title: Downregulation of NOX4 expression by roflumilast N-oxide reduces markers of fibrosis in lung fibroblasts.
abstract: The phosphodiesterase 4 inhibitor roflumilast prevents bleomycin- (BLM-) induced lung fibrosis in animal models. However, its mechanism of action remains unknown. We investigated whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, can modulate in vitro the oxidative effects of BLM on human lung fibroblasts (HLF). In addition, since BLM increases the production of F₂-isoprostanes that have per se fibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F₂-isoprostane 8-epi-PGF₂α was investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF₂α. Proliferation and collagen synthesis were assessed as [(3)H]-thymidine and [(3)H]-proline incorporation. Reactive oxygen species (ROS) and F₂-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLM increased both cell proliferation and collagen synthesis and enhanced ROS and F₂-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF₂α   per se stimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antifibrotic effect of RNO observed in vivo may be related to its ability to mitigate ROS generation via downregulation of NOX4.
authors.affiliation: Department of Dermatology, Harvard Medical School, Wellman Center for Photomedicine, Massachusetts General Hospital, 40 Blossom Street, Boston, MA 02114, USA.
authors.forename: Daniela
authors.initials: D
authors.lastname: Vecchio
authors.forename: Alessandra
authors.initials: A
authors.lastname: Acquaviva
authors.forename: Beatrice
authors.initials: B
authors.lastname: Arezzini
authors.forename: Hermann
authors.initials: H
authors.lastname: Tenor
authors.forename: Piero A
authors.initials: PA
authors.lastname: Martorana
authors.forename: Concetta
authors.initials: C
authors.lastname: Gardi
mesh_terms: D000631:Aminopyridines / Q000494:pharmacology*; D001549:Benzamides / Q000494:pharmacology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D003521:Cyclopropanes / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D015237:Dinoprost / Q000031:analogs & derivatives / Q000378:metabolism; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005355:Fibrosis; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008168:Lung / Q000166:cytology* / Q000473:pathology; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000631:Aminopyridines; D001549:Benzamides; D003521:Cyclopropanes; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; C075750:8-epi-prostaglandin F2alpha; D003094:Collagen; D004247:DNA; D015237:Dinoprost; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C517734:roflumilast N-oxide; D010100:Oxygen
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