Sex-Based Differences Identified in Pulmonary Microvascular Endothelium

Results from a study published in Physiological Genomics revealed important sex-based differences between male and female human pulmonary microvascular endothelial cells (HPMECs). These findings demonstrate a potential driving factor in the sexual dimorphism that arises in forms of pulmonary hypertensive disease.

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Previously, sexual dimorphism has been observed in cases of pulmonary hypertension (PH). PH is a disease that exhibits degrees of sex-based differences, which can be observed in the higher likelihood females have for developing this disease, as well as the higher survival rates females have compared with their male counterparts. The authors of the present study note that past studies evaluating the underlying mechanisms in these discrepancies have been centered on function of sex hormones; however, sex chromosomes and sex-based differences occurring in adult HPMECs have not been sufficiently evaluated in PH.

To address this gap in knowledge, researchers conducted a study to investigate sex-based differences in the human pulmonary microvascular endothelium. They evaluated endothelial cellular responses to normoxia and hypoxia, hypothesizing that HPMEC responses and differences would occur independently of those observed in sex hormone environments. Since human endothelial cells continually undergo shear stress, HPMECs from a limited number of healthy male and female donors (less than 10, with later additions as more donors became available) were cultured under physiological shear stress conditions. Additionally, these samples were analyzed with label-free quantitative proteomics and RNA sequencing.

Of the differentially expressed genes (DEG) in female and male HPMECs—evaluated in normoxia and hypoxia—researchers found the most notable differences expressed in Y-chromosome encoded genes that have been potentially indicated in the development of PH: RPS4Y1, DDX3Y, UTY, ZFY and 228 PRKY. As for X-encoded genes, the most distinguishable difference was detected in the expression of XIST—which was more highly expressed in female HPMECs compared with male. In this assessment, no notable differences were observed in the proteomic analysis on proteins encoded by sex chromosomes.

To identify enriched genetic pathways differentiating the sexes, researchers used a Gene Set Enrichment Analysis (GSEA). After 24 hours of normoxia, the analysis revealed that 3 gene sets in male HPMECs were enriched, compared with 13 in female HPMECs. After 48 hours of normoxia, 3 gene sets in male samples compared with 8 in female samples were enriched. Following 24 hours of hypoxia, researchers found 2 gene sets of male HPMECs enriched compared with 20 in female HPMECs. Responses after 48 hours of hypoxia showed 2 gene sets in male samples and 19 in female samples enriched. Subsequently, researchers conducted this experiment in vitro and observed that male cells had increased proliferation rates compared with female cells in normoxic conditions.

Overall, these results demonstrate, for the first time, that sex-based differences occur in the cellular pathways in the human pulmonary microvascular endothelium—in both RNA and protein expression, as well as normoxic and hypoxic conditions. Most importantly, the authors emphasize, these distinct expressions occurred in HPMECs that were cultured outside of a sex hormone environment.

“Culturing of cells isolated from male and female donors in the absence of the sex hormones is a straightforward model for investigating the sex chromosomal influence on the sex differences,” the authors state, “however, this method does not allow complete elimination of the impact of the sex hormones. Sex hormones have a long-lasting programming effect on cells (82) and the absence of the sex hormones can only eliminate the acute actions of differences in the sex hormone environment to which the cells were exposed prior to isolation.”

They recommend further studies, with large sample sizes, to better understand the association between sex-based differences, pulmonary microvasculature remodeling, and the heighted vascular resistance indicated in the development of PH.

Reference

Kostyunina DSl, Pakhomov NV, Jouida A, Dillon E, Baugh JA, McLoughlin P. Transcriptomics and proteomics revealed sex differences in human pulmonary microvascular endothelial cells. Physiol Genomics. 2023 Dec 4. doi: 10.1152/physiolgenomics.00051.2023