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Administration of bone marrow-derived human mesenchymal stem cells (hMSC) reduces lung inflammation, fibrosis, and mortality in animal models of lung injury, by a mechanism not completely understood. We investigated whether hMSC would prevent epithelial-mesenchymal transition (EMT) induced by hypoxia in primary rat alveolar epithelial cell (AEC). In AEC cultured on semipermeable filters, prolonged hypoxic exposure (1.5% O2 for up to 12 days) induced phenotypic changes consistent with EMT, i.e., a change in cell morphology, a decrease in transepithelial resistance (Rte) and in the expression of epithelial markers [zonula occludens-1 (ZO-1), E-cadherin, AQP-5, TTF-1], together with an increase in mesenchymal markers [vimentin, α-smooth muscle actin (α-SMA)]. Expression of transcription factors driving EMT such as SNAIL1, ZEB1, and TWIST1 increased after 2, 24, and 48 h of hypoxia, respectively. Hypoxia also induced TGF-β1 mRNA expression and the secretion of active TGF-β1 in apical medium, and the expression of connective tissue growth factor (CTGF), two inducers of EMT. Coculture of AEC with hMSC partially prevented the decrease in Rte and in ZO-1, E-cadherin, and TTF-1 expression, and the increase in vimentin expression induced by hypoxia. It also abolished the increase in TGF-β1 expression and in TGF-β1-induced genes ZEB1, TWIST1, and CTGF. Finally, incubation with human recombinant KGF at a concentration similar to what was measured in hMSC-conditioned media restored the expression of TTF-1 and prevented the increase in TWIST1, TGF-β1, and CTGF in hypoxic AEC. Our results indicate that hMSC prevent hypoxiainduced alveolar EMT through the paracrine modulation of EMT signaling pathways and suggest that this effect is partly mediated by KGF. [ABSTRACT FROM AUTHOR]

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal interstitial lung disease of unknown origin. Alveolar epithelial cells (AECs) play an important role in the fibrotic process as they undergo sustained endoplasmic reticulum (ER) stress, and may acquire a mesenchymal phenotype through epithelial-to-mesenchymal transition (EMT), two phenomena that could be induced by localized alveolar hypoxia. Here we investigated the potential links between hypoxia, ER stress and EMT in AECs. Methods: ER stress and EMT markers were assessed by immunohistochemistry, western blot and qPCR analysis, both in vivo in rat lungs exposed to normoxia or hypoxia (equivalent to 8% O2) for 48 h, and in vitro in primary rat AECs exposed to normoxia or hypoxia (1.5% O2) for 2–6 days. Results: Hypoxia induced expression of mesenchymal markers, pro-EMT transcription factors, and the activation of ER stress markers both in vivo in rat lungs, and in vitro in AECs. In vitro, pharmacological inhibition of ER stress by 4-PBA limited hypoxia-induced EMT. Calcium chelation or hypoxia-inducible factor (HIF) inhibition also prevented EMT induction under hypoxic condition. Conclusions: Hypoxia and intracellular calcium are both involved in EMT induction of AECs, mainly through the activation of ER stress and HIF signaling pathways. [ABSTRACT FROM AUTHOR]