Arginase II, A Novel Target in Atherosclerosis

描述(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型低密度脂蛋白(Ox-LDL)与凝集素样氧化型低密度脂蛋白受体-1(LOX-1)的相互作用。我们到目前为止的工作表明，精氨酸酶-2(Arg2)的激活是氧化低密度脂蛋白介导的动脉粥样硬化的关键步骤，可能导致底物L-精氨酸对eNOS的竞争性耗尽，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Arg2池从线粒体释放到胞浆中。这反过来又降低了胞内L-精氨酸的浓度，导致eNOS解偶联。氧化低密度脂蛋白引起的胞浆Arg2活性的变化被Rho-Kinase抑制而减弱，在LOX-1缺失的内皮细胞中根本不会发生这种变化。此外，抑制或siRNA敲除线粒体加工肽酶(MPP)会降低Ox-LDL刺激后细胞内Arg-2的丰度和活性。此外，阻断MPP可减弱OxLDL介导的EC活性氧物种(ROS)和NO的变化。因此，我们假设Ox-LDL-Lox1-Rho激酶信号轴通过MPP介导的Arg2从线粒体到胞浆的分解触发EC胞浆Arg2活性的增加：线粒体MPP被激活，并从Arg2的N端去除线粒体靶向序列(MTS)；Arg2然后移动到胞浆，在那里Arg2活性的增加降低了L-精氨酸的浓度；这通过耗尽eNOS的底物来损害NO的生物可利用性，并增加了ROS(eNOS解偶联)。这些事件导致内皮细胞功能障碍，并促进动脉粥样硬化的形成。在第一个目的中，我们将验证MPP介导的Arg2在MTS位点的切割是导致Arg2体外激活的假设，并测试Ox-LDL诱导的胞浆中Arg2的丰度是否源于MPP活性的增加和线粒体的逆行转运。第二个目标的研究将确定LOX-1信号复合体中潜在合作伙伴在OxLDL刺激下的定位变化，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-MMPs、p27kip1、RhoA、ROCK和mDia1。在第三个目标中，我们将研究Arg2的激活和随后的L-精氨酸底物的耗尽作为eNOS解偶联的机制。在第四个目标中，将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抑制Arg2或基因缺失(ApoE-/-Arg2-/-，双KO)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、血管僵硬、主动脉内膜和中层增厚以及动脉粥样硬化斑块负荷。综上所述，这些研究将使我们能够更好地了解Arg2在动脉粥样硬化的病理生物学中的作用，并确定Arg2是否代表了有效治疗这一疾病过程的新靶点。

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.