Short exposure to hyperoxia causes cultured lung epithelial cell mitochondrial dysregulation and alveolar simplification in mice.

BACKGROUND: Prolonged exposure to high oxygen concentrations in premature infants, although lifesaving, can induce lung oxidative stress and increase the risk of developing BPD, a form of chronic lung disease. The lung alveolar epithelium is damaged by sustained hyperoxia, causing oxidative stress and alveolar simplification; however, it is unclear what duration of exposure to hyperoxia negatively impacts cellular function. METHODS: Here we investigated the role of a very short exposure to hyperoxia (95% O2, 5% CO2) on mitochondrial function in cultured mouse lung epithelial cells and neonatal mice. RESULTS: In epithelial cells, 4 h of hyperoxia reduced oxidative phosphorylation, respiratory complex I and IV activity, utilization of mitochondrial metabolites, and caused mitochondria to form elongated tubular networks. Cells allowed to recover in air for 24 h exhibited a persistent global reduction in fuel utilization. In addition, neonatal mice exposed to hyperoxia for only 12 h demonstrated alveolar simplification at postnatal day 14. CONCLUSION: A short exposure to hyperoxia leads to changes in lung cell mitochondrial metabolism and dynamics and has a long-term impact on alveolarization. These findings may help inform our understanding and treatment of chronic lung disease. IMPACT: Many studies use long exposures (up to 14 days) to hyperoxia to mimic neonatal chronic lung disease. We show that even a very short exposure to hyperoxia leads to long-term cellular injury in type II-like epithelial cells. This study demonstrates that a short (4 h) period of hyperoxia has long-term residual effects on cellular metabolism. We show that neonatal mice exposed to hyperoxia for a short time (12 h) demonstrate later alveolar simplification. This work suggests that any exposure to clinical hyperoxia leads to persistent lung dysfunction.

Heme Oxygenase-1 Supports Mitochondrial Energy Production and Electron Transport Chain Activity in Cultured Lung Epithelial Cells.

Heme oxygenase-1 is induced by many cellular stressors and catalyzes the breakdown of heme to generate carbon monoxide and bilirubin, which confer cytoprotection. The role of HO-1 likely extends beyond the simple production of antioxidants, for example HO-1 activity has also been implicated in metabolism, but this function remains unclear. Here we used an HO-1 knockout lung cell line to further define the contribution of HO-1 to cellular metabolism. We found that knockout cells exhibit reduced growth and mitochondrial respiration, measured by oxygen consumption rate. Specifically, we found that HO-1 contributed to electron transport chain activity and utilization of certain mitochondrial fuels. Loss of HO-1 had no effect on intracellular non-heme iron concentration or on proteins whose levels and activities depend on available iron. We show that HO-1 supports essential functions of mitochondria, which highlights the protective effects of HO-1 in diverse pathologies and tissue types. Our results suggest that regulation of heme may be an equally significant role of HO-1.

Timing and cell specificity of senescence drives postnatal lung development and injury.

Senescence causes age-related diseases and stress-related injury. Paradoxically, it is also essential for organismal development. Whether senescence contributes to lung development or injury in early life remains unclear. Here, we show that lung senescence occurred at birth and decreased throughout the saccular stage in mice. Reducing senescent cells at this stage disrupted lung development. In mice (<12 h old) exposed to hyperoxia during the saccular stage followed by air recovery until adulthood, lung senescence increased particularly in type II cells and secondary crest myofibroblasts. This peaked during the alveolar stage and was mediated by the p53/p21 pathway. Decreasing senescent cells during the alveolar stage attenuated hyperoxia-induced alveolar and vascular simplification. Conclusively, early programmed senescence orchestrates postnatal lung development whereas later hyperoxia-induced senescence causes lung injury through different mechanisms. This defines the ontogeny of lung senescence and provides an optimal therapeutic window for mitigating neonatal hyperoxic lung injury by inhibiting senescence.

Identification of Heme Oxygenase-1 as a Putative DNA-Binding Protein.

