Sexual dimorphism of the pulmonary transcriptome in neonatal hyperoxic lung injury: identification of angiogenesis as a key pathway

Abstract

Bronchopulmonary dysplasia (BPD) is characterized by impaired alveolar secondary septation and vascular growth. Exposure to high concentrations of oxygen (hyperoxia) contributes to the development of BPD. The male sex is considered an independent risk factor for the development of BPD. The reasons underlying sexually dimorphic outcomes in premature neonates are not known. We hypothesized that sex-specific modulation of biological processes in the lung under hyperoxic conditions contributes to sex-based differences. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia [95% [Formula: see text], postnatal day (PND) 1-5: saccular stage of lung development] and euthanized on PND 7 or 21. Pulmonary gene expression was studied using RNA-Seq on the Illumina HiSeq 2500 platform. Analysis of the pulmonary transcriptome revealed differential sex-specific modulation of crucial pathways such as angiogenesis, response to hypoxia, inflammatory response, and p53 pathway. Candidate genes from these pathways were validated at the mRNA level by qPCR. Analysis also revealed sex-specific differences in the modulation of crucial transcription factors. Focusing on the differential modulation of the angiogenesis pathway, we also showed sex-specific differential activation of Hif-1α-regulated genes using ChIP-qPCR and differences in expression of crucial genes (Vegf, VegfR2, and Phd2) modulating angiogenesis. We demonstrate the translational relevance of our findings by showing that our murine sex-specific differences in gene expression correlate with those from a preexisting human BPD data set. In conclusion, we provide novel molecular insights into differential sex-specific modulation of the pulmonary transcriptome in neonatal hyperoxic lung injury and highlight angiogenesis as one of the crucial differentially modulated pathways.

Keywords: RNA-Seq; bronchopulmonary dysplasia; hyperoxia; sex.

Fig. 1.

Expression levels of UTY (ubiquitously transcribed tetratricopeptide repeat containing, Y-linked) and XIST (an X chromosome-specific gene) in male and female neonatal mice lung samples. Lung mRNA samples from mice exposed to hyperoxia [95% $\text{F}{\text{I}}_{{\text{O}}_2}$, postnatal day (PND)1–5] or room air (RA) were submitted for RNA-Seq analysis. The expression levels of UTY (a Y chromosome-specific gene) and XIST (an X chromosome-specific gene) were determined. RNA-Seq signals at the UTY and XIST loci are shown for representative samples from each treatment group and confirm our determination of the sex.

Fig. 2.

Distinct pulmonary transcriptomic responses were noted in male and female neonatal mice after exposure to hyperoxia. A and B: volcano plots of the differentially expressed genes (DEGs; upregulated and downregulated) in male and female neonatal mice in response to hyperoxia exposure compared with room air controls on PND7 (A) and PND21 (B). The genes shaded in yellow are common DEGs between male and female neonatal mice, whereas the genes represented in blue are DEGs exclusive to male neonatal mice, and those in red are exclusive to female neonatal mice. Select differentially regulated genes are highlighted. C and D: Venn diagrams highlighting the differential gene expression on PND7 (C) and PND21 (D) in male and female neonatal mice exposed to hyperoxia. E: 2-way ANOVA of the pulmonary transcriptome showing effect of hyperoxia, sex, and any interaction between the 2 variables.

Fig. 3.

Distinct modulations of pathways are observed between male and female neonatal mice exposed to hyperoxia by gene set enrichment analysis (GSEA) on PND7. Biological processes enriched in the pulmonary transcriptome of hyperoxia-exposed male and female neonatal mice were identified using GSEA. The normalized enrichment score is reported for select enriched pathways (fdr-adjusted q value <0.25). A: top upregulated pathways in both male and female neonatal mice exposed to hyperoxia. B: top downregulated pathways in both male and female neonatal mice. C: pathways that were regulated in opposite directions in male and female mice. PI3K, phosphatidylinositol 3-kinase.

Fig. 4.

