Abstract 14101: CES1 Deficiency is Associated With Oxidative Stress and Endoplasmic Reticulum/Mitochondrial Injury in Pulmonary Endothelial Cells

Abstract

Background: Pulmonary Arterial Hypertension (PAH) is a life-threatening disease characterized by loss of pulmonary microvessels and vascular remodeling. BMPR2 insufficiency in PAH is associated with oxidative stress and mitochondrial dysfunction but mechanism remain incompletely understood. We recently identified CES1 as a downstream target of BMPR2 and key mediator of EC survival. Carboxylesterase1 (CES1) is an endoplasmic reticulum(ER) enzyme responsible for lipid metabolism, cellular detoxification and redox balance. Here, we explore the consequences of CES1 deficiency and its link to EC dysfunction in PAH. Hypothesis: Loss of CES1 promotes EC dysfunction through oxidative stress and disruption of ER-mitochondrial homeostasis. Methods: PECs were transfected with siRNA/plasmid to knockdown/overexpress CES1. To induce oxidative stress, we used Luperox (100 μM). Chronic hypoxia (10% O 2 for 3 weeks) studies were carried out with male wild type and CRISPR-CES1 KO mice. Hemodynamic measurement and vessel morphometry was performed to assess right ventricular (RV) and vascular remodeling. Mitochondrial and ER structure and function was assessed by confocal microscopy and WB assays. Results: Both BMPR2 and CES1-deficient PECs have higher rates of apoptosis in the context of increased ROS production and mitochondrial fragmentation. Lipid staining with OilRedO demonstrated accumulation of lipid droplets in the cytoplasm of CES1-deficient PECs suggesting disruption in lipid metabolism. CES1-deficient PECs also showed an increase in ER stress markers (CHOP, BiP, IRE-1) compared to controls. Overexpression of CES1 was able to protect PECs against ROS accumulation and apoptosis while increasing tube formation in Matrigel assay. Compared to WT, CRISPR-CES1 KO mice demonstrated higher RV systolic pressures, RV and pulmonary microvessel remodeling in normoxia and with chronic hypoxia. Analysis of CES1-KO lung tissue demonstrates high lipid peroxidation, oxidative damage and reduced number of alveolar vessels. Conclusions: CES1 expression is crucial for PMVEC survival by preventing ROS and maintaining mitochondria/ER homeostasis. Therapeutic interventions to restore CES1 expression could serve as novel treatment approaches for PAH.