During postnatal lung development, metabolic changes that coincide with stages of alveolar formation are poorly understood. Responding to developmental and environmental factors, metabolic changes can be rapidly and adaptively altered. The objective of the present study was to determine biological and technical determinants of metabolic changes during postnatal lung development. Over 118 metabolic features were identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS, Sciex QTRAP 5500 Triple Quadrupole). Biological determinants of metabolic changes were the transition from the postnatal saccular to alveolar stages and exposure to 85% hyperoxia, an environmental insult. Technical determinants of metabolic identification were brevity and temperature of harvesting, both of which improved metabolic preservation within samples. Multivariate statistical analyses revealed the transition between stages of lung development as the period of major metabolic alteration. Of 3 distinctive groups that clustered by age, the saccular stage was identified by its enrichment of both glycolytic and fatty acid derivatives. The critical transition between stages of development were denoted by changes in amino acid derivatives. Of the amino acid derivatives that significantly changed, a majority were linked to metabolites of the one-carbon metabolic pathway. The enrichment of one-carbon metabolites was independent of age and environmental insult. Temperature was also found to significantly influence the metabolic levels within the post-mortem sampled lung, which underscored the importance of methodology. Collectively, these data support not only distinctive stages of metabolic change but also highlight amino acid metabolism, in particular one-carbon metabolites as metabolic signatures of the early postnatal lung.
Background and Hypothesis: The pathogenesis of Bronchopulmonary Dysplasia (BPD) is multifactorial leading to inflammation. In BPD, Endothelial-Monocyte Activating Polypeptide II (EMAP II, encoded by Aimp1), a moonlighting pro-inflammatory cytokine, is initially found in bronchiolar club cells followed by intra-alveolar GAL-3+ macrophages. Sustained EMAP II mimics BPD, invoking inflammation, alveolar simplification, and macrophage recruitment. Targeted ablation of EMAP II in the recruited macrophages may dampen innate immune response. Experimental Design: Gender-matched, aged-matched littermate mice with myeloid-cell specific ablation of Aimp1 (Lyz2-Cre;Aimp1flox/flox, denoted as Aimp1Δ/Δ) or without (control) were subjected to lipopolysaccharide (LPS)-endotoxemia. Survival rates of co-housed or singly housed mice were measured over 72 hours following a lethal dose (15 mg/kg). Clinical scores (0-6) based on the integrity of their locomotion, fur, and eyes were assigned every 2 hours. Blood and bone marrow smears, average bodyweights, spleen-weights to bodyweights and liver-weights to bodyweights were analyzed. Results: There were no baseline differences in bodyweight, spleen weight:bodyweight, liver weight:bodyweight (p-val = 0.42, 0.46, 0.64). Representative bone marrow and blood smears showed no notable difference. Aimp1[Symbol]/[Symbol] male mice co-housed (dose 15 mg/kg) but not singly housed survived longer than their littermates (median survival: hours); Aimp1[Symbol]/[Symbol] female mice showed a survival advantage (median survival: hours) with lower clinical scores than their littermates. The kinetics of NFKBIA/I[Symbol]B degradation was similar between Aimp1[Symbol]/[Symbol] and control peritoneal macrophages in response to LPS, although there was a higher basal amount in Aimp1[Symbol]/[Symbol]. Conclusion and Potential Impact: Aimp1/EMAP II does play a positive feedback role in innate immunity, potentially in a metabolically and gender-specific role of Aimp1 which remain to be explored.
IntroductionChanges in dynamic metabolic processes during lung development is not well known, particularly during the saccular and alveolar stages. Lungs of infants prematurely born during either stage have underdeveloped alveolar sacs with arrested growth and frequently require oxygen supplementation, which can result in lung disease of prematurity (bronchopulmonary dysplasia, BPD). Oxidation‐reduction (redox) processes are important in both lung development and pathogenesis; however, clinical studies attempting to limit BPD pathogenesis targeting redox processes have not been promising. In this study, metabolomics were utilized to identify alternative metabolic perturbations during the two lung development stages in an established murine model of hyperoxia‐induced BPD.MethodsPostnatal day (PN) 3 mice from pairs of pregnant dam mice were pooled, randomized, and left at normoxia (room air, 21% O2) or exposed to hyperoxia (85% O2) in a tight‐sealed, plexiglass chamber until PN 15. Nursing dams were rotated every 24 hours to limit oxygen toxicity. Lungs were isolated from mice on PN 1, 3, 5, 7, 10, and 15 within two minutes then snap frozen in liquid nitrogen. Following methanol extraction, metabolites were detected by liquid chromatography‐mass spectrometry. Statistics including partial least squares – discriminant analysis (PLS‐DA) and Variable Importance in Projection (VIP), i.e. a quantitative measure of discriminating metabolites, were performed using Python 2.7/3.0 and R.ResultsA series of analysis were performed to validate, identify, and characterize both significant metabolites and their involved pathways. Samples could be separated by both time and oxygen‐level using PLS‐DA. Enrichment analysis of metabolites with VIP ≥ 1 indicated that both the methionine‐homocysteine degradation cycle (p ≤ 1e‐4) and downstream of it, polyamine synthesis was perturbed (p ≤ 1e‐4). Components of both the arginine and glutathione metabolism pathways were also significantly enriched (p ≤ 1e‐4). Metabolites specifically found in the prior enrichment analysis (e.g., cystathione in glutathione metabolism, spermine, spermidine in polyamine synthesis) were significantly elevated in hyperoxic lung tissues on PN 5, 7, and 10 (one way ANOVA, p ≤ 1e‐2). Metabolites with a fold change of ≥ 1.3 from only day 5 lung tissues were significantly enriched for methylhistidine metabolism (p ≤ 1e‐5).ConclusionSignificant alterations of redox process such as glutathione or arginine metabolism were found which corroborate current studies in lung development and BPD. Yet other metabolic processes such as methionine‐homocysteine degradation and polyamine synthesis were discovered. Deregulation of the recycling of homocysteine into methionine and how it is further metabolized into polyamines is common in other pathogenesis. Further examination to intervene the deregulation in BPD pathogenesis may be attractive.Support or Funding InformationNIH Grant to MAS (R21 HD090227) and fellowship to DL (T32 HL091816‐09)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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