Targeting redox to resorb ECM

项目总结 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 平衡沉积和吸收细胞外基质，但在纤维化中它们会产生过量的 细胞外基质，而ECM降解酶表达下调。这些影响中的许多可能与 由于代谢失调，包括对组织蛋白酶K的抑制，平衡转变到更氧化的状态， 一种胶原酶，对正常的肺功能和发育至关重要。已知的新陈代谢变化 增加活性氧物种，这本身就会改变整个组织的氧化还原(氧化还原)状态。 我们认为，要解决纤维化，治疗应恢复氧化还原平衡并修复缺陷 新陈代谢。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 以及体内纤维化和消退过程中这种状态的转变。我将对Col1a1-GFP进行纵向监测 成纤维细胞通过胸窗并成像NADH和FAD的自体荧光，这两个指标 光学氧化还原比。氧化还原比的变化可以指示ROS的增加。因此，通过使用胸椎 显示细胞氧化还原比的窗口我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到消退阶段的变化。为了进一步探索与中国的关系 成纤维细胞氧化还原状态向纤维化消退，我将探讨这些变化对小鼠模型的加速和 未消退的纤维化。然后，我将机械地测试PGC1a作为氧化还原状态调节器在 并将研究氧化还原比在调节组织蛋白酶K活性中所起的作用，这是 纤维化消退。我假设，将氧化还原状态转变为 更多的还原是恢复成纤维细胞代谢状态所必需的，以促进纤维化的消退。我提议 在结合活体成像、体外细胞培养和精确切割肺的两个特定目标上验证这一假设 切片与选择性激活或抑制所提出的机械途径组件相结合。 总而言之，这些联合研究将测试氧化还原比率作为纤维化消退所需的调节因素。

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.