single cell RNA sequencing of thoracic aorta from Angiotensin II infused control and Sting whole body knockout mice

Background: Sporadic aortic aneurysm and dissection (AAD), caused by progressive aortic smooth muscle cell (SMC) loss and extracellular matrix degradation, is a highly lethal condition. Identifying mechanisms that drive aortic degeneration is a crucial step in developing an effective pharmacologic treatment to prevent disease progression. Recent evidence has indicated that cytosolic DNA and abnormal activation of the cytosolic DNA sensing adaptor STING (stimulator of interferon genes) play a critical role in vascular inflammation and destruction. Here, we examined the involvement of this mechanism in aortic degeneration and sporadic AAD formation. Methods: The presence of cytosolic DNA in aortic cells and activation of the STING pathway were examined in aortic tissues from patients with sporadic ascending thoracic AAD. The role of STING in AAD development was evaluated in Sting-deficient (Stinggt/gt) mice in a sporadic AAD model induced by challenging mice with a combination of a high-fat diet and angiotensin II. We also examined the direct effects of STING on SMC death and macrophage activation in vitro. Results: In human sporadic AAD tissues, we observed the presence of cytosolic DNA in SMCs and macrophages and significant activation of the STING pathway. In the sporadic AAD model, Stinggt/gt mice showed significant reductions in challenge-induced aortic enlargement, dissection, and rupture in both the thoracic and abdominal aortic regions. Single-cell transcriptome analysis revealed that aortic challenge in wild-type mice induced the DNA damage response, the inflammatory response, dedifferentiation and cell death in SMCs, and matrix metalloproteinase expression in macrophages. These changes were attenuated in challenged Stinggt/gt mice. Mechanistically, nuclear and mitochondrial DNA damage in SMCs and the subsequent leak of DNA to the cytosol activated STING signaling, which induced cell death through apoptosis and necroptosis. In addition, DNA from damaged SMCs was engulfed by macrophages in which it activated STING and its target interferon regulatory factor 3, which directly induced matrix metalloproteinase-9 expression. We also found that pharmacologically inhibiting STING activation partially prevented AAD development. Conclusions: Our findings indicate that the presence of cytosolic DNA and subsequent activation of cytosolic DNA sensing adaptor STING signaling represent a key mechanism in aortic degeneration and that targeting STING may prevent sporadic AAD development. Overall design: To understand the role of STING in aorta, we performed single cell RNAseq of thoracic aortic tissues from wild type mice, AngII infused wild type mice, Sting KO mice, AngII infused Sting KO mice, and AngII infusion was applied for 7 days.

背景：散发性主动脉瘤与夹层（aortic aneurysm and dissection, AAD）是一类致死性极高的疾病，其发病机制为进行性主动脉平滑肌细胞（smooth muscle cell, SMC）丢失与细胞外基质降解。明确驱动主动脉退变的机制，是开发有效药物治疗以阻断疾病进展的关键环节。现有研究证据显示，胞质DNA及胞质DNA感知接头蛋白干扰素基因刺激因子（stimulator of interferon genes, STING）的异常激活，在血管炎症与破坏过程中发挥核心作用。本研究旨在探究该机制在主动脉退变及散发性AAD形成中的作用。

方法：我们对散发性升胸主动脉AAD患者的主动脉组织，检测了主动脉细胞内胞质DNA的存在情况及STING通路的激活状态。在高脂饮食联合血管紧张素Ⅱ（angiotensin II, AngII）诱导的散发性AAD小鼠模型中，我们通过STING缺陷型（Stinggt/gt）小鼠评估了STING在AAD发生发展中的作用。此外，我们还在体外实验中探究了STING对SMC死亡及巨噬细胞活化的直接影响。

结果：在人类散发性AAD组织中，我们观察到SMC及巨噬细胞内存在胞质DNA，且STING通路显著激活。在散发性AAD模型中，Stinggt/gt小鼠经造模诱导的胸、腹主动脉区域的扩张、夹层及破裂程度均显著降低。单细胞转录组分析显示，野生型小鼠经造模后，主动脉组织中的SMC出现DNA损伤应答、炎症应答、去分化及细胞死亡，巨噬细胞则出现基质金属蛋白酶表达上调。上述变化在造模后的Stinggt/gt小鼠中得到显著缓解。机制层面，SMC的核DNA与线粒体DNA发生损伤，随后DNA渗漏至胞质并激活STING信号通路，通过凋亡与坏死性凋亡诱导细胞死亡。此外，受损SMC释放的DNA可被巨噬细胞吞噬，进而激活巨噬细胞内的STING及其下游干扰素调节因子3（interferon regulatory factor 3, IRF3），后者可直接诱导基质金属蛋白酶-9（matrix metalloproteinase-9, MMP9）的表达。我们还发现，通过药理学手段抑制STING激活，可部分阻止AAD的发生发展。

结论：本研究结果表明，胞质DNA的存在及其后续激活的胞质DNA感知接头蛋白STING信号通路，是主动脉退变的关键机制，靶向STING或可阻止散发性AAD的发生发展。

整体实验设计：为明确STING在主动脉中的作用，我们对野生型小鼠、血管紧张素Ⅱ输注的野生型小鼠、STING基因敲除（Sting knockout, Sting KO）小鼠，以及血管紧张素Ⅱ输注7天的STING基因敲除小鼠的胸主动脉组织开展了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。