Zhong Lin, Chen Wen Qiang, Ji Xiao Ping, Zhang Mei, Zhao Yu Xia, Yao Gui Hua, Zhang Peng Fei, Zhang Cheng, Zhang Yun
The Key Laboratory of Cardiovascular Remodelling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu Hospital, Jinan, Shandong, China.
J Cell Mol Med. 2008 Dec;12(6A):2362-71. doi: 10.1111/j.1582-4934.2008.00261.x. Epub 2008 Feb 4.
Active inflammation is an important feature of vulnerable plaques, and monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that promotes monocyte-endothelium binding and initiates inflammation. We aimed to determine whether dominant-negative mutation of MCP-1 could reverse atherosclerotic lesion progression and prevent vulnerable plaques from rupture regardless of serum lipid levels. The mutant MCP-1 was produced by deletion of the N-terminal amino acids 2 to 8 (7ND), and a eukaryotic expression vector plRES-EGFP-7ND was constructed. The transwell chamber was used to assay chemotaxis of monocytes in vitro. Thirty New Zealand white rabbits underwent balloon-induced abdominal aortic endothelial injury and were randomly divided into control group without gene intervention (group A, n=10), plRES-EGFP-7ND treatment group (group B, n=10) and empty vector treatment group (group C, n=10). All rabbits were fed a diet of 1% cholesterol for 8 weeks, and then group A rabbits were killed, whereas groups B and C rabbits received an intramuscular injection of plRES-EGFP-7ND and an empty lipofectamine, respectively, and remained on a high cholesterol diet for 4 weeks. At the end of week 12, groups B and C rabbits underwent pharmacological triggering by injection with Chinese Russellis viper venom and histamine. Serum lipids and inflammatory markers were measured, and high-frequency ultra-sonography and intravascular ultrasound imaging were performed. Immunohistochemistry and RT-PCR were used to examine expression of inflammatory markers in the plaques. In vitro transfection of plRES-EGFP-7ND resulted in a significant inhibition of monocyte chemotaxis (P<0.05) and in vivo transfection of plRES-EGFP-7ND significantly increased the thickness of the fibrous caps and decreased plaque vulnerability index. The incidence of plaque rupture in group B was 0% as compared with 56% in the empty vector treatment group (P<0.05). The serum levels and expression of inflammatory markers were significantly reduced in group B. In conclusion, PIRES-EGFP-7ND transfection effectively inhibits plaque inflammation, reverses plaque progression and prevents vulnerable plaques from rupture. These therapeutic effects are independent of serum lipid levels and demonstrate that inhibition of plaque inflammation alone without lipid lowering can stabilize vulnerable plaques.
活动性炎症是易损斑块的一个重要特征，单核细胞趋化蛋白-1（MCP-1）是一种关键的趋化因子，可促进单核细胞与内皮细胞结合并引发炎症。我们旨在确定MCP-1的显性负性突变是否能逆转动脉粥样硬化病变进展，并预防易损斑块破裂，而不考虑血脂水平。突变型MCP-1通过缺失N端第2至8个氨基酸（7ND）产生，并构建了真核表达载体plRES-EGFP-7ND。采用Transwell小室检测体外单核细胞的趋化性。30只新西兰白兔接受球囊诱导的腹主动脉内皮损伤，随机分为未进行基因干预的对照组（A组，n = 10）、plRES-EGFP-7ND治疗组（B组，n = 10）和空载体治疗组（C组，n = 10）。所有兔子喂食含1%胆固醇的饲料8周，然后处死A组兔子，而B组和C组兔子分别肌肉注射plRES-EGFP-7ND和空脂质体，并继续高脂饮食4周。在第12周结束时，对B组和C组兔子注射中华眼镜蛇毒和组胺进行药理激发。检测血脂和炎症标志物，并进行高频超声和血管内超声成像。采用免疫组织化学和RT-PCR检测斑块中炎症标志物的表达。体外转染plRES-EGFP-7ND可显著抑制单核细胞趋化性（P < 0.05），体内转染plRES-EGFP-7ND可显著增加纤维帽厚度并降低斑块易损指数。B组斑块破裂发生率为0%，而空载体治疗组为56%（P < 0.05）。B组炎症标志物的血清水平和表达显著降低。总之，PIRES-EGFP-7ND转染可有效抑制斑块炎症，逆转斑块进展并预防易损斑块破裂。这些治疗效果独立于血脂水平，表明单独抑制斑块炎症而不降低血脂可稳定易损斑块。
