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

Background-Smooth muscle cell migration, in addition to proliferation, cont ributes to a large extent to the neointima formed in humans after balloon a ngioplasty or bypass surgery. Plasminogen activator/plasmin-mediated proteo lysis is an important mediator of this smooth muscle cell migration. Here, we report the construction of a novel hybrid protein designed to inhibit th e activity of cell surface-bound plasmin, which cannot be inhibited by its natural inhibitors, such as alpha (2)-antiplasmin. This hybrid protein, con sisting of the receptor-binding amino-terminal fragment of uPA (ATF), linke d 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 o n neointima formation was tested in human saphenous vein organ cultures. In fection of human saphenous vein segments with Ad.CMV.ATF.BPTI (5 X 10(9) pf u/mL) resulted in 87.5+/-3.8% (mean+/-SEM, n=10) inhibition of neointima fo rmation after 5 weeks, whereas Ad.CMV.ATF or Ad.CMV.BPTI virus had only min imal or no effect on neointima formation. The efficacy of ATF.BPTI in vivo was demonstrated in a murine model for neointima formation. Neointima forma tion 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.B PTI, 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 hybri d protein that binds selectively to the uPA receptor and inhibits plasmin a ctivity directly on the cell surface is a powerful approach to inhibiting n eointima formation and restenosis.