Nrf2 as a critical determinant of smooth muscle function

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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.