P316	Small interfering RNA for in vitro and ex vivo MMP2 gene regulation in vascular smooth muscle cells.
H.Hlawaty, A.San Juan, M-P.Jacob, R.Vranckx, D.Letourneur, L.Feldman, on behalf of Hemostase, Bioingenierie et Remodelages Vasculaires
INSERM U698, CHU X. Bichat, Paris, FR.

Background - Matrix metalloproteinase 2 (MMP2) is constitutively expressed in vascular smooth muscle cells (SMCs) and its active form plays a key role in atherosclerosis and restenosis. The aim of this study was to evaluate the ability of small interfering RNA (siRNA) to silence MMP2 gene expression in SMCs in vitro and ex vivo. Methods and Results - Rabbit aortic SMCs were transfected in vitro with FITC-tagged siRNA against MMP2 mRNA (MMP2-siRNA, 50nM) in presence of jet SI®-ENDO reagent. Cellular uptake of MMP2-siRNA in ~70% of SMCs was demonstrated by confocal microscopy and flow-cytometry analysis. MMP2 activity (gelatin zymography) and lactate dehydrogenase (LDH) concentration (a cytotoxicity index) were measured in the culture medium over time. MMP2 activity (Fig.1) was reduced by 73%, 68% and 55% at 24h, 48h and 72h respectively after transfection (2-way ANOVA, P=0.005, all vs. non-transfected SMCs), without significant effect on cell viability. Using similar conditions, an unrelated siRNA had no effect on MMP2 activity. In a scrape wound assay, SMCs migration was reduced (2-way ANOVA, P<0.0005) in MMP2-siRNA-transfected vs. non-transfected SMCs at 24h (0.17mm±0.071 vs. 0.32mm±0.053) and at 48h (0.37mm±0.071 vs. 0.51mm±0.053). In addition, ex vivo transfection of balloon-injured rabbit aortas with MMP2-siRNA/jet SI®-ENDO (50nM) resulted in efficient cellular uptake and 30% decrease of MMP2 activity in the culture medium 24h after transfection. Conclusions - MMP2-siRNA reduced significantly MMP2 activity and cell migration in rabbit SMCs in vitro. Ex vivo data suggest that RNA interference is a promising technique for in vivo gene silencing in the arterial wall. [SMOOTH MUSCLE CELLS * GENE THERAPY * SMALL INTERFERING RNA* METALLOPROTEASES]

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