TET调控高血压血管重建的作用机制研究

Vascular smooth muscle cells(VSMCs) undergo phenotypic switch during vascular remodeling induced by hypertension, which is modulated by changes of gene expression. 5--hydroxymethylation of methyl-cytosine(5-hmc), which is mediated by TET proteins, plays an important role in regulation of gene expression. However, the contributions of TET and 5-hmc to VSMC phenotypic switch and vascular remodeling under hypertension remain largely unknown. In this project, we detected the different expression of TET and 5-hmc in vascular tissues between spontaneously hypertensive rats (SHR) and wistar kyoto rats (WKY) . And then the different expression of gene modified by 5-hmc in vascular tissues between SHR and WKY is established by the 5-hydroxymethylated DNA immunoprecipitaton sequence. The obtained data are clustered and analyzed by using bioinformatics method. the role and underlying mechanisms of TET proteins and 5-hmc in regulation of phenotypic switch in VSMCs subjected to cyclic mechanical strain by Flexcell-4000 is studied. Finally, the role of TET　in vascular remodeling　is studied in vivo in mice either TET transgene and TET knock down. With the employment of recent advances in technologies, i.e. gene chip、bioinformatics method、mechanical cell biology and molecular biology, the project will achieve successes in illuminating the role of TET proteins and 5-hmc in hypertension-induced vascular remodeling. Furthermore, the accomplishment of the project will provide deep insights into the mechanobiological mechanism and potential targets for hypertension therapy.

高血压血管重建病理基础是血管平滑肌细胞（VSMC）表型转换，受基因表达改变的调控。胞嘧啶羟甲基化是调控基因表达的一种重要机制，由TET蛋白介导完成。但TET蛋白与5羟甲基胞嘧啶(5hmC)在VSMC表型转换和高血压血管重建中的作用机制尚不清楚。本项目拟检测自发性高血压大鼠（SHR）与对照大鼠（WKY）血管TET与5hmC的表达，以羟甲基化DNA免疫沉淀测序技术，建立SHR与WKY血管5hmC修饰基因差异表达谱，生物信息学分析寻找与VSMC表型转换有关的5hmC修饰基因；以细胞张应变加载模型，研究TET蛋白在张应变调控VSMCs表型转换中的作用机制；最后用TET转基因与基因敲除小鼠，在体检测TET在高血压血管重建中的作用。本项目应用基因组学、生物信息学、细胞力学和分子生物学等相关技术，旨在明确TET在高血压血管重建中的作用及其力学生物学机制，为阐明高血压血管重建的发病机理提供力学生物学依据。

TET介导的胞嘧啶羟甲基化在调控基因表达中起重要作用，但TET在高血压血管重建中的作用机制有待深入了解。本研究主要发现有：高通量测序发现自发性高血压大鼠胸主动脉基因组DNA 5-hmc表达有变化，选择基因启动子羟甲基化有变化的基因进一步研究：胞红蛋白在高血压大鼠主动脉和15%张应变刺激平滑肌细胞低表达，过表达胞红蛋白刺激血管平滑肌细胞的增殖与迁移，也抑制血管平滑肌细胞的细胞外基质表达。miR-3571在高血压大鼠主动脉和15%张应变刺激的血管平滑肌细胞中表达降低，miR-3571通过CLDN1抑制血管平滑肌细胞的增殖与迁移。高血压大鼠主动脉和15%张应变刺激血管平滑肌细胞miR-1-3p低表达，miR-1-3p通过ETS-1抑制血管平滑肌细胞的增殖、表型转换和细胞外基质的表达。高通量测序筛选自发性高血压大鼠胸主动脉差异表达的长链非编码RNA，环状RNA与mRNA，研究发现长链非编码RNANONRATT011842在高血压大鼠主动脉高表达，其通过PABPC1调控血管平滑肌细胞的增殖与迁移，参与高血压诱导的颈总动脉血管重建。二甲双胍诱导血管平滑肌细胞TET2的表达，并通过nur77调控血管平滑肌细胞的增殖，迁移和损伤诱导的血管重建。高通量测序检测筛选Crispr -Cas9技术敲除TET2血管平滑肌细胞差异表达的基因组DNA羟甲基化，长链非编码RNA，环状RNA和mRNA，蛋白质组学筛选TET2敲除血管平滑肌细胞差异表达蛋白质，发现TET2在高血压大鼠胸主动脉和15%张应变刺激血管平滑肌细胞低表达，TET2通过GIPR和P4HA3调节血管平滑肌细胞的增殖。TET2敲除小鼠血压与野生型小鼠血压无差异，血管紧张素II诱导高血压TET2敲除小鼠肠系膜动脉对压力的舒张能力减弱。高血压大鼠主动脉及15%张应变刺激的血管平滑肌细胞TET1表达降低，高通量测序检测Crispr-Cas9技术敲除TET1血管平滑肌细胞差异mRNA的表达，进而研究发现TET1通过SPP1与SFRP2调控血管平滑肌细胞的增殖和表型转换。应用静脉移植模型和高通量测序筛选到差异表达的mRNA与miRNA，发现TET1在移植静脉和张应变刺激血管平滑肌细胞表达降低，miR-29a通过TET1调控血管平滑肌细胞表型转换。这些发现提示TET和基因的羟甲基化修饰在调控血管平滑肌细胞功能和血管重建过程中发挥重要作用。