Adventitial delivery of platelet-derived endothelial cell growth factor gene prevented intimal hyperplasia of vein graft
Adventitial delivery of platelet-derived endothelial cell growth factor gene prevented intimal hyperplasia of vein graft
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DOI：
10.1016/j.jvs.2008.07.029
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发表时间：
2008-12-01
影响因子：
4.3
通讯作者：
Ihaya, Akio
中科院分区：
文献类型：
--
作者：
Handa, Mitsuteru;Li, Wei;Ihaya, Akio
背景资料：血小板衍生的内皮细胞生长因子（PD-ECGF），也称为胸苷磷酸化酶（TP），据报道抑制血管平滑肌细胞（VSMC）的迁移和增殖。我们假设，外膜给药的PD-ECGF/TP基因将抑制内膜增生，防止静脉移植failure.Methods：该研究使用了68只雌性家兔。采用袖套法将兔颈静脉移植于颈动脉。为了确定血管壁基因转移效率，将含有编码LacZ基因的质粒载体和不同浓度的胰蛋白酶（0%、0.1%、0.25%和0.5%，每组n = 5）的泊洛沙姆水凝胶（20%）应用于静脉移植物的外膜。7天后通过X-gal染色评价基因转移效率。另外48只兔接受含有0.25%胰蛋白酶和人PD-ECGF/TP基因、LacZ基因或生理盐水的泊洛沙姆水凝胶（20%）。在移植后2周和8周评价内膜厚度（每组在每个时间点的内膜厚度= 8）。通过逆转录-聚合酶链反应、免疫印迹分析和免疫组织化学染色检测转基因表达。免疫组织化学染色也被用来确定VSMC增殖，血红素氧合酶-1的表达，和巨噬细胞infiltration.Results：胰蛋白酶掺入到泊洛沙姆水凝胶显着增加血管壁基因转移。0.25%和0.5%的胰蛋白酶在相同水平下导致更高的基因转移，而不影响内膜增生和炎症;因此，0.25%浓度的胰蛋白酶用于后续实验。与LacZ和生理盐水组相比，接受PD-ECGF/TP基因的移植物在治疗后2周和8周显著降低内膜厚度。PD-ECGF/TP处理组VSMC增殖率较对照组低。PD-ECGF/TP转基因移植物的组织学检查显示血红素加氧酶-1的高表达，这已被报道抑制VSMC增殖，表明血红素加氧酶-1可能在PD-ECGF/TP对VSMC的抑制作用中起重要作用。结论：采用泊洛沙姆水凝胶和低浓度胰蛋白酶，建立了一种安全、高效的基因转移方法。血管移植物外膜应用PD-ECGF/TP基因可显著减少新生内膜增生。我们的数据表明，移植后外膜递送PD-ECGF/TP基因可能是预防静脉移植失败的有希望的方法。（J Vasc Surg 2008;48：1566-74.）
Background: Platelet-derived endothelial cell growth factor (PD-ECGF), also known as thymidine phosphorylase (TP) reportedly inhibits vascular smooth muscle cells (VSMCs) migration and proliferation. We hypothesized that adventitial administration of the PD-ECGF/TP gene will suppress intimal hyperplasia and prevent vein graft failure.Methods: The study used 68 female rabbits. Rabbit jugular vein was autogenously transplanted into carotid artery with a cuff anastomotic technique. To define vascular wall gene transfer efficiency, poloxamer hydrogel (20%) containing plasmid vector encoding the LacZ gene and different concentrations of trypsin (0%, 0.1%, 0.25%, and 0.5%, n = 5 for each group) was applied to the adventitia of the vein graft. Gene transfer efficiency was evaluated 7 days later by X-gal staining. An additional 48 rabbits received poloxamer hydrogel (20%) containing 0.25% trypsin and the human PD-ECGF/TP gene, LacZ gene, or saline. Intima thickness was evaluated at 2 and 8 weeks after grafting (it = 8 for each group at each time point). Transgene expression was examined by reverse transcriptase-polymerase chain reaction, immunoblotting assay, and immunohistochemical staining. Immunohistochemical staining was also used to determine VSMC proliferation, heme oxygenase-1 expression, and macrophage infiltration.Results: Incorporation of trypsin into the poloxamer hydrogel significantly increased vessel wall gene transfer. Trypsin at 0.25% and 0.5% resulted in higher gene transfer at the same level without effecting intimal hyperplasia and inflammation; thus, trypsin at 0.25% concentration was used for subsequent experiments. Compared with the LacZ and saline groups, grafts receiving the PD-ECGF/TP gene significantly reduced intimal thickness at 2 and 8 weeks after treatment. The ratio of proliferative VSMC was lower in PD-ECGF/TP treated grafts. Histologic examination of the PD-ECGF/TP transgene grafts demonstrated high expression of heme oxygenase-1, which has been reported to inhibit VSMC proliferation, suggesting that heme oxygenase-1 may be important in the inhibition effect of PD-ECGF/TP on VSMC. No neoplastic or morphologic changes were found in the remote organs.Conclusions: A safe and highly efficient gene transfer method was developed by using poloxamer hydrogel and a low concentration of trypsin. Neointimal hyperplasia was significantly reduced by adventitial application of the PD-ECGF/TP gene to the vein graft. Our data suggest that adventitial delivery of the PD-ECGF/TP gene after grafting may be promising method for preventing vein graft failure. (J Vasc Surg 2008;48:1566-74.)