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We have demonstrated an association between CELA1 and lung elastin remodeling, and other CELA family members are known to bind CELA1 in a stretch-dependent manner. In this project, fluorophore-labeled recombinant CELA1 will be incubated with ex vivo lung strips on a confocal-compatible stretching device with definition of CELA1 binding kinetics. The non-stretch dependent binding of neutrophil elastase will be demonstrated by similar methods. Using a point mutagenesis strategy, the specificity of CELA1 for tropoelastin exon-14 will be determined by quantitating cleavage of wild type and mutant peptides.
CELA1 mRNA and protein levels change in parallel with increases in lung elastin remodeling except that adult CELA1 protein expression is high. On postnatal day 1 (PND1) CELA1 (+) cells are associated with major airways (gray bars) and vessels (black bars). At later time points CELA1 (+) cells are located in the lung parenchymal (gray bars). Co-staining for CELA1 (red) and elastase activity (green) demonstrates an association. 80% of CELA1 (+) cells are within 10 µm of an elastase signal (green). <10 µm are red balls, ≥10 µm are aqua balls.
We have demonstrated that following closure of the ductus arteriosus, there is an increase in pulmonary arterial elastin remodeling that does not persist, and that doubling of pulmonary arterial blood flow in the adult mouse lung results in changes in arterial wall elastin structure. These changes are consistent with prior reports in rabbit carotid arteries. In this project, changes in elastin structure following pneumonectomy will be quantitated by confocal microscopy, changes in pulmonary arterial compliance following pneumonectomy measured with and without elastase inhibition, and the operative protease or proteases identified.
The ductus arteriosus closes shortly after birth resulting in an increase in pulmonary blood flow. In the first few postnatal days, there is increased elastin remodeling (green) of the pulmonary vessels compared to later time points. Left pneumonectomy results in a doubling of pulmonary blood flow through the right pulmonary artery. 72 hours after pneumonectomy, the elastin matrix of the right pulmonary artery is altered.
We have demonstrated both dynamic regulation of elastin remodeling during alveolar development and alveolar regeneration and an association of CELA1 with lung elastin remodeling during both of these processes. In this project, the role of elastin remodeling in alveolar growth and regulation of lung compliance will be quantitated through morphometric and physiologic techniques. The importance of CELA1 in lung elastin remodeling will be demonstrated by quantitating lung elastase activity in the knockout animal and characterization of the lung phenotype.
Lung elastin remodeling (green) increases throughout saccular (postnatal day 1&3) and alveolar (PND7&14) lung development, and this increase is restricted to the alveolar walls (gray bars) rather than airways and vessels (white bars) or septal tips (black bars). Elastase activity localizes with tropoelastin (red) and is absent in the adult mouse lung. Elastase activity is highly associated with CELA1 expression (data not shown here). Administration of an elastase inhibitor reduces alveolar size at PND14.
We have demonstrated an association of proliferating endothelial cells with elastin remodeling during alveolar lung development and shown a role for both elastin remodeling and CELA1 in angiogenesis in vitro. Using both cell lines and primary endothelial cells, this project will determine whether CELA1 and elastin remodeling exert these effects through the generation of specific elastin degradation products, through activation of protease activated receptors, or through liberation of matrix-bound vascular endothelial growth factor.
Proliferating pulmonary endothelial cells (angiogenin positive, red) are associated with areas of elastin remodeling (green) during the alveolar stage of lung development (PND7&14). Elastase inhibition and CELA1 silencing impair in vitro angiogenesis.
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