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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：
10320952
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/10320952
关键词：
Alanine Amino Acids 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 Transcription Goals Heart Heart failure Homeostasis Human Hydrogen Peroxide Hypertension Image In Vitro Intracellular Membranes Investigation Lead 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 hemodynamics imaging approach in vivo Model mortality new therapeutic target novel programs response shear stress 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发生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，血管内皮这项研究建立在我们最近的工作基础上，我们使用化学遗传学来开发一种新的氧化应激性心肌病动物模型。在这里，我们计划扩大这种化学遗传方法开发一个新的实验程序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，但其潜在的分子机制仍不完全清楚。低水平的稳定ROS过氧化氢（H2 O2）调节NO依赖的生理反应，而较高的ROS水平与高血压有关。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，以确定从生理H2 O2信号传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。我们将继续进行多光谱成像使用高选择性和灵敏度的Hyper 7同时分析H2 O2、NO和Ca 2+的实验， geNop和GECO生物传感器。这些研究将建立嘌呤能P2 Y2受体调节控制血压的H2 O2-、Ca 2 +-和NO依赖性内皮反应。血流动力学切应力导致eNOS激活和内皮ROS增加，生理和病理生理反应。我们发现生理层流剪切应力优先增加内皮细胞核中的H2 O2，而病理性振荡剪切应力增加 H_2O_2在细胞质中的含量较高。我们使用化学发生方法在内皮细胞中产生H2 O2，新的重组构建体，其表达稳健地产生H2 O2的酵母D-氨基酸氧化酶（DAAO）。重组酵母DAAO是静止的，因为血管细胞含有L-而不是D-氨基酸。H2 O2可以是通过向表达重组DAAO的细胞中加入D-丙氨酸来产生。我们的研究表明，H2 O2产生在内皮细胞核中激活Nrf 2调节的转录物，而在胞质溶胶中产生H2 O2 主要增加NF-κ B依赖性转录物。这些不同的转录反应建立了一个 H2 O2的因果作用，并提供了化学发生方法和内皮病理生理学研究在体外，离体和体内模型，我们建议将这些研究从培养的人内皮细胞（目的1）的动脉制剂和转基因小鼠表达的研究内皮中的DAAO（目的2）。这项实验计划可能会导致开发一种新的 “化学遗传”高血压动物模型。这些研究将使用强大的新细胞成像方法，检验细胞内H2 O2代谢的扰动调节内皮细胞反应的假设，正常的脉管系统和高血压以及由氧化应激引起的其它血管疾病状态。