Effect of rapamycin on gene expresssion in mouse model of aortic dissection

Aortic dissection (AD) is a medical emergency that leads to sudden death. Effective medical therapy is not available because molecular mechanism of AD is poorly understood. We performed the transcriptome analysis in mouse AD model that was created by infusion of beta-aminopropionitrile, a collagen crosslink inhibitor, and angiotensin II. Bayesian network analysis of the transcriptome revealed several distinct gene clusters that were tightly co-regulated during AD development. Functional annotation analysis revealed that each gene cluster has distinct function, namely, cell proliferation, inflammation, cell locomotion and adhesion, and muscle differentiation. Among the gene clusters, cell proliferation cluster was the first to be activated during AD development. Cell proliferation before AD development was confirmed by immunoblotting of G1 cyclins. Immunostaining for Ki67 and cell markers revealed the proliferation of smooth muscle cells, fibroblasts, and monocytes in aortic tissue before AD development. Treatment of mice with rapamycin, an mTOR inhibitor, suppressed cell proliferative response in AD model. Rapamycin completely prevented AD when given before AD development, and effectively suppressed progression when given after AD development. Interestingly, transcriptome analysis showed that rapamycin strongly suppressed cell proliferation and muscle differentiation clusters, but its effect on inflammation cluster was modest. We concluded that mTOR constitutes an essential part of the molecular pathogenesis of AD by regulating cell proliferation, and represents a new therapeutic target. Aortic dissection model was created by continuous infusion of 150 mg/kg/day beta-aminopropionitrile, a collagen crosslink inhibitor, and 1 ug/kg/min angiotensin II (B+A). Rapamycin (2 mg/kg/day) or dimethylsulfoxide (DMSO, vehicle) was intraperitoneally administored to mice once a day. Four experimental groups were set; DMSO alone (Pre), rapamycin alone, B+A+DMSO, and B+A+rapamycin, each of which contained 3 mice. Aortic samples were obtained at 3 days of experiment.

主动脉夹层（Aortic dissection, AD）是一种可引发猝死的临床急危重症。目前尚无有效的治疗手段，原因在于人们对主动脉夹层的分子发病机制尚缺乏深入认知。本研究采用β-氨基丙腈（beta-aminopropionitrile，一种胶原交联抑制剂）与血管紧张素II（angiotensin II）持续灌注构建小鼠主动脉夹层模型，并对该模型开展转录组分析。对转录组数据的贝叶斯网络分析显示，在主动脉夹层发生发展过程中，存在多个协同调控的特征性基因簇。功能注释分析表明，各基因簇分别对应独特的生物学功能：细胞增殖、炎症反应、细胞迁移与黏附，以及肌肉分化。在这些基因簇中，细胞增殖簇是主动脉夹层发生过程中最早被激活的基因模块。通过对G1周期蛋白（G1 cyclins）的免疫印迹实验，证实了主动脉夹层发生前的细胞增殖现象。针对Ki67与细胞标志物的免疫染色结果显示，主动脉夹层发生前，主动脉组织内的平滑肌细胞（smooth muscle cells）、成纤维细胞（fibroblasts）及单核细胞（monocytes）均发生增殖。使用雷帕霉素（rapamycin，mTOR抑制剂）处理小鼠，可抑制主动脉夹层模型中的细胞增殖反应。若在主动脉夹层发生前给予雷帕霉素，可完全阻止疾病发生；若在发病后给药，则可有效抑制疾病进展。值得注意的是，转录组分析显示雷帕霉素可显著抑制细胞增殖簇与肌肉分化簇的表达，但对炎症簇的调控作用较为微弱。本研究证实，mTOR通过调控细胞增殖参与主动脉夹层的分子发病过程，是潜在的全新治疗靶点。本研究通过持续灌注150 mg/kg/天的β-氨基丙腈（胶原交联抑制剂）与1 μg/kg/分钟的血管紧张素II（记为B+A组）构建主动脉夹层模型。每日腹腔注射雷帕霉素（2 mg/kg/天）或二甲基亚砜（dimethylsulfoxide, DMSO，溶剂对照）处理小鼠。实验共设置4组：单纯溶剂对照组（Pre组）、单纯雷帕霉素组、B+A+溶剂对照组，以及B+A+雷帕霉素组，每组包含3只小鼠。于实验第3天采集主动脉组织样本。