衰老促进动脉粥样斑块钙化—Sirt1通过抑制EIF2α/ATF4通路保护动脉粥样斑块钙化的机制研究

The incidence of atherosclerotic calcification significantly increased in older adults, which indicates aging to be a potential inducer of atherosclerotic calcification. The expression and activity of Sirt1 reduced during aging. Our preliminary studies found that Sirt1 overexpression inhibits oxLDL-induced osteoblastic differentiation of human aortic smooth muscle cells, while Sirt1 silencing promotes oxLDL-triggered osteoblastic differentiation of the cells. These data suggest that Sirt1 is possible to be involved in aging-induced atherosclerotic calcification. Reports and our data showed EIF2α-ATF4, downstream of endoplasmic reticulum stress pathway, involved in osteoblastic differentiation of human aortic smooth muscle cells. We further found Sirt1 binds with EIF2α. Moreover, Silencing of Sirt1 promotes oxLDL-induced EIF2α phosphorylation and downstream ATF4 expression, but has no effect on other endoplasmic reticulum stress pathways. Based on the deacetylation function of Sirt1, we further hypothesized that Sirt1 may promote EIF2α deacetylation, which further inhibits EIF2α phosphorylation and ATF4 expression, and therefore prevents atherosclerotic calcification. This project will utilize both vascular smooth muscle cell-specific Sirt1 and ATF4 knockout, as well as Sirt1 transgenic mice, and human aortic smooth muscle cells to study the mechanisms of Sirt1 regulating aging-mediated atherosclerotic calcification via EIF2α-ATF4 pathway.

老年人动脉粥样斑块钙化发病显著升高。衰老下调Sirt1，前期发现Sirt1过表达抑制oxLDL诱导的高代人主动脉平滑肌细胞向成骨细胞分化，Sirt1沉默促进oxLDL诱导的细胞向成骨细胞分化，提示Sirt1介导衰老所致动脉粥样斑块钙化。资料及前期结果提示内质网应激下游EIF2α-ATF4促进人主动脉平滑肌细胞向成骨细胞分化。我们发现Sirt1与EIF2α蛋白结合，同时Sirt1沉默促进oxLDL诱导的EIF2α磷酸化及下游ATF4表达，而不影响内质网应激其他通路。鉴于Sirt1的去乙酰化作用，我们推测Sirt1可能通过抑制EIF2α乙酰化，从而抑制EIF2α磷酸化及ATF4表达，抑制动脉粥样斑块钙化。本项目将利用血管平滑肌特异性Sirt1、ATF4敲除及Sirt1过表达小鼠，并在细胞中利用免疫沉淀、蛋白质谱等手段，研究Sirt1通过EIF2α-ATF4调控衰老介导的动脉粥样斑块钙化的机制。

心血管钙化包括主动脉瓣钙化严重影响生命健康，且缺乏有效的干预手段。主动脉瓣钙化发病与年龄相关，提示衰老可能参与主动脉瓣钙化的发生发展。研究人及团队针对衰老与主动脉瓣钙化，进行了相关探索。我们发现主动脉瓣钙化病人标本中Sirt1表达显著下调；予以西方饮食喂养3月可显著诱导老年Apoe-/-小鼠主动脉瓣钙化，但不能显著增加青年Apoe-/-小鼠主动脉瓣钙化，而老年小鼠Sirt1表达显著下调。进一步研究发现与Sirt1激动剂白藜芦醇或Sirt1过表达可显著改善西方饮食诱导的老年Apoe-/-小鼠主动脉瓣钙化。利用Apoe-/-;Sirt1+/-小鼠以及Apoe-/-;Sirt1fl/fl;Tagln-Cre小鼠，我们发现Sirt1全身或肌成纤维细胞特异性敲除可显著促进青年小鼠主动脉瓣钙化的发生，从而证实Sirt1介导了衰老诱导的主动脉瓣钙化的发生发展。机制方面，利用Sirt1沉默的瓣膜间质细胞（VIC）进行转录组测序分析，结果显示Sirt1沉默可显著上调一系列炎症相关基因转录，包括Nlrp3以及Aim2炎症小体mRNA转录。oxLDL可通过促进线粒体氧化应激以及线粒体DNA进入细胞质，激活Nlrp3以及Aim2炎症小体的激活，从而诱导IL-1b的成熟，进而导致VIC向成骨细胞分化。最后，我们利用免疫荧光染色发现Nlrp3以及Aim2在人以及小鼠主动脉瓣钙化中表达升高，且主要定位于肌成纤维细胞中。因此，我们的研究证实衰老引起的Sirt1减少，通过促进Nlrp3以及Aim2炎症小体，诱导了主动脉瓣钙化的发生发展；Sirt1可能作为衰老相关主动脉瓣钙化的潜在治疗靶点。