微囊泡介导M1型巨噬细胞极化促进低剪切力诱导易损斑块形成及Sirt1靶向干预研究

Our preliminary data showed that SIRT1 activator can influence macrophage phagocytosis and apoptosis by regulating autophagy. But whether SIRT1 induces degradation of extracellular matrix and inhibits formation of thin-cap atherosclerotic plaques by regulating macrophage-derived microvesicles and M1 macrophage polarization is unknown. In this study standardized change in shear stress is induced by placing a “cone shape” cast around the mouse carotid artery to established vulnerable and stable plaque models. Using this model, influence of SIRT1 overexpression or gene silencing on the dynamic relationship between plaque vulnerability and macrophage-derived microvesicles, macrophage polarization and the ratio of MMP2/MMP9 expression at different time points in the LDLR-/- mice carotid artery will be carefully investigated. Under low shear stress and oscillatory shear stress condition, effect of recombinant adenoviral SIRT1 or SIRT1 siRNA on microvesicles, macrophage polarization and the ratio of MMP2/MMP9 expression in RAW264.7 cells will also be studied in vitro. This project is aimed to understand the potential therapeutic benefit of SIRT1 in improving the stability of atherosclerotic plaques by reducing microvesicles, promoting M2 programming and inhibition of MMP2/MMP9 activity. Ultimately, this knowledge will lead to new approaches for prevention or treatment of atherosclerosis.

我们前期研究发现SIRT1激动剂通过调控自噬水平影响巨噬细胞吞噬功能和凋亡。SIRT1是否通过调控微囊泡和M1型巨噬细胞极化影响细胞外基质降解抑制薄壁纤维帽斑块形成目前尚无定论。本研究拟于LDLr-/-小鼠颈动脉置入圆锥形铸型套管(Cast)改变局部剪切力构建易损和稳定斑块模型为研究对象，系统观察SIRT1过表达和沉默之后：不同时间点血管斑块稳定性与巨噬细胞源性微囊泡、巨噬细胞亚型M1/M2和MMP2/MMP9表达之间的动态关系。并在体外实验中证实：低剪切力和震荡剪切力环境下，SIRT1腺病毒转染和RNA干扰对巨噬细胞源性微囊泡生成、巨噬细胞亚型M1/M2转化和MMP2/MMP9表达的影响。旨在证实SIRT1靶向干预巨噬细胞源性微囊泡产生，进而激活M2亚型巨噬细胞，抑制MMP2/MMP9活性提高血管斑块稳定性，为防治动脉粥样硬化提供新思路。

SIRT1及SIRT2是否调控巨噬细胞极化影响动脉粥样硬化血管斑块进展尚不明确。按照课题计划，本项目以小鼠腹腔巨噬细胞和高脂饮食诱导的LDLr-/-小鼠动脉粥样硬化模型为研究对象, 分别采用SIRT1/SIRT2过表达及siRNA干扰干预，从体内和体外检测巨噬细胞表型改变在动脉粥样硬化中的作用和机制。重要结果及关键数据：①SIRT1调控巨噬细胞表型转化抑制泡沫巨噬细胞形成和细胞凋亡及增强巨噬细胞胞葬，其作用机制与抑制TXNIP/NLRP3炎症通路有关。②SIRT2过表达调控M1型向M2型巨噬细胞转化抑制血管斑块进展并提高斑块稳定性。③非靶向脂质组学研究发现在M1型巨噬细胞活化过程中，共筛选出183个差异表达脂质分子。代谢通路分析显示差异脂质分子与甘油磷脂代谢通路相关。科学意义：本研究揭示了SIRT1及SIRT2治疗动脉粥样硬化病变的新作用机制，为动脉粥样硬化病变的诊治提供新的思路。