Arginase II, A Novel Target in Atherosclerosis

精氨酸酶 II，动脉粥样硬化的新靶点

基本信息

批准号：
8656386
负责人：
DAN E BERKOWITZ
金额：
$ 40.18万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-15 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8656386
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 endothelial dysfunction 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

项目摘要

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的低密度脂蛋白（OX-LDL）与凝集素样氧化的低密度脂蛋白受体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的关键步骤，可能导致eNOS底物L-精氨酸的竞争耗竭，导致没有生物利用度降低。我们对人主动脉内皮细胞（HAEC）的初步研究表明，OX-LDL暴露从线粒体中释放出ARG2的库池。反过来，这减少了胞质L-精氨酸的浓度，导致eNOS解偶联。胞质ARG2活性的OX-LDL诱发的变化被Rho-激酶抑制作用，并且在LOX-1无效的内皮细胞中根本不会发生。另外，线粒体加工肽酶（MPP）的抑制或siRNA敲低减少了OX-LDL刺激后ARG-2的胞质丰度和活性。此外，阻断MPP会减弱EC活性氧（ROS）和NO的EC介导的变化。因此，我们假设一个OX-LDL-LOX1-RHO激酶信号传导轴通过MPP介导的ARG2通过以下事件启用了从线粒体到细胞质的ARG2的EC胞质ARG2活性的增加，以下事件序列：Mitochrial MPP被激活，并脱离了MITILS的序列（MITILS）（MITILL s）。 arg2;然后，Arg2移至细胞质，在那里增加的ARG2活性降低了L-精氨酸的浓度。这会损害NO的生物利用度，从而耗尽eNOS的底物，并增加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，我们将检验以下假设：MPP介导的MTS位点上的Arg2裂解是负责ARG2体外激活的原因，并测试了由OX-LDL诱导的胞质中ARG2丰度是否是由于MPP活性的增加和MITOCHOCHACHIAR的逆转运输。第二个目标中的研究将定义LOX-1信号传导复合物中潜在伴侣与OXLDL刺激的定位变化，并将检验以下假设：机械传染方法通过LOX-1和以下信号传导中介机构介导OXLDL激活OXLDL激活：MT1-MMP，P27KIP1，Rhoa，Rhoa，Rhoa，Rhoa，Rock和Mdia1。在第三个目标中，我们将研究ARG2激活和随后的L-精氨酸底物作为ENOS解偶联的机理。在第四个目标中，将使用遗传性高胆固醇（APOE - / - ）小鼠的动脉粥样硬化饮食评估用小分子抑制剂或遗传缺失（apoE-/ - / - / - arg2 - / - ，双KO）抑制ARG2的后果。最后一个目标的主要结果变量将包括内皮功能障碍，血管刚度，主动脉内膜和培养基的增厚以及动脉粥样硬化斑块负担。综上所述，这些研究将使我们能够更好地理解Arg2在动脉粥样硬化病理生物学中的作用，并确定ARG2是否代表了有效治疗该疾病过程的新目标。