Sexually dimorphic modulation of pathways is observed between male and female neonatal mice exposed to hyperoxia by gene set enrichment analysis (GSEA) on PND21. Biological processes enriched in the pulmonary transcriptome of hyperoxia-exposed male and female neonatal mice were identified using GSEA. The normalized enrichment score is reported for select enriched pathways (fdr-adjusted q value <0.25). A: top upregulated pathways in male neonatal mice exposed to hyperoxia on PND21. The graph also shows the direction of enrichment in similarly exposed female mice. B: top upregulated pathways in female mice on PND21 and the direction of enrichment in similarly exposed male mice.

Fig. 5.

Sex-specific differences in transcriptional regulator network modulating the response to hyperoxia. A: analysis of upstream transcriptional factors was performed using overrepresentation analysis to identify the key transcription factors modulating the transcriptomic response to hyperoxia (hypergeometric distribution, P < 0.05). Top transcriptional regulators in up- and downregulated genes are depicted as network nodes; edges indicate common gene targets between transcription factors. B–D: an extensive search was carried out for transcriptional regulators that were enriched after hyperoxia exposure (q < 0.25) using GSEA, and the results were clustered by the relative direction of the gene targets in male and female hyperoxia response. B: transcription factors with gene targets enriched in the same direction, either upregulated in both or downregulated in both male and female hyperoxia-exposed neonatal mice at PND7 and PND21. C: transcriptional regulators with a positive normalized enrichment score in females at PND7 acting primarily as transcriptional activators for female mice hyperoxia while having a negative normalized enrichment score in males at PND7 acting primarily as transcriptional repressors for male mice hyperoxia. D: transcriptional regulators with opposite normalized enrichment score in female vs. male hyperoxia response at PND21.

Fig. 6.

Quantitative (q)RT-PCR analysis of mRNA from the lungs of male and female neonatal mice (PND7) exposed to room air or hyperoxia shows differential regulation of gene targets. Fold change over room air levels is represented on the y-axis. Two-way ANOVA was used to assess statistical significance in gene expression among sex and the effect of hyperoxia. Significant differences from room air levels are represented as follows: *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 5/group).

Fig. 7.

Chromatin immunoprecipitation-qPCR analysis of Hif-1α target genes in male and female neonatal mice after exposure to hyperoxia; Hif-1α binding to CA9 (A and C) and VEGFB (B and D) promoter region in lung tissue in hyperoxia-treated male and female mice at PND7 and -21. Data are shown as means ± SE; n ≥ 3 in each group. *P < 0.05 and **P < 0.01 (room air vs. hyperoxia treated); #P < 0.05 and ##P < 0.01 (male vs. female). Two-way ANOVA was used to assess statistical significance in gene expression among genotypes and the effect of hyperoxia.

Fig. 8.

Focused angiogenesis pathway analysis and gene expression of key angiogenic mediators in male and female neonatal mice after hyperoxia exposure. A and B: biological processes enriched in the pulmonary transcriptome of hyperoxia-exposed male and female neonatal mice were identified using gene set enrichment analysis (GSEA) on PND7 (A) and PND21 (B). The normalized enrichment score is reported for select enriched pathways (fdr-adjusted q value <0.25). C: qRT-PCR analysis of mRNA from the lungs of male and female neonatal mice (PND21) exposed to room air or hyperoxia shows differential regulation of key proangiogenic gene targets. Fold change over room air levels is represented on the y-axis. Two-way ANOVA was used to assess statistical significance in gene expression among sex and the effect of hyperoxia. Significant differences from room air levels are represented as follows: *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 5/group).

Fig. 9.

Sexually dimorphic modulation of pathways is observed between male and female human neonates with bronchopulmonary dysplasia (BPD) compared with controls with no BPD. Biological processes enriched in the pulmonary transcriptome of hyperoxia-exposed male and female neonatal mice were identified using gene set enrichment analysis. The normalized enrichment score is reported for select enriched pathways (fdr-adjusted q value <0.25). Gene expression data from babies with severe BPD (n = 15) and controls were used to identify differentially regulated pathways across all time points (PND5, -14, and -28; A) and on PND28 (B) and in male and female premature neonates.