Therapeutic ECM Resorption in Cellular Systems and Precision Cut Lung Slices.

项目摘要 特发性肺纤维化(IPF)的特征是功能性肺泡气体的渐进性替代 用胶原丰富的疤痕交换组织。这种基质的积累、交联化和硬化是 明确疾病的特征，以及有效修复或再生的明确障碍。新出现的证据 表明纤维性瘢痕在适当的条件下仍高度可吸收]，这一过程似乎 患有特发性肺纤维化的人受损或缺失。促进成纤维细胞(和其他 细胞类型)到吸收胶原丰富的瘢痕在很大程度上是未知的。开发方法和途径以达到 解决这一理解上的严重差距是这份U01提案的重点。我们建议开发模型 可在两个原代培养的肺中有效研究ECM沉积和吸收的系统 成纤维细胞和精密肺切片(PCLS)。我们的初步数据显示，在适当的情况下 在刺激下，IPF来源的人肺成纤维细胞可以被促使降解和吸收纤维状胶原。我们 假设在适当的刺激下，成纤维细胞不仅可以吸收富含ECM的胶原 体外，还可以从IPF患者的肺中吸收与瘢痕相关的细胞外基质。我们建议开发和开发 利用新的体外工具来测试这一假说，目的是确定生物途径和 调节生理性胶原蛋白吸收的治疗干预。我们建议追求这些目标 通过两个目标。在第一个目标中，我们将开发文化系统，使我们能够识别 通过肺成纤维细胞促进细胞外基质吸收，阐明胶原吸收的分子机制。 我们将验证这些分析以实现高吞吐量发现，并执行重点筛选作为概念验证 这种方法的价值。我们还将比较固有的ECM沉积和降解特性 IPF和对照成纤维细胞及成纤维细胞亚群。在第二个目标中，我们将开发体外肺组织 检测瘢痕相关IPF ECM的治疗调节和清除机制。我们会 在对照和IPF肺组织中表征基线和刺激引起的胶原蛋白溶解活性，并确定 这种活性与肺细胞类型和组织病理形态的关系。我们还将测试 在AIM 1中确定的候选HITS具有增加靶向纤维胶原降解的能力 原生IPF ECM环境。总之，拟议的研究将建立坚固的ECM沉积模型 原代人肺间质成纤维细胞和体外肺切片中的吸收。这个平台将开辟新的途径 为了识别将IPF中的成纤维细胞转变为基质再吸收状态的信号和机制，生成 为特发性肺纤维化开发先进疗法的新机遇。

Project Summary Idiopathic Pulmonary Fibrosis (IPF) is characterized by progressive replacement of functional alveolar gas exchange tissue with collagen rich scar. The accumulation, crosslinking and stiffening of this matrix are defining features of the disease, and definitive barriers to effective repair or regeneration. Emerging evidence indicates that fibrotic scar remains highly resorbable under appropriate conditions], a process that appears to be impaired or absent in humans with IPF. The conditions and pathways that promote fibroblasts (and other cell types) to resorb collagen rich scar are largely unknown. Development of methods and approaches to address this critical gap in understanding is the focus of this U01 proposal. We propose to develop model systems in which ECM deposition and resorption can be efficiently studied in both primary cultured lung fibroblasts as well as precision cut lung slices (PCLS). Our preliminary data show that under appropriate stimuli, IPF-derived human lung fibroblasts can be prompted to degrade and resorb fibrillar collagen. We hypothesize that under appropriate stimuli fibroblasts can be stimulated to not only resorb collagen rich ECM in vitro, but also resorb scar-associated ECM in the lungs from patients with IPF. We propose to develop and leverage novel in vitro tools to test this hypothesis, with the goal of identifying biological pathways and therapeutic interventions that mediate physiologic collagen resorption. We propose to pursue these goals through two aims. In the first aim we will develop culture systems allowing us to identify the signals that promote ECM resorption by lung fibroblasts and delineate the molecular mechanisms of collagen resorption. We will validate these assays for high-throughput discovery and perform a focused screen as proof of concept of the value of this approach. We will also compare the innate ECM deposition and degradation characteristics of IPF and control fibroblasts and fibroblast subsets. In the second aim we will develop ex vivo lung tissue assays to test therapeutic modulation and mechanisms of clearance of scar-associated IPF ECM. We will characterize baseline and stimulus evoked collagenolytic activity in control and IPF lung tissue, and define the association of this activity with lung cell types and histopathological appearance of the tissue. We will also test candidate hits identified in aim 1 for their capacity to increase targeted fibrillar collagen degradation in the native IPF ECM environment. Together the proposed studies will establish robust models of ECM deposition and resorption in primary human IPF fibroblasts and ex vivo lung slices. This platform will open new avenues for identifying signals and mechanisms that shift fibroblasts in IPF toward a matrix resorbing state, generating new opportunities to develop advanced therapeutics for IPF.