Local Gene Silencing of Monocyte Chemoattractant Protein-1 Prevents Vulnerable Plaque Disruption in Apolipoprotein E-Knockout Mice

Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine (CCL2), has been demonstrated to play important roles in atherosclerosis and becoming an important therapeutic target for atherosclerosis. The present study was undertaken to test the hypothesis that local RNAi of MCP-1 by site-specific delivery of adenovirus-mediated small hairpin RNA (shRNA) may enhance plaque stability and prevent plaque disruption in ApoE−/− mice. We designed an adenovirus-mediated shRNA against mouse MCP-1 (rAd5-MCP-1-shRNA). Male apolipoprotein E-knockout (ApoE−/−) mice (n = 120) were fed a high-fat diet and vulnerable plaques were induced by perivascular placement of constrictive collars around the carotid artery, intraperitoneal injection of lipopolysaccharide and stress stimulation. Mice were randomly divided into RNA interference (Ad-MCP-1i) group receiving local treatment of rAd5-MCP-1-shRNA suspension, Ad-EGFP group receiving treatment of rAd5-mediated negative shRNA and mock group receiving treatment of saline. Two weeks after treatment, plaque disruption rates were significantly lower in the Ad-MCP-1i group than in the Ad-EGFP group (13.3% vs. 60.0%, P = 0.01), and local MCP-1 expression was significantly inhibited in the Ad-MCP-1i group confirmed by immunostaining, qRT-PCR and western blot (P单核细胞趋化蛋白-1（MCP-1）是一种CC趋化因子（CCL2），现已被证实于动脉粥样硬化中发挥重要作用，同时也是动脉粥样硬化的重要治疗靶点。本研究旨在验证下述假说：通过腺病毒介导的短发夹RNA（small hairpin RNA，shRNA）靶向递送实现MCP-1的局部RNA干扰，可增强载脂蛋白E敲除（ApoE−/−）小鼠的斑块稳定性，并阻止斑块破裂。本研究设计了一种针对小鼠MCP-1的腺病毒介导短发夹RNA（rAd5-MCP-1-shRNA）。将120只雄性ApoE−/−小鼠给予高脂饲料喂养，并通过在颈动脉周围血管外放置缩窄环、腹腔注射脂多糖及应激刺激的方式诱导易损斑块形成。将小鼠随机分为三组：RNA干扰组（Ad-MCP-1i），接受rAd5-MCP-1-shRNA混悬液局部治疗；Ad-EGFP组，接受腺病毒介导的阴性对照shRNA治疗；空白对照组（mock组），接受生理盐水治疗。干预两周后，Ad-MCP-1i组的斑块破裂率显著低于Ad-EGFP组（13.3% vs. 60.0%，P=0.01）；经免疫染色、实时定量聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）及蛋白质印迹（western blot）证实，Ad-MCP-1i组的局部MCP-1表达受到显著抑制（P