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A novel urokinase receptor-targeted inhibitor for plasmin and matrix metalloproteinases suppresses vein graft disease

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Author: Eefting, D. · Seghers, L. · Grimbergen, J.M. · Vries, M.R. de · Boer, H.C. de · Lardenoye, J.W.H.P. · Jukema, J.W. · Bockel, J.H. van · Quax, P.H.A.
Institution: TNO Kwaliteit van Leven
Source:Cardiovascular Research, 2, 88, 367-375
Identifier: 425144
doi: doi:10.1093/cvr/cvq203
Keywords: Health · Biomedical Research · Gene therapy · Matrix metalloproteinases · Plasmin(ogen) · Vascular surgery · Vein graft disease · apolipoprotein E3 · aprotinin · hybrid protein · matrix metalloproteinase · matrix metalloproteinase inhibitor · plasmin · plasmin inhibitor · plasminogen activator · tissue inhibitor of metalloproteinase 1 · tissue inhibitor of metalloproteinase 1 amino terminal fragment of urokinase type plasminogen activator bovine pancreas trypsin inhibitor · unclassified drug · urokinase receptor · animal cell · animal experiment · animal model · animal tissue · article · carotid artery · cell surface · controlled study · electroporation · expression vector · female · gene overexpression · gene targeting · genetic transfection · human · human tissue · hypercholesterolemia · in vivo culture · intima · male · mouse · nonhuman · nonviral gene delivery system · priority journal · vein graft disease


Aims 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. Methods and resultsA 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.Conclusion 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. © 2010 The Author.