EFFECTS OF RETROVIRAL-MEDIATED TISSUE-PLASMINOGEN ACTIVATOR GENE-TRANSFER AND EXPRESSION ON ADHERENCE AND PROLIFERATION OF CANINE ENDOTHELIAL-CELLS SEEDED ONTO EXPANDED POLYTETRAFLUOROETHYLENE

Purpose: Seeding prosthetic arterial grafts with genetically modified endothelial cells (ECs) has the potential to substantially improve gra ft function. However, preliminary applications suggest that grafts see ded with retrovirally transduced ECs yield a significantly lower perce nt surface coverage than those seeded with nontransduced ECs. The obje ctive of this study was to test the hypothesis that canine ECs transdu ced with the human tissue plasminogen activator (tPA) gene would have a lower rate of adherence to pretreated expanded polytetrafluoroethyle ne (ePTFE) both in vitro and in vivo and that they would proliferate a t a slower rate on pretreated ePTFE in vitro. Methods: Early passage E Cs derived from canine external jugular vein were transduced with the retroviral MFG vector containing the gene for human tPA. ECs exposed t o media alone served as controls. Iodine 125-labeled ECs were seeded i n vitro onto ePTFE graft segments pretreated with canine whole blood, fibronectin (50 mu g/ml), or media alone, and the percent of ECs adher ent at 1 hour were determined (n = 3). Additional tPA-transduced and - nontransduced ECs were grown for 10 days on either fibronectin (50 mu g/ml)-pretreated ePTFE wafers or tissue culture plastic pretreated wit h gelatin (1%) or fibronectin (50 mu g/ml), and the EC proliferation r ates were determined (n = 3). furthermore, I-125-labeled ECs were seed ed onto fibronectin (50 mu g/ml)-pretreated ePTFE graft segments impla nted as carotid and femoral artery interposition grafts (n = 3). The g rafts were harvested after 1 hour, and the percent of ECs adherent was determined. Results: Human tPA was detected by immunohistochemical st aining 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 less than or equal to 0.05). No significant difference in the rates of adherence or proliferation was seen in vitro between the transduced and nontran sduced ECs. No significant difference in proliferation was found for t he transduced ECs on the three matrices tested in vitro. In contrast, adherence of the transduced ECs in vivo was significantly lower than t hat of nontransduced ECs(64.7% +/- 2.1% vs 73.7% +/- 4.1%, p less than or equal to 0.05) 1 hour after implantation. Conclusions: 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 pr oliferate on the synthetic graft but may result from decreased adheren ce after exposure to in vivo hemodynamic forces.