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.

项目概要 肺间质进行性疤痕形成是许多全身性和原发性肺病的共同特征 包括特发性肺纤维化（IPF）在内的疾病是一种常见疾病，患者被诊断患有这种疾病。 尽管最近获得批准，但这种疾病的发病率很高，中位生存期为 3-5 年。 治疗可以减缓疾病进展的速度，更有效的干预措施可以干扰精确的治疗 迫切需要纤溶酶原激活剂抑制剂-1 (PAI-1) 的疾病机制。 在多种互补动物模型中进行的实验有望成为肺纤维化的治疗靶点 这些研究揭示了该分子的活性与疤痕严重程度之间的直接相关性。 证实 PAI-1 是显着促纤维化刺激（包括 TGF-β 和 尽管大量数据表明 PAI-1 是一种关键的促纤维化蛋白，但 它促进纤维化的机制仍然难以捉摸。 PAI-1 是一种多功能蛋白，通过抑制纤溶酶原激活剂的活性 它调节纤维蛋白溶解和伤口愈合，还与非蛋白酶配体相互作用。 临时基质蛋白玻连蛋白（VTN）。使用缺乏特定功能和转基因的突变蛋白。 在小鼠中，我们证明 PAI-1 的促纤维化作用在很大程度上与其纤溶酶原激活剂无关 抑制活性，并且 PAI-1 不需要玻连蛋白即可完全促进肺部疤痕形成。 结果促使我们进行了一项公正的蛋白质组学研究，以确定受伤者体内新的 PAI-1 结合伙伴 通过这种方法，我们确定分拣蛋白相关受体 1 (SorlA) 是最富集的蛋白质。 SorlA 是一种多结构域受体，参与蛋白质的摄取和细胞内排序。 初步数据证实 PAI-1 与 SorlA 结合，并且这种相互作用被以下突变所阻止： 抑制 PAI-1 的促纤维化活性，我们进一步发现 PAI-1 被关键细胞成分吸收。 重要的是，我们还发现了 SorlA 在肺中以前未被认识到的有效作用。 纤维化，并且使用人类细胞，我们发现 SorlA 表达在纤维化上皮细胞中上调，并且 这些初步数据激发了我们的假设，即 PAI-1 通过一种机制促进肺纤维化。 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 为了质疑这一假设，我们设计了一种结合分子的多方面方法。 生物学技术、患者和小鼠细胞（以及细胞系）的体外实验以及体内实验 使用转基因小鼠、突变 PAI-1 蛋白和肺纤维化互补模型进行的实验。 为了完成我们提出的研究，我们聚集了一支具有以下专业知识的协同调查团队 PAI-1、上皮细胞和成纤维细胞生物学以及动物模型，以及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。