Representative examples of ciliated epithelial cells in the trachea of control and drug-treated mice.

<p>Tissue was evaluated at 100 X and at 40 X magnification for the control (A and B) and drug-treated mice (C and D), respectively. Black arrows point to cilia. Qualitatively, the presence of ciliated epithelial cells in the airways in each of the representive animals is similar.</p

PAS staining of seromucous cells in tracheal glands of vagotomized mice.

<p><b>A.</b> Trachea of mouse treated with immunosuppressive agents. <b>B.</b> Trachea of mouse not treated with immunosuppressive agents. Black arrows point to PAS staining in seromucous cells. Qualitatively, both the drug-treated animals and control mice appeared to have similar PAS staining in the seromucous cells of the tracheal glands.</p

Mean MCC (±SD) from the right lung between 1–1.5 hours and 6–6.5 hours in 7 C57BL/6 control mice (dark bar) and 8 drug-treated C57BL/6 mice (light bars).

<p>There was a trend toward slower clearance in the drug-treated mice, compared to controls, at 1–1.5 hours, but differences in MCC were not statistically significant. Mucociliary clearance was statistically significantly slower in the drug-treated mice, compared to controls, at 6–6.5 hours (p = 0.006).</p

iNOS staining in airways of vagotomized mice.

<p><b>A.</b> Increased iNOS staining (brown) in bronchial epithelium of adult mouse treated with immunosuppressive agents (Black arrow). iNOS staining was qualitatively increased in the bronchial epithelium of five of the five drug-treated mice. <b>B.</b> Minimal iNOS staining in airway of adult mouse that was not treated with immunosuppressive agents. Minimal iNOS staining was observed in five of the five mice not treated with immunosuppressive agents. <b>C.</b> No first antibody control.</p

Effect of E-cigarette emissions on total body weight and mean linear intercept.

<p><b>A.</b> Neonatal mice and their mothers were placed in a chamber and exposed to E-cigarette emissions starting at 24 hours of life. Age-matched control mice were kept in room air. Mice were exposed to either, 0% nicotine/PG or 1.8% nicotine/PG, once a day (400μl cartridge) for the first two days of life, then twice a day for an additional 7 days. Room air mice in trial one and trial two were significantly heavier at days of life 6 and 3 respectively compared to age-matched 1.8% nicotine/PG exposed mice (*<i>p</i><0.05) and remained significantly heavier up through 10 days of life. (n = 5–13 per group, error bars reflect standard deviation). <b>B.</b> Neonatal mice exposed to 1.8% nicotine/PG had a larger MLI, after adjusting for sex and weight compared to mice exposed to room air (Trial 1: <i>p</i><0.054 and Trial 2: <i>p</i><0.006). In trial 2 neonatal mice exposed to 1.8% nicotine/PG had significantly larger MLI than 0% nicotine/PG exposed mice (<i>p</i><0.014). (n = 5–8 per group, error bars reflect standard error of the mean).</p

Decreased cell proliferation in airspaces of neonatal mice exposed to 1.8% nicotine/PG.

<p><b>A.</b> Arrows point to KI67 staining in the airspaces of 10 day old neonatal mice. <b>B.</b> Quantification of KI67 staining showed significantly less KI67 staining in 10 day old neonatal mice chronically exposed to 1.8% nicotine/PG compared to room air and 0% nicotine/PG treated mice. (n = 8 per group, error bars reflect standard error of the mean).</p

Plasma and urine cotinine levels in 10 day old mice.

<p>Neonatal mice exposed to 1.8% nicotine/PG containing E-cigarette emissions for nine consecutive days in trial one had significantly higher levels of plasma and urine cotinine respectively compared to 0% nicotine/PG exposed mice and room air control mice (± standard error of the mean).</p><p>Plasma and urine cotinine levels in 10 day old mice.</p

Hepatocyte Growth Factor, a Determinant of Airspace Homeostasis in the Murine Lung

<div><p>The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor <em>Met</em> in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF–mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.</p> </div

HGF treatment of MLE12 induces prosurvival signaling that protects against alveolar cell death.

<p>A. Representative immunoblots of phosphoproteins in mice treated with HGF for 5 min (pERK and pJNK) or 15 min (pAKT) showing dose response. B. Cleaved caspase 3 immunoblotting in MLE12 cells treated with staurosporine with or without HGF or wortmannin. All experiments performed in triplicate.</p

Generation and characterization of mice deficient in <i>Met</i> expression in alveolar epithelial cells.

<p>A. Right panel-Representative immunohistochemical staining of c-Met in 2 week old mouse lungs. 40× magnification. N = 4–6 mice. Arrows denote expression in type II epithelial cells. Left panel-Representative immunohistochemical staining for HGF in 2 week old mouse lungs. 40× magnification. N = 4–6 mice. Scale bar (L): 25 µm, (R): 50 µm. Arrows denote exclusion of HGF from alveolar epithelial cells with apparent localization to the interstitium. B. Representative fluorescent immunohistochemistry of phosphorylated c-Met in mice deficient in <i>Met</i> and bitransgenic controls. Green-p-Met. 40× magnification. N = 4–6 mice. Scale bar: 25 µm. C. Quantitative immunohistochemistry of p-met expression in the airspace of <i>Met</i>-deleted mice and controls. D. Representative histology of mice deficient in airspace <i>Met</i> expression and controls at two weeks of age. Note patchy airspace enlargement in the targeted mice. Scale bar: 100 µm. E. Airspace dimension by morphometry in <i>Met</i>-deficient mice and controls at 2 and 3 weeks of age. F. Quantitation of SPC+ cells in the airspace by SPC immunohistochemistry in <i>Met</i>-deficient mice compared with control bitransgenic mice. *p<0.05. G. Representative thrombomodulin immunohistochemical staining of the microvascular bed in the lung parenchyma of Met deficient mice compared with controls. Inset shows reduced staining in the alveolar epithelial walls. 40× magnification, inset 100×. N = 5–7 mice per genotype. H. Quantitative immunohistochemistry of thromobomodulin staining of <i>Met</i>-deficient mice and controls. **p<0.01.</p