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(Circulation. 1996;93:1439-1446.)
© 1996 American Heart Association, Inc.

Articles

Seeding of Vascular Grafts With Genetically Modified Endothelial Cells

Secretion of Recombinant TPA Results in Decreased Seeded Cell Retention In Vitro and In Vivo

Peter F. Dunn, MD; Kurt D. Newman, MD; Michael Jones, MD; Izumi Yamada, MD; Vafa Shayani, MD; Renu Virmani, MD; David A. Dichek, MD

From the Molecular Hematology Branch (P.F.D., K.D.N., V.S., D.A.D.) and the Laboratory of Animal Medicine and Surgery (M.J., I.Y.), National Heart, Lung, and Blood Institute, Bethesda, Md; Department of Surgery, Children's National Medical Center, Washington, DC (K.D.N.); Armed Forces Institute of Pathology, Washington, DC (R.V.); and Gladstone Institute of Cardiovascular Disease, San Francisco, Calif (D.A.D.).

Correspondence to David A. Dichek, MD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100. E-mail david dichek@quickmail.ucsf.edu.

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 ß-galactosidase (ß-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 ß-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.

Key Words: carotid arteries • cells • plasminogen activators • viruses

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