Zebrafish models of vascular inflammation and atherosclerosis

描述(申请人提供)：动物实验和人类动脉粥样硬化研究令人信服地证明，低密度脂蛋白(LDL)经历氧化，这大大增强了其致动脉粥样硬化的能力。体内低密度脂蛋白氧化的主要途径之一是由12/15-脂氧合酶(12/15LO)催化的氧化。由于氧化型低密度脂蛋白(OxLDL)可在血管壁上诱导许多炎症反应，动脉粥样硬化病变中高水平的所谓氧化特异性表位往往表明这些病变容易破裂，导致心肌梗死或中风。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。我们小组目前正在开发新的成像方法，使用特异性结合氧化特定表位的抗体。除了成像应用，氧化特异性抗体正在成为动脉粥样硬化的一种治疗方法，因为它们可以防止OxLDL的炎症效应。在这一应用中，我们建议开发新的斑马鱼(Danio Rerio)模型来研究脂蛋白氧化的机制和病理效应，针对活体动物血管中氧化特定表位的新成像技术，以及减少脂蛋白氧化及其病理效应的新治疗策略。具体地说，我们建议：(1)发展成像和分析技术，用于体内和体外研究脂蛋白氧化和检测血管病变中的氧化特异性表位。我们将针对三类常见的氧化特定表位：丙二醛脱皮、氧化磷脂和氧化胆固醇酯。在一种方法中，我们将静脉注射。用荧光标记的抗体标记斑马鱼幼体，并使用共聚焦显微镜检测血管病变中的抗体结合情况。第二种方法是产生有条件表达GFP标记的氧化特异性抗体的转基因斑马鱼。我们还将使用质谱学技术来识别apoB和apoA1脂蛋白中的氧化脂质，以及那些与氧化特异抗体结合的脂类。(2)建立12/15LO诱导的脂蛋白氧化模型，并在体内研究其在血管脂质蓄积和炎症中的作用。将分析转内皮细胞或髓系细胞特异性表达人12/15LO的转基因斑马鱼的血管损伤，以了解氧化脂质的积累、巨噬细胞的募集和泡沫细胞的形成。(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斑马鱼的研究”，我们建议利用斑马鱼模型，包括新的转基因品系以及最先进的成像和质谱学技术，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.