A novel peroxidase in vascular endothelium and the development of atherosclerosis

血管内皮中的新型过氧化物酶与动脉粥样硬化的发展

• 批准号: 7462234
• 负责人: Guangjie Cheng
• 金额: $ 34.86万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7462234
• 关键词: Angiotensin II; Animal Model; Arterial Fatty Streak; Arterial Injury; Atherosclerosis; Bacteria; Biochemistry; Blood; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Cell Line; Cells; Cellular biology; Chemicals; Chloride Ion; Chlorides; Cholesterol; Condition; Development; Disease; Endothelial Cells; Enzymes; Event; Genes; Heart; Heme; Homologous Gene; Host Defense; Human; Hydrogen Peroxide; Hypertension; Immunology; Investigation; Knockout Mice; Lead; Length; Lipid Peroxidation; Lipids; Lipoproteins; Location; Low Density Lipoprotein oxidation; Low-Density Lipoproteins; Mammalian Cell; Mass Spectrum Analysis; Modeling; Modification; Mononuclear; Mus; NADPH Oxidase; Numbers; Oxidants; Oxygen; Pathology; Peroxidase; Peroxidases; Phagocytes; Pharmaceutical Preparations; Physiological; Physiology; Play; Property; Protein Overexpression; Proteins; Public Health; Publishing; RNA Interference; Range; Reaction; Reactive Oxygen Species; Research; Risk Factors; Role; Signal Transduction; Signal Transduction Pathway; Smooth Muscle Myocytes; Source; Substrate Specificity; Techniques; Testing; Therapeutic Intervention; Tissues; Vascular Endothelial Cell; Vascular Endothelium; Vascular Smooth Muscle; Vascular System; atherogenesis; enzyme activity; in vivo; killings; neutrophil; novel; novel diagnostics; oxidation; oxidized low density lipoprotein; peroxidation; protein protein interaction; tool

DESCRIPTION (provided by applicant): Evidence implicates a role for heme-containing peroxidase in atherogenesis due in part to oxidation of low density lipoprotein (LDL). While generally attributed to the phagocyte-specific enzyme myeloperoxidase (MPO), it is unclear whether a peroxidase other than MPO in vascular wall plays a role in atherogenesis. We have now identified and have begun to characterize a new heme-containing peroxidase that is present in heart, vascular smooth muscle and endothelial cells. This Vascular PerOxidase, termed VPO, is highly homologous to MPO, but also contains multiple domains that are likely to participate in protein-protein interactions. We have partially purified VPO from mouse heart and overexpressed cells, and in preliminary studies, some important enzymatic properties are similar to those of MPO. Our earlier investigations focused on novel NADPH- oxidases (Noxes) including Nox1, Nox4 and Nox5, which are major sources of reactive oxygen species in and adjacent to the vessel wall. Nox1 and perhaps other Noxes (but not the phagocyte NADPH-oxidase) are implicated in animal models of Angiotensin II-induced of atherosclerosis and hypertension. VPO, when co- expressed in cells with novel vascular Noxes, utilizes hydrogen peroxide produced by these Noxes to catalyze peroxidative reactions. Like MPO, VPO also generates HOCl from chloride, an important oxidant for the development of atherosclerosis. We hypothesize that VPO can carry out peroxidative reactions in the vascular system (e.g. LDL) that may be involved in the development of atherosclerotic disease. Aim #1 will purify VPO from mammalian cells stably overexpressing VPO and will characterize its basic substrate specificity and enzymatic properties. Aim #2 will investigate the oxidative modification of lipoproteins and lipids by VPO. The ability of purified VPO to oxidize LDL lipoprotein and lipid will be emphasized. Aim #3 will explore the subcellular localization of VPO and compare this with that of the vascular Noxes. In addition, the ability of vascular Noxes to support VPO-dependent oxidation will also be evaluated. Aim #4 will investigate the localization of VPO in intact vasculature and atherosclerotic lesions. These studies are an essential prelude to (patho)physiological studies (e.g., in knockout mice) of the role of VPO in cardiovascular disease. Understanding the role of this new peroxidase in the development of atherosclerosis may provide new diagnostic and/or therapeutic interventions targeted to VPO. PUBLIC HEALTH RELEVANCE: Our research aim is to understand why atherosclerosis, also known as the "hardening of blood vessels," occurs in cardiovascular system. It is clear that chemical change of low density lipoprotein (LDL), which contains "bad cholesterol," is one of the major reasons causing the blood vessels to become harden. Recent findings show that peroxidases are related to cardiovascular diseases including atherosclerosis. Peroxidases are enzymes that catalyze hydrogen peroxide reduction and play important roles in physiology, immunology and pathology such as killing of bacteria, modulation of cell signal transduction pathway and tissue damage. My proposal focuses on relationship between mechanism of cardiovascular disease causing by peroxidases and reactive oxygen species (in general, toxic agents). In the proposal, a novel peroxidase gene has be identified and cloned, called vascular peroxidase (VPO), which mainly exists in cardiovascular system. We will study VPO enzymatic features and in what conditions it changes LDL. First, we will study the properties of this enzyme, including substrate specificity and optimal chemical conditions. Then, we will decide at which part of cell the enzyme is located and explore what causes the increase of the enzyme's activity. We will also evaluate the features of chemical change of LDL and find out the possible ways to block the change. Finally, we will explore whether VPO and other related enzymes exists in intact vasculature and atherosclerotic lesions. Thus, we can elucidate whether the new enzyme is a new risk factor for atherosclerosis. If it is, then we can develop a new diagnostic tool to test the level of this enzyme and its activity in blood, allowing people to find "hardening of blood vessels" earlier. Furthermore, we anticipate this research will lead to the development of a new drug that inhibits the enzyme's activity and blocks the change of LDL.

