DNA羟甲基化酶TET2负性调控内皮-间质转化的力学生物学机制研究

Atherosclerosis is a serious risk to human health. Recently, studies showed that endothelial-to-mesenchymal transition (EndoMT) was induced by disturbed flow shear stress, which plays an important role in atherosclerosis progression. However, the detail mechanism is unclear. Our previous studies showed that Ten Eleven Translocation 2 (TET2), a member of DNA hydroxymethylase family, was downregulated in atherosclerotic lesion. In high fat-fed apoE-/- mice, overexpression of TET2 was contributed to inhibited EndoMT and reduced atherosclerotic lesions . Moreover, TET2 is a mechanica sensitivity protein, which was downregulated by disturbed flow shear stress in cultured endothelial cells. Combination with literatures, we hypothesized that TET2 negatively regulated EndoMT induced by disturbed flow shear stress. To test this hypothesis, disturbed flow shear stress apoE-/- mice with partial carotid ligation and parallel plate flow chamber system were used to detect the effect of TET2 on “flow shear stress- EndoMT”. Then, based on the protein Chip and prediction of bioinformatics, “miR-320-SOX4-Wnt/β-Catenin” pathway was detected to explore the regulative mechanism of TET2. The accomplish of this study will explore the mechanical mechanisms of EndoMT, which will further explore the mechanical mechanisms of atherosclerosis and provide new interventional strategies and targets for the prevention and treatment for atherosclerosis.

动脉粥样硬化(As)严重危害人类健康。近来发现剪切应力诱导内皮-间质转化(EndoMT)且在As进程中起着重要作用，但其调控机制不清楚。本项目前期研究发现As斑块内DNA羟甲基化酶TET2表达明显下调，TET2过表达抑制EndoMT以及高脂饮食apoE-/-小鼠As病变；TET2为切应力敏感蛋白，紊流剪切应力下血管内皮细胞(VEC)TET2表达下调。综合文献，我们推测：TET2负性调控“剪切应力-EndoMT”。为证实该假说，本课题将采用颈总动脉局部结扎apoE-/-小鼠模型及平行平板流动腔系统，探讨TET2对“剪切应力-EndoMT”的影响；再基于基因芯片及生物信息学预测的结果，从“miR-320-SOX4-Wnt/β-Catenin”通路探讨TET2调控“剪切应力-EndoMT”的分子机制。本研究的完成将从新的角度阐明EndoMT剪切应力调控机制，为As有效防治提供新策略和新靶点。

项目的背景.动脉粥样硬化性（atherosclerosis, As）心脑血管疾病严重危害人类健康，As的病理过程表现为动脉对内膜损伤导致的炎性、纤维增生性反应，低剪切应力促As的发生、发展，但其调控机制尚不清楚。.主要研究内容. 以DNA羟甲基酶TET2为切入点，探讨“剪切应力-As”间的关系及其调控机制。 .重要结果.1.低剪切应力下调TET2的表达，进而激活Wnt/β-catenin信号通路促内皮-间质转化（EndMT），TET2 overexpression抑制低剪切应力诱导的EndMT，进而抑制低剪切应力apoE-/-小鼠模型As病变。.2.低剪切应力下调TET2的表达，减少HDAC2的募集，以非DNA去甲基修饰的方式上调琥珀酸脱氢酶B（SDHB）的表达和活性，介导线粒体损伤，增加活性氧（ROS）的产生，促血管内皮细胞焦亡。.3.miR-320 inhibitor通过激活SOX4/β-Catenin 通路调节EndMT，miR-320 mimics抑制低剪切应力诱导Wnt/β-Catenin 信号的活化。.4.miR-320 inhibitor通过上调ROS的生成促血管内皮细胞焦亡，miR-320 mimics改善血管内皮细胞线粒体功能抑制焦亡。.5.琥珀酸（succinate）为三羧酸循环重要的中间代谢产物，琥珀酸通过氧化代谢上调ROS生成，进而促血管内皮细胞焦亡。.科学意义：.低剪切应力下调TET2的表达，进而下调miR-320的表达及损伤线粒体的功能，增强血管内皮EndMT以及焦亡等生物学过程，促As的发生、发展，TET2以及miR-320可能有效防治As的重要靶点分子；EndMT以及焦亡为可逆转的生物学过程，干预EndMT以及焦亡将为As的有效防治提供新的策略和思路。