| BackgroundCoronary 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.Methods1. 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.Results1. 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.Conclusions1.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.
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