High-Throughput Measure of Mitochondrial Superoxide Levels as a Marker of Coronary Artery Disease to Accelerate Drug Translation in Patient-Derived Endothelial Cells Using Opera Phenix® Technology

Abstract

Improved human-relevant preclinical models of coronary artery disease (CAD) are needed to improve translational research and drug discovery. Mitochondrial dysfunction and associated oxidative stress contribute to endothelial dysfunction and are a significant factor in the development and progression of CAD. Endothelial colony-forming cells (ECFCs) can be derived from peripheral blood mononuclear cells (PBMCs) and offer a unique potentially personalised means for investigating new potential therapies targeting important components of vascular function. We describe the application of the high-throughput and confocal Opera Phenix® High-Content Screening System to examine mitochondrial superoxide (mROS) levels, mitochondrial membrane potential, and mitochondrial area in both established cell lines and patient-derived ECFCs simultaneously. Unlike traditional plate readers, the Opera Phenix® is an imaging system that integrates automated confocal microscopy, precise fluorescent detection, and multi-parameter algorithms to visualize and precisely quantify targeted biological processes at a cellular level. In this study, we measured mROS production in human umbilical vein endothelial cells (HUVECs) and patient-derived ECFCs using the mROS production probe, MitoSOXTM Red. HUVECs exposed to oxidized low-density lipoprotein (oxLDL) increased mROS levels by 47.7% (p < 0.0001). A pooled group of patient-derived ECFCs from participants with CAD (n = 14) exhibited 30.9% higher mROS levels compared to patients with no CAD when stimulated with oxLDL (n = 14; p < 0.05). When tested against a small group of candidate compounds, this signal was attenuated by PKT-100 (36.22% reduction, p = 0.03), a novel P2X7 receptor antagonist. This suggests the P2X7 receptor as a valid target against excess mROS levels. As such, these findings highlight the potential of the MitoSOX-Opera Phenix technique to be used for drug discovery efforts in CAD.