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, 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 alpha(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.


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 (5x10(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.
Quax
P H
PH
Gaubius Laboratory TNO-PG, Leiden, Netherlands. pha.quax@pg.tno.nl
Lamfers
M L
ML
Lardenoye
J H
JH
Grimbergen
J M
JM
de Vries
M R
MR
Slomp
J
J
de Ruiter
M C
MC
Kockx
M M
MM
Verheijen
J H
JH
van Hinsbergh
V W
VW
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D007611:Aprotinin / Q000235:genetics / Q000502:physiology*; D001808:Blood Vessels / Q000502:physiology*; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D005263:Femoral Artery / Q000254:growth & development / Q000293:injuries; D005268:Femoral Vein / Q000166:cytology / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D009924:Organ Culture Techniques; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D012501:Saphenous Vein / Q000166:cytology; D014162:Transfection; D017539:Tunica Intima / Q000254:growth & development*; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D007611:Aprotinin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator