Detection of epithelial to mesenchymal transition in airways of a bleomycin induced pulmonary fibrosis model derived from an α-smooth muscle actin-Cre transgenic mouse

observed in patients suffering interstitial pulmonary fibrosis. In vitro studies have also demonstrated that AECs could convert into myofibroblasts following exposure to TGF-beta 1. In this study, we examined whether EMT occurs in bleomycin ( BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells ( BECs) in the EMT. Using an alpha-smooth muscle actin-Cre transgenic mouse (alpha- SMA- Cre/ R26R) strain, we labelled myofibroblasts in vivo. We also performed a phenotypic analysis of human BEC lines during TGF-beta 1 stimulation in vitro. Methods: We generated the alpha-SMA-Cre mouse strain by pronuclear microinjection with a Cre recombinase cDNA driven by the mouse a- smooth muscle actin (alpha-SMA) promoter. alpha-SMA-Cre mice were crossed with the Cre-dependent LacZ expressing strain R26R to produce the double transgenic strain alpha-SMA-Cre/R26R. beta-galactosidase (beta gal) staining, alpha-SMA and smooth muscle myosin heavy chains immunostaining were carried out simultaneously to confirm the specificity of expression of the transgenic reporter within smooth muscle cells ( SMCs) under physiological conditions. BLM-induced peribronchial fibrosis in alpha-SMA-Cre/R26R mice was examined by pulmonary beta gal staining and alpha-SMA immunofluorescence staining. To confirm in vivo observations of BECs undergoing EMT, we stimulated human BEC line 16HBE with TGF-beta 1 and examined the localization of the myofibroblast markers alpha-SMA and F-actin, and the epithelial marker E- cadherin by immunofluorescence. Results: beta gal staining in organs of healthy alpha-SMA-Cre/R26R mice corresponded with the distribution of SMCs, as confirmed by alpha-SMA and SM-MHC immunostaining. BLM-treated mice showed significantly enhanced beta gal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some AECs in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by a- SMA immunostaining. In vitro, addition of TGF-beta 1 to 16HBE cells could also stimulate the expression of alpha-SMA and F-actin, while E- cadherin was decreased, consistent with an EMT. Conclusion: We observed airway EMT in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis.