Project Summary Emerging evidence suggests that inappropriate matrix metalloprotease (MMP) activity may underlie the pathogenesis of atherosclerosis and vascular inflammation. Despite data that MMPs contribute to atherosclerotic lesion remodeling and poor outcomes, essentially nothing is known regarding the role of MMPs as active signaling molecules in controlling the behavior of vascular cells in the context of atherosclerotic disease. We recently made the unanticipated discovery that MMP-1 cleaves and activates protease-activated receptor-1 (PAR1) signaling in blood vessels. This is of major import as it was the first report of a direct signaling function of the principal collagenase in blood vessels. Notably, we found that MMP-1 activates PAR1 by cleaving the receptor at a distinct site from the canonical thrombin cleavage site which generates a longer tethered ligand that is biased towards a different spectrum of G protein signaling pathways. The studies in this proposal will focus on the completely unexplored role of MMP1-PAR1 in the development of atherosclerotic plaques. Furthermore, the involvement of PAR1 in atherosclerosis (regardless of the protease agonist) as a chronic evolving inflammatory disease, is essentially unknown. The central hypothesis to be tested in aim 1 is that endothelial MMP-1 first acts as an active signaling molecule via PAR1 to trigger endothelial inflammation and monocyte entry into early plaques. In advanced lesions, MMP1 from macrophages autostimulates PAR1 to perpetuate a chronic inflammatory and mitogenic state by secretion of MCP-1 and other mediators. We will use both cell-based experiments, and hyperlipidemic mouse models with genetic deficiency of Mmp1a or Par1. We will determine the requirement of MMP1a and PAR1 in mouse endothelial cells for monocyte adhesion and transmigration under shear flow conditions in vitro using mouse heart endothelial cells isolated from of Mmp1a-/- and Par1-/- mice, and monocytes from ApoE-/- mice after high fat diet. Aim 2 will examine the role of the MMP1-PAR1 system on circulating monocytes in subjects with coronary artery disease and acute coronary syndromes undergoing percutaneous coronary interventions (PCI). Monocytes from patients at baseline prior to PCI and from those being treated with a novel PAR1 pepducin, PZ-128, will be used in Parallel Plate (arterial-shear) flow chambers to determine transmigration through endothelial monolayers. We will determine whether subjects with a super-active Mmp1 promoter polymorphism exhibit higher expression of MMP1 that contributes to a `MMP1-PAR1' phenotype vs `TF-PAR1' phenotype on their monocytes. Aim 3 will use adoptive transfer of bone marrow-derived cells from Par1-/-ApoE-/- and Mmp1a-/-ApoE-/- mice to help define the cell- type specific pathobiology (e.g. endothelium vs leukocytes) of the MMP1-PAR1 system in atherogenesis. Our understanding of the pathophysiologic relevance of MMP1-PAR1 signaling on endothelium and monocytes/ macrophages, and the mechanism linking these events to atherosclerotic plaque development will provide a framework to advance future therapies that could halt or reverse the progression of atherosclerosis.

Project Narrative Atherosclerosis and its ischemic complications remain a major cause of death among Americans with estimated healthcare costs approaching a staggering $500 billion. The contribution of endothelial and leukocyte MMP1-PAR1 to life-threatening complications of plaque stenosis/rupture remains of great interest and the work described here may lead to the development of new agents that suppress atherosclerosis.