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme in degrading heme into biliverdin and iron. HO-1 can also enter the nucleus and regulate gene transcription independent of its enzymatic activity. Whether HO-1 can alter gene expression through direct binding to target DNA remains unclear. Here, we performed HO-1 CHIP-seq and then employed 3D structural modeling to reveal putative HO-1 DNA binding domains. We identified three probable DNA binding domains on HO-1. Using the Proteinarium, we identified several genes as the most highly connected nodes in the interactome among the HO-1 gene binding targets. We further demonstrated that HO-1 modulates the expression of these key genes using Hmox1 deficient cells. Finally, mutation of four conserved amino acids (E215, I211, E201, and Q27) within HO-1 DNA binding domain 1 significantly increased expression of Gtpbp3 and Eif1 genes that were identified within the top 10 binding hits normalized by gene length predicted to bind this domain. Based on these data, we conclude that HO-1 protein is a putative DNA binding protein, and regulates targeted gene expression. This provides the foundation for developing specific inhibitors or activators targeting HO-1 DNA binding domains to modulate targeted gene expression and corresponding cellular function.

Hyperoxia causes senescence and increases glycolysis in cultured lung epithelial cells.

Supplemental oxygen and mechanical ventilation commonly used in premature infants may lead to chronic lung disease of prematurity, which is characterized by arrested alveolar development and dysmorphic vascular development. Hyperoxia is also known to dysregulate p53, senescence, and metabolism. However, whether these changes in p53, senescence, and metabolism are intertwined in response to hyperoxia is still unknown. Given that the lung epithelium is the first cell to encounter ambient oxygen during a hyperoxic exposure, we used mouse lung epithelial cells (MLE-12), surfactant protein expressing type II cells, to explore whether hyperoxic exposure alters senescence and glycolysis. We measured glycolytic rate using a Seahorse Bioanalyzer assay and senescence using a senescence-associated ß galactosidase activity assay with X-gal and C12 FDG as substrates. We found that hyperoxic exposure caused senescence and increased glycolysis as well as reduced proliferation. This was associated with increased double stranded DNA damage, p53 phosphorylation and nuclear localization. Furthermore, hyperoxia-induced senescence was p53-dependent, but not pRB-dependent, as shown in p53KO and pRBKO cell lines. Despite the inhibitory effects of p53 on glycolysis, we observed that glycolysis was upregulated in hyperoxia-exposed MLE-12 cells. This was attributable to a subpopulation of highly glycolytic senescent cells detected by C12 FDG sorting. Nevertheless, inhibition of glycolysis did not prevent hyperoxia-induced senescence. Therapeutic strategies modulating p53 and glycolysis may be useful to mitigate the detrimental consequences of hyperoxia in the neonatal lung.

Single-cell transcriptomics reveals lasting changes in the lung cellular landscape into adulthood after neonatal hyperoxic exposure.

Ventilatory support, such as supplemental oxygen, used to save premature infants impairs the growth of the pulmonary microvasculature and distal alveoli, leading to bronchopulmonary dysplasia (BPD). Although lung cellular composition changes with exposure to hyperoxia in neonatal mice, most human BPD survivors are weaned off oxygen within the first weeks to months of life, yet they may have persistent lung injury and pulmonary dysfunction as adults. We hypothesized that early-life hyperoxia alters the cellular landscape in later life and predicts long-term lung injury. Using single-cell RNA sequencing, we mapped lung cell subpopulations at postnatal day (pnd)7 and pnd60 in mice exposed to hyperoxia (95% O2) for 3 days as neonates. We interrogated over 10,000 cells and identified a total of 45 clusters within 32 cell states. Neonatal hyperoxia caused persistent compositional changes in later life (pnd60) in all five type II cell states with unique signatures and function. Premature infants requiring mechanical ventilation with different durations also showed similar alterations in these unique signatures of type II cell states. Pathologically, neonatal hyperoxic exposure caused alveolar simplification in adult mice. We conclude that neonatal hyperoxia alters the lung cellular landscape in later life, uncovering neonatal programing of adult lung dysfunction.