Arginase II, A Novel Target in Atherosclerosis

精氨酸酶 II，动脉粥样硬化的新靶点

• 批准号: 8300883
• 负责人: DAN E BERKOWITZ
• 金额: $ 41万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8300883
• 关键词: Apolipoprotein E; Arginine; Arterial Fatty Streak; Atherogenic Diet; Atherosclerosis; Attenuated; Biological Availability; Blood Vessels; Breeding; Chemicals; Cholesterol; Complex; Coronary artery; Cytosol; Development; Disease; Endothelial Cells; Endothelium; Enzymes; Event; Fibrosis; Functional disorder; Genetic; Human; Infiltration; Investigation; LOX gene; Lead; Lectin; Ligation; Lipids; Low Density Lipoprotein Receptor; MMP14 gene; Mediating; Mitochondria; Mus; Process; Production; Proteins; Reactive Oxygen Species; Rho-associated kinase; Role; Signal Transduction; Site; Small Interfering RNA; Testing; Time; Vascular Diseases; Work; apolipoprotein E-2; arginase; atherogenesis; effective therapy; enzyme activity; improved; in vitro testing; in vivo; inhibitor/antagonist; intima media; macrophage; mitochondrial processing peptidase; mouse arginase II; novel; overexpression; oxidized low density lipoprotein; primary outcome; receptor; retrograde transport; small molecule; tetrahydrobiopterin; vascular endothelial dysfunction

DESCRIPTION (provided by applicant): An early and critical event in the development of endothelial dysfunction in atherosclerosis is the interaction of the oxidized low density lipoprotein (Ox-LDL) with the lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Our work to date has shown that activation of arginase-2 (Arg2) is a key step in Ox-LDL-mediated atherogenesis, likely leading to competitive depletion of the substrate L-arginine for eNOS, leading to decreased NO bioavailability. Our preliminary studies with human aortic endothelial cells (HAEC) suggest that Ox-LDL exposure releases a pre-existing pool of Arg2 into the cytosol from mitochondria. This, in turn, diminishes the concentration of cytosolic L-arginine leading to eNOS uncoupling. Ox-LDL-evoked changes in cytosolic Arg2 activity are muted by Rho-kinase inhibition, and they do not occur at all in LOX-1 null endothelial cells. In addition, inhibition or siRNA knockdown of mitochondrial processing peptidase (MPP) reduce the cytosolic abundance and activity of Arg-2 following Ox-LDL stimulation. Furthermore, blocking MPP attenuates OxLDL-mediated changes in EC reactive oxygen species (ROS) and NO. We therefore hypothesize that an Ox-LDL-LOX1-Rho kinase signaling axis triggers an increase in EC cytosolic Arg2 activity via MPP-mediated decompartmentalization of Arg2 from the mitochondria to cytosol through the following sequence of events: Mitochondrial MPP is activated, and removes the mitochondrial targeting sequence (MTS) from the N-terminus of Arg2; Arg2 then moves to the cytosol where increased Arg2 activity decreases the concentration of L- arginine; this impairs the bioavailability of NO by depleting substrate for eNOS, and also increases ROS (eNOS uncoupling). These events result in EC dysfunction and contribute to atherogenesis. In the first aim, we will test the hypothesis that MPP-mediated cleavage of Arg2 at the MTS site is responsible for the activation of Arg2 in vitro, and test whether Arg2 abundance in the cytosol that is induced by Ox-LDL is due to increased MPP activity and retrograde transport out of mitochondria. Studies in the second aim will define changes in the localization of potential partners in the LOX-1 signaling complex with OxLDL stimulation, and will test the hypothesis that mechanotransduction mediates OxLDL activation of Arg2 through LOX-1 and the following signaling intermediaries: MT1-MMP, p27kip1, RhoA, ROCK, and mDia1. In the third aim we will examine Arg2 activition and subsequent depletion of L-Arginine substrate as mechanisms of eNOS uncoupling. In the fourth aim, an atherogenic diet in genetically hypercholesterolemic (ApoE-/-) mice will be used to evaluate the consequences of inhibiting Arg2 with a small molecule inhibitor, or genetic deletion (ApoE- /- Arg2-/-, double KO). Primary outcome variables for this last aim will include endothelial dysfunction, vascular stiffness, thickening of the aortic intima and media, and the atherosclerotic plaque burden. Taken together, these studies will allow us to better understand the role of Arg2 in the pathobiology of atherosclerosis, and determine whether Arg2 represents a novel target for the effective treatment of this disease process.

