5d,1d,2d,4d,6d,8d,10d,12d,14d,16d,18d,20d,22d,24d,26d,28dto get the time-cumulative drug releasing curve.
3. We cultivated primary adventitial fibroblasts with patched adventitia from abdominal arteries of young Wistar rats sticked to culture flask. When the cells were got successfully in the culture, we qualificated the cells by ICC using vimentin, a-SMA and desmin antibody. We detected the effects of rapamycin layers on cytoactivation of adventitial fibroblasts by MTT technology, while measured the effects on fibroblast migration by transwell method. Finally we measured the expression level of FAK、pFAK、mTOR、pmTOR、MMP2、MMP9、 PCNA in fibroblasts with rapamycin film in DMEM.
4. We positioned the rapamycin layers around graft veins tightly during operation and divided graft models into four groups (control group, blank layer group, low dosage layer group and high dosage layer group).The graft veins harvested4weeks postoperatively were performed HE staining and we measured vascular wall area index(AI) and adventitia area percentage to observe the therapeutical effects on graft stenosis. Finally the expression levels of FAK、ERK1/2、PCNA、mTOR、 pmTOR、MMP2、MMP9were calculated by IHC.
Results
1. The mortality of graft models was10%(6/60),while patency rates were100%1week,88.9%(16/18)4weeks,and77.8%(14/18)8weeks postoperatively respectively. Under echocardiography sonde, the obvious thickness of graft vascular wall were observed8weeks postoperatively. The PI (1.80±0.04)8weeks was obviously higher than PI(1.44±0.03)4weeks (P<0.05),PI(1.17±0.08)1week (P<0.05) and PI(0.24±0.02) control ones (P<0.05),while the PI(1.44±0.03)4weeks was obviously higher than PI(1.17±0.08)1week (P<1.05) and PI(0.24±0.02) control ones (P<0.05),the PI(1.17±0.08)1week was obviously higher than PI(0.24±0.02) control ones (P<0.05).Also, the RI (0.76±0.03)8weeks was obviously higher than RI(0.41±0.03)1week (P<0.05) and RI(0.28±0.02) control ones (P<0.05),while the RI(0.63±0.03)4weeks was obviously higher than RI(0.41±0.03)1week (P<0.05) and PI(0.28±0.02) control ones (P<0.05),but the RI(0.76±0.03)8weeks had no obvious contrast with the PI(0.63±0.03)4weeks. The graft veins harvested postoperatively were performed HE staining and we measured vascular wall area index(AI) and adventitia area percentage.The AI(30.15±9.85%) and adventitia area percentage (18.25±5.23%) in8weeks were both obviously higher than the AI(11.23±3.12%) and adventitia area percentage (8.12±2.05%) in control group(P<0.05), while the AI(23.35±7.12%) and adventitia area percentage (15.23±4.02%) in4weeks were both obviously higher than the AI(11.23±3.12%) and adventitia area percentage (8.12±2.05%) in control group(P<0.05). It indicated that the graft vessel wall, especially the adventitia, was thickened after4and8weeks postoperatively,and the vascular resistance was higher. The expression levels of FAK、vimentin、MMP2、MMP9in adventitia were all higher than those in control group.
2. Electrospun could be performed normally only when the distance was15-20cm and the voltage was10-15kV. SEM images showed that the surface of fibers was smooth and uniform, and, the diameter of the fiber was about3p.m.The loading rate and encapsulation efficiency was4.5%,95.1%in low dosage layer and10.3%,90.6%in high dosage layer. The time-cumulative drug releasing curve showed that rapamycin released41%at8th day and91%at28th day in5%RAPA/PPC film, while40%at6th day and93%at28th day in5%RAPA/PPC film.
3. We observed sporadic cells at7-91day and numerous cells at10-12th day which could transfer into new generations. Then we qualified the cells by ICC using vimentin,α-SMA and desmin antibody. The vimentin was positive while the α-SMA was negative, which indicated the cells cultured were adventitial fibroblasts but not smooth muscle cells. We detected the obvious inhibition of rapamycin layers on cytoactivation and migration of adventitial fibroblasts. Finally we measured the expression levels of FAK、pFAK、mTOR、pmTOR、 MMP2、MMP9、ERK1/2、PCNA in fibroblasts were all depressed by rapamycin layers in DMEM.
4. We positioned the rapamycin-eluting electrospun film around graft veins tightly during operation and the graft veins harvested4weeks postoperatively were performed HE staining. The AI(15.25±2.86%) and adventitia area percentage (9.21±2.03%) in low dosage layer group were both obviously lower than the AI(24.26±7.05%)(P<0.05)and adventitia area percentage (16.08±4.15%) in control group(P<0.05),while the same applied to high dosage layer group(AI13.44±2.55%, adventitia area percentage8.02±1.89%)(P<0.05). However, the high dosage group had no dominant advantage compared with low dosage group. Finally the expression levels of FAK、ERK1/2、PCNA、mTOR、 pmTOR、MMP2、MMP9were all depressed in high and low dosage layer groups.
Conclusions
1.Wistar jugular vein-abdominal artery allograft model performed by the "Cuff" annulation style was successful in researching graft stenosis. The graft vascular wall, especially the adventitia, was thickened after4and8weeks postoperatively, and the vascular resistance was higher, which was in accordance with pathological change of graft stenosis.
2.The electrospun rapamycin layer can release rapamycin steadily, and, can release rapamycin for28days or more. It has evident inhibition on adventitial inflammation and graft stenosis.
3.Adventitial fibroblasts were cultured in vitro successfully, and its proliferation and immigration were obviously depressed by rapamycin-eluting electrospun film, which then inhibited graft stenosis.
4.Rapamycin participated in PI3K/Akt/mTOR signaling pathway in adventitial fibroblasts. Moreover, rapamycin/PPC film cutdawn the expression of MMPs and FAK,the reason of which was unknown.
引文
1. Weintraub, W. S., Grau-Sepulveda, M. V., Weiss, J. M., O'Brien, S. M, Peterson, E. D., Kolm, P., Zhang, Z., Klein, L. W., Shaw, R. E., McKay, C., Ritzenthaler, L. L Popma, J. J., Messenger, J. C., Shahian, D. M., Grover, F. L., Mayer, J. E., Shewan, C. M., Garratt, K. N., Moussa, I. D., Dangas, G. D. and Edwards, F. H. Comparative Effectiveness of Revascularization Strategies. N Engl J Med(Mar 27 2012).
2.李海清,刘志勇.自体移植静脉再狭窄的机制研究及防治进展.现代医学,2006,2(34)：65-67.
3. De Leon, H., Ollerenshaw, J. D., Griendling, K. K. and Wilcox, J. N. Adventitial cells do not contribute to neointimal mass after balloon angioplasty of the rat common carotid artery. Circulation,104,14 (Oct 2 2001),1591-1593.
4.胡维诚.外膜是血管病变的积极参与者.中国动脉硬化杂志,2006,14(1)：1-3.
5.郭淑杰,吴凌云,沈伟利等.血管外膜肌成纤维细胞分化的基因表达谱.ActaPhysiologica Sinica2006,58(4):337-344.
