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

壁内或腔内血凝块通常存在于人类主动脉瘤中，这是一种进展性的动脉瘤。 主动脉的弱化和扩张，与平滑肌细胞（SMC）耗竭、降解 以及炎症细胞的浸润。靶向对血栓至关重要的成分 在小鼠模型中，已经显示出形成可降低动脉瘤的严重性。然而，知识有限， 关于促进动脉瘤内血栓形成的分子机制。初步 研究表明，从人血浆中分离的细胞外囊泡（EV）含有受体， 相互作用蛋白激酶3（RIP 3），一种对SMC坏死至关重要的细胞内信号蛋白。广泛 在体内和体外进行的初步研究表明，RIP 3具有促血栓形成功能 在细胞之外。主动脉瘤患者血浆样本的分析显示， 血浆RIP 3水平与凝血的相关性。两个相关但独立的具体目标是 提出测试中心假设，即受损的主动脉SMC释放富含RIP 3的EV。当 RIP 3释放到细胞外间隙，通过与凝血相互作用刺激血栓形成 件. 在目标1中，我们将使用各种体外方法来解决机制问题，包括RIP 3是如何在体内表达的。 以及细胞外RIP 3如何刺激凝血。目标1a测试RIP 3是否排序为 通过与参与内体分选的蛋白质结合来产生EV。目的1b旨在证明在离体模型中 受血栓影响的主动脉组织促进血浆凝固。目标1c使用定量 蛋白质组学分析以确定由应激SMC释放的EV的“蛋白质特征”。Aim 1D考试 RIP 3是否至少部分地通过与因子IX相互作用来刺激血栓形成，因子IX是RIP 3的丝氨酸蛋白酶之一， 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RIP 3的EV有助于主动脉血栓形成。目的2a确定缺乏EV包装的小鼠是否 因子Rab 27 a/B对血管紧张素II的反应是减少血栓形成和主动脉病变。目标2b将进一步 通过尝试挽救Rip 3-/-的血栓形成缺陷表型，确定EV在动脉瘤中的作用 携带RIP 3 EV的小鼠。作为将基本发现转化为动脉瘤临床管理的一种方式， 目的2c使用现有组织检查血浆RIP 3水平与临床结果之间的关系 和人类主动脉瘤数据库。通过证明RIP 3在凝血中的新的细胞外功能， 该提议将对血栓形成和动脉瘤领域产生范式转换的影响。

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.