Role of Cytochrome P450 (CYP)1A in Hyperoxic Lung Injury: Analysis of the Transcriptome and Proteome

AbstractHyperoxia contributes to lung injury in experimental animals and diseases such as acute respiratory distress syndrome in humans. Cytochrome P450 (CYP)1A enzymes are protective against hyperoxic lung injury (HLI). The molecular pathways and differences in gene expression that modulate these protective effects remain largely unknown. Our objective was to characterize genotype specific differences in the transcriptome and proteome of acute hyperoxic lung injury using the omics platforms: microarray and Reverse Phase Proteomic Array. Wild type (WT), Cyp1a1−/− and Cyp1a2−/− (8–10 wk, C57BL/6J background) mice were exposed to hyperoxia (FiO2 &gt; 0.95) for 48 hours. Comparison of transcriptome changes in hyperoxia-exposed animals (WT versus knock-out) identified 171 genes unique to Cyp1a1−/− and 119 unique to Cyp1a2−/− mice. Gene Set Enrichment Analysis revealed pathways including apoptosis, DNA repair and early estrogen response that were differentially regulated between WT, Cyp1a1−/− and Cyp1a2−/− mice. Candidate genes from these pathways were validated at the mRNA and protein level. Quantification of oxidative DNA adducts with 32P-postlabeling also revealed genotype specific differences. These findings provide novel insights into mechanisms behind the differences in susceptibility of Cyp1a1−/− and Cyp1a2−/− mice to HLI and suggest novel pathways that need to be investigated as possible therapeutic targets for acute lung injury.</jats:p

CDH Spatial Transcriptomics

Differentially expressed gene list</p

Analysis of the transcriptome in hyperoxic lung injury and sex-specific alterations in gene expression.

Exposure to high concentration of oxygen (hyperoxia) leads to lung injury in experimental animal models and plays a role in the pathogenesis of diseases such as Acute Respiratory Distress Syndrome (ARDS) and Bronchopulmonary dysplasia (BPD) in humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. The major goal of this study was to characterize the changes in the pulmonary transcriptome following hyperoxia exposure and further elucidate the sex-specific changes. Male and female (8-10 wk) wild type (WT) (C57BL/6J) mice were exposed to hyperoxia (FiO2>0.95) and gene expression in lung tissues was studied at 48 h. A combination of fold change ≥1.4 and false discovery rate (FDR)<5% was used to define differentially expressed genes (DEGs). Overrepresentation of gene ontology terms representing biological processes and signaling pathway impact analysis (SPIA) was performed. Comparison of DEG profiles identified 327 genes unique to females, 585 unique to males and 1882 common genes. The major new findings of this study are the identification of new candidate genes of interest and the sex-specific transcriptomic changes in hyperoxic lung injury. We also identified DEGs involved in signaling pathways like MAP kinase and NF-kappa B which may explain the differences in sex-specific susceptibility to hyperoxic lung injury. These findings highlight changes in the pulmonary transcriptome and sex-specific differences in hyperoxic lung injury, and suggest new pathways, whose components could serve as sex-specific biomarkers and possible therapeutic targets for acute lung injury (ALI)/acute respiratory distress (ARDS) in humans

Modulation of recovery from neonatal hyperoxic lung injury by sex as a biological variable

Recovery from lung injury during the neonatal period requires the orchestration of many biological pathways. The modulation of such pathways can drive the developing lung towards proper repair or persistent maldevelopment that can lead to a disease phenotype. Sex as a biological variable can regulate these pathways differently in the male and female lung exposed to neonatal hyperoxia. In this study, we assessed the contribution of cellular diversity in the male and female neonatal lung following injury. Our objective was to investigate sex and cell-type specific transcriptional changes that drive repair or persistent injury in the neonatal lung and delineate the alterations in the immune-endothelial cell communication networks using single cell RNA sequencing (sc-RNAseq) in a murine model of hyperoxic injury. We generated transcriptional profiles of >55,000 cells isolated from the lungs of postnatal day 1 (PND 1; pre-exposure), PND 7, and PND 21neonatal male and female C57BL/6 mice exposed to 95 % FiO2 between PND 1–5 (saccular stage of lung development). We show the presence of sex-based differences in the transcriptional states of lung endothelial and immune cells at PND 1 and PND 21. Furthermore, we demonstrate that biological sex significantly influences the response to injury, with a greater number of differentially expressed genes showing sex-specific patterns than those shared between male and female lungs. Pseudotime trajectory analysis highlighted genes needed for lung development that were altered by hyperoxia. Finally, we show intercellular communication between endothelial and immune cells at saccular and alveolar stages of lung development with sex-based biases in the crosstalk and identify novel ligand-receptor pairs. Our findings provide valuable insights into the cell diversity, transcriptional state, developmental trajectory, and cell-cell communication underlying neonatal lung injury, with implications for understanding lung development and possible therapeutic interventions while highlighting the crucial role of sex as a biological variable

Fold change for the selected genes in the microarray and qPCR experiment.

<p><i>Notes: </i><b><i>C</i></b><i>: Common to both male and female animals; </i><b><i>F</i></b><i>: In females only; </i><b><i>M</i></b><i>: In males only.</i></p

List of pulmonary DEGs after hyperoxia exposure.

<p>List of pulmonary DEGs after hyperoxia exposure.</p