6.Cattaneo, D., Merlini, S., Pellegrino, M., Carrara, F., Zenoni, S., Murgia, S., Baldelli, S., Gaspari, F., Remuzzi, G. and Perico, N. Therapeutic drug monitoring of sirolimus: effect of concomitant immunosuppressive therapy and optimization of drug dosing. Am J Transplant,4,8 (Aug 2004),1345-1351.
7. Blatt, J., Stavas, J., Moats-Staats, B., Woosley, J. and Morrell, D. S. Treatment of childhood kaposiform hemangioendothelioma with sirolimus. Pediatr Blood Cancer, 55,7 (Dec 15 2010),1396-1398.
8. Rajathurai, T., Rizvi, S. I., Lin, H., Angelini, G. D., Newby, A. C. and Murphy, G. J. Periadventitial rapamycin-eluting microbeads promote vein graft disease in long-term pig vein-into-artery interposition grafts. Circ Cardiovasc Interv,3,2 (Apr 2010), 157-165.
9. Ishiwata, S., Verheye, S., Robinson, K. A., Salame, M. Y., de Leon, H., King, S. B., 3rd and Chronos, N. A. Inhibition of neointima formation by tranilast in pig coronary arteries after balloon angioplasty and stent implantation. J Am Coll Cardiol,35,5 (Apr 2000),1331-1337.
10. Wang, X. L., Du, F. G., Jiao, J., Meng, Y. Z. and Li, R. K. Preparation and properties of biodegradable polymeric blends from poly(propylene carbonate) and poly(ethylene-co-vinyl alcohol). J Biomed Mater Res B Appl Biomater,83,2 (Nov 2007),373-379.
11. Li, Y. and Shimizu, H. Compatibilization by homopolymer:significant improvements in the modulus and tensile strength of PPC/PMMA blends by the addition of a small amount of PVAc. ACS Appl Mater Interfaces,1,8 (Aug 2009), 1650-1655.
12. Wang, W., Itoh, S., Konno, K., Kikkawa, T., Ichinose, S., Sakai, K., Ohkuma, T. and Watabe, K. Effects of Schwann cell alignment along the oriented electrospun chitosan nanofibers on nerve regeneration. J Biomed Mater Res A,91,4 (Dec 15 2009),994-1005.
13. Prabhakaran, M. P., Venugopal, J. R. and Ramakrishna, S. Mesenchymal stem cell differentiation to neuronal cells on electrospun nanofibrous substrates for nerve tissue engineering. Biomaterials,30,28 (Oct 2009),4996-5003.
14. Acosta-Jaquez, H. A., Keller, J. A., Foster, K. G., Ekim, B., Soliman, G. A., Feener, E. P., Ballif, B. A. and Fingar, D. C. Site-specific mTOR phosphorylation promotes mTORC1-mediated signaling and cell growth. Mol Cell Biol,29,15 (Aug 2009), 4308-4324.
15. Moorman, N. J. and Shenk, T. Rapamycin-resistant mTORC1 kinase activity is required for herpesvirus replication. J Virol,84,10 (May 2010),5260-5269.
16. Zaborowska, I. and Walsh, D. PI3K signaling regulates rapamycin-insensitive translation initiation complex formation in vaccinia virus-infected cells. J Virol,83,8 (Apr 2009),3988-3992.
17. Hannan, E. L., Racz, M. J., Walford, G., Jones, R. H., Ryan, T. J., Bennett, E., Culliford, A. T., Isom, O. W., Gold, J. P. and Rose, E. A. Long-term outcomes of coronary-artery bypass grafting versus stent implantation. N Engl J Med,352,21 (May 26 2005),2174-2183.
18. Montalescot, G., White, H. D., Gallo, R., Cohen, M., Steg, P. G., Aylward, P. E., Bode, C., Chiariello, M., King, S. B.,3rd, Harrington, R. A., Desmet, W. J., Macaya, C. and Steinhubl, S. R. Enoxaparin versus unfractionated heparin in elective percutaneous coronary intervention. N Engl J Med,355,10 (Sep 7 2006),1006-1017.
19. Park, S. J., Kim, Y. H., Park, D. W., Yun, S. C., Ahn, J. M., Song, H. G., Lee, J. Y, Kim, W. J., Kang, S. J., Lee, S. W., Lee, C. W., Park, S. W., Chung, C. H., Lee, J. W., Lim, D. S., Rha, S. W., Lee, S. G., Gwon, H. C., Kim, H. S., Chae, I. H., Jang, Y, Jeong, M. H., Tahk, S. J. and Seung, K. B. Randomized trial of stents versus bypass surgery for left main coronary artery disease. N Engl J Med,364,18 (May 5 2011), 1718-1727.
20. Mauri, L. Why We Still Need Randomized Trials to Compare Effectiveness. N Engl J Med(Mar 27 2012).
21. Serruys, P. W., Morice, M. C., Kappetein, A. P., Colombo, A., Holmes, D. R., Mack, M. J., Stahle, E., Feldman, T. E., van den Brand, M., Bass, E. J., Van Dyck, N., Leadley, K., Dawkins, K. D. and Mohr, F. W. Percutaneous coronary intervention versus coronary-artery bypass grafting for severe coronary artery disease. N Engl J Med,360,10 (Mar 5 2009),961-972.
22. Petry, J. J., Burstein, S., Chang, W. H., Wortham, K., Sedor, C. and Hunter, S. A. Prostacyclin production by vein grafts in the arterial circulation:a study in rats. Prostaglandins Leukot Med,9,5 (Nov 1982),511-516.
23. Westerband, A., Crouse, D., Richter, L. C., Aguirre, M. L., Wixon, C. C., James, D. C., Mills, J. L., Hunter, G. C. and Heimark, R. L. Vein adaptation to arterialization in an experimental model. J Vasc Surg,33,3 (Mar 2001),561-569.
24. Muller, V., Ott, R., Tannapfel, A., Hohenberger, W. and Reck, T. Arterialization of the portal vein in liver transplantation:a new microsurgical model in the rat. Transplantation,71,7 (Apr 15 2001),977-981.
25. Borin, T. F., Miyakawa, A. A., Cardoso, L., de Figueiredo Borges, L., Goncalves, G. A. and Krieger, J. E. Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21(cip1) in vascular remodelling during vein arterialization in the rat. Int J Exp Pathol,90,3 (Jun 2009),328-337.
26. Kelly, B. S., Heffelfinger, S. C., Whiting, J. F., Miller, M. A., Reaves, A., Armstrong, J., Narayana, A. and Roy-Chaudhury, P. Aggressive venous neointimal hyperplasia in a pig model of arteriovenous graft stenosis. Kidney Int,62,6 (Dec 2002),2272-2280.
27. Swedberg, S. H., Brown, B. G., Sigley, R., Wight, T. N., Gordon, D. and Nicholls, S. C. Intimal fibromuscular hyperplasia at the venous anastomosis of PTFE grafts in hemodialysis patients. Clinical, immunocytochemical, light and electron microscopic assessment. Circulation,80,6 (Dec 1989),1726-1736.
