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

• 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
• 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

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 体内活性氧簇(ROS)调节一氧化氮(NO)信号转导途径的机制 血管内皮细胞。拟议的研究建立在我们使用化学遗传学开发的最新工作的基础上 氧化应激所致心肌病的新动物模型。在这里，我们计划扩大这种化学发生 开发一种新的研究内皮功能障碍和高血压的实验方案。 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 分子机制仍不完全清楚。低水平稳定的ROS过氧化氢(H_2O_2) 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来鉴定 生理性H_2O_2信号转导到高血压和高血压发生的机制 其他与病理性氧化应激相关的血管疾病状态。我们将进行多光谱成像 使用高选择性和高灵敏度的Hyper7同时分析过氧化氢、一氧化氮和钙离子的实验， Genop和GECO生物传感器。这些研究将建立嘌呤能P2Y2受体的机制 调节控制血压的依赖于过氧化氢、钙和一氧化氮的内皮反应。 血流动力学切应力导致eNOS激活和内皮细胞ROS增加，从而促进 包括生理反应和病理生理反应。我们发现生理层流剪应力 优先增加内皮细胞核中的过氧化氢，同时病理性振荡切应力增加 细胞胞浆中有更多的过氧化氢。我们使用一种化学发生方法在内皮细胞中产生过氧化氢，使用 表达酵母D-氨基酸氧化酶(DAAO)的新型重组构建物，该酵母菌能旺盛地产生过氧化氢。 重组酵母DAAO是静止的，因为维管细胞中含有L-而不是D-氨基酸。双氧水可以 通过将D-丙氨酸加入到表达重组DAAO的细胞中而产生。我们的研究表明，过氧化氢会产生 在内皮细胞核中激活Nrf2调节的转录本，而在胞浆中产生过氧化氢 主要增加依赖于核因子-kB的转录本。这些不同的转录反应建立了 过氧化氢的因果作用，并在化学发生途径和内皮细胞之间提供了强有力的联系 病理生理学。在体外、体外和体内模型的研究中，我们建议将这些研究从培养的 人内皮细胞(Aim 1)动脉制备及转基因小鼠表达的研究 血管内皮细胞中的DAAO(目标2)。这一实验计划可能会导致开发一种新的 高血压的“化学遗传学”动物模型。这些研究将使用强大的新细胞成像方法来 验证这样一种假设，即细胞内过氧化氢代谢的扰动调节内皮反应 正常的血管系统和高血压，以及其他由氧化应激引起的血管疾病状态。