Intramural or intraluminal blood clots are commonly present in human aortic aneurysms, a progressive weakening and dilatation of aorta that is associated with depletion of smooth muscle cells (SMCs), degradation of matrix proteins, and infiltration of inflammatory cells. Targeting constituents that are vital for thrombus formation have been shown to reduce aneurysm severity in mouse models. However, limited knowledge exists with regard to the molecular mechanisms that promote thrombus formation within aneurysms. In preliminary studies, we demonstrated that extracellular vesicles (EVs) isolated from human plasma contained receptor interacting protein kinase 3 (RIP3), an intracellular signaling protein that is critical to SMC necrosis. Extensive preliminary studies, performed both in vivo and in vitro, demonstrated that RIP3 has a pro-thrombotic function outside of cells. Analysis of plasma samples from aortic aneurysm patients showed a significant linear correlation between plasma RIP3 levels and coagulation. Two related, yet independent specific aims are proposed to test the central hypothesis that injured aortic SMCs release EVs that are rich in RIP3. When discharged to the extracellular space, RIP3 stimulates thrombosis by interacting with coagulation components.
In Aim 1, we will use various in vitro approaches to address mechanistic questions, including how RIP3 is packed inside EVs and how extracellular RIP3 stimulates coagulation.
Aim 1 a tests whether RIP3 is sorted into EVs by binding to proteins involved in endosomal sorting.
Aim 1 b seeks to demonstrate in an ex vivo model that aneurysm-affected aortic tissues promote plasma to undergo coagulation.
Aim 1 c uses quantitative proteomic analysis to determine the ?protein signature? of EVs released by stressed SMCs.
Aim 1 d exams whether RIP3 stimulates thrombosis at least in part by interacting with Factor IX, one of the serine proteases of the coagulation system.
In Aim 2, using a preclinical model of aortic aneurysm, we will test the hypothesis that RIP3-carrying EVs contribute to aortic thrombosis.
Aim 2 a determines whether mice lacking the EV packing factor Rab27a/b respond to angiotensin II with diminished thrombosis and aortic pathology.
Aim 2 b will further establish the role of EVs in aneurysm by attempting to rescue the deficient thrombotic phenotype of Rip3-/- mice with RIP3-carrrying EVs. As a way to translate basic findings to the clinical management of aneurysm, Aim 2c examines the relationship between plasma RIP3 levels and clinical outcomes using an existing tissue and data bank of human aortic aneurysm. By proving the novel extracellular function of RIP3 in coagulation, this proposal will have a paradigm-shifting impact on the field of thrombosis and aneurysm.
Aortic aneurysm is a major cause of death and disability in the United States, but current treatment relies on detection, monitoring, lifestyle changes, and surgery. Therefore, there is a critical need to understand the pathogenesis of aortic aneurysm for the development of pharmacological treatments and methods of prevention. The proposed research aims to investigate the molecular and cellular underpinnings that cause aneurysm development, growth, and rupture, which will help in identifying targets for therapeutic intervention.
