Role of Inflammation in the Pathobiology of Abdominal Aortic Aneurysm

描述(由申请人提供)： 腹主动脉瘤(AAA)是一种常见且可能危及生命的血管疾病，在美国退伍军人中很常见。主动脉管壁炎症导致AAA的两个基本结构改变：弹性蛋白降解和主动脉中膜内平滑肌细胞(SMC)的丢失。动脉瘤样变性与弹性蛋白特异的蛋白水解酶，特别是基质金属蛋白酶(MMPs)的合成和活性增加有关。虽然巨噬细胞和SMC是AAAs发病机制中的关键细胞介质，但触发炎性级联反应的上游刺激导致基质改变仍不清楚。晚期糖基化终末产物受体(RAGE)的激活既会导致急性炎症反应，又会导致组织损伤的慢性、持续性炎症状态的持续存在。此外，RAGE与内源性金属蛋白酶组织抑制物(TIMPs)一起，上调包括MMP9在内的MMPs的表达和活性，MMP9是巨噬细胞表达的一种对AAA形成至关重要的蛋白酶。SMC的凋亡与RAGE和透明蛋白1(MDIA-1)的相互作用有关。可以使用RAGE的配体结合诱饵--可溶性RAGE(SRAGE)以及RAGE缺失的小鼠来阻断RAGE。在SMCs表达由SM22启动子驱动的显性阴性(DN)RAGE的转基因小鼠中，可以评估RAGE激活的细胞特异性贡献。我们假设RAGE改变巨噬细胞极化和SMC凋亡，导致作为AAA发展基础的基质变性。因此，这项建议的具体目标是：目标1：确定RAGE介导的信号是否改变了AAA促炎病理生理基础上的巨噬细胞激活和极化。我们假设RAGE介导巨噬细胞从M2(修复性)到M2(炎症性)的极化，M2(炎症性)表型传递损伤信号并有助于AAA。我们将描述弹性酶灌注后RAGE零、sRAGE治疗和未治疗小鼠的主动脉组织中的M1和M2亚型。我们将通过使用RAGE配体刺激的原代骨髓来源的巨噬细胞的体外研究来证实我们的发现，以确定RAGE对基质金属蛋白酶和TIMP活性以及巨噬细胞极化的影响。我们将用RAGE充足的骨髓重建RAGE缺失的小鼠，以确定骨髓来源的白细胞上RAGE的存在是否足以使弹性蛋白酶灌流的小鼠主动脉中的巨噬细胞极化为M1亚型。目的#2：研究RAGE介导的主动脉壁内SMC凋亡是否导致AAA的发病。我们推测，RAGE通过与MDIA-1的相互作用，介导了主动脉壁内SMC的凋亡和增殖。我们将利用SMC DN RAGE小鼠来评估弹性蛋白酶诱导的AAA中SMC特异性激活RAGE的作用。使用Ki67和TUNEL染色，我们将定量弹性蛋白酶灌流的野生型、RAGE零和SRAGE处理的野生型小鼠主动脉中SMC的增殖和凋亡指数。在体外，我们用RAGE配体刺激原代培养的主动脉SMC，并检测RAGE对SMC增殖和凋亡指数的影响。我们将使用抑制性RNA技术来确定MDIA-1是否是RAGE效应所必需的。这一建议是创新的，因为我们将使用体内和体外模型来了解RAGE介导的巨噬细胞极化和SMC凋亡的变化如何导致AAA的发生。该提案中描述的研究可能最终导致新的诊断和治疗策略用于预防和治疗。退伍军人人群中AAA发病率的增加和目前可用的治疗选择有限，突显了这一建议的临床相关性。

DESCRIPTION (provided by applicant): Abdominal aortic aneurysms (AAAs) are a common and potentially life-threatening vascular condition prevalent among US veterans. Aortic wall inflammation results in two fundamental structural changes which underlie AAA; elastin degradation and loss of smooth muscle cells (SMC) within the aortic media. Aneurysmal degeneration is related to increased elaboration and activity of elastin-specific proteinases, particularly matrix metalloproteinases (MMPs). While macrophages and SMC are key cellular mediators in the pathogenesis of AAAs; the upstream stimulus which triggers the inflammatory cascade responsible for matrix changes remains unknown. Activation of the receptor for advanced glycation end products (RAGE) leads to both an acute inflammatory response and perpetuation of a chronic, sustained inflammatory state leading to tissue damage. Additionally, along with endogenous tissue inhibitors of metalloproteinases (TIMPs), RAGE upregulates the expression and activity of MMPs including MMP-9, a protease expressed by macrophages that is critical to AAA formation. SMC apoptosis has been linked to the interaction of RAGE with diaphanous 1 (mDia-1). RAGE blockade may be effected using soluble RAGE (sRAGE), a ligand-binding decoy of RAGE, as well as RAGE null mice. Transgenic mice in which SMCs express dominant negative (DN) RAGE driven by the SM22¿ promoter allow assessment of cell-specific contributions of RAGE activation. We hypothesize that RAGE alters macrophage polarization and SMC apoptosis, leading to matrix degeneration fundamental to AAA development. Thus, the specific aims of this proposal are: Aim #1: Determine if RAGE-mediated signaling alters macrophage activation and polarization underlying the proinflammatory pathophysiology of AAA. We hypothesize that RAGE mediates the polarization of macrophages from an M2 (reparative) to an M2 (inflammatory) phenotype that transmits a damage signal and contributes to AAA. We will delineate the M1 and M2 subtypes in the aortic tissues of RAGE null, sRAGE-treated, and untreated mice following elastase perfusion. We will confirm our findings with in vitro studies using primary bone marrow derived macrophages stimulated with RAGE ligands to determine the effects of RAGE on MMP and TIMP activity and macrophage polarization. We will reconstitute RAGE null mice with RAGE-sufficient bone marrow to determine whether the presence of RAGE on bone marrow derived leukocytes is sufficient to polarize macrophages into an M1 subtype in the aorta of elastase-perfused mice. Aim #2: Characterize if RAGE-mediated SMC apoptosis within the aortic wall leads to the pathogenesis of AAA. We hypothesize that RAGE, through its interaction with mDia-1, mediates SMC apoptosis and proliferation within the aortic wall. We will utilize SMC DN RAGE mice to assess SMC-specific contributions of RAGE activation in elastase-induced AAA. Using Ki67 and TUNEL staining, we will quantify proliferative and apoptotic indices in SMCs of elastase-perfused aorta of wild-type, RAGE null, and sRAGE- treated wild-type mice. In vitro, we will stimulate primary aortic SMC with RAGE ligands and measure effects of RAGE on SMC proliferative and apoptotic indices. We will determine if mDia-1 is necessary for RAGE's effects using inhibitory RNA technology. This proposal is innovative in that we will use in vivo and in vitro models to understand how RAGE-mediated alterations in macrophage polarization and SMC apoptosis result in AAA development. The studies described in this proposal may eventually lead to novel diagnostic and therapeutic strategies for prevention and treatment. The clinical relevance of this proposal is underscored by the increasing incidence of AAAs among the VA population and the limited treatment options that are currently available.