28. Tennant, M. and McGeachie, J. K. Adaptive remodelling of smooth muscle in the neo-intima of vein-to-artery grafts in rats:a detailed morphometric analysis. Anat Embryol (Berl),187,2 (Feb 1993),161-166.
29. Davies, M. G., Owens, E. L., Mason, D. P., Lea, H., Tran, P. K., Vergel, S., Hawkins, S. A., Hart, C. E. and Clowes, A. W. Effect of platelet-derived growth factor receptor-alpha and -beta blockade on flow-induced neointimal formation in endothelialized baboon vascular grafts. Circ Res,86,7 (Apr 14 2000),779-786.
30. Graaf, R., Tintu, A., Stassen, F., Kloppenburg, G, Bruggeman, C. and Rouwet, E. N-acetylcysteine prevents neointima formation in experimental venous bypass grafts. Br J Surg,96,8 (Aug 2009),941-950.
31. Karacagil, S., Narbani, A., Almgren, B., Bowald, S. and Bergqvist, D. Modified vein cuff technique for distal polytetrafluoroethylene graft anastomoses:how we do it. Eur J Surg,161,1 (Jan 1995),47-48.
32. Misra, S., Fu, A. A., Puggioni, A., Karimi, K. M., Mandrekar, J. N., Glockner, J. F., Juncos, L. A., Anwer, B., McGuire, A. M. and Mukhopadhyay, D. Increased shear stress with upregulation of VEGF-A and its receptors and MMP-2, MMP-9, and TIMP-1 in venous stenosis of hemodialysis grafts. Am J Physiol Heart Circ Physiol, 294,5 (May 2008), H2219-2230.
33. de Paula, C. A., Coulson-Thomas, V. J., Ferreira, J. G, Maza, P. K., Suzuki, E., Nakahata, A. M., Nader, H. B., Sampaio, M. U. and Oliva, M. L. Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways. J Biol Chem,287,1 (Jan 2 2012),170-182.
34. Huang, S., Bjornsti, M. A. and Houghton, P. J. Rapamycins:mechanism of action and cellular resistance. Cancer Biol Ther,2,3 (May-Jun 2003),222-232.
35. Kawatsu, S., Oda, K., Saiki, Y., Tabata, Y. and Tabayashi, K. External application of rapamycin-eluting film at anastomotic sites inhibits neointimal hyperplasia in a canine model. Ann Thorac Surg,84,2 (Aug 2007),560-567; discussion 567.
36. Zou, J., Zhang, X., Yang, H., Zhu, Y., Ma, H. and Wang, S. Rapamycin-loaded nanoparticles for inhibition of neointimal hyperplasia in experimental vein grafts. J Cardiothorac Surg,62011),69.
37. Peterson, R. T., Beal, P. A., Comb, M. J. and Schreiber, S. L. FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions. J Biol Chem,275,10 (Mar 10 2000), 7416-7423.
38. Mothe-Satney, I., Brunn, G J., McMahon, L. P., Capaldo, C. T., Abraham, R. T. and Lawrence, J. C., Jr. Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-specific antibodies. J Biol Chem, 275,43 (Oct 27 2000),33836-33843.
39. Peterson, R. T., Desai, B. N., Hardwick, J. S. and Schreiber, S. L. Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. Proc Natl Acad Sci U S A,96,8 (Apr 13 1999), 4438-4442.
40. Lohmann, C. H., Schwartz, Z., Niederauer, G. G., Carries, D. L., Jr., Dean, D. D. and Boyan, B. D. Pretreatment with platelet derived growth factor-BB modulates the ability of costochondral resting zone chondrocytes incorporated into PLA/PGA scaffolds to form new cartilage in vivo. Biomaterials,21,1 (Jan 2000),49-61.
41. Telemeco. T. A., Ayres, C., Bowlin, G. L., Wnek, G. E., Boland, E. D., Cohen, N., Baumgarten, C. M., Mathews, J. and Simpson, D. G. Regulation of cellular infiltration into tissue engineering scaffolds composed of submicron diameter fibrils produced by electrospinning. Acta Biomater,1,4 (Jul 2005),377-385.
42. Shen, Y., Qian, Y., Zhang, H., Zuo, B., Lu, Z., Fan, Z., Zhang, P., Zhang, F. and Zhou, C. Guidance of olfactory ensheathing cell growth and migration on electrospun silk fibroin scaffolds. Cell Transplant,19,2 2010),147-157.
43. Qiu, X. F., Dong, N. G, Sun, Z. Q., Hu, P., Su, W. and Shi, J. W. [Controlled release of siRNA nanoparticles loaded in a novel external stent prepared by emulsion electrospinning attenuates neointima hyperplasia in vein grafts]. Zhonghua Yi Xue Za Zhi,89,41 (Nov 10 2009),2938-2942.
44. Liu, K., Cao, G., Zhang, X., Liu, R., Zou, W. and Wu, S. Pretreatment with intraluminal rapamycin nanoparticle perfusion inhibits neointimal hyperplasia in a rabbit vein graft model. Int JNanomedicine,52010),853-860.
45. Takechi, Y, Tanaka, H., Kitayama, H., Yoshii, H., Tanaka, M. and Saito, H. Comparative study on the interaction of cell-penetrating polycationic polymers with lipid membranes. Chem Phys Lipids,165,1 (Jan 2012),51-58.
46. Owida, A. A., Do, H. and Morsi, Y. S. Numerical analysis of coronary artery bypass grafts:An over view. Comput Methods Programs Biomed(Jan 2 2012).
47. Yang, W., Zhang, J., Wang, H., Gao, P., Singh, M., Shen, K. and Fang, N. Angiotensin Ⅱ downregulates catalase expression and activity in vascular adventitial fibroblasts through an AT1R/ERK1/2-dependent pathway. Mol Cell Biochem,358,1-2 (Dec 2011),21-29.
48. Coen, M., Gabbiani, G. and Bochaton-Piallat, M. L. Myofibroblast-mediated adventitial remodeling:an underestimated player in arterial pathology. Arterioscler Thromb Vasc Biol,31,11 (Nov 2011),2391-2396.
49. Busnelli, M., Froio, A., Bacci, M. L., Giunti, M., Cerrito, M. G., Giovannoni, R. Forni, M., Gentilini, F., Scagliarini, A., Deleo, G, Benatti, C., Leone, B. E., Biasi, G. M. and Lavitrano, M. Pathogenetic role of hypercholesterolemia in a novel preclinical model of vascular injury in pigs. Atherosclerosis,207,2 (Dec 2009),384-390.
50. Barrett, J. M., Rovedo, M. A., Tajuddin, A. M., Jilling, T., Macoska, J. A., MacDonald, J., Mangold, K. A. and Kaul, K. L. Prostate cancer cells regulate growth and differentiation of bone marrow endothelial cells through TGFbeta and its receptor, TGFbetaRⅡ. Prostate,66,6 (May 1 2006),632-650.
51. Kadono, H., Furuzono, T., Masuda, M., Okada, M., Ueki, M., Takamizawa, K., Tanaka, R., Miyatake, K., Koyama, Y. and Takakuda, K. In vivo evaluation of hydroxyapatite nanocoating on polyester artificial vascular grafts and possibility as soft-tissue compatible material. ASAIO J,56,1 (Jan-Feb 2010),61-66.
