Bioluminescence imaging of atherosclerosis associated reactive oxygen species

描述(由申请人提供)：近一个世纪以来，心血管疾病(CVD)一直是美国人的主要死亡原因。心血管疾病的不良后果通常是动脉粥样硬化斑块破裂所致。防治心血管疾病最有效的战略是二级预防：亚临床疾病的诊断和治疗。非侵入性评估动脉粥样硬化性炎症是斑块不稳定的原因，是这一策略的重要组成部分。传统的成像方式不能评估斑块的易损性，因此预测价值有限。相比之下，分子成像将解剖结构与斑块的生化特征一起可视化。目前用于CVD的分子成像技术存在成本高和患者固有风险等缺点。生物发光成像(BLI)确实存在以下问题：氧化敏感的探针，如鲁米诺及其衍生物L-012，在与吞噬细胞产生的活性氧(ROS)接触时发出无害的可见光谱光。这些吞噬细胞在CVD患者的动脉壁中丰富，主要是在炎症斑块及其周围，并直接导致其破裂。这项提议的目标是开发BLI来检测动物模型中这些细胞产生的ROS，从而允许在体内对动脉粥样硬化性炎症进行成像。这一目标被集中于三个具体的目标：在目标1中，将分析三个已知的BLI探针的化学发光性质，以用于单细胞生物发光显微镜(BLM)。首先，将测试每个探针的ROS特异性和化学信号增强潜力。这些发现在细胞环境中的重复性将通过从动物模型中分离出的吞噬细胞进行测试。通过这些实验确定的优化条件将用于在单个吞噬细胞启动ROS合成级联时，用BLM对它们进行可视化。在目标2中，BLI将用于测量体外和体内动物模型的炎症。首先，BLI将被评估为目前临床检测血清髓过氧化物酶的一种廉价替代方法，髓过氧化物酶是心血管疾病的可溶酶标记物，它能产生最有效的生理性ROS，次氯酸。接下来，鲁米诺衍生物L-012将用于对载脂蛋白E缺陷小鼠的动脉粥样硬化进行成像。BLI信号将与斑块炎症相关，如免疫组织化学所定量。AIM 3包括一项合成新的BLI探针的计划，该探针具有更好的体内使用性能，即能够穿透更深层次组织的红移发射光谱。我们将在类似于AIMS 1和2中概述的实验中，针对当前的化合物对所建议的连接物进行评估。这些连接物的支架将允许合成大量变体，并有可能成为下一代BLI探针。 公共卫生相关性：心血管疾病是美国最主要的死亡原因，每3例死亡中就有1例以上是心血管疾病造成的。这项研究项目将开发使动脉斑块发出可见光的技术，医生可以用高灵敏度的数码相机拍摄。这可以使更早的诊断和治疗，从而挽救更多的生命，所有这些都没有任何有害的辐射或高度侵入性的程序。

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) has consistently been the leading cause of death in the United States for nearly a century. Adverse outcomes of CVD usually result from rupturing atherosclerotic plaques. The most effective strategy for combating CVD is secondary prevention: diagnosis and treatment of subclinical disease. Non-invasively assessing atherosclerotic inflammation, the cause of plaque instability, is an important element of this strategy. Traditional imaging modalities cannot assess plaque vulnerability and thus are of limited predictive value. In contrast, molecular imaging visualizes the anatomy together with the biochemical signature of plaques. Current molecular imaging techniques used for CVD have setbacks, e.g., high cost and inherent risk to the patient. Bioluminescence imaging (BLI) does share these problems: oxidation-sensitive probes such as luminol and its derivative L-012 emit harmless, visible-spectrum light upon contact with reactive oxygen species (ROS) produced by phagocytes. These phagocytes are enriched in the arterial walls of CVD patients, mainly in and around inflamed plaques, and directly contribute to their rupture. The goal of this proposal is to develop BLI to detect ROS produced by these cells in animal models, thus allowing atherosclerotic inflammation to be imaged in vivo. This goal has been focused into three Specific Aims: In Aim 1, the chemiluminescence properties of three known BLI probes will be analyzed for use in single- cell bioluminescence microscopy (BLM). First, each probe will be tested for ROS specificity and chemical signal enhancement potential. The reproducibility of these findings in cellular environments will be tested with phagocytes isolated from animal models. Optimized conditions determined by these experiments will be used to visualize individual phagocytes with BLM as they initiate the ROS-synthesis cascade. In Aim 2, BLI will be used to measure inflammation of animal models both ex vivo and in vivo. First, BLI will be evaluated as an inexpensive alternative to current clinical assays for serum myeloperoxidase, a soluble enzyme marker for CVD and that creates the most potent physiological ROS, hypochlorous acid. Next, luminol derivative L-012 will be used to image atherosclerosis in apolipoprotein E-deficient mice. BLI signal will be correlated with plaque inflammation as quantified by immunohistochemistry. Aim 3 consists of a plan to synthesize new BLI probes that have improved properties for use in vivo, namely red-shifted emission spectra that are capable of penetrating deeper tissues. The proposed conjugates, oxazine and squaraine derivates, will be evaluated against current compounds in experiments similar to those outlined in Aims 1 and 2. The scaffolds of these conjugates will allow for a large number of variants to be synthesized with the possibility of becoming next-generation BLI probes. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is the top cause of mortality in the United States, responsible for more than 1 out of every 3 deaths. This research project will develop techniques to make arterial plaques glow with visible light which doctors can photograph with highly sensitive digital cameras. This could allow earlier diagnosis and treatment and thus save more lives, all without any harmful radiation or highly invasive procedures.