Sex-Based Angiogenic Phenotypic Differences in Congenital Diaphragmatic Hernia Endothelial Cells

Introduction:: Congenital Diaphragmatic Hernia (CDH) is a fetal developmental disorder affecting one in 2,500 to 4,000 infants each year [1]. Dysregulation of diaphragm formation produces a hole through which the abdominal organs herniate into the thoracic cavity. This leads to decreased space for lung growth resulting in both pulmonary hypoplasia and pulmonary hypertension (CDH-PH) [2]. CDH-PH is associated with changes in the pulmonary vasculature architecture, including increased deposition of the vessel extracellular matrix (ECM) [3], thickening of the vessel walls [4], and decreased number of vessels in the lung [2]. There is a higher incidence rate of CDH in males in some clinical cohorts, but the impact of sex is not well understood [5]. We sought to understand if primary endothelial cells from normal and CDH donors demonstrated sexually dimorphic phenotypic differences in a three-dimensional (3D) in vitro angiogenesis assay.


Materials and Methods:: Bead sprouting assays were performed to quantify 3D angiogenic vessel formation. Primary human umbilical vein endothelial cells (HUVECs) from both CDH and healthy donors (n=3 per group) were individually used in each assay to generate four test groups: male CDH patients, female CDH patients, male control patients, and female control patients. Collagen beads were coated with HUVECs and embedded into a fibrin clot within wells of a 48 well plate, where they were able to proliferate and sprout for 3 days in EGM2 medium. After 3 days, the wells were fixed with 4% paraformaldehyde and 0.1% Triton and stained with hoechst and phalloidin to visualize the nuclei and actin cytoskeleton, respectively. Each well was then imaged via epifluorescent microscopy, and these images were processed through a custom MATLAB image analysis software to determine the number of sprouts per bead, length of the longest sprout, and percentage of beads that had sprouted. A Poisson dispersion test was used to analyze differences in the number of sprouts per bead in each sex between disease states and a two-way ANOVA was used to determine differences in the length of the longest sprout per bead in each sex between disease states.


Results, Conclusions, and Discussions:: In culture, CDH cells are clearly distinguishable from healthy donors cells, whereby CDH HUVECs are more spread and elongated in static culture with a tendency to form less confluent monolayers. Control and CDH donors were able to generate angiogenic sprouting in a 3D in vitro assay with ~80% of the total beads that were seeded with HUVECs generating at least one angiogenic sprout in all experimental groups. Male cells from healthy donors had significantly more sprouts and longer sprouts than those from healthy female donors. Interestingly, cells from male CDH donors produced the same number of sprouts as cells from male healthy donors, except the sprouts were shorter. Although healthy female cells produced fewer and shorter sprouts than healthy male cells, there was no observed difference in the number or length of sprouts between healthy and CDH female cells. Some of these results may be the result of differential responses to pro-angiogenic factors in the culture medium as has been shown in other endothelial cells [6]. These studies demonstrate an in vitro assay that can be used to dissect sex-based and CDH-based mechanisms that underpin differences observed in the vascular development from clinical studies.

Acknowledgements (Optional): :

References (Optional): : [1] W.-Y. Teo, et al. PEDN. 2020 [2] P. K. Chandrasekharan, et. al. Matern. Health Neonatol. 2017 [3] M. N. Monroe, et. al. Pediatr. Pulmonol. 2020 [4] R. Tenbrinck, et. al. PEDN. 1992 [5] S. R. Sferra, et. al. J Pediatr. 2023 [6] B. Hayward-Piatkovskyi, et. al. Am. J. Physiol. 2022