Abstract
Alterations in microRNA (miRNA) expression may contribute to COPD pathogenesis. In COPD, lung fibroblast repair functions are altered in multiple ways, including extracellular mediator release. Our prior study revealed miR-503 expression is decreased in COPD lung fibroblasts, although the exact role played by miR-503 is undetermined. The current study examined a role of miR-503 in cytokine, growth factor and fibronectin production by lung fibroblasts from patients with and without COPD. Primary adult lung fibroblasts were isolated from patients with or without COPD. MiR-503 expression and interleukin (IL)-6, -8, PGE2, HGF, KGF, VEGF and fibronectin release were examined with or without inflammatory cytokines, IL-1β and tumor necrosis factor (TNF)-α. MiR-503 expression was decreased in COPD lung fibroblasts. The expression of miR-503 was positively correlated with %FVC, %FEV1, and %DLco as well as IL-6, -8, PGE2, HGF, KGF, and VEGF in the absence or presence of IL-1ß/TNF-α. In addition, IL-8 and VEGF release from COPD lung fibroblasts were increased compared to those from control. Exogenous miR-503 inhibited VEGF release from primary adult and fetal lung fibroblasts but not IL-8 release. As expected, COPD fibroblasts proliferated more slowly than control fibroblasts. MiR-503 did not affect proliferation of either control or COPD lung fibroblasts. MiR-503 inhibition of VEGF protein production and mRNA was mediated by direct binding to the 3’ untranslated region of VEGF mRNA. Endogenous miR-503 was differently regulated by exogenous stimulants associated with COPD pathogenesis, including IL-1ß/TNF-α, TGF-ß1 and PGE2. Endogenous miR-503 inhibition augmented VEGF release by IL-1ß/TNF-α and TGF-ß1 but not by PGE2, demonstrating selectivity of miR-503 regulation of VEGF. In conclusions, reduced miR-503 augments VEGF release from lung fibroblasts from patients with COPD. Since VEGF contributes to disturbed vasculature in COPD, altered miR-503 production might play a role in modulating fibroblast-mediated vascular homeostasis in COPD.
Highlights
Chronic obstructive pulmonary disease (COPD) was projected to be the third leading cause of death worldwide by 2020 [1], but achieved this distinction several years early [WHO Factsheet
We further examined associations between miR-503 expression and fibroblast production of IL-6, -8, prostaglandin (PG)E2, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF) and fibronectin, all of which have been suggested to contribute to COPD pathophysiology [8, 11, 18,19,20,21]
MO, USA); hsa-miRNA-503 Mimic, hsa-miRNA-503 Inhibitor and microRNA Negative Control (25 nM) were from Dharmacon (Lafayette, CO, USA); anti-human VEGF antibody (AF293-NA), biotinylated anti-human VEGF antibody (BAF293), anti-human IL-8 antibody (MAB208), biotinylated anti-human IL-8 antibody (BAF208), recombinant human TGF-ß1, IL-1ß, tumor necrosis factor (TNF)-α, Human IL-6 DuoSet ELISA kit, Human HGF DuoSet ELISA kit, Human and KGF/FGF-7 DuoSet ELISA were from R&D Systems (Minneapolis, MN, USA); horseradish peroxidase (HRP)-streptavidin conjugate was from Zymed Laboratories (South San Francisco, CA, USA); and Prostaglandin E2 (PGE2) assay kit (EIA) was from Cayman (Ann Arbor, MI, USA)
Summary
Chronic obstructive pulmonary disease (COPD) was projected to be the third leading cause of death worldwide by 2020 [1], but achieved this distinction several years early [WHO Factsheet. A principal cause of COPD is smoking that induces chronic inflammation, tissue destruction and inhibits tissue repair in the lungs [2]. Lung fibroblasts compose 20–40% of alveolar cells and contribute to the repair, maintenance and integration of lung structures [4,5,6,7]. In COPD, functions of lung fibroblasts are altered in multiple ways [8,9,10,11] and altered lung fibroblast production of inflammatory cytokines, growth factors and fibronectin could contribute the pathogenesis of COPD [6]
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