RNA‐Seq Analysis Revealed Sex Differences in Pulmonary Microvascular Endothelial Cells in the Response to Hypoxia

Abstract

Pulmonary hypertension (PH) is characterized by the abnormal elevation of mean pulmonary arterial pressure which may lead to right ventricle (RV) failure and death. PH is classified into five groups, four of them have been reported to have sex dimorphism with females showing better RV functioning than males. The most marked sex dimorphism is observed in Group 1, pulmonary arterial hypertension (PAH), which is 2‐4 times more common in females than in males. It has only recently been discovered that sex chromosomes may play a role in the sex differences in PH that is independent of sex hormones, although the exact mechanisms are still unknown (1). One of the approaches to get insight into the role of the sex chromosomes in vitro is isolated cells, cultured in the absence of sex hormones. The aim of this study was to reveal sex differences in human pulmonary microvascular endothelial cells, using hypoxia as an in vitro model of pulmonary hypertension.Human pulmonary microvascular endothelial cells (HPMEC) from female (n=4) and male (n=3) healthy donors were exposed to hypoxia (1% O2) for 48 hours. RNA from these samples was sequenced on the Illumina NovaSeq 6000. Differentially expressed genes (DEGs) were identified using DESeq2 (Wald test, p(adj.)<0.05) (2). Gene Set Enrichment Analysis (GSEA, Hallmark gene set collection, FDR<0.05) was used to find enriched pathways (3,4).Hypoxia induced changes in the expression of 79 genes in male HPMEC and of 34 genes in female HPMEC compared to normoxic controls. Among them 19 genes were common between the two sexes, 69 genes changed in males only, 24 genes changed in females only in response to hypoxia. GSEA revealed that in both male and female HPMEC hypoxia induced the enrichment of gene sets “Hypoxia”, “Glycolysis”, “Epithelial Mesenchymal Transition”, “MTORC1 Signaling”, and “Myogenesis”. One gene set, “MYC Targets V1”, was changed in opposite directions in the two sexes: it was upregulated in hypoxia in male HPMEC and downregulated in hypoxia in female HPMEC. Comparisons between male and female HPMEC basally (in normoxia) and in the hypoxic conditions revealed that 11 Y‐encoded genes were expressed more highly in male HPMEC than in female HPMEC (DDX3Y, KDM5D, USP9Y, ZFY, EIF1AY, UTY, RPS4Y1, NLGN4Y, PRKY, TXLNGY, TTTY14). Two X‐encoded genes (XIST, RPS4X) were expressed more highly in female HPMEC in normoxia than in male HPMEC, and one X‐coded gene (XIST) was expressed more highly in female HPMEC in hypoxia than in male HPMEC in hypoxia.According to RNA‐seq analysis, sex differences exist in male and female HPMEC. Some of the identified DEGs and pathways changed in response to hypoxia in the opposite directions in male compared to female HPMEC. Sex differences were induced by sex hormone independent mechanisms (i.e. sex chromosomes), since HPMECs were cultured in the absence of sex hormones. Further research is needed to explore the contribution of the genes and pathways reported here in the development of sex differences in PH. Kostyunina, D. S., & McLoughlin, P. (2021). Antioxidants, 10(5):779; Love, M. I., Huber, W., & Anders, S. (2014). Genome Biology, 15(12), 1–21; Subramaniana et al.(2005). PNAS, 102(43), 15545–15550; Liberzon et al. (2015). Cell Syst, 1(6), 417–425.