Sex-related external factors influence pulmonary vascular angiogenesis in a sex-dependent manner

Abstract

Bronchopulmonary dysplasia (BPD) is a disease with a significant sexual dimorphism where males have a disadvantage compared with their female counterparts. Although mechanisms behind this sexual dimorphism are poorly understood, sex differences in angiogenesis have been identified as one possible source of the male disadvantage in BPD. Pulmonary angiogenesis was assessed in vitro using a bead sprouting assay with pooled male or female human pulmonary microvascular endothelial cells (HPMECs, 18-19 wk gestation, canalicular stage of human lung development) in standard (sex-hormone containing) and hormone-stripped medium. We identified sex-specific phenotypes in angiogenesis where male HPMECs produce fewer but longer sprouts compared with female HPMECs. The presence of sex hormones from standard culture medium modifies the male HPMEC phenotype with shorter and fewer sprouts but does not influence the female phenotype. Using a conditioned medium model, we further characterized the influence of the sex-specific secretome. Male and female HPMECs secrete factors that increase the maximum length of sprouts in female, but not male HPMECs. The presence of sex hormones abolishes this response. The male HPMEC secretome inhibits angiogenic sprouting in male HPMECs in the absence of sex hormones. Taken together, these results demonstrate that the pulmonary endothelial cell phenotypes are influenced by sex hormones and sex-specific secreted factors in a sex-dependent manner.NEW & NOTEWORTHY We identified a sex-specific phenotype wherein male HPMECs produce fewer but longer sprouts than females. Surprisingly, the presence of sex hormones only modifies the male phenotype, resulting in shorter and even fewer sprouts. Furthermore, we found the sex-specific secretome has a sex-dependent influence on angiogenesis that is also sex-hormone sensitive. These new and surprising findings point to the unappreciated role of sex and sex-related exogenous factors in early developmental angiogenesis.

Keywords: angiogenesis; pulmonary endothelial; sex hormone; sex specific; sexual dimorphism.

Figure 1.

Male HPMECs produce fewer, but longer, sprouts compared with female HPMECs. A: male- and female-coated beads cultured in HFM and SM. Dashed yellow circles outline beads, arrow denotes sprouts. B: percentage of beads that produced at least one sprout, means ± SD, n = 3 wells. C: average number of sprouts per bead, n = 72–86 beads. D: average maximal length of sprouts per bead, n = 72–86 beads. *P < 0.05, **P < 0.01, ****P < 0.0001. HFM, hormone-free medium; HPMEC, human pulmonary microvascular endothelial cell; SM, standard medium.

Figure 2.

HPMEC secretome influences angiogenesis in a sex-dependent manner. A: male- and female-coated beads cultured in HFM conditioned by male or female HPMECs. Dashed yellow circles outline beads, arrows denote sprouts. B: percentage of beads that produced at least one sprout, means ± SD, n = 3 wells. C: average number of sprouts per bead, n = 72–104 beads. D: average maximal length of sprouts, n = 72–104 beads. C denotes control, *P < 0.05 and ****P < 0.0001 compared with the sex-matched control. HFM, hormone-free medium; HPMEC, human pulmonary microvascular endothelial cell.

Figure 3.

HPMEC response to the secretome is sex-hormone dependent. A: male- and female-coated beads cultured in SM (hormone containing) conditioned by male or female HPMECs. Dashed yellow circles outline beads, arrows denote sprouts. B: percentage of beads that produced at least one sprout, means ± SD, n = 3 wells. C: average number of sprouts per bead, n = 59–86 beads. D: average maximal length of sprouts, n = 59–86 beads. C denotes control, *P < 0.05 compared with the sex-matched control. HPMEC, human pulmonary microvascular endothelial cell; SM, standard medium.

Figure 4.

Sex-specific angiogenesis phenotype and the influence of exogenous factors. A: baseline phenotype: male HPMECs produce longer but fewer sprouts than females. B: male HPMECs produce shorter and fewer sprouts in the presence of sex hormones. C: female HPMECs produce longer sprouts in the presence of a female secretome. D: sex hormones and a female secretome increase sprout length in male HPMECs but eliminate changes from the female secretome alone in female HPMECs. E: male HPMECs produce fewer sprouts, whereas female HPMECs produce longer sprouts in the presence of a male secretome. F: sex hormones in combination with the male secretome nullify the effects of either source independently, with male and female angiogenesis resembling the baseline phenotype. HPMEC, human pulmonary microvascular endothelial cell. Created with Biorender.com and published with permission.