Sex-differences in pulmonary vascular endothelial development: factors that influence angiogenesis
Date
2023
Authors
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Publisher
University of Delaware
Abstract
Extremely preterm birth (<32 weeks gestation) requires life-saving treatments, specifically mechanical ventilation with oxygen therapy, which creates a hyperoxic environment within the still-developing lung. This leads to disrupted alveolarization, increased inflammation, tissue damage, and disrupted angiogenesis, which can all contribute to developing bronchopulmonary dysplasia (BPD). Despite the uniform treatment approach on extremely preterm infants, BPD is a disease with a significant sexual dimorphism where males are disadvantaged compared to 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. ☐ Proper lung development in the alveolar stage heavily depends on pulmonary angiogenesis, a complex process of forming new blood vessels. Recent studies have shown significant sex differences in endothelial cell expression profiles, behavior, and angiogenesis. Sex differences can arise from intrinsic (chromosomal) or extrinsic sources. Sex hormones are a commonly studied extrinsic factor, specifically the male-associated hormone testosterone and the female-associated hormone estrogen. During normal human gestation, testosterone peaks between 10 and 17 weeks while estrogen steadily increases starting at 20 weeks gestation through birth. This estrogen spike is abolished in prematurely born infants. Sex hormones have been implicated in angiogenesis, a complex, multi-cellular process that requires significant changes in cellular behavior and metabolism. Proliferator-activated receptor gamma (PPARγ) is a transcription factor that sits in the center of these complex behavior changes, as well as many other cellular responses. Additionally, PPARγ is a cofactor with estrogen receptors and has been implicated in BPD and pulmonary hypertension. These factors make PPARγ an ideal target to study in the context of sex differences in pulmonary angiogenesis related to BPD. This approach has not previously been taken. ☐ This dissertation is divided into three main aims that characterize the sexual dimorphism in pulmonary angiogenesis and the factors contributing to these differences. ☐ In Aim 1, we characterized the sexual dimorphism in pulmonary angiogenesis, specifically identifying a sex phenotype. Pulmonary angiogenesis was assessed in vitro using a bead sprouting assay with pooled male or female human pulmonary microvascular endothelial cells in standard (sex-hormone containing) and hormone-stripped medium. We identified a sex-specific angiogenesis phenotype, specifically that male HPMECs produce fewer but longer sprouts than female HPMECs. This male phenotype was also sex hormone-sensitive, while the female phenotype was not. The sex-specific secretome could also influence the sex phenotype in a sex-specific way. Both male and female HPMECs secrete factors that increase female HPMEC sprout length, which is abolished when sex hormones are present. 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. ☐ In Aim 2, we identified sex differences in the proliferative capacity of pulmonary endothelial cells and investigated the role sex hormones play in angiogenesis. Using a Boyden chamber assay, we found there is no sexual dimorphism in HPMEC migration, a cellular process that was also found to be sex hormone insensitive. Pulmonary endothelial cell proliferation was sexually dimorphic, female HPMECs were significantly more proliferative than male HPMECs, but this process was also sex hormone insensitive. These findings suggest that the source of the sexual dimorphism in HPMEC proliferation is intrinsic to the cells. We used a bead sprouting assay to assess sex hormone influence on angiogenesis directly. Female HPMECs produced more sprouts when exposed to estradiol (E2), while male HPMECs produced fewer. This is an interesting finding that warrants further investigation. In contrast, dihydrotestosterone (DHT) treatment resulted in robust and significant increases in angiogenic properties in both male and female HPMECs. While both male and female HPMECs responded positively to DHT, the female response was attenuated compared to the male response. Taken together, sex hormones heavily influence angiogenesis, and the magnitude of the response may be due to intrinsic sex differences. ☐ In aim 3, we characterized pulmonary endothelial cell metabolism, determined the expression profile of PPARγ, and identified the role PPARγ plays in pulmonary angiogenesis. Using the seahorse assay, we found a sexual dimorphism in the total ATP production where male HPMECs produce significantly more ATP/min/cell compared to female HPMECs. Interestingly both male and female HPMECs generate their ATP equally from glycolysis and mitochondrial sources. Further, almost no ATP was generated from FAO, possibly due to a lack of substrate in a hormone-free medium. We also found that these metabolic profiles are estrogen insensitive in male and female HPMECs. Next, we found a sexual dimorphism in PPARγ expression using western blotting, where female HPMECs had a higher PPARγ expression than males. BPD is associated with decreased expression of PPARγ. Male HPMECs exposed to PPARγ agonist upregulated glycolysis for ATP production. Both PPARγ agonist and antagonist significantly increased angiogenesis in both male and female HPMECs. These findings demonstrate that PPARγ is involved in pulmonary angiogenesis and could provide a therapeutic target in the context of BPD. ☐ In summary, the work in this dissertation includes a thorough investigation into the sex differences in pulmonary angiogenesis, the role sex hormones play in this process, and how cellular metabolism in the context of PPARγ influences pulmonary endothelial cell biology as it relates to angiogenesis.
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Keywords
Angiogenesis, Bronchopulmonary dysplasia, Metabolism, Pulmonary endothelial cells, Sex differences, Sex hormones