Bronchopulmonary dysplasia (BPD) is a chronic lung disease characterized by arrested alveolarization, a complication of premature birth. The gasotransmitter hydrogen sulfide (H2S) is emerging as a mediator of lung physiology and disease. In this study, the impact of systemic application of H2S on post-natal alveolarization was evaluated in a mouse BPD model. Exposure of newborn mice to 85% O2 for 10 days decreased the total number of alveoli in the lung by 56% and increased mean alveolar septal wall thickness by 29%, assessed by stereological analysis. Systemically administration of GYY4137, the slow-release H2S donor, for 10 days improved lung alveolarization in mouse pups breathing 85% O2, compared with vehicle-treated littermates. Though without effect on lung oxidative status, systemic H2S administration reduced leukocyte infiltration into alveolar airspaces caused by hyperoxia, and normalized lung interleukin (IL)-10 levels that were else diminished by 85% O2. The rapid-release H2S donor NaHS was used to treat primary mouse alveolar type II (ATII) cells; NaHS had no impact on cell viability but stimulated ATII cell migration. Glibenclamide attenuated the impact of NaHS on ATII cell migration, implicating ion channels; and was accompanied by activation of Akt, hinting at two possible mechanisms of H2S action. Although exposure of ATII cells to 85% O2 produced substantial changes in gene expression, exposure to either GYY4137 or NaHS had no impact on ATII cell gene expression, as determined by microarray suggesting that the effects observed were independent of changes in gene expression. H2S can be generated endogenously by cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth). In this study it is demonstrated that the expression of Cbs and Cth in mouse lungs is dynamically regulated during lung alveolarization, and that alveolarization is impaired in Cbs-/- and Cth-/- mouse pups, where a 50% reduction in the total number of alveoli was observed, with no influence on mean alveolar septal wall thickness. Immunofluorescence staining and laser-capture micro dissection revealed that CBS and CTH expression was present in the lung vessels and in the airway epithelium. Vessel remodeling occurred in the absence of Cbs and Cth, since it led to a 100 500% increase in vessel muscularization of small- and medium- size lung vessels. Inhibition of CBS or CTH expression in endothelial cells, using either small interfering RNA or via pharmacological inhibition (propargylglycine) respectively, diminished angiogenic capacity resulting in a 50% decrease in number of tubes formed, and a 30-40% decrease in tube length. On the other hand, administration of GYY4137, promoted endothelial tube formation. These data support the further investigation of H2S as a candidate interventional strategy to limit the impaired alveolarization associated with BPD, also suggests a key role for the H2S generating enzymes Cbs and Cth in lung alveolarization and pulmonary vascular development and homeostasis.
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