Pro-inflammatory Pyroptotic Cell Death in Aortic Degeneration

项目摘要 升主动脉瘤和夹层(ATAAD)，与遗传疾病或 自发性作为散发性ATAAD，是一种极其致命的疾病，通常表现为外科紧急情况。 不幸的是，没有临床证明的药物可以防止零星的ATAAD进展。有一个 迫切需要制定有效的药理学策略来治疗ATAAD。散发性ATAAD的特点 是进行性的主动脉平滑肌细胞(SMC)耗竭和细胞外基质(ECM)破坏导致 主动脉扩张，夹层，最终破裂。我们的长期目标是提高我们对 散发性ATAAD的分子发病机制希望开发新的药理学策略来预防 疾病的发展。我们的初步研究表明，SMC中受损的DNA激活了一种成孔作用 Gasdermin D(GSDMD)蛋白，可导致SMC下垂，这是一种炎性形式的程序性细胞死亡 这可能是导致主动脉SMC丧失的常见途径。因此，此应用程序的目标是 探讨GSDMD介导的SMC下垂在ATAAD发病中的作用及机制。 胺胞浆DNA传感和上睑下垂代表ATAAD治疗靶点的程度。 我们的中心假设是，DNA损伤触发的传感器通路激活GSDMD介导的SMC 上睑下垂，导致主动脉变性、夹层和破裂。在目标1中，我们将确定 GSDMD介导的ATAAD形成的SMC下垂。我们将检验GSDMD介导的假设 SMC下垂与主动脉退行性变、生物力学衰竭和ATAAD的发生密切相关。 在Gsdmd-/-小鼠和我们建立的可诱导的SMC特异性Gsdmd基因敲除小鼠中检测这些方面 零星的ATAAD模型。在目标2中，我们将研究GSDMD激活的机制- 介导的SMC上睑下垂。在一系列体外和体内实验中，我们将检验细胞- Solic DNA触发的cGAS-STING传感器信号，通过TBK1/IKK2/DNA-PK激酶，直接磷酸化- 延缓GSDMD并促进其在质膜上的激活和孔形成，导致线粒体 损害并最终导致SMC死亡。在目标3中，我们将确定抑制细胞周期的程度- 胞质DNA传感和嗜焦性细胞死亡将阻止ATAAD的发生和进展。我们将测试 假设同时阻断cGAS刺痛通路和上睑下垂可以预防主动脉脱垂。 我们的散发性ATAAD模型中的结构和疾病进展。我们希望这项工作将决定如何 DNA损伤导致上睑下垂，从而损害升主动脉的结构和功能 墙。这项工作的积极影响将是更好地理解 在主动脉退行性变的发展中引起SMC下垂，为治疗提供了一个令人兴奋的新方向-- 预防ATAAD形成及其致命后遗症的治疗策略。关于DNA的新机制 损伤诱导的嗜酸性细胞死亡在许多其他心血管疾病中也有广泛的意义。 1

Project Summary Ascending thoracic aortic aneurysms and dissections (ATAAD), either associated with genetic conditions or spontaneous as sporadic ATAAD, are extremely lethal diseases that often present as surgical emergencies. Unfortunately, no clinically proven medication is available to prevent sporadic ATAAD progression. There is a critical need to develop effective pharmacological strategies to treat ATAAD. The hallmark of sporadic ATAAD is progressive aortic smooth muscle cell (SMC) depletion and extracellular matrix (ECM) destruction leading to aortic dilatation, dissection, and ultimately rupture. Our long-term goal is to improve our understanding of the molecular pathogenesis of sporadic ATAAD in hope of developing new pharmacological strategies to prevent disease progression. Our preliminary studies suggest that damaged DNA in SMCs activates a pore-forming protein, gasdermin D (GSDMD), that drives SMC pyroptosis, an inflammatory form of programmed cell death that may represent a common pathway to aortic SMC loss. Therefore, the objectives of this application are to determine the role and mechanisms of GSDMD-mediated SMC pyroptosis in ATAAD development and to ex- amine the extent to which cytosolic DNA sensing and pyroptosis represent therapeutic targets against ATAAD. Our central hypothesis is that sensor pathways triggered by damaged DNA activate GSDMD-mediated SMC pyroptosis, leading to aortic degeneration, dissection, and rupture. In Aim 1, we will determine the role of GSDMD-mediated SMC pyroptosis in ATAAD formation. We will test the hypothesis that GSDMD-mediated SMC pyroptosis is critically involved in aortic degeneration, biomechanical failure, and ATAAD development by examining these aspects in Gsdmd-/-mice and inducible SMC-specific Gsdmd knockout mice in our established sporadic ATAAD model. In Aim 2, we will investigate the mechanisms underlying the activation of GSDMD- mediated SMC pyroptosis. In a series of in vitro and in vivo experiments, we will test the hypothesis that cyto- solic DNA-triggered cGAS-STING sensor signaling, through TBK1/IKK2/DNA-PK kinases, directly phosphory- lates GSDMD and promotes its activation and pore formation in plasma membranes, leading to mitochondrial damage and ultimately pyroptotic SMC death. In Aim 3, we will determine the extent to which suppressing cy- tosolic DNA sensing and pyroptotic cell death will prevent ATAAD development and progression. We will test the hypothesis that simultaneously blocking the cGAS-STING pathway and pyroptosis will prevent aortic de- struction and disease progression in our sporadic ATAAD model. We expect that this work will determine how damaged DNA induces pyroptosis and thereby compromises the structure and function of the ascending aortic wall. The positive impact of this work will be an improved understanding of the molecular mechanisms that cause SMC pyroptosis in the development of aortic degeneration, providing an exciting new direction for treat- ment strategies for preventing ATAAD formation and its fatal sequelae. The novel mechanisms regarding DNA damage-induced pyroptotic cell death will also have broad implications in many other cardiovascular diseases. 1