Transcriptomic and Proteomic Changes Driving Pulmonary Fibrosis Resolution in Young and Old Mice.

Bleomycin-induced pulmonary fibrosis in mice mimics major hallmarks of idiopathic pulmonary fibrosis. Yet in this model, it spontaneously resolves over time. We studied molecular mechanisms of fibrosis resolution and lung repair, focusing on transcriptional and proteomic signatures and the effect of aging. Old mice showed incomplete and delayed lung function recovery 8 weeks after bleomycin instillation. This shift in structural and functional repair in old bleomycin-treated mice was reflected in a temporal shift in gene and protein expression. We reveal gene signatures and signaling pathways that underpin the lung repair process. Importantly, the downregulation of WNT, BMP, and TGFß antagonists Frzb, Sfrp1, Dkk2, Grem1, Fst, Fstl1, and Inhba correlated with lung function improvement. Those genes constitute a network with functions in stem cell pathways, wound, and pulmonary healing. We suggest that insufficient and delayed downregulation of those antagonists during fibrosis resolution in old mice explains the impaired regenerative outcome. Together, we identified signaling pathway molecules with relevance to lung regeneration that should be tested in-depth experimentally as potential therapeutic targets for pulmonary fibrosis.

Olodaterol shows anti-fibrotic efficacy in in vitro and in vivo models of pulmonary fibrosis.

BACKGROUND AND PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by excessive fibroblast activation ultimately leading to scarring of the lungs. Although, the activation of ß2 -adrenoceptors (ß2 -AR) has been shown to inhibit pro-fibrotic events primarily in cell lines, the role of ß2 -adrenoceptor agonists has not yet been fully characterized. The aim of our study was to explore the anti-fibrotic activity of the long-acting ß2 -adrenoceptor agonist olodaterol in primary human lung fibroblasts (HLF) and in murine models of pulmonary fibrosis. EXPERIMENTAL APPROACH: We assessed the activity of olodaterol to inhibit various pro-fibrotic mechanisms, induced by different pro-fibrotic mediators, in primary HLF from control donors and patients with IPF (IPF-LF). The in vivo anti-fibrotic activity of olodaterol, given once daily by inhalation in either a preventive or therapeutic treatment regimen, was explored in murine models of lung fibrosis induced by either bleomycin or the overexpression of TGF-ß1. KEY RESULTS: In both HLF and IPF-LF, olodaterol attenuated TGF-ß-induced expression of α-smooth muscle actin, fibronectin and endothelin-1 (ET-1), FGF- and PDGF-induced motility and proliferation and TGF-ß/ET-1-induced contraction. In vivo olodaterol significantly attenuated the bleomycin-induced increase in lung weight, reduced bronchoalveolar lavage cell counts and inhibited release of pro-fibrotic mediators (TGF-ß, MMP-9 and tissue inhibitor of metalloproteinase-1). Forced vital capacity was increased only with the preventive treatment regimen. In the TGF-ß-overexpressing model, olodaterol additionally reduced the Col3A1 mRNA expression. CONCLUSION AND IMPLICATIONS: Olodaterol showed anti-fibrotic properties in primary HLF from control and IPF patients and in murine models of lung fibrosis.

The SK-N-MC cell line expresses an orexin binding site different from recombinant orexin 1-type receptor.

Orexin A and B (also known as hypocretins), two recently discovered neuropeptides, play an important role in food intake, sleep/wake cycle and neuroendocrine functions. Orexins are endogenous ligands of two G-protein-coupled receptors, termed OX1 and OX2. This work presents the first short orexin A and B analogues, orexin A 23-33 and orexin B 18-28, with high affinity (119 +/- 49 and 49 +/- 23 nm) for OX1 receptors expressed on SK-N-MC cells and indicates the importance of the C-terminal part of the orexin peptides for this ligand-receptor interaction. However, these C-terminal fragments of orexin did not displace the 125I-labelled orexin B from the recombinant orexin 1 receptor stably expressed in Chinese hamster ovary cells. To examine the role of the shortened orexin A 23-33 in feeding, its effects in mimicking or antagonizing the effects of orexin A were studied in rats after administration via the lateral hypothalamus. In contrast with orexin A, which potently induced feeding up to 4 h after administration, orexin A 23-33 neither induced feeding nor inhibited orexin A-induced feeding. Modafinil (Vigil), which was shown earlier to activate orexin neurons, displayed binding neither to the orexin receptor expressed on SK-N-MC cells nor to the recombinant orexin 1 receptor, which indicates that modafinil displays its antinarcoleptic action via another yet unknown mechanism. PCR and subsequent sequencing revealed expression of the full-length orexin 1 receptor mRNA in SK-N-MC and NT-2 cells. Interestingly, sequencing of several cDNA clones derived from RNA of both SK-N-MC and NT-2 cells differed from the published nucleotide sequence at position 1375. Amino acid prediction of this A -->G change results in an isoleucine --> valine substitution at the protein level, which may provide evidence for an editing process.