Adenoviral expression of a urokinase receptor - Targeted protease inhibitor inhibits neointima formation in murine and human blood vessels

Abstract

Background - Smooth muscle cell migration, in addition to proliferation, contributes to a large extent to the neointima formed in humans after balloon angioplasty or bypass surgery. Plasminogen activator/plasmin-mediated proteolysis is an important mediator of this smooth muscle cell migration. Here, we report the construction of a novel hybrid protein designed to inhibit the activity of cell surface-bound plasmin, which cannot be inhibited by its natural inhibitors, such as α2-antiplasmin. This hybrid protein, consisting of the receptor-binding amino-terminal fragment of uPA (ATF), linked to the potent protease inhibitor bovine pancreas trypsin inhibitor (BPTI), can inhibit plasmin activity at the cell surface. Methods and Results - The effect of adenovirus-mediated ATF.BPTI expression on neointima formation was tested in human saphenous vein organ cultures. Infection of human saphenous vein segments with Ad.CMV.ATF.BPTI (5 × 109 pfu/mL) resulted in 87.5±3.8% (mean±SEM, n=10) inhibition of neointima formation after 5 weeks, whereas Ad.CMV.ATF or Ad.CMV.BPTI virus had only minimal or no effect on neointima formation. The efficacy of ATF.BPTI in vivo was demonstrated in a murine model for neointima formation. Neointima formation in the femoral artery of mice, induced by placement of a polyethylene cuff, was strongly inhibited (93.9±2%) after infection with Ad.CMV.mATF.BPTI, a variant of ATF.BPTI able to bind specifically to murine uPA receptor; Ad.CMV.mATF and Ad.CMV.BPTI had no significant effect. Conclusions - These data provide evidence that adenoviral transfer of a hybrid protein that binds selectively to the uPA receptor and inhibits plasmin activity directly on the cell surface is a powerful approach to inhibiting neointima formation and restenosis.