Novel Role of Thrombospondin-1 in Protection against Rupture of Abdominal Aortic Aneurysm

腹主动脉瘤（AAA）是主动脉的进行性弱化和扩张。大幅 在对动脉瘤破裂的分子机制的理解方面存在知识差距， 是AAA患者死亡的主要原因。根据我们先前关于血小板反应蛋白-1（TSP 1）升高的报告， 在人类和小鼠垂体组织中，我们进行了单细胞RNA测序（scRNA-seq）分析， 确定巨噬细胞（M β）是小鼠动脉瘤主动脉中升高的TSP 1的主要来源。我们 随后通过将Lyz 2-Cre与我们的新的Thbs 1基因敲除小鼠杂交， 构建Thbs 1flox/flox小鼠。当经受血管紧张素II（Ang II）与 在高胆固醇血症中，超过60%的Thbs 1 β M患者死于AAA破裂，发生率高2.6倍 Thbs1wt.有趣的是，存活至28天Ang II输注结束的Thbs 1 ️ M小鼠显示动脉瘤较少 膨胀比Thbs 1 wt.当Thbs 1 β M β小鼠被激发时，也发现了较小的淋巴瘤扩张。 血管周围应用CaCl 2，AAA模型不产生破裂。我们提出两个具体目标 阐明M β特异性Thbs 1基因缺失差异影响主动脉粥样硬化的机制， 扩张和破裂，重点是AAA破裂。具体目标1致力于建立断裂- M β TSP 1的预防功能。具体地说，我们将确定致命性破裂前的主动脉反应， 雄性和雌性Thbs 1 β M β小鼠在Ang II模型中，随后鉴定破裂相关分子 通过scRNA-seq、原位杂交和免疫染色的特征。此外，我们将研究 使用不同的小鼠模型在晚期阶段产生破裂的M β特异性Thbs 1敲除的影响 的动脉瘤，这是更相关的人AAA比早期破裂产生的血管紧张素II模型。具体 目的2探讨动脉瘤破裂的分子机制。初步研究表明， 与野生型相比，Thbs 1-/- M β s迁移或吞噬凋亡细胞的能力也显著降低 中性粒细胞胞外陷阱（NETs）。我们将测试M β TSP 1是否通过CD 47促进NET清除， 介导的肌动蛋白聚合。接下来，我们将建立受损的M-12迁移的因果关系， Thbs 1巨噬细胞吞噬作用对Thbs 1巨噬细胞吞噬作用的影响。我们将首先确定NET负担是否增加 Thbs 1组中，M细胞断裂死亡，NET与M细胞的空间分布关系。第二，我们将测试 通过过继转移策略，恢复Thbs 1-Thb M中的M迁移减少了NET积累。而且我们 将检查增强或减弱NET清除率是否会影响Thbs 1型颅内动脉瘤破裂。最后我们 将分析TSP 1的表达及其与破裂和非破裂组织中M β和NET积聚的相关性， 人AAA组织。通过剖析多方面的功能，M神经元通过TSP 1操纵，该项目 将对动脉瘤破裂的认识产生重大影响。

Abdominal aortic aneurysm (AAA) is the progressive weakening and dilation of the aorta. A substantial knowledge gap exists in the understanding of molecular mechanisms responsible for aneurysm rupture, the major cause of mortality among AAA patients. Following our prior report of elevated thrombospondin-1 (TSP1) in human and mouse aneurysmal tissues, we conducted single-cell RNA sequencing (scRNA-seq) analysis and identified macrophages (Mɸs) being the primary source of elevated TSP1 in mouse aneurysmal aorta. We subsequently generated Mɸ-specific Thbs1 knockout mice (Thbs1∆Mɸ) by crossing Lyz2-Cre with our newly constructed Thbs1flox/flox mice. When subjected to aneurysm induction by angiotensin II (Ang II) coupled with hypercholesterolemia, over 60% of Thbs1∆Mɸ died due to AAA rupture, an incidence that was 2.6 times higher than Thbs1wt. Intriguingly, Thbs1∆Mɸ mice that survived to the end of 28-day Ang II infusion showed less aneurysm dilation than Thbs1wt. Smaller aneurysmal expansion was also found when Thbs1∆Mɸ mice were challenged with perivascular application of CaCl2, an AAA model that does not produce rupture. We propose two specific aims to delineate the mechanisms through which Mɸ-specific Thbs1 gene deletion differentially affects aortic dilation and rupture with an emphasis on AAA rupture. Specific Aim 1 devotes to establishing the rupture- preventive function of Mɸ TSP1. Specifically, we will determine the aortic responses proceeding lethal rupture in male and female Thbs1∆Mɸ mice in the Ang II model followed by identifying rupture-associated molecular signatures through scRNA-seq, in situ hybridization and immunostaining. Furthermore, we will examine the effects of Mɸ-specific Thbs1 knockout using a different murine model that produces rupture in advanced stages of aneurysm, which is more relevant to human AAA than the early rupture produced by the Ang II model. Specific Aim 2 focuses on investigating molecular mechanisms of aneurysm rupture. Preliminary studies showed that compared to wildtype, Thbs1-/- Mɸs had significantly reduced ability to migrate or to engulf apoptotic cells as well as neutrophil extracellular traps (NETs). We will test whether Mɸ TSP1 promotes NET clearance through CD47- mediated actin polymerization. Next, we will establish the causal effect of impaired Mɸ migration and phagocytosis on aneurysm rupture of Thbs1∆Mɸ mice. We will first determine whether NET burden is increased in Thbs1∆Mɸ died from rupture, and the spatial relationship between NETs and Mɸs. Second, we will test whether restoring Mɸ migration in Thbs1∆Mɸ reduces NET accumulation via adoptive transfer strategies. Furthermore, we will examine whether enhancing or attenuating NET clearance affect aneurysm rupture in Thbs1∆Mɸ. Lastly, we will analyze TSP1 expression and its association with Mɸ and NET accumulation in ruptured and non-ruptured human AAA tissues. By dissecting the multifaceted functions of Mɸs through TSP1 manipulations, this project will produce significant impact on the understanding of aneurysm rupture.