Nrf2 as a critical determinant of smooth muscle function

Nrf2 作为平滑肌功能的关键决定因素

• 批准号: 8260499
• 负责人: Steven S An
• 金额: $ 40.8万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 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/8260499
• 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; Public Health; 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

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. PUBLIC HEALTH RELEVANCE: In the past 20 years, asthma cases have more than doubled. Asthma is a debilitating airway disorder and affects some 20 million people in the United States, but remains unexplained. Here we focus on the structure and function of airway smooth muscle - the end organ that leads to airway constriction - and the role for a transcription factor (Nrf2) in the context of airway wall remodeling in chronic asthma.

描述（由申请人提供）：慢性哮喘的病理学表现为气道壁的显著结构变化，特别是细胞外基质（ECM）的改变和气道平滑肌（ASM）的增厚。然而，这些结构变化如何影响哮喘气流阻塞还不清楚。我们提出了一种全新的实验方法来阐明这种结构-功能关系，这种关系是基于氧化应激的功能失调调节。我们推断ASM可能是氧化应激的主要靶点。为了支持这种模式，我们已经证明，小鼠，与一个转录因子（Nrf 2），调节几个抗氧化基因的缺失，更容易受到氧化应激和气道高反应性，高反应性主要是由于增加ASM收缩。基于初步数据显示Nrf 2依赖性细胞保护基因的差异诱导和健康与哮喘的人ASM细胞的力生成，我们假设慢性哮喘中的气流阻塞是由于Nrf 2定向调节氧化应激缺陷导致ECM重塑异常和ASM细胞收缩性增加。此外，我们假设萝卜硫素刺激Nrf 2介导的抗氧化途径可以恢复ECM对ASM的细胞保护作用，并完全抑制ASM收缩。为了解决这些假设，我们将使用UV辅助毛细力光刻来制造微米和纳米地形定义的基质，更好地概括ECM结构和气道壁的弹性。使用这些仿生基质与ECM模式和刚度的可变分布，我们将量化人类ASM细胞的结构和功能的变化。对于定量结构分析，我们将实现高通量定制的微流体装置，其允许用于培养活细胞的芯片内设计。在单细胞水平，我们将测量ASM刚度的变化，使用磁扭转细胞仪（MTC），收缩力使用傅立叶变换牵引显微镜（FTTM），和离散分子水平的重塑动态的细胞骨架使用自发纳米级示踪剂运动（SNTM）。通过这些技术创新，我们将探索ASM内物理力的内部网络，以确定：（目的1）影响ASM收缩的生化和机械因素;（目的2）ASM收缩与Nrf 2介导的氧化应激调节之间的分子联系;（目的3）靶向Nrf 2途径消除ASM收缩的功效。最后，作为概念验证，我们将验证萝卜硫素（以西兰花芽提取物的形式）在由屋尘螨（HDM）诱导的气道高反应性小鼠模型中的治疗有效性。HDM模型与人类致敏性更相关，从而在我们可能在小鼠模型中发现的与人类疾病之间提供了更紧密的联系。如果成功，从这些研究中获得的知识有可能将我们的方法重新导向哮喘研究和治疗，并可能导致新的干预策略的发展。 公共卫生相关性：在过去的20年里，哮喘病例增加了一倍多。哮喘是一种使人衰弱的气道疾病，在美国影响着大约2000万人，但仍然无法解释。在这里，我们专注于气道平滑肌的结构和功能-导致气道收缩的终末器官-以及转录因子（Nrf 2）在慢性哮喘气道壁重塑中的作用。