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 (PROTOMAP) 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. 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. PROTOMAP did not detect robust increases in proteolytic fragments of extracellular-matrix 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.

The tandem stenosis mouse model: Towards understanding, imaging, and preventing atherosclerotic plaque instability and rupture

“…One example would be the production of MMPs, predominantly by macrophages, which can degrade the fibrous cap of atherosclerotic plaques (Amento et al, 1991; Galis et al, 1994; Mach et al, 1997). A recent study conducting parallel proteomic analysis of human and mouse plaques, focusing on the comparison between stable and unstable atherosclerosis, has further strengthened links between proteolytic degradation, inflammation, and the rupture of atherosclerotic plaques (Vaisar et al, 2020). Consequently, an immune cell‐driven bias towards extracellular matrix degradation in atherosclerosis is considered a likely key step in driving plaque instability and rupture.…”

“…Therefore, the ability to model plaque instability and rupture in animals is a critically required tool for cardiovascular research to allow for the discovery and dissection of the fundamental biological mechanisms underpinning cardiovascular events. Indeed, transcriptomic and proteomic comparisons between the regions displaying stable and unstable atherosclerosis indicate that both are different disease entities (Chen et al, 2013; Vaisar et al, 2020). With this in mind, we will now discuss the TS model, which our laboratory designed and validated to provide a reliable, flexible, and widely applicable model of plaque instability and rupture.…”

Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes

“…Replicating the observed DBD kidney key degradation profiles of talin-1 in human kidney immortalized podocyte cells revealed a role of the calpain-1-TGFβ axis in proteolytic activation and cytoskeletal dysregulation that may deem grafts susceptible to subsequent injury-a causal link that further studies should aim to establish.To investigate the donor kidney degradome we analyzed deceased and living donor kidney biopsies obtained with identical procurement protocols to minimize preanalytical confounding factors. This stringent experimental selection, based on paired transplant outcomes, also increased our confidence in obtaining meaningful data that predominantly relates to donor minimizing the impact or bias from surgical or posttransplant factors.We, and more recently others, have shown that the application of the PROTOMAP technique to complex clinical samples provides a comprehensive profiling of both N-and C-terminal proteoforms with sufficient sequence coverage to determine epitope coverage by commercial antibodies, thereby allowing validation by immunoblotting 25,41,42. Initial, unbiased PROTOMAP degradation profiling and pathway analysis mapped α -actinin, synaptopodin, talin, laminin β2, integrin α1, and utrophin as a cluster of cytoskeletal proteins associated with focal adhesion in podocytes had undergone enhanced proteolytic processing in DBD kidneys with SO posttransplant function.…”

Deceased donor kidney degradomics indicates cytoskeletal proteolytic alterations impacting post-transplant function

“…We, and more recently others, have shown that the application of the PROTOMAP technique to complex clinical samples provides a comprehensive profiling of both N-and C-terminal proteoforms with sufficient sequence coverage to determine epitope coverage by commercial antibodies, thereby allowing validation by immunoblotting. 27,47,48 . Initial, unbiased PROTOMAP degradation profiling and pathway analysis mapped α -actinin, Synaptopodin, Talin, Laminin β2, Integrin α1 and Utrophin as a cluster of cytoskeletal proteins associated with focal adhesion in podocytes had undergone enhanced proteolytic processing in DBD kidneys with SO posttransplant function.…”