Untargeted metabolomics and proteomics reveal the versatile effects of myeloperoxidase-oxidized LDL on endothelial cells.

Background: Myeloperoxidase-oxidized LDLs (Mox-LDLs) trigger endothelial cells and contribute to the development of atherosclerosis. However, the mechanisms underlying Mox-LDL-induced stimulation remain elusive. In this study, we applied untargeted metabolomics and proteomics approaches to investigate human umbilical vein endothelial cells (HUVECs) following exposure to Mox-LDLs.

Methods: HUVECs were exposed for 24 h to Mox-LDLs (0 or 100 μg/ml) with or without native LDLs (0 or 1 mg/ml). Supernatants and cell lysates were analyzed by liquid chromatography coupled to mass spectrometry. Using Workflow4Metabolomics work environment, MZmine, SIRIUS and MetGem, we selected and identified key metabolites influenced by Mox-LDL treatment. For the proteomics analysis, we used DIA-NN and the FragPipe-Analyst application to detect proteins differentially expressed after Mox-LDL treatment.

Results: Metabolomics analysis revealed increased levels of sphingolipids, phospholipids and oxidized-cholesterol derived compounds in HUVECs following Mox-LDL exposure. We also detected an increase in small peptides, likely reflecting Mox-LDLs catabolism. Mox-LDL treatment of HUVECs also altered the expression of proteins involved in hemostasis, cell adhesion, angiogenesis, inflammation and stress responses. In addition, proteins from the mitochondrial respiratory chain were upregulated after Mox-LDL treatment. Finally, a trihydroxy-unsaturated fatty acid was secreted by HUVECs exposed to Mox-LDLs and could serve as a biomarker of Mox-LDL exposure.

Conclusions: Our findings suggest that Mox-LDLs are internalized and degraded by HUVECs. They seem to induce increased mitochondrial activity and oxidative stress, likely mediated by reactive oxygen species. We believe that HUVECs activate cytoprotective antioxidant coping mechanisms (glutathione synthesis, heme oxygenase-1…) to survive. Mox-LDLs may also modulate hemostasis and inflammatory responses.