Supplementary Material for: Smooth Muscle Cell Notch2 Is Not Required for Atherosclerotic Plaque Formation in ApoE Null Mice

<b><i>Introduction:</i></b> We previously identified Notch2 in smooth muscle cells (SMC) in human atherosclerosis and found that signaling via Notch2 suppressed human SMC proliferation. Thus, we tested whether loss of Notch2 in SMC would alter atherosclerotic plaque progression using a mouse model. <b><i>Methods:</i></b> Atherogenesis was examined at the brachiocephalic artery and aortic root in a vascular SMC null (inducible smooth muscle myosin heavy chain Cre) Notch2 strain on the ApoE^−/− background. We measured plaque morphology and size, as well as lipid, inflammation, and smooth muscle actin content after Western diet. <b><i>Results:</i></b> We generated an inducible SMC Notch2 null on the ApoE^−/− background. We observed ∼90% recombination efficiency with no detectable Notch2 in the SMC. Loss of SMC Notch2 did not significantly change plaque size, lipid content, necrotic core, or medial area. However, loss of SMC Notch2 reduced the contractile SMC in brachiocephalic artery lesions and increased inflammatory content in aortic root lesions after 6 weeks of Western diet. These changes were not present with loss of SMC Notch2 after 14 weeks of Western diet. <b><i>Conclusions:</i></b> Our data show that loss of SMC Notch2 does not significantly reduce atherosclerotic lesion formation, although in early stages of plaque formation there are changes in SMC and inflammation.

<b><i>Introduction:</i></b> 我们先前在人类动脉粥样硬化的平滑肌细胞（smooth muscle cells, SMC）中鉴定出Notch2（Notch2），并发现Notch2信号通路会抑制人类SMC的增殖。为此，我们构建小鼠模型，旨在探究平滑肌细胞中Notch2缺失是否会改变动脉粥样硬化斑块的进展进程。<b><i>Methods:</i></b> 我们在ApoE^−/−背景的小鼠中，采用诱导型平滑肌肌球蛋白重链Cre重组酶系统，构建了平滑肌细胞特异性Notch2敲除模型，并分别对头臂动脉与主动脉根的动脉粥样硬化发生情况进行检测。在小鼠接受西方饮食干预后，我们对斑块的形态与大小、脂质含量、炎症水平及平滑肌肌动蛋白的表达量进行了定量分析。<b><i>Results:</i></b> 我们成功构建了ApoE^−/−背景下的诱导型平滑肌细胞特异性Notch2敲除模型，经检测其SMC中Notch2的重组效率约为90%，且未检测到残留的Notch2表达。平滑肌细胞Notch2缺失并未显著改变斑块大小、脂质含量、坏死核心面积或血管中膜面积。但在西方饮食干预6周后，平滑肌细胞Notch2缺失会降低头臂动脉斑块中的收缩型SMC比例，并升高主动脉根斑块的炎症浸润水平；而在西方饮食干预14周后，上述变化则不再出现。<b><i>Conclusions:</i></b> 本研究数据表明，平滑肌细胞Notch2缺失并不会显著抑制动脉粥样硬化斑块的形成，不过在斑块形成的早期阶段，会出现SMC表型及炎症水平的相关改变。