52. Cherian, S. M., Bobryshev, Y. V., Liang, H., Inder, S. J., Wang, A. Y, Lord, R. S., Tran, D., Pandey, P., Halasz, P. and Farnsworth, A. E. Immunohistochemical and ultrastructural evidence that dendritic cells infiltrate stenotic aortocoronary saphenous vein bypass grafts. Cardiovasc Surg,9,2 (Apr 2001),194-200.
53. Kim, T. H., Shin, J. H., Oh, S. J., Park, I. K., Woo, C. W., Han, K. H. and Dong, K. R. Inhibition of neointimal hyperplasia after stent placement with rhenium 188-filled balloon dilation in a canine iliac artery model. J Vasc Interv Radiol,21,7 (Jul 2010), 1066-1070.
54. Li, G., Chen, S. J., Oparil, S., Chen, Y. F. and Thompson, J. A. Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries. Circulation,101,12 (Mar 28 2000),1362-1365.
55. Schaller, M. D. Cellular functions of FAK kinases:insight into molecular mechanisms and novel functions. J Cell Sci,123, Pt 7 (Apr 1 2010),1007-1013.
56. Chan, K. T., Bennin, D. A. and Huttenlocher, A. Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK). J Biol Chem,285,15 (Apr 9 2010),11418-11426.
57. Guan, J. L. Integrin signaling through FAK in the regulation of mammary stem cells and breast cancer. IUBMB Life,62,4 (Apr 2010),268-276.
58. Lichtenstein, M. P., Madrigal, J. L., Pujol, A. and Galea, E. JNK/ERK/FAK Mediate Promigratory Actions of Basic Fibroblast Growth Factor in Astrocytes via CCL2 and COX2. Neurosignals,20,2 2012),86-102.
59. Valiente, M., Ciceri, G., Rico, B. and Marin, O. Focal adhesion kinase modulates radial glia-dependent neuronal migration through connexin-26. J Neurosci,31,32 (Aug 10 2011),11678-11691.
60. Shi, C., Lu, J., Wu, W., Ma, F., Georges, J., Huang, H., Balducci, J., Chang, Y. and Huang, Y. Endothelial cell-specific molecule 2 (ECSM2) localizes to cell-cell junctions and modulates bFGF-directed cell migration via the ERK-FAK pathway. PLoS One,6,62011), e21482.
61. Janostiak, R., Tolde,O., Bruhova, Z., Novotny, M., Hanks, S. K., Rosel, D. and Brabek, J. Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and invasiveness. Mol Biol Cell,22,22 (Nov 2011),4256-4267.
62. Deramaudt, T. B., Dujardin, D., Hamadi, A., Noulet, F., Kolli, K., De Mey, J., Takeda, K. and Ronde, P. FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion. Mol Biol Cell,22,7 (Apr 2011), 964-975.
63. Nugent, H. M., Sjin, R. T., White, D., Milton, L. G, Manson, R. J., Lawson, J. H. and Edelman, E. R. Adventitial endothelial implants reduce matrix metalloproteinase-2 expression and increase luminal diameter in porcine arteriovenous grafts. J Vasc Surg,46,3 (Sep 2007),548-556.
64. Wilcox, J. N. and Scott, N. A. Potential role of the adventitia in arteritis and atherosclerosis. Int J Cardiol,54 Suppl(Aug 1996), S21-35.
65. Mirzayans, R., Scott, A., Andrais, B., Pollock, S. and Murray, D. Ultraviolet light exposure triggers nuclear accumulation of p21(WAF1) and accelerated senescence in human normal and nucleotide excision repair-deficient fibroblast strains. J Cell Physiol,215,1 (Apr 2008),55-67.
66. Shi, Y, Patel, S., Niculescu, R., Chung, W., Desrochers, P. and Zalewski, A. Role of matrix metalloproteinases and their tissue inhibitors in the regulation of coronary cell migration. Arterioscler Thromb Vasc Biol,19,5 (May 1999),1150-1155.
67. Osman, B., Akool el, S., Doller, A., Muller, R., Pfeilschifter, J. and Eberhardt, W. Differential modulation of the cytokine-induced MMP-9/TIMP-1 protease-antiprotease system by the mTOR inhibitor rapamycin. Biochem Pharmacol, 81,1 (Jan 1 2011),134-143.
68. Liu, Z., Li, X., Simoneau, A. R., Jafari, M. and Zi, X. Rhodiola rosea extracts and salidroside decrease the growth of bladder cancer cell lines via inhibition of the mTOR pathway and induction of autophagy. Mol Carcinog(Apr 22 2011).
69. Gangoiti, P., Arana, L., Ouro, A., Granado, M. H., Trueba, M. and Gomez-Munoz, A. Activation of mTOR and RhoA is a major mechanism by which Ceramide 1-phosphate stimulates macrophage proliferation. Cell Signal,23,1 (Jan 2011),27-34.
70. Zheng, M., Wang, Y. H., Wu, X. N., Wu, S. Q., Lu, B. J., Dong, M. Q., Zhang, H., Sun, P., Lin, S. C., Guan, K. L. and Han, J. Inactivation of Rheb by PRAK-mediated phosphorylation is essential for energy-depletion-induced suppression of mTORC1. Nat Cell Biol,13,3 (Mar 2011),263-272.
71. Liu, C., Huang, S. J. and Qin, Z. L. Inhibition of periostin gene expression via RNA interference suppressed the proliferation, apoptosis and invasion in U2OS cells. Chin Med J (Engl),123,24 (Dec 2010),3677-3683.
72. Singh, S., Srivastava, S. K., Bhardwaj, A., Owen, L. B. and Singh, A. P. CXCL12-CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells:a novel target for therapy. Br J Cancer,103,11 (Nov 23 2010),1671-1679.
73. Chang, C. H., Tsai, W. C., Lin, M. S., Hsu, Y. H. and Pang, J. H. The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration. JAppl Physiol,110,3 (Mar 2011),774-780.
74. Park, J. H., Yoon, J. H., Lim, Y. S., Hwang, H. K., Kim, S. A. and Ahn, S. G. TAT-Hsp27 promotes adhesion and migration of murine dental papilla-derived MDPC-23 cells through beta1 integrin-mediated signaling. Int J Mol Med,26,3(Sep 2010),373-378.
75. Sebai, S. C., Cribier, S., Karimi, A., Massotte, D. and Tribet, C. Permeabilization of lipid membranes and cells by a light-responsive copolymer. Langmuir,26,17 (Sep 72010),14135-14141.
76. Chen, C. H. and Sarbassov dos, D. The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2. J Biol Chem,286,46 (Nov 18 2011), 40386-40394.
77. Lin, X., Zhang, Y., Liu, L., McKeehan, W. L., Shen, Y., Song, S. and Wang, F. FRS2alpha is essential for the fibroblast growth factor to regulate the mTOR pathway and autophagy in mouse embryonic fibroblasts. Int J Biol Sci,7,8 2011),1114-1121.
