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: 8458580
• 负责人: DAN E BERKOWITZ
• 金额: $ 39.03万
• 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/8458580
• 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）：动脉粥样硬化中内皮功能障碍发展的早期和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（Arg 2）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的底物L-精氨酸竞争性耗尽，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Arg 2从线粒体释放到胞质溶胶中。这反过来又减少了导致eNOS解偶联的胞质L-精氨酸的浓度。氧化低密度脂蛋白引起的变化，胞质精氨酸2活性减弱的Rho激酶抑制，他们不发生在所有的LOX-1空内皮细胞。此外，线粒体加工肽酶（MPP）的抑制或siRNA敲低降低了Ox-LDL刺激后Arg-2的胞质丰度和活性。此外，阻断MPP减弱了OxLDL介导的EC活性氧（ROS）和NO的变化。因此，我们假设Ox-LDL-LOX 1-Rho激酶信号传导轴通过MPP介导的Arg 2从线粒体到细胞溶质的去部分化，通过以下事件序列触发EC细胞溶质Arg 2活性的增加：线粒体MPP被激活，并从Arg 2的N-末端去除线粒体靶向序列（MTS）; Arg 2然后移动到胞质溶胶，其中增加的Arg 2活性降低L-精氨酸的浓度;这通过耗尽eNOS的底物而损害NO的生物利用度，并且还增加ROS（eNOS解偶联）。这些事件导致EC功能障碍并促进动脉粥样硬化形成。在第一个目标中，我们将测试的假设，MPP介导的切割Arg 2在MTS网站负责Arg 2在体外的激活，并测试是否Arg 2丰度在胞质溶胶中，是由Ox-LDL诱导的是由于MPP活性增加和逆行转运出线粒体。第二个目标的研究将定义LOX-1信号复合物中潜在伴侣在OxLDL刺激下的定位变化，并将测试机械转导通过LOX-1和以下信号中介介导Arg 2的OxLDL活化的假设：MT 1-MMP，p27 kip 1，RhoA，ROCK和mDia 1。在第三个目标中，我们将研究Arg 2激活和随后的L-精氨酸底物耗尽作为eNOS解偶联的机制。在第四个目的中，将使用遗传性高胆固醇血症（ApoE-/-）小鼠中的致动脉粥样硬化饮食来评价用小分子抑制剂抑制Arg 2或遗传缺失（ApoE- /-Arg 2-/-，双KO）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荷。总之，这些研究将使我们能够更好地了解Arg 2在动脉粥样硬化病理生物学中的作用，并确定Arg 2是否代表有效治疗这种疾病过程的新靶点。