Rap1介导炎症反应在动脉粥样硬化中的作用

Repressor activator protein 1 (Rap1) was identified more than a decade ago as a protein that reside in the nucleus, associated to the telomeres. But recently, a ground-breaking study revealed that Rap1 could also stably reside in the cytoplasm of human cells. In the cytoplasm, Rap1 serve as an adaptor for the IκB kinase (IKK) to help to recruit and phosphorylate the p65 subunit of NFκB and enhances NFκB signaling that mediates a myriad of gene expression events, including many genes involved with the inflammatory responses. ..Atherosclerosis, formerly considered as a simple lipid storage disease that caused pathology by arterial obstruction, actually involves ongoing inflammatory responses. Deregulation of the NFkB signaling pathway is responsible, at least in part, for the underlying inflammatory process. It is tempting to speculate that telomere length affects the ratio between telomere-bound Rap1 and cytoplasmic-Rap1 that is available for the activation of NFκB pathway. A very exciting possibility is that telomere shortening (a process that is accelerated during atherosclerosis) releases more cytoplasmic-Rap1 and directly contributes to inflammation in atherosclerosis...The current project proposes to establish the importance of Rap1 in atherosclerosis. We hypothesize telomere shortening dispatches Rap1 into the cytoplasm and results in the activation of NFκB-dependent gene expression and thereby drive atherosclerosis-associated inflammation. We will investigate whether or not: 1) knockdown of Rap1 attenuates stimuli-induced NFkB-mediated genes in macrophages, endothelial and smooth muscle cells (the major cell types involved in atherosclerosis); 2) peritoneal macrophages from Rap1 knockout mice have impaired NFkB signaling when compared with wild-type macrophages; 3) the absence of Rap1 protects against atherosclerosis in ApoE knockout mice. Using cell lines, mice deficient in Rap1 and other molecular techniques, we hope to demonstrate that the release of Rap1 aggravates inflammation during atherosclerosis. The proposed study to examine non-telomeric function of Rap1 during atherosclerosis would provide novel scientific knowledge and uncover the underlying causes of inflammation during the process of atherosclerosis.

端粒结合蛋白Rap1不仅仅存在于细胞核内发挥着维护端粒正常功能的作用，同时也存在于细胞质中，并且能够结合IKK复合体，促进NF-kB的亚基p65的磷酸化，进而调控着NF-kB信号通路。而作为引发炎症的重要信号通路，NF-kB信号通路的失调在动脉粥样硬化疾病发展中起着非常重要的作用。解析Rap1蛋白与动脉粥样硬化病变的关系有着重大意义。因此本项目以Rap1蛋白为研究对象，通过运用基因干扰技术在巨噬细胞、内皮细胞、平滑肌细胞中敲除Rap1基因，来研究NF-kB信号通路是否因此受到抑制；同时首次建立Rap1/ ApoE基因双敲除小鼠模型，并运用免疫组织化学、酶联免疫吸附等技术研究敲除Rap1 基因是否能够抵抗或缓解ApoE基因敲除小鼠模型中动脉粥样硬化疾病的发生。本项目的完成将为寻找动脉粥样硬化防治新的炎症靶点奠定科学基础，同时也为其他动脉粥样硬化新药研究提供可借鉴的依据。

端粒结合蛋白Rap1 不仅仅在核内发挥着维护端粒长度及正常功能的作用，同时也具有非端粒的功能，即在肿瘤细胞的细胞质中Rap1能够很大程度上激活NFkB信用通路。作为引发炎症的重要信号通路，NFkB信号通路的失调在动脉粥样硬化疾病发展中起着非常重要的作用。细胞质中的Rap1介导了炎症反应从而最终是否造成了动脉粥样硬化疾病的产生是本课题研究的重点。由于巨噬细胞、内皮细胞、平滑肌细胞在动脉粥样硬化疾病发展过程中具有非常重要的作用，因此在本项目中，我们利用RNA干扰技术，在巨噬细胞系（THP-1），内皮细胞系（HUVEC）和平滑肌细胞系（HASMC）中建立了相对应的Rap1基因敲除的细胞模型，实验结果发现，只有在THP-1巨噬细胞中，敲除Rap1基因能显著抑制脂多糖LPS激活的IkBα，p65的蛋白磷酸化，从而抑制NFkB信号通路的活性并同时抑制了NFkB介导的相关促炎性细胞因子的分泌。而在HUVEC、HASMC中，并未观察到敲除Rap1能抑制LPS，肿瘤坏死因子 TNFα，或白介素 IL-1β激活的NFkB信号通路的活性。除此以外，我们同样从Rap1 基因敲除小鼠以及野生型小鼠中提取另外两种巨噬细胞：腹腔巨噬细胞以及骨髓分化巨噬细胞，并以它们作为研究对象，研究发现敲除Rap1基因的原代巨噬细胞显著抑制了LPS激活的NFkB信号通路及NFkB调控的炎症因子。而在人体动脉粥样硬化病变的样本中，免疫组织化学结果也显示Rap1蛋白大量存在与动脉粥样硬化病变处巨噬细胞丰富的区域，并且Rap1蛋白随着动脉粥样硬化的病变严重程度逐级增多。综上所述，本项目的研究发现为动脉粥样硬化的防治提供了理论依据及新的治疗途径。