Upregulating carnitine palmitoyltransferase 1 attenuates hyperoxia-induced endothelial cell dysfunction and persistent lung injury.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants that may cause long-term lung dysfunction. Accumulating evidence supports the vascular hypothesis of BPD, in which lung endothelial cell dysfunction drives this disease. We recently reported that endothelial carnitine palmitoyltransferase 1a (Cpt1a) is reduced by hyperoxia, and that endothelial cell-specific Cpt1a knockout mice are more susceptible to developing hyperoxia-induced injury than wild type mice. Whether Cpt1a upregulation attenuates hyperoxia-induced endothelial cell dysfunction and lung injury remains unknown. We hypothesized that upregulation of Cpt1a by baicalin or L-carnitine ameliorates hyperoxia-induced endothelial cell dysfunction and persistent lung injury. METHODS: Lung endothelial cells or newborn mice (< 12 h old) were treated with baicalin or L-carnitine after hyperoxia (50% and 95% O2) followed by air recovery. RESULTS: We found that incubation with L-carnitine (40 and 80 mg/L) and baicalin (22.5 and 45 mg/L) reduced hyperoxia-induced apoptosis, impaired cell migration and angiogenesis in cultured lung endothelial cells. This was associated with increased Cpt1a gene expression. In mice, neonatal hyperoxia caused persistent alveolar and vascular simplification in a concentration-dependent manner. Treatment with L-carnitine (150 and 300 mg/kg) and baicalin (50 and 100 mg/kg) attenuated neonatal hyperoxia-induced alveolar and vascular simplification in adult mice. These effects were diminished in endothelial cell-specific Cpt1a knockout mice. CONCLUSIONS: Upregulating Cpt1a by baicalin or L-carnitine ameliorates hyperoxia-induced lung endothelial cell dysfunction, and persistent alveolar and vascular simplification. These findings provide potential therapeutic avenues for using L-carnitine and baicalin as Cpt1a upregulators to prevent persistent lung injury in premature infants with BPD.

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.

Loss of the transcriptional repressor Rev-erbα upregulates metabolism and proliferation in cultured mouse embryonic fibroblasts.

The transcriptional repressor Rev-erbα is known to down-regulate fatty acid metabolism and gluconeogenesis gene expression. In animal models, disruption of Rev-erbα results in global changes in exercise performance, oxidative capacity, and blood glucose levels. However, the complete extent to which Rev-erbα-mediated transcriptional repression of metabolism impacts cell function remains unknown. We hypothesized that loss of Rev-erbα in a mouse embryonic fibroblast (MEF) model would result in global changes in metabolism. MEFs lacking Rev-erbα exhibited a hypermetabolic phenotype, demonstrating increased levels of glycolysis and oxidative phosphorylation. Rev-erbα deletion increased expression of hexokinase II, transketolase, and ribose-5-phosphate isomerase genes involved in glycolysis and the pentose phosphate pathway (PPP), and these effects were not mediated by the transcriptional activator BMAL1. Upregulation of oxidative phosphorylation was not accompanied by an increase in mitochondrial biogenesis or numbers. Rev-erbα repressed proliferation via glycolysis, but not the PPP. When treated with H2O2, cell viability was reduced in Rev-erbα knockout MEFs, accompanied by increased ratio of oxidized/reduced NADPH, suggesting that perturbation of the PPP reduces capacity to mount an antioxidant defense. These findings uncover novel mechanisms by which glycolysis and the PPP are modulated through Rev-erbα, and provide new insights into how Rev-erbα impacts proliferation.

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.