Poster U3 - Response of Pulmonary Artery Endothelial Cells to Shear Stress Conditions in Fontan Patients

Introduction: The objective of this research is to investigate the effects of altered hemodynamics in Fontan patients on endothelial cell (EC) dysfunction leading to pulmonary vascular remodeling (PVR). People born with a congenital heart defect (CHD) resulting in only one functional ventricle will undergo the Fontan operation, that diverts blood flow from the vena cavas to the pulmonary arteries [1-2]. Over time, this procedure can cause complications referred to as “Fontan Failure”, such as PVR, which in turn, reduces cardiac output [3]. PVR is not completely understood; however, vascular wall shear stress (WSS) has been hypothesized to play a major role. These pathological WSSs can lead to EC dysfunction, where various biomarkers are induced and released [4]. These biomarkers may include proteins, lipids, and metabolites, which can be visualized through mass spectrometry imaging (MSI) due to its versatility in analysis and spatial distribution [5].

Materials and

Methods: Human pulmonary arterial endothelial cells (HPAEC) were obtained from PromoCell and grown in Endothelial Growth Medium (EGM) supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin Streptomycin solution, and 1% Trypsin EDTA solution in 25 cm3 culture flasks. Cells will be harvested for experiments at passages 16-20 at 80-90% confluence and suspended in a concentration of 105 cells/ml. The cells will be placed in large 750 ml flasks for sterile gas exchange and rotated on an orbital shaker in the incubator for 48 hours to simulate the WSS of Fontan patients. For MSI analysis, the cell cultures will be prepared, by embedding in gelatin and sectioning/mounting on glass slides. A dihydroxybenzoic acid (DHB) matrix will either be sprayed on the glass slides (HTX Sprayer) or mixed directly with cell pellets. MSI will be utilized in both positive and negative ion modes to characterize the changes in lipids and metabolites induced by the Fontan WSS conditions. The phenotypic signatures will be mapped to identify the spatial changes in each sample and correlated with the local hemodynamics.

Results, Conclusions, and Discussions: The expected results are that there will be an increased antioxidant response, increased production of proinflammatory cytokines, and a dysregulated endothelin-1 (ET-1) signaling. Additionally, it is hypothesized that there will be the induction of ether-linked lipids [6]. Specifically, it is anticipated that cholesterol and eTG will decrease, while ceramides and diacylglycerol will increase [7]. With an increasing number of Fontan patients aging into adulthood it is necessary to understand the mechanics of Fontain failure to ensure quality of life [8]. By using patient specific WSS conditions and spatial multi-omic maps of ECs with MSI, potential targets of PVR and failing Fontan diagnosis may be discovered.

Acknowledgements (Optional): Thank you to the people of the Crouch and Good Labs.

References
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