Objective To construct vectors that express phosphatidylinositol-3-kinase, catalytic, beta polypeptide （PIK3cb） shRNA in eukaryon plasmid catalyzed by PI3K in rat, then test their effects on intimal hyperplasia in transplanted vein graft. Methods One hundred and fifty SD rats were randomly divided into six groups （n=25, in each group）: blank (25% Pluronic F-127), shRNA-1, shRNA-2, 1/2 （shRNA-1+shRNA-2）, negative control (pGenesil-1 scramble shRNA) and positive control （wortmannin） group. The jugular vein in rats were interpositioned autologously into the common carotid artery. shRNA and 25% Pluronic F-127 were mixed and coated around the transplanted vein in three PIK3cb shRNA groups. Every 5 samples were removed according to the time point (1, 3, 7, 14 and 28 days after operation), respectively. The thickness of intima and neointima area were calculated and analyzed by computer system. The PCNA expression was detected by Western blot and SP immunohistochemistry. Results The intimal thickness of three PIK3cb shRNA groups were lower than those in the blank group and negative control group on day 3, 7, 14, 28 after operation (P＜0.05); The neointima area in three PIK3cb shRNA groups (except shRNA-2 group on day 3, 7) began to decrease significantly from day one (P＜0.05). The protein expression of PCNA in three PIK3cb shRNA groups on day 3 after operation were decreased compared with blank group and negative group (P＜0.05). The percentage of the PCNA positive cells area in three PIK3cb shRNA groups were significantly lower than those in blank group and negative control group in each time point (Plt;0.05). There were no significant differences between blank and negative control group in different time points (Pgt;0.05). Conclusion The PIK3cb shRNA can effectively inhibit the proliferation of vascular smooth muscle cell, which may provide a new gene therapy for the prevention of vein graft restenosis after bypass grafting.
Objective To investigate the development and significance of the expression of early growth response gene-1 （EGR-1） in autogenous vein graft in rats and detect the role of it in intimal hyperplasia. Methods Autogenous vein graft model was established in 90 Wistar rats, transplanting the right jugular vein to infra renal abdominal aorta by microsurgical technique. The vein graft samples were harvested at hour 1, 2, 6 and 24, day 3, 7,14, 28 and 42 after procedure. Normal vein as control group. Egr-1 mRNA was measured by reverse transcription-PCR and in situ hybridization. Western blot and immunohistochemistry were used to detect the protein expression of Egr-1. Results Intimal hyperplasia reached peak at day 28 after autogenous vein graft surgery. Egr-1 mRNA and Egr-1 protein hadn’t been found in the normal vein. The expressions of Egr-1 mRNA and Egr-1 protein had biphasic changes. By reverse transcription-PCR and in situ hybridization, we found that the level of Egr-1 mRNA rose at 1 hour after graft, the expression of Egr-1 mRNA was (35±7)%. Decline at hour 6， 24 and day 3, the positive rates of Egr-1 mRNA were (8±2)%, (8±6)% and (8±4)% respectively. Reincrease at day 7, a peak at day 28, the positive rate of Egr-1 mRNA was (45±6)% (compared with other phase, P＜0.01). At day 42, the expression of Egr-1 mRNA declined again. Immunohistochemical staining and Western blot revealed Egr-1 protein had expressed at hour 2 early phase, the expression of Egr-1 protein was (30±5)%, and until to hour 6. The level of Egr-1 protein was decrease at hour 24 and day 3, the positive rates were (7±3)% and (7±8)% respectively. A peak at day 28, the positive rate of Egr-1 protein was (40±9)% (compared with other phase, P＜0.01). We found that immu-noreative Egr-1 located vascular smooth muscle cells （VSMCs） and monocytes/macrophages in tunica media at the early phase of day 7 and 14, and in neointimal and medial VSMCs at later phase of day 28. Egr-1 was also present in the endoluminal endothelial cells. Conclusion In autogenous vein graft, Egr-1 plays an important role in the proliferation of VSMCs. Egr-1 may become a new target for the prevention and therapy of intimal hyperplasia, stenosis and emphraxis after vein graft.
