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 α 2 -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 (5×10 9 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.