PROJECT SUMMARY Abdominal aortic aneurysms (AAA) formation and subsequent aortic rupture can lead to sudden death and is a significant clinical problem with no currently known medical treatments available. The hallmarks of patient AAA include thrombus formation and cell death mechanisms such as apoptosis and neutrophil extracellular traps (NETs). The clearance of dead cell debris is mediated via the process of efferocytosis, by which apoptotic tissue is recognized for engulfment by professional phagocytes (e.g., macrophages) and non-professional phagocytes (e.g., endothelial cells; ECs), and remains to be elucidated in the pathogenesis of AAA. Thus, our hypothesis focuses on the dysregulation of inflammation-resolution pathways that lead to to defective efferocytosis and promote chronic aortic tissue inflammation and vascular remodeling. Our recent study has demonstrated a critical role orchestrated by EC-dependent pannexin-1 (Panx1) channels in causing aortic inflammation and AAA formation. The scientific premise of this proposal focuses on the dysregulation of EC- mediated efferocytosis causing an imbalance of inflammation-resolution via Panx1 activation in AAA progression. Therefore, the central hypothesis in this proposal is that EC efferocytosis is dysregulated due to cleavage of MerTK, a cell surface tyrosine kinase receptor that recognizes apoptotic cells, leading to accumulation of dead cell debris and thrombus formation. Second, our mechanistic studies will dissect the dynamic communication between ECs and macrophages, involving defective EC-mediated efferocytosis leading to excessive iron-mediated cell death (ferroptosis) in macrophages, that collectively feedbacks to cause Panx1 activation and an chronic inflammatory loop. Our supportive data demonstrates that resolution of aortic inflammation is associated with increased EC-dependent MerTK expression and efferocytosis of neutrophils. Furthermore, defective EC-mediated efferocytosis exacerbates ferroptosis in macrophages (via SLC7A11 and Nrf2-signaling) that feedbacks to cause EC-Panx1 activation and eATP release. Collectively, our results suggest that dysregulation of EC-mediated efferocytosis and macrophage-dependent ferroptosis creates a break in the inflammation-resolution process during AAA formation and aortic rupture. We will delineate the proposed studies using the murine elastase-treatment AAA and our innovative aortic rupture model, as well as by analysis of human AAA tissue from our biorepository. Using novel inducible cell-specific genetic knockout mice such as Cdh5Cre-ERT2/MerTKfl/fl, MerTKCR (cleavage-resistant), and Cx3CR1Cre- ERT2/SLC7A11fl/fl mice, we will delineate the previously unknown mechanisms of dysregulated efferocytosis and ferroptosis in activation of Panx1 channels during AAA formation. Our studies will provide novel insight into mechanisms of molecular signaling interactions between ECs and macrophages to define the inflammatory loop between efferocytosis/f...