血管平滑肌细胞KLF5基因表达缺失促进小鼠腹主动脉瘤的形成及其抗凋亡机制

Abdominal aortic aneurysm (AAA) is a kind of vascular disease with localized dilatation of abdominal aorta. The mortality was higher in the middle and old aged people. Vascular smooth muscle cells (VSMCs) are the major cell types in the aorta, and the apoptosis of VSMCs is the key to the formation of AAA. Transcription factor KLF5 plays an important role in the process of vascular remodeling, but its molecular mechanism in AAA formation has not yet been elucidated. We found that the KLF5 expression of VSMCs in human AAA tissues was significantly higher than that in normal aorta tissue, and the VSMCs-specific KLF5 knockout could promote the CaPO4-induced AAA formation in mice. The results of gene chip screening and qRT-PCR experiments showed that the mRNA expression of apoptosis-related Dapk1 and Apaf1 was significantly increased in the AAA tissues of VSMC-specific KLF5 knockout mice compared with in the AAA tissues of WT mice. Bioinformatics analysis showed that there are a plurality of KLF5 binding sites in promoter of Dapk1 and Apaf1 gene. Together, we hypothesized that the KLF5 expression was upregulated in CaPO4-induced AAA tissue, which downregulated the expression of Dapk1 and Apaf1 protein, and that inhibited the apoptosis of VSMCs and the formation of AAA. In the present research, we will study the anti-apoptotic mechanism of KLF5 in the VSMCs and the inhibition of AAA formation. This research will provide a new therapeutic target for the prevention and treatment of abdominal aortic aneurysm.

腹主动脉瘤（AAA）在中老年人群中病死率较高，但其形成机制尚未阐明。血管平滑肌细胞（VSMCs）凋亡是AAA形成的关键环节。KLF5在血管重塑过程中发挥重要作用，但其在AAA形成中的分子机制研究鲜有报道。我们前期研究发现，VSMCs源KLF5在人AAA组织中表达升高；VSMCs KLF5基因敲除促进CaPO4诱导的小鼠AAA的形成；基因芯片和PCR实验发现，VSMCs KLF5基因敲除小鼠AAA组织中凋亡相关蛋白Dapk1和Apaf1的mRNA表达显著增加。生物信息学分析显示，Dapk1和Apaf1基因启动子上存在多个KLF5结合的位点。我们猜测，在CaPO4诱导的血管损伤后，VSMCs中KLF5表达升高，KLF5通过下调Dapk1和Apaf1的表达，抑制VSMCs的凋亡和AAA的形成。本项目将对KLF5在VSMCs抗凋亡功能和抑制AAA形成的机制进行研究，为AAA的防治提供新的分子靶点。

腹主动脉瘤（AAA）在中老年人群中病死率较高，但其形成机制尚未阐明。血管平滑肌细胞（VSMC）凋亡是AAA形成的关键环节，KLF5在血管重塑过程中发挥重要作用，深入了解KLF5表达的调控机制，从而为早期预防、诊断和治疗AAA提供分子靶点。. 本研究以临床AAA患者组织样本为研究切入点，采用免疫印迹法、免疫组化和免疫荧光染色进行分子病理观察；通过杂交饲养ApoE-/-VSMCKlf5-/-双基因敲除小鼠，并构建AngII灌注，高脂饲养诱导的AAA的模型探索血管平滑肌细胞KLF5基因表达的分子机制；结合体外原代培养小鼠VSMC以及人、小鼠VSMC细胞株实验，采用分子生物学检测方法结合荧光染色方法研究KLF5、靶基因和线粒体形态共定位分析，电镜实验辅助确证体内体外VSMC线粒体形态动力学变化，从人、动物和细胞三个层面探讨VSMC中Klf5表达调控翻译起始因子Eif5a及其下游线粒体融合蛋白Mfn1与分裂蛋白Drp1表达的动态调节的分子机制及其在衰老和AAA形成过程中的作用。另外，探索了Klf5表达上游的调控机制可能与肥胖相关基因FTO表达上调参与KLF5 mRNA’非翻译区m6A去甲基化修饰，稳定其mRNA表达相关。研究结果显示：（1）VSMC中Klf5的下调与年龄相关的血管疾病AAA破裂有关；在VSMCKlf5-/-小鼠中通过促进活性氧（ROS）的形成而加剧了血管衰老，并导致加重Ang II诱导的AAA表型。细胞水平研究发现，Klf5敲低促进线粒体裂变，而Klf5过表达能够抑制其裂变。（2）分子生物学实验机制研究证实，Klf5通过与真核翻译起始因子（eIF5a）的启动子结合而激活其转录，而eIF5a启动子又通过与线粒体融合蛋白1（Mfn1）相互作用保持线粒体完整性。因此，由Klf5下调引起的eIF5a表达降低导致线粒体裂变和过量的ROS产生。而抑制线粒体裂变会降低ROS的产生和VSMC的凋亡、衰老。（3）采用高通量RNA甲基化测序和Western blot实验技术分析AngII刺激VSMCs的结果显示，肥胖相关基因（FTO）表达升高与Klf5的mRNA终止密码子附近1457位点m6A甲基化修饰水平显著降低相关，增加了mRNA的稳定性，解释了AngII诱导Klf5表达升高的又一新的分子机制。. 本项目组已完成全部科研计划，相关结果已发表国内外论文6篇。