Targeting redox to resorb ECM

项目摘要 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 平衡沉积和再吸收细胞外基质，但在纤维化中，它们产生过量的 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， 一种对正常肺功能和发育必不可少的胶原分解酶。众所周知，代谢变化 增加活性氧物质，其本身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，治疗应该恢复氧化还原平衡和修复缺陷， 新陈代谢.在这个项目中，我将集中在确定机制调节氧化还原状态的成纤维细胞 以及在纤维化和消退期间在体内这种状态的转变。我将纵向监测Col 1a 1-GFP+ 通过胸窗观察成纤维细胞，并对NADH和FAD的自发荧光进行成像，这两种荧光的指标是 光学氧化还原比氧化还原比的变化可以指示增加的ROS。因此，通过使用胸部 我可以实时识别细胞应激的变化，使我能够 直接比较纤维化进展与消退阶段的变化。为了进一步探讨 成纤维细胞氧化还原状态对纤维化的解决，我将探讨这些变化在小鼠模型的加速和 未消退的纤维化然后，我将机械地测试PGC 1a作为氧化还原状态调节剂的作用， 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，作为一个关键环节， 纤维化消退。我假设，内源性或外源性机制，改变氧化还原状态， 还原性更强对于恢复成纤维细胞代谢状态以促进纤维化消退是必需的。我建议 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中测试该假设 切片结合选择性激活或抑制所提出的机制途径组分。 这些联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。

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.