78. Yuan, J., Mehta, P. P., Yin, M. J., Sun, S., Zou, A., Chen, J., Rafidi, K., Feng, Z., Nickel, J., Engebretsen, J., Hallin, J., Blasina, A., Zhang, E., Nguyen, L., Sun, M., Vogt, P. K., McHarg, A., Cheng, H., Christensen, J. G., Kan, J. L. and Bagrodia, S. PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity. Mol Cancer Ther,10,11 (Nov 2011),2189-2199.
79. Qin, L., Wang, Y., Tao, L. and Wang, Z. AKT down-regulates insulin-like growth factor-1 receptor as a negative feedback. JBiochem,150,2 (Aug 2011),151-156.
80. Xie, X., Zhang, D., Zhao, B., Lu, M. K., You, M., Condorelli, G., Wang, C. Y. and Guan, K. L. IkappaB kinase epsilon and TANK-binding kinase 1 activate AKT by direct phosphorylation. Proc Natl Acad Sci USA,108,16 (Apr 19 2011),6474-6479.
81. Rodriguez, A., Catalan, V., Gomez-Ambrosi, J., Garcia-Navarro, S., Rotellar, F., Valenti, V., Silva, C., Gil, M. J., Salvador, J., Burrell, M. A., Calamita, G., Malagon, M. M. and Fruhbeck, G. Insulin- and leptin-mediated control of aquaglyceroporins in human adipocytes and hepatocytes is mediated via the PI3K/Akt/mTOR signaling cascade. J Clin Endocrinol Metab,96,4 (Apr 2011), E586-597.
82. Hollander, M. C., Maier, C. R., Hobbs, E. A., Ashmore, A. R., Linnoila, R. I. and Dennis, P. A. Aktl deletion prevents lung tumorigenesis by mutant K-ras. Oncogene, 30,15 (Apr 14 2011),1812-1821.
83. Zha, X., Wang, F., Wang, Y., He, S., Jing, Y, Wu, X. and Zhang, H. Lactate dehydrogenase B is critical for hyperactive mTOR-mediated tumorigenesis. Cancer Res,71,1(Jan 1 2011),13-18.
84. Liu, J. L., Mao, Z., Gallick, G. E. and Yung, W. K. AMPK/TSC2/mTOR-signaling intermediates are not necessary for LKB1-mediated nuclear retention of PTEN tumor suppressor. Neuro Oncol,13,2 (Feb 2011),184-194.
85. Saiki, S., Sasazawa, Y, Imamichi, Y., Kawajiri, S., Fujimaki, T., Tanida, I., Kobayashi, H., Sato, F., Sato, S., Ishikawa, K., Imoto, M. and Hattori, N. Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition. Autophagy,7,2 (Feb 2011),176-187.
86. Hughes, D., Fu, A. A., Puggioni, A., Glockner, J. F., Anwer, B., McGuire, A. M., Mukhopadhyay, D. and Misra, S. Adventitial transplantation of blood outgrowth endothelial cells in porcine haemodialysis grafts alleviates hypoxia and decreases neointimal proliferation through a matrix metalloproteinase-9-mediated pathway--a pilot study. Nephrol Dial Transplant,24,1 (Jan 2009),85-96.
2.李海清,刘志勇.自体移植静脉再狭窄的机制研究及防治进展.现代医学,2006,2(34)：65-67.
3. De Leon, H., Ollerenshaw, J. D., Griendling, K. K. and Wilcox, J. N. Adventitial cells do not contribute to neointimal mass after balloon angioplasty of the rat common carotid artery. Circulation,104,14 (Oct 2 2001),1591-1593.
4.胡维诚.外膜是血管病变的积极参与者.中国动脉硬化杂志,2006,14(1)：1-3.
5.郭淑杰,吴凌云,沈伟利等.血管外膜肌成纤维细胞分化的基因表达谱.ActaPhysiologica Sinica2006,58(4):337-344.
6.Cattaneo, D., Merlini, S., Pellegrino, M., Carrara, F., Zenoni, S., Murgia, S., Baldelli, S., Gaspari, F., Remuzzi, G. and Perico, N. Therapeutic drug monitoring of sirolimus: effect of concomitant immunosuppressive therapy and optimization of drug dosing. Am J Transplant,4,8 (Aug 2004),1345-1351.
7. Blatt, J., Stavas, J., Moats-Staats, B., Woosley, J. and Morrell, D. S. Treatment of childhood kaposiform hemangioendothelioma with sirolimus. Pediatr Blood Cancer, 55,7 (Dec 15 2010),1396-1398.
8. Rajathurai, T., Rizvi, S. I., Lin, H., Angelini, G. D., Newby, A. C. and Murphy, G. J. Periadventitial rapamycin-eluting microbeads promote vein graft disease in long-term pig vein-into-artery interposition grafts. Circ Cardiovasc Interv,3,2 (Apr 2010), 157-165.
9. Ishiwata, S., Verheye, S., Robinson, K. A., Salame, M. Y., de Leon, H., King, S. B., 3rd and Chronos, N. A. Inhibition of neointima formation by tranilast in pig coronary arteries after balloon angioplasty and stent implantation. J Am Coll Cardiol,35,5 (Apr 2000),1331-1337.
10. Wang, X. L., Du, F. G., Jiao, J., Meng, Y. Z. and Li, R. K. Preparation and properties of biodegradable polymeric blends from poly(propylene carbonate) and poly(ethylene-co-vinyl alcohol). J Biomed Mater Res B Appl Biomater,83,2 (Nov 2007),373-379.
11. Li, Y. and Shimizu, H. Compatibilization by homopolymer:significant improvements in the modulus and tensile strength of PPC/PMMA blends by the addition of a small amount of PVAc. ACS Appl Mater Interfaces,1,8 (Aug 2009), 1650-1655.
12. Wang, W., Itoh, S., Konno, K., Kikkawa, T., Ichinose, S., Sakai, K., Ohkuma, T. and Watabe, K. Effects of Schwann cell alignment along the oriented electrospun chitosan nanofibers on nerve regeneration. J Biomed Mater Res A,91,4 (Dec 15 2009),994-1005.
13. Prabhakaran, M. P., Venugopal, J. R. and Ramakrishna, S. Mesenchymal stem cell differentiation to neuronal cells on electrospun nanofibrous substrates for nerve tissue engineering. Biomaterials,30,28 (Oct 2009),4996-5003.
14. Acosta-Jaquez, H. A., Keller, J. A., Foster, K. G., Ekim, B., Soliman, G. A., Feener, E. P., Ballif, B. A. and Fingar, D. C. Site-specific mTOR phosphorylation promotes mTORC1-mediated signaling and cell growth. Mol Cell Biol,29,15 (Aug 2009), 4308-4324.
15. Moorman, N. J. and Shenk, T. Rapamycin-resistant mTORC1 kinase activity is required for herpesvirus replication. J Virol,84,10 (May 2010),5260-5269.
16. Zaborowska, I. and Walsh, D. PI3K signaling regulates rapamycin-insensitive translation initiation complex formation in vaccinia virus-infected cells. J Virol,83,8 (Apr 2009),3988-3992.
