Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture

Abstract

Rationale: Plaque rupture is the proximate cause of most myocardial infarctions and many strokes. However, the molecular mechanisms that precipitate plaque rupture are unknown. Objective: By applying proteomic and bioinformatic approaches in mouse models of protease-induced plaque rupture and in ruptured human plaques, we aimed to illuminate biochemical pathways through which proteolysis causes plaque rupture and identify substrates that are cleaved in ruptured plaques. Methods and Results: We performed shotgun proteomics analyses of aortas of transgenic mice with macrophage-specific overexpression of urokinase (SR-uPA +/0 mice) and of SR-uPA +/0 bone marrow transplant recipients, and we used bioinformatic tools to evaluate protein abundance and functional category enrichment in these aortas. In parallel, we performed shotgun proteomics and bioinformatics studies on extracts of ruptured and stable areas of freshly harvested human carotid plaques. We also applied a separate protein-analysis method (protein topography and migration analysis platform) to attempt to identify substrates and proteolytic fragments in mouse and human plaque extracts. Approximately 10% of extracted aortic proteins were reproducibly altered in SR-uPA +/0 aortas. Proteases, inflammatory signaling molecules, as well as proteins involved with cell adhesion, the cytoskeleton, and apoptosis, were increased. ECM (Extracellular matrix) proteins, including basement-membrane proteins, were decreased. Approximately 40% of proteins were altered in ruptured versus stable areas of human carotid plaques, including many of the same functional categories that were altered in SR-uPA +/0 aortas. Collagens were minimally altered in SR-uPA +/0 aortas and ruptured human plaques; however, several basement-membrane proteins were reduced in both SR-uPA +/0 aortas and ruptured human plaques. Protein topography and migration analysis platform did not detect robust increases in proteolytic fragments of ECM proteins in either setting. Conclusions: Parallel studies of SR-uPA +/0 mouse aortas and human plaques identify mechanisms that connect proteolysis with plaque rupture, including inflammation, basement-membrane protein loss, and apoptosis. Basement-membrane protein loss is a prominent feature of ruptured human plaques, suggesting a major role for basement-membrane proteins in maintaining plaque stability.