A novel urokinase receptor-targeted inhibitor for plasmin and matrix metalloproteinases suppresses vein graft disease.
OBJECTIVE
Matrix metalloproteinases (MMP) and plasminogen activator (PA)/plasmin-mediated proteolysis, especially at the cell surface, play important roles in matrix degeneration and smooth muscle cell migration, which largely contributes to vein graft failure. In this study, a novel hybrid protein was designed to inhibit both protease systems simultaneously. MMP and plasmin activity were inhibited at the cell surface by this hybrid protein, consisting of the receptor-binding amino-terminal fragment (ATF) of urokinase-type PA, linked to both the tissue inhibitor of metalloproteinases (TIMP-1) and bovine pancreas trypsin inhibitor (BPTI), a potent protease inhibitor. The effect of overexpression of this protein on vein graft disease was studied.


RESULTS
A non-viral expression vector encoding the hybrid protein TIMP-1.ATF.BPTI was constructed and validated. Next, cultured segments of human veins were transfected with this vector. Expressing TIMP-1.ATF.BPTI in vein segments resulted in a mean 36 ± 14% reduction in neointima formation after 4 weeks. In vivo inhibition of vein graft disease by TIMP-1.ATF.BPTI is demonstrated in venous interpositions placed into carotid arteries of hypercholesterolaemic APOE*3Leiden mice. After 4 weeks, vein graft thickening was significantly inhibited in mice treated with the domains TIMP-1, ATF, or BPTI (36-49% reduction). In the TIMP-1.ATF.BPTI-treated mice, vein graft thickening was reduced by 67±4%, which was also significantly stronger when compared with the individual components.


CONCLUSIONS
These data provide evidence that cell surface-bound inhibition of the PA and MMP system by the hybrid protein TIMP-1.ATF.BPTI, overexpressed in distant tissues after electroporation-mediated non-viral gene transfer, is a powerful approach to prevent vein graft disease.
Eefting
Daniel
D
Department of Vascular Surgery, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
Seghers
Leonard
L
Grimbergen
Jos M
JM
de Vries
Margreet R
MR
de Boer
Hetty C
HC
Lardenoye
Jan-Willem H P
JW
Jukema
J Wouter
JW
van Bockel
J Hajo
JH
Quax
Paul H A
PH
D000818:Animals; D053318:Apolipoprotein E3 / Q000235:genetics; D007611:Aprotinin / Q000096:biosynthesis / Q000235:genetics; D050197:Atherosclerosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002339:Carotid Arteries / Q000601:surgery; D002417:Cattle; D002460:Cell Line; D049109:Cell Proliferation; D004195:Disease Models, Animal; D018274:Electroporation; D005341:Fibrinolysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D006083:Graft Occlusion, Vascular / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D006801:Humans; D006937:Hypercholesterolemia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006965:Hyperplasia; D008297:Male; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012501:Saphenous Vein / Q000378:metabolism* / Q000473:pathology / Q000601:surgery; D013997:Time Factors; D046509:Tissue Culture Techniques; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics; D014684:Venae Cavae / Q000378:metabolism* / Q000473:pathology / Q000637:transplantation
D053318:Apolipoprotein E3; D061965:Matrix Metalloproteinase Inhibitors; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D007611:Aprotinin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D020782:Matrix Metalloproteinases