Chronic Lung Allograft Dysfunction: Role for Tumor Suppressor LKB1 in Exosomes

慢性肺同种异体移植功能障碍：肿瘤抑制因子 LKB1 在外泌体中的作用

基本信息

批准号：
10516866
负责人：
THALACHALLOUR MOHANAKUMAR
金额：
$ 40.7万
依托单位：
ST. JOSEPH'S HOSPITAL AND MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-15 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10516866
关键词：
5&apos -AMP-activated protein kinase Acute Aldehydes Allografting Automobile Driving Binding Biological Markers Biological Models Biopsy Bronchiolitis Obliterans CD80 gene CD86 gene CDC37 gene Cell Culture Techniques Cell Line Cells Chronic Clinical Cyclic AMP-Dependent Protein Kinases Data Development Diagnosis Down-Regulation Early identification Epithelial Epithelial Cells Family Fibrosis Flow Cytometry Functional disorder Genetic Transcription Goals Heat-Shock Proteins 90 Human Immunologics In Vitro Institutes Intervention Liver Lung Lung Transplantation Lung diseases MHC Class II Genes Mediating Mesenchymal Metabolism Methods Modeling Molecular Morphology Pathogenesis Pathology Pathway interactions Patients Phosphorylation Phosphotransferases Plasma Platelet-Derived Growth Factor Receptor Play Property Prostaglandin D2 Protein Kinase Receptor Signaling Regulation Reporting Risk Risk Factors Role STK11 gene Sampling Signal Pathway Signal Transduction Syndrome TNFRSF5 gene Testing Therapeutic Transforming Growth Factor beta Transplant Recipients Transplantation Tumor Suppressor Genes Tumor Suppressor Proteins Up-Regulation Viral Respiratory Tract Infection Western Blotting airway epithelium base cell growth clinical diagnosis donor-specific antibody epithelial to mesenchymal transition exosome graft dysfunction high risk in vitro Model knock-down liver function lung allograft multicatalytic endopeptidase complex nano-string novel prevent primary endpoint receptor expression receptor upregulation secondary endpoint statistics therapeutically effective transcription factor

项目摘要

Project Summary/Abstract Lung transplantation (LTx) is a therapeutic option for patients with advanced lung diseases. Long‐term survival after LTx is, however, limited by chronic lung allograft dysfunction (CLAD). CLAD most commonly manifests itself as bronchiolitis obliterans syndrome (BOS) and about 50% of recipients (LTxRs) will develop BOS within 5 years post-LTx. Epithelial-mesenchymal-transition (EMT) and fibrosis have been implicated in the pathogenesis of BOS. We demonstrated that liver kinase 1 (LKB1), a tumor suppressor gene, is downregulated in BOS but not in stable biopsies using both western blotting and aldehyde bead conjugated exosomes by flow cytometry. We also demonstrated that LKB1 knockdown induces exosome release from airway epithelial cell line, BEAS-2B, and another human airway epithelial cell line, HPBEC. Exosomes released from LTxRs with BOS also induces EMT which was regulated by LKB1 in BEAS-2B and HPBEC. NanoString analyses identified LKB1 knockdown induced PDGFRβ expression in human airway epithelial cells. We also demonstrated that biopsies from BOS LTxRs had reduced LKB1 and increased PDGFβR with inverse correlation. These novel findings indicate an important role for the tumor suppressor gene LKB1 in the regulation of PDGFβR and, therefore, fibrosis development. Studies proposed using both clinical samples and in vitro cell culture model, we will define the mechanism by which exosomes with downregulated LKB1 released from transplanted lungs mediate EMT leading to CLAD. Aim 1 of the proposal is to determine serially whether inactivation of LKB1 in exosomes isolated from plasma from LTxRs with known risk factors (primary graft dysfunction [PGD]), acute rejection [AR] and respiratory viral infections [RVI]) can be useful as a non- invasive biomarker for LTxRs at risk for CLAD. Our hypothesis is that persistent downregulation of LKB1 in exosomes will be a biomarker for LTxRs at risk for developing CLAD. The second goal is to determine and quantitate exosomes with LKB1/AMPK1 using serial retrospectively stored plasma from LTxRs with known clinical diagnosis will be a more sensitive marker for CLAD and to determine its potential to differentiate restrictive allograft syndrome and BOS by defining their immunological and molecular properties. Our third goal is to define the mechanisms by which loss of LKB1 results in EMT and upregulation of PDGFRβ and promotes the pathogenesis of CLAD. Towards this; a) we will define the regulatory mechanisms suppressing LKB1 in LTxRs with PGD, AR and RVI, risk factors for CLAD, and b) we will determine the mechanisms by which LKB1 downregulation leads to upregulation of PDGFRβ and its signaling pathways which contributes towards development of fibrosis. Results from these studies will provide novel information for the role of LKB1, in EMT and fibrosis related pathologies including CLAD following LTx.

项目概要/摘要肺移植（LTx）是晚期肺病患者的一种治疗选择。然而，LTx 后最常见的表现是慢性肺同种异体移植功能障碍 (CLAD)。本身就是闭塞性细支气管炎综合征 (BOS)，大约 50% 的接受者 (LTxR) 会在体内出现 BOS LTx 后 5 年，上皮间质转化 (EMT) 和纤维化与此有关。我们证明了肝激酶 1 (LKB1)（一种肿瘤抑制基因）是 BOS 的发病机制。在 BOS 中下调，但在使用蛋白质印迹和醛珠缀合的稳定活检中未下调我们还通过流式细胞术证明了 LKB1 敲除诱导外泌体释放。气道上皮细胞系 BEAS-2B 和另一种人类气道上皮细胞系 HPBEC 释放。来自带有 BOS 的 LTxR 也诱导 EMT，这在 BEAS-2B 和 HPBEC 中受 LKB1 调节。分析发现，LKB1 敲低可诱导人气道上皮细胞中的 PDGFRβ 表达。证明 BOS LTxR 的活检减少了 LKB1 并增加了 PDGFβR，且呈反比这些新发现表明抑癌基因 LKB1 在肿瘤细胞中发挥着重要作用。 PDGFβR 的调节以及纤维化发展的研究建议使用临床样本和在体外细胞培养模型中，我们将定义 LKB1 下调的外泌体的机制移植肺释放的 EMT 介导导致 CLAD 该提案的目标 1 是连续确定。从具有已知危险因素的 LTxR 血浆中分离的外泌体中 LKB1 是否失活（主要移植物功能障碍 [PGD]）、急性排斥反应 [AR] 和呼吸道病毒感染 [RVI]）可用作非 LTxR 的侵入性生物标志物面临 CLAD 的风险，我们的假设是 LKB1 的持续下调。外泌体将成为有 CLAD 风险的 LTxR 的生物标志物。使用已知的 LTxR 连续回顾性储存的血浆，用 LKB1/AMPK1 定量外泌体临床诊断将成为 CLAD 更敏感的标志物，并确定其区分的潜力我们的第三个目标是通过定义其免疫学和分子特性来识别限制性同种异体移植综合征和 BOS。旨在明确 LKB1 缺失导致 EMT 和 PDGFRβ 上调并促进为此，我们将定义抑制 LKB1 的调控机制。 LTxR 与 PGD、AR 和 RVI、CLAD 的风险因素，以及 b) 我们将确定 LKB1 的机制下调导致 PDGFRβ 及其信号通路上调，从而有助于这些研究的结果将为 LKB1 在 EMT 中的作用提供新的信息。以及纤维化相关病理，包括 LTx 后的 CLAD。