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

项目摘要/摘要 肺移植(LTX)是晚期肺部疾病患者的一种治疗选择。长期生存 然而，LTX后受限于慢性肺移植功能障碍(CLAD)。穿着最常见的货单 自身为闭塞性毛细支气管炎综合征(BOS)和约50%的受者(LTxRs)将在 LTX术后5年。上皮-间充质转化(EMT)和纤维化被认为与 BOS的发病机制。我们证明了肿瘤抑制基因肝脏激酶1(LKB1)是 免疫印迹和乙醛珠联用在BOS中下调，但在稳定的活检中不下调 用流式细胞术检测外切体。我们还证明了LKB1基因敲除后可诱导外切体释放。 呼吸道上皮细胞系BEAS-2B和另一株人呼吸道上皮细胞系HPBEC。释放的Exosome BOS还可诱导BEAS-2B和HPBEC中受LKB1调控的EMT。纳米线 分析证实，LKB1基因敲除可诱导人呼吸道上皮细胞中PDGFRβ的表达。我们也 证实BOSLTxR的活检组织降低了LKB1，增加了PDGFRβR。 相关性。这些新的发现表明肿瘤抑制基因LKB1在肺癌中的重要作用。 调节血小板衍生生长因子βR，因此，纤维化的发展。建议同时使用临床样本和 在体外细胞培养模型中，我们将确定外切体下调LKB1的机制 从移植肺中释放的物质参与了EMT导致包膜的过程。该提案的目标1是连续确定 已知危险因素的LTxR患者血浆中分离的外切体中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])可以作为一种非 LTxRs的侵入性生物标志物存在包裹体的风险。我们的假设是LKB1的持续下调在 Exosome将成为LTxRs的生物标志物，有可能发展为包膜。第二个目标是确定和 用LKB1/AMPK1对已知的LTxR连续回溯保存血浆中的外切体进行定量 临床诊断将是一个更敏感的标志，并确定其潜在的鉴别 限制性同种异体移植综合征和BOS通过定义它们的免疫学和分子特性。我们的第三个目标 目的是明确LKB1基因缺失导致EMT和PDGFRβ上调及促进的机制 CLAD的发病机制。为此；a)我们将定义抑制LKB1的调控机制 LTxRs与PGD、AR和RVI，包裹性心脏病的危险因素，以及b)我们将确定LKB1 下调导致PDGFRβ及其信号通路上调，从而促进 纤维化的发展。这些研究结果将为LKB1在EMT中的作用提供新的信息 与肝纤维化相关的病理改变，包括LTX后的包膜。

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.