O215	Gene silencing of MMP-2 or MMP-9 inhibits human saphenous vein smooth muscle cell invasion.
K.E.Porter, N.A.Turner, K.T.Hall
University of Leeds, Leeds, GB.

Introduction: Neointimal hyperplasia in saphenous vein (SV) bypass grafts is a major clinical problem that results in patency rates of ~50% after 10 years. A prerequisite for neointima formation is activation of matrix metalloproteinases (MMPs), enzymes that remodel the extracellular matrix. Gelatinases (MMP-2 and MMP-9) are able to degrade all the components of the basement membrane, an important requirement for smooth muscle cell (SMC) invasion and neointima formation. Animal studies using knockout or overexpression techniques support a role for both MMP-2 and -9 in SMC invasion and neointima formation. Methods: We designed small inhibitory RNA molecules (siRNA) to investigate the effect of MMP-2 and -9 gene silencing on human SV-SMC invasion in vitro. 21-mer siRNA oligonucleotides were transfected (>95% efficiency) into cultured SV-SMC using Lipofectamine-2000. Using a modified Boyden chamber assay, in which degradation of Matrigel basement membrane matrix is a prerequisite for SMC invasion, we investigated the effect of transfection with either control siRNA oligonucleotides or siRNA oligonucleotides specific for either MMP-2 or -9 upon cell invasion towards a chemotactic stimulus (15 ng/ml PDGF-BB + 20 ng/ml IL-1α) over a 24h period. Membranes were fixed and stained to quantify invasive cells and gelatin zymography was performed on cell supernatants to quantify MMP-2 and -9 secretion. Results: In SMC transfected with either MMP-2 or -9 siRNA, secretion of MMP-2 and -9 was selectively decreased to 12.1±0.6% and 7.5±2.5% of that observed in mock-transfected cells, respectively. Importantly, MMP-2 siRNA did not reduce MMP-9 secretion, and MMP-9 siRNA did not reduce MMP-2 secretion. Silencing of either MMP-2 or -9 decreased cell invasion to 40.5±15.5% and 10.7±5.4% of that observed in mock-transfected cells respectively. SMC transfected with control siRNA showed no reduction in secreted MMP-2 or -9 (161±39.3% and 109.8±18.2% of mock-transfected, respectively) and no reduction in cellular invasion (97.6±12.4% and 86.2±8.9% respectively). Conclusions: Selective silencing of either MMP-2 or -9 gene expression is sufficient to significantly inhibit human SV-SMC invasion in vitro. Our data highlight the potential of targeted MMP-2 or -9 gene silencing as a therapeutic strategy for prevention of bypass graft failure.

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