Hlawaty Hanna, San Juan Aurélie, Jacob Marie-Paule, Vranckx Roger, Letourneur Didier, Feldman Laurent J
INSERM U698, Bioingénierie Cardiovasculaire, CHU Bichat Université Paris 13, 46 rue Henri Huchard, 75877 Paris Cedex 18, France.
J Gene Med. 2009 Jan;11(1):92-9. doi: 10.1002/jgm.1275.
Small interfering RNA (siRNA) delivery is a promising approach for the treatment of cardiovascular diseases. Matrix metalloproteinase (MMP) 2 over-expression in the arterial wall has been implicated in restenosis after percutaneous coronary intervention, as well as in spontaneous atherosclerotic plaque rupture. We hypothesized that in vivo local delivery of siRNA targeted at MMP2 (MMP2-siRNA) in the balloon-injured carotid artery of hypercholesterolemic rabbits may lead to inhibition of MMP2 expression.
Two weeks after balloon injury, 5 micromol/l of Tamra-tagged MMP2-siRNA, scramble siRNA or saline was locally injected in the carotid artery and incubated for 1 h.
Fluorescent microscopy studies showed the circumferential uptake of siRNA in the superficial layers of neointimal cells. MMP2 mRNA levels, measured by the real-time reverse transcriptase-polymerase chain reaction, was decreased by 79 +/- 25% in MMP2-siRNA- versus scramble siRNA-transfected arteries (p < 0.05). MMP2 activity, measured by gelatin zymography performed on the conditioned media of MMP2-siRNA versus scramble siRNA transfected arteries, decreased by 53 +/- 29%, 50 +/- 24% and 46 +/- 14% at 24, 48 and 72 h, respectively (p < 0.005 for all). No effect was observed on MMP9, pro-MMP9 and TIMP-2 levels.
The results obtained in the present study suggest that significant inhibition of gene expression can be achieved with local delivery of siRNA in the arterial wall in vivo.
小干扰RNA（siRNA）递送是一种有前景的心血管疾病治疗方法。动脉壁中基质金属蛋白酶（MMP）2的过表达与经皮冠状动脉介入术后再狭窄以及自发性动脉粥样硬化斑块破裂有关。我们假设，在高胆固醇血症兔球囊损伤的颈动脉中体内局部递送靶向MMP2的siRNA（MMP2-siRNA）可能会抑制MMP2表达。
球囊损伤两周后，将5微摩尔/升的Tamra标记的MMP2-siRNA、乱序siRNA或生理盐水局部注入颈动脉并孵育1小时。
荧光显微镜研究显示，新生内膜细胞表层有siRNA的周向摄取。通过实时逆转录-聚合酶链反应测量，MMP2-siRNA转染的动脉与乱序siRNA转染的动脉相比，MMP2 mRNA水平降低了79±25%（p<0.05）。通过对MMP2-siRNA与乱序siRNA转染动脉的条件培养基进行明胶酶谱分析来测量MMP2活性，在24、48和72小时时分别降低了53±29%、50±24%和46±14%（所有p<0.005）。未观察到对MMP9、前MMP9和TIMP-2水平有影响。
本研究获得的结果表明，在体内动脉壁中局部递送siRNA可实现对基因表达的显著抑制。
