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型的化学发生方法来鉴定分子活性氧（ROS）调节一氧化氮（NO）信号通路的机制血管内皮。拟议的研究以最新的工作为基础，我们使用化学遗传学来发展由氧化应激引起的一种新的心肌病动物模型。在这里，我们计划扩大这种化学生成开发新的实验计划来研究内皮功能障碍和高血压的方法。许多研究已经在内皮功能障碍和高血压中实施了氧化物应激，但基础分子机制仍然不完全理解。低水平的稳定的ROS过氧化氢（H2O2）调节无依赖性物理反应，而较高的ROS水平与高血压有关。提出的实验利用了化学发生和生物传感器技术的最新进展，以鉴定从物理H2O2信号转移到高血压和高血压发展的基础机制其他血管疾病状态与病理氧化物胁迫有关。我们将追求多光谱成像只需使用高选择性和敏感的Hyper7，可以简单地分析H2O2，NO和Ca2+的实验， Genop和Geco生物传感器。这些研究将建立嘌呤能P2Y2受体的机制调节控制血压的H2O2-，Ca2+ - 和无依赖性内皮反应。血液动力学剪切应力会导致eNOS激活并增加内皮ROS，可以促进生理学和病理生理反应。我们发现生理层流剪应力优先增加内皮细胞核中H2O2，而病理振荡剪切应力增加细胞胞质中的H2O2更多。我们使用化学遗传方法在内皮细胞中生成H2O2，使用表达酵母D-氨基酸氧化酶（DAAO）的新型重组构建体可稳健地产生H2O2。重组酵母Daao是静止的，因为血管细胞含有L-但不含D-氨基酸。 H2O2可以通过将D-丙氨酸添加到表达重组DAAO的细胞中产生。我们的研究表明H2O2产生了在内皮细胞核中，激活NRF2调节的转录本，而在细胞质中产生H2O2 主要增加NF-KB依赖性转录本。这些差分转录响应建立了 H2O2的因果作用，并在化学发生方法与内皮之间提供了牢固的联系病理生理学。在体外研究，体内和体内模型，我们建议从培养的人内皮细胞（目标1）对动脉制剂的投资和表达的转基因小鼠的投资 Daao在内皮中（AIM 2）。这个实验计划可能会导致新的发展高血压的“化学发生”动物模型。这些研究将使用强大的新细胞成像方法检验以下假设：细胞内H2O2代谢中的扰动调节内皮反应均正常的脉管系统和高血压，以及其他血管疾病状态，由氧化应激引起。