Zebrafish models of vascular inflammation and atherosclerosis

描述（由申请人提供）：动物实验和人体动脉粥样硬化研究令人信服地证明低密度脂蛋白（LDL）发生氧化，这大大增强了其动脉粥样硬化性。体内LDL氧化的主要途径之一是由12/15-脂氧合酶（12/15LO）催化氧化。由于氧化LDL （OxLDL）在血管壁上诱导了许多炎症反应，动脉粥样硬化病变中所谓的高水平氧化特异性表位往往表明这些病变容易破裂，诱发心肌梗死或中风。识别易破裂的动脉粥样硬化斑块是心血管成像的主要挑战，因为目前的成像技术提供的斑块组成信息很少。我们的团队目前正在开发新的成像方法，使用特异性结合氧化特异性表位的抗体。除了成像应用外，氧化特异性抗体正成为动脉粥样硬化的一种治疗方法，因为它们可以防止OxLDL的炎症作用。在这一应用中，我们建议建立新的斑马鱼模型来研究脂蛋白氧化的机制和病理效应，在活体动物血管中靶向氧化特异性表位的新成像技术，以及减少脂蛋白氧化及其病理效应的新治疗策略。具体而言，我们建议：(1)开发用于体内和体外脂蛋白氧化研究和血管病变中氧化特异性表位检测的成像和分析技术。我们将针对三种常见的氧化特异性表位：丙二醛脱皮，氧化磷脂和氧化胆固醇酯。在一种方法中，我们将向斑马鱼幼虫静脉注射荧光标记的抗体，并使用共聚焦显微镜检测血管病变中的抗体结合。第二种方法是产生转基因斑马鱼，有条件地表达gfp标记的氧化特异性抗体。我们还将使用质谱技术来鉴定载脂蛋白ob和载脂蛋白a1中的氧化脂质以及与氧化特异性抗体结合的脂质。(2)建立12/ 15lo诱导的脂蛋白氧化模型，在体内研究其在血管脂质积累和炎症中的作用。在内皮细胞或髓细胞特异性表达人12/15LO的转基因斑马鱼的血管病变中，将分析氧化脂质积累、巨噬细胞募集和泡沫细胞形成。(3)检测氧化特异性抗体、12/15LO抑制剂和抗氧化剂的治疗潜力。综上所述，为了“用研究工具和技术加强对斑马鱼的研究”，我们建议使用斑马鱼模型，包括新的转基因系以及最先进的成像和质谱技术，来研究一个重要的动脉粥样硬化过程-脂蛋白氧化。

DESCRIPTION (provided by applicant): Animal experimental and human atherosclerosis studies have convincingly demonstrated that low- density lipoprotein (LDL) undergoes oxidation, which greatly enhances its atherogenicity. One of the major pathways of LDL oxidation in vivo is the oxidation catalyzed by 12/15-lipoxygenase (12/15LO). Because oxidized LDL (OxLDL) induces many inflammatory responses in the vascular wall, high levels of so-called oxidation-specific epitopes in atherosclerotic lesions often indicate that these lesions are prone to rupture, inducing myocardial infarction or stroke. Identification of vulnerable atherosclerotic plaques prone to rupture is a major challenge for cardiovascular imaging, as current imaging techniques provide little information on plaque composition. Our group is currently developing new imaging approaches using antibodies that specifically bind oxidation-specific epitopes. In addition to imaging applications, oxidation-specific antibodies are emerging as a therapeutic treatment of atherosclerosis because they prevent OxLDL's inflammatory effects. In this application, we propose to develop new zebrafish (Danio rerio) models to study mechanisms and pathologic effects of lipoprotein oxidation, new imaging techniques targeting oxidation-specific epitopes in the vasculature of live animals, as well as novel therapeutic strategies to diminish lipoprotein oxidation and its pathologic effects. Specifically, we propose: (1) To develop imaging and analytic techniques for in vivo and ex vivo study of lipoprotein oxidation and detection of oxidation-specific epitopes in vascular lesions. We will target three common classes of oxidation-specific epitopes: malondialdehide, oxidized phospholipids and oxidized cholesteryl esters. In one approach, we will inject i.v. zebrafish larvae with fluorescently labeled antibodies and use a confocal microscope to detect antibodies binding in vascular lesions. The second approach will be to generate transgenic zebrafish with conditional expression of GFP-labeled oxidation-specific antibodies. We will also use mass spectrometry techniques to identify oxidized lipids in apoB and apoA1 lipoproteins as well as those bound to oxidation-specific antibodies. (2) To create a model of 12/15LO-induced lipoprotein oxidation and to study in vivo its role in vascular lipid accumulation and inflammation. Vascular lesions in transgenic zebrafish with endothelial or myeloid cell specific expression of human 12/15LO will be analyzed for accumulation of oxidized lipids, macrophage recruitment and foam cell formation. (3) To test the therapeutic potential of oxidation-specific antibodies, 12/15LO inhibitors and antioxidants. In summary, in order to contribute to "enhancing zebrafish research with research tools and techniques", we propose to use the zebrafish model, including new transgenic lines as well as state-of-art imaging and mass spectrometry techniques, to study an important atherogenic process - lipoprotein oxidation. PUBLIC HEALTH RELEVANCE: In this application we propose to create a zebrafish model of vascular inflammation and atherosclerosis, the disease that causes heart attack and stroke. The optical transparency of zebrafish larvae will enable dynamic monitoring of the processes of lipoprotein oxidation, vascular lipid accumulation and inflammation in a live animal. Successful development of a zebrafish model of atherosclerosis will help significantly enhance mechanistic studies of atherosclerosis as well as radically advance screening for new therapies.