ObjectiveTo detect the inhibitory effect of early growth response gene-1 DNA enzyme (EDRz) on proliferation of vascular smooth muscle cell (VSMC) and intimal hyperplasia, and confirm the effect of gene therapy on stenosis and occlusion after vein transplantation. MethodsEDRz was constructed, and autogenous vein graft model was established with Wistar rats, transplanting the right jugular vein to infra renal abdominal aorta by microsurgical technique. EDRz was transfected to the graft veins and the vein graft samples were harvested at hour 1, 2, 6, 24 and on day 3, 7, 14, 28, 42 after grafting, 10 Wistar rats were randomly selected in every time. The expression of EDRz in transfected vein graft was detected by fluorescent microscope. Egr-1 mRNA was measured by reverse transcription-PCR (RT-PCR) and in situ hybridization, respectively. The protein expression of Egr-1 was detected by Western blot and immunohistochemistry, respectively. HE stained vein grafts were observed under microscope. Results① The results of EDRz transfected vein graft: At hour 1 after grafting, EDRz was mainly located in adventitia, tunica media, and partial endothelial cells of vein graft; At hour 2, 6, and 24, EDRz was located in tunica media of vein graft; and on day 7, it was mainly located in intima of vein graft. There wasn’t EDRz in vein grafts on day 14, 28, and 42. ② The results of expression of Egr-1 mRNA: Detection by RT-PCR: At hour 1 after transfecting, the expression of Egr-1 mRNA arrived at the peak, and declined at hour 2, 6, and 24. The expression was tenuity on day 3. Egr-1 mRNA expression was not found on day 7, 14, 28, and 42. The expression of Egr-1 mRNA at hour 1 was significantly higher than that of the other time point (Plt;0.01). The result of in situ hybridization was coincident with RT-PCR. ③ The results of expression of Egr-1 protein: The result of Western blot: There was no expression of Egr-1 protein in normal veins. At hour 2 after grafting, expression of Egr-1 protein was found, and declined at hour 6, 24, and on day 3. There was no expression of Egr-1 protein at hour 1, and on day 7, 14, 28, and 42. The expression of Egr-1 protein at hour 2 was significantly higher than that of the other time point (Plt;0.01). The result of immunohistochemistry was coincident with Western blot. ④The degree of VSMC hyperplasia and intimal thickness were lighter in EDRz transfected vein grafts than that in nottransfected vein grafts contemporarily. ConclusionsEDRz could reduce the expression of Egr-1 in autogenous vein graft, and could effectively restrain VSMC proliferation and intimal hyperplasia, and prevent vascular stenosis and occlusion after vein grafting.
Objective To investigate the effect of adenovirus vector mediated transfer of human herpes simplex virus thymidine kinase (HSVtk) gene inhibits intimal hyperplasia of vein grafts. Methods Auto vein graft models of Wistar rats were established. Adenovirus vector dwelled in cervical veins which were transplanted into inferior renal abdominal aorta. The combination of HSVtk (4×109 plaque forming units) and ganciclovir (GCV) was applied to test the inhibition effect. GCV was infused 〔60 mg/(kg·d), IP, Bid〕 from day 3 to day 21 after transplantation. Vein samples were harvested and the existence of HSVtk DNA was measured by PCR and the mRNA of it was studied by in situ hybridization. Van gieson (VG) and proliferating cell nuclear antigen (PCNA) stains were carried out in paraffin sections to study the thickness of neointima and smooth muscle cells (SMCs) proliferation with a computer-assisted analysis system. The apoptosis of SMCs also was detected by TUNEL. Results The existence of HSVtk gene in veins and its transcription were demonstrated. Morphometric analysis demonstrated a reduced intima thickness in the group receiving combination therapy (HSVtk/GCV) compared with HSVtk alone 〔(17.2±3.2) μm versus (31.1±2.5) μm, P＜0.05〕. GCV per se had no effect on intimal hyperplasia after vein transplantation. The apoptosis of SMCs increased significantly and expression of PCNA decreased in HSVtk/GCV gene therapy group versus blank control group 〔(9.1±2.3)% vs (28.7±3.6)%, P＜0.05; (38.7±5.6)%vs (18.5±2.6)%, P＜0.05〕. Conclusion GCV conditions reduction of intimal hyperplasia after intraluminal delivery of HSVtk in transplanting vena veins involving SMCs apoptosis.
Objective To investigate the influence of RNA interference targeting c-Jun gene on the proliferation of rat vascular smooth muscle cells (VSMCs). Methods The experiment was performed with c-Jun siRNA （c-Jun siRNA group）, control reverse sequence siRNA (control siRNA group) or no siRNA (control group). VSMCs were transfected with siRNA targeting c-Jun gene by liposome. Effects of c-Jun siRNA on mRNA and protein expressions of c-Jun were examined by RT-PCR analysis and Western blot respectively. MTT test and 3H-TdR incorporation were used to detect VSMCs proliferation. Cell cycle analysis of VSMCs in vitro was determined by flow cytometer. Results The expression levels of mRNA and protein of c-Jun in c-Jun siRNA group were significantly lower than those in control group （P＜0.05， P＜0.01). There was no significant difference between control group and control siRNA group （Pgt;0.05）. Proliferation activity of VSMCs decreased significantly in c-Jun siRNA group compared with that in control group （P＜0.05） and VSMCs was blocked in the G0/G1 phase of cell cycle significantly （P＜0.05）. There was no significant difference between control group and control siRNA group （Pgt;0.05）. Conclusion c-Jun gene silenced by RNA interference can inhibit VSMCs proliferation effectively in vitro.
