(G-278) Sex-Based Angiogenic Phenotypic Differences in Congenital Diaphragmatic Hernia Endothelial Cells

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.

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.