Targeting redox to resorb ECM

靶向氧化还原再吸收 ECM

• 批准号: 10382514
• 负责人: Patrick Link
• 金额: $ 6.76万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-03 至 2023-01-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382514
• 关键词: Agonist; Antibodies; Biogenesis; Biology; Bleomycin; Cell Culture Techniques; Cellular Stress; Characteristics; Chest; Collagen; Data; Deposition; Detection; Diagnosis; Disease; Dopamine; Dopamine Agonists; Dopamine D1 Receptor; Enzymes; Equilibrium; Extracellular Matrix; Fibroblasts; Fibrosis; Flavin-Adenine Dinucleotide; Goals; Grant; Histology; Human; Hydroxyproline; Image; Impairment; Implant; In Situ; In Vitro; Life Expectancy; Link; Lung; Measures; Mediating; Metabolic; Metabolism; Microscopic; Mitochondria; Modeling; Monitor; Mus; Nicotinamide adenine dinucleotide; Optics; Oxidation-Reduction; Oxidative Stress; Oxides; PPAR gamma; Pathway interactions; Phase; Phenotype; Play; Proteins; Pulmonary Fibrosis; Reactive Oxygen Species; Resolution; Role; Signal Transduction; Slice; System; Techniques; Testing; Therapeutic; Time; Tissues; Work; base; cathepsin K; cellular imaging; cofactor; collagenase; enzyme activity; fatty acid oxidation; fibrotic lung; genetic manipulation; human disease; idiopathic pulmonary fibrosis; imaging modality; improved; in vivo; inhibitor; insight; intravital imaging; lung development; lung repair; mouse model; novel; novel strategies; prevent; pulmonary function; repaired; response; two photon microscopy; two-photon; unpublished works

PROJECT SUMMARY Idiopathic Pulmonary Fibrosis (IPF) is a rapidly progressing and incurable disease. In healthy tissue, fibroblasts balance depositing and resorbing extracellular matrix, yet in fibrosis they produce excessive amounts of extracellular matrix while ECM degrading enzymes are downregulated. Many of these effects may be linked to a shift in balance to a more oxidative state due to dysregulated metabolism, including inhibition of Cathepsin K, a collagenolytic enzyme essential to normal lung function and development. Metabolic shifts are known to increase reactive oxygen species, which themselves alter the overall tissue oxidation-reduction (redox) state. We believe that to resolve fibrosis, therapeutics should restore the redox balance and repair defective metabolism. In this project, I will focus on identifying mechanisms regulating the redox state of the fibroblasts and transitions in this state in vivo during fibrosis and resolution. I will longitudinally monitor Col1a1-GFP+ fibroblasts through a thoracic window and image the autofluorescence of NADH and FAD, two indicators of the optical redox ratio. Changes to the redox ratio can be indicative of increased ROS. Therefore, by using a thoracic window to image the cellular redox ratio I can identify changes to the cellular stress in real-time, allowing me to directly compare changes during fibrosis progression to resolution phases. To further explore the relationship of fibroblast redox state to fibrosis resolution, I will explore these changes in mouse models of accelerated and non-resolving fibrosis. I will then mechanistically test the role of PGC1a as a regulator of the redox state in fibroblasts and will investigate the role the redox ratio plays in regulating Cathepsin K activity as a key link to fibrosis resolution. I hypothesize that endogenous or exogenous mechanisms that shift the redox state to be more reductive are essential to restore the fibroblast metabolic state to promote fibrosis resolution. I propose to test this hypothesis in two specific aims combining intravital imaging, in vitro cell culture, and precision cut lung slices in combination with selective activation or inhibition of proposed mechanistic pathway components. Together these combined studies will test the redox ratio as a required regulator of fibrosis resolution.

项目摘要 特发性肺纤维化（IPF）是一种快速进展且无法治愈的疾病。在健康组织中，成纤维细胞 平衡沉积和吸收细胞外基质，但在纤维化中，它们产生过多的 ECM降解酶时细胞外基质被下调。这些效果中的许多可能与 由于新陈代谢失调，包括抑制组织蛋白酶K，，平衡转移到更氧化状态的状态 正常肺功能和发育必不可少的胶原式解释性酶。已知代谢转移已知 增加活性氧本身会改变整体组织氧化还原（氧化还原）状态。 我们认为，要解决纤维化，治疗剂应恢复氧化还原平衡并修复缺陷 代谢。在这个项目中，我将专注于确定调节成纤维细胞氧化还原状态的机制 并在纤维化和分辨率期间在体内的过渡。我将纵向监视Col1a1-GFP+ 成纤维细胞通过胸部窗口，并为NADH和FAD的自动荧光图像，这是两个指标 光学氧化还原比。氧化还原比的变化可以表明ROS增加。因此，使用胸腔 窗口图像细胞氧化还原比I可以实时识别细胞应力的变化，使我能够 直接比较纤维化进展过程中的变化与分辨率阶段。进一步探索 成纤维细胞氧化还原状态至纤维化的分辨率，我将探索这些变化的加速和 非分辨纤维化。然后，我将机械地测试PGC1A作为氧化还原状态的调节剂的作用 成纤维细胞并将研究氧化还原比在调节组织蛋白酶活性中的作用 纤维化分辨率。我假设将氧化还原状态转移的内源性或外源机制 还原性对于恢复成纤维细胞代谢状态以促进纤维化的分辨率至关重要。我建议 在两个具体的目的中检验该假设，结合了浸润成像，体外细胞培养和精确切割肺 切片结合选择性激活或抑制所提出的机械途径成分。 这些合并的研究将共同​​测试氧化还原比作为纤维化分辨率所需的调节剂。