17. Hannan, E. L., Racz, M. J., Walford, G., Jones, R. H., Ryan, T. J., Bennett, E., Culliford, A. T., Isom, O. W., Gold, J. P. and Rose, E. A. Long-term outcomes of coronary-artery bypass grafting versus stent implantation. N Engl J Med,352,21 (May 26 2005),2174-2183.
18. Montalescot, G., White, H. D., Gallo, R., Cohen, M., Steg, P. G., Aylward, P. E., Bode, C., Chiariello, M., King, S. B.,3rd, Harrington, R. A., Desmet, W. J., Macaya, C. and Steinhubl, S. R. Enoxaparin versus unfractionated heparin in elective percutaneous coronary intervention. N Engl J Med,355,10 (Sep 7 2006),1006-1017.
19. Park, S. J., Kim, Y. H., Park, D. W., Yun, S. C., Ahn, J. M., Song, H. G., Lee, J. Y, Kim, W. J., Kang, S. J., Lee, S. W., Lee, C. W., Park, S. W., Chung, C. H., Lee, J. W., Lim, D. S., Rha, S. W., Lee, S. G., Gwon, H. C., Kim, H. S., Chae, I. H., Jang, Y, Jeong, M. H., Tahk, S. J. and Seung, K. B. Randomized trial of stents versus bypass surgery for left main coronary artery disease. N Engl J Med,364,18 (May 5 2011), 1718-1727.
20. Mauri, L. Why We Still Need Randomized Trials to Compare Effectiveness. N Engl J Med(Mar 27 2012).
21. Serruys, P. W., Morice, M. C., Kappetein, A. P., Colombo, A., Holmes, D. R., Mack, M. J., Stahle, E., Feldman, T. E., van den Brand, M., Bass, E. J., Van Dyck, N., Leadley, K., Dawkins, K. D. and Mohr, F. W. Percutaneous coronary intervention versus coronary-artery bypass grafting for severe coronary artery disease. N Engl J Med,360,10 (Mar 5 2009),961-972.
22. Petry, J. J., Burstein, S., Chang, W. H., Wortham, K., Sedor, C. and Hunter, S. A. Prostacyclin production by vein grafts in the arterial circulation:a study in rats. Prostaglandins Leukot Med,9,5 (Nov 1982),511-516.
23. Westerband, A., Crouse, D., Richter, L. C., Aguirre, M. L., Wixon, C. C., James, D. C., Mills, J. L., Hunter, G. C. and Heimark, R. L. Vein adaptation to arterialization in an experimental model. J Vasc Surg,33,3 (Mar 2001),561-569.
24. Muller, V., Ott, R., Tannapfel, A., Hohenberger, W. and Reck, T. Arterialization of the portal vein in liver transplantation:a new microsurgical model in the rat. Transplantation,71,7 (Apr 15 2001),977-981.
25. Borin, T. F., Miyakawa, A. A., Cardoso, L., de Figueiredo Borges, L., Goncalves, G. A. and Krieger, J. E. Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21(cip1) in vascular remodelling during vein arterialization in the rat. Int J Exp Pathol,90,3 (Jun 2009),328-337.
26. Kelly, B. S., Heffelfinger, S. C., Whiting, J. F., Miller, M. A., Reaves, A., Armstrong, J., Narayana, A. and Roy-Chaudhury, P. Aggressive venous neointimal hyperplasia in a pig model of arteriovenous graft stenosis. Kidney Int,62,6 (Dec 2002),2272-2280.
27. Swedberg, S. H., Brown, B. G., Sigley, R., Wight, T. N., Gordon, D. and Nicholls, S. C. Intimal fibromuscular hyperplasia at the venous anastomosis of PTFE grafts in hemodialysis patients. Clinical, immunocytochemical, light and electron microscopic assessment. Circulation,80,6 (Dec 1989),1726-1736.
28. Tennant, M. and McGeachie, J. K. Adaptive remodelling of smooth muscle in the neo-intima of vein-to-artery grafts in rats:a detailed morphometric analysis. Anat Embryol (Berl),187,2 (Feb 1993),161-166.
29. Davies, M. G., Owens, E. L., Mason, D. P., Lea, H., Tran, P. K., Vergel, S., Hawkins, S. A., Hart, C. E. and Clowes, A. W. Effect of platelet-derived growth factor receptor-alpha and -beta blockade on flow-induced neointimal formation in endothelialized baboon vascular grafts. Circ Res,86,7 (Apr 14 2000),779-786.
30. Graaf, R., Tintu, A., Stassen, F., Kloppenburg, G, Bruggeman, C. and Rouwet, E. N-acetylcysteine prevents neointima formation in experimental venous bypass grafts. Br J Surg,96,8 (Aug 2009),941-950.
31. Karacagil, S., Narbani, A., Almgren, B., Bowald, S. and Bergqvist, D. Modified vein cuff technique for distal polytetrafluoroethylene graft anastomoses:how we do it. Eur J Surg,161,1 (Jan 1995),47-48.
32. Misra, S., Fu, A. A., Puggioni, A., Karimi, K. M., Mandrekar, J. N., Glockner, J. F., Juncos, L. A., Anwer, B., McGuire, A. M. and Mukhopadhyay, D. Increased shear stress with upregulation of VEGF-A and its receptors and MMP-2, MMP-9, and TIMP-1 in venous stenosis of hemodialysis grafts. Am J Physiol Heart Circ Physiol, 294,5 (May 2008), H2219-2230.
33. de Paula, C. A., Coulson-Thomas, V. J., Ferreira, J. G, Maza, P. K., Suzuki, E., Nakahata, A. M., Nader, H. B., Sampaio, M. U. and Oliva, M. L. Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways. J Biol Chem,287,1 (Jan 2 2012),170-182.
34. Huang, S., Bjornsti, M. A. and Houghton, P. J. Rapamycins:mechanism of action and cellular resistance. Cancer Biol Ther,2,3 (May-Jun 2003),222-232.
35. Kawatsu, S., Oda, K., Saiki, Y., Tabata, Y. and Tabayashi, K. External application of rapamycin-eluting film at anastomotic sites inhibits neointimal hyperplasia in a canine model. Ann Thorac Surg,84,2 (Aug 2007),560-567; discussion 567.
36. Zou, J., Zhang, X., Yang, H., Zhu, Y., Ma, H. and Wang, S. Rapamycin-loaded nanoparticles for inhibition of neointimal hyperplasia in experimental vein grafts. J Cardiothorac Surg,62011),69.
37. Peterson, R. T., Beal, P. A., Comb, M. J. and Schreiber, S. L. FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions. J Biol Chem,275,10 (Mar 10 2000), 7416-7423.
38. Mothe-Satney, I., Brunn, G J., McMahon, L. P., Capaldo, C. T., Abraham, R. T. and Lawrence, J. C., Jr. Mammalian target of rapamycin-dependent phosphorylation of PHAS-I in four (S/T)P sites detected by phospho-specific antibodies. J Biol Chem, 275,43 (Oct 27 2000),33836-33843.
