Inhibition of MMP-2 gene expression with small interfering RNA in rabbit vascular smooth muscle cells.
Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs). Using small interfering RNA (siRNA), we evaluated the effect of MMP-2 inhibition in VSMCs in vitro and ex vivo. Rabbit VSMCs were transfected in vitro with 50 nmol/l MMP-2 siRNA or scramble siRNA. Flow cytometry and confocal microscopy showed cellular uptake of siRNA in approximately 80% of VSMCs. MMP-2 mRNA levels evaluated by real-time RT-PCR, pro-MMP-2 activity from conditioned culture media evaluated by gelatin zymography, and VSMC migration were reduced by 44 +/- 19%, 43 +/- 14%, and 36 +/- 14%, respectively, in MMP-2 siRNA-transfected compared with scramble siRNA-transfected VSMCs (P < 0.005 for all). Ex vivo MMP-2 siRNA transfection was performed 2 wk after balloon injury of hypercholesterolemic rabbit carotid arteries. Fluorescence microscopy showed circumferential siRNA uptake in neointimal cells. Gelatin zymography of carotid artery culture medium demonstrated a significant decrease of pro-MMP-2 activity in MMP-2 siRNA-transfected compared with scramble siRNA-transfected arteries (P < 0.01). Overall, our results demonstrate that in vitro MMP-2 siRNA transfection in VSMCs markedly inhibits MMP-2 gene expression and VSMC migration and that ex vivo delivery of MMP-2 siRNA in balloon-injured arteries reduces pro-MMP-2 activity in neointimal cells, suggesting that siRNA could be used to modify arterial biology in vivo.
Hlawaty
Hanna
H
Institut National de la Santé et de la Recherche Médicale U698, Université Paris 7, F75018 Paris, France.
San Juan
Aurélie
A
Jacob
Marie-Paule
MP
Vranckx
Roger
R
Letourneur
Didier
D
Feldman
Laurent J
LJ
D000800:Angioplasty, Balloon / Q000009:adverse effects; D000818:Animals; D020212:Carotid Artery Injuries / Q000201:enzymology / Q000209:etiology / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D002460:Cell Line; D002465:Cell Movement; D017077:Culture Media, Conditioned / Q000378:metabolism; D004195:Disease Models, Animal; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005240:Feasibility Studies; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic; D015316:Genetic Therapy / Q000379:methods*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000201:enzymology* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000201:enzymology* / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D011817:Rabbits; D013997:Time Factors; D014162:Transfection
D017077:Culture Media, Conditioned; D004792:Enzyme Precursors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020778:Matrix Metalloproteinase 2