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

腹主动脉瘤(AAA)是指主动脉的进行性变弱和扩张。一大笔钱 在了解导致动脉瘤破裂的分子机制方面存在知识差距， AAA患者死亡的主要原因。在我们先前报告血栓反应蛋白-1(TSP1)升高之后 在人和小鼠动脉瘤组织中，我们进行了单细胞RNA测序(scRNA-seq)分析和 巨噬细胞(Mɸ，S)是小鼠主动脉瘤TSP 1升高的主要来源。我们 随后将ɸ-Cre与我们的新基因杂交，获得了M只Thbs1特异性Thbs1基因敲除小鼠(Thbs1∆Mɸ 构建Thbs1 FLOX/FLOX小鼠。血管紧张素II(Ang II)联合血管紧张素II(Ang II)诱导动脉瘤时 高胆固醇血症，超过60%的Thbs1∆Mɸ死于腹主动脉破裂，发病率是前者的2.6倍 比Thbs1wt.有趣的是，存活到28天血管紧张素II输注结束的Thbs1∆Mɸ小鼠显示出较少的动脉瘤 膨胀比Thbs1wt。当Thbs1∆Mɸ小鼠接受 血管周围应用CaCl2，这是一种不会产生破裂的AAA模型。我们提出了两个具体目标 M-ɸ特异性Thbs1基因缺失对主动脉影响的机制探讨 扩张和破裂，重点是AAA破裂。具体目标1致力于确定破裂- MɸTSP1的预防功能。具体地说，我们将确定发生致命性破裂的主动脉反应。 在血管紧张素Ⅱ模型中雄性和雌性Ths1∆Mɸ小鼠随后识别破裂相关分子 通过scRNA-seq、原位杂交和免疫染色进行标记。此外，我们还将研究 M-ɸ特异性Thbs1基因敲除对晚期破裂小鼠模型的影响 与Ang II模型造成的早期破裂相比，这与人类AAA的相关性更大。特定的 目的2重点研究动脉瘤破裂的分子机制。初步研究表明， 与野生型相比，Thbs1-/-MɸS的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MɸTSP1是否通过CD47促进净清算- 介导的肌动蛋白聚合。接下来，我们将确定M-ɸ迁移受损和 巨噬细胞对Thbs1∆Mɸ小鼠动脉瘤破裂的影响我们将首先确定净负担是否增加 在Thbs1中，∆Mɸ死于破裂，以及篮网和MɸS之间的空间关系。第二，我们将测试 在Thbs1ɸM∆中恢复Mɸ迁移可通过采用迁移策略减少净积累。此外，我们 将检查增强或减弱净清除是否影响Thbs1∆Mɸ中的动脉瘤破裂。最后，我们 将分析破裂型和非破裂型TSP1的表达及其与M-ɸ和净蓄积的关系 人体AAA组织。本项目通过对MɸS的TSP1操作，对其多方面的功能进行剖析 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.