39. Peterson, R. T., Desai, B. N., Hardwick, J. S. and Schreiber, S. L. Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. Proc Natl Acad Sci U S A,96,8 (Apr 13 1999), 4438-4442.
40. Lohmann, C. H., Schwartz, Z., Niederauer, G. G., Carries, D. L., Jr., Dean, D. D. and Boyan, B. D. Pretreatment with platelet derived growth factor-BB modulates the ability of costochondral resting zone chondrocytes incorporated into PLA/PGA scaffolds to form new cartilage in vivo. Biomaterials,21,1 (Jan 2000),49-61.
41. Telemeco. T. A., Ayres, C., Bowlin, G. L., Wnek, G. E., Boland, E. D., Cohen, N., Baumgarten, C. M., Mathews, J. and Simpson, D. G. Regulation of cellular infiltration into tissue engineering scaffolds composed of submicron diameter fibrils produced by electrospinning. Acta Biomater,1,4 (Jul 2005),377-385.
42. Shen, Y., Qian, Y., Zhang, H., Zuo, B., Lu, Z., Fan, Z., Zhang, P., Zhang, F. and Zhou, C. Guidance of olfactory ensheathing cell growth and migration on electrospun silk fibroin scaffolds. Cell Transplant,19,2 2010),147-157.
43. Qiu, X. F., Dong, N. G, Sun, Z. Q., Hu, P., Su, W. and Shi, J. W. [Controlled release of siRNA nanoparticles loaded in a novel external stent prepared by emulsion electrospinning attenuates neointima hyperplasia in vein grafts]. Zhonghua Yi Xue Za Zhi,89,41 (Nov 10 2009),2938-2942.
44. Liu, K., Cao, G., Zhang, X., Liu, R., Zou, W. and Wu, S. Pretreatment with intraluminal rapamycin nanoparticle perfusion inhibits neointimal hyperplasia in a rabbit vein graft model. Int JNanomedicine,52010),853-860.
45. Takechi, Y, Tanaka, H., Kitayama, H., Yoshii, H., Tanaka, M. and Saito, H. Comparative study on the interaction of cell-penetrating polycationic polymers with lipid membranes. Chem Phys Lipids,165,1 (Jan 2012),51-58.
46. Owida, A. A., Do, H. and Morsi, Y. S. Numerical analysis of coronary artery bypass grafts:An over view. Comput Methods Programs Biomed(Jan 2 2012).
47. Yang, W., Zhang, J., Wang, H., Gao, P., Singh, M., Shen, K. and Fang, N. Angiotensin Ⅱ downregulates catalase expression and activity in vascular adventitial fibroblasts through an AT1R/ERK1/2-dependent pathway. Mol Cell Biochem,358,1-2 (Dec 2011),21-29.
48. Coen, M., Gabbiani, G. and Bochaton-Piallat, M. L. Myofibroblast-mediated adventitial remodeling:an underestimated player in arterial pathology. Arterioscler Thromb Vasc Biol,31,11 (Nov 2011),2391-2396.
49. Busnelli, M., Froio, A., Bacci, M. L., Giunti, M., Cerrito, M. G., Giovannoni, R. Forni, M., Gentilini, F., Scagliarini, A., Deleo, G, Benatti, C., Leone, B. E., Biasi, G. M. and Lavitrano, M. Pathogenetic role of hypercholesterolemia in a novel preclinical model of vascular injury in pigs. Atherosclerosis,207,2 (Dec 2009),384-390.
50. Barrett, J. M., Rovedo, M. A., Tajuddin, A. M., Jilling, T., Macoska, J. A., MacDonald, J., Mangold, K. A. and Kaul, K. L. Prostate cancer cells regulate growth and differentiation of bone marrow endothelial cells through TGFbeta and its receptor, TGFbetaRⅡ. Prostate,66,6 (May 1 2006),632-650.
51. Kadono, H., Furuzono, T., Masuda, M., Okada, M., Ueki, M., Takamizawa, K., Tanaka, R., Miyatake, K., Koyama, Y. and Takakuda, K. In vivo evaluation of hydroxyapatite nanocoating on polyester artificial vascular grafts and possibility as soft-tissue compatible material. ASAIO J,56,1 (Jan-Feb 2010),61-66.
52. Cherian, S. M., Bobryshev, Y. V., Liang, H., Inder, S. J., Wang, A. Y, Lord, R. S., Tran, D., Pandey, P., Halasz, P. and Farnsworth, A. E. Immunohistochemical and ultrastructural evidence that dendritic cells infiltrate stenotic aortocoronary saphenous vein bypass grafts. Cardiovasc Surg,9,2 (Apr 2001),194-200.
53. Kim, T. H., Shin, J. H., Oh, S. J., Park, I. K., Woo, C. W., Han, K. H. and Dong, K. R. Inhibition of neointimal hyperplasia after stent placement with rhenium 188-filled balloon dilation in a canine iliac artery model. J Vasc Interv Radiol,21,7 (Jul 2010), 1066-1070.
54. Li, G., Chen, S. J., Oparil, S., Chen, Y. F. and Thompson, J. A. Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries. Circulation,101,12 (Mar 28 2000),1362-1365.
55. Schaller, M. D. Cellular functions of FAK kinases:insight into molecular mechanisms and novel functions. J Cell Sci,123, Pt 7 (Apr 1 2010),1007-1013.
56. Chan, K. T., Bennin, D. A. and Huttenlocher, A. Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK). J Biol Chem,285,15 (Apr 9 2010),11418-11426.
57. Guan, J. L. Integrin signaling through FAK in the regulation of mammary stem cells and breast cancer. IUBMB Life,62,4 (Apr 2010),268-276.
58. Lichtenstein, M. P., Madrigal, J. L., Pujol, A. and Galea, E. JNK/ERK/FAK Mediate Promigratory Actions of Basic Fibroblast Growth Factor in Astrocytes via CCL2 and COX2. Neurosignals,20,2 2012),86-102.
59. Valiente, M., Ciceri, G., Rico, B. and Marin, O. Focal adhesion kinase modulates radial glia-dependent neuronal migration through connexin-26. J Neurosci,31,32 (Aug 10 2011),11678-11691.
60. Shi, C., Lu, J., Wu, W., Ma, F., Georges, J., Huang, H., Balducci, J., Chang, Y. and Huang, Y. Endothelial cell-specific molecule 2 (ECSM2) localizes to cell-cell junctions and modulates bFGF-directed cell migration via the ERK-FAK pathway. PLoS One,6,62011), e21482.
61. Janostiak, R., Tolde,O., Bruhova, Z., Novotny, M., Hanks, S. K., Rosel, D. and Brabek, J. Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and invasiveness. Mol Biol Cell,22,22 (Nov 2011),4256-4267.
62. Deramaudt, T. B., Dujardin, D., Hamadi, A., Noulet, F., Kolli, K., De Mey, J., Takeda, K. and Ronde, P. FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion. Mol Biol Cell,22,7 (Apr 2011), 964-975.
63. Nugent, H. M., Sjin, R. T., White, D., Milton, L. G, Manson, R. J., Lawson, J. H. and Edelman, E. R. Adventitial endothelial implants reduce matrix metalloproteinase-2 expression and increase luminal diameter in porcine arteriovenous grafts. J Vasc Surg,46,3 (Sep 2007),548-556.
