Hydrogen peroxide in endothelial function and dysfunction

过氧化氢在内皮功能和功能障碍中的作用

基本信息

批准号：
10543765
负责人：
Thomas Michel
金额：
$ 44.22万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10543765
关键词：
Alanine Amino Acids Animal Genetics Animal Model Antioxidants Biosensor Blood Pressure Blood Vessels Cardiomyopathies Cardiovascular Diseases Catalysis Caveolae Cell Nucleus Cell Surface Receptors Cells Clinical Trials Country Cytosol D-Amino Acid Dehydrogenase Development Diabetes Mellitus Disease Endothelial Cells Endothelium Experimental Models Functional disorder Generations Genetic Genetic Transcription Goals Heart Heart failure Homeostasis Human Hydrogen Peroxide Hypertension Image In Vitro Intracellular Membranes Investigation Learning Metabolism Modeling Molecular Morbidity - disease rate NF-kappa B NOS3 gene Nitric Oxide Nitric Oxide Signaling Pathway Oxidants Oxidation-Reduction Oxidative Stress P2Y2 receptor Pathologic Pathway interactions Pharmaceutical Preparations Phenotype Phosphorylation Physiological Physiology Post-Translational Protein Processing Preparation Proteins Public Health Purinoceptor Reactive Oxygen Species Recombinants Research Role Signal Pathway Signal Transduction Technology Testing Transcript Transgenic Mice Vascular Diseases Vascular Endothelium Work Yeasts blood pressure control cellular imaging cofactor endothelial dysfunction experimental study genetic approach hemodynamics imaging approach in vivo Model mortality new therapeutic target novel programs response shear stress spectrograph time use transcriptome

项目摘要

The proposed studies will use new biosensors and novel chemogenetic approaches to identify the molecular mechanisms whereby reactive oxygen species (ROS) regulate nitric oxide (NO) signaling pathways in the vascular endothelium. The proposed studies build on recent work in which we used chemogenetics to develop a new animal model of cardiomyopathy caused by oxidative stress. Here we plan to expand this chemogenetic approach to develop a new experimental program to study endothelial dysfunction and hypertension. Many studies have implicated oxidative stress in endothelial dysfunction and hypertension, yet the underlying molecular mechanisms remain incompletely understood. Low levels of the stable ROS hydrogen peroxide (H2O2) modulate NO-dependent physiological responses, while higher ROS levels are associated with hypertension. The proposed experiments exploit recent advances in chemogenetic and biosensor technologies to identify the mechanisms underlying the transition from physiological H2O2 signaling to the development of hypertension and other vascular disease states associated with pathological oxidative stress. We will pursue multispectral imaging experiments that will simultaneously analyze H2O2, NO and Ca2+ using highly selective and sensitive HyPer7, geNOp, and GECO biosensors. These studies will establish the mechanisms whereby purinergic P2Y2 receptors modulate H2O2-, Ca2+-, and NO-dependent endothelial responses that control blood pressure. Hemodynamic shear stress leads to eNOS activation and to increases in endothelial ROS that can promote both physiological as well as pathophysiological responses. We found that physiological laminar shear stress preferentially increases H2O2 in the endothelial cell nucleus, while pathological oscillatory shear stress increases H2O2 more in the cell cytosol. We used a chemogenetic approach to generate H2O2 in endothelial cells, using novel recombinant constructs expressing a yeast D-amino acid oxidase (DAAO) that robustly produces H2O2. The recombinant yeast DAAO is quiescent since vascular cells contain L- but not D-amino acids. H2O2 can be generated by adding D-alanine to cells expressing recombinant DAAO. Our studies showed that H2O2 generated in the endothelial cell nucleus activates Nrf2-modulated transcripts, whereas generation of H2O2 in the cytosol principally increases NF-kB-dependent transcripts. These differential transcriptional responses establish a causal role for H2O2 and provide a strong connection between chemogenetic approaches and endothelial pathophysiology. Studying in vitro, ex vivo, and in vivo models, we propose to extend these studies from cultured human endothelial cells (Aim 1) to the investigation of arterial preparations and transgenic mice expressing DAAO in the endothelium (Aim 2). This experimental program may lead to the development of a new “chemogenetic” animal model of hypertension. These studies will use powerful new cell imaging approaches to test the hypothesis that perturbations in intracellular H2O2 metabolism modulate endothelial responses both in the normal vasculature and in hypertension, and in other vascular disease states caused by oxidative stress.

拟议的研究将使用新的生物传感器和新的化学遗传学方法来识别分子活性氧（ROS）调节一氧化氮（NO）信号通路的机制所提出的研究建立在我们最近使用化学遗传学进行开发的工作的基础上。在这里，我们计划扩展这种由氧化应激引起的心肌病的新动物模型。方法开发一个新的实验计划来研究内皮功能障碍和高血压。许多研究表明氧化应激与内皮功能障碍和高血压有关，但潜在的原因稳定的 ROS 过氧化氢 (H2O2) 水平较低的分子机制仍不完全清楚。调节 NO 依赖性生理反应，而较高的 ROS 水平与高血压相关。拟议的实验利用化学遗传学和生物传感器技术的最新进展来识别从生理性 H2O2 信号转导到高血压发展的潜在机制与病理性氧化应激相关的其他血管疾病状态我们将寻求多光谱成像。使用高选择性和灵敏的 HyPer7 同时分析 H2O2、NO 和 Ca2+ 的实验， geNOp 和 GECO 生物传感器将建立嘌呤能 P2Y2 受体的机制。调节 H2O2-、Ca2+- 和 NO 依赖性内皮反应来控制血压。血流动力学剪切应力导致 eNOS 激活和内皮 ROS 增加，从而促进我们发现生理层流剪切应力。优先增加内皮细胞核中的 H2O2，同时病理性振荡剪切应力增加 H2O2 更多地存在于细胞质中我们使用化学遗传学方法在内皮细胞中产生 H2O2。表达酵母 D-氨基酸氧化酶 (DAAO) 的新型重组结构，可强力产生 H2O2。重组酵母 DAAO 是静止的，因为维管细胞含有 L-氨基酸但不含有 D-氨基酸。通过向表达重组 DAAO 的细胞中添加 D-丙氨酸而产生。我们的研究表明，会产生 H2O2。内皮细胞核中的 Nrf2 激活 Nrf2 调节的转录本，而细胞质中 H2O2 的产生主要增加 NF-kB 依赖性转录本。 H2O2 的因果作用并提供化学遗传学方法和内皮细胞之间的紧密联系通过研究体外、离体和体内模型，我们建议将这些研究扩展到培养模型。人内皮细胞（目标 1）用于动脉制剂和转基因小鼠表达的研究内皮细胞中的 DAAO（目标 2）。这些研究将使用强大的新细胞成像方法来研究高血压的“化学遗传学”动物模型。检验细胞内 H2O2 代谢的扰动调节内皮反应的假设正常脉管系统和高血压以及氧化应激引起的其他血管疾病状态。