Objective To investigate the effects of Rho-kinase inhibitor——fasudil hydrochloride hydrate on vein graft intimal hyperplasia in vivo. Methods Twenty-four healthy rabbits (2.3-2.5 kg) were randomly divided into two groups(n=12). Fasudil hydrochloride hydrate (experimental group) and normal sodium (control group) were given 3 days beforeoperation with 30 mg/kg by intravenous injection everyday and continued until the end of the experiment. After a longitudinal incision, the femoral vein and the famoral artery were exposed about 3 cm. An approximately 2.5 cm segment of the famoral vein was harvested for the reversed-vein graft. The femoral artery was removed 1 cm segment and replaced by the harvested femoral vein. At 2 and 4 weeks after operation, the grafts were stained with HE to observe the thickness of the intima. Furthermore, the prol iferating cell nuclear antigen (PCNA) and transmission electron microscope was used to study the prol iferation of smooth muscle cell. In situ apoptosis was detected by TUNEL assay. Results All rabbits survived till the end of the experiment. The color Doppler imaging examination showed that all grafts were patency. At 2 and 4 weeks after the operation, HE staining showed that the intimal hyperplasia were obvious in the two groups. There were lots of cells in the intima, and more fusiform smooth muscle cells in the media. At 2 and 4 weeks, the intimal thickness were (30.33 ± 3.23) μm and (43.11 ± 4.92) μm in experimental group and were (44.83 ± 3.53) μm and (66.16 ± 8.45) μm in control group. The rates of PCNA positive cell were 14.28% ± 2.76% and 7.61% ± 1.06% in experimental group and were 20.08% ± 3.56% and 8.73% ± 1.35% in control group. The rates of TUNEL positive cell were 3.55% ± 0.36% and 1.22% ± 0.18% in experimental group and were 1.11% ± 0.31% and 0.55% ± 0.11% in control group. There were significant differences (P lt; 0.05) between the two groups at 2 weeks or 4 weeks, between2 weeks and 4 weeks within group. Conclusion Intravenous injection of fasudil hydrochloride hydrate is an effective method for prevention of vein graft intimal hyperplasia of rabbit.
Abstract: Objective To investigate the effects of haemopoietic stem cell mobilization on vein graft patency and intimal hyperplasia of anastomosis. Methods Twentyfour New Zealand rabbits were randomly divided into experimental group and control group, 12 rabbits in each group. A double side of carotid arteryvein transplantation model was made in each rabbit. One side of vein graft was digested by 0.25% trypsin for complete endothelial denudation before transplantation. Recombinant human granulocyte colonystimulating factor was given by subcutaneous injection 24 hours after operation, once per day in successive 10 days in experimental group, saline was given in the same way in control group. Bone marrow stem cells mobilization was observed after operation, including karyote counts and mononuclear cell proportion in peripheral blood. The patency rate of vein grafts and the degree of anastomosis intimal hyperplasia were observed too. Results The karyote counts (t=8.406,P=0.000)and mononuclear cell proportion(t=31.267,P=0.000) in peripheral blood of experimental group increased significantly 5 days after operation than those in control group. The vein grafts with intact endothelium had higher patency rate in both groups. In the vein grafts with complete endothelial denudation, the patency rate were obviously lower, but it was higher in experimental group than those in control group (67% vs. 30%). In the end of experiment, the pulsatility index of the vein grafts anastomosis with complete endothelial denudation was lower in experimental group than that in control group(t=2.958,P=0.009). Pathological examination showed that various degrees of intimal hyperplasia in all anastomoses of vein grafts were observed 4 weeks after operation. The degree of anastomosis intimal hyperplasia was more severe in vein grafts with complete endothelial denudation. Compared with control group, re-endothelization occurred completely in vein grafts with complete endothelial denudation of experimental group and the degree of anastomosis intimal hyperplasia was relatively lower (Plt;0.05). Conclusion Haemopoietic stem cell mobilization can provide protective effects on vein grafts by accelerating reendothelization which might increase vein grafts patency rate in the near future after operation and reduce anastomosis restenosis caused by intimal hyperplasia.
