Figure 1. EVs accumulate in lung fibrosis, initiate lung remodeling, and impair alveolar epithelial cell function.

(A) C57BL/6J mice were exposed to orotracheal bleomycin or NaCl (control). Lung function was assessed and lung tissue and BALF were collected over the indicated time course. EVs were concentrated from BALF and characterized. BLM, bleomycin. (B) Quasistatic compliance and (C) hydroxyproline level of the corresponding experiments are shown. (D) BALF-EVs were observed by electron microscopy (scale bars indicate 600 nm) and (E) numbered by Nanosight (data expressed as number of particles per BALF). (F) EV quantification according to particle diameters (expressed in nm) at each time point after bleomycin exposure (mean ± SD). (G) Correlation between EV number and quasistatic compliance is depicted. (B–G) Each point corresponds to a mouse (n = 5–8 for NaCl groups, n = 13–20 for BLM groups). (H) BALF-EVs were isolated from mice with pulmonary fibrosis (14 days after bleomycin) or control mice and used for functional assays. (I and J) PCLS from normal C57BL/6J were cultured with the abovementioned BALF-EVs. After 7 days, the expression of fibrosis-related genes was assessed by qPCR. Data are representative of PCLS from individual mice exposed to control- (n = 4 PCLS) or fibrotic EVs (n = 6 PCLS). Gene expression was normalized to Hprt expression. (K) Murine EpCAM-positive cells and CCL-206 fibroblasts in Matrigel were exposed to BALF-EVs for 14 days. Representative images (left panel, scale bar = 1 mm or 500 μm for region of interest [ROI] zoom) and quantification (right panel, n = 4 control EVs, n = 4 fibrotic EVs) of the organoid formation efficiency. Statistical analysis by Kruskal-Wallis followed by Dunn’s multiple comparisons tests (B–D), Spearman’s correlation test (G), or nonparametric Mann-Whitney test (I–K). P values are indicated for each comparison.