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.
ObjectiveTo evaluate the effect of human heme oxygenase 1 augmentation in vein grafts by adenoviral mediated gene transfer of heme oxygenase 1 (Ad hHO 1) on intimal hyperplasia.MethodsTwenty one Japanese white rabbits were divided into three groups: control group, Ad null control group, and Ad hHO 1 group(each group 7 rabbits). During the operation of rabbits jugular vein into carotid artery interposition grafting, harvested rabbit jugular vein segments were exposed for 30min at room temperature to heparin saline, recombinant replication deficient adenovirus encoding hHO 1(Ad hHO 1, 1× 10 9pfu/ml), and nude recombinant replication deficient adenovirus (Ad null, 1×10 9pfu/ml). Quantitative histological studies of the vein segments were performed 28 days after operation. Protein of hHO 1 was detected with method of immunohistochemical staining(S P) in 14 days and 28 days after operation.ResultsThe average intimal thickness, medial thickness and intimal to medial(I/M) ratio were calculated for each group 28 days after bypass operation. Compared to intimal thickness, I/M ratio of control group veins and Ad null group veins,Ad hHO 1 group veins decreased significantly( P lt;0.01). There was no statistically difference in medial thickness ( P gt;0 05). Strong staining of hHO 1 was detected in vein grafts wall of Ad hHO 1 group.ConclusionAd hHO 1 gene therapy may inhibit intimal hyperplasia of vein grafts in rabbits.
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 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.
In order to investigate the effect of vascular beds on the vascular wall of autogenously grafted vein, femoral veins were reversely placed in between the cut ends of collateral femoral arteries in 11 dogs with atraumatic technique. The grafted veins were covered with vivid muscle or skin respectively after being assured to be patent, and investigated by histomorphologic method and computerized image analysis technique at postoperative intervals of 1 week, 4 weeks and 16 weeks. The results showed that: 1. One graft developed pseudoaneurysm at 1 week, and two grafts were occulded in skin-covered group, whereas, no complications occurred in muscle-covered group. 2. Intimal thickening of grafts in skin-covered group was much more obvious than that in the muscle-covered group (P lt; 0.05). 3. The relative contents of microstructural components of the graft wall showed no significant difference quantitatively between the two groups. So, the conclusion was: 1. Subcutaneous transplantation appeared to be a potential causative factor in inducing short-term excessive dilatation and long-term intimal hyperplasia of vein graft. 2. Muscular covering is of priority in blood vessel graft.
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.
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.
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 assess the effect of topical appl ication of 5-fluorouracil (5-FU) on intimal hyperplasia in rabbit vein graft. Methods Sixty-four male New Zealand white rabbits, aged 5 months and weighing 2.8-3.0 kg, were randomly divided into group A, B, C, and D (n=16 rabbits per group). Artery defect model was establ ished by cutting about 1 cm artery from the middle part of the dissociated left common carotid artery. A section about 3 cm was cut from the right external jugular vein, and the harvested vein was inverted and end-to-end anastomosed to the artery defect with 9-0 non-traumatic suture. After anastomosis, the extima of the grafted veins in group A, B, and C was completely wrapped with cotton sheet (12 mm × 30 mm × 1 mm in size) immersed by 5-FU at a concentration of 50.0, 25.0, and 12.5 mg/mL, respectively, and eachvein was treated 5 times (1 minute at a time). In group D, the extima of the graft veins was treated with normal sal ine instead of 5-FU. The grafted veins were obtained 1, 2, 4, and 6 weeks after operation, HE staining and Masson staining were preformed for histological changes of grafted vein wall, prol iferating cell nuclear antigen (PCNA) immunohistochemistry staining and TUNEL label ing staining were conducted for prol iferation and apoptosis of smooth muscle cell of the grafted vein, and transmission electron microscope observation was performed for cellular ultrastructure. Results The HE staining, Masson staining, and PCNA immunohistochemistry staining showed that the thickness of intima in group A and B was obviously less than that in group C and D at 1, 2, 4, and 6 weeks after operation, and the prol iferation cells in group A and B were less than that in group C and D at 1, 2, and 4 weeks after operation. The thickness of the intima, the degree of intima hyperplasia, the degree of vessel lumen stenosis of four groups at different time points were as follows: at 1 week after operation, group A [(12.69 ± 1.68) μm, 0.73 ± 0.05, 0.025 ± 0.003], group B [(17.52 ± 2.01) μm, 0.86 ± 0.06, 0.027 ± 0.004], group C [(21.92 ± 1.85) μm, 1.06 ± 0.09, 0.036 ± 0.006] and group D [(26.45 ± 3.86) μm, 1.18 ± 0.08, 0.041 ± 0.005]; at 2 weeks after operation, group A [(24.61 ± 2.91) μm, 0.86 ± 0.06, 0.047 ± 0.003], group B [(37.28 ± 2.78) μm, 1.17 ± 0.09, 0.060 ± 0.004], group C [(46.52 ± 2.25) μm, 1.44 ± 0.08, 0.073 ± 0.003], and group D [(52.07 ± 3.29) μm, 1.45 ± 0.05, 0.081 ± 0.006]; at 4 weeks after operation, group A [(61.09 ± 6.84) μm, 1.38 ± 0.08, 0.106 ± 0.007], group B [(63.61 ± 8.25) μm, 1.40 ± 0.07, 0.107 ± 0.010], group C [(80.04 ± 7.65) μm, 1.64 ± 0.07, 0.129 ± 0.011], and group D [(84.45 ± 9.39) μm, 1.68 ± 0.10, 0.139 ± 0.014]; at 6 weeks after operation, group A [(65.27 ± 5.25) μm, 1.46 ± 0.07, 0.113 ± 0.005], group B [(65.82 ± 7.12) μm, 1.45 ± 0.05, 0.112 ± 0.011], group C [(84.45 ± 9.39) μm, 1.69 ± 0.09, 0.135 ± 0.007], and group D [(87.27 ± 8.96) μm, 1.76 ± 0.05, 0.140 ± 0.012]. Group A and B were inferior to group C and D in terms of the above three parameters and cell prol iferation index 1, 2 and 4 weeks after operation (P lt; 0.05). Group A and B were superior to group C and D in terms of cell apoptosis index of intima and media 1 and 2 weeks after operation (P lt; 0.05). Transmission electron microscope observation showed that the synthetic cell organelles such as rough endoplasmic reticulum, golgi apparatus, and ribosome in group A and B were obviously less than those in group C and D (P lt; 0.05). Conclusion Topicalappl ication of 5-FU can effectively inhibit intima hyperplasia of the vein grafts.