Novel Approaches for Identification of Idiopathic Pulmonary Fibrosis Therapy Targets Using Microscale Collagen Hydrogels

使用微型胶原水凝胶识别特发性肺纤维化治疗靶点的新方法

• 批准号: 10376721
• 负责人: Katherine Anne Cummins
• 金额: $ 3.99万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-02 至 2023-03-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376721
• 关键词: Animal Model; Automobile Driving; Behavior; Biological; Biological Assay; Blocking Antibodies; Cell Count; Cell Separation; Cell Size; Cell Volumes; Cell physiology; Cell surface; Cells; Characteristics; Cicatrix; Clinical Research; Collagen; Collagen Fiber; Contracts; Coupled; Data; Deposition; Development; Devices; Diagnosis; Disease; Disease Progression; Disease model; Drug Targeting; ECM receptor; Encapsulated; Extracellular Matrix; FDA approved; Fibroblasts; Fibrosis; Fluorescence-Activated Cell Sorting; Gases; Gel; Genetic; Goals; HMMR gene; Heterogeneity; Hydrogels; Impairment; In Vitro; Inhalation; Integrins; Investigation; Life; Link; Lung; Lung Capacity; Lung Transplantation; Methodology; Methods; Microfluidic Microchips; Microfluidics; Microspheres; Modeling; Molecular; Myofibroblast; Pathogenicity; Pathway interactions; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physiological; Population; Population Heterogeneity; Process; Production; Prognosis; Proteins; Pulmonary Fibrosis; Reagent; Respiration; Role; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Source; Stratification; Surface; Survival Rate; System; Techniques; Therapeutic Agents; Tissues; base; cell behavior; collagenase; drug discovery; effective therapy; exhaustion; experimental study; idiopathic pulmonary fibrosis; improved; inhibitor/antagonist; interest; interstitial; miniaturize; nanolitre; novel; novel strategies; patient stratification; physical separation; prevent; receptor; receptor expression; receptor for advanced glycation endproducts; response; single-cell RNA sequencing; targeted treatment; therapeutic development; therapeutic target; therapeutically effective; therapy outcome; transcriptome; transcriptome sequencing; transcriptomics; treatment strategy

Project Summary/Abstract Idiopathic pulmonary fibrosis (IPF) impairs respiration through scarring of the interstitial space, resulting in (1) a stiffened lung that prevents inhalation and (2) limited gas exchange to adjacent pulmonary vasculature. Patients rely on lung transplants to extend their life beyond the median 3-year survival post-diagnosis as the only therapeutic agents used for IPF treatment slow disease progression are unable to cease or reverse fibrosis. The deficiency of pharmaceutical treatment strategies coupled with poor lung transplant survival rates indicates an obvious need for development of effective treatment options. Unfortunately, drug discovery has largely been restricted by physiologically irrelevant animal models and by reagent exhaustive, minimally informative in vitro platforms. The main goal of this proposal is to demonstrate our ability to identify pathways of importance in fibrotic progression and, by probing the IPF disease space, ultimately aid in therapeutic development. The outlined methodology enables biological discovery by systematically probing heterogenous populations of IPF cells by investigating functional behaviors of fibrotic cells from two complementary approaches. The first proposed methodology stratifies cell populations prior to encapsulation in collagen hydrogels to enable studies on how specific surface markers effect fibrotic behavior. Using flow sorting techniques, primary fibroblasts derived from IPF patients are separated into high and low expressing populations of specific surface receptors, a method that enhances the rarity and value of these precious cells. Stratified fibroblasts are then encapsulated in miniature collagen hydrogels using reagent-efficient microfluidic devices, reducing total cell volumes required for each condition and maximizing the number of surface receptors that can be investigated from a given cell source. Effects of low and high receptor expression will then be quantified and compared using several metrics of fibrotic function previously optimized. The second approach identifies potential therapeutic targets by functionally sorting and sequencing single fibrotic cells based on their contractility. Single fibroblasts are encapsulated into the previously described microgels and cultured to allow for spreading, adhering, and contracting of the cells into the surrounding matrix. As the cells exert force on the nearby collagen fibers, they compact the gel into a smaller sphere. Due to heterogeneity in the contraction ability of these fibroblasts, constructs are greatly varied in size and size-based sorting mechanisms are employed to segregate the most and least contractile cells. RNA sequencing is then used to directly compare transcriptomic profiles to fibroblast contractility and to identify upregulated pathways of interest. To validate sequencing hits as important mechanisms driving fibrotic functions, function-blocking antibodies and pharmaceutical inhibitors are used to confirm loss in fibrotic function for fibrogenic subpopulations.

项目总结/摘要 特发性肺纤维化（IPF）通过间质空间瘢痕化损害呼吸，导致（1） 阻止吸入的僵硬的肺和（2）与相邻肺脉管系统的有限的气体交换。患者 依靠肺移植来延长他们的生命，超过诊断后的中位3年生存期， 用于IPF治疗的治疗剂减缓疾病进展不能停止或逆转纤维化。的 药物治疗策略的缺乏加上肺移植存活率低表明， 显然需要制定有效的治疗方案。不幸的是，药物发现在很大程度上 受限于生理学上不相关的动物模型和试剂详尽，体外信息量极小 平台该提案的主要目标是证明我们有能力识别重要的途径， 纤维化进展，并通过探测IPF疾病空间，最终有助于治疗开发。的 概述的方法通过系统地探测IPF的异质性群体实现生物学发现 通过两种互补的方法研究纤维化细胞的功能行为。 第一种提出的方法在包封在胶原蛋白水凝胶中之前对细胞群进行分层， 研究特定的表面标志物如何影响纤维化行为。使用流排序技术， 将来自IPF患者的成纤维细胞分成特异性表面活性剂的高表达群体和低表达群体， 受体，一种提高这些珍贵细胞的稀有性和价值的方法。分层的成纤维细胞， 使用试剂高效微流体装置封装在微型胶原蛋白水凝胶中， 每种条件所需的体积，并最大限度地增加可以研究的表面受体的数量 从给定的细胞来源。然后使用以下方法定量和比较低和高受体表达的影响： 先前优化的纤维化功能的几个度量。第二种方法确定潜在的治疗 通过基于纤维化细胞的收缩性对单个纤维化细胞进行功能性分选和测序来靶向它们。单个成纤维细胞 被封装到前述微凝胶中并培养以允许扩散、粘附和 细胞收缩到周围的基质中。当细胞对附近的胶原纤维施加力时， 将凝胶压实成更小的球体。由于这些成纤维细胞收缩能力的异质性， 构建体的大小变化很大，并且采用基于大小的分选机制来分离最多的 和最少收缩的细胞。然后使用RNA测序直接比较成纤维细胞的转录组学谱。 收缩性，并鉴定感兴趣的上调途径。验证测序命中是否重要 驱动纤维化功能的机制、功能阻断抗体和药物抑制剂用于 证实纤维化亚群的纤维化功能丧失。