Huber T S, Welling T H, Sarkar R, Messina L M, Stanley J C
Jobst Research Laboratories, Department of Surgery, University of Michigan Medical School, Ann Arbor, USA.
J Vasc Surg. 1995 Dec;22(6):795-803. doi: 10.1016/s0741-5214(95)70071-4.
Seeding prosthetic arterial grafts with genetically modified endothelial cells (ECs) has the potential to substantially improve graft function. However, preliminary applications suggest that grafts seeded with retrovirally transduced ECs yield a significantly lower percent surface coverage than those seeded with nontransduced ECs. The objective of this study was to test the hypothesis that canine ECs transduced with the human tissue plasminogen activator (tPA) gene would have a lower rate of adherence to pretreated expanded polytetrafluoroethylene (ePTFE) both in vitro and in vivo and that they would proliferate at a slower rate on pretreated ePTFE in vitro.
Early passage ECs derived from canine external jugular vein were transduced with the retroviral MFG vector containing the gene for human tPA. ECs exposed to media alone served as controls. Iodine 125-labeled ECs were seeded in vitro onto ePTFE graft segments pretreated with canine whole blood, fibronectin (50 micrograms/ml), or media alone, and the percent of ECs adherent at 1 hour were determined (n = 3). Additional tPA-transduced and -nontransduced ECs were grown for 10 days on either fibronectin (50 micrograms/ml)-pretreated ePTFE wafers or tissue culture plastic pretreated with gelatin (1%) or fibronectin (50 micrograms/ml), and the EC proliferation rates were determined (n = 3). Furthermore, 125I-labeled ECs were seeded onto fibronectin (50 micrograms/ml)-pretreated ePTFE graft segments implanted as carotid and femoral artery interposition grafts (n = 3). The grafts were harvested after 1 hour, and the percent of ECs adherent was determined.
Human tPA was detected by immunohistochemical staining in 61% +/- 5% of the transduced ECs and was expressed at 35.4 +/- 12.9 ng/hr/10(6) cells. Fibronectin and whole blood pretreatment of the ePTFE grafts led to greater EC adherence in vitro than did media alone (90.9% +/- 5.3% vs 77.8% +/- 5.8% vs 4.7% +/- 1.1%, p < or = 0.05). No significant difference in the rates of adherence or proliferation was seen in vitro between the transduced and nontransduced ECs. No significant difference in proliferation was found for the transduced ECs on the three matrices tested in vitro. In contrast, adherence of the transduced ECs in vivo was significantly lower than that of nontransduced ECs (64.7% +/- 2.1% vs 73.7% +/- 4.1%, p < or = 0.05) 1 hour after implantation.
Lower rates of surface endothelialization by genetically modified ECs in vivo do not appear to be due to an impaired capacity to initially adhere or proliferate on the synthetic graft but may result from decreased adherence after exposure to in vivo hemodynamic forces.
用基因修饰的内皮细胞（ECs）接种人工动脉移植物有显著改善移植物功能的潜力。然而，初步应用表明，用逆转录病毒转导的ECs接种的移植物的表面覆盖率明显低于用未转导的ECs接种的移植物。本研究的目的是检验以下假设：用人类组织型纤溶酶原激活剂（tPA）基因转导的犬ECs在体外和体内对预处理的膨体聚四氟乙烯（ePTFE）的黏附率较低，并且它们在体外预处理的ePTFE上的增殖速度较慢。
用含有人类tPA基因的逆转录病毒MFG载体转导源自犬颈外静脉的早期传代ECs。仅暴露于培养基的ECs作为对照。将碘125标记的ECs体外接种到用犬全血、纤连蛋白（50微克/毫升）或仅用培养基预处理的ePTFE移植物段上，并测定1小时时黏附的ECs百分比（n = 3）。将额外的经tPA转导和未转导的ECs在纤连蛋白（50微克/毫升）预处理的ePTFE薄片或用明胶（1%）或纤连蛋白（50微克/毫升）预处理的组织培养塑料上培养10天，并测定ECs增殖率（n = 3）。此外，将125I标记的ECs接种到作为颈动脉和股动脉间置移植物植入的纤连蛋白（50微克/毫升）预处理的ePTFE移植物段上（n = 3）。1小时后收获移植物，并测定黏附的ECs百分比。
通过免疫组织化学染色在61%±5%的转导ECs中检测到人类tPA，其表达量为35.4±12.9纳克/小时/10⁶细胞。ePTFE移植物的纤连蛋白和全血预处理导致体外ECs黏附比仅用培养基时更多（90.9%±5.3%对77.8%±5.8%对4.7%±1.1%，p≤0.05）。在体外，转导和未转导的ECs在黏附率或增殖率上没有显著差异。在体外测试的三种基质上，转导的ECs的增殖没有发现显著差异。相比之下，植入后1小时，转导的ECs在体内的黏附明显低于未转导的ECs（64.7%±2.1%对73.7%±4.1%，p≤0.05）。
基因修饰的ECs在体内较低的表面内皮化率似乎不是由于在合成移植物上最初黏附或增殖的能力受损，而是可能由于暴露于体内血流动力学力后黏附减少所致。
