A Novel PAI-1 Function Drives Lung Fibrosis

PAI-1 的新功能可驱动肺纤维化

• 批准号: 10605479
• 负责人: Daniel A Lawrence
• 金额: $ 69.67万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605479
• 关键词: Afibrinogenemia; Aging; Alzheimer' s Disease; Animal Model; Atherosclerosis; Attenuated; Automobile Driving; Binding; Biology; Biotin; Bleomycin; Cell Line; Cell Surface Receptors; Cells; Cessation of life; Chimeric Proteins; Cicatrix; Confocal Microscopy; Cytoplasm; Data; Diagnosis; Disease; Disease Pathway; Disease Progression; Endocytosis; Epithelial Cells; Essential Amino Acids; Fibrinolysis; Fibroblasts; Fibrosis; Foundations; Gene Deletion; Heart; Human; In Vitro; Incidence; Kidney; Knock-in Mouse; Label; Lesion; Ligands; Ligase; Liver; Lung; Lung diseases; Mediating; Mediator; Modeling; Molecular Biology Techniques; Morbidity - disease rate; Mus; Mutation; Organ; Pathway interactions; Patients; Phage Display; Pharmaceutical Preparations; Phenotype; Plasminogen Activator; Plasminogen Activator Inhibitor 1; Plasminogen Activator Interaction; Profibrotic signal; Protein Sortings; Proteins; Proteomics; Pulmonary Fibrosis; Regulation; Role; Severities; Shortness of Breath; Skin; Stimulus; Surface Plasmon Resonance; Testing; Therapeutic; Tissues; Transforming Growth Factor beta; Transgenic Mice; Urokinase; Vitronectin; alveolar epithelium; design; efficacious intervention; efficacious treatment; experience; experimental study; fibrogenesis; fibrotic lung; idiopathic pulmonary fibrosis; in vivo; inhibitor; lung development; lung injury; mortality; mosaic; mouse model; mutant; mutation screening; new therapeutic target; novel; overexpression; prevent; protein reconstitution; prototype; receptor; small molecule; sortilin; therapeutic target; uptake; validation studies; wound healing

Project Summary Progressive scarring of the lung interstitium is a common feature of many systemic and primary lung diseases including Idiopathic Pulmonary Fibrosis (IPF). IPF is a common disorder, and patients diagnosed with this disorder experience substantial morbidity and a median survival of 3-5 years. Although recently approved therapies slow the rate of disease progression, more efficacious interventions that interfere with precise disease mechanisms are desperately needed. Plasminogen activator inhibitor-1 (PAI-1) holds substantial promise as a therapeutic target for lung fibrosis as experiments in multiple complementary animal models reveal a direct correlation between the activity of this molecule and the severity of scarring. These studies substantiate PAI-1 as a critical down-stream mediator of prominent pro-fibrotic stimuli including TGF-β and matrix stiffness. Despite the large amount of data implicating PAI-1 as a critical pro-fibrotic protein, the mechanism by which it promotes fibrosis remains elusive. PAI-1 is a multifunctional protein with inhibitory activity against the plasminogen activators through which it regulates fibrinolysis and wound healing. PAI-1 also interacts with non-protease ligands including the provisional matrix protein vitronectin (VTN). Using mutant proteins lacking specific functions and transgenic mice, we demonstrated that the pro-fibrotic action of PAI-1 is largely independent of its plasminogen activator inhibitory activity and that vitronectin is not required for PAI-1 to fully promote scarring of the lung. These results led us to perform an unbiased proteomic study to identify novel PAI-1 binding partners within the injured lung. With this approach, we identified sortilin related receptor-1 (SorlA) as the most enriched protein. SorlA is a multi-domain receptor implicated in the uptake and intracellular sorting of proteins. Our preliminary data confirm that PAI-1 binds to SorlA and that this interaction is prevented by mutations that suppress the profibrotic activity of PAI-1. We further find that PAI-1 is taken up by key cellular constituents of the fibrotic lesion. Importantly, we also identify a previously unrecognized and potent role for SorlA in lung fibrosis, and using human cells, we find that SorlA expression is upregulated in fibrotic epithelial cells and fibroblasts. These preliminary data motivate our hypothesis that PAI-1 promotes lung fibrosis through an interaction with SorlA that leads to cell uptake, cytosolic localization, and a profibrotic alteration in cell phenotype. To interrogate this hypothesis, we have designed a multifaceted approach incorporating molecular biology techniques, in vitro experiments with cells from patients and mice (as well as cell lines), and in vivo experiments using transgenic mice, mutant PAI-1 proteins, and complementary models of lung fibrosis. To accomplish our proposed studies, we have brought together a synergistic investigative team with expertise in PAI-1, epithelial cell and fibroblast biology, and animal modeling, and the results from our studies promise to elucidate previously unexplored mechanisms and novel therapeutic targets.

项目摘要 进行性瘢痕形成的肺结节是一个共同的特点，许多系统性和原发性肺 疾病，包括特发性肺纤维化（IPF）。IPF是一种常见疾病，诊断为IPF的患者 这种疾病的发病率很高，中位生存期为3-5年。虽然最近批准 治疗减缓疾病进展的速度，更有效的干预措施，干扰精确的 疾病机制是迫切需要的。纤溶酶原激活物抑制剂-1（派-1）在细胞内具有显著的 有望作为肺纤维化的治疗靶点，在多种互补动物模型中进行实验 揭示了这种分子的活性与疤痕严重程度之间的直接相关性。这些研究 证实派-1是显著促纤维化刺激物（包括TGF-β和 基体刚度尽管大量数据表明派-1是一种关键的促纤维化蛋白， 它促进纤维化的机制仍然是难以捉摸的。 派-1是一种多功能蛋白质，通过抑制纤溶酶原激活剂， 它调节纤维蛋白溶解和伤口愈合。派-1还与非蛋白酶配体相互作用，包括 临时基质蛋白玻连蛋白（VTN）。利用缺乏特定功能的突变蛋白和转基因 我们证明派-1的促纤维化作用在很大程度上不依赖于其纤溶酶原激活剂 抑制活性，且派-1完全促进肺瘢痕形成不需要玻连蛋白。这些 结果使我们进行了一项无偏见的蛋白质组学研究，以确定新的派-1结合伙伴在受伤的 肺。通过这种方法，我们鉴定了分拣蛋白相关受体-1（SorlA）作为最富集的蛋白质。 SorlA是涉及蛋白质的摄取和细胞内分选的多结构域受体。我们 初步数据证实派-1与SorlA结合，这种相互作用被突变阻止， 抑制派-1的促纤维化活性。我们进一步发现派-1被细胞的关键成分摄取， 纤维化病变重要的是，我们还确定了SorlA在肺组织中以前未被认识到的有效作用。 使用人细胞，我们发现SorlA表达在纤维化上皮细胞中上调， 成纤维细胞这些初步的数据激发了我们的假设，即派-1通过抑制肺纤维化来促进肺纤维化。 与SorlA相互作用，导致细胞摄取、胞质定位和细胞内促纤维化改变 表型为了质疑这一假设，我们设计了一种多方面的方法， 生物学技术，用来自患者和小鼠的细胞（以及细胞系）进行的体外实验，以及体内实验， 使用转基因小鼠、突变派-1蛋白和肺纤维化的互补模型进行的实验。到 为了完成我们提出的研究，我们召集了一个具有以下专长的协同调查小组： 派-1、上皮细胞和成纤维细胞生物学以及动物模型，我们的研究结果有望 阐明以前未探索的机制和新的治疗靶点。