(A) EMAPII detected by Western blot in BALF (24 hours) and lung lysates after induction with doxycycline (3 weeks); vinculin was used as loading control. Lanes 1, 2, and 3 represent independent lines from either ST (CCSP-rtTA) or DT (EMAPII/CCSP-rtTA) mice. (B) Schematic of transgenic induction (top). EMAPII expression detected in lung cryosections by specific EMAPII polyclonal antiserum immunostaining (green) and DAPI (nuclei, blue) after transgenic induction in ST versus DT mice (3 weeks) (bottom). Note marked alveolar EMAPII staining (arrows) in the DT mice. Scale bar: 50 μm. (C) Caspase-3 activity in lung lysates of ST or DT mice (4 weeks of induction; *P < 0.05, ANOVA). (D) Macrophage counts in the BALF of ST mice or DT mice (6 months; mean + SEM; *P < 0.05, Student’s t test). AM, alveolar macrophage. (E) Representative photomicrographs of Richardson-stained glycol methacrylate lung sections (scale bar: 300 μm) of non–EMAPII-expressing ST CCSP-rtTA (STc) or ST EMAPII (STe) mice compared with EMAPII/CCSP-rtTA mice (DT) after 3 and 6 months of induction, respectively. Note the increase of airspace diameters, consistent with emphysema-like simplification of alveolar structures in DT mice but not in control mice. (F) Morphometry of H&E-stained lungs from ST and DT mice induced for 6 months. Acinar airspace size was expressed as MLI (*P = 0.01, ANOVA; n = 5–6 at 3 months; n = 7–8 at 6 months). (C and F) Lines within the boxes show medians; bounds of the boxes show 25th and the 75th percentiles of the data, respectively; and whiskers show outliers, if applicable (5th and 95th percentiles, respectively).

(A and B) EMAPII expression after CS exposure. (A) Production of EMAPII (pro and mature forms) in lung lysates in mice exposed to CS (4 weeks) compared with that in control mice exposed to ambient air (air control [AC]), assessed by Western blot (mean densitometry units [DUs] normalized to vinculin ± SEM; *P < 0.05 versus control; n = 5/group). (B) EMAPII localization in the lung parenchyma detected by coimmunofluorescence with EMAPII antiserum (green), CD11b antibody (red), and DAPI (blue). In controls, EMAPII expression is sparse, and EMAPII colocalized with macrophages (arrowheads) (left panel). After CS exposure (2 months), EMAPII expression is increased both cellularly and extracellularly, less prominently colocalized with macrophages (arrowheads, middle), and increasingly expressed in parenchyma cells (arrows; right). Scale bar: 100 μm. (C–F) Effect of neutralizing EMAPII (E-AB) or control antibody (IgG) administered by nebulization (50 μg/100 μl) during month 3 of CS exposure on CS-induced lung injury at 4 months. (C) Treatment protocol. (D) Apoptosis detected by caspase-3 activity in lung lysates (caspase units normalized by protein; mean + SEM; *P < 0.05, ANOVA). (E) Number of cells in BALF, and (F) lung static compliance (mean + SEM; *P < 0.01, ANOVA). (G) Representative H&E-stained lung sections (scale bar: 100 μm) showing simplification of lung alveolar structures in response to CS but preserved alveolar architecture when treated with neutralizing EMAPII. (H) Morphometric measurement of MLIs (mean + SEM; *P < 0.05, ANOVA; n = 5–12).

(A) Immunoblot of lung EMAPII 4 weeks after VEGF receptor inhibitor treatment (VEGFR-inh, 20 mg/kg), (B) quantified as densitometry units normalized to vinculin (mean ± SEM; *P < 0.05 versus control; n = 3/group). Ctrl, control. (C) Lung EMAPII production following active caspase-3 (Casp-3) i.t. (0.8 U; 48 hours) or vehicle treatment (mean densitometry units ± SEM; *P < 0.05 versus control; n = 4/group). (D) Lung caspase-3 activity after recombinant mature EMAPII nebulization (EMAPII i.t.; 50 μg/100 μl; 2 weeks) and caspase inhibitor (Q-VD-OPH, 25 μg/100 μl i.p.) or vehicle (DMSO) treatment, relative to that of controls (saline i.t.). Mean + SEM. *P < 0.05 versus control; ^#P < 0.05 versus EMAPII plus vehicle; n = 4; ANOVA. (E) Lung EMAPII (proform) production in lung-specific mature EMAPII-overexpressing mice (DT) treated with vehicle or caspase inhibitor 3 times weekly (8 weeks; mean + SEM; *P < 0.05 versus vehicle; n = 4; t test). (F and G) Synergistic effect of EMAPII and CS on lung in EMAPII DT or control (ST) mice induced for 12 weeks, of which the last 4 weeks were combined with CS exposure. (F) EMAPII (mean densitometry units + SEM) and (G) ceramide 16:0 expression normalized by lipid phosphorus (Pi). *P < 0.01; ^†P < 0.05; n = 4–5. Lines within boxes show medians; bounds of boxes show 25th and 75th percentiles, respectively; and whiskers show 5th and 95th percentiles, respectively. (H) Schematic of apoptosis-anchored feed-forward loop of EMAPII production.

(A) Secreted EMAPII in the acellular BALF from otherwise healthy active smokers (triangles; n = 9) compared with that from nonsmokers (circles; n = 10). Mean arbitrary units measured by densitometry were normalized by protein concentration (AU/μg/ml; mean ± SD, *P = < 0.01, t test). (B) Box plot of secreted mature EMAPII in acellular BALF from healthy nonsmokers, ex-smokers without COPD, or ex-smokers with COPD shown in arbitrary units normalized by vinculin densitometry units (*P < 0.05, ANOVA; n = 15/group). Demographic and clinical data are shown in Supplemental Table 1. (C) EMAPII detection by immunostaining with polyclonal EMAPII antibody (red, arrows; DAPI-nuclei are stained in blue) in lung parenchyma of patients with emphysema (bottom panels) compared with subjects without lung disease (normal lung, top panel). Note intense EMAPII staining in the alveolar wall (alv; inset) in emphysematous lungs. IF, immunofluorescence. Scale bar: 50 μm. (D) Box plot of lung EMAPII expression in lung sections from subjects without lung disease (normal; n = 4), with emphysema (emphys; n = 9), or with usual interstitial pneumonitis (UIP; n = 13) (immunohistochemistry staining arbitrary units; *P < 0.05 versus normal, ANOVA). (B and D) Lines within the boxes show medians; bounds of the boxes show 25th and the 75th percentiles of the data, respectively; and whiskers show outliers (5th and 95th percentiles, respectively).