Nrf2 as a critical determinant of smooth muscle function

Nrf2 作为平滑肌功能的关键决定因素

基本信息

批准号：
8434873
负责人：
Steven S An
金额：
$ 38.84万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-05-01 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8434873
关键词：
Address Adhesions Affect Agonist Allergens Antioxidants Asthma Biochemical Biological Markers Biomimetics Blood capillaries Broccoli - dietary Cells Chronic Contracts Custom Cytometry Cytoskeleton Data Development Disease Dose Elasticity Enzymes Extracellular Matrix Fourier Transform Gene Deletion Gene Expression Gene Targeting Generations Genes Glutathione Disulfide Human Hydrogen Peroxide Individual Intervention Knock-out Knowledge Lead Life Link Magnetism Measures Mechanics Methods Microfluidic Microchips Microscopy Modeling Molecular Motion Mus Muscle Cells Muscle Contraction Muscle function Nitric Oxide Synthase Nitrogen Obstruction Organ Oxidation-Reduction Oxidative Stress Oxidative Stress Pathway Oxygen Pathology Pathway interactions Pattern Peroxonitrite Phosphorylation Population Protein Isoforms Pyroglyphidae Regimen Regulation Research Role Signal Pathway Signal Transduction Smooth Muscle Smooth Muscle Myocytes Structure Structure-Activity Relationship Sulforaphane T-Lymphocyte Therapeutic Studies Tracer Traction United States airway hyperresponsiveness asthmatic airway asthmatic patient base capillary constriction design human disease innovation lithography mouse model nano nanoscale nuclear factor-erythroid 2 oxidant stress public health relevance respiratory smooth muscle response restoration small hairpin RNA therapeutic effectiveness transcription factor

项目摘要

DESCRIPTION (provided by applicant): The pathology of chronic asthma shows prominent structural changes in the airway wall, specifically alteration in the extracellular matrix (ECM) and thickening of the airway smooth muscle (ASM). However, how these structural changes affect asthmatic airflow obstruction is not well understood. We are proposing an entirely new experimental approach to elucidate this structure-function relationship that is based on dysfunctional regulation of oxidative stress. We reason that ASM may be a major target of oxidative stress. In support of this paradigm, we have demonstrated that mice, with a deletion of a transcription factor (Nrf2) that regulates several antioxidant genes, are more susceptible to oxidative stress and airway hyperresponsiveness, and that the hyperresponsiveness is primarily attributable to increased ASM contractility. Based on preliminary data showing differential induction of Nrf2-dependent cytoprotective genes and force generation by healthy versus asthmatic human ASM cells, we hypothesize that airflow obstruction in chronic asthma is attributable to defective Nrf2-directed regulation of oxidative stress that leads to abnormal ECM remodeling and increased contractility of the ASM cell. In addition, we hypothesize that stimulation of Nrf2-directed antioxidant pathways by sulforaphane can restore the cytoprotective status conferred by the ECM on ASM and inhibit ASM contraction altogether. To address these hypotheses, we will use UV-assisted capillary force lithography to fabricate micro- and nano-topographically defined substrata that better recapitulate ECM structure and elasticity of the airway wall. Using these biomimetic substrata with variable distribution of ECM patterns and rigidities, we will quantify changes in both structure and function of human ASM cells. For quantitative structural analysis, we will implement the high-throughput custom-built microfluidic devices that allow the in- chip design for culturing live cells. At the single cell level, we will measure changes in ASM stiffness using Magnetic Twisting Cytometry (MTC), contractile force using Fourier Transform Traction Microscopy (FTTM), and discrete molecular-level remodeling dynamics of the cytoskeleton using Spontaneous Nanoscale Tracer Motions (SNTM). With these technical innovations, we will probe the internal network of physical forces within ASM to determine: (Aim 1) the biochemical and mechanical factors that affect ASM contraction; (Aim 2) the molecular link between ASM contraction and Nrf2-directed regulation of oxidative stress; and (Aim 3) the efficacy of targeting Nrf2 pathways to eliminate ASM contraction. Finally, as a proof-of-concept, we will validate the therapeutic effectiveness of sulforaphane (in the form of broccoli sprout extract) in a mouse model with airway hyperresponsiveness induced by house dust mite (HDM). The HDM model is much more relevant to human sensitization and thereby provides a tighter link between what we might discover in the mouse model to human disease. If successful, the knowledge gained from these studies has the potential to redirect our approach to asthma research and therapy, and may lead to the development of new intervention strategies.

描述（由申请人提供）：慢性哮喘的病理显示气道壁的显着结构变化，特别是细胞外基质（ECM）的变化以及气道平滑肌（ASM）的增厚。但是，这些结构变化如何影响哮喘气流阻塞。我们提出了一种全新的实验方法，以阐明基于功能失调的氧化应激调节的结构功能关系。我们认为ASM可能是氧化应激的主要目标。为了支持这种范式，我们证明了调节几种抗氧化基因的转录因子（NRF2）的小鼠更容易受到氧化应激和气道高反应性的影响，并且高反应性主要可归因于增加的ASM降低性。 Based on preliminary data showing differential induction of Nrf2-dependent cytoprotective genes and force generation by healthy versus asthmatic human ASM cells, we hypothesize that airflow obstruction in chronic asthma is attributable to defective Nrf2-directed regulation of oxidative stress that leads to abnormal ECM remodeling and increased contractility of the ASM cell.此外，我们假设通过硫烷对NRF2定向的抗氧化途径的刺激可以恢复ECM在ASM上赋予的细胞保护状态并完全抑制ASM收缩。为了解决这些假设，我们将使用UV辅助的毛细血管光刻来构建微观和纳米图表的基质，从而更好地概括了气道壁的ECM结构和弹性。使用这些仿生基底层具有ECM模式和刚性的可变分布，我们将量化人ASM细胞的结构和功能的变化。为了进行定量的结构分析，我们将实施高通量定制的微流体设备，以允许用于培养活细胞的芯片设计。在单细胞水平上，我们将使用傅立叶变换牵引显微镜（FTTM）（FTTM）以及使用自发的纳米级纳米级Tracer Tracer Motions（SNTM）来测量使用磁性扭曲细胞仪（MTC），使用傅立叶变换牵引显微镜（FTTM）的收缩力的变化。通过这些技术创新，我们将探究ASM内部力量内部力量的内部网络，以确定：（目标1）影响ASM收缩的生化和机械因素；（AIM 2）ASM收缩与NRF2定向的氧化应激调节之间的分子联系；（目标3）靶向NRF2途径以消除ASM收缩的功效。最后，作为概念验证，我们将在小鼠模型中验证硫磺烷（以西兰花发芽提取物的形式）的治疗有效性，该模型具有由房屋粉尘螨（HDM）引起的气道高反应性。 HDM模型与人类的敏感性更为相关，从而在小鼠模型中与人类疾病中发现的东西之间提供了更紧密的联系。如果成功的话，从这些研究中获得的知识将有可能重定向我们的哮喘研究和治疗方法，并可能导致发展新的干预策略。