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A2900 - Exosome Mediated Signaling of Fibroblast Invasion Is Dependent on Surface Expression of Fibronectin
Author Block: D. Chanda1, K. P. Hough1, E. Otoupalova1, M. L. Locy1, J. Deshane1, R. D. Sanderson2, V. J. Thannickal1; 1Medicine, University of Alabama at Birmingham, Birmingham, AL, United States, 2Pathology, University of Alabama at Birmingham, Birmingham, AL, United States.
Rationale
Exosomes are nanosize vesicles derived from endocytosis and released extracellularly. Exosome cargo primarily include nucleic acid, protein and lipid mediators. Recent evidence indicates a role for exosomes in cellular homeostasis, modification of extracellular matrix (ECM), and intercellular communication. Fibroblast migration and invasion are critical to tissue injury repair, including the lung. Fibronectin (FN) is a major ECM protein, and acts as a chemoattractant for fibroblast invasion. In this study, we investigated whether FN expressed on the surface of human lung fibroblasts-derived exosomes mediates fibroblast invasion.
Methods
Exosomes were purified from the culture medium of human lung fibroblasts (IMR90) by differential centrifugation. Size and concentration of purified vesicles were determined by Nanoparticle Tracking Analysis using NanoSight NS300 (Malvern Instruments). An exosome peptide library was created by SWATH-MS (SCIEX). Exosome surface expression of FN was determined by flow cytometry (BD LSR II), and Image-Stream Mark II imaging flow cytometry (EMD Millipore). Fibroblast invasion assay was performed in Matrigel (Corning) Trans-Well invasion chambers.
Results
Seven hundred eighteen unique peptides were detected in the exosomes by SWATH-MS, including the principal exosome markers CD9, CD63, CD81, and HSP70. Presence of FN was abundant in the IMR90 fibroblasts-derived exosomes. Exosomes were fluorescently dual-labeled with antibodies against FN (PE), and exosome marker CD63 (eFluor450) and analyzed by flow cytometry to validate surface expression of FN. To demonstrate the role of exosomes in fibroblast invasion, exosomes were seeded in matrigel prior to fibroblast invasion. Results indicated an exosome dose-dependent invasion of fibroblasts. When exosomes were pre-treated with FN antibody before seeding in matrigel, fibroblast invasion was inhibited. Senescent lung fibroblasts secreted significantly higher number of exosomes compared to non-senescent fibroblasts, and the majority of exosomes were trapped in the ECM. Similar findings were noted in fibroblasts obtained from human subjects with idiopathic pulmonary fibrosis (IPF), and non-senescent fibroblasts treated with TGF-β1.
Conclusions
This study demonstrates a role of exosomes in cell non-autonomous fibroblast invasion. We identified FN on the surface of exosomes as a critical mediator of fibroblast invasion. Additionally, we found a higher concentration of exosomes that are bound to the ECM of senescent lung fibroblasts. These findings provide potential links between the invasive phenotype of fibroblasts and age-related predisposition to fibrosis.