Single-cell transcriptomic analysis of Smad3-deficent mice

To analyze both early and late events associated with Smad3-inactivation, and to identify how specific smooth muscle cell (SMC) phenotypes may confer increased susceptibility to disease in the aortic root, we performed single-cell transcriptomic analysis on aortic cells from control (Smad3+/+; Myh11-CreER; + or – EGFP-L10a mice are refered to as Smad3+/+) and Smad3-deficient (Smad3lox/lox; Myh11-CreER; + or - EGFP-L10a are referred to as Smad3SmKO) mice at 10- and 18-weeks post-deletion, when aortic dilation is undetectable and moderate, respectively. Analysis of SMC-specific transcriptional changes show that, in addition to the expected decrease in TGF-β -induced expression of extracellular matrix components, Smad3-inactivation resulted in a broad reduction in the expression of components and modulators of focal adhesions, including that of several integrins, anchoring proteins, adaptors and modulators. Examination of the aortic transcriptome of mice with postnatal, smooth-muscle specific deletion of Smad3 at two different timeponts, 10- and 18-weeks post-deletion when dilation is undetectable and moderate, respectively.

为分析Smad3失活相关的早期与晚期事件，并明确特定平滑肌细胞（smooth muscle cell, SMC）表型如何使主动脉根部更易罹患疾病，我们对对照小鼠（Smad3+/+; Myh11-CreER; 携带或不携带EGFP-L10a的小鼠记为Smad3+/+）及Smad3缺陷小鼠（Smad3lox/lox; Myh11-CreER; 携带或不携带EGFP-L10a的小鼠记为Smad3SmKO）在基因缺失后10周和18周的主动脉细胞开展了单细胞转录组分析——此时小鼠分别尚未出现可检测到的主动脉扩张，以及出现中度主动脉扩张。对平滑肌细胞特异性转录变化的分析结果显示，除了预期的转化生长因子β（transforming growth factor-β, TGF-β）诱导的细胞外基质成分表达下调外，Smad3失活还导致黏着斑（focal adhesions）相关成分及调节因子的表达广泛降低，涵盖多种整合素（integrins）、锚定蛋白、接头蛋白及调节因子。本研究同时针对出生后经平滑肌细胞特异性敲除Smad3的小鼠，在基因缺失后10周和18周这两个时间点（此时主动脉扩张分别尚未可测与呈中度程度）的主动脉转录组展开了检测分析。