6, 0.001, Fig. that elastin remodeling regulates angiogenesis, and data supporting developmental regulation of CELA1 expression, we hypothesized that lung elastin remodeling changes in a developmentally regulated manner, that elastin remodeling is usually associated with both angiogenesis and CELA1, and that both elastase Rabbit Polyclonal to AML1 activity and CELA1 regulate in vitro angiogenesis. MATERIALS AND METHODS Mouse model of lung development. C57BL/6J mice from Jackson Laboratories were used at gestational ages 15.5 days [(E) (PND) values of 0.05 were considered significant. All error bars depict SEs. RESULTS Temporal-spatial changes in elastin remodeling during saccular and alveolar lung development. Since human (10, 11) and animal (8, 9) data suggested a physiological role for elastin remodeling in lung development, we sought to quantitate and localize elastin remodeling during pseudoglandular, saccular, and alveolar stages. To do so, we used elastin in situ zymography, desmosine ELISA, and a fluorometric elastase assay to quantify and localize lung elastin remodeling. By elastin in situ zymography, lung elastase activity increased throughout lung development, increasing 24-fold from E15.5 to PND14. The elastin in situ zymography signal decreased 33-fold between PND14 and PN 8 wk (Fig. 1 0.05 by 1-way ANOVA). By fluorometric elastase assay, lung elastase activity was increased 1.5- to 2-fold (not statistically significant). By way of comparison, the fluorometric elastase signal of PN 8 wk pancreas homogenate was 600-fold higher than the elastase signal of PND14 lung homogenate. These data demonstrate that lung elastase activity is usually increased during the saccular and alveolar stages of lung development compared with the pseudoglandular stage and the adult lung. Open in a separate windows Fig. 1. Elastin remodeling during lung development. (PND) and increasing activity at PND7 and PND14 compared with RVX-208 earlier time points. Adult mouse lung had very little elastin remodeling. half of image). 0.05, ** 0.01, and *** 0.001 by one-way ANOVA. To define the location of elastase activity RVX-208 during the RVX-208 saccular and alveolar stages of lung development, we immunostained elastin in situ zymography sections for tropoelastin and localized elastase activity. In the PND1 lung, the zymography signal was equally distributed between nonseptal tip elastin and major airway and vascular structures. In PND3, PND7, and PND14 lung sections, elastase activity was predominantly located within nonseptal tip elastin. Remodeling of nonseptal tip elastin increased sevenfold between PND1 and PND14 (Fig. 1numbers denote mean number of CELA1+ cells/image. One-way ANOVA statistical comparisons were made between identical compartments at different time points. For clarity, notations of differences between compartments at the same time period are omitted. 0.05, ** 0.01, and *** 0.001 by one-way ANOVA. Temporal-spatial changes in CELA1 expression correlate RVX-208 with RVX-208 changes in elastin remodeling. To define the temporal pattern of CELA1 expression during lung development, we quantitated lung mRNA and protein levels at sequential stages of development. Compared with the canalicular stage of lung development, CELA1 mRNA levels were 12-fold higher during the saccular and early alveolar stages of lung development, and mRNA levels decreased at PND14 and PN 8 wk (Fig. 2and and and and 0.01 and *** 0.001 by one-way ANOVA. Validation of CELA1 antibody. Several experiments exhibited antibody specificity. First, CELA1 Western blot demonstrated no nonspecific bands (Fig. 5and 0.001, Fig. 6, 0.001, Fig. 6, 0.05, ** 0.01, and *** 0.001 by em t /em -test (two groups) or one-way ANOVA (three groups). DISCUSSION We have for the first time described the temporal-spatial pattern of elastin remodeling during lung development, demonstrated that this remodeling is usually mediated by serine proteases, and shown an association between lung elastin remodeling and CELA1 expression. Elastin remodeling localized to major arteries and airways on PND1 with increasing remodeling of distal airspace.
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- Binding was detected with biotinylated goat anti-human IgM -chain specific antibodies (Jackson Immunoresearch), followed by streptavidin conjugated to phycoerythrin (PE) (BD Biosciences)
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- Furthermore, most serum antibodies are made by plasma cells generated in prior immune replies, and so are not made by the plasma or plasmablasts cells giving an answer to the immunogenic antigen appealing
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