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...