Seeding of vascular grafts with genetically modified endothelial cells. Secretion of recombinant TPA results in decreased seeded cell retention in vitro and in vivo

Abstract

Background: Seeding of small-diameter vascular grafts with endothelial cells (ECs) genetically engineered to secrete fibrinolytic or antithrombotic proteins offers the potential to improve graft patency rates.

Methods and results: Sheep venous ECs were transduced with a retroviral vector encoding human tissue plasminogen activator (TPA). The ECs were seeded onto 4-mm-ID synthetic (Dacron) grafts. Retention of the seeded ECs was measured 2 hours after placement of the seeded grafts both in vitro in a nonpulsatile flow system and in vivo (in sheep) as femoral and carotid interposition grafts. On exposure to flow in vitro, ECs transduced with TPA were retained at a significantly lower rate (median, 67%) than either untransduced ECs (81%) or ECs transduced with a control retroviral vector producing beta-galactosidase (beta-Gal) (80%) (P < .05 for TPA versus either control). On implantation in vivo, ECs transduced with TPA were retained at a very low rate (median, 0%), significantly less than the retention of ECs transduced with the beta-Gal vector (32%; P < .00001). Decreased in vivo retention of ECs transduced with TPA correlated modestly with increased in vitro cellular passage level (r2 = .48; P < .0001) but not with in vivo blood flow rate (P = .45). Addition of the protease inhibitor aprotinin to the cell culture and graft perfusion media resulted in a significant (P < .05) increase in in vitro retention of ECs transduced with TPA.

Conclusions: Increased TPA expression significantly decreases seeded EC adherence in vitro and in vivo. Gene therapy strategies for decreasing graft thrombosis may require expression of antithrombotic molecules that lack proteolytic activity.