Submitted on April 30, 2007
Accepted on September 17, 2007
Inhibition of MMP2 Gene Expression with Small Interfering RNA in Rabbit Vascular Smooth Muscle Cells
Hanna Hlawaty1, Aurelie San Juan1, Marie-Paule Jacob1, Roger Vranckx1, Didier Letourneur1, and Laurent Feldman2*
1 INSERM U698; CHU Bichat, Universite Paris 7; Universite Paris 13, Paris, France
2 AP-HP CHU Bichat, INSERM U698, Universite Paris 7, Paris, France
* To whom correspondence should be addressed. E-mail: laurent.feldman{at}bch.ap-hop-paris.fr.
Matrix metalloproteinase 2 (MMP2) is constitutively expressed in vascular smooth muscle cells (VSMCs). We evaluated the effect of MMP2 inhibition in VSMCs in vitro and ex vivo using small interfering RNA (siRNA). Rabbit VSMCs were transfected in vitro with 50 nmol/L MMP2-siRNA or scramble siRNA. Flow cytometry and confocal microscopy showed cellular uptake of siRNA in ~80% of VSMCs. MMP2 mRNA levels (real-time RT-PCR), pro-MMP2 activity in VSMC-conditioned culture media (gelatin zymography), and VSMC migration were reduced by 44±19%, 43±14%, and 36±14%, respectively, in MMP2-siRNA- vs. scramble siRNA-transfected VSMCs (P<0.005 for all). Two weeks after balloon injury of hypercholesterolemic rabbit carotid arteries, ex vivo MMP2-siRNA transfection was performed. Fluorescence microscopy showed circumferential siRNA uptake in neointimal cells. Gelatin zymography of carotid artery culture media demonstrated a 27±7% decrease of pro-MMP2 activity in MMP2-siRNA- vs. scramble siRNA-transfected arteries (P<0.01). In vitro MMP2-siRNA transfection in VSMCs markedly inhibits MMP2 gene expression and VSMC migration. Ex vivo delivery of MMP2-siRNA in balloon-injured arteries reduced pro-MMP2 activity in neointimal cells, suggesting that siRNA could be used to modify arterial biology in vivo.
