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进行性替代 交换富含胶原蛋白的疤痕组织。这种基质的积累、交联和硬化是 定义疾病的特征，以及有效修复或再生的明确障碍。新出现的证据 表明纤维化瘢痕在适当条件下保持高度可吸收性]，这一过程似乎 在患有IPF的人类中受损或不存在。促进成纤维细胞（和其他细胞）生长的条件和途径 细胞类型）再吸收富含胶原的瘢痕的能力在很大程度上是未知的。制定方法和方针， 解决这一认识上的关键差距是U01提案的重点。我们建议开发模型 系统，其中ECM沉积和再吸收可以有效地研究在原代培养的肺 成纤维细胞以及精密切割肺切片（PCLS）。我们的初步数据显示，在适当的 刺激，可促使IPF衍生的人肺成纤维细胞降解和再吸收纤维状胶原。我们 假设在适当刺激下，成纤维细胞不仅可以被刺激吸收富含胶原的ECM， 体外，而且还吸收IPF患者肺中的瘢痕相关ECM。我们建议发展和 利用新的体外工具来测试这一假设，目的是确定生物学途径， 介导生理性胶原再吸收的治疗干预。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，使我们能够识别信号， 促进肺成纤维细胞的ECM再吸收，并阐明胶原再吸收的分子机制。 我们将验证这些检测方法的高通量发现，并进行重点筛选作为概念验证 这种方法的价值。我们还将比较先天ECM沉积和降解特性 的IPF和对照成纤维细胞和成纤维细胞亚群。在第二个目标中，我们将开发离体肺组织 用于测试瘢痕相关IPF ECM的治疗调节和清除机制的测定。我们将 表征对照和IPF肺组织中的基线和刺激诱发的胶原溶解活性，并定义 该活性与肺细胞类型和组织的组织病理学外观的关联。我们还将测试 在目标1中鉴定的候选命中物，其具有增加靶向纤维胶原降解的能力， 原生IPF ECM环境。共同提出的研究将建立强大的模型ECM沉积 和原代人IPF成纤维细胞和离体肺切片中的再吸收。这个平台将开辟新的途径 用于鉴定使IPF中的成纤维细胞向基质再吸收状态转变的信号和机制， 开发IPF先进疗法的新机会。

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.