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

• Published in: Cardiovascular research 2010-11, Vol.88 (2), p.367-375
• Main Authors: Eefting, Daniel, Seghers, Leonard, Grimbergen, Jos M., de Vries, Margreet R., de Boer, Hetty C., Lardenoye, Jan-Willem H.P., Jukema, J. Wouter, van Bockel, J. Hajo, Quax, Paul H.A.
• Format: Article
• Language: English
• Subjects: Animals; Apolipoprotein E3 - genetics; Aprotinin - biosynthesis; Aprotinin - genetics; Atherosclerosis - genetics; Atherosclerosis - metabolism; Atherosclerosis - pathology; Biological and medical sciences; Cardiology. Vascular system; Carotid Arteries - surgery; Cattle; Cell Line; Cell Proliferation; Disease Models, Animal; Electroporation; Fibrinolysin - antagonists & inhibitors; Fibrinolysin - metabolism; Gene therapy; Genetic Therapy - methods; Graft Occlusion, Vascular - genetics; Graft Occlusion, Vascular - metabolism; Graft Occlusion, Vascular - pathology; Graft Occlusion, Vascular - prevention & control; Humans; Hypercholesterolemia - genetics; Hypercholesterolemia - metabolism; Hypercholesterolemia - pathology; Hyperplasia; Male; Matrix Metalloproteinase Inhibitors; Matrix metalloproteinases; Matrix Metalloproteinases - metabolism; Medical sciences; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Plasmin(ogen); Receptors, Urokinase Plasminogen Activator - metabolism; Recombinant Fusion Proteins - biosynthesis; Saphenous Vein - metabolism; Saphenous Vein - pathology; Saphenous Vein - surgery; Surgery (general aspects). Transplantations, organ and tissue grafts. Graft diseases; Time Factors; Tissue Culture Techniques; Tissue Inhibitor of Metalloproteinase-1 - biosynthesis; Tissue Inhibitor of Metalloproteinase-1 - genetics; Transfection; Urokinase-Type Plasminogen Activator - biosynthesis; Urokinase-Type Plasminogen Activator - genetics; Vascular surgery; Vascular surgery: aorta, extremities, vena cava. Surgery of the lymphatic vessels; Vein graft disease; Venae Cavae - metabolism; Venae Cavae - pathology; Venae Cavae - transplantation
• ISSN: 0008-6363; 1755-3245
• DOI: 10.1093/cvr/cvq203

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 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. 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.