Figure 1.
Generation and characterization of mice deficient in Met expression in alveolar epithelial cells.
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 Met 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 Met-deleted mice and controls. D. Representative histology of mice deficient in airspace Met 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 Met-deficient mice and controls at 2 and 3 weeks of age. F. Quantitation of SPC+ cells in the airspace by SPC immunohistochemistry in Met-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 Met-deficient mice and controls. **p<0.01.
Figure 2.
Increased oxidative stress, reduced alveolar cell proliferation, and increased inflammation in lungs of Met–deficient mice.
A. Representative immunohistochemical staining for proliferation marker Ki67 in Met-deficient mice compared with controls. Arrows show increased alveolar cell proliferation in control lungs compared with mutant lungs. Arrowheads denote increased proliferation signal in alveolar macrophages in mutant lungs. Scale bar: 50 µm (top panel), 25 µm (bottom panel). B. Quantitative immunohistochemistry of Ki67 staining in the alveolar epithelium of Met-deficient mice and controls. C. Representative immunohistochemical staining (brown) for nitrotyrosine (NiTyr) in lungs of control and SPCMetf/f mice. Arrowheads denote positive staining. 20× magnification. N = 4 mice per genotype. Scale bar: 50 µm. D. Quantitative immunohistochemistry of nitrotyrosine staining in SPCMetf/f mice compared with controls. E. Macrophage abundance per Mac3 immunohistochemistry in Met-deficient lungs and controls at 2 and 3 weeks of age. Note increasing macrophage influx in mutant lungs. N = 4–6 mice per genotype. F. Representative immunohistochemical staining (brown) for macrophages (Mac3) in lungs of control and SPCMet f/f mice at 3 weeks of age. Arrowheads denote positive staining. 40× magnification. N = 4–6 mice per genotype. Scale bar: 25 µm.
Figure 3.
HGF signaling induces proliferation and scattering of MLE12 cells.
A. Proliferation dose response of HGF and EGF treatment of MLE12 cells demonstrating a significant induction of proliferation by HGF. *p<0.05. B. Proliferation response of MLE12 cells transfected with MET or a control vector showing increased proliferation resulting from MET transfection. C. Cell dispersion images of MLE12 cells treated with EGF or HGF compared to media control. D. Effect of HGF treatment on staurosporine induced caspase 3 cleavage. ST-staurosporine. All cell experiments performed in triplicate. CC3-cleaved caspase 3. E. Densitometric quantitation of effect of HGF treatment on staurosporine induced caspase 3 cleavage. F. Survival time-course of primary alveolar epithelial cells from control and Met-deficient mice. *p<0.05. G. Relative expression of Gclc and Nqo1 with HGF treatment of primary murine alveolar epithelial cells. *p<0.05 compared with control conditions.
Figure 4.
HGF treatment improves airspace caliber in TSK/+ mice.
A. Serum HGF levels in mice treated with HGF infusion pumps at 50 µg/d. **p<.001 B. Phospho-Met immunofluorescent staining in lungs of mice treated with HGF infusion compared with PBS carrier infusion. Arrow in inset denotes phosphorylated c-Met (p-Met, green) in alveolar epithelial cells of HGF treated mice. Scale bar: 50 µm. C. Histology of TSK/+ lung treated with HGF compared with untreated controls. 20× magnification. Scale bar 100 µm. D. Morphometric assessment of airspace dimension in mice treated with HGF for 2 weeks compared with wild-type mice and untreated controls. Lo HGF-50 µg/d. Hi HGF-100 µg/d. E. Representative staining for oxidative stress marker nitrotyrosine in lungs of TSK/+ mice treated with HGF and controls. Arrow denotes staining in alveolar epithelial cells. 40× magnification. Scale bar 50 µm. F. Quantitative immunohistochemistry of nitrotyrosine staining in TSK/+ lungs treated with saline or HGF by infusion pump compared to wild-type controls. G. Representative immunoblotting of normalized phosphomediator levels (akt and stat3) in two TSK/+ mice treated with HGF compared with saline treated mice. N = 4–8 mice per group and treatment.
Figure 5.
HGF treatment of MLE12 induces prosurvival signaling that protects against alveolar cell death.
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.