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.
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 evaluate the effect of low power red laser illumination on the intimal proliferative response in vein graft models.MethodsAutogenous vein graft models were established in 80 rats by transplanting jugular vein to carotid artery by end to end anastomosis, and were randomized into two groups: control group (graft nonilluminated), laser illumination group (0.9 J/cm2).The grafted veins were harvested at 3,7,14 or 28 day respectively after operation. IH (intimal hyperplasia) and SMC (smooth muscle cell) proliferations were pathologically and immunohistochemically observed and analyzed by computer digitizing system. ResultsThere were no significant differences in the intimal average thickness and the areas of lumen between two groups for 3 day. Laser group was significantly lower than the control in both the intimal average thickness and the stenosis of lumen at 7 day,14 day and 28 day (P＜0.05).Immunohistochemical analysis of PCNA indicate the decreased positive cell in laser group compared with the control group (P＜0.01).ConclusionThese preliminary results demonstrate that a certain density of low power red laser illumination in vein graft inhibits SMC proliferation and neointimal hyperplasia in rat.
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.
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.
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.
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.
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.
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 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.