描述（由申请人提供）：有证据表明含血红素过氧化物酶在动脉粥样硬化形成中的作用，部分原因是低密度脂蛋白（LDL）的氧化。虽然通常归因于吞噬细胞特异性酶髓过氧化物酶 (MPO)，但目前尚不清楚血管壁中除 MPO 之外的过氧化物酶是否在动脉粥样硬化形成中发挥作用。我们现在已经鉴定并开始表征一种存在于心脏、血管平滑肌和内皮细胞中的新型含血红素过氧化物酶。这种血管过氧化物酶，称为 VPO，与 MPO 高度同源，但也包含多个可能参与蛋白质-蛋白质相互作用的结构域。我们从小鼠心脏和过表达的细胞中部分纯化了VPO，并且在初步研究中，一些重要的酶学特性与MPO相似。我们早期的研究重点是新型 NADPH 氧化酶 (Noxes)，包括 Nox1、Nox4 和 Nox5，它们是血管壁内及其附近活性氧的主要来源。 Nox1 和可能其他 Nox（但不包括吞噬细胞 NADPH 氧化酶）与血管紧张素 II 诱导的动脉粥样硬化和高血压动物模型有关。当VPO与新型血管Nox在细胞中共表达时，VPO利用这些Nox产生的过氧化氢来催化过氧化反应。与 MPO 一样，VPO 也会从氯化物中产生 HOCl，这是动脉粥样硬化发展的重要氧化剂。我们假设 VPO 可以在血管系统（例如 LDL）中进行过氧化反应，这可能参与动脉粥样硬化疾病的发展。目标#1将从稳定过表达VPO的哺乳动物细胞中纯化VPO，并表征其基本底物特异性和酶性质。目标 #2 将研究 VPO 对脂蛋白和脂质的氧化修饰。将强调纯化的 VPO 氧化 LDL 脂蛋白和脂质的能力。目标 #3 将探索 VPO 的亚细胞定位，并将其与血管 Noxes 进行比较。此外，还将评估血管 Noxes 支持 VPO 依赖性氧化的能力。目标 4 将研究 VPO 在完整脉管系统和动脉粥样硬化病变中的定位。这些研究是 VPO 在心血管疾病中作用的（病理）生理学研究（例如，在基因敲除小鼠中）的重要前奏。了解这种新的过氧化物酶在动脉粥样硬化发展中的作用可能会提供针对 VPO 的新的诊断和/或治疗干预措施。公共健康相关性：我们的研究目的是了解为什么心血管系统会发生动脉粥样硬化（也称为“血管硬化”）。显然，含有“坏胆固醇”的低密度脂蛋白（LDL）的化学变化是导致血管硬化的主要原因之一。最近的研究结果表明，过氧化物酶与包括动脉粥样硬化在内的心血管疾病有关。过氧化物酶是催化过氧化氢还原的酶，在生理学、免疫学和病理学中发挥重要作用，例如杀死细菌、调节细胞信号转导途径和组织损伤。我的建议重点关注过氧化物酶引起的心血管疾病的机制与活性氧（通常是有毒物质）之间的关系。该提案鉴定并克隆了一种新的过氧化物酶基因，称为血管过氧化物酶（VPO），主要存在于心血管系统中。我们将研究 VPO 酶的特性以及它在什么条件下改变 LDL。首先，我们将研究这种酶的特性，包括底物特异性和最佳化学条件。然后，我们将确定该酶位于细胞的哪个部位，并探讨是什么原因导致该酶的活性增加。我们还将评估LDL的化学变化特征，并找出阻止这种变化的可能方法。最后，我们将探讨VPO和其他相关酶是否存在于完整的脉管系统和动脉粥样硬化病变中。因此，我们可以阐明这种新酶是否是动脉粥样硬化的新危险因素。如果是的话，那么我们就可以开发一种新的诊断工具来测试血液中这种酶的水平及其活性，让人们更早地发现“血管硬化”。此外，我们预计这项研究将导致一种新药的开发，这种新药可以抑制酶的活性并阻止低密度脂蛋白的变化。