PET Imaging of Myocardial Oxidative Stress in T2DM

摘要 活性氧（reactive oxygen species，ROS）是多种疾病发病机制中的重要介质，如癌症、神经认知和神经退行性疾病以及糖尿病。此外，继发于ROS增加的氧化应激是各种心血管疾病包括动脉硬化、高血压和心力衰竭的发病机制的中心。ROS包括自由基超氧阴离子，其引发其他自由基物质的产生。当ROS产生超过细胞防御机制时，它与DNA相互作用，导致突变，蛋白质导致缺陷或功能失调的酶，脂质导致细胞膜损伤。鉴于超氧化物在疾病病理生理学中的核心作用，本提案的目的是开发一种PET放射性示踪剂，该示踪剂将允许非侵入性测量氧化应激，特别是在心血管疾病中。为了实现这一目标，我们将采用由两个并行执行但在补助金结束时收敛的具体目标组成的研究设计。在特定目标1（SA 1）中，我们将开发并验证PET成像方法，以测量心肌超氧阴离子水平作为氧化应激的指标。在此过程中，我们将合成[18] F标记的二氢乙锭甲氧基类似物（[18]F-DHE）类似物用于超氧化物水平成像，通过体外测定筛选类似物，对选择的最佳试剂进行代谢物分析，并在2种动物模型sadriamycin-诱导的ROS和基于特异性目的2的结果的生理模型中验证[18]F-DHE类似物，通过与光学和共焦显微镜测量的相关性。在具体目标2（SA 2）中，我们将描述2型糖尿病啮齿动物模型中心肌病病理进展中性别、心肌FA代谢和ROS产生之间的关系。我们将通过各种干预来操纵心肌脂肪酸代谢的动态范围，以描述脂肪酸代谢和ROS产生的性别差异以及与心肌能量学和功能的相互作用。为此，我们将进行心肌耗氧量的体内测量（使用[11] C-乙酸酯）和FA利用氧化和酯化（使用[11] C-棕榈酸酯）与PET;心肌能量学，通过磷酸肌酸和三磷酸腺苷浓度及其比值和肌酸激酶通量（使用[31]P-MRS）定义;和左心室结构/工作通过MRI和超氧化物水平通过DHE氧化的光学成像。此外，将进行相关基因/蛋白质表达和酶测定，以支持目标结果。如前所述，目标与PET方法收敛，PET方法用于根据SA 2的结果测量超氧化物的产生。最有前途的化合物将进行毒性研究，为未来临床使用所需的探索性新药（xIND）申请做准备。通过完成这些目标，我们能够很好地在人体中评估一种能够测量组织超氧化物水平的新PET成像方法。此外，我们将获得重要的机制见解性别和糖尿病心肌病变过程之间的联系，以及如何增加脂肪代谢和氧化应激之间的关系是贡献。因此，我们现在将能够在人类中进行类似的研究，并获得关键的信息，这些信息将对糖尿病患者以及氧化应激起重要作用的其他疾病的管理产生有利的影响。

ABSTRACT Reactive oxygen species (ROS) are important mediators in the pathogenesis of a wide range of diseases such as cancer, neurocognitive and neurodenerative disorders, and diabetes mellitus. Moreover, oxidative stress secondary to increased ROS is central to pathogenesis various cardiovascular diseases including arteriosclerosis, hypertension, and heart failure. ROS include the free radical superoxide anion ,which initiates the production of other free radical species. When ROS production surpasses cellular defensive mechanisms it interacts with DNA causing mutations, proteins causing defects or dysfunctional enzymes, and lipids causing damage to cellular membranes. In light of the central role superoxide in the pathophysiology of disease, the objective of this proposal is to develop a PET radiotracer that will permit the noninvasive measurement of oxidative stress, in particular in cardiovascular disease. To meet this goal, we will employ a research design composed of two Specific Aims that are performed in parallel but converge by the end of grant. In Specific Aim 1 (SA 1), we will develop and validate a PET imaging approach to measure myocardial superoxide anion levels as an indicator of oxidative stress. In doing so, we will synthesize [18]F-labeled methoxyanalogs of dihydroethidium ([18]F-DHE) analogs for imaging superoxide levels, screen analogs through in-vitro assays, perform metabolite analysis of select optimal agents, and validate the [18]F-DHE analog in 2 animal modelsadriamycin- induced ROS and a physiological model based on results from Specific Aim 2-by correlation to optical and confocal microscopy measurements. In Specific Aim 2 (SA2), we will delineate the relationship between the gender, myocardial FA metabolism and ROS production in the pathological progression of cardiomyopathy in a rodent model of type-2 diabetes mellitus . We will manipulate the dynamic range of myocardial fatty acid metabolism through various interventions to delineate gender differences in fatty acid metabolism and ROS production and the interplay with myocardial energetics and function. To that end, we will perform in-vivo measurements of myocardial oxygen consumption (using [11]C-acetate) and FA utilization oxidation and esterification (using [11]C-palmitate) with PET; myocardial energetics as defined by the phosphocreatine and adenosine triphosphate concentrations and their ratio and creatine kinase flux using [31]P-MRS; and left ventricular structure/ work by MRI and superoxide levels by optical imaging of DHE oxidation. In addition, relevant gene/protein expression and enzymatic assays will be performed to support the results of the aim. As suggested earlier, the aims converge with PET method being used to measure superoxide production based on results from SA2. The most promising compound will then undergo toxicity studies in preparation for an exploratory new drug (xIND) application necessary for future clinical use. By completing these Aims we well positioned to evaluate in humans a new PET imaging approach capable of measuring tissue superoxide levels. Moreover, we will have gained important mechanistic insights into the nexus between gender and the diabetic cardiomyopathic process and how the relationship between increased FA metabolism and oxidative stress are contributory. As a consequence, we will now be capable of performing similar studies in humans and gaining key pieces of information that should favorably impact the management of the diabetic patient as well as in other diseases where oxidative stress plays an important role.