O1042: Alterations of endothelial cell bioenergetics in human congenital diaphragmatic hernia

Background: Pulmonary vasculature in congenital diaphragmatic hernia (CDH) is characterized by significant pathologic remodeling, which contributes to pulmonary hypertension (PH). Recent data suggest that changes in endothelial cell (EC) mitochondrial metabolism are important to pathogenesis of PH. Mitochondria are semiautonomous organelles that govern cellular bioenergetic capacity, alter cellular redox states through production of reactive oxygen species (ROS), and control apoptosis. As such, mitochondria regulate many downstream effectors of hypoxic pulmonary vasoconstriction and vascular remodeling and could be exploited for novel diagnostics and therapeutics in CDH.

Methods: Human umbilical vein endothelial cells were taken from healthy newborns (n=21) and those with CDH (CDH EC, n=12) to use as a model of CDH vasculature. We applied an extracellular flux analyzer to compare mitochondrial bioenergetics between groups and correlate bioenergetic changes with clinical outcomes. Mitochondrial and cellular functions were evaluated via flow cytometry. Finally, single cell RNA sequencing (scRNA-seq) was employed to determine whether gene expression patterns supported changes in pulmonary EC mitochondria from the nitrofen rat model of CDH.

Results: CDH EC mitochondria demonstrated higher basal and maximal oxygen consumption rates (OCR) compared to control ECs (Fig.1A-B). Maximal respiration was highest in CDH patients with MRI observed/expected total lung volumes (O/E TLV) < 40% (compared to >40%) and in those with >20 day hospital length of stay (LOS) or death compared to surviving patients with LOS < 20 days (Fig.1C-D). CDH EC also displayed increased non-mitochondrial oxygen consumption (Fig.1E), increased proton leak (Fig.1F), and decreased ATP coupling efficiency (Fig.1G), suggesting that CDH EC generate higher ROS and require more oxygen to generate ATP (Fig.1H). Flow cytometry confirmed that these bioenergetic changes were associated with elevated superoxide production, increased cellular ROS, and decreased inner mitochondrial membrane mass (Fig.1I-K). Mitochondrial membrane potential and baseline NO levels were unchanged. Transcriptomic data from the nitrofen model at E21.5 (late fetal stage) also indicated that there are alterations in pulmonary endothelial cell bioenergetics and increased ROS production based on findings of decreased RNA expression of multiple electron transport chain (ETC) complexes and genes that regulate mitochondrial fission and mitophagy (Fig.2A-C).

Conclusion: Our data show that human CDH EC exhibit alterations in bioenergetics that correlate with more severe CDH and that are associated with increased oxidative stress. Observed changes in inner mitochondrial membrane mass imply changes in mitochondrial structure and/or mitophagy. Transcriptomic changes in the nitrofen model further suggest decreased expression of ETC complexes and mitochondrial dysfunction in CDH pulmonary endothelial cells. Together, these findings highlight mitochondrial dysfunction as a potential contributor to pulmonary hypertension in CDH and a promising target for future therapies.