Gas6调控SIRT6修复DNA损伤抗内皮衰老的作用及机制

Endothelial senescence induces vascular endothelial disorders which would lead to the development and progression of atherosclerosis (AS). Accumulating evidence has shown that overexpression of Sirtuin 6 (SIRT6) could delay vascular endothelial senescence through inactivating NF-κB and restoration of DNA damage via directly binding to DNA double-strand breaks. Nevertheless, there are few key factors which could effectively activate intranuclear SIRT6. Our previous studies indicated that Growth arrest-specific protein 6 (Gas6) promoted vascular endothelium repair and enhanced SIRT6 expression in myocardium. However, the specific mechanisms of Gas6-mediated SIRT6 upregulation in endothelium senescence are still unknown. Based on the knowledge of our preliminary studies, we are trying to identify the definite associations among Gas6, SIRT6 and endothelium senescence by evaluating sera levels of Gas6 and expression of SIRT6 in circulating endothelial cells, as well as the endothelium functions of the elderly. With models of Gas6-/- aging mice and senescent HUVECs in vitro, we will further clarify the anti-senescence role of Gas6 in modulating SIRT6-mediated restoration of DNA damage via administration of exogenous human/mice recombinant Gas6 protein or Gas6 gene interference RNA. Moreover, we will elucidate the molecular mechanisms of Gas6 in regulating the expression of SIRT6 with Axl/PI3K/Akt/FoxO3a signal inhibitors and report gene analysis to evaluate the activation of SIRT6 promoter. This program will provide novel targets for treatments of AS by anti-endothelial senescence from the perspective of maintaining the genome stability.

内皮衰老等所致的内皮功能障碍是动脉粥样硬化（AS）发生的始动环节。SIRT6通过抑制NF-κB过度激活并直接与DNA断端结合修复DNA损伤可延缓内皮衰老，但目前尚缺乏有效激活核内SIRT6的因子。我们前期研究提示生长停滞特异性蛋白6（Gas6）可促进内皮修复；预实验提示Gas6可促进SIRT6的表达，但Gas6可否上调SIRT6抗内皮衰老尚未见报道。本项目拟在前期研究基础上，通过观察老年人外周血Gas6和循环内皮细胞中SIRT6水平及内皮功能的变化，探讨Gas6和SIRT6与内皮衰老的关系；构建各年龄代Gas6敲除小鼠及人脐静脉内皮细胞衰老模型，使用Gas6重组蛋白、RNA干扰、信号通路关键分子抑制剂以及报告基因分析等技术，阐明Gas6调控SIRT6修复DNA损伤抗内皮衰老的作用和分子机制，这将为从源头上通过抗内皮衰老来干预AS提供新靶点。

背景：SIRT6通过抑制NF-κB过度激活并直接与DNA断端结合修复DNA损伤可延缓内皮衰老，但目前尚缺乏有效激活核内SIRT6的因子。我们前期研究提示生长抑制特异性蛋白6（Growth arrest-specific 6，Gas6）可促进内皮修复；预实验提示Gas6可促进SIRT6的表达，但Gas6可否上调SIRT6抗内皮衰老尚未见报道。.研究内容：本项目拟在前期研究基础上，通过观察老年人外周血Gas6和循环内皮细胞中SIRT6水平及内皮功能的变化，探讨Gas6和SIRT6与内皮衰老的关系；构建各年龄代Gas6敲除小鼠及人脐静脉内皮细胞衰老模型，使用Gas6重组蛋白、RNA干扰、信号通路关键分子抑制剂以及报告基因分析等技术，阐明Gas6调控SIRT6修复DNA损伤抗内皮衰老的作用和分子机制，这将为从源头上通过抗内皮衰老来干预AS提供新靶点。.结果：通过细胞实验我们证明：建立过氧化氢诱导的内皮细胞衰老模型，并予β半乳糖苷酶染色验证。予以不同浓度Gas6干预模型组与对照组，证实Gas6抗内皮衰老的作用以及获取干预最适浓度。Gas6过表达促进缺氧缺血骨髓间充质干细胞存活及促进骨髓间充质干细胞旁分泌减轻心肌细胞凋亡。高糖缺氧条件下，过表达Axl通过调控PI3K/Akt/mTOR/p70S6K通路增加HIF-1α蛋白合成从而保护脐静脉内皮细胞的成管能力。缺氧下调了脐静脉内皮细胞SIRT6的表达及过表达SIRT6可以显著增强缺氧条件下脐静脉内皮细胞的增殖和迁移。通过动物实验我们证明：在心梗小鼠模型中，外源性给予Gas6蛋白能上调SIRT6及Axl蛋白的表达。心肌内移植Gas6过表达的MSCs可显著改善心肌梗死后的心功能并限制梗死面积，提高骨髓间充质干细胞治疗心肌梗死的疗效。.意义：本课题组阐明了Gas6抗内皮衰老的作用及Gas6在缺血性衰老心肌中的作用和机制，可为动脉粥样硬化等疾病的防治提供新思路，有着较大的临床应用价值。