Objective To investigate the effect of survivin antisense oligodeoxyribonucleotides (survivin ASODNs) on intimal hyperplasia (IH) in vein graft in rats. Methods Autogenous vein graft models were established in 60 Wistar rats by transplanting the interior jugular vein to the common jugular artery using microsurgical technique. The rats were divided into 5 groups according to random digits table, including survivin ASODNs 50 μg group and 200 μg group, scramble ODNs 200 μg group (ODNs group）, Lipofectin+pluronic group and control group. Vein graft samples were collected on 7 d and 14 d after transplantation, respectively. The degrees of hyperplasia were determined and then compared by histomorphology between different groups. The expression of survivin mRNA was measured by RT-PCR and immunohistochemistry. The relevant protein products were detected by Western blot and immunohistochemistry was also used to detect the expression of PCNA. Apoptosis of VSMC was measured by TUNEL.Results Day 7 and 14 were the days that intimal hyperplasied most in control group, ODNs group and Lipofectin+pluronic group, there was no significant difference among these groups yet (Pgt;0.05). The IH could be suppressed by locally transfecting 50 μg of survivin ASODNs (P＜0.05), and it showed a better inhibiting effect in 200 μg of survivin ASODNs group (P＜0.05). The expression of survivin mRNA increased significantly in control group. The expressions of both survivin and PCNA in VSMC significantly decreased in survivin ASODNs group (P＜0.05), whereas the positive cells of TUNEL increased significantly (P＜0.05). Conclusion Transfection of survivin ASODNs may inhibit the IH after vein graft through suppressing the hyperplasia and stimulating the apoptosis of VSMC, and inhibiting the expression of survivin.
ObjectiveTo assess the inhibitory ability of sarpogrelate on neointimal hyperplasia of carotid artery in rat balloon-injuried model, and to compare the proliferation of vascular smooth muscle cell (VSMC) by monitoring the expression of proliferative cell nuclear antigen (PCNA). MethodsTwenty-four male SD rats (SPF, 8 weeks) were allocated prospectively and randomly into 3 groups: blank group, sarpogrelate group, and clopidogrel group. Each group included 8 rats. All the rats were fed high-fat diet for 1 week before the operation. No drug was fed in the blank group, and sarpogrelate 100 mg/(kg·d) or clopidogrel 20 mg/(kg·d) was fed in the sarpogrelate group or clopidogrel group respectively. The carotid artery of rat was dilated by the Forgarty balloon catheter. The rats were killed 2 weeks later and the samples were got in the balloon-injuried carotid arteries. Histomorphological analysis and immunohistochemical analysis were proceeded. The thickness ratio and area ratio of intima and media, the ratio of PCNA positive cells and PCNA absorbance were calculated among the three groups. ResultsCompared with the blank group, the average intimal thickness, average intimal area, thickness ratio of intima and media, area ratio of intima and media, PCNA absorbance, and ratio of PCNA positive cells were significant decreased in the sarpogrelate group (P < 0.001) and the clopidogrel group (P < 0.001), but which had no significant differences between the sarpogrelate group and the clopidogrel group (P > 0.05). There was no significant difference in the average media thickness or area among the 3 groups (P > 0.05). ConclusionSarpogrelate and clopidogrel could significantly reduce the thickness or area of intima, the absorbance of PCNA and the ratio of PCNA positive cells.
【Abstract】ObjectiveSome studies have demonstrated that recombinant adenoviruses are efficient vectors for gene transfer to the venous wall and AdCMV.tk encoded thymidine kinase can be used to reduce restenosis. In this study AdCMV.tk was apply to human vein smooth muscle cells (SMC) and organ cultured saphenous veins to study its effects on proliferation of SMCs and reduction of intimal hyperplasia. MethodsThe adenovirus vector transferred tk gene and mark gene lacZ to the SMC of human saphenous veins and organ cultured vein segments. Various concentrations ganciclovir (GCV) were contained in culture media. The efficiency of gene transfer was studied by using Xgal staining. The proliferation of SMC was monitored by the method of trypan blue exclusion. The bystander effect was observed by mixed cell culture. After vein segments treated by AdCMV.tk+GCV and cultured for 14 days, HE and VG staining were carried out and intimal thickness was analysis by computer image system. ResultsAdenovirus vector could infect saphenous vein SMC efficiently both in cultured SMCs and organ cultured vein segments. Gene expression sustained 14 d at least. The inhibition of SMCs proliferation in vitro was a positive correlation in GCV concentrations and the levels of tk expression. The proliferation of SMCs transfectered lacZ wasn’t restrained by GCV (P＜0.05). In mixed cell experiment there was at least 55% reduction in total cell number when as few as 10% of the cells express tk. Assessment of this “suicide gene strategy” in saphenous vein organ culture model demonstrated that veins treated with AdCMV.tk+GCV had a significant reduction at 14 days in the intimal thickness compared to control group (P＜0.01). ConclusionThe results suggest that adenovirusmediated gene transfer of tk along with GCV administration may be a useful strategy to treat the proliferation of intimal hyperplasia of transplanting saphenous veins. Bystander effects are amplified by AdCMV.tk/GCV gene therapy system.