Calpains and Abdominal Aortic Aneurysms

摘要： 腹主动脉瘤（AAA）是腹主动脉的永久性扩张，在手术后超过80%的死亡率。 破裂目前的治疗仅限于手术修复，突出了探索机械见解的必要性 发展出有效的非手术疗法在AAA患者中，主动脉结构完整性 由于平滑肌细胞（SMC）收缩丝与细胞外基质的分离，壁被破坏 蛋白酶这种结合是由细胞骨架连接蛋白介导的。然而，机制 AAA形成中血管壁结构完整性的潜在损失尚不清楚。近日，利用 在血管紧张素II（AngII）诱导的AAA动物模型中，我们发现， 主动脉钙蛋白酶，血管平滑肌组织中的钙依赖性半胱氨酸蛋白酶。此外，钙蛋白酶抑制 显著减弱AngII诱导的AAA形成。钙蛋白酶是唯一已知的靶向蛋白酶。 维持主动脉结构完整性的一系列细胞骨架蛋白，包括细丝蛋白。使用钙蛋白酶-1或 -2，两种主要的普遍存在的同种型特异性缺陷小鼠，我们确定钙蛋白酶-2的遗传缺陷， 但钙蛋白酶-1不能完全抑制AngII诱导的AAA形成。钙蛋白酶-2缺乏减弱血管紧张素Ⅱ- 诱导细胞骨架蛋白、细丝蛋白A和talin片段化。人和小鼠AAA的免疫染色 显示钙蛋白酶-2蛋白由浸润的巨噬细胞和主动脉外膜成纤维细胞（AoAF）表达。 小鼠巨噬细胞特异性钙蛋白酶-2缺乏对AngII诱导的AAA形成没有影响， 提示血管壁主要是AoAF来源钙蛋白酶-2的关键作用。在培养的AoAF中，AngII促进ASK-1/ NF-κ B通过激活钙蛋白酶-2介导炎性细胞因子诱导。此外，钙蛋白酶-2沉默 通过TGF-β / Rho激酶信号传导抑制（肌）成纤维细胞分化和迁移。基于 在上述背景下，我们将检验钙蛋白酶-2激活促进血管紧张素II诱导的AAAs的假设， 加速外膜成纤维细胞衍生的NF-κ B/ASK-1介导的外膜炎症，和 （肌）成纤维细胞迁移到主动脉中膜，这反过来又通过上调细胞骨架蛋白而引起中膜破坏。 SMC中的丝断裂。为了检验这一假设，提出了以下目标： 成纤维细胞衍生的钙蛋白酶-2在AngII诱导的AAA中的作用。A.成纤维细胞中钙蛋白酶-2缺乏 减弱AngII诱导的AAA形成？B。钙蛋白酶-2调节TGF-β/Rho A促进成纤维细胞生长 通过LTBP 3/RhoGDI 1？C. AoAF中钙蛋白酶-2缺失是否减弱AngII诱导的细胞骨架 体外主动脉平滑肌细胞中的蛋白质片段化？目的2：确定钙蛋白酶-2促进 AngII诱导的外膜炎症？A.钙蛋白酶-2是否参与了ASK-1/ NF-kB的激活并与之相关 与AAA发展过程中的外膜炎症有关？B。calpain-2对血管紧张素II诱导的ASK-1/NF-kB的调节作用 在AoAF中通过TRX/IkB激活？C.人腹主动脉瘤组织中的细胞骨架蛋白断裂是否与 用钙蛋白酶吗目的3：确定钙蛋白酶-2缺乏对已建立的AAA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.