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）一直是美国的主要死亡原因。心血管疾病的不良后果通常由动脉粥样硬化斑块破裂引起。对抗心血管疾病最有效的策略是二级预防：诊断和治疗亚临床疾病。无创评估动脉粥样硬化性炎症（斑块不稳定的原因）是该策略的重要组成部分。传统的成像方式不能评估斑块易损性，因此预测价值有限。相比之下，分子成像可以可视化斑块的解剖结构和生化特征。目前用于心血管疾病的分子成像技术存在缺陷，例如成本高，对患者存在固有风险。生物发光成像（BLI）确实存在这些问题：氧化敏感探针，如发光氨及其衍生物L-012，在与吞噬细胞产生的活性氧（ROS）接触时，会发出无害的可见光谱光。这些吞噬细胞富集于CVD患者的动脉壁，主要在炎症斑块内和周围，并直接导致斑块破裂。本提案的目标是在动物模型中开发BLI来检测这些细胞产生的ROS，从而使动脉粥样硬化炎症在体内成像。这一目标已经集中在三个特定的目标：在Aim 1中，将分析三种已知的BLI探针的化学发光特性，以用于单细胞生物发光显微镜（BLM）。首先，每个探针将测试ROS特异性和化学信号增强电位。这些发现在细胞环境中的可重复性将用从动物模型中分离的吞噬细胞进行测试。这些实验确定的优化条件将用于可视化单个吞噬细胞与BLM，因为它们启动ros合成级联。在Aim 2中，BLI将用于测量动物模型的离体和体内炎症。首先，BLI将作为目前临床检测血清髓过氧化物酶（CVD的一种可溶性酶标记物，能产生最有效的生理活性氧次氯酸）的廉价替代方法进行评估。接下来，鲁米诺衍生物L-012将用于载脂蛋白e缺乏小鼠的动脉粥样硬化成像。BLI信号将通过免疫组织化学量化与斑块炎症相关。目标3包括计划合成新的BLI探针，这些探针具有改进的体内使用性能，即能够穿透更深组织的红移发射光谱。提议的偶联物，恶嗪和方卡因衍生物，将在类似目标1和2中概述的实验中与现有化合物进行评估。这些缀合物的支架将允许合成大量的变体，并有可能成为下一代BLI探针。

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.