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: 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
• 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

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 血管内皮。拟议的研究建立在我们最近使用化学遗传学开发的工作的基础上 氧化应激引起的心肌病的新动物模型。在这里，我们计划扩展这种化学遗传学 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 分子机制仍不完全清楚。低水平的稳定 ROS 过氧化氢 (H2O2) 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。我们将追求多光谱成像 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。这些研究将建立嘌呤能 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 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 H2O2，同时病理性振荡剪切应力增加 H2O2较多存在于细胞质中。我们使用化学遗传学方法在内皮细胞中产生 H2O2，使用 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 D-氨基酸。 H2O2 可以 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 H2O2 产生 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。这些差异转录反应建立了 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 病理生理学。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 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）。这个实验计划可能会导致新的开发 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 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。