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A2915 - The Fibrotic Extracellular Matrix Induces Exosomal Pro-Fibrotic miRNA Production of Fibrocytes
Author Block: S. Sato, C. B. Upagupta, T. Yanagihara, M. R. Kolb; Firestone Institute for Respiratory Health, McMaster University, Hamilton, ON, Canada.
[Rationale] Exosomes are small lipid vesicles, and the exosomal miRNAs are considered important for biological processes and cell-to-cell communication. Fibrocytes are circulating bone marrow-derived cells that are reported to migrate into the injured lungs, and contribute to fibrogenesis. We have previously reported that the expression of miR-21-5p, which was reported as a pro-fibrotic miRNA, was increased in fibrocytes from fibrotic lungs compared with those from normal lungs. Furthermore, we also reported that fibroblasts uptake more fibrocyte-derived exosomes when compared to fibroblast-derived exosomes (AJRCCM 2017;195:A7566). In this study, we examined the effect of the extracellular matrix (ECM) of fibrotic lungs on the expression of miR-21-5p in fibrocytes. [Methods] Pulmonary fibrosis was induced in rats by intratracheal administration of an adenoviral gene vector encoding active TGF-β1 or control vector. Primary rat fibrocytes and fibroblasts were cultured from rat lungs, and were sorted by anti-CD45 magnetic beads. Human mononuculear cells isolated from peripheral blood of healthy donors were cultured for seven days on fibronectin-coated dish, and the adherent cells were used in the following experiments as human fibrocytes. Fibrocytes were cultured on different stiffness plates (1, 50, 107kPa), and were also cultured in decellularized rat lung scaffolds generated from fibrotic lung and control lung. We also determined whether or not dissolved ECM composition, recombinant TGF-β1, and nintedanib affect the cellular and exosomal miRNA expression of fibrocytes. Exosomal miRNAs were extracted by using two commercial kits (Invitrogen total exosome isolation kit, and Invitrogen total exosome RNA isolation kit). [Results] The exosomes of fibrocytes derived from fibrotic rat lung significantly upregulated the expression of col1a1 of fibroblasts. However, the exosomes of fibrocytes derived from control rat lungs and those derived from fibrotic fibrocytes pretreated with miR-21 inhibitor showed no similar effect. Culturing on 107kPa plate or fibrotic decellularized lung scaffolds increased miR-21-5p expression compared with 1kPa plate or normal lung scaffolds in both cellular and exosomal expression levels. Dissolved ECM composition collected from fibrotic lung and recombinant TGF-β1 increased miR-21-5p expression on fibrocytes, but these effects were attenuated when cells were cultured on the 1kPa plate. Nintedanib downregulated miR-21-5p expression of fibrocytes in a dose dependent manner. These results were observed on both human and rat fibrocytes. [Conclusions] Our results indicate that ECM plays a crucial role in fibrogenesis through their effect on miRNA expression within fibrocytes, and may serve as a potential target for the treatment of pulmonary fibrosis.