Integrative investigation of lipidome and signal pathways in human endothelial cells under oxidative stress

Abstract

Phospholipids in human endothelial cells (ECs), cell line EA.hy926, were profiled by a novel lipidomics approach, combining liquid chromatography (LC)–ion trap mass spectrometry (MS) and LC–tandem quadrupole MS. More than 200 species of phospholipids were quantified. Twenty-eight were identified as the most discriminant species in response to different levels of oxidative stress induced by hydrogen peroxide (H₂O₂). H₂O₂ treatment induced phosphorylation of cytosolic phospholipase A₂ (cPLA₂) via the activation of extracellular-regulated kinase 1/2 (ERK1/2), increasing the production of lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC). The release of arachidonic acid (AA, 20 : 4) increased from no H₂O₂ exposure to 1 h exposure, decreased from 1 h to 2 h, and increased again from 2 h to 4 h exposure time. The particular increase seen of phosphatidylcholine (PC) species that include AA chains from 1 h to 2 h indicates that the released AA is reincorporating into PC molecules to reduce the extension of the AA cascade. The change in free AA levels seen suggests possible defense mechanisms to oxidative injury in ECs. We further verified nine species as potential biomarkers by adding inhibitor and demonstrated direct correlation to the activity of the cPLA₂–AA pathway. The oxidative injury to cell line EA.hy926 provided a novel application for a combined lipidomics and signal transduction approach. This combined approach has enabled future investigations for possible therapeutic interventions in phospholipids and cPLA₂ activity for defense against oxidative cellular stress.