O63	Non-viral, electroporation mediated gene transfer of TIMP-1.ATF, a cell-surface directed MMP inhibitor, suppresses intimal hyperplasia in vein grafts more efficiently than TIMP-1 in vivo.
1D.Eefting, 2M.De Vries, 2J.Grimbergen, 1H.Van Bockel, 2P.Quax
1Leiden University Medical Center, Leiden, NL; 2TNO Quality of Life, Leiden, NL.

Proteases of the plasminogen activator (PA) system and of the matrix metalloproteinase (MMP) system play a pivotal role in SMC migration. SMC migration and proliferation are key players in the development of intimal hyperplasia, the major cause of vein graft failure. In earlier studies we demonstrated that inhibition of both protease systems simultaneously with viral gene delivery of the hybrid protein TIMP-1.ATF, reduces SMC migration and neointima formation in vitro more efficiently than TIMP-1 separately (human saphenous vein model). The hybrid protein TIMP-ATF consists of the TIMP-1 domain, as a MMP inhibitor, linked to the uPA-receptor binding aminoterminal fragment of urokinase (ATF). By binding to the uPA-receptor this protein not only will anchor the TIMP-1 moiety directly to the cell surface, it also will prevent the local activation of plasminogen by blocking the binding of uPA to its receptor. Non-viral gene transfer by electroporation was used to avert major disadvantages of viral gene delivery such as immune responses and short-term expression. Here, we have used electroporation mediated delivery of TIMP-1.ATF and TIMP-1 plasmids to reduce intimal hyperplasia in a murine model for vein graft disease, in which a venous interposition of a donor mouse is placed in the carotid artery of a recipient mouse. pcDNA3.1-TIMP-1, TIMP-1.ATF or a Luciferase control plasmid was electroporated in both calf muscles of hypercholesterolemic ApoE3Leiden mice (n=8), one day prior to vein graft surgery. Four weeks after electroporation and surgery, vein grafts were harvested and intimal hyperplasia was measured. Intimal hyperplasia was inhibited with 63% in the TIMP-1.ATF treated animals, whereas TIMP-1 reduced intimal hyperplasia with 53% (p<0.001 and p=0.002, respectively). Conclusion These data show that intramuscular electroporation of TIMP-1 and TIMP-1.ATF inhibits neointima formation in distant vein grafts in the carotid artery in the mouse. Furthermore, binding of TIMP.ATF hybrid protein to the uPA receptor at the cell surface enhances the inhibitory effect of TIMP-1 on neointima formation in vitro as well as in vivo.

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