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. 2023 Sep 15;208(10):1115–1125. doi: 10.1164/rccm.202303-0471OC

Figure 2.

Figure 2.

Extracellular vesicle (EV)-driven damage is mediated by neutrophil elastase (NE) and matrix metalloproteinase 12 (MMP12). (A) Quantification of EV surface expression of neutrophil elastase (NE) using bead-based flow cytometry. Representative histograms shown are pooled airway EVs acquired from mice exposed to air or cigarette smoking (CS) for 3-months. (B) Analysis of NE activity of CS-exposed and air-exposed EVs using a NE-specific fluorescence resonance energy transfer (FRET) assay. Data are representative of 2 or more independent FRET assays. (C) Summary of our model of EV acquisition and transfer. Airway EVs from Wild-type or NE-deficient (NEKO) mice receiving cigarette CS for 1 month were harvested, purified, and transferred intratracheally to syngeneic, Wild-type BL/6 mice. Mice were treated with one dose of EVs (107) and their lung damage was quantified one-week post-treatment. (n = 5 per group). (D) Cumulative Mean Linear Intercept (Lm) scoring following transfer of Wild-type or NEKO EVs was determined as a means of quantifying alveolar enlargement. (n = 5 per group). (E) Representative images (hematoxylin and eosin) one week following EV transfer. Scale bars: 100 μm. (F) Quantification of EV surface expression of MMP12 using bead-based flow cytometry. Representative histograms shown are pooled airway EVs acquired from mice exposed to air or CS for 3-months (G) Analysis of MMP12 activity of CS-exposed and air-exposed EVs using an MMP12-specific FRET assay. Data are representative of 2 or more independent FRET assays. (H) Cumulative Lm scoring following transfer of NEKO EVs was determined as a means of quantifying alveolar enlargement. For some conditions, EVs were pretreated with varying concentrations of an MMP12 inhibitor (MMP408) (n ⩾ 5 per group). (I) Representative Images (hematoxylin and eosin) one week following EV transfer. Scale bars, 100 μm. The quantified results are expressed in terms of mean ± SEM. Statistical significance was determined by Mann-Whitney test for all comparisons except in (H), where the Kruskal-Wallis test was used. **P < 0.01, *P < 0.05. ns = not significant.