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<sup>®</sup> Technology

Weiqian E. Lee; Marie Besnier; Elijah Genetzakis; Owen Tang; Katharine A. Kott; Stephen T. Vernon; Michael P. Gray; Stuart M. Grieve; Michael Kassiou; Gemma A. Figtree

doi:10.3390/ijms25010022

International Journal of Molecular Sciences (Dec 2023)

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<sup>®</sup> Technology

Weiqian E. Lee,
Marie Besnier,
Elijah Genetzakis,
Owen Tang,
Katharine A. Kott,
Stephen T. Vernon,
Michael P. Gray,
Stuart M. Grieve,
Michael Kassiou,
Gemma A. Figtree

Affiliations

Weiqian E. Lee: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia
Marie Besnier: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia
Elijah Genetzakis: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia
Owen Tang: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia
Katharine A. Kott: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia
Stephen T. Vernon: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia
Michael P. Gray: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia
Stuart M. Grieve: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia
Michael Kassiou: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia
Gemma A. Figtree: Kolling Institute, University of Sydney, Sydney, NSW 2006, Australia

DOI: https://doi.org/10.3390/ijms25010022
Journal volume & issue: Vol. 25, no. 1
p. 22

Abstract

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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 n = 14) exhibited 30.9% higher mROS levels compared to patients with no CAD when stimulated with oxLDL (n = 14; p 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.

Published in International Journal of Molecular Sciences

ISSN: 1661-6596 (Print); 1422-0067 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Biology (General); Science: Chemistry
Website: http://www.mdpi.com/journal/ijms

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