低剪切应力促As新机制：DNA羟甲基酶TET2-/-引发血管内皮细胞焦亡

The activation and inflammatory phenotype of vascular endothelial cell induced by low shear stress is an important step of atherosclerosis (As), but its potential regulatory mechanisms are not fully explored. Recent studies showed that pyroptosis (proinflammatory cell programmed death) is a mechanical response biological process and exists throughout the initiation and progression of As. Our previous study showed that low shear stress downregulates endothelial cell DNA hydroxymethylase TET2 expression. Moreover, the succinate dehydrogenase (SDH) expression was inhibited and pyroptosis was promoted in TET2 shRNA-treated endothelial cell. Combination with literatures and previous experiments, we propose that TET2-/- induces ECs pyroptosis, which mediates the low shear stress-promoted As. In this study, we first use the low shear stress apoE-/- mice model and parallel plate flow chamber system to explore the relationship of TET2 endothelial cell pyroptosis under low shear stress. Then, based on the gene Chip and prediction of bioinformatics, “SDHB-succinate/GPR91-NLRP3” pathway was detected to explore the regulative mechanism of TET2-/- on endothelial cell pyroptosis via gene transfection, CRISPR/Cas9, amino acid site-directed mutagenesis and mass spectra technologies. The accomplish of this study will broaden the understanding of the biomechanical mechanism of As and provide new strategies and targets for the effective prevention and control of As.

低剪切应力诱发血管内皮细胞（ECs）炎症是动脉粥样硬化（As）的重要环节，但其调控机制尚不清楚。新近研究提示，焦亡（促炎性程序性细胞死亡）是剪切应力敏感的生物学过程并贯穿As病变进程。本项目前期研究发现，低剪切应力下调ECs DNA羟甲基酶TET2表达，TET2 shRNA抑制琥珀酸脱氢酶B（SDHB）表达并促ECs焦亡。综合文献和前期实验，我们推测“TET2-/-引发ECs焦亡，介导低剪切应力促As”。 本课题将采用低剪切应力apoE-/-小鼠模型及平行平板流动腔系统，探讨低剪切应力下TET2表达与ECs焦亡的关系；再基于基因芯片及生物信息学预测结果，结合基因转染、CRISPR/Cas9、氨基酸定点突变、质谱等方法和技术，从“SDHB-琥珀酸/GPR91-NLRP3”通路探讨和验证TET2-/-引发焦亡的分子机制。本研究将拓宽对As生物力学机制的认识，为As的有效防治提供新策略和靶点。

低剪切应力诱发血管内皮细胞炎症是动脉粥样硬化发生、发展的重要环节，但其调控机制尚不清楚。我们的研究表明，焦亡（促炎性程序性细胞死亡）是剪切应力敏感的生物学过程，低剪切应力促血管内皮细胞焦亡。机制上，低剪切应力下调血管内皮细胞TET2的表达，从而通过SDHB途径促血管内皮细胞内三羧酸循环重要的代谢中间体-琥珀酸的蓄积，上调其特异性受体GPR91的表达及活性，激活PKC信号通路，介导NLRP3 295位点丝氨酸磷酸化修饰，促血管内皮细胞焦亡。外源性补充琥珀酸类似物促高脂饮食apoE-/-小鼠循环血液中琥珀酸含量增加，加剧动脉粥样硬化病变；内皮细胞特异性GPR91 shRNA慢病毒减轻高脂饮食apoE-/-小鼠动脉粥样硬化病变，这些结果表明低剪切应力通过“TET2-琥珀酸/GPR91-PKC-NLRP3”途径促血管内皮细胞焦亡及动脉粥样硬化病变。NLRP3的295位丝氨酸磷酸化修饰为NLRP3炎症小体激活的重要途径；干预琥珀酸/GPR91 信号通路以及焦亡可能为有效防治动脉粥样硬化病变提供新的策略。. 此外，代谢组学的结果表明，TET2 shRNA组血管内皮细胞的N-乙酰-L-赖氨酸含量明显降低,SDHB过表达血管内皮细胞羟脯氨酸的含量明显降低；蛋白组学的结果表明，GPR91过表达血管内皮细胞差异表达的基因主要集中在流动剪切应力与动脉粥样硬化等生物学过程，其结构域重要集中在伴侣蛋白TCP-1家族，差异表达的蛋白主要为核蛋白，多组学深入探讨TET2-SDHB-琥珀酸/GPR91途径在动脉粥样硬化硬化中的作用及其调节机制，将进一步拓展动脉粥样硬化的力学调控机制。