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model; novel; novel strategies; prevent; pulmonary function; repaired; response; two photon microscopy; two-photon; unpublished works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.

项目概要 特发性肺纤维化（IPF）是一种进展迅速且无法治愈的疾病。在健康组织中，成纤维细胞 平衡细胞外基质的沉积和再吸收，但在纤维化过程中，它们会产生过量的 细胞外基质，而 ECM 降解酶下调。其中许多影响可能与 由于代谢失调（包括组织蛋白酶 K 的抑制）导致平衡向氧化性更强的状态转变， 一种对正常肺功能和发育至关重要的胶原蛋白分解酶。众所周知，代谢变化 增加活性氧，其本身会改变整个组织的氧化还原（氧化还原）状态。 我们认为，为了解决纤维化，治疗应该恢复氧化还原平衡并修复缺陷 代谢。在这个项目中，我将重点研究调节成纤维细胞氧化还原状态的机制 以及纤维化和消退过程中体内这种状态的转变。我将纵向监测 Col1a1-GFP+ 通过胸腔窗口观察成纤维细胞，并对 NADH 和 FAD 的自发荧光进行成像，这两个指标是 光学氧化还原比。氧化还原比的变化可以表明活性氧增加。因此，通过使用胸椎 细胞氧化还原比率成像的窗口 我可以实时识别细胞压力的变化，使我能够 直接比较纤维化进展到消退阶段期间的变化。为了进一步探讨其中的关系 成纤维细胞氧化还原状态到纤维化消退，我将探讨加速和纤维化小鼠模型中的这些变化 未解决的纤维化。然后我将在机制上测试 PGC1a 作为氧化还原态调节剂的作用 成纤维细胞，并将研究氧化还原比在调节组织蛋白酶 K 活性中的作用作为关键环节 纤维化消退。我假设将氧化还原态转变为内源性或外源性机制 更多还原对于恢复成纤维细胞代谢状态以促进纤维化消退至关重要。我建议 通过结合活体成像、体外细胞培养和精密切割肺的两个具体目标来检验这一假设 切片与所提出的机制途径成分的选择性激活或抑制相结合。 这些综合研究将共同​​测试氧化还原比作为纤维化消退所需的调节剂。