PROJECT SUMMARY Alterations in hemodynamic shear stress at atherosclerosis-prone sites promotes endothelial activation, characterized by nuclear factor-B (NF-B)-driven expression of cell adhesion molecules that mediate leukocyte homing. In addition to flow patterns, proinflammatory cytokines (e.g. IL-1) promote NF-B-dependent endothelial activation. While the recent CANTOS trial using an IL-1 antagonist highlights the potential for treating atherogenic inflammation beyond lipid lowering therapies alone, our understanding of how endothelial activation contributes to this effect remains limited. During the previous funding period, my laboratory identified the signaling adaptor Nck1 as a critical regulator of atherogenic endothelial activation. Endothelial cells deficient in Nck1 lack flow-induced NF-B activation and proinflammatory gene expression both in vitro and in vivo, and domain swap experiments and point mutation analysis suggest a critical role for the Nck1 SH2 domain and the first Nck1 SH3 domain in mediating these effects. To understand how Nck1 mediates atherogenic endothelial activation, we isolated Nck1’s proinflammatory functions to the Nck1 SH2 domain and fist SH3 domain. Additionally, we demonstrated an interaction between Nck1 and IL-1 signaling in response to disturbed flow, showing that disturbed flow activates the IL-1 receptor (IL1R1) signaling partner IRAK-1 in a Nck1-dependent manner both in vitro and in vivo. IRAK-1 depletion inhibited the proinflammatory response to disturbed flow, but the mechanism by which disturbed flow stimulates IRAK-1 signaling remains unknown. The Nck family of signaling adaptors (Nck1 and Nck2) play redundant roles during vascular development and retinal neovascularization, with defects only observed when both isoforms are inhibited. Consistent with our cell culture studies, global Nck1 knockout mice show reduced atherosclerosis characterized by diminished macrophage and smooth muscle incorporation, whereas endothelial Nck2 knockouts do not. While bone marrow chimeras suggest non-hematopoietic Nck1 mediates this response, questions concerning the relative roles of endothelial and smooth muscle Nck1 in this response persist. A recent GWAS analysis linked Nck1 to coronary artery disease, underscoring its importance to human disease. Therefore, we hypothesize that Nck1 adaptors mediate the formation of IL-1-related signaling complexes to induce atherogenic endothelial activation, and selective inhibition of Nck1 or its critical domains will allow efficient inhibition of atherogenic endothelial activation without affecting ischemic vascular remodeling. We will determine the mechanisms by which Nck1 regulates disturbed flow-induced IRAK-1 activation (Aim 1), characterize the interplay between Nck1 and IL-1-associated signaling in disturbed flow-induced endothelial activation in vivo (Aim 2), and assess whether selective Nck1 inhibition differentially affects atherosclerosis and ischemic angiogene...