描述（由申请人提供）：动脉粥样硬化内皮功能障碍发展的早期关键事件是氧化低密度脂蛋白（Ox-LDL）与凝集素样氧化低密度脂蛋白受体-1（LOX-1）的相互作用。迄今为止，我们的工作表明，精氨酸酶 2 (Arg2) 的激活是 Ox-LDL 介导的动脉粥样硬化形成的关键步骤，可能导致 eNOS 底物 L-精氨酸的竞争性消耗，从而导致 NO 生物利用度降低。我们对人主动脉内皮细胞 (HAEC) 的初步研究表明，Ox-LDL 暴露会将预先存在的 Arg2 库从线粒体释放到细胞质中。这反过来又降低了胞质 L-精氨酸的浓度，导致 eNOS 解偶联。 Ox-LDL 引起的胞质 Arg2 活性变化可通过 Rho 激酶抑制而减弱，并且在 LOX-1 缺失的内皮细胞中根本不会发生。此外，抑制或 siRNA 敲低线粒体加工肽酶 (MPP) 会降低 Ox-LDL 刺激后 Arg-2 的胞质丰度和活性。此外，阻断 MPP 可减弱 OxLDL 介导的 EC 活性氧 (ROS) 和 NO 的变化。因此，我们假设 Ox-LDL-LOX1-Rho 激酶信号传导轴通过 MPP 介导的 Arg2 从线粒体到细胞质的去区室化，通过以下事件序列触发 EC 胞质 Arg2 活性的增加：线粒体 MPP 被激活，并从线粒体 N 末端去除线粒体靶向序列 (MTS)。 精氨酸2;然后，Arg2 移动到细胞质，其中 Arg2 活性增加，L-精氨酸浓度降低；这会消耗 eNOS 的底物，从而损害 NO 的生物利用度，并且还会增加 ROS（eNOS 解偶联）。这些事件导致 EC 功能障碍并导致动脉粥样硬化。在第一个目标中，我们将测试 MPP 介导的 MTS 位点 Arg2 裂解导致 Arg2 体外激活的假设，并测试 Ox-LDL 诱导的细胞质中 Arg2 丰度是否是由于 MPP 活性增加和逆行转运出线粒体所致。第二个目标的研究将定义 OxLDL 刺激下 LOX-1 信号复合物中潜在伙伴的定位变化，并将检验机械转导通过 LOX-1 和以下信号中介介导 OxLDL 激活 Arg2 的假设：MT1-MMP、p27kip1、RhoA、ROCK 和 mDia1。在第三个目标中，我们将研究 Arg2 的激活和随后 L-精氨酸底物的消耗作为 eNOS 解偶联的机制。第四个目标是，使用遗传性高胆固醇血症 (ApoE-/-) 小鼠的致动脉粥样硬化饮食来评估用小分子抑制剂或基因缺失（ApoE-/- Arg2-/-，双 KO）抑制 Arg2 的后果。最后一个目标的主要结果变量包括内皮功能障碍、血管僵硬度、主动脉内膜和中膜增厚以及动脉粥样硬化斑块负担。总而言之，这些研究将使我们能够更好地了解 Arg2 在动脉粥样硬化病理学中的作用，并确定 Arg2 是否代表了有效治疗该疾病过程的新靶点。