Figure 4. Fibroblast-specific deletion of α₆ protects mice against bleomycin injury-induced experimental lung fibrosis.

(a) Time-dependent deletion of α₆-expression in lung fibroblasts in conditional α₆^−/− mice following tamoxifen (Tam) administration. Levels of α₆-protein in cell lysates and on the cell surface were determined by immunoblot and flow cytometry. (b) Schematic shows the design of animal experiments. (c) Frozen lung tissue sections from bleomycin-treated mice were double-stained for α₆ (green) and αSMA (red). Nuclei were stained by DAPI (blue). Confocal immunofluorescent images were overlaid to show α₆-expression in αSMA-positive lung myofibroblasts. Epithelial α₆-expression in tam-treated mice was shown in the inset. Scale bar, 20 μm. (d) Representative images for trichrome staining of collagens in paraffin-embedded lung tissue sections. Scale bar, 150 μm. (e) Quantification of hydroxyproline contents in right lungs of mice from four mouse groups: Sal+C.O., Sal+Tam, Bleo+C.O. and Bleo+Tam. (f) Quantification of fibronectin (FN) and αSMA protein expression in left lungs by immunoblot. Shown are representative blots. (g) Shown are representative images for ex vivo mid-lung transaxial μCT scans. The average percentages of aerated lung volumes of mice in four groups (n=5 per group) are shown in the bar graph. (h) Immunohistochemical staining of two adjacent lung sections shows Mmp-2 expression in the areas of αSMA-expressing lung myofibroblasts. Nuclei were stained by hematoxylin (blue). Scale bar, 100 μm. (i) Frozen lung tissue sections were stained for laminin (green) (a component of the BMs) and αSMA (red). Nuclei were stained by DAPI (blue). Inset shows laminin and αSMA staining in the relatively normal area of the same lung section. Scale bar, 20 μm. (j) Lung (myo)fibroblasts (FB and MFB) were isolated from mice in four groups. The ability of (M)FBs to invade the BM matrices was determined by invasion assay. Results are the means±s.d. of three separate experiments, each performed in triplicates; *P<0.05 and **P<0.01; one-way analysis of variance. Bleo, bleomycin; C.O., corn oil; Sal, saline.