Dynamic tissue-specific modulation of redox stress using chemogenetics

这项R33研究计划建议表征和验证新的转基因小鼠品系 化学遗传学创建强大的模型，将用于确定预防和治疗的新靶点 治疗心力衰竭和主动脉瘤。最新的心力衰竭和主动脉瘤动物模型 形成受到方法学复杂性的限制，许多模型与人类疾病的相关性有限。 病理生理学。几乎所有的人类心力衰竭和大多数动脉疾病状态都与高血压有关。 受影响组织中的活性氧(ROS)水平。因此，我们开发了一种新的化学遗传学 利用酵母D-氨基酸氧化酶动态调节体内氧化还原应激的方法 产生ROS过氧化氢(H_2O_2)。DAAO被D-氨基酸激活，但不被L-氨基酸激活 哺乳动物细胞中发现的酸。我们用重组的嗜心性腺相关病毒感染小鼠或大鼠 表达DAAO的同型9(AAV9)。当动物被提供D-氨基酸时，DAAO会产生过氧化氢 在心脏中，动物迅速发展为心力衰竭--这可以通过药物治疗来逆转。虽然 这种方法是信息性的，一个主要的限制是需要一次一个地用病毒感染动物。此外， 基于病毒的方法受到体内大多数病毒载体有限的组织选择性的限制。因此，我们 产生了新的转基因小鼠系，旨在在选定的心血管组织中表达DAAO。我们有 制定了严格的绩效衡量标准，以定量评估我们在以下拟议研究中的进展 这些新的转基因品系。目标1建议对三个独立的转基因创始人系进行实验研究 在心脏中表达DAAO(由MYH6启动子驱动)将创建一个强大且经济高效的模型 确定预防和/或治疗心力衰竭的新药理靶点和新药。在目标2中，我们建议 将这一方法扩展到研究血管壁氧化还原应力。我们最近培育出了两种新的转基因 含有DAAO的方正小鼠系，其中loxP位点两侧的终止密码子被克隆到编码中 达奥地区。通过用精选的商业可获得的品系培育这些“开花”的DAAO转基因品系 在组织特异性启动子的控制下表达Cre重组酶，我们将能够动态调节 氧化还原应激在不同的小鼠组织中，允许研究氧化还原的疾病状态的广泛范围 应激与疾病的发生有关。在这里，我们将重点研究DAAO在血管内皮细胞中的表达 (使用VE-钙粘蛋白-Cre)和血管平滑肌细胞(使用MYH11-Cre)，以鉴定 氧化应激导致主动脉瘤的机制和寻找新的药物靶点以预防或 治疗动脉瘤。在心脏和主动脉中，我们将进行最先进的单细胞RNA测序，以跟踪 伴随着氧化还原压力的发展和消退而产生的细胞群体和转录图谱的变化。 拟议的研究将导致产生强大的新模型系统，从而导致识别 治疗和预防心力衰竭和主动脉瘤的新靶点和新药。

This R33 research program proposes to characterize and validate novel transgenic mouse lines that exploit chemogenetics to create powerful models that will be used to identify new targets for the prevention and treatment of heart failure and aortic aneurysms. Most current animal models of heart failure and aortic aneurysm formation are limited by methodological complexities, and many models have limited relevance to human disease pathophysiology. Nearly all human heart failure and most arterial disease states are associated with high levels of reactive oxygen species (ROS) in affected tissues. We therefore developed a novel chemogenetic approach that dynamically modulates redox stress in vivo by exploiting a yeast D-amino acid oxidase (DAAO) that generates the ROS hydrogen peroxide (H2O2). DAAO is activated by D-amino acids, but not by the L-amino acids found in mammalian cells. We infected mice or rats with a recombinant cardiotropic adeno-associated virus isotype 9 (AAV9) that expresses DAAO. When animals are provided with D-amino acids, DAAO generates H2O2 in the heart, and the animals rapidly develop heart failure– which can be reversed by drug treatments. Although this approach is informative, a major limitation is the need to infect animals one at a time with the virus. Moreover, virus-based methods are constrained by the limited tissue selectivity of most viral vectors in vivo. Therefore, we generated new transgenic mouse lines designed to express DAAO in selected cardiovascular tissues. We have developed rigorous Performance Measures to quantitatively assess our progress in the proposed studies of these new transgenic lines. Aim 1 proposes experiments studying three independent transgenic founder lines expressing DAAO in the heart (driven by the MYH6 promoter) that will create a robust and cost-effective model to identify new pharmacological targets and new drugs to prevent and/or treat heart failure. In Aim 2, we propose to expand this approach to study redox stress in the vascular wall. We recently produced two new transgenic founder mouse lines containing DAAO in which a stop codon flanked by LoxP sites was cloned into the coding region of DAAO. By breeding these “floxed” DAAO transgenic lines with selected commercially-available lines expressing Cre recombinase under control of tissue-specific promoters, we will be able to dynamically regulate redox stress in different mouse tissues, permitting the study of the broad range of disease states in which redox stress is associated with pathogenesis. Here we will focus on DAAO expression in vascular endothelial cells (using VE-cadherin-Cre) and vascular smooth muscle cells (using MYH11-Cre) in order to identify the mechanisms whereby oxidative stress causes aortic aneurysms and to identify new drug targets to prevent or treat aneurysms. In both heart and aorta, we will perform state-of-the-art single cell RNA sequencing in order to track the changes in cell populations and transcriptional profiles that accompany development and regression of redox stress. The proposed studies will lead to the generation of powerful new model systems that will lead to the identification of novel targets and new drugs for the treatment and prevention of heart failure and aortic aneurysms.