64. Wilcox, J. N. and Scott, N. A. Potential role of the adventitia in arteritis and atherosclerosis. Int J Cardiol,54 Suppl(Aug 1996), S21-35.
65. Mirzayans, R., Scott, A., Andrais, B., Pollock, S. and Murray, D. Ultraviolet light exposure triggers nuclear accumulation of p21(WAF1) and accelerated senescence in human normal and nucleotide excision repair-deficient fibroblast strains. J Cell Physiol,215,1 (Apr 2008),55-67.
66. Shi, Y, Patel, S., Niculescu, R., Chung, W., Desrochers, P. and Zalewski, A. Role of matrix metalloproteinases and their tissue inhibitors in the regulation of coronary cell migration. Arterioscler Thromb Vasc Biol,19,5 (May 1999),1150-1155.
67. Osman, B., Akool el, S., Doller, A., Muller, R., Pfeilschifter, J. and Eberhardt, W. Differential modulation of the cytokine-induced MMP-9/TIMP-1 protease-antiprotease system by the mTOR inhibitor rapamycin. Biochem Pharmacol, 81,1 (Jan 1 2011),134-143.
68. Liu, Z., Li, X., Simoneau, A. R., Jafari, M. and Zi, X. Rhodiola rosea extracts and salidroside decrease the growth of bladder cancer cell lines via inhibition of the mTOR pathway and induction of autophagy. Mol Carcinog(Apr 22 2011).
69. Gangoiti, P., Arana, L., Ouro, A., Granado, M. H., Trueba, M. and Gomez-Munoz, A. Activation of mTOR and RhoA is a major mechanism by which Ceramide 1-phosphate stimulates macrophage proliferation. Cell Signal,23,1 (Jan 2011),27-34.
70. Zheng, M., Wang, Y. H., Wu, X. N., Wu, S. Q., Lu, B. J., Dong, M. Q., Zhang, H., Sun, P., Lin, S. C., Guan, K. L. and Han, J. Inactivation of Rheb by PRAK-mediated phosphorylation is essential for energy-depletion-induced suppression of mTORC1. Nat Cell Biol,13,3 (Mar 2011),263-272.
71. Liu, C., Huang, S. J. and Qin, Z. L. Inhibition of periostin gene expression via RNA interference suppressed the proliferation, apoptosis and invasion in U2OS cells. Chin Med J (Engl),123,24 (Dec 2010),3677-3683.
72. Singh, S., Srivastava, S. K., Bhardwaj, A., Owen, L. B. and Singh, A. P. CXCL12-CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells:a novel target for therapy. Br J Cancer,103,11 (Nov 23 2010),1671-1679.
73. Chang, C. H., Tsai, W. C., Lin, M. S., Hsu, Y. H. and Pang, J. H. The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration. JAppl Physiol,110,3 (Mar 2011),774-780.
74. Park, J. H., Yoon, J. H., Lim, Y. S., Hwang, H. K., Kim, S. A. and Ahn, S. G. TAT-Hsp27 promotes adhesion and migration of murine dental papilla-derived MDPC-23 cells through beta1 integrin-mediated signaling. Int J Mol Med,26,3(Sep 2010),373-378.
75. Sebai, S. C., Cribier, S., Karimi, A., Massotte, D. and Tribet, C. Permeabilization of lipid membranes and cells by a light-responsive copolymer. Langmuir,26,17 (Sep 72010),14135-14141.
76. Chen, C. H. and Sarbassov dos, D. The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2. J Biol Chem,286,46 (Nov 18 2011), 40386-40394.
77. Lin, X., Zhang, Y., Liu, L., McKeehan, W. L., Shen, Y., Song, S. and Wang, F. FRS2alpha is essential for the fibroblast growth factor to regulate the mTOR pathway and autophagy in mouse embryonic fibroblasts. Int J Biol Sci,7,8 2011),1114-1121.
78. Yuan, J., Mehta, P. P., Yin, M. J., Sun, S., Zou, A., Chen, J., Rafidi, K., Feng, Z., Nickel, J., Engebretsen, J., Hallin, J., Blasina, A., Zhang, E., Nguyen, L., Sun, M., Vogt, P. K., McHarg, A., Cheng, H., Christensen, J. G., Kan, J. L. and Bagrodia, S. PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity. Mol Cancer Ther,10,11 (Nov 2011),2189-2199.
79. Qin, L., Wang, Y., Tao, L. and Wang, Z. AKT down-regulates insulin-like growth factor-1 receptor as a negative feedback. JBiochem,150,2 (Aug 2011),151-156.
80. Xie, X., Zhang, D., Zhao, B., Lu, M. K., You, M., Condorelli, G., Wang, C. Y. and Guan, K. L. IkappaB kinase epsilon and TANK-binding kinase 1 activate AKT by direct phosphorylation. Proc Natl Acad Sci USA,108,16 (Apr 19 2011),6474-6479.
81. Rodriguez, A., Catalan, V., Gomez-Ambrosi, J., Garcia-Navarro, S., Rotellar, F., Valenti, V., Silva, C., Gil, M. J., Salvador, J., Burrell, M. A., Calamita, G., Malagon, M. M. and Fruhbeck, G. Insulin- and leptin-mediated control of aquaglyceroporins in human adipocytes and hepatocytes is mediated via the PI3K/Akt/mTOR signaling cascade. J Clin Endocrinol Metab,96,4 (Apr 2011), E586-597.
82. Hollander, M. C., Maier, C. R., Hobbs, E. A., Ashmore, A. R., Linnoila, R. I. and Dennis, P. A. Aktl deletion prevents lung tumorigenesis by mutant K-ras. Oncogene, 30,15 (Apr 14 2011),1812-1821.
83. Zha, X., Wang, F., Wang, Y., He, S., Jing, Y, Wu, X. and Zhang, H. Lactate dehydrogenase B is critical for hyperactive mTOR-mediated tumorigenesis. Cancer Res,71,1(Jan 1 2011),13-18.
84. Liu, J. L., Mao, Z., Gallick, G. E. and Yung, W. K. AMPK/TSC2/mTOR-signaling intermediates are not necessary for LKB1-mediated nuclear retention of PTEN tumor suppressor. Neuro Oncol,13,2 (Feb 2011),184-194.
85. Saiki, S., Sasazawa, Y, Imamichi, Y., Kawajiri, S., Fujimaki, T., Tanida, I., Kobayashi, H., Sato, F., Sato, S., Ishikawa, K., Imoto, M. and Hattori, N. Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition. Autophagy,7,2 (Feb 2011),176-187.
86. Hughes, D., Fu, A. A., Puggioni, A., Glockner, J. F., Anwer, B., McGuire, A. M., Mukhopadhyay, D. and Misra, S. Adventitial transplantation of blood outgrowth endothelial cells in porcine haemodialysis grafts alleviates hypoxia and decreases neointimal proliferation through a matrix metalloproteinase-9-mediated pathway--a pilot study. Nephrol Dial Transplant,24,1 (Jan 2009),85-96.