Calpains and Abdominal Aortic Aneurysms

摘要： 腹主动脉瘤(Aaa)是腹主动脉永久性扩张，术后死亡率超过80%。 破裂。目前的治疗仅限于外科修复，强调了探索机械洞察力的必要性。 致力于有效的非手术疗法的发展。在AAA患者中，主动脉的结构完整性 由于平滑肌细胞(SMC)收缩细丝从细胞外基质中解离，壁被破坏 由蛋白水解酶。这种联系是由细胞骨架连接蛋白介导的。然而，这种机制 AAA地层中血管壁结构完整性的潜在损失尚不清楚。最近，使用 在血管紧张素II(AngII)诱导的AAA动物模型上，我们发现AAA的蛋白质和活性显著增加。 主动脉钙蛋白酶，动脉瘤组织中的一种钙依赖性半胱氨酸蛋白酶。此外，钙蛋白酶抑制 显著抑制血管紧张素转换酶诱导的AAA形成。钙蛋白酶是唯一已知的靶向于 一系列维持主动脉结构完整性的细胞骨架蛋白，包括细丝蛋白。使用Calain-1或 -2，这两种普遍存在的同种异型特异性缺陷小鼠，我们确定了calain-2的遗传缺陷， 但不是Calain-1，它完全钝化了Angii诱导的AAA形成。钙蛋白酶-2缺乏可减弱血管紧张素转换酶 诱导细胞骨架蛋白、细丝素A和塔林的碎裂。人和小鼠AAA的免疫染色 浸润性巨噬细胞和主动脉外膜成纤维细胞(AoAFs)表达Calain-2蛋白。 小鼠巨噬细胞特异性钙蛋白酶-2缺乏对血管紧张素Ⅱ诱导的AAA的形成没有影响 提示血管壁起着关键作用，主要是AoAFs衍生的calain-2。在培养的AoAFs中，AngII促进ASK-1/ 核因子-kB通过激活calain-2诱导炎性细胞因子。此外，Calain-2沉默 通过转化生长因子-β/Rho激酶信号通路抑制成纤维细胞的分化和迁移。基于 描述的背景，我们将检验Calain-2激活促进Angii诱导的AAA的假设 促进外膜成纤维细胞来源的NF-kB/ASK-1介导的外膜炎症，以及 (Myo)成纤维细胞迁移到主动脉中层，进而通过上调细胞骨架而导致中层破坏 SMC中的细丝断裂。为了检验这一假设，提出了以下目标：目标1：确定 成纤维细胞衍生的钙蛋白酶-2在血管紧张素Ⅱ诱导的腹主动脉瘤中的作用。A.在成纤维细胞中是否存在钙蛋白酶-2缺乏 抑制血管紧张素转换酶诱导的AAA形成？B.Calain-2是否调节转化生长因子-β/Rho A促进成纤维细胞 通过LTBP3/RhoGDI1实现差异化？C.AoAFs中Calain-2的缺失是否会减弱Angii诱导的细胞骨架 体外培养的主动脉平滑肌细胞的蛋白质碎裂？目标2：确定Calain-2促进的机制 血管紧张素转换酶诱导的外膜炎症？A.Calain-2是否参与ASK-1/NF-kB的激活及其相互关系 在AAA发展过程中有外膜炎症吗？B.Calain-2对血管紧张素Ⅱ诱导的ASK-1/NF-kB有调节作用吗 在AoAFs中通过TRX/IKB激活？C.人AAA组织中的细胞骨架蛋白片段化是否与 和Calain一起？目的3：确定钙蛋白酶-2缺乏对已建立的腹主动脉硬化进展的影响。

ABSTRACT: Abdominal aortic aneurysm (AAA) is a permanent dilation of the abdominal aorta with over 80% mortality after rupture. The current therapy is restricted to surgical repair, highlighting the need to explore mechanic insights into the development of effective, non-surgical therapeutics. In AAA patients, structural integrity of the aortic wall is disrupted due to dissociation of smooth muscle cell (SMC) contractile filaments from extracellular matrix by proteases. This association is mediated by cytoskeletal linker proteins. However, the mechanism underlying loss of vessel wall structural integrity in AAA formation is not understood. Recently, using Angiotensin II (AngII)-induced animal model of AAA, we found a profound increase in protein and activity of aortic calpain, a calcium dependent cysteine protease in aneurysmal tissue. In addition, calpain inhibition significantly attenuated AngII-induced AAA formation. Calpains are the only known proteases targeting an array of cytoskeletal proteins that maintain structural integrity of the aorta, including filamin. Using calpain-1 or -2, the two major ubiquitous isoform-specific deficient mice, we determined that genetic deficiency of calpain-2, but not calpain-1, completely blunted AngII-induced AAA formation. Calpain-2 deficiency attenuated AngII- induced fragmentation of cytoskeletal proteins, filamin A, and talin. Immunostaining of human and mouse AAA revealed expression of calpain-2 protein by infiltrated macrophages and aortic adventitial fibroblasts (AoAFs). Macrophage specific calpain-2 deficiency in mice had no influence on AngII-induced AAA formation, which suggest a critical role for vessel wall mainly AoAFs derived-calpain-2. In cultured AoAFs, AngII promotes ASK-1/ NF-kB mediated inflammatory cytokine induction via activation of calpain-2. Further, calpain-2 silencing suppressed (myo)fibroblast differentiation and migration via TGF-β / Rho kinase signaling. Based on the described background, we will test the hypothesis that calpain-2 activation promotes AngII-induced AAAs by accelerating adventitial fibroblast-derived NF-kB/ASK-1 mediated adventitial inflammation, and (myo)fibroblasts migration to aortic media, which in turn causes medial destruction by upregulating cytoskeletal filament fragmentation in SMCs. To test this hypothesis, the following aims are proposed: Aim 1: Determine the contribution of fibroblasts derived-calpain-2 in AngII-induced AAA. A. Does calpain-2 deficiency in fibroblasts attenuate AngII-induced AAA formation? B. Does calpain-2 regulate TGF-β/Rho A in promoting fibroblasts differentiation via LTBP3/RhoGDI1? C. Does calpain-2 depletion in AoAFs attenuate AngII-induced cytoskeletal protein fragmentation in aortic SMCs in vitro? Aim 2: Define the mechanism by which calpain-2 promotes AngII-induced adventitial inflammation? A. Does calpain-2 contribute to ASK-1/ NF-kB activation and correlate with adventitial inflammation during AAA development? B. Does calpain-2 regulate AngII-induced ASK-1/NF-kB activation via TRX/IkB in AoAFs? C. Does cytoskeletal protein fragmentation in human AAA tissue associate with calpain? Aim 3: Determine the effect of calpain-2 deficiency on progression of established AAAs.