Wednesday, April 24, 2024
4/24/2024
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 PDGFRß expression in human airway epithelial cells. We also demonstrated that biopsies from BOS LTxRs had reduced LKB1 and increased PDGFßR with inverse correlation. These novel findings indicate an important role for the tumor suppressor gene LKB1 in the regulation of PDGFßR 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 PDGFRß 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 PDGFRß 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.
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