A novel peroxidase in vascular endothelium and the development of atherosclerosis

描述（由申请方提供）：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归因于低密度脂蛋白（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），但尚不清楚血管壁中MPO以外的过氧化物酶是否在动脉粥样硬化形成中起作用。我们现在已经确定，并已开始表征一种新的血红素含过氧化物酶，是目前在心脏，血管平滑肌和内皮细胞。这种血管过氧化物酶，称为VPO，与MPO高度同源，但也包含可能参与蛋白质-蛋白质相互作用的多个结构域。我们已经从小鼠心脏和过表达细胞中部分纯化了VPO，在初步研究中，一些重要的酶性质与MPO相似。我们早期的研究集中在新型NADPH氧化酶（Noxes），包括Nox 1，Nox 4和Nox 5，它们是血管壁内和邻近血管壁的活性氧的主要来源。Nox 1和其他可能的Nox（但不是吞噬细胞NADPH氧化酶）与血管紧张素II诱导的动脉粥样硬化和高血压的动物模型有关。当VPO在细胞中与新的血管Noxes共表达时，其利用由这些Noxes产生的过氧化氢来催化过氧化反应。像MPO一样，VPO也从氯化物产生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（如LDL）中进行过氧化反应，这可能与动脉粥样硬化疾病的发展有关。目的#1将从稳定过表达VPO的哺乳动物细胞中纯化VPO，并将表征其基本底物特异性和酶性质。目的#2将研究VPO对脂蛋白和脂质的氧化修饰。纯化的VPO氧化LDL脂蛋白和脂质的能力将被强调。目的#3将探索VPO的亚细胞定位，并将其与血管Noxes的亚细胞定位进行比较。此外，还将评估血管Noxes支持VPO依赖性氧化的能力。目的#4将研究VPO在完整血管和动脉粥样硬化病变中的定位。这些研究是（病理）生理学研究（例如，在基因敲除小鼠中）的VPO在心血管疾病中的作用。了解这种新的过氧化物酶在动脉粥样硬化发展中的作用，可能会提供新的诊断和/或针对VPO的治疗干预措施。公共卫生相关性：我们的研究目的是了解为什么动脉粥样硬化，也被称为“血管硬化”，发生在心血管系统。很明显，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。最近的研究表明，过氧化物酶与心血管疾病，包括动脉粥样硬化。过氧化物酶是催化过氧化氢还原的酶，在生理、免疫和病理学中发挥重要作用，如杀菌、调节细胞信号转导通路和组织损伤等。我的建议集中在过氧化物酶引起的心血管疾病的机制和活性氧（一般来说，有毒物质）之间的关系。本研究拟鉴定并克隆一种主要存在于心血管系统的新的过氧化物酶基因，命名为血管过氧化物酶（VPO）。我们将研究VPO的酶特性以及在何种条件下它会改变LDL。首先，我们将研究这种酶的性质，包括底物特异性和最佳化学条件。然后，我们将确定酶位于细胞的哪个部分，并探索是什么导致酶的活性增加。我们还将评估LDL化学变化的特征，并找出可能的方法来阻止这种变化。最后，我们将探讨VPO和其他相关酶是否存在于完整的血管和动脉粥样硬化病变。因此，我们可以阐明这种新的酶是否是动脉粥样硬化的一个新的危险因素。如果是，那么我们就可以开发一种新的诊断工具来测试这种酶的水平及其在血液中的活性，让人们更早发现“血管硬化”。此外，我们预计这项研究将导致开发一种新的药物，抑制酶的活性和阻止LDL的变化。

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.