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Role of RIP3-laden extracellular vesicles in thrombosis and aortic aneurysm

负载 RIP3 的细胞外囊泡在血栓形成和主动脉瘤中的作用

基本信息

批准号：
10630195
负责人：
Bo Liu
金额：
$ 47.28万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-15 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10630195
关键词：
Address Affect Aneurysm Angiotensin II Aorta Aortic Aneurysm Aortic Injury Binding Biogenesis Biological Markers Blood Platelets Blood Vessels Blood coagulation Cause of Death Cells Cessation of life Chest Clinical Clinical Data Clinical Management Coagulation Process Complex Data Detection Development Dilatation - action Endosomes Enzymes Event Extracellular Matrix Extracellular Space Factor IX Fibrin Fibrinogen Functional disorder Generations Growth Human In Vitro Inflammatory Infiltrate Intracellular Signaling Proteins Knockout Mice Knowledge Life Style Ligation Literature Medial Medical center Membrane Methods Modeling Molecular Monitor Mus Mutant Strains Mice Necrosis Operative Surgical Procedures Outcome Pathogenesis Pathologic Pathology Pathway interactions Patients Pharmacological Treatment Phenotype Phosphorylation Phosphotransferases Plasma Play Pre-Clinical Model Prevention Protein Kinase Interaction Proteins Proteomics Publishing RIPK1 gene RNA Recombinants Reporting Research Role Rupture Sampling Serine Protease Severities Signal Transduction Smooth Muscle Myocytes Sorting Stress Structure System Testing Therapeutic Intervention Thrombin Thrombophilia Thrombosis Thrombus Time Tissue Banks Tissue Sample Tissues Translating United States Venous biobank data repository disability experimental study extracellular extracellular vesicles in vivo inhibitor mouse model nanoscale novel pre-clinical receptor function small molecule targeted treatment thrombotic translational study vascular inflammation vesicular release

项目摘要

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 1a tests whether RIP3 is sorted into EVs by binding to proteins involved in endosomal sorting. Aim 1b seeks to demonstrate in an ex vivo model that aneurysm-affected aortic tissues promote plasma to undergo coagulation. Aim 1c uses quantitative proteomic analysis to determine the “protein signature” of EVs released by stressed SMCs. Aim 1d 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 2a determines whether mice lacking the EV packing factor Rab27a/b respond to angiotensin II with diminished thrombosis and aortic pathology. Aim 2b 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.

动脉壁内或管腔内血栓常见于人类主动脉瘤，进行性发展。主动脉变弱和扩张，与平滑肌细胞(SMC)耗尽、降解有关基质蛋白和炎性细胞的渗透。靶向对血栓至关重要的成分在小鼠模型中，形成动脉瘤已被证明可以降低动脉瘤的严重程度。然而，有限的知识存在关于促进动脉瘤内血栓形成的分子机制。在预赛中研究表明，从人血浆中分离的细胞外小泡含有受体。相互作用蛋白激酶3(RIP3)，一种细胞内信号蛋白，在SMC坏死中起关键作用。广泛性在体内和体外进行的初步研究表明，RIP3具有促血栓功能在牢房外。对主动脉瘤患者血浆样本的分析显示，血浆RIP3水平与凝血功能的相关性两个相互关联但又相互独立的具体目标是提出了一项中心假设，即受损的主动脉SMCs会释放富含RIP3的EV。什么时候释放到细胞外空间，RIP3通过与凝血相互作用刺激血栓形成组件。在目标1中，我们将使用各种体外方法来解决机械问题，包括RIP3是如何包装在EVS内，以及细胞外RIP3如何刺激凝血。Aim 1a测试RIP3是否归入 EVS通过与参与内体分选的蛋白质结合。Aim 1b试图在体外模型中证明受动脉瘤影响的主动脉组织促进血浆进行凝血。AIM 1c使用量化蛋白质组学分析，以确定应激状态下SMC释放的电动汽车的“蛋白质特征”。Aim 1D考试 RIP3是否至少部分通过与凝血因子IX相互作用来刺激血栓形成凝血系统。在目标2中，使用主动脉瘤的临床前模型，我们将检验以下假设携带RIP3的EV会导致主动脉血栓形成。Aim 2a确定小鼠是否缺乏EV包装因子Rab27a/b对血管紧张素II有反应，血栓形成和主动脉病变减少。目标2b将进一步试图挽救RIP3-/-缺陷的血栓表型以确定EVS在动脉瘤中的作用携带RIP3电动汽车的小鼠。作为一种将基本发现转化为动脉瘤临床治疗的方法，目的2c利用现有的组织研究血浆RIP3水平和临床结果之间的关系。和人体主动脉瘤数据库。通过证明RIP3在凝血中的新的细胞外功能，这一提议将对血栓和动脉瘤领域产生翻天覆地的影响。