Extracellular matrix (ECM) formation and remodeling play a central role in the processes of lung alveolarization and secondary septation. A key component of ECM structure is a complex collagen and elastin network of fibers. Formation and maintenance of this crucial structure is perturbed in aberrant lung development in patients with bronchopulmonary dysplasia (BPD) as well as in experimental animal models of BPD. Lysyl oxidases comprise a family of five members, which facilitate the covalent cross-linking of collagen and elastin molecules, thus controlling ECM structural homeostasis. Moreover, alternative non-matrix gene regulatory roles for these enzymes have been proposed. Recently, lysyl oxidases have been implicated in the pathogenesis of various lung diseases, including cancer, fibrosis, pulmonary hypertension and BPD. Although several studies report perturbations to lysyl oxidase expression, and elastin and collagen deposition associated with the arrest of alveolarization in BPD, a causal role for lysyl oxidases in this processes has not yet been fully addressed. In the study presented here, lysyl oxidase activity was neutralized in vivo in a murine hyperoxia-based model of BPD. In addition, non-matrix roles for three of the five family members, Lox, Loxl1 and Loxl2 were explored on the primary mouse fibroblasts background. In the first part of the study, an arrest in alveolarization in developing mouse lungs was induced by hyperoxia exposure. Damage to alveolar formation was accompanied by an increase in lysyl oxidase activity, and an increase in the abundance of collagen and collagen cross-links. In contrast, the abundance of the insoluble elastin and elastin cross-links desmosine and isodesmosine was decreased, resulting in a substantial shift in collagen-toelastin ratio. Normalization of lysyl oxidase catalytic activity partially restored normal levels of collagen. The abundance of elastin cross-links and the formation of elastin foci was also improved. However, no significant improvement in lung alveolarization was observed. In the second part of the study, a microarray analysis after small interfering (si)RNA knockdown in primary mouse lung fibroblasts revealed a dysregulation of expression of a large group of genes, including several ECM-relevant players, such as Mmp3, Mmp9, Rarres1 or Eln. Moreover, the expression patterns of these genes in an in vivo animal model of BPD correlated with observations made in vitro. Importantly, the gene regulatory roles of lysyl oxidases were independent of lysyl oxidase catalytic activity.
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