Enhanced in vivo antithrombotic effects of endothelial cells expressing recombinant plasminogen activators transduced with retroviral vectors

Abstract

Background: The effects of regulating endothelial cell (EC) plasminogen activator production on thrombus accumulation in vivo are incompletely understood. By overexpressing plasminogen activators in ECs via gene transfer, the hypothesis was tested that increased levels of plasminogen activators inhibit the accumulation of thrombus in vivo.

Methods and results: Cultured baboon ECs transduced with human cDNAs for wild-type tissue plasminogen activator (TPA) or for glycosylphosphatidylinositol-anchored urokinase-type plasminogen activator (a-UPA) were seeded onto collagen-coated segments of vascular graft (collagen segments) and exposed overnight to flow using an in vitro perfusion circuit. The antigenic levels of TPA and UPA each increased 10-fold in the media perfusing the corresponding transduced ECs compared with untransduced ECs (P < or = .05 in both cases). In baboons the antithrombotic effects of TPA-transduced or a-UPA-transduced ECs were measured as 111In-platelet deposition and 125I-fibrin accumulation on collagen segments bearing sparsely attached ECs (tarnsduced versus untransduced) interposed in exteriorized arteriovenous femoral shunts. Platelet-rich thrombus formed on the collagen segments with fibrin-rich thrombus propagated distally. The presence of TPA-transduced or a-UPA-transduced ECs on collagen segments at a density of 25,000 ECs/cm2 decreased 111AIn-platelet deposition and 125I-fibrin accumulation on collagen surfaces compared with untransduced ECs present at equivalent density (P < .05 for platelet deposition with TPA-transduced ECs and P < .05 for platelet deposition on the propagated tail, as well as fibrin accumulation on the graft with a-UPA-transduced ECs). The systemic levels of fibrinopeptide A, thrombin-antithrombin complex, D-dimer, and both local and systemic levels of TPA and UPA were not increased by transduced ECs compared with untransduced ECs. The focal antithrombotic effects of transduced ECs appear to be due to local enhancement of thrombolysis.

Conclusions: ECs transduced with recombinant TPA and a-UPA enhance local antithrombotic activity in vivo. This strategy of attaching transduced ECs overexpressing plasminogen activators may be therapeutically useful by preventing thrombo-occlusive failure of implanted cardiovascular devices or mechanically denuded vessels.