Pro-inflammatory Pyroptotic Cell Death in Aortic Degeneration

项目摘要 升胸主动脉瘤和夹层（ATAAD），与遗传条件或 自发性ATAAD，如散发性ATAAD，是非常致命的疾病，通常表现为外科急诊。 不幸的是，没有临床证明的药物可用于预防偶发性ATAAD进展。有一个 迫切需要开发有效的药理学策略来治疗ATAAD。散发性ATAAD的特征是 是进行性主动脉平滑肌细胞（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，以防止 疾病进展。我们的初步研究表明，SMC中受损的DNA激活了成孔蛋白， 一种蛋白质，gasdermin D（GSDMD），驱动SMC焦亡，一种程序性细胞死亡的炎性形式 这可能代表了主动脉SMC丢失的共同途径。因此，本申请的目的是 确定GSDMD介导的SMC焦亡在ATAAD发展中的作用和机制， 细胞质DNA传感和焦亡代表针对ATAAD的治疗靶标的程度。 我们的中心假设是受损DNA触发的传感器通路激活GSDMD介导的SMC 导致主动脉变性、夹层和破裂。在目标1中，我们将确定 ATAAD形成中GSDMD介导的SMC焦亡。我们将检验GSDMD介导的 SMC焦亡与主动脉变性、生物力学失效和ATAAD的发展密切相关， 在Gsdmd-/-小鼠和可诱导SMC特异性Gsdmd敲除小鼠中检测这些方面， 散发性ATAAD模型。在目标2中，我们将研究GSDMD激活的机制。 介导的平滑肌细胞凋亡。在一系列的体外和体内实验中，我们将测试细胞- solDNA触发的cGAS-STING传感器信号传导，通过TBK 1/IKK 2/DNA-PK激酶，直接磷酸化， 使GSDMD延迟并促进其活化和质膜孔的形成，导致线粒体 最终导致平滑肌细胞死亡。在目标3中，我们将确定在多大程度上抑制cy- 溶解的DNA感应和焦变性细胞死亡将阻止ATAAD的发展和进展。我们将测试 同时阻断cGAS-STING通路和焦亡将防止主动脉脱髓鞘的假设， 结构和疾病进展。我们希望这项工作将决定如何 损伤的DNA诱导细胞凋亡，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对分子机制的更好理解， 在主动脉退行性变的发展过程中引起SMC的热凋亡，为治疗提供了一个令人振奋的新方向- 预防ATAAD形成及其致命后遗症的治疗策略。关于DNA的新机制 损伤诱导的细胞凋亡在许多其它心血管疾病中也具有广泛的意义。 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