Alveolar hypoplasia is the phenotypic hallmark of bronchopulmonary dysplasia. Although several animal models recapitulate arrested alveolar development through hyperoxia exposure, the exact mechanisms driving this process remain poorly understood. Based on mounting evidence that environmental triggers exert changes in lung architecture through altered epigenetic mechanisms (9
), we hypothesized that hyperoxia exposure disrupts alveolar development through imbalance of histone deacetylases (HDACs) and their signaling mediators. Our results demonstrate that hyperoxia decreases expression of HDAC1 and HDAC2 and increases expression of the cyclin-dependent kinase p21, resulting in cell cycle arrest. To our knowledge, this is the first report detailing the effects of hyperoxia in arresting alveolar development through altered HDAC activity in neonatal mice.
To date, studies describing the effects of hyperoxia on alveolar formation have focused on inflammation and oxidative mechanisms that cause lung injury and disrupt alveolar architecture (5
). Elevated oxygen levels induce superoxide radicals and pro-inflammatory cytokines responsible for neutrophil recruitment into the lung. Minimal data exist, however, on whether alveolar changes are a direct result of lung inflammation and injury or due to permanent disruption of normal developmental signaling pathways. We found no evidence of persistent inflammation or increased cytokine expression following 15 days of exposure to 80% oxygen consistent with previous reports (24
). These observations raise the possibility that alveolar changes due to hyperoxia may be due to epigenetic regulation of developmental genes rather than ongoing inflammation or injury.
HDACs have the ability to deacetylate non-histone proteins such as nuclear factor κB (NF-κB) that are central to inflammatory signaling pathways. Decreased HDAC1 expression has been shown in bronchial biopsies of patients with asthma, thus favoring increased expression of inflammatory genes. Similarly, markedly decreased HDAC2 activity has been shown in lungs of patients with chronic obstructive pulmonary disease (COPD) and in a mouse model of emphysema due to oxidative and nitrative stress from exposure to cigarette smoke (16
). We determined the localization and abundance of HDAC1 and HDAC2 in lung sections exhibiting hypoplasia in response to hyperoxia. Our results showed that total HDAC activity and HDAC1 and HDAC2 abundance in alveolar and airway epithelium significantly decreased following hyperoxia. In addition to transcription factor repression, class I HDACs, and especially HDAC1, also repress genes involved in cell differentiation and proliferation including the cyclin-dependent kinase inhibitor p21WAF1/CIP1
). Decreased HDAC activity with inhibition of cell proliferation may thus promote a state of cell cycle arrest and inhibit alveolar growth.
Our results demonstrated that hyperoxia in newborn pups increased p21 and p53 levels at 3 days and 15 days, consistent with data in adult mice showing increased p53 gene transcription, protein levels, and activity following hyperoxia (27
). Though recent reports indicate that the p53 pathway can elicit hyperoxia-induced senescence independent of p21 activation (29
), the exact role of p53 in modulating cellular response to hyperoxia remains to be investigated. p21 may thus act as a molecular switch involved in triggering a ‘danger’ signal that attempts to protect the cell from further damage by inducing irreversible cell cycle growth arrest.
Azithromycin (AZM) is a macrolide antibiotic previously shown to suppress activation of NF-κB by tumor necrosis factor-alpha (TNF-α) in cultured tracheal aspirate cells from premature infants (30
). Studies in neonatal rats showed that daily treatment with AZM during exposure to > 95% oxygen from day of life 4 to 14 improved survival and decreased lung damage and inflammation when compared to saline controls (11
). Finally, a prospective double-blind, randomized, placebo-controlled pilot study in preterm humans investigated whether AZM treatment conferred protection from developing BPD. Although no difference was seen in incidence of BPD or mortality, a significant reduction in need for postnatal steroids and mechanical ventilation was noted in the treatment group (31
). Results from our study showed no difference in hyperoxia-induced alveolar hypoplasia between animals treated with AZM versus saline controls. Moreover, treatment with AZM may confer a potential negative impact on lung growth homeostasis as uncovered by the finding that p21, and therefore cell senescence/mitosis, is induced by AZM (32
Several possibilities may account for the difference between our study and the previous study in neonatal rats. First, it is possible that species-specific differences related to AZM efficacy may render mice less protected than rats. Second, the level of oxygen concentration differed between the two studies. We chose 80% oxygen based on minimal mortality for pups and lactating dams as compared to significant toxicity seen with 95% oxygen. Lower oxygen concentration is also associated with a lower degree of lung inflammation (24
). Since immunomodulatory properties of AZM confer protection from inflammation, it is possible that any protective effect of AZM was less at 80% oxygen compared to 95% oxygen. A limitation of our study is that we have focused on a limited panel of molecules that participate in epigenetic regulation. Several additional classes of HDAC including sirtuins as well as HAT (histone acetyltransferase) family members and co-activators may also influence these developmental signaling pathways. Furthermore, our experimental animal design using hyperoxia serves only as a surrogate model for human BPD and represents one component of a complex and multifactorial pathologic process. In-depth studies on how varying oxygen levels and exposure times affect various signaling pathways may lead to additional interventions to prevent hyperoxic injury (33
In summary, we have utilized an established newborn mouse model to provide novel evidence that the mechanism, in part, by which hyperoxia induces alveolar hypoplasia is through decreased HDACs and increased p21. Alveolar hypoplasia thus results from arrested lung growth and cell cycle arrest induced by hyperoxia. Further understanding of these epigenetic mechanisms in alveolar senescence may help to identify new molecular targets to ultimately prevent and treat human BPD. Future studies with these goals using HDAC inducers such as caffeine/theophylline (methylxanthine derivates) in protecting against hyperoxia-induced alveolar hypoplasia are currently underway.